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The Dementias Early Diagnosis and Evaluation

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The Dementias Early Diagnosis and Evaluation

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The Dementias

The Dementias
Early Diagnosis and Evaluation

edited by
Karl Herholz
Wolfson Molecular Imaging Centre
University of Manchester
Manchester, U.K.

Daniela Perani
Vita-Salute San Raffaele University
San Raffaele Scientific Institute
Milan, Italy

Chris Morris
Wolfson Unit of Clinical Pharmacology
University of Newcastle upon Tyne
Newcastle upon Tyne, U.K.

New York London

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Preface

Our motivation to compile this book stems from our exposure to a number of
issues that are difficult to reconcile. Most people probably welcome the increase
in longevity that is observed in many countries today, but this also brings with it a
substantial increase in dementia with prevalence and incidence rising steeply
beyond 70 years of age. It is widely acknowledged that this imposes severe
stress on families and caregivers and risks the failure of social welfare systems.
Dementia may therefore become one of the most difficult challenges for societies
in this century. Yet, we are far away from a medical solution. Even if a drug that
could slow or halt the progression of dementing diseases was made available
tomorrow, it probably would have only limited effect since too much brain
function has been lost to degenerative brain disease once the clinical symptoms of
dementia become clearly evident. Restoration of normal cognition will therefore
remain an elusive goal. Thus, there is the danger that any available drug would
either be given too late to prevent dementia and its consequences, or it would be
given indiscriminately to people with nonspecific complaints or even without any
symptoms. Since only a small proportion of these individuals are likely to
develop dementia within the next few years, precious medical resources would be
wasted. Early and accurate diagnosis of dementing diseases before the onset
of dementia therefore becomes of paramount importance if we are to avoid such
a situation and use preventative treatment strategies efficiently once they become
available.
Over the past few years, many new methods and techniques have been
developed to improve early diagnosis. Most of these require special training and
expert knowledge for application and an understanding of their potential and
limitations. In this book, active researchers who are experts in their respective
fields present and evaluate these methods to provide a guide to their use in early

iii
iv Preface

dementia diagnosis and assessment. It is, however, quite clear that no single
method can cover all aspects that are relevant for early diagnosis. With this
overview we hope to provide comprehensive information to clinicians,
researchers, and the pharmaceutical industry about which methods are the most
effective ones for screening, early diagnosis of symptomatic subjects, and
distinguishing between different dementing diseases. While we cannot yet provide
definitive answers to all these questions, we hope that the evaluation will provide
guidance for further research.
Clinical evaluation and care are the basis of dementia diagnosis and
treatment, and we therefore put the chapter on clinical issues at the beginning.
Neuropsychology provides methods for the objective and quantitative assessment
of symptoms and behavioral and cognitive deficits, and therefore this naturally
takes its place close to clinical evaluation. Next comes cerebrospinal fluid and
blood biomarkers that hold promise in complementing symptom-based diagnosis
by adding biochemical information on the underlying disease process. Chapters
on genetics and novel molecular targets provide the latest information on risk
factors and molecular mechanisms with their perspectives for early diagnosis.
After that we enter the wide arena of in vivo imaging methods, which have
undergone rapid development and bewildering diversification in recent years,
ranging from structural imaging with computed tomography and magnetic
resonance imaging to functional and molecular imaging with functional magnetic
resonance imaging, magnetic resonance spectroscopy, single-photon emission
computed tomography, and positron emission tomography. Most of these are
ready to be applied in the clinical arena, but quite often their actual value and
efficacy is still under dispute. Authors therefore explain the techniques and
provide information on diagnostic sensitivity and specificity when available. The
methods chapters are then concluded with a view from neuropathology, which
still provides the most detailed methods for tissue examination and therefore
sets the diagnostic gold standard, although the significance of post-mortem
studies in early dementia is limited because of the lack of clinical follow-up data,
for obvious reasons. We did not want to leave our readers with just a compilation
of methods and data but felt that an overview and final evaluation, although
necessarily somewhat subjective, should conclude the book. We also address
some of the wider implications of improved early diagnosis that need considera-
tion when we enter this extremely important clinical field, which may hold
dangers, but hopefully many surprises as well.

Karl Herholz
Daniela Perani
Chris Morris
Acknowledgments

We are very grateful to the authors, who are all busy researchers but dedicated
a significant amount of their time to the book. Their contribution cannot be
overestimated. This interdisciplinary project would probably not have been
possible without previous scientific collaboration of the editors and some of the
authors, which was supported by the European Commission in their framework
programs 4 to 6. Last but not least, we thank the publisher’s editorial and
production team, especially Geoff Greenwood and Dana Bigelow, for their
continuous encouragement and support. The publication of this book was
generously supported by an educational grant from GE Healthcare.

v
Contents

Preface . . . . iii
Acknowledgments . . . . v
Contributors . . . . xiii

1. The Clinical Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1


Norman L. Foster
Introduction . . . . 1
Dementia Evaluation . . . . 2
Difficult Diagnostic Crossroads and Decision
Points . . . . 14
Summary . . . . 25
References . . . . 26

2. Neuropsychological Screening and Advanced


Neuropsychological Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Daniela Perani
Introduction . . . . 33
Neuropsychological Assessment . . . . 34
Assessment of Psychological and Behavioral
Disturbances . . . . 45
Functional Scales . . . . 46
The Role of Neuropsychological Assessment of
Dementia . . . . 48

vii
viii Contents

Advanced Tests and Future Research


Approaches . . . . 56
Conclusions . . . . 60
References . . . . 60

3. Biomarkers in Blood and Cerebrospinal Fluid . . . . . . . . . . 73


Harald Hampel and Katharina Buerger
Introduction . . . . 73
b-Amyloid Peptide 40 and 42 . . . . 74
Amyloid Protein Precursor . . . . 77
Tau Proteins . . . . 78
Apolipoprotein E . . . . 82
Isoprostanes . . . . 83
a1-Antichymotrypsin . . . . 83
The Soluble Interleukin-6 Receptor
Complex . . . . 84
C-Reactive Protein . . . . 85
C1q . . . . 85
Homocysteine . . . . 86
Oxysterols and Cholesterol
Metabolism . . . . 87
3-Nitrotyrosine . . . . 88
14-3-3 Protein in Creutzfeldt–Jakob
Disease . . . . 89
Perspective of Biomarker Research in AD:
b-Amyloid Antibodies . . . . 89
Discussion . . . . 90
References . . . . 94

4. Genetics: Facts and Perspectives . . . . . . . . . . . . . . . . . . . . . . . 109


Sandro Sorbi, Paolo Forleo, Francesca Massaro,
and Andrea Ginestroni
Introduction . . . . 109
Summary . . . . 129
References . . . . 129
Contents ix

5. Approaches to the Identification of Novel Diagnostic


and Therapeutic Targets in Dementia . . . . . . . . . . . . . . . . . . 137
Kate E. Wilson and Chris Morris
Introduction . . . . 137
Functional Genomics . . . . 138
Microarray-Based Gene Expression
Profiling . . . . 138
Proteomics . . . . 142
Fluid Analysis . . . . 147
Conclusions . . . . 150
References . . . . 151

6. Quantitative and Functional Magnetic Resonance


Imaging Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Giovanni B. Frisoni and Nicola Filippini
Image Acquisition: Technical Features . . . . 157
Quantitative Magnetic Resonance Imaging Tools to Rate
Regional Atrophy and Cerebrovascular
Disease . . . . 163
Quantitative Magnetic Resonance Imaging for the
Clinical Diagnosis . . . . 170
Microstructural Imaging: Diffusion and Magnetization
Transfer Imaging . . . . 179
Functional Magnetic Resonance Imaging in Dementias
and Mild Cognitive Impairment . . . . 182
References . . . . 188

7. Perfusion Imaging with Single Photon Emission


Computed Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Nadine J. Dougall and Klaus P. Ebmeier
Single Photon Emission Computed
Tomography . . . . 197
Blood Flow Tracers . . . . 198
Alzheimer’s Dementia . . . . 199
Techniques to Improve Diagnostic Accuracy . . . . 202
Cohort Studies . . . . 204
Vascular Dementia . . . . 204
x Contents

Frontotemporal Dementia . . . . 210


Dementia with Lewy Bodies . . . . 215
Mild Cognitive Impairment . . . . 215
Conclusion . . . . 222
References . . . . 222

8. FDG PET: Imaging Cerebral Glucose Metabolism


with Positron Emission Tomography . . . . . . . . . . . . . . . . . . . 229
Karl Herholz
Positron Emission Tomography . . . . 229
Measurement of Local Cerebral Glucose
Metabolism . . . . 230
Main Findings in Alzheimer’s Disease . . . . 231
Longitudinal Studies and Trials . . . . 235
Early Diagnosis of Alzheimer’s Disease . . . . 236
Clinical Diagnostic Use of 18F-2-Fluoro-2-Deoxy-
D-Glucose Positron Emission Tomography in
Alzheimer’s Disease . . . . 238
Dementia with Lewy Bodies . . . . 238
Frontotemporal Dementia . . . . 238
Vascular Dementia . . . . 240
Creutzfeldt-Jakob Disease . . . . 241
Summary . . . . 241
References . . . . 242

9. Imaging of Neurotransmitter Systems in Dementia . . . . 253


Steen G. Hasselbalch and Gitte M. Knudsen
Introduction . . . . 253
Normal Aging . . . . 255
Alzheimer’s Disease . . . . 257
Lewy Body Dementia . . . . 266
Frontotemporal Dementia . . . . 268
Cognitive Dysfunction Due to Cerebrovascular
Disease . . . . 269
Conclusions . . . . 270
References . . . . 270
Contents xi

10. Development and Application of b-Amyloid Imaging


Agents in Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . 279
William E. Klunk, Robert D. Nebes, Nicholas Tsopelas,
Brian J. Lopresti, Julie C. Price, Steven T. DeKosky,
and Chester A. Mathis
Introduction . . . . 279
Development of Amyloid Imaging
Technologies . . . . 282
Future Applications of Amyloid Imaging . . . . 297
Summary . . . . 302
References . . . . 303

11. A View on Early Diagnosis of Dementias from


Neuropathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Kurt A. Jellinger
Introduction . . . . 311
Classification of Dementia Disorders . . . . 312
Basic Pathology of Major Dementing
Disorders . . . . 312
Early Pathology of AD and Clinical
Correlates . . . . 352
Early Morphological Changes in Dementia Disorders
and Relation to Biomarkers . . . . 367
Correlation of Clinical and Pathological
Criteria . . . . 373
Effects of Concurrent Pathologies . . . . 373
Neuropathology of AD and Clinical
Relevance . . . . 374
Conclusions . . . . 375
Abbreviations . . . . 375
References . . . . 378

12. Guideline and Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429


Karl Herholz, Daniela Perani, and Chris Morris
Introduction . . . . 429
Diagnosis of Mild Manifest Dementia . . . . 430
xii Contents

Nondemented Subjects with Cognitive


Impairment . . . . 433
Asymptomatic Subjects . . . . 436
Monitoring Treatment . . . . 437
Conclusion . . . . 439
References . . . . 439

Index . . . . 443
Contributors

Katharina Buerger Alzheimer Memorial Center and Geriatric Psychiatry


Branch, Department of Psychiatry, Ludwig-Maximilian University,
Munich, Germany

Steven T. DeKosky Departments of Psychiatry and Neurology, University


of Pittsburgh, Pittsburgh, Pennsylvania, U.S.A.

Nadine J. Dougall Division of Psychiatry, Gordon Small Center for


Research in Old Age Psychiatry, University of Edinburgh, Edinburgh, U.K.

Klaus P. Ebmeier Division of Psychiatry, Gordon Small Center for


Research in Old Age Psychiatry, University of Edinburgh, Edinburgh, U.K.

Nicola Filippini Laboratory of Epidemiology Neuroimaging and


Telemedicine (LENITEM), IRCCS San Giovanni di Dio FBF—The National
Center for Research and Care of Alzheimer’s Disease, Brescia, Italy

Paolo Forleo Department of Neurological and Psychiatric Sciences,


University of Florence, Florence, Italy

Norman L. Foster Imaging and Research, Center for Alzheimer’s Care,


and Department of Neurology and The Brain Institute, University of Utah,
Salt Lake City, Utah, U.S.A.

Giovanni B. Frisoni Laboratory of Epidemiology Neuroimaging and


Telemedicine (LENITEM), IRCCS San Giovanni di Dio FBF—The National
Center for Research and Care of Alzheimer’s Disease, Brescia, Italy

xiii
xiv Contributors

Andrea Ginestroni Department of Neurological and Psychiatric Sciences,


University of Florence, Florence, Italy
Harald Hampel Alzheimer Memorial Center and Geriatric Psychiatry
Branch, Department of Psychiatry, Ludwig-Maximilian University,
Munich, Germany
Steen G. Hasselbalch Neurobiology Research Unit and Memory Disorders
Research Group, Copenhagen University Hospital, Rigshospitalet,
Copenhagen, Denmark

Karl Herholz Wolfson Molecular Imaging Centre, University of


Manchester, Manchester, U.K., and Department of Neurology, University
of Cologne, Cologne, Germany
Kurt A. Jellinger Institute of Clinical Neurobiology, Vienna, Austria

William E. Klunk Department of Psychiatry, University of Pittsburgh,


Pittsburgh, Pennsylvania, U.S.A.

Gitte M. Knudsen Neurobiology Research Unit, Copenhagen University


Hospital, Rigshospitalet, Copenhagen, Denmark

Brian J. Lopresti Department of Radiology, University of Pittsburgh,


Pittsburgh, Pennsylvania, U.S.A.

Francesca Massaro Department of Neurological and Psychiatric Sciences,


University of Florence, Florence, Italy

Chester A. Mathis Departments of Radiology, Pharmacology, and


Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh,
Pennsylvania, U.S.A.
Chris Morris Wolfson Unit of Clinical Pharmacology, Chemical Hazards
and Poisons Division–Newcastle, Health Protection Agency, and School of
Neurology, Neurobiology, and Psychiatry, The Medical School, University of
Newcastle upon Tyne, Newcastle upon Tyne, Tyne and Wear, U.K.
Robert D. Nebes Department of Psychiatry, University of Pittsburgh,
Pittsburgh, Pennsylvania, U.S.A.
Daniela Perani Departments of Neuroscience and Nuclear Medicine,
Vita-Salute San Raffaele University and San Raffaele Scientific Institute,
Milan, Italy
Julie C. Price Department of Radiology, University of Pittsburgh,
Pittsburgh, Pennsylvania, U.S.A.
Contributors xv

Sandro Sorbi Department of Neurological and Psychiatric Sciences,


University of Florence, Florence, Italy
Nicholas Tsopelas Department of Psychiatry, University of Pittsburgh,
Pittsburgh, Pennsylvania, U.S.A.
Kate E. Wilson Institute for Ageing and Health, University of Newcastle,
Newcastle General Hospital, Newcastle upon Tyne, Tyne and Wear, U.K.
1
The Clinical Issues

Norman L. Foster
Imaging and Research, Center for Alzheimer’s Care, and Department
of Neurology and The Brain Institute, University of Utah, Salt Lake City,
Utah, U.S.A.

INTRODUCTION
The diagnosis and management of dementia will be a major challenge for health
care systems in the coming decades. As the world’s population ages, the
prevalence of dementia will grow dramatically (1). Alzheimer’s disease (AD),
the most common cause of dementia, accounts for much of the increasing
prevalence of dementia with age (2). However, many other causes of dementia,
such as stroke and Parkinson’s disease, also increase dramatically as people
become older. The care of those with dementia causes great burdens on caregivers
and society. The number of individuals with dementia will grow most rapidly in the
developing world, but dementing diseases will have their greatest social impact in
Japan, Europe, and North America, where their proportion of the population will
be highest. Prevention and improved diagnosis and treatment of cognitive
impairment and dementia offer the only hope to alter this grim equation.
Fortunately, there has been an explosion of information about neuro-
degenerative diseases, and dementia has moved from being a backwater to the
vanguard of neuroscience research. New insights and conceptual breakthroughs
have led to improved detection and evaluation of dementia, and there are many
more new approaches now in development. There is every reason to expect these
advances will continue and be translated into clinical practice. Awareness and
application of these recent developments provide great opportunities for
physicians to help patients and society.

1
2 Foster

DEMENTIA EVALUATION
The Role of Dementia Evaluation
Patients and families have many reasons for seeking a physician’s advice when
memory loss or cognitive impairments appear (Table 1). The hope of treatment that
improves symptoms is a major motivation, but other important concerns also
should be addressed. Physicians too often focus solely on treatment and fail to
recognize the great value patients and families place on accurate diagnosis. There
is comfort in understanding the cause of symptoms and in knowing that everything
that should be done has been done. An accurate and specific diagnosis also
empowers physicians. Once the cause of dementia is determined, the physicians
can use their specialized knowledge of the disease to guide management and
coordinate care. It becomes possible to develop a rational and evidence-based care
plan appropriate for that disease. Family members also may be seeking help for
themselves. An authoritative dementia evaluation can resolve conflicting opinions
among family members and clarify their role in care. With a detailed management
plan and prognostic information, patients, families, and physicians can plan for the
future together.
Physicians often are reluctant to evaluate dementia because it opens a
“Pandora’s box” of concerns they aren’t prepared to address (3). Many physicians
lack the experience and confidence to perform an evaluation. Even if an evaluation
is performed, physicians may be reluctant to inform patients and families of their
diagnosis (4). Information about prognosis also may be withheld, even though it
greatly benefits patients and their families (5). Usually patients and families aren’t
satisfied until they learn the specific cause of cognitive impairment; generalities,
platitudes, and nostrums aren’t sufficient. In one study, caregivers reported that a
correct diagnosis of AD was provided in only 38% of initial physician
consultations, causing them to seek care from someone else (6). Most recognize
the limits of medicine, and appreciate a definitive diagnosis, even when few drug
treatments can be offered. Patients with suspected neurological disease have less
distress about their symptoms and less anxiety when they receive a definitive
diagnosis (7). Physicians therefore should use whatever effective means are
available to accurately determine the cause of dementia.

Table 1 Motivations for Seeking a Dementia Evaluation


Determine whether perceived memory loss or cognitive decline is truly present
Seek a specific medical explanation for the cause of symptoms
Find a treatment that can improve symptoms
Understand what can be expected in the future and how to prepare for it now
Learn whether other family members are at risk of developing similar problems
Know when and how to obtain help
Hope for continued support and better treatments in the future
The Clinical Issues 3

Dementia evaluations require complex medical decision-making. There are


no shortcuts or easy simplifications. Dozens of diseases that cause dementia must
be distinguished (8). Clinical medicine offers no more difficult exercise in
differential diagnosis. It is important not to underestimate the effort required for
an accurate assessment. One must provide adequate time to obtain a detailed
history and to perform a comprehensive mental status and neurological
examination. Although daunting, physicians can find great satisfaction and
intellectual reward in uncovering and treating cognitive impairment.
The evaluation of cognitive impairment and dementia consists of three
distinct steps: (1) the recognition of cognitive deficits, (2) an initial assessment to
decide whether dementia is present, and (3) when impairment is confirmed, the
determination of its cause. Physicians often claim to have “diagnosed” dementia
after completing only one of these steps. However, diagnosis is incomplete until
all three are accomplished. Each step has distinct challenges and requires
considerable clinical judgment.

Recognizing Impairment in Cognition


It is relatively easy to recognize sudden or severe cognitive loss, but substantial
effort can be required when deficits are subtle or gradually progressive. Patients
and families may identify explicit cognitive problems, but at least as often
complaints are nonspecific or symptoms become evident to the physician only
indirectly. Health professionals should be alert to “triggers” that suggest
cognitive impairment (Table 2). This is necessary because patients often fail to
mention that they have cognitive symptoms. This may be out of fear that they will
lose their independence. Some patients are unable to recognize deficits as a result
of their disease (anosognosia). Others may find it embarrassing to discuss

Table 2 Situations that Should Trigger a Dementia Evaluation


Patient complaints or concerns of others
Persistent and progressive memory loss
Inability to learn and retain new information
Difficulty handling complex tasks
Poor reasoning ability
Trouble with spatial ability and orientation
Difficulty with language
Change in behavior
Observations during medical care
Difficulty following directions
Inability to provide an accurate medical history
Vague or evasive responses to direct questions
Appearing dependent on others for information or financial assistance
Non-compliance or confusion about appointments and treatment
Irritable or indifferent when asked direct questions
4 Foster

cognitive complaints, particularly since for many there continues to be a


considerable social stigma attached to mental problems. Family members also
may fail to reveal their concerns about a patient’s cognitive problems, either out
of respect for their relative’s dignity or reluctance to interfere. To overcome these
barriers, physicians must establish rapport and be compassionate and sensitive to
these issues. Often a simple and direct question, such as “Any trouble with
memory?”, is sufficient to identify a subtle cognitive impairment.
Current methods of screening for cognitive impairment are too imprecise
and time-consuming to apply to the general population, or even to all elderly
people (9). However, elderly individuals seeking medical care have a much higher
prevalence of dementia, and the routine use of a brief cognitive screen in the
physician’s office may be appropriate. When there are complaints or “triggers”
suggesting cognitive problems, an initial assessment for dementia is essential (10).
The evaluation of dementia must consider different factors in each
individual. A change in cognitive abilities can be determined only when a
patient’s literacy, education level, occupation, and usual activities are considered.
Once concern about cognitive impairment is raised, a careful history from the
patient and at least one collateral source (family member, friend or employer) is
needed to determine whether there truly has been a change and whether it is
progressive. A collateral source is necessary since patients may not be able to
recognize their problems or report them accurately or with sufficient detail. When
the reliability of patient responses appears uncertain, it often is prudent to defer a
medical history until another source is available to confirm facts and to avoid
basing judgments on misinformation. Interpreting a history of cognitive
impairment requires considerable clinical skill. Depressed patients often perceive
more memory loss than they truly have (11). Unrecognized visual problems or
hearing loss may cause inaccurate responses that are mistaken for cognitive
impairment. Confirming cognitive impairment can be particularly difficult in
patients with legal or financial motives for initiating an assessment. A single visit
may be insufficient, and serial examination may be required to confirm that the
clinical course is stable or progressive.

Deciding Whether Dementia Is Present


After identifying cognitive impairment, the second step in evaluation is to
determine whether dementia is present. Dementia is defined as a decline in two or
more cognitive domains, such as memory, language, visuospatial processing,
abstraction, and judgment, that is sufficient to cause impairments in everyday
activities. The requirement for functional impairment means that dementia
represents a more severe change in cognition and is more certain to be due to a
true brain disease. Furthermore, the standard of functional impairment caused by
cognition provides an independent measure of significant decline with face
validity. By requiring impairment in at least two cognitive domains, the definition
of dementia implies an extensive involvement of brain function. Deficits in a
The Clinical Issues 5

single cognitive domain, such as aphasia, apraxia or alexia, can be caused by


damage in discrete areas in a single cerebral hemisphere, and alone do not
constitute dementia. Memory loss is a particularly important sign in dementia.
Although amnesia alone is insufficient to establish dementia, it is an indication
that several brain regions may be affected, since memory loss is not localizable to
a single cerebral hemisphere.
A careful mental status assessment is needed to be certain that symptoms
cannot be explained by damage in a single anatomical region or a single cognitive
domain. Intellectual function is multifaceted, and occasionally the physician and
patient are so drawn to a single component of cognition that they miss the full
extent of impairment. Several methods of assessing each cognitive domain
should be used. For example, one should test both verbal and non-verbal stimuli
to make sure that memory problems can’t be attributed solely to aphasia.
Determining whether there truly is functional impairment and if it can be
explained by cognitive loss require astute clinical judgment. Since cognitive
difficulties usually develop insidiously, patients and their families often adapt to
them easily. As a result, they may not recognize the loss in performance, or they
may attribute it to factors other than dementia. For example, spouses may
gradually take over cooking or family finances without considering that this was
necessitated by changes in their mate. Likewise, inability to shop independently
may be misattributed to arthritis, depression, or simply disinterest, rather than to
cognitive loss. The physician faces the challenge of analyzing the evidence
without simply accepting explanations patients and families provide, and of
deciding whether functional decline is due to cognitive disability, physical
disability, or both. It is important to consider typical daily activities and
occupation throughout life to assess whether current deficiencies truly represent a
change in abilities sufficient for dementia.
Cognitive impairment can be clinically significant, yet insufficient to cause
functional limitation or represent dementia. Patients with problems in a single
cognitive domain often avoid functional impairments by compensating with
strengths in other cognitive areas. Stroke is a common cause of circumscribed
cognitive deficits. For example, patients with Broca’s aphasia due to a left
hemisphere stroke may have severe aphasia, yet remain independent. Difficulties
compensating for deficits that seem limited to a single cognitive domain suggest
that problems are actually more pervasive than suspected; the patient may
have dementia.
It is logical to expect that patients developing dementia initially would have
impairment only in a single cognitive domain. Likewise, impairments insufficient
to affect everyday activities should occur before dementia develops. These
precepts underlie recent efforts to recognize prodromal dementia. There have
been many attempts to define characteristics that might identify individuals who
will eventually develop dementia. Currently, the terms most widely accepted for
this situation are mild cognitive impairment (MCI) (often used in a clinical
context) and cognitive impairment, not demented (CIND) (frequently used in
6 Foster

epidemiological studies). Criteria for these conditions are not consistent from
report to report, and their use should be scrutinized carefully. The prevalence of
cognitive impairment insufficient to cause functional impairment appears to be
greater than the prevalence of dementia, but more studies are needed (12).
Despite the difficulties of definition, recognizing these conditions has more than
heuristic value. They represent a group of patients with a significantly increased
risk of developing dementia over subsequent years and are an appropriate target
for new therapies (13).
It may be helpful to distinguish different kinds of cognitive impairment
insufficient to be classified as dementia. Since different dementing diseases have
predominant deficits in different cognitive domains, it is logical to assume that
their initial symptoms also differ. For example, isolated memory impairment
would be expected to precede the development of dementia in AD, where memory
loss usually is the most prominent symptom. Likewise, progressive aphasia may
precede frontotemporal dementia (FTD) (14). Thus individuals with amnestic
MCI and predominant or isolated memory loss are believed to be more likely to
progress to dementia caused by AD, and those with non-amnestic MCI may
precede other forms of dementia (15). It also may be useful to classify patients by
presumed etiology, since this can have prognostic significance (16,17).
Deriving reliable criteria to identify prodromal dementia based purely upon
clinical grounds turns out to be more difficult than it might first appear. Not only
must multiple areas of cognition be considered, but boundaries of both normal
cognition for age and impaired cognition sufficient to represent dementia also must
be defined. Detailed neuropsychological testing provides age-appropriate norms to
guide clinical judgment and explicit operational criteria for clinical trials. MCI
differs from CIND by requiring a voiced complaint of impairment. An expressed
memory complaint is important because it is a strong predictor of AD and a face
valid criterion for a change in ability (18). Individuals categorized as having MCI
are more likely than those with CIND to progress to unequivocal dementia (13,19).
In a diverse clinical population where diagnosis and management must be
provided to everyone, the early recognition of prodromal dementia must rely on
physician judgment and interpretation, rather than solely on neuropsychological
test results or strict application of criteria.

Determining the Cause of Cognitive Impairment and Dementia


The final and critical step in a dementia evaluation is to determine the specific cause
of significant cognitive impairment. This is valuable for many reasons (Table 3).
There are a large number of potential causes to consider. Neurodegenerative
diseases are the most common, but systemic medical illnesses and many other
neurological disorders also can alter brain function and lead to dementia.
A complete review of all dementing diseases is beyond the scope of this chapter,
but the physician must consider all categories of disease during a dementia
The Clinical Issues 7

Table 3 Benefits of Determining the Specific Cause of Dementia


Choose drugs that are known to be effective in that particular disease
Avoid drugs that are not appropriate for that disease, known to be ineffective, or are
contraindicated
Know what disease complications to expect
Avoid unnecessary evaluations when a new symptom consistent with the disease develops
Plan for future care based upon the known clinical course of the specific disease
Educate caregivers about the cause of the disease, its symptoms, and recent research
advances
Answer (sometimes unspoken) questions about family risk based upon genetics of the
disease

evaluation, including genetic, metabolic, infectious, neoplastic, and vascular


disorders (8).
There is a great opportunity and need to improve the methods used to
recognize specific causes of cognitive impairment. Currently, we must rely on the
clinical approaches of evaluating the medical history and examination and a few
informative laboratory studies. The medical history is used to reconstruct the
clinical course of the illness, noting the appearance and timing of symptoms.
Physical and mental status examinations then are used to confirm and expand the
character of the illness. Finally, this information and the results of laboratory
testing and brain imaging are compared to typical clinical features of specific
dementing illnesses (Table 4). The best match between a patient’s findings and
the characteristic features of a disease identifies the specific diagnosis, with the
degree of congruence generally indicating the level of diagnostic certainty.
Consensus diagnostic criteria and clinical guidelines are available for MCI and
the most common dementing disorders and can be very helpful. Criteria are
particularly valuable in communicating diagnostic information among health
providers and in establishing a defined, homogeneous group of patients for
research studies. However, clinical criteria provide only guidelines and are not
applicable in all situations; clinical judgment is still necessary.
Some dementing diseases cause such distinctive abnormalities that they
markedly assist diagnosis (Table 5). Other physical abnormalities are less
specific. However, performance of a detailed neurological examination is critical,
because it can identify subtle and asymmetric motor signs and gait abnormalities
that suggest focal lesions or limit the disorders under consideration. Some routine
laboratory tests should be obtained in every patient with dementia (28). This
testing is warranted because it easily can identify common medical illnesses that
cause or amplify dementia (Table 6). Particularly in the elderly, medical illnesses
can cause cognitive impairment before physical symptoms are evident. When
there is reason for clinical suspicion, further laboratory testing can help identify
less common dementing disorders (Table 7).
The routine use of structural brain imaging for the evaluation of dementia
has been more controversial, but is now generally accepted (28). It is indisputable
8

Table 4 Characteristic Presentation and Course of Mild Cognitive Impairment and the Most Common Dementing Diseases
Disease Usual features Diagnostic guidelines

Mild cognitive impairment Insidious onset of progressive decline in cognitive abilities ADCS criteria (13); International Workshop
insufficient to cause impairment in everyday activities; criteria (20)
must be associated with a complaint; several subtypes
have been proposed: the amnestic form in which
memory loss is predominant appears to be most likely to
progress to Alzheimer’s disease
Alzheimer’s disease Insidious onset of gradually progressive memory loss; NINCDS-ADRDA
memory loss remains the most prominent feature as criteria (21)
aphasia, apraxia, impaired judgment, and behavior
change develop; seizures and incontinence late in the
disease
Vascular dementia Sudden onset, stepwise progression of dementia associated NINDS-AIREN criteria (22); Modified
with ischemic stroke; usually with focal motor and Hachinski Ischemic Score (23)
sensory deficits, although these deficits may resolve;
cognitive deficits variable and correspond to the location
of strokes; frontal lobe impairment may be most obvious
with subcortical stroke; seizures and incontinence may
occur early in the course
Foster
Dementia with Lewy bodies Insidious onset of gradually progressive dementia with Consensus second workshop criteria (24)
parkinsonism (either within a year of developing
Parkinson’s disease or alternatively the development of
parkinsonism concurrent with dementia or soon there-
after); spontaneous visual hallucinations, unexplained
fluctuations in symptom severity; visuo-spatial deficits
The Clinical Issues

and memory loss prominent


Parkinson’s disease with Idiopathic Parkinson’s disease, followed by the insidious Consensus workshop
dementia onset of progressive dementia more than a year later criteria (25)
Frontotemporal dementia Insidious onset of progressive language or behavior Consensus criteria (26); work group criteria
disturbance, often with personality change; relative (27)
preservation of functional abilities at first; subsequently
more pervasive progressive dementia with memory loss
while language and behavior changes remain most
prominent; often progresses to mutism; later develop-
ment of parkinsonism common; may be associated with
motor neuron disease
Abbreviations: ADCS, Alzheimer’s Disease Cooperative Study; NINCDS-ADRDA, National Institute of Neurological and Communicative Disorders and Stroke–
Alzheimer’s Disease and Related Disorders Association; NINDS-AIREN, National Institute of Neurological Disorders and Stroke–Association Internationale pour la
Recherche et l’Enseignement en Neurosciences.
9
10 Foster

Table 5 Distinctive Features on Examination that Suggest a Specific Cause of Dementia


Abnormality Disease most likely

Supranuclear gaze palsy Progressive supranuclear palsy


Alien hand Corticobasal degeneration
Chorea Huntington’s disease
Tendon xanthomas Cerebrotendinous xanthomatosis
Dystonia Wilson’s disease
Keyser–Fleisher ring Wilson’s disease
Asterixis Liver or renal failure
Icterus, ascites, hepatomegaly Liver failure
Muscle fasciculations and atrophy Frontotemporal dementia with motor neuron
Pupils nonreactive to light disease
Neurosyphilis

that computed tomography (CT) and magnetic resonance imaging (MRI) aid the
recognition of many neurological illnesses causing dementia (Table 8). Although
many of these diseases often cause focal abnormalities, the neurological
examination alone may be insensitive for early detection or when patients are
unable to adequately participate in a detailed assessment.
Simply identifying an abnormal laboratory test or brain scan is insufficient;
one must interpret the results and their clinical significance. It is important to
realize that “Occam’s razor,” or the search for a single unifying diagnosis to
explain symptoms and signs, is not always appropriate when evaluating older
patients (29). Since it is common for a medical illness to exacerbate an
underlying dementing illness or for dementing diseases to co-exist and interact,
the principle of parsimony of diagnosis may lead one astray (30,31). It is more
appropriate to consider the possibility that multiple pathologies are causing
cognitive decline.
It can be very difficult to be sure when two or more conditions are
contributing to a patient’s dementia, yet this has major implications for deciding
on treatment and assessing response. Serial examinations may help disentangle

Table 6 Laboratory Studies Appropriate for Routine Use in Dementia Evaluations


Test Causes of dementia detected

Complete blood count Severe anemia, systemic infection, leukemia


Electrolyte panel Electrolyte disturbances
Glucose and calcium Hypoglycemia, hypocalcemia, hypercalcemia
Chemistry panel Hepatic or renal disease
Thyroid function tests Hypothyroidism or hyperthyroidism
Vitamin B12 level B12 deficiency, pernicious anemia
The Clinical Issues 11

Table 7 Ancillary Laboratory Studies for Dementia Evaluations


Test Rationale and indications

Tests for syphilis Neurosyphilis (in endemic areas, exposure,


unreactive pupils)
HIV AIDS (risk factors identified)
Urinalysis Urinary tract infection (incontinence or urinary
complaints)
Sedimentation rate Vasculitis, temporal arteritis, chronic infection
Blood gases Hypoxia, hypercarbia (lung disease, carbon
monoxide exposure)
Drug screen Alcohol, prescription and recreational drugs
(exposure)
Heavy metals Lead and arsenic poisoning (exposure, peripheral
neuropathy)
Hu antibody Paraneoplastic limbic encephalitis (history of
relevant cancer)
Ceruloplasmin Wilson’s disease (liver disease, young age,
dystonia)
Homocysteine Risk factor for vascular dementia and perhaps
Alzheimer’s disease
Carbohydrate deficient transferrin Clinically unapparent alcoholism
Apolipoprotein E genotype Genetic risk factor for Alzheimer’s disease
Presenilin 1 genotype Familial early-onset Alzheimer’s disease
Folate Nutritional deficiency

symptoms and reveal which of several conditions is the predominant cause of the
dementia. For instance, in a patient with mixed dementia due to AD and stroke,
gradual progression of dementia without further strokes on MRI suggests that AD
has caused the worsening of symptoms. By contrast, if there is sudden worsening

Table 8 Causes of Dementia that Can Be Detected Early with Structural Brain Imaging
Primary and metastatic brain tumors
Ischemic stroke
Intracerebral hemorrhage
Subarachnoid hemorrhage
Subdural hematoma
Multiple sclerosis
Brain abscess
Progressive multifocal leukoencephalopathy
Traumatic brain injury
Normal pressure hydrocephalus
Creutzfeldt–Jakob disease (diffusion-weighted and FLAIR MRI only)
Abbreviation: FLAIR MRI, fluid attenuated inversion recovery magnetic resonance imaging.
12 Foster

of dementia associated with new focal deficits, stroke is implicated as the cause of
worsening and is likely to be more important. Unfortunately, awaiting serial
observations entails delaying diagnosis and appropriate interventions.
In most cases, the cause of dementia currently can be determined with a
high degree of accuracy, but not complete certainty. Consensus diagnostic
criteria are available for several dementing diseases and can help, but they cannot
address all clinical situations. Physicians must approach diagnosis with
conviction and humility, recognizing the limits of current knowledge. The
cause of cognitive impairment always should be considered open to review and
possible revision until it is confirmed by pathologic examination (usually
impossible during life) or the identification of a causative genetic mutation
(relevant only in early-onset familial dementia). It is appropriate for physicians to
recommend that families always obtain a postmortem examination to confirm
clinical diagnoses, particularly because the results may have substantial
implications for other family members.

The Rationale for Early Recognition and Evaluation


It is an accepted principle in medicine that early treatment is more effective than
waiting and trying to treat the complications and full ravages of a disease. Of
course, this assumes that the disease and its appropriate treatment are known.
Otherwise, there is a risk that treatments will cause more harm than benefit. As
already outlined, it is not easy to recognize cognitive impairment early and
determine its cause. Consequently, some have argued that the evaluation and
diagnosis of dementia should be delayed as long as possible to minimize
diagnostic errors and avoid the adverse social consequences of labeling a person
with dementia (32). It is true that the diagnosis of a dementing disease can lead to
unwarranted rationing of medical care, unnecessary restrictions in activities,
social stigma, discrimination by insurance companies, and altered perception of
family and friends. Nevertheless, early recognition and treatment of dementia has
many benefits that more than offset these disadvantages (Table 9). These benefits
are particularly compelling when the rights of patient autonomy are considered
and treatments become more effective. Medical paternalism has lost favor; we
increasingly recognize that patients have the right to know what their physicians
know about their condition, and the right to be involved in decisions that affect
them (4). Societal misunderstanding and discrimination against people with
cognitive impairment need to be addressed, but it is spurious to believe that the
best medical response is to delay diagnosis or interventions. Individuals seeking
medical attention for cognitive impairment already may have great distress, and
there are social consequences of cognitive impairment regardless of medical care.
Furthermore, unless early evaluation of dementia is undertaken, patients risk
unnecessary disease complications and may lose the support of family and friends
who misinterpret problems as volitional.
The Clinical Issues 13

Table 9 Benefits of Early Recognition and Evaluation of Cognitive Impairment


Drug treatments are more effective if begun early
Permanent neuronal damage can be prevented in reversible dementias
Coexisting illnesses that may amplify symptoms can be treated
Complications of inappropriate treatments can be avoided
Disease complications can be prevented or identified and treated early
Home and driving safety can be assessed to prevent problems
Social supports can be provided to assist family members and permit patient to remain
at home
Patients can participate in decision making and plan for their own future care

Utilizing New Diagnostic Technologies


New biomarkers and technology will develop as we learn more about the
biochemical and anatomic pathology of specific dementing disorders. These
advances will provide a great opportunity to improve diagnostic accuracy. It will
be a significant challenge to determine how to use this new diagnostic technology
most effectively. Diagnostic testing can be extremely valuable, but it also can be
misused (Table 10). The proper application of new technology to dementia care
always must involve individualized clinical assessment and treatment.
There is concern that new technology and preferential reimbursement for
procedures have undervalued and damaged the therapeutic relationship patients
develop with their physicians. Clinicians will need to integrate new diagnostic
methods without losing the traditional humanistic values of clinical medicine. The
professional relationship that develops with a patient is especially important in
dementia care, because dementia alters even a patient’s unique intellect and
personality. To provide optimal dementia care, a physician must know very
personal information about daily habits, and inner thoughts and feelings. The
astute physician also must expand the professional relationship, which is
traditionally with the patient alone, to include family members and caregivers
(3). Such a therapeutic triad is a major asset during evaluation to provide a
knowledgeable informant and to help explain the purpose and results of diagnostic

Table 10 Potential Misuses of Diagnostic Technology


Overuse—cost of needlessly repeated studies
Overdependence—using test results to decide whether there is dementia when clinical
assessment is reliable; using test results to make a diagnosis without utilizing other
relevant clinical data
Overinterpretation—assigning cause of dementia to clinically insignificant test results
Misinterpretation—failing to recognize the presence of clinically significant abnormal-
ities, or mistaking a result as abnormal when it is not
Omission—failure to incorporate significant imaging findings in diagnosis
14 Foster

testing. Good communication between physician and caregivers also is critical to


the success of ongoing care as symptoms continue to evolve.
Incorporating new diagnostic techniques will add greater complexity to the
already intellectually demanding task of dementia evaluation. Test results cannot
be considered in isolation. Instead, they have to be considered along with other
evidence to decide the cause of symptoms. Sensitivity, specificity, predictive
value, and the circumstances of testing must be taken into account following the
principles of evidence-based medicine (33). Genetic testing, which can be more
definitive than most diagnostic tests, adds further complexity to a dementia
evaluation since ethical and legal implications for family members must be
considered (34).
In addition to threatening traditional values and increasing the complexity
of evaluations, new diagnostic tests will increase the costs of a dementia
evaluation. Furthermore, invasive tests could cause physical harm. We should use
new diagnostic methods wisely. The best strategy is to know their benefits and
limitations, and use them only when clinical history and examination fail tests
seem unlikely to be of much provide a highly confidant, accurate, and specific
diagnosis. Technological advances that overcome current limitations will
improve care and simultaneously justify their expense and burden for patients.

DIFFICULT DIAGNOSTIC CROSSROADS


AND DECISION POINTS
The assessment of cognitive impairment and dementia requires a series of
difficult decisions (Table 11). Some decision points will benefit from new
technologies, others will not. New diagnostic tests seem unlikely to be of much
benefit when deciding whether to initiate a dementia evaluation. Cognitive
complaints, especially of memory lapses, are almost universal, and physicians
will need to continue to rely upon their clinical judgment that further
investigations are warranted. Patient and family complaints will continue to be
important indicators of a significant problem, because medical advice about

Table 11 Difficult Diagnostic Crossroads and Decision Points


Identifying prodromal dementia in patients with cognitive impairment
Distinguishing single and multiple domain cognitive deficits
Distinguishing neurodegenerative disease from psychiatric illness
Identifying coexisting neurodegenerative disease
Identifying the cause of rapidly progressive dementia
Distinguishing AD and mimics of AD
Distinguishing dementias with parkinsonism
Recognizing mixed dementia
Selection and monitoring of treatment
Abbreviation: AD, Alzheimer’s disease.
The Clinical Issues 15

cognitive complaints is usually sought only when symptoms seem unusual,


persistent, or severe. Deciding whether a patient has dementia or not also appears
unlikely to benefit from new diagnostic technology. The threshold for
determining that cognitive impairments are sufficient to impair everyday
activities will continue to require much clinical information that must be judged
based upon individual circumstances. The limitations of laboratory tests for this
purpose are easily demonstrated. A brain scan can identify a stroke, but cannot
tell whether it is causing symptoms. Likewise, while the genetic test for
Huntington’s disease can be definitive, it cannot be used to decide whether a
person has chorea.
There is also a risk of overdependence on new diagnostic technology.
Conventional laboratory testing and neuroimaging are already very good at
identifying focal mass lesions, medical illnesses, and most dementing disorders
that begin suddenly. Cognitive impairment caused by medication is also readily
discernible and easily remedied without further diagnostic studies. As new
methods become available, we should not overlook or abandon these already
successful components of the dementia evaluation.
Several inevitable diagnostic decision points or crossroads are reached that
would benefit significantly from new diagnostic approaches and biomarkers.
Some reflect the challenges of diagnosing an illness well before its full extent is
evident. Others involve the difficulty in unambiguously identifying common
dementing diseases that lack distinctive clinical, laboratory, or imaging features
or when multiple conditions are occurring simultaneously. Each of these decision
points presents its own challenges and demands a different solution.

Identifying Prodromal Dementia in Patients


with Cognitive Impairment
The earliest possible clinical recognition of dementing diseases requires
identifying individuals currently without functional deficits whose cognitive
impairment later will progress to frank dementia. Such individuals could be
categorized as having MCI, CIND, or might be judged to be performing as
expected for their age or for their background. Our current ability to recognize
patients with prodromal dementia is unknown and varies immensely depending
upon physician expertise and interest. Individual characteristics of patients and
families undoubtedly play a crucial role in whether such individuals are
recognized or brought to clinical attention. Even when cognitive complaints do
not appear to be significant, it is appropriate to reassess patients later since they
may be aware of changes that have not yet become clinically evident (10).
As currently constituted, MCI and CIND both include a heterogeneous
group of disorders, some progressive and others stable. Not everyone with MCI
or CIND has prodromal dementia, although everyone with progressive dementia
that began insidiously first must have had cognitive impairment insufficient to
interfere with everyday function. There is no easy solution for dealing with the
16 Foster

issue of prodromal dementia. Some have suggested that physicians should


assume that cognitive impairment represents early dementia to avoid delaying
treatment (35). This approach is attractive for its simplicity and practicality, but it
ignores the reality of a complex situation. Some individuals with MCI, and up
to 5% to 10% of individuals with CIND, are found to revert to normal on re-
examination (19). Thus, there would be many serious diagnostic errors with
potential adverse consequences to patients and families if everyone classified as
MCI or CIND were presumed to have prodromal dementia. The preferable
alternative is to indicate the increased risk of a dementing disease and
recommend close observation (36). However, this approach is unsatisfying to
physician and patient alike and brings with it other problems. Patients and
families must endure uncertainty and are unable to plan for the future. We already
know from people at-risk for Huntington’s disease that the uncertainty of having
a serious illness in the future has its own morbidity and burden (37). Serial
examinations raise issues of their own. What represents a significant change?
When is test variability explained by normal fluctuations in performance? Our
current inability to recognize when cognitive impairment alone represents a
dementing disease delays diagnosis and treatment. We urgently need new
diagnostic methods to address this important clinical decision point.

Distinguishing Single and Multiple Domain Cognitive Deficits


Impairment in only a single cognitive domain has special diagnostic and
prognostic implications. A mild deficit in a single cognitive area can be benign
and represent a previously unsuspected learning disability or a normal variation
in ability. On the other hand, some neurodegenerative disorders characteristically
have a focal onset. In these cases, demonstration of a circumscribed cognitive
deficit can be helpful in early diagnosis. For example, FTD often begins as a
progressive aphasia or semantic dementia, and corticobasal degeneration often
starts with a strongly lateralized apraxia and motor disturbance (26,38). Deficit in
a single cognitive domain also justifies a careful search for an explanatory small
stroke or mass lesion.
By contrast, multiple domain cognitive deficits are more likely to represent
prodromal dementia. MCI patients with cognitive deficits in multiple domains are
more likely to progress to dementia over two years than those with only memory
impairment (39). Neurodegenerative diseases typically cause pervasive deficits,
even if there is a single predominant symptom. For example, patients with AD
may have predominant language deficits or visuospatial deficits (40).
It often is not obvious whether cognitive impairment involves a single or
multiple cognitive domains. An adept mental status examination, the skills of a
speech pathologist, and standardized neuropsychological assessment can help,
but may not be definitive. Clinical findings are not always reliable because they
can be highly subjective and must be interpreted. Several functional brain
imaging techniques offer the potential to more objectively assess whether there
The Clinical Issues 17

are abnormalities in brain areas serving specific cognitive domains. These


imaging methods and objective biomarkers could significantly aid the difficult
clinical decision crossroad of characterizing the extent of brain dysfunction.

Identifying When Cognitive Impairment Is Due


to Psychiatric Illness
Mood and behavior disturbance often leads to a diagnostic conundrum. Both
psychiatric disease and dementing disorders frequently cause cognitive
impairment with behavioral symptoms. Families commonly report that behavior
changes were an early symptom of what later clearly became a dementing
disease. Many people find personality and behavior changes especially
disturbing and they may be more likely to motivate an evaluation than memory
problems. Many behavioral symptoms commonly are seen in AD (41). Apathy
is probably the most common behavioral symptom, which often leads
physicians and families to misinterpret symptoms as being due to depression.
Furthermore, mood clearly can be affected when individuals recognize
progressive problems with their memory. Since major depression can develop
de novo in the course of AD, it should also be considered a complication of
dementia (42).
It is characteristic for behavioral disturbance to be the initial and most
prominent symptom in FTD. Behaviors may be so unusual that FTD is more
likely to be misdiagnosed as a psychiatric disorder rather than as another
dementing disorder. Symptoms like disinhibition, stereotypy, emotional
shallowness, mental inflexibility, perseveration, and compulsions are sufficiently
uncommon in dementing disorders that they are useful in diagnostic criteria and
symptom checklists (26,43). Psychiatric disease is more common than dementia
in younger age groups, and FTD often has an early age of onset. So it is no
surprise that it is often difficult to distinguish FTD from psychiatric illness, until
other cognitive deficits become apparent.
In psychiatric illness, there can be objective evidence of cognitive
problems or they can be more perceived than real. There is increasing evidence
that chronic schizophrenia can cause progressive dementia (44). Psychosis and
mood disturbance due to any psychiatric disease sometimes are severe enough to
interfere with cognitive performance and everyday activities, yet resolve when
treated with psychotropic medication. Substance abuse also can cause memory
loss and cognitive impairment, both when patients are under the influence of a
drug, and as a long-term complication, such as Wernicke’s disease (45).
Depression is especially easily mistaken for a dementing disease because it, like
neurodegenerative diseases, often develops late in life. Typical features of major
depression also often are not expressed fully in the elderly (46). Depression
frequently causes memory complaints. While patients with dementing diseases
generally complain of less difficulty than family members, the reverse is typically
the case for patients with depression. Distinguishing depression can be
18 Foster

particularly difficult when cognitive complaints are accompanied by social


withdrawal and lack of interest that alter everyday function. Diagnostic
guidelines and criteria for depression may be helpful, but they still require the
subjective and sometimes unreliable interpretation of symptoms.
The usual recommendation is to identify and treat depression and
psychiatric illness before proceeding further with a dementia evaluation. This
approach is appropriate given current options, but it may delay recognition and
appropriate treatment of an underlying dementing disorder, especially if the
response to antidepressant treatment is ambiguous. Since the biochemical and
anatomic abnormalities in psychiatric and neurodegenerative diseases differ so
markedly, it is reasonable to expect that future technologies will distinguish them.

Identifying Coexisting Neurodegenerative Disease


Even when an evaluation identifies a potential cause of cognitive impairment,
there still is uncertainty that it accounts for a patient’s symptoms. Laboratory
studies may detect a medical illness, but treatment often does not completely
reverse symptoms (47). This can be because the illness has already caused
permanent neuronal injury, and stabilization of symptoms is the most that can be
expected. Alternatively, the medical illness might persist despite the best
treatment. For example, in some cases chronic renal failure and hepatic failure
may be incurable, and symptoms would be expected to continue and fluctuate as
the underlying illness varies. A final possible explanation is that there is a
coexisting dementing disease. Currently, it is impossible to know which
explanation is correct without serial assessments to see if impairments improve,
are stable, fluctuate along with the medical condition, or are progressive. If
symptoms are progressive despite effective treatment, there clearly is an additional
dementing disorder whose diagnosis and treatment has been delayed greatly. In
disorders where treatments are ineffective or only partially effective, recognition
of an unrelated, clearly progressive dementing disorder often is delayed even
longer until dementia is very severe.
Uncertainty in the outcome of delirium poses a similar problem and
illustrates how common it is for causes of cognitive impairment to coexist.
Medical care for dementia commonly begins with an episode of delirium. One
study found that 40% of adults with dementia had a superimposed delirium on
admission to the hospital. On the other hand, only 25% of those with delirium were
found to have dementia, once their acute mental status changes had resolved (48).
Usually a medical condition causing the delirium can be identified; nevertheless, a
coexisting dementing disorder still must be considered. Patients with dementia are
not only particularly prone to delirium, but also may have a prolonged period of
recovery from delirium. This problem often appears in hospitalized patients. Pain
medications, anesthetic agents, and sedatives frequently induce a protracted
postoperative delirium in elderly patients. If not recognized and treated
The Clinical Issues 19

appropriately, delirium can cause either a misplaced presumption of dementia or


delay the evaluation of an underlying dementing disease.
The possibility of medication induced cognitive impairment also can delay
the recognition of a neurodegenerative disease. Medications commonly cause
cognitive side effects, especially with aging that alters the metabolism of many
drugs. The list of medications with potential to cause cognitive impairment is
long. Some individual medications and classes of medications are particularly of
concern (49). Psychotropic and sedative medications are common culprits, but
drugs given primarily for systemic illnesses also can cause cognitive problems.
Sometimes, it is possible to temporally relate the onset of symptoms with the first
use of the medication. In other cases, the role of medication is proven when it is
stopped and cognitive symptoms resolve. When discontinuance of medication
cannot be medically justified, there may be little recourse except for serial
observations. In any case, when medications are a potential factor, considerable
clinical judgment is required and there is a delay in the final resolution of
causality and treatment.
Even finding a serious neurological disease does not assure that the cause of
cognitive impairment is established, and a progressive dementia may later be
recognized. Brain atrophy caused by dementing diseases increases the risk of a
subdural hematoma, and amyloid angiopathy of AD can cause intracerebral
hemorrhage (50). Seizures beginning late in life also can be diagnostically
ambiguous. Seizures cause memory lapses; in particular, partial complex seizures
may be misdiagnosed as a dementing disease, especially because they also can
cause behavioral changes. A suspected seizure disorder can be confirmed readily
with an EEG. However, stroke and AD may explain the seizure disorder, making
several explanations of dementia possible even when seizures appear to account
for symptoms. The use of anticonvulsants adds another possible explanation.
Clearly, we need diagnostic methods that can identify specific neurodegenerative
diseases even when other causes of cognitive impairment are present.

Identifying the Cause of Rapidly Progressive Dementia


Dementia that becomes severe in less than a year requires urgent evaluation.
Diseases that cause such rapidly progressive dementia differ from those with a
more indolent course (Table 12). In addition to the clinical assessment and
laboratory and imaging studies performed in a usual dementia evaluation, such
cases warrant further testing, including a spinal fluid examination and an EEG.
Many causes of rapidly progressive dementia are readily detected. Intracranial
mass lesions are more likely, and delirium also must be considered. Bacterial,
tuberculous, fungal, and viral infections can be identified with blood and spinal
fluid cultures and testing for specific antigens. Paraneoplastic antibodies and
spinal fluid cytology can help detect malignancies that are not identified on a
physical examination or imaging studies. An EEG can identify non-convulsive
20 Foster

Table 12 Causes of Rapidly Progressive Dementia


Creutzfeldt–Jakob disease
Meningitis/encephalitis (including TB, fungal, herpes, carcinomatous, paraneoplastic)
Vasculitis
CNS lymphoma, primary and metastatic brain tumors
Subdural hematoma
Non-convulsive status epilepticus
Multi-infarct dementia (occasionally)
Alzheimer’s disease (rarely)
Abbreviations: TB, tuberculosis; CNS, central nervous system.

status epilepticus. However, despite such extensive testing, it still may be difficult
to determine the cause of rapidly progressive dementia.
It is particularly important to identify Creutzfeldt–Jakob disease (CJD) so
proper precautions can be used to prevent the potential iatrogenic trans-
mission (51). Definitively identifying CJD is difficult, but several clinical
findings and laboratory tests often are abnormal in CJD and can aid diagnosis.
Sequential EEGs may demonstrate the evolution of typical periodic discharges.
MRI can reveal focal abnormalities in the cerebral cortex or striatum with
diffusion-weighted or FLAIR sequences (52). High levels of the 14-3-3 protein
usually can be detected in the spinal fluid (53). Clinical criteria for CJD have been
developed incorporating these features (54).
Despite such clues, the diagnosis of CJD can remain elusive. Although
diagnosis can often be made easily when typical features have evolved, early
definitive identification of CJD is not easy, and a substantial group of patients
have atypical features. CJD can have a surprising variety of clinical
presentations. There can be initial memory loss that suggests AD, focal cognitive
deficits that suggest a focal lesion, or a gait disorder that suggests a
neurodegenerative disease. Some of these clinical phenotypes can be explained
by variations in prion protein glycotype and its interaction with prion genotype,
but this information currently is available only after a brain biopsy or
autopsy (55). New variant CJD associated with bovine spongiform encephalo-
pathy also has atypical features (56). Current laboratory testing for CJD is also
fallible and cannot be relied upon when symptoms are atypical. While a spinal
fluid 14-3-3 protein can be helpful if it is elevated, it is not always abnormal in
CJD, and it can be elevated in several other disorders associated with rapid brain
destruction (57). Brain biopsy is sometimes the only way to avoid the serious
consequences of misdiagnosis or treatment delay.
Rapidly progressive dementia illustrates how clinical methods and
diagnostic technology sometimes must be reconciled. The serious implications
of a rapidly progressive dementia must be weighed against the costs and real risks
of brain biopsy to the patient, surgical team, and pathology staff. The quandary of
The Clinical Issues 21

whether to perform this invasive testing is further complicated if there is difficulty


in determining whether the illness really is progressing rapidly. There may not be
informants, or their stories may differ. The physician then must consider all the
evidence and judge its quality. Because of the need for urgent intervention, any
suspicion must favor brain biopsy if no cause has been identified. However, if
there is suspicion that historical information is inaccurate, it may be appropriate
to defer brain biopsy until the rapid decline is confirmed by serial examinations.
Such situations illustrate why there is still considerable need for practical
biomarkers to identify disorders causing rapidly progressive dementia.

Distinguishing Alzheimer’s Disease and Mimics


of Alzheimer’s Disease
AD was not recognized as the most common cause of dementia until systematic
autopsy studies were performed in the 1960s and 1970s (58,59). Early
investigations found that the clinical diagnosis of AD was frequently wrong.
Subsequently, with the development of explicit criteria, comprehensive
evaluation methods, and brain imaging, the clinical diagnosis of AD has become
significantly more accurate, yet diagnostic errors remain (60,61). Today most
errors are due to “AD mimics,” illnesses that are clinically indistinguishable from
AD (Table 13).
AD mimics can occur when there are atypical presentations of a dementing
disorder that usually does not resemble AD. This may happen when such a
disorder’s typically distinctive features have not yet appeared. For example,
familial CJD may have a course of many years that is very similar to AD and lack
periodic EEG discharges (62). Likewise, progressive supranuclear palsy may
lack its typical eye movement abnormalities (63).
Most AD mimics are disorders that ordinarily share so many characteristics
with AD that they are difficult to distinguish. FTD and dementia with Lewy
bodies (DLB) are the most important examples. Patients with FTD character-
istically have particularly prominent behavioral or language disturbances, yet
they frequently also meet diagnostic criteria for AD (64). Behavioral and
language symptoms are common in both AD and FTD. Impaired judgment and
insight is frequent in both AD and FTD, and probably reflects involvement of the

Table 13 Alzheimer’s Disease and Its Mimics


Alzheimer’s disease
Frontotemporal dementia, including Pick’s disease
Dementia with Lewy bodies
Vascular dementia
Progressive supranuclear palsy
Familial Creutzfeldt–Jakob disease
Corticobasal degeneration
22 Foster

frontal association cortex. Aphasia also is not unique to FTD, and has long been
considered a cardinal feature of AD as indicated in diagnostic criteria for
AD (21). Diagnostic criteria suggest FTD can be distinguished from AD by the
relatively early onset of behavioral and language symptoms, but this requires
considerable subjective medical judgment. It also may be helpful to identify
behavioral and language symptoms often seen in FTD but uncommon in AD,
such as disinhibition, apathy, perseverative behavior, stereotypy of speech,
echolalia, and mutism. However, their reliability and specificity for FTD are
uncertain, and improvements in clinical criteria for FTD are still needed (65).
Nevertheless, such differences in type, predominance, and evolution of
symptoms are the basis for behavioral inventories meant to aid clinical
diagnosis (43). Structural brain imaging may help distinguish AD and FTD by
contrasting the severity of atrophy in involved brain regions (66). Probably the
most frequent pathologic abnormality in FTD is nonspecific neuronal loss and
gliosis with or without vacuolization. However, FTD sometimes is caused by
distinctive abnormalities in tau, ubiquitin, or neurofilament protein (67,68).
Eventually these should permit the development of biomarkers that identify
histopathological subtypes of FTD.
DLB is another important AD mimic that now appears to be the second most
common cause of dementia in the United States. It accounted for approximately
20% of all dementia cases in recent autopsy series (69). Although DLB always has
pathologic changes characteristic of Parkinson’s disease, it may not have
noticeable motor signs or other distinctive features (70). DLB has the same
biochemical signature of dopamine deficiency typical of Parkinson’s disease and
also has characteristic deposits of ubiquitin in cortical Lewy bodies; it is realistic to
expect new diagnostic methods could exploit these features and aid in diagnosis.

Distinguishing the Cause of Dementia with Parkinsonism


Dementia is often accompanied by the same slowing of gait, rigidity, impaired
balance, and bradykinesia usually associated with Parkinson’s disease.
Parkinsonism can precede the onset of dementia, develop concurrently, or
appear only after many years of cognitive impairment. Clinicians now have
considerable difficulty in distinguishing the several disorders causing dementia
with parkinsonism (Table 14).
The thoughtful clinician faces a diagnostic quandary when confronting a
demented patient with parkinsonism. With the exception of unusual cases where
parkinsonism is caused by strategically placed cerebral infarcts, conventional
laboratory studies and structural imaging provide little assistance. Unlike the
pathologist who can identify, quantify, and localize neuronal loss and
characteristic inclusions, the clinician is relegated to basing diagnostic judgments
solely on clinical history and examination. In an admirable effort to achieve
clarity and uniformity in diagnostic classification, consensus clinical criteria have
been developed that are heavily dependent upon the relative timing of motor and
The Clinical Issues 23

Table 14 Causes of Dementia with Parkinsonism


Dementia with Lewy bodies
Parkinson’s disease
Alzheimer’s disease
Frontotemporal dementia, including frontotemporal dementia with parkinsonism linked to
chromosome 17 (FTDP-17)
Progressive supranuclear palsy
Corticobasal degeneration
Vascular dementia (rarely)

cognitive symptom onset (24). The criteria indicate that when spontaneous motor
symptoms precede the onset of dementia, the cause is Parkinson’s disease with
dementia. If motor symptoms and dementia occur simultaneously early in the
illness, the clinical diagnosis is DLB. However, if motor symptoms develop late
in a progressive dementia, the cause is AD. Additional criteria address other
symptoms and amplify these categories, but symptom onset remains critical to
classification. Unfortunately, rules of symptom onset are arbitrary and require
subjective interpretation of historical information that sometimes is limited or
unreliable. These clinical criteria understandably have only modest ability to
predict pathology (71). New technology to replicate biochemical and anatomic
information used by pathologists for classification would aid diagnosis and
development of more specific treatments enormously.
The challenges of clinical diagnosis of parkinsonism and dementia are
understandable. Because continued use of drugs may be clinically necessary, it
may be impossible to determine the onset of motor symptoms or whether they are
spontaneous, as required by criteria. Extrapyramidal symptoms also are
frequently seen in AD, although they usually begin later than in DLB. In one
study that monitored motor symptoms prospectively, over half of patients with
moderately severe AD develop new parkinsonian signs over two years (72).
Consequently, many disorders causing parkinsonism also are AD mimics. For
example, FTD may cause parkinsonism. This is particularly relevant in FTD with
parkinsonism linked to chromosome 17 (FTDP-17), an early-onset, autosomal
dominant disorder. In some families with FTDP-17, parkinsonism even may be
the presenting symptom, and much more evident than dementia (73).
The consideration of additional symptoms considered in criteria for DLB
may not resolve the difficulty with clinical diagnosis. For example, consensus
criteria use visual hallucinations and fluctuations to distinguish DLB. However,
medications and AD may cause visual hallucinations (74). Family reports of
fluctuations can be difficult to interpret, and subjective clinical judgment is
required to know when fluctuations are significant.
Pathologic studies also suggest why clinical diagnosis is so challenging.
Cerebral Lewy bodies are accompanied by pathological changes of AD about
two-thirds of the time (75). Thus some have called this “Lewy body variant
24 Foster

of AD” (76). Much additional work will be needed to clarify these difficult
clinical issues.

Recognizing Mixed Dementia


The dictum to avoid overdependence upon Occam’s razor is amply demonstrated
by the evidence that vascular and AD pathology frequently coexist. This co-
occurrence of AD and vascular pathology is often termed “mixed dementia” (77).
Mixed dementia is particularly frequent in the “oldest-old” (age 85 or older), the
age group that will increase the most over the next several decades (2). We
greatly need to improve our ability to diagnose mixed dementia. Current brain
imaging often identifies vascular lesions, yet there is no easy way to determine
how they contribute to patient symptoms. We know that vascular disease and AD
interact in important ways to increase the likelihood of clinically significant
cognitive decline (78,79). However, most reports on dementia focus on separate,
“pure” clinical entities, and most effort has been focused on distinguishing
vascular dementia from AD, rather than determining when both are present and
clinically important.
Traditional approaches have suggested that the extent of vascular lesions
on MRI scan might be useful, but recent studies have found this inaccurate (80).
A requirement that dementia develop in association with a clinically recognized
stroke has also been suggested by NINDS-AIREN criteria for vascular
dementia (22). However, accurate historical information may be difficult, or
cognitive changes may have been overlooked in the face of major motor deficits.
Perhaps as a consequence, the requirement for time linkage appears to cause
insensitivity for vascular dementia (80). The demonstration of focal motor or
sensory deficits on the neurological examination might appear sufficient, but they
may resolve, or strokes might affect areas of the brain associated with cognition
without damaging motor or sensory pathways. In other cases, individual strokes
may be too small to recognize clinically. Furthermore, subtle focal deficits may
be difficult to elicit if the patient is evaluated only when severely demented.
Evidence of impairment of the cerebral cortex on functional imaging
studies, rather than the number or location of subcortical infarcts, appears to be
the best predictor of dementia (81). The effects of subcortical strokes can be mild,
and the most prominent symptoms and metabolic abnormalities often reflect
frontal lobe dysfunction (82). The cognitive deficits of strokes appear to be more
than additive in their effects, and they may become noticeable only several
months later (83,84).

Selection and Monitoring of Treatment


Although treatment is not the focus of this monograph, the selection and
monitoring of treatment also can be a diagnostic decision point. A rational
therapeutic plan can only be based upon a specific diagnosis. Otherwise,
treatment is merely guesswork and outcomes are left to chance. Since imaging
The Clinical Issues 25

aids in identifying the specific cause of dementia, it plays an important role in the
selection of initial treatment. Perhaps less well recognized is how the results of
diagnostic brain imaging can be incorporated into subsequent treatment
decisions. A specific diagnosis helps to predict treatment outcomes. When the
response to appropriate disease specific therapy is unexpected, the accuracy of
the clinical diagnosis should be questioned. For example, an explanation other
than depression should be sought when severe apathy fails to improve with an
anti-depressant. In this situation, FTD should be considered. Likewise, the motor
symptoms of idiopathic Parkinson’s disease almost always respond to treatment
with dopaminergic agents. If they have never been beneficial, the diagnosis of
Parkinson’s disease with dementia is difficult to sustain, and another dementing
disorder is more likely. In diagnostic reassessments like these, additional brain
imaging methods can be considered or a repeat study that may reveal new or
evolving abnormalities can help provide critical confirmatory information.
Since definitive diagnostic test results are usually lacking in dementing
disorders, selection of treatment also is not definitive. Diagnostic confidence
inevitably modifies physician judgments about treatment. The results of brain
imaging can alter diagnostic confidence and influence therapy, particularly as the
effects of initial treatments are monitored. For example, treatment with
cholinesterase inhibitors might be aggressively pursued when clinical and
imaging features are typical of AD, but discontinued if side effects develop when
clinical and imaging findings are discordant for this diagnosis. Likewise, a
therapeutic trial of drugs used to treat AD sometimes may be warranted when the
diagnosis is uncertain. However, they might be avoided or discontinued if
imaging finds predominant frontal abnormalities, since cholinesterase inhibitors
provide little benefit and may worsen symptoms in FTD (85).
There is also great interest in using brain imaging to evaluate new drug
therapies. There is evidence that longitudinal changes in MRI and glucose
metabolism measured by positron emission tomography may permit more rapid
and cost-effective clinical trials in dementia (86,87). However, validation of an
imaging surrogate biomarker for disease progression is a major undertaking.
Further investigations are needed before it is clear whether imaging results are
reliable and can be appropriately interpreted in complex clinical trials. For
example, one recent randomized trial of immunotherapy for AD found surprising
imaging results that appear to be discordant with clinical outcomes (88). In the
future, it may be possible with molecular imaging to design therapies for
dementing diseases based upon individual biochemical profiles and disease
staging, just as it is now starting to be used in clinical oncology (89,90).

SUMMARY
The evaluation of dementia is challenging, but worthwhile. Our ability to
determine the cause of dementia has improved, but much remains to be done.
There are many opportunities to refine our current methods at critical and difficult
26 Foster

diagnostic decision points. The accuracy of early diagnosis is particularly


important. Recent advances in knowledge about neurodegenerative disease and
new technology offer the realistic hope that we will achieve significant progress
in dementia diagnosis. However, we must carefully evaluate new imaging
methods and biomarkers to assure that they are used appropriately, efficiently,
and effectively.

ACKNOWLEDGMENTS

Supported in part by NIH grants RO1-AG22394 and P50-AG08671 (the


Michigan AD Research Center), and the Donald W. Reynolds Foundation.

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2
Neuropsychological Screening and
Advanced Neuropsychological Tests

Daniela Perani
Departments of Neuroscience and Nuclear Medicine, Vita-Salute San
Raffaele University and San Raffaele Scientific Institute, Milan, Italy

Any supermarket cashier can make the diagnosis of Alzheimer’s disease.


—Yearbook of Neurology and Neurosurgery (1990)

INTRODUCTION
Dementia is a syndrome characterized by the reduction of cognitive abilities
(most often memory) to the point that impairment in daily activities occurs.
Clinical assessment, supported by the results of neuropsychological tests, is at
present the only in vivo non-invasive screening and diagnostic tool for Alzheimer’s
disease (AD) and other disorders associated with dementia. Dementia diagnosis,
indeed, is based exclusively on clinical, behavioral, and neuropsychological
findings according to DSM-IV (1). The same principle applies to the diagnosis of
the most common causes of dementia: AD (2), vascular dementia (VaD) (3), and
frontotemporal dementia (FTD) (4). According to NINCDS-ADRDA criteria,
neuropsychological testing is required to confirm the presence of dementia,
established on clinical grounds.
The recent development of symptomatic pharmacological treatment for AD
and the possible introduction of new therapies in the near future make the
assessment of dementia at its different stages a great scientific and public health

33
34 Perani

challenge. Further, as dementia is a clinical diagnosis, there is no doubt that


cognitive assessment plays a crucial role in the detection of early dementia in
particular. Cognitive decline is not an inevitable outcome of aging, and can be the
result of an incipient, unrecognized pathology. The term mild cognitive impairment
(MCI) is reserved for patients whose impairment is documented by neuropsycho-
logical testing, but does not affect activities of daily living.
This chapter reviews the principles of neuropsychological assessment of
dementia, including the application of standard batteries of neuropsychological
tests and of short screening tests. It also analyzes the tests to be applied to detect
and assess the specific deficits found in the different forms of dementia, and
discusses the advantages and flaws of current screening and diagnostic tests of
dementia. Not all patients with dementia evolve and decline in a similar fashion.
On this ground, neuropsychological tests can provide a quantitative assessment of
specific patients’ profiles. Future research should determine the possible
predictive value of these profiles.

NEUROPSYCHOLOGICAL ASSESSMENT
An ideal neuropsychological test should be psychometrically adequate. It should
thus be reliable, valid, and have a high sensitivity and high specificity.
Reliability refers to the consistency of the information provided by the test,
if administered to the same subject by two different examiners, or by the same
examiner on two different occasions. An important issue, in particular for follow-
up and therapeutic studies, is the availability of alternate forms, to avoid test-retest
effects in the case of repeated examinations of the same subjects. The concept of
validity is complex and has multiple facets. Validity refers to the function to be
assessed in absolute terms (construct validity), or in relation to other tests, which
are considered to be valid from the former point of view (concurrent validity).
Moreover, diagnostic validity refers to the ability to correctly classify affected and
unaffected subjects. The proportion of patients who are actually classified as
demented by a test reflects its sensitivity. The sensitivity of a test decreases as a
function of the proportion of cases of dementia, which the test is unable to classify
(false negatives). The drawback of increasing the sensitivity of a test is the parallel
increase of false positives, i.e., patients erroneously classified as demented.
Therefore, a second index is crucial, i.e., specificity. Specificity reflects the
proportion of non-demented subjects, which are correctly classified by the test. At
present, the majority of diagnostic tests for dementia show very good sensitivity
and specificity in the moderate stage of the disease, but not in the early stage. The
Mini-Mental State Examination (MMSE) (5), for example, shows a sensitivity of
87% and a specificity of 92% for scores of 23–24 (6). Another issue is the need for
tests with high sensitivity and specificity, not only in the very early, even
“preclinical,” and moderate stages of the disease, but also during the late stages
(severe dementia).
Neuropsychological Screening and Advanced Tests 35

A necessary step in order to fulfill the criteria for psychometric adequacy is


the standardization of the testing procedures, and the availability of normative
data, which allows to control for the effects of age, gender, and education.
In principle, socioeconomic status and cultural background are also known to
affect the performance on neuropsychological testing.
On practical grounds, there are a number of approaches to the cognitive
assessment of dementia. A basic distinction is between clinical cognitive
assessment, the application of intelligence tests, screening tests, and formal
neuropsychological assessment, based on psychometric tests with normative
values.

Clinical Assessment
The clinical evaluation of mental function is a part of the neurological and
psychiatric examination. It consists of a series of “paper and pencil” tests that are
aimed at investigating the main cognitive areas with only limited supportive
material. The patient’s performances are not measured quantitatively, but on the
basis of the clinical experience of the examiner. There are several published
guides to the neuropsychological examination at the “patient’s bedside” (7–9).
This kind of cognitive evaluation is the only level of assessment that is required
by most diagnostic criteria, such as DSM-IV. It is, however, recommended to
complete the assessment with brief quantitative screening tests. The clinical assess-
ment can be standardized using rating scales, such as the Global Deterioration Scale
(GDS) (10) and Clinical Dementia Rating (CDR) (11), which also include an
assessment of functional status.
Medical history even in mildly affected patients should always include
information from both patient and caregiver. In particular, the assessment of
awareness of cognitive dysfunction might help in the distinction between AD and
MCI. While most MCI patients tend to overestimate cognitive deficits when
compared to their caregiver’s assessment, AD patients in the early stages of
disease underestimate cognitive dysfunction (12). Anosognosia can thus be
regarded as a characteristic symptom at a stage of very mild AD, but not MCI.

Intelligence Tests
Tests that have been developed for the standardized evaluation of intelligence
level in normal subjects have been traditionally used to assess cognitive decline
due to neurological dysfunction. The intelligence quotient (IQ) provided, for
example, by the Wechsler Adult Intelligence Scale (WAIS) battery has been used
to diagnose and assess dementia. The IQ evaluates the multidimensionality of
mental ability and provides a global score that characterizes an individual’s
performance as a whole, as well as a measure of aggregate intelligence that is
composed of elements or abilities that are qualitatively differentiable (13). An
early observation is that discrepancies between some verbal and performance
36 Perani

tasks are evident in normal aging and accentuate in early dementia. Wechsler
himself noticed that some subtests tend to “Hold” (Vocabulary, Information,
Object Assembly, and Picture Completion) opposed to “Don’t Hold” tests (Digit
Span, Similarities, Digit Symbol, and Block Design). On this basis, he proposed a
deterioration quotient (Hold–Don’t Hold/Hold), which has been used as an index
of dementia.
The length of the administration procedures, as well as the development of
tests that are directly related to the cognitive impairments and that are specific to
dementia, has progressively reduced the diffusion of the diagnostic application of
intelligence tests. However, it should be noted that the use of the WAIS subtests
with an emphasis on the quality of the responses rather than on the quantity is a
source of interesting supplementary information for the clinician (14,15). There
are abbreviated versions of the WAIS (16), which continue to be used in AD
patients (17).

Screening Tests
These include a number of tests that can be administered in a limited amount of
time and provide a brief examination of multiple aspects of cognition. Adequate
psychometric information is available for some of these measures, such as
the MMSE.

Mini-Mental State Examination


The MMSE is the most widely used short screening instrument for dementia.
Since it has been translated into many languages, it represents an almost universal
way of assessing the severity of dementia in individuals as well as in population
samples. It tests a limited number of cognitive functions and can be administered
in about 10 to 15 minutes. The score ranges from 0 to 30, and the “cut-off score”
below which dementia can be suspected is 24. Normative data, which allow
correction of raw values on the basis of age and education, are available for
several languages. The test has good sensitivity at scores lower than 24, but it is
influenced by age and educational variables, and shows less sensitivity in other
neurological diseases such as Huntington’s disease, multiple sclerosis, and
Parkinson’s disease. Huntington’s disease patients showed distinct cognitive
profiles in comparison to AD with better orientation for date and recall of words
(18). The profile differences were independent of the severity of dementia and
were able to classify patients as AD or Huntington’s disease with 84% accuracy.
A modified version of the MMSE has been proposed for patients with Parkinson’s
disease (19).

The Short Mental Status Test


This is another short screening test, including an improved memory test, which
has been recently shown to increase sensitivity in comparison to MMSE in
patients with MCI (20).
Neuropsychological Screening and Advanced Tests 37

Clinical Dementia Rating Scale


Each patient with dementia presents with different progression rates. It might be
important to obtain a clinical subdivision in dementia stages, without considering
disease progression as a fixed and stereotyped model. CDR represents the most
diffuse method of evaluation both in clinics and in research (21,22). CDR score is
obtained by information provided by a caregiver or an operator and by a cognitive
examination that assesses memory, judgment, abstract reasoning, and spatial and
temporal orientation, plus evaluation of social and working activity, hobbies, and
activities of daily living. The scores are 0 (normal), 0.5 (possible impairment),
1 (mild impairment), 2 (moderate impairment), and 3 (severe impairment). The
scale has been extended to 4 and 5, in order to classify the most advanced stages
of the disease (21).

Mattis Dementia Rating Scale


This scale assesses five cognitive domains: attention, initiation and perseveration,
construction, conceptual thinking, and memory. The most difficult items are
presented first, in contrast to many other tests in which items are presented in
ascending order of difficulty. The total score clearly discriminates AD patients
from normal control subjects. It also differentiates patients with dementia from
patients whose cognitive functioning is compromised by psychiatric illness and
from healthy community elderly subjects with limited education (23). The scale
has been used with success in patients with conditions other than AD, including
VaD (24) and AIDS (25). The subscales of the battery, however, might vary in
sensitivity (26). In particular, attention and conceptual thinking do not
discriminate controls from mildly impaired patients.

Other Tests
It must be stressed that even though these screening tests are frequently employed
in clinical practice for the detection and diagnosis of dementia and AD, most of
them lack sensitivity and specificity. Even if the MMSE is the most widely used
by frontline physicians, difficulties with the MMSE in detecting early dementia
have been reported, making it lengthy and potentially cumbersome to use (27).
The main goal for future research in this domain is to provide clinicians with
simple and brief tests, based on solid theoretical and experimental data, with high
sensitivity and specificity.
The Blessed-Roth test, which includes an Information, Memory, and
Orientation test (28), is largely used in dementia assessment.
The Clock Drawing test, widely appreciated for its simplicity and ease of
administration, rendering it a popular tool in both clinical and research practices, is
a brief measure used to detect cognitive decline associated with a variety of
neurobehavioral disorders. It correlates highly with the MMSE and other measures
of global cognitive decline. However, a challenging issue has been identifying the
neuropsychological and neuroanatomic substrates underlying impaired
38 Perani

performance on clock drawing in dementia (29). Cosentino and colleagues found


that the drawing-on-command condition places high demands on executive control
functioning, since patients are required to perform in a novel context, whereas the
copy condition provides a purer measure of “visuoconstructional” ability. Their
data suggest that heavy demands on executive control associated with the
interruption of large-scale cortical–subcortical neural networks underlie impair-
ment in clock drawing in mild dementia.
The 7 Minute Test (30) represents an attempt in that direction. Four brief
tests (Enhanced Cued Recall, Temporal Orientation, Verbal Fluency, and Clock
Drawing) are included, which showed a mean sensitivity of 92% and a mean
specificity of 96%.
There are other promising new tools such as the Montreal Cognitive
Assessment (MoCA), developed to screen patients who present with mild
cognitive complaints and usually perform in the normal range on the MMSE (31).
This study assessed the sensitivity and specificity of the MoCA in patients with
MCI and AD and normal elderly controls. It showed sensitivities of 78%
(MMSE) versus 100% (MoCA) in AD, and 18% (MMSE) versus 90% (MoCA) in
MCI. The MoCA is a simple, stand-alone cognitive screening tool with superior
sensitivity. It covers important cognitive domains, can be administered in
10 minutes, and fits on one page.
Another screening test aimed at identifying patients with MCI and patients
with dementia in the early stages of the disease is the DemTect (32). It showed
high sensitivities of 80% and 100% to detect MCI and mild AD, respectively. It is
short (eight to 10 minutes), easy to administer, and its transformed total score
(maximum 18) is independent of age and education. The DemTect helps in
deciding whether cognitive performance is adequate for age (13–18 points), or
whether MCI (9–12 points) or dementia (8 points or below) should be suspected.

Neuropsychological Batteries
Several batteries have been designed to test dementia. They comprise
standardized, structured interviews, tests, and examinations for diagnosing
common mental disorders in later life, with special reference to the dementias.
Their principal aim is to incorporate in a single instrument all the
information required to make an accurate clinical diagnosis of dementia.

Cambridge Examination for Mental Disorders of the Elderly


The Cambridge Examination for Mental Disorders of the Elderly (CAMDEX)
(33) is a standardized, structured interview and quantitative evaluation of a
wide range of cognitive functions. The principal goal is to establish the type
of dementia, e.g., AD type, VaD, dementia with Lewy bodies (DLB), FTD, or
dementia secondary to physical disease. It can provide key features common to
dementia and other psychiatric syndromes of later life that are likely to overlap
with and cause difficulty in the differential diagnosis from dementia. The battery
Neuropsychological Screening and Advanced Tests 39

is also aimed to diagnose dementia at an early stage and to provide an estimate of


the clinical severity of dementia. It is applicable to both clinical and community-
based studies. CAMDEX incorporates several components to establish personal
and family history, and concise neuropsychological tests to assess all the
cognitive deficits specified in operational diagnostic criteria, i.e., memory
impairment, aphasia, apraxia, agnosia, and disturbances in executive function.
Items within a cognitive domain are graded in difficulty to permit assessment of
the full range of cognitive ability. Scores can be calculated for each of these broad
areas of cognitive function or added to give a total score.
Among several screening measures of CAMDEX, the CAMCOG is the
only significant predictor for the clinical diagnosis of dementia. To gain
efficiency, the screening measures of the CAMDEX protocol may be restricted to
the CAMCOG (34).
A new version, CAMDEX-R, has been developed in order to increase its
ability to diagnose different forms of dementia, including DLB and FTD, and to
make clinical diagnoses based on DSM-IV and ICD-10 criteria.

Consortium to Establish a Registry for Alzheimer’s Disease


The Consortium to Establish a Registry for AD (CERAD) (35) proposed a
diagnostic battery that has now been translated and used in several countries.
The main CERAD battery consists of a relatively brief neurological
evaluation designed to help clinicians to make reliable and valid diagnosis of
probable AD and a brief neuropsychological battery intended to assess the
severity of the overall dementia and the extent to which various cognitive domains
are affected. So far the diagnostic accuracy for AD in the early phase is 86%.
The CERAD battery includes the CDR, which relies on subjective
assessment by the neurologist. Its administration requires training, and its scoring
has been simplified by using the “sum of boxes.” The neuropsychological battery,
which has a high inter-rater reliability and high re-test reliability, has been
augmented by the addition of further measures of delayed verbal recall, Trails A
and B, and a simple test of verbal intelligence aimed at estimating the subject’s
premorbid IQ. One study (36) found that it could distinguish even the mildest
cases of AD from nondemented controls. However, in some of the countries that
use CERAD, the battery has not yet been extensively validated.
The CERAD 1 includes: Word Fluency (category-animal), the Boston
Naming Test, the MMSE, Word List Memory, Constructional Praxis, Word List
Delayed Recall, Word List Recognition, plus a behavioral rating scale. A second
version of the test, CERAD 2, has been proposed. It includes the Shipley-
Hartford Vocabulary, Verbal Paired Associate Learning Test, Recall of
Constructional Praxis items, Verbal Paired Associate Recall, Trails A and B,
Nelson Adult Reading Test, Word Fluency (letter: “F” and “P”), Clock Drawing,
and Finger Tapping.
40 Perani

Alzheimer’s Disease Assessment Scale


The Alzheimer’s Disease Assessment Scale (ADAS) was designed to measure
the severity of the most important symptoms of AD (37). Its subscale ADAS-cog
is the most popular cognitive testing instrument used in clinical pharmacological
trials for AD. It consists of tasks measuring disturbances of memory, language,
praxis, and attention. A review has concluded that in comparison with other
rating scales, the ADAS-cog is regarded as more comprehensive, although it is
not a substitute for an extensive neuropsychological test study (38). Age and
education norms are available for ADAS-cog. It must be remembered that ADAS
was not designed for diagnostic use (it assumes that the diagnosis of probable AD
has already been reached), and that it does not provide any type of individual or
group profile. In addition, it does not take into account psychiatric or behavioral
manifestations of AD. Finally, the standardization is limited, which restricts the
comparability of results in multicenter international research studies.

Psychometric Assessment of Specific Cognitive Domains


Ideally, a neuropsychological battery should be a collection of reliable, valid tests
with known specificity and sensitivity. The tests should be standardized with
reference to normal individuals matched for sex, age, education, and also
socio-economic characteristics. Notwithstanding the fundamental role of neuro-
psychological methods in the diagnosis and in treatment evaluation for dementia,
validated psychometric measurements are still not extensively used in dementia
assessment. We will briefly describe some of the available tests, subdivided by
cognitive area.
Memory
It is commonly accepted that memory is not a unitary cognitive function, but can
be fractionated in multiple components with different disease susceptibility. In
dementia conditions the evaluation of the different memory processes
is mandatory.

Short-term and working memory: Short-term memory (STM) is a


system that allows the retention of a limited number of various kinds of
information to be retained over short periods of time. This retention process is
typically involved in carrying out various types of cognitive tasks, an aspect
which is captured by the concept of working memory (39). Baddeley defined
working memory “as the system for the temporary maintenance and manipulation
of information, necessary for the performance of such complex cognitive
activities as comprehension, learning, and reasoning” (40). Working memory is
composed of three subsystems: the Phonological Loop, the Visuospatial
Sketchpad, and the Central Executive (39). The Phonological Loop enables
phonological material in a phonological store to be maintained by a recycling
process. The Visuospatial Sketchpad is involved in storing a limited quantity of
Neuropsychological Screening and Advanced Tests 41

visuospatial information for a limited time. The Central Executive, which is an


attention system, is responsible for the control of the other slave subsystems. It is
generally agreed that patients with dementia, in particular AD and FTD, show
early in their disease a deficit of executive functions that are underpinned by the
Central Executive system (41). In the very earliest stage of the disease, there
might be a clear dissociation between the impairment of Central Executive
system and the relative preservation of the articulatory loop and the Visuospatial
Sketchpad (42). Patients at this stage of the disease, for example, might show
great difficulty in performing two different tasks simultaneously and in
manipulating information maintained in working memory.
Considering these cognitive aspects, it is not surprising that a number of
studies have addressed the assessement of STM in AD and other dementias. The
more commonly used measure of STM in AD is the memory span. Two tests are
widely used in clinical practice: the digit span and the visuospatial span. In the
digit span test, subjects are presented with a series of digits and are required to
reproduce immediately the series in the correct order. The number of digits the
subject can correctly recall constitutes his digit span. The visuospatial span is
usually measured with the Corsi test (43). This consists of nine cubes fastened in
a random order to a board. Each time the examiner taps the blocks in a
prearranged sequence, the patient must attempt to copy this tapping pattern. In
AD patients, memory span is generally reduced for words (44,45), letters
(44,46,47), digits (44,46,47), and spatial location (48–50). Although reduced
compared to normal old subjects, the memory span in early and in moderate AD
is relatively preserved compared to their performance on tasks tapping explicit
long term memory. Another approach has been to examine the free-recall
accuracy as a function of the serial position of the stimulus after giving the
subjects lists of words. Normal adults, both young and old, typically show a
u-shaped function with a recency (better recall for the first items of the list) and a
primacy (better recall for the last items) effect (51). The recency and the primacy
effects concern STM and LTM, respectively. In AD patients there is a mild
decrease in the recency portion of the curve and a major decrease in the primacy
portion. These results suggest that STM is relatively spared in AD compared to
LTM (52).

Explicit long-term memory: The evaluation of long-term memory


systems is mandatory in dementia. This should include the assessment of episodic
memory, i.e., the memory system involved in remembering specific episodes of
one’s own past (53), whose involvement is often the earliest symptom of AD, and
the assessment of semantic memory, i.e., encyclopedic knowledge, and the
meaning of words. The presence of episodic memory impairment is actually
required for the diagnosis of dementia (1,2). Episodic memory impairment is also a
powerful predictor of future dementia in individuals at risk (54–58). The early
detection of episodic memory impairment is therefore crucial for the diagnosis of
42 Perani

dementia and for the detection of persons that are likely to develop dementia in
the future.
There is a general agreement (59,60) that measures of delayed recall are the
most effective for the early diagnosis of AD compared to measures of immediate
recall. A non-progressive decrease in delay recall is a frequent finding in normal
aging, but it is also very common in AD, in such a way to render it unusable for
the evaluation of disease progression. The most used tests are learning of a word
list and the “story recall test” logical memory (LM), based on the evaluation of
the encoding of a written piece immediately after its reading by the examiner and
after ten minutes delay with different interferences. The California Verbal
Learning Test (CVLT) (61), for the evaluation of short- and long-term memory in
dementia using local normative values, is widely applied as part of batteries in
multicenter studies (62–64). A deficit in delay recall is typical in the initial phase
of dementia. The performances are indeed severely affected in early mild
dementia. In the non-verbal domain, the recall of Rey’s figure and Corsi’s
supraspan learning are among the most popular measures.
The theoretical principle of “encoding specificity” underlines the strict
dependence between the encoding and retrieval conditions. Indeed, an effective
retrieval requires specific encoding, because “what is perceived determines what
is stored, and what is stored determines what retrieval cues are effective in
providing access to what is stored” (65). It is well known that the encoding
specificity effect is lacking or is very weak even in the early stage of AD (54).
Most tests currently used for the diagnosis of dementia do not take into account
the relationship between encoding and retrieval. One exception is represented by
the Free and Cued Selective Reminding Test (FCSRT) (66). The FCSRT shows
high sensitivity and specificity for the diagnosis of early dementia (57,67).
However, as pointed out by Buschke et al. the FCSRT has two major limitations
(54). First, the power of the test is limited by ceiling effects. These ceiling effects
make important bias in the comparison of the effect of encoding specificity for
aged with dementia and aged without dementia. Buschke and co-workers
developed a test, the Double Memory Test (DMT), to remedy the limitations of
the FCSRT. The DMT includes two memory tests, one with and one without
coordinated acquisition and retrieval. The Category Cued Recall (CCR) memory
test optimizes encoding specificity by using the same category cues for
acquisition as well as retrieval. Buschke and co-workers compared the DMT
with two standard memory tests, Paired Associated (PA) and LM (68), in the
diagnosis of early dementia. They found that the CCR had much higher
sensitivity (93%) and specificity (99%) than PA (68%, 91%) and LM (48%,
92%). CCR had the greatest advantage in the mildest cases. These results show
that tests based on encoding specificity are much more powerful for the diagnosis
of early dementia than traditional learning tests. For the screening of dementia,
Buschke and co-workers have recently devised a test, the Memory Screen Test,
which is a brief, four-item delay free and cued recall memory test (69). This test
Neuropsychological Screening and Advanced Tests 43

showed good sensitivity and specificity for the screening of dementia (70),
providing efficient, reliable, and valid screening for AD and other dementias.
Attention
Attention is also a non-unitary function. Tests that evaluate vigilance and
selective focused attention include the verbal and non-verbal span and letter
cancellation tests. Selective divided attention is measured by the Brown-
Peterson, Trail B, and Stroop tests. The test most widely used to evaluate
selective attention abilities is the “cancellation” test. In this test, of which many
variants exist, the subjects are required to detect as quickly as possible a given
series of stimuli, for example a letter or a digit, among a great number of
distracters. Another widely used test of selective attention is the Stroop test (71).
Here the subjects are exposed to ambiguous stimuli, namely names of colors
written in an incongruous ink color (for example the word BLUE written in red).
In this task, the subjects are required to name as fast as possible the color of the
ink in which each word is written and to ignore the word itself.
Executive Functions
The most widely used tests in clinical practice are verbal fluency, Trail Making
Test (TMT), and Wisconsin Card Sorting Test (WCST). In the verbal fluency
tasks, the subject is required to retrieve as fast as possible words belonging to a
specific category (e.g., animals) or beginning with a given letter. Another widely
used test of executive functions and mental flexibility is the TMT (72). This test is
given in two parts, A and B. The subject must first draw lines to connect
consecutively numbered and lettered circles on one work sheet (Part A), and then
connect the same number of consecutively numbered and lettered circles on
another worksheet by alternating between the two sequences (Part B). The WCST
is probably the most widely used test to assess executive functions, in particular
“abstract behavior” and “shift of set” (73). In this test, the subject is given a pack
of 60 cards on which are printed one to four symbols, triangle, star, cross, or
circle, in red, green, yellow, or blue. The patient’s task is to place them by one
under four-stimulus cards according to a principle that the patient must deduce
from the pattern of the examiner responses to the patient’s placement of the cards.
Language
Language impairments are a prominent feature of AD (74) and FTD. The
mechanisms underlying the language deficits found in persons with AD are often
attributed to the direct result of the cognitive deficits, particularly those related to
memory. Impaired naming is the major change in language functioning in early
AD, and reflects the impairment of semantic memory. Word-finding difficulties
tend to be prominent, and their assessment plays a role in the differential
diagnosis of the dementias, especially AD (75,76). Naming tests such as the
Boston Naming Test (77) are therefore commonly used; as mentioned above it is
also part of the CERAD battery. Normative data for normal aging have recently
44 Perani

become available (78). Besides naming, other disorders in accessing semantic


information or complete loss of semantic knowledge have been described in AD
(79). Many studies have also shown impairment in syntactic processing. This
deficit has been particularly evident in comprehension of syntactically complex
sentences. Again, the poor syntactic processing in the AD population has been
ascribed to reduced working memory and difficulty in integrating information
(80). Due to its integrative nature, discourse processing, either in production and
comprehension, poses great demand in AD. Picture description is used for this
aim with a reported decrease in the number of content units and conciseness with
increasing severity (81). As the disease progresses, the language impairments
become more profound and might resemble fluent aphasia. There are significant
auditory and reading comprehension deficits, and language production is fluent
and paraphasic. In late stages of dementia, decreasing verbal output often
resulting in mutism is the rule. Tests such as the Token Test for the evaluation of
language comprehension (82,83) and the Reporter’s Test (82) have been used in
many studies aimed in particular at differentiating various types of dementia (84).
Special tests have also been used to assess repetition (85).

Praxis
Another cognitive deficit encountered in all stages of dementia is apraxia. An
evaluation battery proposed by De Renzi and Lucchelli has been used in AD (86).
The authors showed that both ideational apraxia and, to a lesser extent, ideomotor
apraxia (IMA) are present in AD. Apraxia is a crucial feature of corticobasal
degeneration. Spatial orientation and limb praxis are usually spared in FTD (87).
In AD, all aspects of visuoconstruction, including copying, drawing, and
construction in 3D, are impaired. The immediate reproduction of Rey’s Complex
Figure (88) and the Constructional Apraxia test (89) test visuospatial competence
as well as planning and perceptual organization. The WAIS-R block design task is
impaired in AD (90). Another possible source of constructional difficulties is
impaired executive function and global or local attention processes.

Visuospatial Abilities
In AD there are severe impairments of spatial abilities (91). AD patients have
difficulties in scanning the visual field in order to locate objects, and may present
with a deficit in spatial exploration and Balint’s syndrome (gaze apraxia, optic
ataxia, and simultagnosia) (92). These impairments lead to deficits in everyday
life including problems in writing, drawing, driving, following lines of text when
reading, and telling the time from a watch. Some patients, though rarely, may
present with spatial hemineglect. There is a disruption of personal and
extrapersonal orientation: inability to follow unfamiliar route, but also becoming
lost in familiar surroundings. Tests of the ability to undertake the “road map test”
in relation to one’s own body position showed a prevalence deficit in AD (93).
Neuropsychological Screening and Advanced Tests 45

The two aspects of spatial cognition that are most frequently evaluated in AD
are mental rotation and judgement of line orientation. Tests of detection of object
rotation or object rotation to solve object matching problems (94,95) show
significant impairment. The TMT B (96), described above, is also used to measure
the visuomotor planning. Street’s test (89) also measures visual and perceptual
abilities.

ASSESSMENT OF PSYCHOLOGICAL AND


BEHAVIORAL DISTURBANCES
The importance of evaluating non-cognitive aspects of dementia is increasingly
appreciated. In particular, the measurement of behavioral disturbances in
dementia represents a methodological and clinical challenge (97). Coexistence
of cognitive deficits with behavioral changes and psychotic symptoms makes it
difficult to obtain a proper characterization of the individual aspects. Several
instruments have been developed for the assessment of global, as well as of
specific, behavioral symptoms (98). Direct observation is limited to patients in
specialized institutions, whereas interview of the caregivers is possible for
patients at home. There is a tendency by the caregivers to provide either an above
or an under estimation of the deficits, according to the relationship with the
patient and the stress level (99).

Neuropsychiatric Inventory
The Neuropsychiatric Inventory (NPI) scale was developed in order to gather
data on the presence and nature of psychopathological disorders in patients with
cerebral pathologies (100), and addresses particularly patients with AD or other
dementias. It is based on caregiver’s information obtained by a questionnaire and
provides measurements of frequency and severity of behavioral disturbances. The
scale relies on the caregiver and evaluates twelve common psychiatric disorders,
from delusion, hallucinations, agitation, and depression to anxiety, disinhibition,
and apathy. Each single item is correlated to further sub-items that provide more
detailed information. Administration time is about 20 minutes. NPI is a valid
instrument for behavioral evaluation in AD, VaD, and FTD and may also help in
the differential diagnosis of dementia (101).

Clinical Insight Rating Scale


The evaluation of subjective experience is of great value in a patient with
dementia, in particular the awareness of the functional, behavioral, and cognitive
deficits. There is maintenance of insight, at least in the initial and intermediate
phases of disease progression (99). The awareness of disease seems to depend
upon the type and severity of dementia, due to the selective involvement of
specific brain regions (frontal lobes and right hemisphere, in particular) (102).
The Clinical Insight Rating scale represents a standardized approach to achieve
46 Perani

this aim for patient examination. It is a semi-structured interview preceded by an


interview with the caregiver that should provide information on the reasons for
the request for medical examination, the duration of disease, the onset and
progression of cognitive symptoms, and their impact on functional and daily
activities (103,104).

Assessment of Depression
A particular concern is the evaluation of depression. This is usually achieved with
the application of rating scales, such as the Geriatric Depression Scale, but also the
Hamilton, Beck, and Cornell scales. The latter properly addresses the evaluation
of depressive symptoms in dementia. It is the only scale validated in patient
populations, even at moderate-severe stages. It consists of a series of standardized
items obtained by patient and caregiver’s interviews. It is also based on the
patient’s own observations and does not require direct answers by the patient to a
standardized questionnaire. There are 19 items with graduate scale points from 0
(absence of symptom) to 2 (severe symptom).

FUNCTIONAL SCALES
It should be underlined that all diagnostic criteria require that the cognitive
deficits, assessed clinically and quantified by neuropsychological tests, have an
impact on the activities of daily living. Several functional scales are used with the
aim of evaluating working, social, and relational capacity in patients with
dementia (105). These scales present with a series of specific problems, which is
mainly concerned with the limited information about their validity and
sensitivity. A functional impairment depends also on occupational status and
personal attitudes, and it is additionally dependant on the subjective evaluation
and clinical interview (1). Nevertheless, standardized examinations are largely
used because they can give answers when it is necessary to compare different
phases of the disease, thereby measuring disease progression. They are also
useful for determining the impact of rehabilitative and pharmacological
treatments (106).
More precisely, a functional evaluation is aimed at measuring the
individual capacities in concrete activities and in social roles (107).

Living-Instrumental Activities of Daily Living


These rating scales, which have not been developed specifically for dementia, are
widely used for functional assessment, and form the basis for more targeted
instruments, such as the Activities of Daily Living (ADL) questionnaire (108).
ADL consists of instrumental ADL (IADL), which evaluates complex skills, such
as managing money, and basic ADL (BADL), which assesses self-maintenance
skills. The Barthel Index within the BADL provides valuable measurements of
walking, bathing, eating, dressing, and use of toilet. A patient is independent,
Neuropsychological Screening and Advanced Tests 47

dependent or completely dependent when he/she can exert control over all of
these functions, needs assistance or is completely dependent on nurse or
caregiver help. The ADL are widely used but are influenced by the cognitive
status, by the social and frequent usage of the subject, and also by the physical
conditions, such as for the BADL (106). A complete autonomy reflects the
capacity for the subject to live independently at home. In dementia there is a rapid
loss of IADL, and a certain sparing of the BADL, but in the more advanced
stages, all the activities are lost. A major problem, however, mainly concerns the
limited sensitivity in the early phase of dementia, in particular in individuals with
large social and relational interests. To solve this problem the Advanced
Activities of Daily Living (AADL) have been introduced. These represent
complex and demanding aspects of daily living such as travelling, hobbies, and
recreational and social activities (107). The AADL can reveal behavioral
modifications that, while not indicative of a specific cognitive impairment,
indicate the likelihood of incipient dementia.
A serious problem is the floor effect, since the lowest level of these
scales includes individuals with very different degrees of disability. The use
of the Bedford Alzheimer Assessment Nursing Scale, for severe disability,
allows a precise evaluation of the functional level in each individual, with the
possibility of monitoring also the effect of rehabilitative and therapeutic
approaches (109).
These indirect functional evaluations, all based on information provided by
the patient or very often by the caregiver, have some limits linked to variables
that might influence a correct report on the real capacity of the patients. To
overcome this problem, there are more direct evaluations based on objective
observation of functional capacity of the patient to perform a series of
standardized tasks that mimic the basic daily activities. Among these the
Physical Performance Test and the Direct Assessment Functional Scale formerly
used in the elderly have been applied in the evaluation of patients with dementia
(110,111). Finally, it is useful in monitoring of crucial survival abilities, such as
walking capacity, equilibrium, and feeding by standardized instruments (Tinetti
Scale) (112).

Communicative Abilities of Daily Living


Communicative Abilities of Daily Living (CADL) is a functionally oriented
battery which has been proposed to assess aphasic patients in an ecologically
valid fashion (113). CADL has been used to investigate functional communi-
cation skills in subjects with mild and moderate AD and their performance
compared with that of Wernicke’s aphasics, normal elderly, and elderly
depressed subjects (114). Subjects with mild and moderate AD were impaired
on the CADL, and the impairment was more severe in the moderately demented
group than in the mildly demented group. Language skills were also impaired in
elderly depressed subjects as compared with normal elderly controls; however,
48 Perani

the elderly depressed subjects were less impaired than were the mild AD subjects.
Finally, although performance of the moderate AD and Wernicke’s aphasic
subjects did not differ in terms of total CADL score, the performance of these
groups’ subtests was markedly different.

THE ROLE OF NEUROPSYCHOLOGICAL


ASSESSMENT OF DEMENTIA
The pattern of neuropsychological impairment in patients with different forms of
dementia reflects the location of brain damage rather than its underlying
pathology. Specific pathological conditions are associated with a relatively
selective topographical pattern of brain involvement, thus leading to relatively
typical neuropsychological pictures. However, there are many exceptions. For
example, typical AD is characterized by the early involvement of medial
temporal cortex and associative temporoparietal areas; typically, frontal
involvement is less severe or delayed. The situation is more complicated in the
case of dementias associated with focal lobar involvement in the temporal and
frontal lobes. Neuropsychological assessment provides different patient profiles
that might help in the differential diagnosis.
The fundamental applications of neuropsychology for the evaluation of
dementia are the following:
1. Differential diagnosis between cognitive changes in normal aging and
conversion to dementia (early diagnosis)
2. Differential diagnosis among different dementia conditions
3. Quantitative evaluation of disease progression
4. Assessment of severe dementia
5. Monitoring the effect of pharmacological treatments.

Early Diagnosis
It is well accepted that the natural course of dementia from a condition of full
health to an established dementia status may last several years. At the beginning
the patient might have a deficit limited to memory or to attention (not both),
without any disorder of instrumental and daily activities. The cognitive
impairment then proceeds to a degree that allows the diagnosis of dementia. In
the transitional state between normal aging and mild dementia is fitted the
term MCI.
The term MCI is an operational diagnostic term used to describe subjects at
risk of developing AD or in the pre-clinical stage of the disease (115). It is
reserved for subjects whose impairment is objectively demonstrable but is not
pronounced in more than one domain of cognition and does not seriously affect
activities of daily living. Neuropsychological assessment in MCI subjects shows
impairment in memory tests, especially delayed recall, a possible index of an
Neuropsychological Screening and Advanced Tests 49

incipient dementia process, thus supporting the role of cognitive testing in


identifying early or pre-clinical AD (116). Identification of pre-clinical AD
among MCI subjects might be important for the timely detection of patients to
whom currently available treatment options should be offered (117).
Nevertheless, it has been suggested that the proposed criteria for MCI may
apply to a heterogeneous population in which memory complaints could be due to
somatic diseases, drug-induced states, affective disorders, or other neurological
conditions, rather than to an ongoing AD-related process (118). Important aspects
in this respect are which kind of memory tests should be used to define impairment
(sensitivity and specificity) and if patients with other cognitive non-mnestic
deficits should be excluded. The different inclusion and exclusion criteria might
account for the reported large variable conversion rate to AD (119). In fact, narrow
inclusion criteria might fail to capture the heterogeneity of clinical AD
presentations, such as progressive visuospatial or language deficit. The problem
is even more complex because the differential diagnosis of these variants might
include a greater likelihood of dementia syndromes such as DLB and FTD. MCI
has been shown to convert to AD at 10% to 15% a year (115). 20% to 50% of MCI
subjects, according to different studies however, may remain stable (115,120).
Amnesic MCI usually evolves to AD, whereas MCI with deficits in a single
cognitive domain may convert to AD or to FTD, DLB, VaD, primary progressive
aphasia, or Parkinson’s disease. An MCI in several cognitive domains usually
reflects possible AD, VaD, or even normal aging.
The evaluation of memory is the basis for the differential diagnosis between
the early onset of dementia, and cognitive changes related to normal aging. The
initial phase of AD is marked by a progressive deterioration of episodic memory.
When the process advances, the impairment spreads to other cognitive functions,
such as semantic memory, language, and visuospatial ability.
A range of cognitive functions other than episodic memory, including
semantic memory (knowledge- or fact-based memory), attention, and executive
function (high-level control processes), is significantly impaired in the mild
stages of AD (118,121,122), and indeed, tests of many different cognitive
capacities have been shown to be predictive of future AD diagnosis among
memory-impaired subjects (56,59).
An ideal neuropsychological tool for the early detection of dementia should
be sensitive enough to differentiate dementia from normal aging and other brain
disorders that can mimic dementia, in particular depression. Recent neuropsy-
chological longitudinal research studies have been used in detecting individuals
at risk of developing dementia: community-based studies of elderly subjects,
studies of MCI with high risk of developing AD, and in presymptomatic
individuals with known genetics for autosomal dominant familial AD. These
approaches showed that episodic memory deficits might precede the onset of
clinical dementia by at least five years (123). Other cognitive domains, such as
mental speed, attention, and executive functions can be affected in advance, thus
becoming indicators of incipient dementia (59,124). In autosomal dominant AD,
50 Perani

subgroups of converters showed low IQ scores and lower verbal memory scores
than non-converters (125). In MCI and very early AD, impairment on tests of
episodic memory is generally the most predictive measure (57,115), but deficits
in semantic memory [semantic knowledge of famous people is also lost in very
early AD (126,127)], attention, and mental speed also predict conversion to AD
(15,128,129). Longitudinal studies revealed the role of a spatial learning test from
the CANTAB computer battery, the PA Learning (PAL) test, which is sensitive in
detecting the earliest stage of AD and is also able to distinguish patients with
depression (130). In the early phase of AD several extensive cognitive batteries
have been designed showing considerable promise (131).

Differential Diagnosis of Dementia


Neuropsychological evaluation should be considered an ancillary tool for the
differential diagnosis of dementia within the context of other clinical evidence. It
is of secondary role with respect to neuroradiological investigations in the
differential diagnosis of AD and cerebro-VaD, and with respect to clinical
examination for the differential diagnosis of AD and “subcortical dementia”
(such as in the case of Parkinson’s disease and Huntington’s disease).

Vascular Dementia
VaD is the second most common type of dementia; however, it is increasingly
being recognised that VaD is actually a heterogeneous syndrome and that several
vascular pathologies can lead to cognitive deterioration (132). In contrast to the
striking deficits produced by cortical infarcts, lesions of the subcortical white
matter are mainly associated with a non-specific slowing of behavior.
Cerebrovascular disease also plays an important role in forms of cognitive
decline other than dementia, and as such, it appears to be no less prevalent in old
age than AD. In addition, AD and VaD represent the extremes of the clinical
spectrum of dementia conditions that includes also various combinations of
cerebrovascular and degenerative pathology. It is thus evident how classical
diagnostic criteria are often of reduced clinical value (133). The relationship
between cerebrovascular lesions, as shown by neuroimaging, and focal
neuropsychological signs is difficult to define during clinical follow-up. There
is a common belief that the cognitive impairment should be ascribed to mixed
vascular and AD types of dementia (134). The prevalence of this kind of
dementia is high in the elderly population. A large multi-center study for the
treatment of CVD and AD showed a slower rate of progression of cognitive
impairment in “pure” VaD in comparison to AD (135) using the ADAS-cog
assessment in a two-year follow-up study.
A specific neuropsychological profile, characterized by a prominent
impairment of executive and visuospatial functions associated with a relative
sparing of episodic memory, has been suggested to be typical of subcortical
VaD (136).
Neuropsychological Screening and Advanced Tests 51

Frontotemporal Lobar Degenerations


Frontotemporal lobar degeneration (FTLD/FTD) is characterized by a relatively
selective degeneration of frontal and/or temporal cortical areas. According to
widely accepted diagnostic criteria (4), three different clinical syndromes are
delineated. The frontal variant of FTD is characterized by prevalent behavioral
symptoms, with early change in personality and difficulty in modulating behavior,
often resulting in inappropriate responses and activities; semantic dementia is
associated with prevalent semantic/cognitive impairment; and progressive
aphasia is characterized by early non-fluent aphasia (137,138). The differential
diagnosis from AD is unreliable on the basis of NINCDS-ADRDA criteria (139).
Improved reliability may be achieved by means of clinical features and
neuropsychological tests (140). Memory disorder and even dementia are not
paramount as early symptoms of FTD, so that at least in the early stages, this
syndrome has to be differentiated from psychiatric disorders more than from other
types of dementia. Thus, measurements of behavioral impairments are
mandatory. A behavioral frontotemporal dysfunction assessment scale validated
on patients at the mild stage (MMSE O18) helps to distinguish between FTD,
AD, and VaD (141). The items of this structured interview are classified into four
classes: self-monitoring dyscontrol, self-neglect, self-centered behavior, and
affective behavior. A score of 1 is given for the class if at least one symptom is
present. A total score of 3 or more gives a sensitivity of 100%, a specificity of
93%, and a diagnostic accuracy for FTD of 97%. The assessment of
neuropsychiatric symptoms with other standardized scales or inventory is useful
in distinguishing dementia patients with FTD and AD (142,143). The Frontal
Behavioral Inventory Scale (144) provides a tool to distinguish between the
different clinical variants of FTD.
In the frontal variant of FTD it is possible to identify two opposite
behavioral profiles (145). Apathy, psychomotor inhibition, lack of emotional
reactions, and defective social functioning characterize the more common
“apathetic” behavior, whereas impulsiveness, perseverations, disinhibition, and
lack of concern for others are associated with the less common “disinhibited”
behavior (146).
Diagnostic criteria for FTD are based on the following clinical findings:
insidious onset and gradual progression, early decline in social interpersonal
conduct, early emotional blunting, and loss of insight. One or more of the
following can be associated: oral and manual exploration, hyperphagia, echolalia,
impulsive behavior, jocularity, and inappropriate actions or words. A
characteristic aspect is the discrepancy between judgment and behavioral
alteration that is out of proportion with the memory disorder. Neuropsychology
shows significant impairment on frontal lobe tests in the absence of severe
amnesia, aphasia, or perceptual disorders. In FTD, an extensive neuropsycho-
logical test battery, aimed at assessing the different cognitive domains, including
in particular the executive functions, is suggested: the MMSE (147) as a global
52 Perani

measure of dementia severity, Tower of London Test, WCST for frontal function,
and Raven’s Colored Progressive Matrices (148) for abstract reasoning. Also,
there are the CVLT (61), Story Recall, and Word Learning (149) tests to assess
verbal recall and verbal learning, the recall of Rey’s Complex Figure (88) for
visual long-term memory, and the Digit Span forward and reverse test (150) to
evaluate STM. The pattern of memory decline differs from that of AD: memory
performances benefit from cues and from the provision of multiple-choice
alternative responses. A deficit of retrieval strategies more than a storage problem
has been suggested (84). The TMT A is used to test attention/concentration of the
patients, and the TMT B, which also measures visuomotor planning, is used for
the assessment of divided attention (96). The immediate reproduction of Rey’s
Complex Figure (88) and the Constructional Apraxia test for visuospatial
competence, as well as planning and perceptual organization and the Street’s test
(89) for visual and perceptual abilities, are also used. Language ability should be
tested not only for the classic naming, repetition, reading, and writing
competences, but also for phonological, semantic, and grammatical aspects, as
well as language comprehension (e.g., Token Test) (89). With this aim, the
BADA Italian standardized aphasia protocol (151) has been used in Italy. In FTD
there is an economy of language output, hypophonia, and loss of prosody.
Perseveration, echolalia, and stereotypies occur in the course of the disease.
Mutism usually ensues in the final stages. Patients have no perceptuospatial
difficulty, and skills are preserved even in advanced disease. There is no spatial
disorientation, which is an important distinguishing feature from AD. Due to
reduced cooperation and concentration deficits, it might be difficult to administer
the whole neuropsychological battery to some patients.
Primary progressive aphasia usually presents with insidious and gradual
progression of anomia or word-comprehension impairment. Intact premorbid
language and absence of a “specific” cause, stroke in particular, are also
hallmarks. A non-fluent aphasia is typical. Acalculia and IMA may be present, but
there is an absence of significant forgetfulness and apathy for the initial two years.
ADL impairment, when present, should be ascribed to language deficits.
Semantic dementia presents with anomia, impairment in word comprehen-
sion, and loss of knowledge on semantic tasks (152). A fluent aphasia of the
transcortical type is a hallmark. There might be also reading difficulties (surface
dyslexia) with inability to read irregularly spelled words, but intact syntactic
functions. The Pyramids and Palms test can be used to evaluate the impairment of
semantic knowledge (153).
In a series of comparative studies, patients with the clinical diagnosis of
probabile AD and FTD showed significantly different neuropsychological profiles.
In an object and action naming test, FTD patients were particularly impaired in the
latter (154); during word generation on a phonemic cue (f, p, l as initial letters) or a
semantic cue (animal, fruit names), FTD patients showed major impairment with
the phonemic fluency, whereas AD patients were impaired in semantic fluency.
Neuropsychological Screening and Advanced Tests 53

A brief neuropsychological assessment for the differential diagnosis


between FTD and AD has been designed by Siri and coworkers (155). They
found considerable overlap between AD and FTD. Four tests (Rey figure recall,
phonemic fluency, oral apraxia, and cube analysis) achieved 70% sensitivity and
80% specificity for the correct classification of patients in the FTD or AD group.
The conclusion is to include these four tests, as well as other measures sensitive
to the frontal impairment, since they can be useful in the differential diagnosis.
Dementia with Lewy Bodies
DLB is diagnosed in vivo on the basis of a series of clinical criteria, which do not
include a differential pattern of cognitive impairment in comparison with AD (156).
Others instead reported differences in cognitive profiles. Mental status, compared to
AD, shows a disproportionately severe attention, fluency, visuoperceptual, as well as
visuoconstructive, and visuospatial impairment. Fluctuations are the rule,
particularly for attentional aspects. Neuropsychological features are characterized
by mild memory deficits, attentional deficits, impaired working memory, and slowed
processing speed. The DLB profile was reported to resemble the pattern of dementia
found in PD (157). These results are compatible with the view that cognitive
dysfunction in DLB reflects combined subcortical and cortical involvement with
relative sparing of the medial temporal lobes. A strong predictor of accelerated
cognitive decline in DLB is the APOE E4 allele.
The clock-face drawing has been used to differentiate DLB and AD type
dementia syndromes, with a higher score for Draw than Copy in AD, a pattern not
specific for DLB (158).
Alzheimer’s Disease and Depression
Neuropsychological deficits such as poor episodic memory are a consistent
feature of the early course AD, but they overlap with cognitive impairments in
other disorders such as depression, making differential diagnosis difficult.
Contributing to this is the fact that some episodic memory tests that are very
sensitive to AD, particularly “effortful” tasks such as free recall, are also
vulnerable in depression (159,160). It is very important that patients without AD
are not falsely “detected” by such a test.
Computerised and traditional tests of memory, attention, and executive
function were given to mild AD, questionable dementia, and major depression
patients (161). Only a restricted set of neuropsychological measures was sensitive to
impairment in questionable dementia compared to the depressed and control group,
in particular, tests for episodic and semantic memory and category fluency. A
visuospatial associative learning test (the PAL test, see 130) accurately distinguished
AD from depressed subjects and revealed a subgroup of questionable dementia
patients who performed like AD patients. Elements of contextual and cued recall
may account for the task’s sensitivity and specificity for AD. The PAL test is thus
sensitive and specific for AD, and it may also be sensitive to the specific memory
deficits of prodromal AD.
54 Perani

Because of its requirement to combine object-based and contextual


information, PAL may be particularly sensitive to the entorhinal/hippocampal
damage sustained in AD, and the provision of supportive recall cues may explain its
relative insensitivity to strategic, or effortful processes, which may underlie poor
memory function in the elderly and in depression. This might be relevant for clinical
practise. All the tasks of attention and executive function are of no value in
differentiating individuals with questionable dementia and those who are depressed.
As a whole, these results suggest that prodromal-early AD differs from depressed
and elderly healthy subjects significantly in terms of memory test scores.

Evaluation of Disease Progression


In this field, psychometric instruments should have characteristics that are
different with respect to the ones employed in the early diagnosis. First of all, they
should not present “floor effects,” in particular for episodic memory measure-
ments, nor should they be insufficiently sensitive to impairments in cognitive
functions partially spared by the pathological processes (i.e., apraxia, implicit
memory). In this respect, visuoperceptive and language tests, in other words, the
fundamental assessment of right and left hemispheric cortical function also need
appropriate norms and criteria (see “Advanced Tests and Future Research
Approaches” section).
Since in the advanced phase of disease all the traditional neuropsycholo-
gical tests show a “floor effect” and become useless, in the evaluation of severe
cognitive impairment, new neuropsychological batteries have been developed
(see “Severe Dementia” section).
In a follow-up evaluation, consideration should be given to determining
cognitive deficits, functional level in daily activity, and non-cognitive symptoms.
According to the severity as assessed by the CDR, the following schema can be
followed:
CDR 0.5: MMSE, extensive psychometric examination, IADL, Barthel
Index.
CDR 1: MMSE, extensive psychometric examination (optional), IADL,
Barthel Index.
CDR 2: MMSE, IADL, Barthel Index, NPI, Tinetti Scale.
CDR 3: MMSE, Barthel Index, NPI, Tinetti Scale, BANNS Scale.
CDR 4: Barthel Index, NPI, Tinetti Scale, BANNS Scale.

Severe Dementia
The great increase of the prevalence of age-associated dementias, particularly AD,
has become a major health problem. The majority of cases evolve towards a stage
of marked severity, which can last many years. Severe dementia is accompanied by
loss of autonomy and impairment in activity of daily living ADL, and severe
Neuropsychological Screening and Advanced Tests 55

cognitive deficits in several cognitive domains (memory, language, attention,


praxis, and visuospatial behavior). This stage of dementia is operationally defined
on the basis of scores on global scales such as stage 6–7 on GDS, or 3 or more on
CDR. The MMSE threshold for severe dementia is considered below 10. The
advanced phases of dementia are very often associated with behavioral and
psychiatric symptoms. These aspects, which might appear acutely or sub-acutely
in patients who are often old or very old, should always be considered in order to
rule out associated pathologies (pneumonia, acute pain, urinary retention, etc.).
Aggressiveness is the most frequent disorder (estimated between 30% and 55%)
(162). Keene et al. 1999 (163) found that in AD or VaD, aggression is related to
more severe dementia and loss of personal care. Sometimes aggressive behavior is
associated with conflicts with the family and caregivers. Depression has a
prevalence of 17–35% in the literature, the highest percentage for a population of
patients with severe dementia (164); it may be also highly correlated with anxiety
(165). The best scale for measuring depression in severe dementia is the Cornell
Scale (166). Hallucinations, motor disorders, and neurological signs should be
considered in the differential diagnosis with AD and other dementias (LBD, FTD,
CBD, etc.) (167).
Despite its size, the population of patients with severe dementia has been
studied relatively little. In addition, the few studies have dealt almost
exclusively with AD, and, as a consequence, little is known about the advanced
stages of other dementias. Evaluation tools currently used for dementia are, in
most cases, diagnostic instruments designed for patients who are in the early
and moderate stages of their disease, and they are therefore generally
unsatisfactory for assessing patients with severe dementia (168). For instance,
the MMSE often yields a floor effect in cases of advanced dementia so that
preserved capacities in these individuals may be overlooked. Researchers
investigating advanced dementia have used instruments such as behavioral
scales (169) and coma scales (170). The Katz ADL Index is used in the
evaluation of functional status and is designed to be used in institutions by a
physician or a nurse.
While these scales are based on the observation of behaviors and
symptoms, they do not provide a precise evaluation of the cognitive profiles of
the patient (171). In recent years, several batteries specifically aimed at testing
patients with severe dementia have been proposed. One such scale is the
Hierarchic Dementia Scale (172) that includes 20 subtests which cover the entire
range of cognitive and motor functions. Other batteries such as the Severe
Cognitive Impairment Profile (173) and the Test for Severe Impairment Battery
(SIB) are reliable, validated measures of neuropsychological functioning in
severely demented patients with AD, though they are not validated outside of the
United States. The SIB has been developed keeping into account the specific
cognitive and behavioral features of patients with severe dementia (174). This
scale is very appropriate for severe dementia since, provided the patients agree to
take it, it almost always yields scores that are above 0. Administration time is
56 Perani

around 30 minutes and includes evaluation of all cognitive functions plus


evaluation of social interaction derived from the CADL. It includes very simple
tasks, which are presented with gestual indices. It has the advantage of having
been validated not only in its original US version, but also in several languages
including French (175,176), Italian (177), and Spanish (178,179).

Monitoring the Effect of Pharmacological Treatments


With regard to monitoring the effects of pharmacological treatments, short
neuropsychological batteries, such as ADAS and CAMDEX, are frequently used.
They provide quantitative information on the main cognitive areas within a
limited time frame, together with information on affective and behavioral
disturbances. Clinically, it is more relevant to measure changes in functional
impairment than changes in task performances. It is thus necessary to use not only
the classic measurements of cognitive deficits but also “ecologic” tasks and
functional scales. As for cognitive assessment, the available instruments are
limited: Rivermead Behavioral Memory Test (180), and attention and executive
function tests for daily living (still to be standardized in many countries). The
limits of functional scales have already been discussed.

ADVANCED TESTS AND FUTURE RESEARCH APPROACHES


Neuropsychology is an important asset in the study and treatment of cognitive
decline, but must be embedded in a multidisciplinary context. In addition, in
order to understand the different symptoms of different diseases in the elderly,
one should keep in mind the relevant principles in the neuropsychological
assessment of older individuals and the complex relationship between aging and
cognition. Neuropsychological assessment aims to identify cognitive impair-
ments in a maximally objective manner. To this end, patients are confronted with
standardized tasks, preferably well documented with regard to reliability,
validity, and norms. It should be remembered, however, that testing “oldest”
elderly people is lacking for many instruments, in particular for normative data.
Neuropsychological assessment is a powerful aid in the detection of early
dementia, but has certain limitations. Firstly, the test results will be useful only if
the subject is fully cooperative. Secondly, individual variation in cognitive
performance is—especially in the elderly—considerable, even among people
with the same demographic characteristics (age, sex, education); therefore, a
wide margin of uncertainty must be allowed before impairment can be inferred.
Even so, the impairment may be due to many causes besides brain dysfunction.
Finally, a good deal of interpretation may be required in deciding what functional
disturbance(s) underlie a deviant test result. For example, the term MCI is
reserved for patients whose impairment is objectively demonstrable, but is not
pronounced in more than one domain of cognition, and does not seriously affect
activities of daily living (181). In MCI, however, while current criteria provide a
Neuropsychological Screening and Advanced Tests 57

convenient clinical procedure for identifying people at risk of developing


dementia, there are numerous limitations. Firstly, even strict criteria still allow
considerable latitude in assessment methods, for instance, in the quality and
number of tests used. In connection with this, studies of the prevalence of MCI
and its rate of conversion to dementia are poorly comparable and yield highly
variable results. It would be very important to reconsider neuropsychological
batteries, by implementing new tests, and obtaing normative data in elderly
normal individuals with well-defined social, demographic, and educational
characteristics. More sensitive instruments for cognitive decline evaluation will
be thus available.
Furthermore, different types of dementia may not be equally recognizable by
their early symptoms (182). For example, VaD actually consists of a heterogeneous
collection of clinical features (132). The combined occurrence of AD and VaD is
probably more common than it has been assumed. Lacunar infarcts and white
matter lesions in subcortical areas lead to cognitive effects usually quite different
from those of AD. Patients with white matter lesions demonstrate a clinical
syndrome of subcortical dementia. Their cognitive performance remains largely
adequate, though conspicuously slowed. This slowing is accompanied by
relatively mild impairments of memory (mostly affecting reproduction) and
executive function (mainly reflected by loss of mental flexibility and difficulty in
dealing with complex situations). The patients are usually well aware of their
limitations. It is thought that subcortical ischaemia contributes substantially to
“normal” age-related decline (183,184). On the other hand, multi-infarct dementia,
a syndrome caused by large cortical infarcts, leads to multiple functional and
cognitive disturbances. An obvious example is the effect of single cortical infarcts,
which consist of circumscribed functional disturbances corresponding with the
location of the infarct. Assessment of the principal deficit might not be enough, and
an extended neuropsychological evaluation is mandatory to define the complete
cognitive profile. In addition, unlike AD, cerebrovascular disease is not necessarily
progressive. It usually can cause more limited and static cognitive impairment.
This requires neuropsychological assessment that is able to define cognitive
stability and distinguish a real worsening from fluctuations associated with
temporary variables. The problem of evaluation of cognitive fluctuations is crucial
in the neuropsychological assessment of DLB. Together with standardized
neuropsychological batteries, it can be useful to administer scales for the
evaluation of fluctuation in behavioral and cognitive symptoms (185).
Different profiles according to the disease stages should be considered in
the evaluation of dementia. For example, when an extensive battery of
visuospatial perception tests was administered to mild probable AD patients in
very early stage, these were not impaired (186). They were impaired only in an
object-naming task. After eight months, the performance in the subtests of object
perception was unchanged, while there was a significant decline in the total score
of the items investigating space perception. A significant worsening was also
observed in the Rey’s figure copy score and was correlated with the decrease in
58 Perani

the spatial perception score. This study confirmed that impairment in visual
perceptual tests requiring access to semantic and lexical knowledge is present in
the earliest phase of AD, whereas visuospatial and constructional impairments
became evident only later. The authors suggest that this pattern of progression
may represent the clinical correlate of increasing pathological involvement of
posterior associative cortex.
There is also a characteristic cognitive profile and course of dementia in
FTD. Nonetheless, cognitive test performance does not clearly distinguish FTD
from AD in the early phase. A cross-sectional and longitudinal study was
specifically instituted to address the cognitive characteristics of FTD, with the
aim of determining which features distinguish FTD from AD. A large series of
patients were included, some with a pathologically verified diagnosis of Pick’s
disease. Information regarding the initial symptoms of dementia were obtained
from each patient’s caregiver, global dementia severity was estimated by the
BDS and the ADL scales, and specific cognitive domains were assessed by
administering tests of memory, language, visuospatial, and reasoning abilities
and selective attention. Among initial symptoms reported by caregivers,
personality change and language impairment were significantly more common
in FTD than AD. At initial cognitive testing, deficits in memory were common in
both groups, but more prevalent in AD. Patients with FTD had greater
impairments on the ADL Scale. During the course of illness, patients with FTD
declined significantly faster than those with AD on language tests and on global
measures of dementia severity (187). These results in this large co-hort of
patients with dementia underline the importance of determining cognitive
profiles at the beginning and during the course of the disease, for the
differential diagnosis.
The same is supported by another study that suggests a selection of
neuropsychological tests are sensitive in the differential diagnosis of AD (155).
Neuropsychological performance (including measures of language, semantic
memory, visual and spatial perception, and executive functions) was compared in
two groups of patients with clinical diagnosis of probable AD or FTD. The aim was
to identify a specific cognitive profile for FTD to be used as a sensitive short
evaluation for the differential diagnosis with AD. Both groups were severely
impaired in most tasks, but interestingly also in “frontal lobe” tests that have been
suggested to play an important role in the differential diagnosis. Noteworthy,
significant differences were found for a minority of tests that assessed oral praxis,
visuospatial perception, and verbal fluency. The authors suggest a shortened testing
procedure based on four tests (Rey Complex Figure recall, phonemic fluency, cube
analysis, and oral apraxia test), together with other measures sensitive to frontal
impairment as a useful tool in the differential diagnosis between AD and FTD.
New research approaches in the neuropsychological evaluation of dementia
might derive from neuroimaging studies. Normal subjects executing specific
cognitive tasks during functional magnetic resonance imaging (fMRI) or positron
emission tomography (PET) acquisitions have shown the activation of complex
Neuropsychological Screening and Advanced Tests 59

neural systems. These studies provide evidence for the neural correlates of
cognition that go far beyond classical neuropsychological correlates in brain-
damaged patients. For example, they suggest that whereas the left temporal
neocortex plays a crucial role in all tasks involving lexical-semantic processing,
some regions of the left prefrontal convexity are selectively recruited during verb
processing. Repetitive transcranial magnetic stimulation (rTMS) also showed
different neural correlates for noun and verb processing in the human brain (188).
In fact, a shortening of naming latency for actions was observed only after
stimulation of left prefrontal cortex. On this basis, noun and verb processing in
different dementia types were assessed (154). Object and action naming was
tested in probable AD patients with mild to moderate dementia and in a group of
FTD patients. AD and FTD patients were impaired in naming compared with
control subjects; action naming being more severely impaired. However, the
discrepancy between object and action naming was significantly greater in FTD
than in AD patients, independent of the severity of dementia or of overall
language impairment. The latter finding is compatible with the hypothesis that
the frontal lobe plays a crucial role in action naming. The authors suggest a
relatively selective impairment in action naming as a characteristic neuropsy-
chological feature of FTD.
New interesting research approaches aimed at identifying specific
personality and behavioral profiles in dementia have recently emerged. In
FTD, diagnostic criteria agree that alterations in personality and social conduct
are a central clinical feature of the disease (4,137). However, quantitative
instruments have not been used to systematically measure these changes.
Similarly, specific social deficits in the FTD syndrome have not been isolated to
either the frontal or temporal variants of the disease. A promising approach is the
study of “theory of mind,” a key aspect of social cognition related to the
prefrontal cortex, which has been reported to be affected in the frontal variant of
FTD (189). One source of confusion may be that clinicians and researchers have
focused on discrete behavioral changes (138,142,190), since they do not have the
tools to observe a patient’s social processing on a higher level. Since personality
becomes fixed by early adulthood and remains constant throughout old age,
significant changes in personality during adulthood typically have a neuropatho-
logic etiology (191). Because the primary brain areas affected in FTD are the
frontal lobes, in particular the orbitofrontal and frontal medial cortex, the anterior
temporal lobes, the amygdala, and ventral striatum (63), the observed personality
changes ostensibly arise from a disruption of these structures. In contrast, early in
the disease, social functioning remains preserved in AD, most likely because of
the relative sparing of these anterior structures (192,193). It therefore seems
important to include investigations of how particular social deficits correlate with
neuropsychological functioning and areas of pathology on functional brain scans.
It will become possible to further characterize a patient’s personality change as a
function of the timing of the disease progression. A recent paper suggests the
Interpersonal Adjectives Scales, since they were able to differentiate frontal and
60 Perani

temporal variants of FTD from patients with AD, who remained within the
normal range on all scores, on the basis of both degree and direction of
personality change (194,195). On a similar basis, the Frontal Behavioral
Inventory Scale is a useful tool for the assessment of behavioral changes in
FTD patients.
The identification of clinical subtypes of probable AD, such as the so-called
posterior cortical atrophy variant, raises the issue of a more focused
neuropsychological assessment. Posterior cortical atrophy presents with prevalent
visuospatial deficits, thus evaluation of visuoperceptual abilities and also praxis is
necessary. Two batteries have been proposed so far, the Visual Object and Space
Perception Battery and the Birmingham Object Recognition Battery. They are
both time-demanding, and therefore considering the patient’s compliance, a
useful evaluation can be achieved by a using a shorter task administration, such as
Benton’s Line Judgment Orientation and Benton’s Face Test.

CONCLUSIONS
Not long ago, cognitive aging was viewed as a general deterioration of cognition
(reflected by a decreasing IQ) and dementia as an acceleration of this process.
Currently, we know that cognitive decline occurs in various forms, marked by
considerable differences in the nature and order of development of behavioral
symptoms. This knowledge is an important asset in the early detection,
management, and treatment of dementia. The neuropsychological assessment
of dementia has a central role and is concerned with all disease stages. The
current implicit assumption that all patients with AD tend to evolve and decline in
a similar fashion needs to be critically re-examined. Neuropsychological tests
allow one to determine various patients’ profiles. Future research should
determine the possible predictive value of these profiles, which has important
implications for therapeutic trials. Clinically, neuropsychological instruments are
crucial for the diagnosis, prognosis, and monitoring of pharmacological
treatments. In the field of research, its role is even more clear-cut: a correct
clinical diagnosis is mandatory in instrumental and pharmacological trials.
However, the neuropsychological approach to dementia certainly has limitations
and is at its proper place only in a multidisciplinary context. The concerted effort
of all branches of neuroscience is required for patient care, as well as research on
cognitive decline in aging individuals and in neurological diseases.

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3
Biomarkers in Blood and
Cerebrospinal Fluid

Harald Hampel and Katharina Buerger


Alzheimer Memorial Center and Geriatric Psychiatry Branch, Department
of Psychiatry, Ludwig-Maximilian University, Munich, Germany

INTRODUCTION
Alzheimer’s disease (AD) is the most common neurodegenerative disorder and
afflicts about 10% of the population over 60. Biomarker research in neuro-
degenerative disorders using blood and cerebrospinal fluid (CSF) is focused on
specific targets: early detection, differential diagnosis (classification), tracking of
disease progression, and evaluation of therapeutic strategies. With the currently
approved antidementia drugs and with the development of novel disease
modifying therapeutic strategies (secondary prevention), it is of particular
clinical interest to establish early diagnostic and preclinical prognostic
biomarkers of AD.
Criteria for a useful biomarker were proposed by an international consensus
group on molecular and biochemical markers of AD in 1998 (1). According to
these guidelines, a biomarker for AD should detect important aspects of the
fundamental neuropathology and be validated in neuropathologically confirmed
cases. Its sensitivity for detecting AD should exceed 85% and its specificity in
differentiating between AD and other dementias should be at least 75%. Ideally, a
biomarker should also be reliable, reproducible, non-invasive, simple to perform,
and inexpensive.

73
74 Hampel and Buerger

Until now, a large number of biomarker studies in AD have been


reported (2–4). Relevance for clinical practice and feasibility, however, is
questionable for the majority of markers that have been studied so far.
Recently, based on accumulating data, the biological markers working group of
the NIA neuroimaging in AD initiative suggested a selected set of 13
biomarker candidates as feasible core markers of AD for large-scale multi-
center studies (4). Feasibility was determined by the availability of a validated
assay for the biological marker in question, with properties that include high
precision and reliability of measurement, as well as good description of
reagents and standards. Core analytes were those judged by the initiative to
have reasonable evidence for association with key mechanisms of pathology
implicated in AD. There could be several different reasons to support
measuring a biochemical marker, ranging from increasing diagnostic accuracy,
enhancing the prediction of progression from mild cognitive impairment (MCI)
to clinical AD, or providing insight into a pathway influenced by drug
treatment for AD. It is likely that no single marker could serve all these
utilities, hence the need for a panel of measures.
In this chapter, currently available data on these 13 putative biomarkers of
AD are presented and their potential applicability for clinical use is discussed.
Moreover, results on protein 14-3-3 are described briefly, a biomarker that has
been established for the diagnosis of primary dementia disorders, such as
Creutzfeldt–Jakob disease (CJD). As a perspective for further biomarker
research, recent studies on naturally occurring antibodies against beta-amyloid
(Ab) peptide are presented.

b-AMYLOID PEPTIDE 40 AND 42


Extracellular senile plaques consisting of Ab are one of the histopathological
hallmarks of AD (5). Secreted soluble Ab is found in various body fluids
including plasma and CSF (6).

Ab in Cerebrospinal Fluid
Initial reports on Ab in CSF as a biomarker for AD were disappointing, with
results ranging form a slight decrease in AD, with a large overlap, to no change
(7–9). In these studies, total Ab in CSF was examined. More recent studies have
shown two major C-terminal variants of Ab, with either 40 (Ab40) or 42 (Ab42)
amino acids. Different ELISA tests have been developed for the measurement of
Ab42 in CSF and plasma (10,11). There are commercially available ELISA-kits
for the measurement of Ab42 and Ab40 in CSF (e.g., The Genetics, Schlieren,
Switzerland; Innogenetics, Gent, Belgium). Several studies reported that Ab42
concentrations are decreased in CSF of AD patients (12–17). The specificity to
distinguish patients with AD from controls has varied from 42% to 88%, and the
Biomarkers in Blood and Cerebrospinal Fluid 75

sensitivity has varied from 72% to 100% in these studies. CSF levels for Ab42
seem to be unrelated to age. Based on recent data, a cutoff-level of O500 pg/ml
has been suggested to discriminate AD from normal aging (18). Further, it has
been reported that Ab42 levels were lower in AD patients with the APOE 34
allele than those without APOE 34 allele (19). The sensitivity of Ab42
measurement was 83.6% for AD patients carrying an APOE 34 allele, whereas in
AD patients without APOE 34 allele it was 54.2%. Decreased CSF Ab42 levels
are not specific for AD since studies showed that half of the patients with vascular
dementia (VaD) had decreased CSF Ab42 (16).
It has been hypothesized that a decrease of Ab42 in CSF indicates an early
stage of AD before clinically overt dementia is detectable. A significant decrease
of CSF Ab42 in MCI subjects compared to controls has been shown, but this
study had no follow-up measure (20). In a further study investigating MCI
patients, Ab42 levels did not differ significantly from age-matched normal
controls (21). Control subjects in this study, however, were subjects with memory
complaints without neuropsychological impairment. Since it has been reported
that individuals with memory complaints have a higher risk of dementia (22–24),
the control group in this study might represent a subgroup with subclinical AD
pathology reflected in altered CSF Ab42 levels. More recently, it has been shown
that Ab42 protein may be an indicator of early identification of AD in MCI
subjects when taking potential confounding factors into account such as age,
severity of cognitive decline, time of observation, APOE E 34 (APOE 34) carrier
status, and gender (25).
Studies correlating CSF Ab42 protein concentrations with cognitive
performance in AD have been contradictory. Cross-sectionally, the concentration
of Ab42 protein and cognitive measures were either inversely correlated (26–28)
or no significant correlation was found (12,15,16,29). In a longitudinal study, a
decrease in CSF Ab42 protein has been found within a three year follow-up (30).
A highly significant correlation between low CSF concentrations at baseline and
one year serial follow-up was demonstrated. In a separate study, no correlation
was found between CSF levels and duration or severity of AD (12). The potential
value of Ab42 during the course of AD progression should be further evaluated.
Several studies have been performed on the potential of CSF Ab42 to
differentiate AD from other neurodegenerative disorders. Compared to control
subjects with other neurological conditions, a slight decrease has been described
in non-AD dementias (31). Normal or decreased levels of Ab42 were reported in
Parkinson’s disease (PD) (11,32). In Lewy body dementia, a disorder also
characterized by the presence of senile plaques, low levels of Ab42 protein have
been detected. The range of Ab42 protein concentrations found in Lewy body
dementia overlaps with Ab42 concentrations found in AD patients (20,33–35).
Furthermore, low CSF Ab42 protein is found in a relatively large percentage of
patients with frontotemporal dementia (FTD) and VaD (16,36). In summary, CSF
Ab42 does not seem to significantly support the differential diagnosis of AD.
76 Hampel and Buerger

A satisfying explanation for the decrease of the Ab42 level in CSF to about
40–50% of control levels in AD (14) is still lacking. One suggested mechanism is
that it is caused by the deposition of Ab42 in plaques, with lower amounts of Ab
being free to diffuse into CSF (11). This explanation is also supported by the
finding of a strong correlation between low Ab42 in ventricular CSF and high
numbers of plaques in the neocortex and hippocampus (37). However,
subsequent studies also found a marked reduction in CSF Ab42 in disorders
without b-amyloid plaques, such as CJD (38), amyotrophic lateral sclerosis (39),
and multiple system atrophy (40). This points to other mechanisms, such as
disturbances in Ab formation and breakdown in AD. Interestingly, one study
reported a significant increase of Ab42 protein in patients with early stage of AD
followed by a steady decrease concentration (10). This might be explained by
different sets of antibodies and protocols used, as well as by differences in the
stage of the disease when the patients were included.
Several CSF studies have shown that concentrations of another amyloid
b peptide species, Ab40, were similar in AD and controls (11,17,41,42). In one
study (43), however, a decrease in CSF Ab40 values was found with significant
overlap between the groups. Another study reported that Ab40 is deposited later
in the disease and is prominent in vascular amyloid deposits (44).
In addition to Ab42 and Ab40 in CSF alone, the ratio of Ab42 to Ab40 has
been investigated. The ratio of Ab42 to Ab40 is suggested to be superior to the
concentration of Ab42 alone in discriminating patients with AD from normal
controls (42). Furthermore, a recent study showed that for the neurochemical
diagnosis of AD, the number of correctly classified patients turned out to be
slightly higher compared to all groups (non-Alzheimer dementia and control
subjects) when the Ab peptide ratio was used instead of the Ab42 concentration
alone (correct discrimination of AD and controls increases from 86.7% to 94%
when Ab42 is replaced with Ab peptide ratio); however, this effect failed to reach
significance (17).
Ab in Plasma
Some studies suggest that plasma Ab40 and Ab42 levels were two- to threefold
higher in patients with familial AD and with presenilin mutations than in subjects
with sporadic AD and controls (45). Further, it has been reported that Ab levels are
approximately 100-fold lower in plasma than CSF (45). Some studies
demonstrated that plasma Ab40 and Ab42 levels are similar in AD and control
groups (46,47). However, others have shown that plasma Ab40 levels are
increased in AD patients with APOE 34 allele compared to those without this allele
and age-matched controls (46). Because of the potential overlap between the AD
patients and controls, measurement of plasma Ab40 levels is not useful as a
diagnostic tool to distinguish patients with sporadic AD from elderly non-
demented (ND) controls (46,47). A longitudinal study of unrelated individuals
reported that those who subsequently developed AD had higher plasma Ab42
levels at entry than those who did not develop dementia (48). These results indicate
Biomarkers in Blood and Cerebrospinal Fluid 77

that elevated plasma Ab42 levels may be detected several years before onset of
symptoms, supporting the role of extracellular Ab42 in the pathogenesis of AD.
However, although blood is easy to obtain, it is still unclear if there are
systemic changes specific for AD, and to what extent changes in blood
composition reflect pathological changes seen in the brain since plasma Ab42
levels showed no difference between AD and controls, whereas data with Ab40
are controversial (with either an increase in AD or no change). One longitudinal
study has shown that elevated plasma Ab42 levels occur before the onset of MCI
in some individuals (48).
In summary, Ab1–42 comes close to fulfilling the criteria for a useful AD
diagnostic test as recently summarized by an expert review (49). It is, however, of
limited value in differentiating AD from other primary dementias. Further studies
will be required to evaluate the whether the ratio of Ab42 to Ab40 is superior to
the concentration of Ab42 alone (17).

AMYLOID PROTEIN PRECURSOR


Amyloid b protein is derived through proteolytic processing of a larger
membrane bound glycoprotein, the amyloid protein precursor (APP) (50).
APP is ubiquitously expressed as an integral membrane protein and is cleaved by
proteases called a-, b-, and g-secretase (51). The sequential cleavage by b- and
g-secretase generate amyloidgenic Ab peptides and simultaneously soluble
b-APP [b-secreted APP (sAPP)]. In contrast, a-secretase cleavage precludes Ab
formation and generates soluble a-sAPP (52).
The total amounts of sAPP, or specifically cleaved forms of APPa and
sAPPb, can be measured and are abundant in CSF. Results of studies measuring
the total level of sAPP in AD compared to healthy controls are contradictory,
ranging from no significant change (53) to a decrease (7,54). Studies on APP in
AD from human brain tissue are also inconsistent. In two studies it was shown
that APP levels did not differ between AD and normal human brains (55,56).
However, in another study a decline of APP in specific brain areas in AD has been
reported (57). In a previous study, the a- and b-secretase cleaved APP was
investigated in patients with AD compared to healthy controls and patients with
MCI. No significant changes were found for CSF a-sAPP or CSF b-sAPP
between patients with AD and healthy controls (52). However, the level of
CSFb-sAPP was significantly increased in patients with MCI compared to
healthy controls (52), which might be due to disturbances in APP metabolism.
Furthermore, another recent study showed no significant differences in the levels
of CSF b-sAPP between AD patients and controls, but the CSF a-sAPP, and total
sAPP levels, were significantly lower in AD patients compared to controls (58).
Taken together, b-sAPP in CSF does not currently seem to be an adequate
marker for sporadic AD. Nevertheless it is of importance to investigate b-sAPP in
larger samples of familial and sporadic AD (58).
78 Hampel and Buerger

TAU PROTEINS
Neurofibrillary tangles are one of the major pathological hallmarks of AD. They
consist of paired helical filaments (PHF), derived from abnormally hyper-
phosphorylated microtubule-associated protein tau (59). Physiologically, tau is
located in the neuronal axons and a component of the cytoskeleton and
intracellular transport systems. Due to alternative splicing of tau mRNA, there
are six isoforms ranging in size from 352 to 441 amino acids, with molecular
weights ranging from 50 to 65 kDa (60), encoded by a single gene consisting of
16 exons on chromosome 17q21. Furthermore, at least 30 phosphorylation sites,
either threonine or serine, exist on tau extracted from human brain (61). Due to
hyperphosphorylation, tau loses its ability to bind to the microtubules and to
stimulate their assembly, and shows an increased tendency to aggregate (62).
Total tau (t-tau) and truncated forms of monomeric and phosphorylated tau are
released and can be measured in the CSF.
The first promising report on CSF t-tau as a biomarker for AD was
published in 1993. An ELISA with a polyclonal reporter antibody was used (63).
After this, ELISA methods based on monoclonal antibodies have been developed
that detect all isoforms of tau independent of phosphorylation sites (64,65) to
measure total and phosphorylated CSF tau protein (66–68).

Total Tau
T-tau, a general marker of neuronal destruction, has been intensively studied in
more than 30 studies on 2000 AD patients and 1000 age-matched elderly controls
over the last 5–10 years (69). The most consistent finding is a statistically
significant increase in CSF t-tau protein in AD. The mean level of CSF t-tau
protein concentration is about 300% higher in AD compared to elderly controls.
Across the reviewed studies, sensitivity and specificity levels varied due to the
different control groups and statistical methods used. Specificity levels were
between 65% and 86% and sensitivity between 40% and 86% (14). In several
studies, a significant elevation was also found in patients with very early
dementia (31,70,71). Overall, in mild dementia, the potential of CSF t-tau protein
to discriminate between AD and normal aging is high, with a mean sensitivity of
75% and a specificity of 85% (69). An age-associated increase of t-tau protein has
been shown in ND subjects (72). Therefore, the effect of age should be considered
when t-tau protein levels are diagnostically employed. Age-dependent reference
values for t-tau protein have already been established: for subjects between
21–50 years old at !300 pg/ml, between 51–70 years old at !450 pg/ml, and
between 70–93 years old at !500 pg/ml (18).
MCI is a major risk factor for AD. Ten to fifteen percent of patients with
MCI have been reported to convert to AD in a year (73). In patients suffering
from MCI who converted to AD during follow-up, elevated t-tau levels at
baseline were found in a relatively high number of individuals (20,74). Memory
impaired subjects who later developed AD could be discriminated by high
Biomarkers in Blood and Cerebrospinal Fluid 79

CSF t-tau from those who did not progress with 90% sensitivity and 100%
specificity (75). Longitudinally, elevated CSF levels of t-tau in MCI subjects
were found and still remained elevated after conversion to clinical AD. Another
study showed that 88% of patients with MCI had elevated t-tau concentrations
and/or low CSF Ab1–42 levels at baseline (13). Thus, elevated CSF t-tau in MCI
may have the potential to predict AD, a finding that was supported by a recent
study (2). Cross-sectional studies correlating CSF t-tau concentrations with
cognitive status in AD have shown a correlation between elevation of t-tau and
cognitive decline (26,27,76), though others have found no systematic effect
(16,77–80). Longitudinal studies of t-tau in mildly to moderately demented AD
patients showed no statistically significant correlation with progression during
follow-up (12,81,82). CSF t-tau remained elevated for up to two years in mild to
moderate AD. Initial and follow-up levels of t-tau correlated strongly, suggesting
a stable rate of neurodegeneration during this time period. It could be
hypothesized that CSF t-tau will decrease over time if treatment of AD achieves
disease-modification and neuroprotection (83).
An increase of CSF t-tau has also been found in a proportion of cases with
other dementia disorders. In VaD, FTD, and dementia with Lewy bodies (DLB),
elevated CSF t-tau has been found (20,33,69,74,78,84–88). Other studies,
however, found normal levels compared to controls in these disorders
(14,16,36,81,89,90). The potential of CSF t-tau, however, is limited in its ability
to discriminate AD from other relevant dementia disorders. At a sensitivity level
of 81%, CSF t-tau reached a specificity level of only 57% in distinguishing AD
from other dementias (16,91). Therefore, t-tau has not been suggested as a marker
for the differential diagnosis of AD.
T-tau rather reflects non-specific processes of axonal damage and neuronal
degeneration. This notion is further supported by an increase in CSF t-tau in
disorders with extensive and/or rapid neuronal degeneration such as CJD (92,93).
A highly significant increase of 580% was documented in CJD compared to AD
patients. At a cut-off level of 2130 pg/ml, t-tau yielded a sensitivity of 93% and
a specificity of 100% between AD and CJD (94). An elevation of CSF t-tau,
correlating with clinical severity, has been shown in normal pressure
hydrocephalus (95). Moreover, a marked transient increase of CSF t-tau has
been demonstrated after acute stroke. The transient increase of CSF t-tau
correlated with infarct size measured by cranial computed tomography (96).
Elevated levels of CSF t-tau in patients with diffuse axonal damage after
traumatic brain injury have been found, thus decreasing with clinical
improvement (68).
The differential diagnoses of AD, alcoholic dementia, PD, progressive
supranuclear palsy, corticobasal degeneration, and other psychiatric disorders
show normal CSF t-tau (20,36,64,97,98), with elevated CSF t-tau concentrations
only occasionally being reported (14,36,74,99,100).
In geriatric major depression (MD), an important psychiatric condition
in the differential diagnosis of AD, CSF t-tau has also been investigated.
80 Hampel and Buerger

Subgrouping a sample of AD patients, healthy controls, and patients with MD


according to age resulted in a correct classification rate of 94.5% in the “young
old” subjects (!70 years of age) compared to only 68.4% in the “old old”
(70 years of age). This report supports the notion that elevated CSF t-tau
particularly in subjects younger than 70 years of age is highly indicative of a
neurodegenerative process (72,101).

Phosphorylated Tau
Promising efforts are under way to establish phosphorylated tau (p-tau) in CSF as a
putative biological marker for AD. Several ELISA methods have been developed
specifically detecting phosphorylation of tau protein at different epitopes, such
as threonine 181 and 231 (p-tau181C231) (66), threonine 181 (p-tau181) (67),
threonine 231 and serine 235 (p-tau231C235) (102), serine 199 (p-tau199)
(102), threonine 231 (p-tau231) (103), and serine 396 and 404 (p-tau396C404) (104).
Although there is no doubt that tau phosphorylation differs in AD, it is hard
to speculate why this should be the case. There have been few studies of p-tau199
and p-tau181 in the human brain. With the exception of one study (105) all that is
known about these sites is that they are phosphorylated in advanced AD
neuropathological changes (106). Furthermore, it is well established that p-tau231
appears early in the pathological development of the disease, even before the
formation of PHF in neurons of the hippocampus (105,107). Phosphorylation at
both threonine 181 and serine 199 occurs later, and these are only found to any
appreciable extent in intracellular tangles (105).
One question, yet to be resolved, is which site of abnormal phosphorylation
is most useful for the differentiation between AD and other disease groups.
A comparative study examining the different tests (p-tau181 vs. p-tau231 vs.
p-tau199) in the same subjects and controls have shown that, applied as single
markers, p-tau231 and p-tau181 reached specificity levels O75% between AD and
the combined non-AD group when sensitivity was set at R85%. With respect to
these data and with respect to the fact that the majority of data on CSF p-tau in
AD is available for p-tau181 and p-tau231, studies on p-tau181 and p-tau231 will
now be described in more detail. Moreover, there is a commercially available
assay for p-tau181, and a commercial assay for p-tau231 will soon become
available.
CSF p-tau231 distinguished between AD-patients and subjects with other
neurological disorders (OND) with a sensitivity of 85% and a specificity of 97%
showing elevated CSF p-tau231 in AD (103). In this first study describing the
assay, a total of 39 CSF samples were prepared and analyzed from individuals
with AD, a number of different dementias, other neurological conditions, and
controls. The data suggest that the assay can distinguish AD from other
dementias (103). In a subsequent study on differential diagnosis of AD using CSF
p-tau231 in an independent sample, a sensitivity level of 90.2% and a specificity
Biomarkers in Blood and Cerebrospinal Fluid 81

level of 80.0% between AD and non-AD disorders was reported (108).


Furthermore, p-tau231 significantly improved differential diagnosis compared to
t-tau between AD and other non-AD groups, particularly FTD (108). In AD versus
FTD, p-tau231 correctly allocated 91% of subjects compared to only 66% using
t-tau (108). In the differentiation between AD and MD, p-tau231 levels were found
to be significantly increased in AD patients compared to geriatric MD and healthy
controls subjects (109). Results of this study further indicated that p-tau231 also
has the potential to differentiate between very mild possible AD and MD, even if
Mini-Mental State Examination (MMSE) score did not (109).
Recently, the positive and the negative predictive value was calculated for
p-tau231 in the differential diagnosis of AD (110). A positive predictive value of
77.1% and a negative predictive value of 91.7% were found. The high negative
predictive value means that a negative test rules out AD with 90% probability.
Another promising value of p-tau231 may be its ability to predict cognitive
decline in MCI patients. A longitudinal study showed elevated levels for p-tau231
in 77 MCI patients in comparison to healthy controls at baseline (111). High CSF
p-tau231 levels at baseline significantly correlated with subsequent cognitive
decline and conversion to AD. This study suggests that high p-tau231 may be a
predictor variable for progressive cognitive decline in subjects with MCI. A one-
year longitudinal MCI study showed progressive elevation of p-tau231
concentrations in MCI subjects compared to healthy controls (112). In a six-year
longitudinal serial CSF study, p-tau231, but not t-tau, concentrations decreased
linearly over time during the clinical progression of AD (113). The decrease of
CSF p-tau231 with AD progression might reflect the increasing sequestration of
p-tau into the tangle, suggesting that p-tau becomes more insoluble rather than
entering into the CSF, whereas solubility of t-tau remains unaffected.
CSF p-tau181 was elevated in AD compared to other dementias and healthy
controls and has been proposed as a potential marker for discriminating AD
patients from patients suffering from DLB (87). Focussing on the differentiation
between AD and DLB, specificity at a given sensitivity level was improved by
p-tau181 compared to t-tau (34,35). In a study with 101 subjects comparing p-tau181
and t-tau in different diagnostic subgroups, p-tau181 was increased in patients with
probable and possible AD compared with VaD, and dementia in PD (18).
Compared with FTD, PD, VaD, and normal aging, both p-tau181 and t-tau were
increased in probable AD. In possible AD, p-tau181 was increased compared to
FTD and VaD.
A recent study directly compared the diagnostic performance of p-tau231,
p-tau181, and p-tau199 in the same patient cohort, including a large series of
patients with AD, DLB, FTD, VaD, and OND (106). The p-tau231 and p-tau181
assays performed nearly equally well in the discrimination of AD from ND
controls, whereas the p-tau199 assay showed a weaker discrimination (106).
Discrimination between AD and DLB was maximized using p-tau181 at a
sensitivity of 94% and a specificity of 64%, while p-tau231 maximized group
separation between AD and FTD with a sensitivity of 88% and a specificity
82 Hampel and Buerger

of 92% (106). Thus, differences in the phosphorylation of specific tau epitopes


between dementia disorders may be reflected in the CSF level of the
corresponding p-tau variant (106).
Interestingly, despite a very marked increase in t-tau in CJD, there is only a
slight elevation of p-tau181 (114). Furthermore, p-tau181 does not change after
acute stroke in contrast to t-tau (96). These findings suggest that CSF p-tau is not
simply a marker for neuronal damage, like CSF t-tau, but might specifically
reflect the phosphorylation state of tau, and thus possibly the formation of tangles
in AD.
In summary, p-tau proteins come closest to fulfilling the criteria for a useful
biological marker of AD (106). There is a tendency for p-tau proteins to perform
differently in the discrimination of AD from other primary dementia disorders.

APOLIPOPROTEIN E
Apolipoprotein E (APOE, gene; ApoE, protein) is the major apolipoprotein in the
central nervous system where it is involved in the mobilisation and redistribution
of cholesterol, necessary for the maintenance of myelin and neuronal membranes
during development and following injury (115). However, APOE is of special
interest in AD research since the presence of the APOE 34 allele has been
suggested to be a major risk factor for the development of late-onset AD (116).
APOE polymorphism influences levels of amyloid deposits in the brain of AD
patients and elderly ND controls (117).
Previous studies on CSF APOE in AD patients and controls, however, have
shown conflicting results. While in some investigations elevated CSF APOE
levels have been found (118,119), others have reported reduced APOE
concentrations in CSF (120). Interestingly, in one study APOE levels in CSF
were reduced in AD patients without the APOE 34 allele, but increased in AD
patients and APOE 3 4 allele carriers (120). However, another study reported
decreased CSF APOE concentrations in APOE 3 4 allele carriers (118,121);
Lindh and colleagues also reported increased levels of APOE in CSF in patients
with MCI and other dementia disorders than AD compared to healthy controls
(118). To investigate whether APOE levels in CSF differ with various ages at
onset of AD, one study examined APOE in early- and late-onset AD, showing
significantly lower APOE levels in early-onset AD patients and higher in the late-
onset group compared to controls (122).
Concerning serum ApoE in AD patients in comparison to controls, studies
are still inconclusive. Some studies report decreased serum ApoE levels (123),
whereas others have observed similar or elevated serum ApoE levels between AD
and controls (119,124).
Taken together, levels of ApoE in CSF and serum seem not to fulfill the
criteria for a useful biomarker of AD.
Biomarkers in Blood and Cerebrospinal Fluid 83

ISOPROSTANES
Several studies have suggested a role for oxidative damage in the pathogenesis of
different neurodegenerative diseases, especially AD and amyotrophic lateral
sclerosis (ALS) (125–127). Oxidative damage to CNS tissue prominently manifests
as lipid peroxidation (LPO).
Isoprostanes are prostaglandin isomers that are producted exclusively from
free-radical-catalyzed peroxidation of arachidonic acid (128). Isoprostanes are
biochemically stable end-products of LPO that are released by phospholipases,
circulate in plasma, and are excreted in urine (129). Therefore, analysis of LPO by
measuring isoprostane levels has been performed in brain, CSF, serum, and urine
(130–133). Special attention has been focussed on isomers of the F2-isoprostanes
(F2a-iPs), especially 8,12-iso-iPF2a-VI. In different studies, it has been reported
that 8,12-iso-iPF2-VI levels are elevated in urine, blood, and CSF of AD patients
and that these values correlate with memory impairment, CSF tau levels, and the
number of APOE 4 alleles (130–132,134). The results, however, for blood and
urine, in contrast to CSF, have been conflicting since other studies found no
elevated F2-isoprostanes levels (135,136). In addition to AD, in early
Huntington’s disease, meningoencephalitis, and stroke, elevated levels of
isoprostanes were also found (137,138), while in ALS, PD, and schizophrenia
no increase of isoprostanes has been observed (139,140). Furthermore, CSF
F2-isoprostanes concentrations in AD patients are significantly correlated with
global indices of brain degeneration such as decreasing brain weight and degree of
cerebral cortical atrophy, but not with APOE genotype or the tissue density of
neuritic plaques or neurofibrillary tangles (140).
In summary, although additional studies are needed to confirm and extend
these findings in larger cohorts of MCI and AD patients, F2-isoprostanes seem to
be an interesting marker in AD, not only in CSF, but also in serum and urine.

a1-ANTICHYMOTRYPSIN
It has been suggested that a number of molecules associated with inflammation
are involved in the pathogenesis of AD. Antichymotrypsin (ACT), one of the
serine proteinase inhibitors, plays an important role in inflammation.
Interestingly, elevated levels of ACT were found in the brains of patients with
AD and were also described as one of the components of senile plaques (141).
ACT has been investigated in CSF and serum in several studies, but the
results have been controversial. While in some studies no difference in ACT
levels between AD and controls have been found (142), others have described
higher levels of ACT in AD than controls (143–145). A recently performed study
found that ACT levels correlate with the severity of dementia (146). Further
studies are essential to confirm these findings. In addition, investigations on MCI
are needed since ACT has not been studied in MCI so far.
84 Hampel and Buerger

In summary, serum and CSF levels of ACT might be independently


upregulated in AD. The measurement of ACT in serum could be useful as a
screening marker in AD. Further independent studies, however, are still needed.

THE SOLUBLE INTERLEUKIN-6 RECEPTOR COMPLEX


It has been shown that amyloid within senile plaques is associated with activated
microglia and astrocytes that express inflammatory proteins and neuroregulatory
factors such as interleukin-6 (IL-6). IL-6 has been consistently detected in the
frontal, parietal, and occipital cortex and hippocampus of AD patients but not of
ND elderly subjects. In further investigations, IL-6 has been demonstrated in
diffuse early plaques without neuritic pathology in isocortical (frontal temporal
and parietal cortex) and hippocampal brain samples of AD patients. IL-6
immunoreactivity was rare in classical plaques and absent in compact or burned-
out plaques. Therefore, it has been suggested that IL-6 expression may appear
before neuritic changes rather than follow neuritic degeneration.
Basic studies show that IL-6 exerts its biological actions only by complex
interactions with specific soluble or membrane bound receptors, forming the
biologically active IL-6 receptor complex (IL-6RC). The component proteins, in
addition to the 19.5 kDa cytokine IL-6, are two membrane glycoproteins, an
80 kDa protein referred to as the ligand-binding a-subunit (gp80, IL-6R, or
CD126) and a 130 kDa protein referred to as the non-ligand binding, affinity
converting, and signal transducing b-receptor (gp130 or CD130). All members of
the IL-6 cytokine family [IL-6, IL-11, oncostatin M, leukemia inhibitory factor,
ciliary neurotrophic factor, and cardiotrophin-1 (CT-1)] share gp130 as a
component critical for signal transduction. In the nervous system, IL-6 can be
secreted by microglia, astroglia, neurons, and endothelial cells, the IL-6R by
neurons, and gp130 by all cells. Gp130 neuropil immunoreactivity was observed
in telencephalic structures including the hippocampus, cerebral cortex, and
caudate-putamen. Activation of membrane bound gp130 by IL-6 and the soluble
IL-6R was reported to generate a neuronal differentiation signal. Soluble forms of
the two receptors (sIL-6R, sgp130) arise by limited proteolysis (shedding) or
differential splicing (sIL-6R of 38 kDa and sgp130 of 68kDa). It has been reported
that this soluble complex (sIL-6RC) forms a hexameric structure in solution,
consisting of the three different proteins with a 2:2:2 stoichiometry. There is a
complex regulatory interaction between all sIL-6R components. sIL-6R enhances
IL-6 effects by making the ligand accessible to the membrane-bound signal-
transducing b-subunit; however, it has also been shown to augment the action of
sgp130, which neutralizes IL-6 signals. There are commercially available
bioassays (ELISA) to detect the IL-6RC in biological fluids. Using such an assay,
significantly decreased CSF concentrations of sIL-6R (101) and sgp130 (147), in
the presence of unchanged IL-6 concentrations (148), in AD-patients compared
to healthy age-matched controls were reported. In addition, these data indicate
that the application of multivariate discriminant analysis using combined CSF
Biomarkers in Blood and Cerebrospinal Fluid 85

t-tau protein and sIL-6RC components may add more certainty to the diagnosis of
AD (147). The reported method, however, needs to be extended to an
independent group of AD patients, other neurodegenerative conditions, and
control subjects to assess the true diagnostic applicability. Interpretation of the
relationship between CSF and brain levels of the IL-6RC at present remain
speculative and require studies based on simultaneous measurement of
corresponding CSF and brain samples.

C-REACTIVE PROTEIN
C-reactive protein (CRP) is a pentraxin acute phase reactant synthesized in the
liver. Its plasma level can increase up to 1000-fold during the acute-phase
response (149). CRP is not normally found in the brain, but previous studies have
demonstrated the presence of CRP in senile plaques and neurofibrillary tangles in
the brains of AD patients (150,151). It is up-regulated in AD brains compared to
samples from ND individuals (152). In a follow-up study, examining over 1000
cases, it has been observed that men in the upper three quartiles for serum high-
sensitivity CRP had a 3-fold, significantly increased risk for all dementias
combined, AD, and VaD (153). Remarkably, the serum samples that were
investigated had been taken and stored 20–25 years earlier, long before onset of
dementia symptoms in any subject. In contrast, another recent study of only 11
AD and 11 ND patients found comparable levels of CRP in AD and ND patients.
In summary, CRP might play a causal role, or merely be a marker of
inflammatory processes. Whether the association proves to be direct or indirect,
CRP measurements may turn out to be an important adjunct for global risk
assessment of dementia (153).

C1Q
C1q, a subcomponent of C1, the first component of the classical complement
pathway, is associated with neuritic plaques and with neurons in the hippocampus
of AD brain.
There is evidence that C1q, in addition to its normal production in the liver,
is also synthesized in the brain by pyramidal neurons and glial cells (154–156).
b-pleated, fibrillar Ab and, more recently, tau-containing neurofibrillary tangles
have been found to directly and fully activate the classical complement pathway
in vitro in an antibody-independent fashion, (157,158). Of the many different
inflammatory mediators that enhance Ab aggregation, C1q is one of the most
potent (159).
In several studies, abundantly elevated C1q levels have been observed in
the brains of AD patients compared to unaffected brains (160,161). In CSF,
significantly lower levels of C1q have been observed in AD compared to control
patients, thus correlating with cognitive deficits (162). In summary, these results
86 Hampel and Buerger

support the hypothesis that complement plays a role in the pathogenesis of AD


potentially by triggering local inflammation (163).

HOMOCYSTEINE
Homocysteine is a precursor of methionine and cysteine. Folate and vitamin B12
are needed for the conversion of homocysteine to methionine, and vitamin B6 is
essential for the conversion of homocysteine to cysteine (164). Deficiencies of
folate or vitamin B12, and vitamin B6, result in increased levels of homocysteine.
There are a variety of assays to measure homocysteine levels, including immuno-
assays and high pressure liquid chromatography (HPLC)-based methods, which
have similar performance and high precision (165,166).
Hyperhomocysteinemia is considered a potentially important risk factor
for heart disease, carotid atherosclerosis, and stroke (167–170). Atherosclerosis
and stroke, in turn, increase the risk for AD (171,172). However, in AD, VaD,
and cognitive deterioration, increased levels of homocysteine in serum in
combination with decreased vitamin B12 or folic acid are considered as
potential risk factors for the development of cognitive impairment (173). A
significant increase in total homocysteine levels were demonstrated in a large
case control study of 164 patients with AD compared to 108 controls (174).
Total homocysteine levels were stable over a three-year follow-up period, and
homocysteine levels did not correlate with duration of symptoms. A recent
longitudinal study has shown an association between hyperhomocysteinemia
and a higher risk of AD (175). In this study, plasma homocysteine levels greater
than 14 umol/L almost doubled the risk of AD (175). In another study it has
been demonstrated that, independent of low folate levels, higher levels of
homocysteine were associated with cognitive decline in a large group of older
subjects (176). In contrast, no relation between increased homocysteine
concentrations and cognitive decline was observed in a large study with 702
individuals with a mean follow-up duration of 2.7 years (177). A recent MRI
study suggested that increased homocysteine was a risk factor for cerebro-
vascular disease independent of AD (178). This leads to the question of whether
homocysteine is directly linked to mechanisms of AD or indirectly, via
cerebrovascular disease.
Besides homocysteinemia, low vitamin B12 and folate levels were also
found in AD. Since supplementation with B group vitamins lowers homocysteine
levels by up to 30% (174), dietary interventions are now being tried in current
studies to explore the benefit on cognitive outcome (4). Furthermore, in the US,
fortification of cereal grain products with folic acid was mandated in the late 1990s,
and there has already been a decrease in mean values of homocysteine in older
individuals. This will make it more difficult to evaluate whether further
supplementation has benefits in dementia (4).
Studies of folate supplementation in depression, one of several neuropsy-
chiatric diseases also associated with low folate serum levels, have shown benefits
Biomarkers in Blood and Cerebrospinal Fluid 87

even when folate status was not low (179). A potential explanation might be that
serum folate concentrations do not reflect concentrations in the CNS or that folate
requirements are increased in neuropsychiatric diseases (180).
In summary, elevated homocysteine levels in plasma seem to be a strong,
independent risk factor for the development of dementia and AD. Further large-
scale studies are currently in progress.

OXYSTEROLS AND CHOLESTEROL METABOLISM


The brain is the most cholesterol-rich organ in the human body. Cholesterol
metabolism in the CNS is postulated to be regulated independently and
potentially by a unique mechanism because CNS is segregated from the systemic
circulation by the blood-brain barrier (181). It is known that cholesterol is
synthesised in the brain in situ and that extracerebral cholesterol does not
contribute significantly to brain cholesterol content (173,182–184). Converging
evidence links cholesterol metabolism and AD (185). It has been hypothesised
that increased removal of cholesterol from brain occurs during neurodegenerative
processes (173,186). Accumulation of excess cholesterol in hippocampal neurons
promotes the cleavage of the APP into amyloidogenic components with
consequent acceleration of neuronal degeneration. Conversion of cholesterol to
24S-hydroxycholesterol mediated by cholesterol 24S-hydroxylase is the major
pathway for the elimination of brain cholesterol and the maintenance of brain
cholesterol homeostasis (173,181,187).
Concentrations of 24S-hydroxycholesterol in plasma and CSF are
significantly higher in AD and vascular demented patients at early stages of
the disease compared to healthy subjects. Variation in genetic background, time
of disease onset, and severity of dementia are potential sources of variance
(187–190). In a recent study, an influence of APOE 34 allele on CSF
24S-hydroxycholesterol concentrations, and a gene-dosage effect was observed
which might point to the existence of a link between the established AD, risk
factor, APOE 34 allele, and CNS cholesterol metabolism. Also, in patients with
MCI, increased CSF 24S-hydroxycholesterol levels were found suggesting that
high levels of 24S-hydroxycholesterol appear to occur early in the disease
process (187).
The relationship between serum cholesterol and lipid levels and the risk of
AD is not clear. In one prospective study, high serum cholesterol in midlife
appeared to increase the risk of incident AD (189), while another population-
based study found no clear relationship between cholesterol levels and AD (191).
Cholesterol-lowering drugs (HMG-CoA reductase inhibitors, or statins)
decrease the prevalence of AD (192). Statins reduce the generation of the
amyloid precursor protein, the neuronal secretion of beta-amyloid, and de novo
cholesterol synthesis (193–196). Recent epidemiological studies indicate that the
prevalence of diagnosed AD and VaD is reduced among people taking statins for
a longer period of time. High-dose simvastatin treatment (80 mg/day) in patients
88 Hampel and Buerger

with hypercholesterolemia leads to a significant decrease in brain-specific serum


24S-hydroxycholesterol concentrations, indicating diminished cholesterol
metabolism in the brain (188). As statin treatment not only reduces total and
LDL cholesterol levels, but also increases high-density lipoprotein (HDL) levels,
statin treatment may help reduce the risk of AD by increasing serum HDL and
CSF cholesterol levels. HDL is critical for the maturation of synapses and the
maintenance of synaptic plasticity (197,198).
Taken together, although measures of cholesterol metabolism do not appear
to be diagnostically useful in AD, they may serve as indices of treatment effects and
mechanisms in clinical trials. The mounting evidence implicating cholesterol
pathways in AD has led to preliminary studies of statins in patients with AD
(183,199). Following treatment with a statin, CSF levels of 24S-hydroxy-
cholesterol decreased, but CSF levels of Ab were not found to be altered.
To study the correlation between cholesterol metabolism and the
processing of APP, the effect of statin treatment (simvastatin or atorvastatin)
on Ab in humans was tested (200). Treatment with both statins reduced total
plasma cholesterol levels by 56% (p !0.001). No significant change of plasma
Ab40, Ab42, and total Ab was found questioning the effect of statins on the
processing of APP in humans (200).

3-NITROTYROSINE
In the presence of reactive oxygen species and nitric oxide, 3-nitrotyrosine (3NT)
may be formed in constituent proteins in the brain. The predominant pathway
appears to involve nitric oxide in the presence of superoxide ion to form
peroxynitrite. Peroxynitrite in turn reacts with tyrosine residues in proteins or
with free tyrosine to form 3NT (201). 3NT can be found in CSF in specific
proteins, e.g., superoxide dismutase (202), or can be found as free 3NT. With
normal aging, 3NT concentrations in CSF increase modestly from about 0.75 nM
at 40 years to about 2 nM at 80 years (203).
In the brains of patients with AD, regionally specific increases in 3NT have
been found in the neocortex and cerebellum (204). In the CSF of patients with
AD, the concentration of 3NT is reported (203) to be 11.4 nM, or about sixfold
higher than age-matched controls. In this study, concentrations of 3NT in CSF
were inversely correlated with MMSE scores, but did not correlate with duration
of disease.
Analysis of 3NT is relatively straightforward when done by HDL with
electrochemical detection (HPLC/ED) (201). CSF analysis requires only 400 ul,
and acid precipitation of proteins is sufficient for the analysis of free 3NT
(201,203). The stability of 3NT in plasma proteins, however, is not well
established, and thus the significance of 3NT concentrations in this compartment
is less clear (201,205). Increased concentrations of 3NT in CSF, along with
changes in isoprostanes, and 8OHDG as outlined in this review, are consistent
with the suggestion that oxidative stress may play an important role in the
Biomarkers in Blood and Cerebrospinal Fluid 89

pathogenesis of AD. It is not yet clear whether the increase in reactive oxygen
species with oxidative stress is proximal or distal in the pathophysiological
cascade leading to cell death. As with a number of proposed biomarkers for AD,
longitudinal studies are necessary to determine if changes in 3NT in CSF increase
monotonically with disease progression, or might have a more complex
relationship with disease severity. Even in the absence of a linear relationship
with disease severity, measurement of 3NT and other markers of oxidative stress
may provide an indirect marker of drug efficacy in subacute clinical trials using
putative disease-modifying agents.

14-3-3 PROTEIN IN CREUTZFELDT–JAKOB DISEASE


Several studies have reported an elevation of 14-3-3 protein in CSF of sporadic
CJD (206). Levels of this protein are high in 95% of sporadic CJD patients (207).
However, although the increase of the 14-3-3 protein in CSF is used as a
diagnostic test in CJD, sensitivity and specificity of the 14-3-3 protein test varies
between the different subtypes of sporadic CJD, distinguished by electrophoretic
mobility of proteinase K-resistent protein (PrPsc) and genotype at codon 129 of
the prion protein gene (206,208). The sensitivity of the 14-3-3 test has been
shown to be higher in patients with molecular features of classic sporadic CJD
rather than in patients with the nonclassic CJD subgroups (94% vs. 77%). The
difference appears to be related to the PrPsc type (208).
Furthermore, it should be mentioned that in the differential diagnosis of
neurodegenerative disorders, only rare cases other than CJD are positive for
14-3-3. Single cases are reported in AD, stroke, cerebral neoplasia, Hashimoto’s
encephalopathy, inflammation, and some other rare conditions (206).
In summary, 14-3-3 protein test is a useful CSF test for the clinical
diagnosis of CJD, but only in a subgroup of the CJD cases. Disease duration,
dependent on the PrPsc genotype, should therefore be taken into consideration
(208,209). Concerning discrimination CJD from other forms of dementia, 14-3-3
protein has been proved to be a useful marker (206).

PERSPECTIVE OF BIOMARKER RESEARCH IN AD:


b-AMYLOID ANTIBODIES
Naturally occurring antibodies against Ab have been detected in CSF and blood
of patients with AD, neurological diseases, and healthy controls (5,210–212). In a
study of Ab-antibody in CSF by ELISA, Du and colleagues found a statistically
significant decrease of antibody levels in patients with AD compared to age-
matched healthy control subjects. Brettschneider and colleagues assessed the
diagnostic value of serum Ab42-antibodies for AD. Ab42-antibody levels
were measured using a newly developed immuno-precipitation assay with
radiolabelled amyloid b1–42 peptide (213). A highly significant decrease of Ab42-
antibody levels in AD patients was found independently of age, cognitive status,
90 Hampel and Buerger

and APOE 34 carrier status. Ab42 antibody levels were correlated with gender
only in AD, with a higher level occurring in women. When Ab42 antibody
sensitivity (specificity) was set at O80%, specificity (sensitivity) was below 50%
in correctly allocating patients and healthy controls. These data indicate a
potentially pathophysiological decrease of serum Ab42-antibodies in AD. Ab42-
antibodies in the serum alone, however, appear not to be useful as a diagnostic
marker of AD.
So far, very little is known about Ab-antibodies with respect to their
function, induction, specificity, and role in disease processes. However,
considering data from recent investigations in transgenic AD mouse models,
there is evidence for a therapeutic impact of Ab-antibodies. A reduction of
cerebral plaque load and cognitive impairment was observed after active and
passive immunization resulting in increased production or administration of
Ab-antibodies in these animal models (214–220). Unfortunately, the phase II
clinical trial of the Ab vaccination approach had to be withdrawn because of
signs consistent with meningo-encephalitis in an increasing number of patients
(221). Setbacks in active immunization are shifting the focus to passive
administration of antibodies. Dodel and colleagues detected naturally occurring
Ab-antibodies in commercially available immunoglobulin G products. In a
clinical approach they investigated patients with different neurological diseases
treated with intravenous immunoglobulin (IVIG) preparations. A significant
decrease of total Ab and Ab1–42 in CSF compared to baseline values was
observed after treatment. In serum, a significant increase of total Ab without
change in the Ab1–42 level was observed (222). These data are in agreement with
observations in transgenic mouse models (220,223). A clinical trial involving the
administration of IVIG to five patients with AD with simultaneous measurements
of CSF and serum Ab levels has shown a decrease of total CSF Ab levels by 30%
following IVIG. Total Ab increased in the serum by 230%. No significant change
was found in CSF/ serum Ab42 levels. ADAS-cog was improved by 3.7 points
(G2.9), though MMSE scores remained essentially unchanged. Although the
sample size of this pilot study is too small to draw a clear conclusion, the study
provides evidence for a more detailed investigation of IVIG for the treatment of
AD (224). Due to the very preliminary nature of the findings, the question
of whether soluble Ab-antibodies in serum and CSF has potential value as a
biomarker of biological disease activity or a potential surrogate endpoint for
clinical trials cannot be finally answered (225).

DISCUSSION
Results and overall direction of change, respectively, that have been reported so
far on the biomarkers as discussed in this chapter are given in Table 1. The
majority of studies presented here refer to Ab42, t-tau, and p-tau in the CSF and
yield the most convincing evidence for these core biomarkers to be useful either
Biomarkers in Blood and Cerebrospinal Fluid 91

Table 1 Results/Directions of Change Reported so far on CSF and Blood Biomarkers as


Discussed in this Chapter

Cerebrospinal fluid

Alzhei- Mild cogni- Frontotem-


mer’s tive impair- poral Creutzfeldt–
Biomarker disease ment VaD dementia DLB Jakob disease

Ab42 Y Y4 Y Y Y Y
Ab40 4Y 4 4 4 4 4
APP Y4 [ 4 4 4
t-tau [ [ [4 [4 [4 [[[
p-tau [[ [ ([) 4 ([) 4
ApoE [Y4 [ [4 [4 [
Isoprostanes [ 4 [
ACT [4 4
Interleukin-6 Y4
receptor complex
C1q Y
Oxysterols and [ [
cholesterol
3-Nitrotyrosine [
14-3-3 Protein [ [ [[[

Serum

Ab42 4[ Y[
Ab40 4[ Y[4
APP
t-tau
p-tau
ApoE Y4 4
Isoprostanes 4[ 4[
ACT 4[
Interleukin-6 Y
receptor complex
CRP [ [
C1q
Homocysteine 4[ 4[
Oxysterols and Y[ Y[ 4
cholesterol
Overall direction of change of biomarkers in CSF and serum of patients with Alzheimer’s disease, mild
cognitive impairment, VaD, frontotemporal dementia, LBD, and Creutzfeldt–Jakob disease as far as
data are available. Blanks indicate that the issue has not been addressed so far. Please note that the
database for this overview, i.e., the number of studies investigating a particular biomarker, varies
considerably between markers.
Key: [, increased; 4, not different; Y, decreased in comparison to non-demented controls, respectively.
Abbreviations: VaD, vascular dementia; DLB, dementia with Lewy bodies.
92 Hampel and Buerger

as diagnostic, classificatory, or prognostic markers of AD. A large number of


studies have demonstrated that tests based on CSF t-tau protein and CSF Ab42
have reasonable specificity and sensitivity when differentiating AD from normal
aging. A smaller number of studies show similar accuracy when distinguishing
AD from MD. One main criticism of these studies is that few have included
postmortem confirmation of diagnosis. Nevertheless, provisional work hints that
these tests may also be useful in detecting incipient AD in MCI patients.
Unfortunately, the value of these biomarkers to clinicians is limited, as they are
not specific enough to accurately separate AD from other common forms of
dementia, such as VaD and DLB. The combination of both CSF t-tau protein and
CSF Ab42 does not markedly improve their individual sensitivity.
CSF p-tau, based on different phosphorylation epitopes of tau protein, has
now been examined in a number of independent studies. Initial results are
extremely promising, showing that different p-tau protein epitopes may
substantially contribute to improved diagnostic accuracy of AD compared with
healthy aged controls, elderly depressed patients, and those with other types of
dementia. Compared with CSF t-tau protein and CSF Ab42, CSF p-tau is more
specific and less influenced by covariables such as age or degree of cognitive
decline. This has important implications for the value of CSF p-tau to clinicians.
If the marker is abnormal very early in the course of disease relatively
independently from the degree of cognitive decline, then the marker may be ideal
as a diagnostic test. If, however, the marker is closely linked to current or future
cognitive decline, then it may be better suited as a prognostic tool. However,
conceptually the two areas may overlap in the case of MCI. Roughly 50% of MCI
patients deteriorate to AD over five years, and this group may be considered to
have a very early form of AD at baseline. However, about 40% of MCI subjects
actually improve over time, suggesting an absence of neurodegenerative
pathophysiology. Thus at baseline, it is hoped that an accurate diagnostic test
of AD would differentiate between these subgroups, which would also inform
prognosis for MCI sufferers as a whole.
Studies of all possible biomarkers to date in AD suggest p-tau comes
closest to an ideal diagnostic marker. However, different epitopes of
phosphorylated tau may have different strengths and weaknesses. CSF p-tau231
may be the most useful in distinguishing AD from FTD, and CSF p-tau181 may
improve separation between AD and DLB. In addition, CSF p-tau231 may be the
most useful prognostic marker candidate that predicts cognitive decline to AD in
MCI subjects. Further studies are needed to decide whether detection of multiple
phospho-epitopes may allow a distinct representation of AD related pathology at
different stages of the disease, based on the “evolutionary” model of a sequential
phosphorylation pattern of tau protein (105).
In assessing the clinical significance of these findings, several confounding
factors have to be taken into account. An important factor is the uncertainty of the
clinical diagnostic criteria against which these markers have been tested.
Neuropathological studies suggest that high proportions (30–50%) of clinically
Biomarkers in Blood and Cerebrospinal Fluid 93

diagnosed patients with VaD have notable concomitant AD pathology. Similarly,


a high proportion of those with clinically diagnosed AD have evidence of
vascular pathology (226). For example, in a health maintenance organization
dementia registry (90), only 36% of patients had pathologically definite AD and
no other findings, while 45% had pathologically definite AD plus coexistent
vascular pathological features and 22% had pathological findings of AD plus
DLB pathological features. This may be due to the influence of vascular risk
factors on the onset and progression of AD, since our clinical criteria may be, at
least in part, inadequate, or because the diagnosis of mixed dementia is typically
overlooked. Whatever the explanation, in patient samples based on clinical
diagnosis, it is difficult to achieve high specificity for CSF biomarkers because
the gold standard itself is not completely stable. The only way to resolve this
question is to study the markers in neuropathologically verified subjects. There is
a related issue with elderly comparison groups. Even if such individuals are
asymptomatic and age-matched, it is possible that they harbour presymptomatic
AD brain lesions (227). In large samples this will invariably reduce the specificity
of even the most accurate AD biomarkers. The true value of a marker to
practicing clinicians can only be assessed using representative and heterogeneous
populations. Most, if not all, studies to date have been evaluated putative markers
in highly selected subgroups, representing relatively pure forms of the disease.
Finally, there are several practical consideration. For example, storage time
and freeze-thaw cycles of CSF aliquots may influence results. Equally important,
clinicians (neurologists as well as psychiatrists) need to consider a lumbar
puncture as a routine investigation. Studies have demonstrated lumbar puncture
in geriatric patients as safe and tolerable.
In the near future novel methodological approaches in the characterization
and quantification of proteins in biofluids might reveal additional information and
potentially new candidate biomarkers. For example, the peptide pattern of a
sample can be depicted as a multi-dimensional peptide mass fingerprint with each
peptide’s position being characterized by its molecular mass and chromato-
graphic behaviour. Such a fingerprint of a CNS sample consists of more than
6000 different signals. First data on this promising new approach to analyse CNS
diseases on the peptide level have been recently reported (39).
In conclusion, the newly established immunoassays detecting tau-proteins
and Ab-proteins, as well as the rapidly developing modern structural and
functional brain imaging methods (such as MRI, DTI, MRS, fMRI, and PET)
open up exciting avenues for early and accurate diagnosis of AD. Beyond
diagnosis, it is hoped that markers of prognosis will enable clinicians to monitor
whether new treatments of AD are working effectively and inexpensively. The
accuracy of any diagnostic test in AD is likely to be increased by the cumulative
information from clinical and neuropsychological examination, as well as genetic
testing and brain imaging (98). Large international dementia consortia are
currently investigating potential AD biomarkers in large-scale multi-center trials.
94 Hampel and Buerger

The reviewed CSF measures may gain a potential clinical utility as


biomarkers of disease. However, the preliminary and retrospective nature of the
majority of findings, the absence of assay standardization, and the partial lack of
comparison patient populations must be addressed in future studies testing the
usefulness of these CSF measures, particularly for predictive, diagnostic, or
treatment evaluation purposes.

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4
Genetics: Facts and Perspectives

Sandro Sorbi, Paolo Forleo, Francesca Massaro,


and Andrea Ginestroni
Department of Neurological and Psychiatric Sciences, University
of Florence, Florence, Italy

INTRODUCTION
There has been a major expansion in recent years on research into the genetic
causes of dementia, and a simple search on the PubMed database including the
terms “Dementia” and “Genetics” in March 2005 identified more than 13,000
references from 1964. Following the description of Alzheimer’s first case, the
first suggestion that genetic factors may play a role in Alzheimer’s disease (AD)
were reported in a paper of Lua (1920) and subsequently in a case report by
Flugel (1922), both speculating a possible genetic inheritance of the disease.
Since then, from 1930 to 1990, more than 50 pedigrees with a familial form of
AD have been described, suggesting that familial aggregation is a common
feature of AD. This observation has been successively confirmed by several
epidemiological studies (1,2). The analysis of these collected families and the
possible genotype-phenotype correlations, together with the outstanding progress
of molecular biological techniques, have made the field of dementia genetics in
the past 15 years an area of intense research and fruitful discoveries. This
information assembled in the past few years has led to the principal finding that
analysis of the genetic mechanisms underlying familial clustering of the disease
is likely to be directly relevant to the pathogenesis of the common and apparently
sporadic forms of AD. In addition, today’s knowledge of genetic factors is
currently evolving and leading to the progressive definition of a putative cascade

109
110 Sorbi et al.

of biochemical events that, applied to early diagnosis, therapeutic trials,


treatment, and preventive approaches, could provide fundamental advances to
prevent and cure AD and other dementias.

Alzheimer’s Disease
AD is a genetically complex and heterogeneous neurodegenerative disorder
characterized by memory loss that leads to progressive and irreversible cognitive
decline until death.
The neuropathology of AD is characterized by widespread neuronal
degeneration, the abundant presence of neuritic plaques, containing beta-
amyloid, and neurofibrillary tangles, mainly composed of tau aggregates,
dystrophic cortical neurites, and amyloid microangiopathy. Senile plaques are
located in the extracellular space of the brain and are mainly formed by a highly
hydrophobic peptide that contains a 42 amino acid residues (A beta 1–42),
produced from its precursor amyloid precursor protein (APP) through proteolytic
processing (see Chapter 11).
Since 1991, the results of genetic studies have led to the identification of
gene mutations and polymorphisms that can either cause AD or substantially
increase the risk of developing the disease. Mutations in three genes (Table 1),
APP, located on chromosome 21, and presenilin-1 (PSEN1) and presenilin-2
(PSEN2), located on chromosomes 14 and 1 respectively, result in familial AD
(FAD). The FAD cases account for approximately 5% to 10% of all early onset
AD cases, and mutations in PSEN1 are the most frequent. Although more than
140 different PSEN1 mutations are described in more than 200 families with
different ethnic origins, only 10 different mutations among 13 families have been
reported for the PSEN2 gene, and only about 20 families with APP mutations are
known (3,49).

Amyloid Precursor Protein


Following the identification of b-amyloid (Ab) and APP, there was considerable
speculation as to the causation of AD as being genetic. In the next few years,
several different missense mutations were found in exons 16 and 17 of APP in
families with early onset AD. All mutations are missense mutations lying within
or close to the domain encoding beta-amyloid peptide.

Table 1 Genes Involved in Alzheimer’s Disease


Chromosomal Identified genetic Effect of mutation on Alzheimer’s
Gene name location mutations disease pathogenesis

APP 21q21 18 [ Ab production and aggregation


PSEN1 14q24 142 [ Ab 42/40 ratio
PSEN2 1q42 10 [ Ab 42/40 ratio
Genetics: Facts and Perspectives 111

The APP mutations account for a very small proportion (2–3%) of all
published cases of FAD and 5–7% of reported cases of early onset FAD. The
pathogenicity of these mutations has been strongly supported by the fact that they
are virtually 100% penetrant in FAD kindreds where they occur in affected or
at-risk individuals, but are absent in age-matched controls. Swedish and Flemish
mutations at codon 670/671 and at codon 692 are rare; mutations at codon 717
have been described in about 20 families worldwide from different ethnic origins,
including Anglo-Saxon, Italian, and Japanese subjects (4–6). Mutations in APP
have been shown to affect the release of Ab in transfected cells and patient
fibroblasts. Transgenic mice expressing the APP V717F mutation produce
numerous Ab deposits in the form of classical senile plaques, and the brains of
these animals exhibit other neuropathological features of AD including neuronal
and synaptic loss and gliosis (6,7).While the APP codon 717 mutations are
associated with overproduction of Ab1–42, the Swedish double missense mutant
leads to an increase in total Ab secretion. The Swedish mutant involves the
substitution of the two N-terminal amino acids of the Ab domain, presumably
rendering APP more susceptible to b-secretase activity. Therefore, FAD
mutations in APP appear to affect both Ab release and the intracellular
trafficking of APP.

Presenilins
Mutations in PSEN1 (8) and in the related PSEN2 (9,10) are found to be causative
in about 50% of kindreds with FAD. The coding region of PSEN1 is derived from
10 exons (numbered 3–12). So far, mutations have been found in six of the 10
coding exons, with exons 5 and 8 accounting for 65% of the mutations. As some
mutations result in a later onset age, it cannot yet be excluded that mutations in
PSEN1 may also result in late onset forms (O65 years) of the disease.
The FAD mutations in PSEN1 are missense mutations causing single
amino acid changes or, rarely, exonic deletions and appear to be 100% penetrant
and are best classified as autosomal dominant “causative” gene defects. In
contrast, only ten PSEN2 mutations have been identified, suggesting that
mutations on PSEN2 gene are a rare but possible cause of disease and that FAD
mutations are considerably more frequent in PSEN1 than in PSEN2.
In 1995 a large Italian AD kindred was identified with a mutation in PSEN2
consisting of a methionine to valine substitution at residue 239 (9).This mutation
is characterized by some peculiarities of the clinical and neuropathologic
phenotype compared to sporadic AD. In the autopsy analysis (11) of two
probands, in addition to neurofibrillary changes and Ab deposits, ectopic neurons
in the subcortical white matter containing neurofibrillary tangles were observed.
Furthermore, an unusually high number of ghost tangles in the cerebral cortex
were found. The major peculiarity of this family was the clinical onset with
epileptic seizures before the dementia (Fig. 1).
112 Sorbi et al.

Figure 1 An example of familial Alzheimer’s disease pedigree of a large Italian family


carrying PSEN2 Met239Val mutation. * Indicates the affected members bearing the
mutation. Source: From Ref. 11.

The combination of molecular and biochemical studies in the familial


forms of Alzheimer’s dementia supported the “amyloid hypothesis” as the main
mechanism involved in the pathogenesis.
Mutations in the APP and presenilin genes invariably increase production
of Ab42 leading to enhanced deposition of this toxic fragment in the brain
parenchyma (12–14) and consequently neuronal death. Abnormal accumulation
of Ab peptide could be considered a possible trigger of the disease and, together
with neurofibrillary tangle formation and neuronal dysfunction, lead to the
classical clinical picture of severe memory decline and loss of higher cortical
functions. The mechanism by which Ab1–42 accumulates in the brain has been
partially clarified in the last few years.
Formation of Ab42 requires proteolytic processing of APP. APP is a type I
transmembrane protein with a largely undefined function that undergoes
a primary glycosylation step in the Golgi apparatus and endoplasmic reticulum,
which is then transported to the plasma membrane where it is partly internalized
via endocytosis through a clathrin-dependent pathway. During these metabolic
steps, one of two proteolytic processes occurs. In the major metabolic pathway
the APP molecule is cleaved by alpha-secretase into two fragments, a large
N-terminal soluble form of APP (APPs alpha), which is eventually released into
the extracellular fluid, and a C-terminal fragment (CTF) termed C83 bound to the
membrane. An alternative minor pathway involves cleavage of APP at position -1
of the Ab portion of APP by beta-secretase leading to the formation again of a
large soluble protein (APPs beta) and a CTF termed C99 (Fig. 2).
Cleavage by an enzyme, gamma-secretase, of C83 leads to the formation of
p3 (amino acids 16–42 of Ab), precluding the formation of intact Ab.
Metabolism of C99 by gamma-secretase releases peptides Ab1–40 and
pathogenic Ab1–42.
Alpha-secretase processing of APP does not result in the formation of
amyloidogenic fragments and is therefore not implicated in the formation of Ab
or in the development of AD. Several different enzymes appear to account for
Genetics: Facts and Perspectives 113

Figure 2 Amyloid precursor protein (APP) cleavage and a- and b-secretase pathways and
production of b-amyloid peptide. Abbreviations: AICD, APP intracellular domain; CTF,
C-terminal fragment; sAPP, soluble APP; TM, transmembrane. Source: From Ref. 15.

Alpha-secretase activity including enzymes of the disintegrin and metallopro-


tease class [ADAM 10 and ADAM 17 or tumor necrosis factor-a converting
enzyme (TACE)], with these enzymes showing alpha-secretase activity regulated
by proteolytic processing and phosphorylation (16).
b-secretase is involved in the first step of Ab generation, and inhibition of
this protease provides a possible target for drugs against AD (17). Several lines
of research have identified the transmembrane aspartyl protease beta-site APP
cleaving enzyme (BACE1) as the putative beta-secretase. BACE1 is a type I
transmembrane protein, primarily expressed in neurons and localized in
intracellular endosomes and in the trans-Golgi network where the interaction
occurs. Experimental enhancement of BACE1 expression (18) leads to the
generation of C99, C89, and APPs beta, and a decrease in the levels of alpha-
secretase cleavage products. This phenomenon has recently been demonstrated
also in sporadic AD (19), but the role in the pathogenesis of the sporadic form of the
disease is still to be elucidated. In contrast, antisense inhibition of BACE1
decreases b-secretase activity and Ab production. A homologue of
BACE1, BACE2, has been identified by genetic database comparison and mapped
to chromosome 21 (20). The speculation of a possible involvement of BACE2 in
the processing of APP and ultimately in the generation of Ab, given its proximity
to the obligate Down syndrome region, has though failed to be confirmed, since
114 Sorbi et al.

major expression of BACE2 is localized outside of the brain and inhibition studies
using antisense tools or protease overexpression does not lead to variation in
Ab production.
Gamma-secretase catalyzes the intramembrane proteolysis of APP, and this
process is closely linked to the development of Ab1–42 peptide. This protease
cleaves APP to produce C83 and C99 and releases the APP intracellular domain
(AICD) into the cytoplasm, which can bind to cytoplasmic adaptor proteins and
translocate to the nucleus where it may regulate gene expression. The gamma-
secretase activity has several substrates in addition to APP, perhaps the most
important of which is Notch, a family of cell-surface receptors which are
essential for correct embryonic development. Recent findings suggest that
gamma-secretase is a complex of at least four integral membrane proteins:
presenilin, nicastrin, Aph-1, and Pen-2 (21), with the presenilins being the active
site of gamma-secretase.
Sequence analysis of presenilins demonstrates two intramembrane
aspartate residues, closely associated with the hydrophobic region that undergoes
endoproteolysis leading to the formation of the two active presenilin
heterodimers. Mutation of the two aspartate residues results in the blockade of
gamma-secretase cleavage of the C99 peptide, leading to a reduction of Abeta
production with simultaneous accumulation of C83 and C99 fragments and
simultaneous abolition of presenilin endoproteolytic processing (22). In addition
there is a close intracellular relationship between APP and PSEN1, which
coprecipitate together in small amounts to form complexes and are located in the
same intracellular vesicle compartments.
The evidence presented above supports the notion that a complex flow of
events, known as “amyloid cascade theory,” triggered by as yet unknown stimuli
or promoted by a single genetic defect, increases Ab42 production. A chronic
imbalance between the production and clearance of fibrillogenic Ab peptide with
a tendency to misfold and aggregate progressively reduces the efficacy of
synaptic transmission, promotes a microglial and astrocytic activation, and
disrupts neuronal membrane homeostasis and potentiates oxidative stress to the
cell. Recent findings suggest that the toxicity of Ab does not lie in the insoluble
fibrils that accumulate but rather in the soluble oligomeric intermediates (23).
Soluble Ab oligomers are found in human AD cerebrospinal fluid, and their total
amount correlates with the severity of the disease better than senile plaques (24).
Evidence suggests that the soluble oligomers can directly compromise synaptic
function and that senile plaques may function as reservoirs of fibrous polymers
that are in equilibrium with the diffusible species (25).
Because APP is axonally transported and processed in presynaptic
terminals, synapses are sites where oligomers of Ab may accumulate in high
amounts. Soluble oligomers of Ab42 inhibit long term potentiation in the
hippocampus of rodents, which may suggest that these oligomers are responsible
for memory impairment in AD patients. In transgenic mouse models of AD,
synaptic dysfunction and memory impairment can occur in the absence of any
Genetics: Facts and Perspectives 115

evidence of Ab deposition or neuronal degeneration (26). Evidence for frank


cellular apoptosis in AD is controversial, but there is a growing recognition that
apoptotic mechanisms may play a role in disease pathogenesis. Experimental
studies suggest that Ab can activate caspase through the extrinsic pathway,
implicating binding of extracellular Ab to cell receptors, while other studies
suggest that the intrinsic pathway may be more relevant. Accumulation of Ab in
the endoplasmic reticulum or endosomes may activate apoptotic mechanisms
through the unfolded protein response or endoplasmic reticulum stress;
alternatively, intracellular Ab may bind to alcohol dehydrogenase within
mitochondria and activate apoptosis through mitochondrial stress. One of the
consequences of caspase activation is cleavage of tau protein. Fragments of tau,
particularly those that contain the microtubule binding domain which is critical
for self interaction, more readily aggregate into fibrils than full length tau, but the
precise way in which Ab accumulation induces a cascade of neuronal metabolic
changes that includes the hyperphosphorylation of wild type tau molecules in AD
is the subject of active study, and several kinases have been proposed as possible
candidates (Fig. 3).

Genetic Analysis in Alzheimer’s Disease


With the identification of mutations in APP, PSEN1, and PSEN2 as causes of AD,
there is the possibility of using genetic testing to give a positive diagnosis of AD in
individuals with AD dementia and also the potential for predictive testing
in at-risk individuals. The majority of cases of dementia associated with mutations
in APP, PSEN1, and PSEN2 are early onset (generally age at onset of less than
65 years), and almost without exception where a family history is known, carriers
will develop disease, though there are cases where PSEN1 mutations are not fully
penetrant (28). These cases of FAD are, however, rare (29), and not necessarily
seen except in large specialist centers, and therefore the possibility of genetic
testing is only applicable to a very small minority of dementia cases. Identification
of a genetic basis for early onset dementia has, though, similar implications to that
found for Huntington’s disease, and therefore almost identical procedures
will apply.
When presented with an individual with early onset dementia, patients and
families are naturally anxious to know what the diagnosis is, and also if there is
any likelihood that the disorder may be inherited. Frequently, evidence is brought
forward by families of dementia in a previous generation or of dementia in a
sibling. It should be stressed here that a positive family history of dementia is
often apparent in a great many cases of AD, and some studies show up to 40% of
AD cases with an affected relative (30). These cases are often though, of late
onset, and not necessarily suitable for any genetic testing. In such circumstances,
taking a brief family history is warranted, with particular attention being paid to
the age at onset of symptoms in affected family members, as within families with
early onset autosomal dominant dementia, age at onset is frequently very similar
116 Sorbi et al.

Dominantly inherited Nondominant forms of Alzheimer's disease


forms of Alzheimer's disease (including "sporadic" Alzheimer's disease)

Missense mutations in the APP or Failure of Aβ clearance mechanisms


presenilin 1 or 2 genes (e.g., inheritance of ApoE4, ?faulty Aβ degradation)

Increased Aβ42 production Gradually increasing Aβ42 levels in brain


throughout life

Accumulation and oligomerization of Aβ42 in limbic and association cortices

Subtle effects of Aβ oligomers on synaptic efficacy

Gradual deposition of Aβ42 oligomers as diffuse plaques

Microglial and astrocylic activation and attendant inflammatory responses

Altered neuronal ionic homeostasis: oxidative injury

Altered kinase and phosphatase activities lead to tangles

Widespread neuronal and synaptic dysfunction and selective neuronal loss,


with attendant neurotransmitter deficits

DEMENTIA

Figure 3 A hypothetical sequence of the pathogenetic steps of Alzheimer’s disease based


on currently available evidence. Source: From Ref. 27.

and may be one indication that a mutation in a particular gene may be present and
require further investigation (29,31–35). A patient’s and family’s knowledge of a
family history of dementia, however, brings with it certain anxieties, and
appropriate specialist counselling measures should be instituted prior to, and
during, any investigations (36,37). If an individual is, however, presented with
Genetics: Facts and Perspectives 117

early onset dementia suggesting AD, genetic analysis can be instituted if access
to facilities and appropriate technical support is present. In the first instance, only
the proband should be tested for the most likely mutations which will entail
sequencing the PSEN1 gene, followed by exons 16 and 17 of APP, and then, if
available, all coding exons of PSEN2, since such mutations of the latter are rare.
Given the fact that in many families with suspected AD, there is no evidence of
mutations in APP, PSEN1, and PSEN2 (29), it should be noted that families may
face a degree of uncertainty over the findings of any testing, and therefore
following testing, appropriate support should be made readily available (37).
If mutations in APP, PSEN1, or PSEN2 are identified, then confirmation of
diagnosis can be made, appropriate care and treatment of the patient provided,
support and counselling given to the family (36,38), and the possibility of
predictive testing undertaken. Predictive testing, however, has certain issues
associated with it (38) and should not be undertaken lightly due to the impact
upon life choices for the individual, and should only be undertaken in those
families where a mutation has been identified in an affected individual. Once
again, extensive counselling and support should be provided. In those found to be
at risk of developing AD because of the presence of a defined mutation,
additional support could be given in the form of longitudinal neuropsychiatric
assessment to determine if there are signs of cognitive impairment. Longitudinal
magnetic resonance imaging (MRI) studies (39,40) and possibly positron
emission tomography (PET) scanning may predict cognitive changes and give the
possibility of disease slowing treatments including cholinesterase inhibitors (41)
or vaccination against Ab, if it is possible given the side effects (42), then further
supportive measures can be undertaken.

Apolipoprotein E and Other Genes Potentially Associated


with Alzheimer’s Disease
The current knowledge, mainly derived from studies on familial AD, points out
that a single genetic defect occurring in these pathways could lead to dementia.
Despite the increasing number of APP, PSEN1, and PSEN2 mutations detected so
far, research on novel candidate genes or molecules implicated in the sporadic
form of AD has not reached a sufficient power or level of evidence compared to
the only recognized risk factor to date, Apolipoprotein E.
Since 1993, over 150 association studies have replicated and confirmed the
early findings (43) reporting an increased frequency of the Apolipoprotein E E4
allele in AD and the association of this allele with late onset AD (LOAD) and
sporadic forms of AD. Apolipoprotein E (ApoE, protein; APOE, gene) is a
37 KDa protein involved in cholesterol transport in the brain and periphery. The
three major isoforms of human ApoE (ApoE E2, ApoE E3, ApoE E4) are coded
by the E2, E3, and E4 alleles, ApoE E3 being the most frequent isoform. ApoE
E4 differs from ApoE E3 in a Cys/Arg change at position 112. It is now widely
accepted that APOE E4 allele confers, in terms of relative risk, an increased risk
118 Sorbi et al.

of AD, whereas the E2 allele appears to have a protective effect. The risk for AD
conferred by APOE E4 allele increases in a dose-dependent fashion, being eight
times more likely for carriers of a double dose of E4 allele compared to APOE E3
carriers. Further epidemiological studies and genotype-phenotype correlations
have suggested that E3/E4 and E4/E4 genotypes are more frequent in female
patients with AD, with a compelling dose effect, and also that particular ethnic
populations (i.e., African Americans) show a higher frequency of the APOE E4
allele, and therefore have an increased risk of developing AD. In addition, several
studies, although controversial, have shown a role for ApoE in the mechanistic
processes that lead to brain injury and ultimately to dementia. ApoE may
contribute to amyloid and tau deposition in AD, binding to amyloid in a isoform
specific manner enhancing aggregation, and in a similar way, interfering with tau
binding to microtubules. APOE E4 is strongly associated with increased neuritic
plaques, and the presence of this allele increases the odds ratio for cerebral
amyloid angiopathy. In addition, APOE E4 carriers have shown reduced glucose
metabolism and choline acetyltransferase activity in selected brain regions,
demonstrate an accelerated cognitive decline, and are poor responders to
memory-enhancing drugs prescribed for AD.
Several studies have shown that the E4 allele is also overrepresented in
mild cognitive impairment (MCI), and an increased frequency of the allele is a
strong predictor of clinical progression from MCI to AD. Although the E4 allele
alone does not imply conversion to AD in MCI, the combination of genetic
assessment with functional brain imaging is now seen as a promising preclinical
AD detection strategy (see Chapter 8). Recent PET studies have demonstrated an
association between the E4 allele and an abnormal reduction of glucose
metabolism in normal individuals carrying the E4 genotype in the same brain
regions as found in AD patients. It remains unknown if this is a predictor of future
cognitive decline. In addition, there is evidence that the E4 allele leads to greater
longitudinal metabolic decline in healthy elderly persons converting to MCI.
Nonetheless, no study has been carried out to assess the impact of the E4 allele on
brain physiology in the conversion from MCI to AD (44).
Several studies have addressed the utility of APOE genotyping in the
diagnosis of AD, and the suggestion has been made that the presence of the E4
allele is indicative of AD in a patient presenting with dementia (45). The presence
of an APOE E4 allele is, however, neither sufficient or specific to support a
clinical diagnosis of AD on the basis of genetic testing alone (46), and currently it
offers little significant gain over currently used diagnostic tools. For prediction of
dementia development, there is almost universal agreement that APOE
genotyping should not be offered or used (47,48), and therefore it remains a
tool for academic use only.
APOE E4 allele frequency has also been associated with a number of
neurological diseases, mainly degenerative, in which the major pathogenic
step is represented by proteinaceous aggregates in the brain. This has suggested
a putative role of ApoE in the clearance and internalization of small peptides that
Genetics: Facts and Perspectives 119

tend to accumulate in the brain. An increased E4 frequency was shown in patients


with traumatic brain injury with a poor neurologic recovery, possibly due to
enhanced b-amyloid deposition. The role of ApoE has also been suggested in
increasing oxidative stress and in altering inflammation-related astrocyte and
glial activation, and given the fact that ApoE is the major component of very-low
density lipoproteins, a function in determining intracellular cholesterol
distribution and homeostasis has been proposed.
However, despite all the evidence presented above, APOE remains a
genetic risk factor that is neither sufficient nor necessary to cause AD, and only
increases the risk of devloping disease.
Since mutations in APP, PSEN1, and PSEN2 genes account for less than
10% of early onset FAD cases, and the presence of the APOE E4 allele is not a
causative determinant for AD, several other genetic factors predisposing or causing
the disease must exist.
Other Genetic Loci for Alzheimer’s Disease
The current strategies to identify novel candidate genes (Table 2) or loci that
could be related to sporadic AD or other dementias are today principally based on
linkage analysis studies that provide new chromosomal regions to investigate,
and on genetic association studies (family based or case-control designed),
applied to detect genetic variants (polymorphisms) in molecules implicated in the
disease-specific pathways or in biologically disease-related genes.
In comparison with early-onset forms of AD, the genetic basis of LOAD
appears much more complex. Most LOAD cases are sporadic, with no family
history of the disease. Genetic susceptibility at multiple genes and interaction
between them and/or environmental factors are likely to be responsible for the
aetiology of LOAD. Linkage analysis narrows down a chromosomal candidate
region which may harbour dozens of genes (positional candidate genes) and
consequently hundreds or thousands of single nucleotide polymorphisms. Some
of these genes may be functional candidates with respect to pathogenic
mechanisms of interest. Besides the chromosome 19 region harbouring APOE,
multiple genome scans and linkage analyses have identified important
chromosomal regions that potentially harbour additional LOAD genes on
chromosomes 1, 5, 9, 10, 12, and 21 (containing APP), in addition to several loci
with possible linkage (50). Although there are disagreements among linkage
reports on the locations of putative AD genes on these chromosomes, given the
overwhelming linkage evidence, it now seems logical to subject the relevant
linkage regions to an exhaustive gene hunt. The application of refined methods
that account for disease phenotype heterogeneity and the inclusion of informative
covariates accounting for locus heterogeneity (age at onset, gender, APOE
genotype, etc.) would be useful in either reducing or eliminating the background
noise, allowing the identification of genes related to a defined stratum of LOAD.
Replicating linkage for complex diseases represents a challenging prospect, and
usually requires a sample far larger than the original to see a similar signal.
120 Sorbi et al.

Table 2 Susceptibility Genes for Alzheimer’s Disease


Association with
Gene Chromosomal location Alzheimer’s disease

APOE 19q32.2 C
ACE 17q23 C/K
ACT 14q32.1 C/K
BACE 1 11q23 C/K
BChE 3q26.1–q26.2 C/K
BH 17q11.1–q11.2 C/K
CATD 11p15.5 C/K
CST3 20p11.2 C/K
CTNNA3 10q21 C/K
5-HTT 17q11.1–q12 C/K
IDE 10q23–q25 C/K
LRP 1 12q13.1–13.3 C/K
NCSTN 1q23 C/K
NOS3 7q35 C/K
PEN2 19q13 C/K
PLAU 10q22 C/K
PS1 promoter 14q24 C/K
TGF-b1 19q13.1–q13.3 C/K
UBQLN1 9q21.2–q21.3 C/K
VLDL-R 9pter–p23 C/K
a2M 12p13.3–12.3 C/K
C/K indicates the positive and negative association studies. For specific indications on association
studies of a specific gene, visit the “Alzgene” database (http://www.alzforum.org/res/com/gen/alz-
gene/default.asp).
Source: From Ref. 3.

The collection of additional families for linkage studies may be useful in


addressing the issue of power. However, there is no guarantee that this would
provide more meaningful information about the linkage regions than we already
have from the existing linkage data. In addition, possible candidate genes should
be confirmed with case-control association analyses, although often giving
inconsistent results on populations with different ethnic background and if
limited to a relatively small number of cases and controls. Carefully designed
high-resolution large linkage genome scans together with association studies on
relatively large case-control cohorts may provide useful additional and/or
confirmatory information on the locations of putative AD genes.
In addition, genome-wide linkage or association studies can only identify
potentially important chromosomal regions that may harbour functional genes,
but the next steps must be focused to investigate these regions with fine or linkage
mapping and to perform functional tests on these plausible biological and
positional candidate genes.
Genetics: Facts and Perspectives 121

A recent full genome scan indicated two major regions of interest on


chromosome 19q13 and on chromosome 11q25 related to early onset FAD (51).
These regions contain, apart from APOE, two interesting candidates, namely the
PEN2 gene, encoding a protein relevant to gamma-secretase activity, and the
BACE1 gene. To date, association studies on small populations with newly
discovered polymorphisms in nicastrin and BACE1 have yielded contrasting or
negative results (52).
Numerous proteins, enzymes, and proteases involved in the mechanism of
degradation, clearance, and toxicity of Ab have been investigated as possible
candidate genes of susceptibility for developing AD. In particular, recent studies
suggest new susceptibility loci on chromosome 10q. Over 240 genes in this
region are thought to be strong positional and biological candidates able to
interfere with AD-related biochemical pathways such as the urokinase-
plasminogen activator (PLAU) gene and the insulin degrading enzyme (IDE).
PLAU is a serine protease (chromosome 10q22.2) that converts the inactive
zymogen plasminogen into its active form plasmin. Although the plasmin
proteolytic cascade is traditionally involved in fibrinolysis and cell migration, the
plasmin system seems to be relevant to Ab clearance. Ledesma and colleagues
(53) report that plasminogen and its proteolytic fragment plasmin are present on
the membrane of cultured mature hippocampal neurons where plasmin could
cleave APP at the alpha site precluding the formation of APP, and could promote
Ab degradation. Recently a positive association with a single C/T nucleotide
polymorphism of PLAU (P141L) and LOAD has suggested a possible effect on
the risk of developing AD, but these results have been not replicated by other
studies (54–57).
IDE gene (located on chromosome 10q24) encodes for an enzyme able to
degrade Ab monomers, thereby modulating cerebral Ab levels. Indeed loss of
IDE function in knockout mice and a rat mutant model leads to increased cerebral
Ab levels. Moreover, IDE appears to degrade the AICD. Despite initial reports of
significant linkage of IDE in a family-based sample, negative data have been
found in successive case-control studies (58–61).
Other known Ab degrading enzymes such as neprilysin and endothelin
converting enzyme-2 (ECE2) encoded by genes on chromosome 3, and
plasminogen and the tissue-type plasminogen activator encoded by genes on
chromosome 6, despite their potential and clinically relevant roles on AD
neuropathogenesis, have not been positively associated with AD in case control
or family based studies. Finally, Bertram, and Hiltunen have recently suggested
an association with the ubiquilin 1 (UBQLN1) gene (located on chromosome
9q22) because of its potential role in the proteasomal degradation of proteins and
its interaction with PS1 and PS2 (62). These data have, however, not been
replicated yet.
Regarding the Ab clearance, increasing evidence suggests that the low-
density lipoprotein receptor-related protein (LRP) mediates the efflux of Ab from
the brain to the periphery. LRP is a scavenger receptor that can bind a variety of
122 Sorbi et al.

ligands, including ApoE, APP, and alpha2 macroglobulin. Ab can form a


complex with ApoE or alpha-2-macroglobulin and, after binding to LRP, is
internalized to endosomes for degradation or directly exported into the plasma.
Several studies have tested alpha 2 macroglobulin and LRP1 genes in patients
with AD with contrasting results (63–66). In summary, the alpha-2-macro-
globulin gene might be considered a risk factor only in LOAD cases with a family
history (67), while study of the LRP1 gene performed to date does not support
any association with disease.
The exact mechanism of Ab toxicity is unclear. One hypothesis appears to
involve the induction of apoptosis, but to date there is no evidence for an
association with specific apoptosis candidate genes. Another hypothesis is based
on the role of inflammation in AD: although inflammation and the up-regulation
of inflammatory mediators is observed in the AD brain around senile plaques,
none of the various members of cytokine family (such as interleukin 1-alpha,
interleukin 1-beta, interleukin-6) have been reported to be associated with disease
with additional evidence of genetic linkage. However, interesting data based on a
number of full-genome screens in AD have shown positive genetic linkage and
association with the TNF alpha gene (50).

Perspectives
The knowledge of genes involved in the amyloid cascade has moved towards the
development of specific targets for possible therapeutic approach. There is a great
interest in identifying small molecular inhibitors of the beta and gamma
secretases, although problems have arisen with these strategies because these
proteases also process other critical substrates (e.g., gamma secretase processes
Notch). Possible selective inhibitors for APP include nonsteroidal anti-
inflammatory drugs and cholesterol-lowering drugs (20).
An alternative approach is to prevent aggregation of the Ab peptide and/or
promote its clearance. In this field, early work in APP transgenic mice
demonstrates that immunization with Ab significantly attenuates AD-like
pathology and ameliorates memory deficits (68,69). Although the mechanism
by which brain Ab is reduced following vaccination remains controversial, recent
preliminary reports indicate that Ab immunization may lower senile plaque load
and stabilize behavioral decline in humans with AD (70,71). However, the
findings of an adverse effect involving meningoencephalitis in approximately 6%
of active Ab-vaccinated patients in early clinical trials conducted in 300 patients
(42) raise questions about the safety of this approach. The development of
efficacious and safe immunogens with careful antigen and antibody selection, as
well as of new vaccination techniques for immuntherapy of AD, can be expected
in the future to maximize efficacy and minimize serious adverse events.
An alternative active approach that may carry a lower risk of adverse events is
mucosal (nasal or oral) vaccination. This method has been tested in APP
Genetics: Facts and Perspectives 123

transgenic mice and has led to significant decreases in plaque burden, Ab levels,
neuritic dystrophy, and gliosis (72).
For the identification of AD genes it would be useful to combine the genetic
map information with gene expression profiling data, which in itself provides a
powerful instrument to rapidly identify a set of genes differently expressed
between normal and diseased tissues (73). Only the integration of the information
from genetic maps (location of genes) and genomic maps based on gene
expression and indicating specific genes may lead to the rapid identification of
causal genes. The combination of these two strategies may prove to be useful for
AD, and this is already evidenced by preliminary studies (74).
Another important aspect that should be considered in order to gain an
understanding of the genetic basis of complex disorders is gene–gene interaction.
With the exception of APOE, which is universally recognized to have a high risk
associated with AD, the general understanding is that several genes, each with a
moderate effect, are involved in the aetiology of a complex disease such as AD.
Therefore it has been hypothesized that other possible candidate genes, having a
small effect on the risk of AD, may not be detectable in all association studies due
to power or sample heterogeneity issues, but that they may show a considerable
effect in conjunction with other candidate genes. Much larger case-control
samples are required for gene–gene interaction studies as stratification of the data
reduces the power considerably, and this represents a limitation. With the
exception of a few published cases-controls studies that have utilized more than
1000 subjects, most genetic studies use a smaller sample, raising questions about
the meaningfulness of the association data rather than providing promising future
aims. Only significant results from appropriately powered studies could be
considered for replication in other samples from different populations.

Frontotemporal Dementias
Frontotemporal dementias (FTD) are complex clinical entities, characterized
neuropathologically by glial and neuronal tau deposition with widespread
neuronal loss and gliosis (see Chapter 11), with a marked clinical and genetic
heterogeneity. The nosological classification is still under debate, even though
Consensus Criteria have been published (75). Common clinical features include
presenile age of onset (35 to 75 years age range) and psychotic symptoms at onset
with late memory impairment. Often extrapyramidal signs or motor neuron
involvement is present.
The disorder is quite rare, with a prevalence of 10–15 per 100,000
individuals aged less than 65 years, and representing about 15–20% of all the
presenile dementing syndromes. At least three clinical syndromes have been
described in the past: a frontal variant of FTD, with pronounced changes in
behavior and personality, semantic dementia, and primary progressive
(nonfluent) aphasia. These latter forms of the disease are purely sporadic, but
124 Sorbi et al.

approximately 40% of all FTD cases are familial, with a large clinical variability
between and within kinships with FTD.
Linkage analysis studies, trying to homogenize different families with
diverse clinical patterns, have led, more or less simultaneously worldwide, to the
identification of a significant locus on chromosome 17, in the same region as the
tau protein gene (TAU). Initial sequencing of TAU in chromosome 17-linked
families showed negative results. An early report of an intronic mutation in 1998
led to the subsequent identification of TAU defects as the cause of FTD in some
families. The reason for this initial delay could be related to the structure of TAU,
which is composed of 15 exons, of which 11 are expressed in the adult brain.
Exons 2, 3, and 10 are subject to alternative splicing, producing six different
isoforms of the protein. These six isoforms are characterized by the presence of
two diverse functional domains, the first being composed of variable NH2-
terminal inserts that interact with plasma membranes and the second by three or
four COOH-terminal microtubule binding repeats that ensure binding with
tubulin and promote microtubule assembly (Fig. 4).
The first molecular variation found in TAU was an intronic mutation (77).
Further identification of novel mutations clustering in exon 10 and nearby in
intron 10, where the mutations affects tau splicing resulting in an altered 3/4
repeat ratio, provided direct evidence that intronic mutations could alter gene
structure with loss of protein function. In fact, alterations in splice regulation
caused by these mutations may disrupt a putative stem-loop structure or impair
the binding of regulatory proteins to the repeat region (78). Such a different
conformation of mutated tau could promote excessive deposition of the protein
(also in absence of hyperphosphorylation) due to microtubule malfunction, and
slowing intraneuronal transport of substrates. Thus, precipitation of tau
aggregates and subsequent NFT formation could be interpreted as the
consequence of this process.
To date more than 100 families with FTD have been identified with over 30
different mutations in TAU (78). Nucleotide changes include missense mutations,
deletions, and splice site mutations. A direct correlation between the site of
mutation and a specific cellular pathology has been predicted by early studies,
suggesting that, for example, tau aggregates predominantly composed by
filaments with four repeats were more common in patients with exon or intron 10
mutations. Today, with the discovery of novel pathogenic mutations, this is no
longer confirmed, since mutations not clustering in the exon-intron 10 region
could not necessarily lead to a specific neuronal or glial pathology or lead to a
selective deposition of a particular isoform, (i.e., aberrant insoluble tau filaments
lacking isoforms with a specific number of NH2-terminal inserts).
Most missense mutations in the microtubule-binding region reduce the
ability of tau to interact with microtubules interfering with correct microtubule
assembly. The primary effect of these mutations may be equivalent to a partial
loss of function, with resultant microtubule destabilisation and deleterious effects
on cellular processes, such as rapid axonal transport.
Genetics: Facts and Perspectives 125

Figure 4 Mechanism of neurofibrillary degeneration. Tau is a major microtubule-


associated protein of a normal mature neuron where it stimulates assembly and establishes
microtubules. In a normal brain, tau contains 2–3 mol phosphate/mol of the protein for its
optimal activity. Hyperphosphorylation of tau depresses this normal biological activity. In
Alzheimer’s disease and other tauopathies, tau becomes abnormally hyperphosphorylated.
The tangles are ubiquitinated for degradation by the non-lysosomal ubiquitin pathway, but
apparently this degradation, if any, is minimal. Source: From Ref. 76.

The intronic mutations and most coding region mutations in exon 10


increase the splicing of exon 10, thus changing the ratio between three- and four-
repeat isoform and resulting in the overproduction of four-repeat tau. The
possible existence of different binding sites on microtubules for three-repeat and
four-repeat tau could explain the cytoplasmic accumulation of the overproduced
isoform with the effect of a “gain of toxic function” mechanism that leads to
neurodegeneration (79,80).
The heterogeneity of pathological manifestation of tau mutations is also
reflected in clinical phenotypes in mutated families. As far as is known, there are
no clear relationship between the clinical presentation and the position of the
mutation, although early reports speculated on a more close association between
126 Sorbi et al.

particular clinical features and mutation location. Additional clinical features of


TAU mutation families described so far include seizures at onset, pure AD
phenotype or late onset dementia, progressive supranuclear palsy, and
predominant motor neurone involvement with acute respiratory failure. More-
over different phenotypes can be expressed in a single family such as a
pathological diagnosis of FTD in the father, and corticobasal degeneration in the
offspring. These findings support the notion that additional genetic or
environmental factors may account for this marked phenotypic heterogeneity.
There are, however, families with an FTD-like presentation that do not
show mutation in TAU, and in a recent study the relevance of TAU mutations in
FTD and related dementias was addressed (81). Mutations in TAU gene were only
identified in just over 6% of familial or sporadic cases, with 25% of cases with a
positive family history of FTD showing mutations. Even cases with FTD showing
linkage to chromosome 17 often do not show mutation in TAU, suggesting that
other genes may cause FTD (82). In apparently sporadic individuals only 4–5% of
cases show mutation of TAU, again suggesting that these are rare causes of FTD.
Other rare forms of FTD occur with specific additional phenotypes, and
these may provide indications in the early differential diagnosis of FTD coupled
with genetic testing. A large Danish pedigree has been described which initially
showed the pathology of DLDH (OMIM 600795) though subsequently showed
tau-positive inclusions in neurones and glia (83). This family demonstrates
linkage to a small region on the short arm of chromosome 3p11, and the causative
gene is suggested to be CHMP2B (84). Other forms of FTD with linkage to
chromosome 9q21–22 and a clinical and pathological feature of ALS (Hosler
et al. 2000:OMIM 105550) occur, and in these the feature of ALS would suggest
a differential diagnosis, but again the causative gene is unknown. Early onset
FTD in the thirties with the additional features of inclusion body myopathy and
Paget’s disease shows linkage to chromosome 9p13 (OMIM#167320), and Watts
and colleagues have identified mutations in the Valosin-containing protein gene
(VCP) to be the cause of this disorder (85). In these cases, frontal cortical and
cerebellar atrophy with ubiquitin and VCP-positive inclusions in neurons are a
particular neuropathological feature.The clinical features of a progressive
myopathy linked with Paget’s disease in approximately half of cases and FTD
in about one-third would suggest a diagnosis of this disorder and would warrant
referral for genetic testing. Although tauopathies have been considered to result
from genetic defects, screening for tau gene mutations in sporadic cases, FTD3,
FTD/ALS9, FTD/CBD9, and familial PSP is not likely to identify pathogenic
mutations (86).

Dementia with Lewy Bodies


Dementia with Lewy bodies (DLB), which neuropathologically is diagnosed by
an abnormal aggregation of alpha-synuclein, is not now thought to be a rare cause
of familial neurodegenerative disease. Alpha-synucleinopathies are a group of
Genetics: Facts and Perspectives 127

neurodegenerative disorders typically characterized by the loss of selective


neuronal populations associated with the accumulation of pathological
aggregates of alpha-synuclein, a normal synaptic protein that has been implicated
in neurotransmission or in the organization and regulation of synaptic vesicles.
This heterogenous group also includes also Parkinson’s disease, dementia that
occurs during the course of Parkinson’s disease, and primary autonomic failure.
In 1996 consensus guidelines were proposed for the clinical and
neuropathological diagnosis of DLB (87). The core clinical features for a
diagnosis of probable and possible DLB are: fluctuating cognitive impairment,
recurrent visual hallucinations, and parkinsonism. The consensus criteria
arbitrarily suggest that if dementia occurs within 12 months of the onset of
extrapyramidal motor symptoms, the patient should be diagnosed with possible
DLB, while if the clinical history of parkinsonism is longer than 12 months, the
diagnosis of Parkinson’s disease dementia should be more appropriate. Lewy
bodies (LB) are relatively large spherical eosinophilic cytoplasmic intraneuronal
inclusion bodies that range from approximately 4 to 30 m in diameter, first
described by Frederich Lewy in 1912 in the substantia innominata and the dorsal
motor nucleus of the vagus in a case of Parkinson’s disease. These proteinaceous
inclusions are a defining hallmark of Parkinson’s disease and dementia with LB,
but there are as yet no definite pathological criteria that separate the different
alpha-synucleopathies either from each other or from AD and vascular pathology.
Consensus criteria for DLB include ubiquitin immunohistochemistry for LB
identification, although the recently developed alpha-synuclein immunochem-
istry is a better marker for visualizing LB and also shows previously under-
recognised neuritic pathology, termed Lewy neurites (88). Pathological alpha-
synuclein aggregates can constitute the fibrils of typical classical LB in the
pigmented nuclei of the brainstem, in neocortex and amygdala, or Lewy neurites
in the Ammon’s horn.
Alpha synuclein is a dynamic molecule: it adopts an unfolded random coil
structure in aqueous solution, shows an alpha helical structure upon binding to
acid phospholipid vesicles, and is able to form insoluble fibrils with a high beta-
sheet content resembling the filaments found in LB. Although mutations in alpha
synuclein facilitate the transformation to a beta sheet structure, wild type alpha
synuclein can adopt a beta sheet conformation possibly due to saturation effects,
unknown protein-protein interaction, or environmental or genetic factors. Indeed,
several studies suggest that the age-related increase in oxidative damage to
membranes promotes the aggregation of alpha synuclein (Fig. 5). APP,
chromogranin A, synphilin 1, and synaptophysin are axonally transported
proteins that have been reported in LB (see Chapter 11). Even neurofilament and
tubulin seem to be implicated in LB formation as immunohistochemical analysis
shows their presence. It is possible that the presence of Lewy neurites and
neurotransmitter deficits are more likely to be directly associated with clinical
symptoms than density of cortical LB.
128 Sorbi et al.

Figure 5 (See color insert.) Molecular pathophysiology of a-synuclein (aSN) mono-


mers converting into partially folded, toxic, and/or aggregated forms. Putative toxic
forms of aSN include fibrillar aggregates (Lewy bodies, Lewy neurites, cytoplasmic glial
inclusions); amorphous aggregates; oligomers; protofibrils; soluble forms including
partially folded intermediates, Ser-129-phosphorylated aSN, nitrosylated, and ubiquity-
lated. Source: From Ref. 89.

Currently, the mechanisms responsible for the abnormal aggregation of


alpha-synuclein in insoluble and toxic forms remain to be explained. One
possibility is that there is a pathological upregulation of normal, wild-type alpha-
synuclein due to mRNA hyperexpression. Another is that alpha-synuclein could
become insoluble or more able to aggregate. Alternatively, an inefficient
proteasome system could process toxic protofibrils of the alpha-synuclein
proteins, which are then sequestered into LB (90). The last mechanism could
represent an effort by neurons to counteract biological stress inside the cell.
Sporadic and familial cases of DLB have been reported. Genetic
investigation of familial DLB has been very limited and in most cases negative.
Genetics: Facts and Perspectives 129

Recently a novel genetic mutation, E46K, has been found in the alpha synuclein
gene (SNCA) in a Basque family with autosomal dominant parkinsonism and the
typical pathological hallmarks of DLB (91), suggesting that genetic causes of
DLB share common mechanisms with genetic forms of PD (92,93). In the
recently described family with triplication of SNCA (94), some family members
presented with a clinical picture that could be considered to fulfil criteria for
DLB. However multiplication of SCNA has been investigated in pathologically
confirmed patients with DLB, without positive results (95). Beta synuclein is a
member of the synuclein family, and is highly homologous to alpha synuclein,
both being involved in synaptic function. In a recent report, Ohtake and
Limprasert suggest that mutations in beta-synuclein gene may predispose to
DLB (96). The recent identification of the dardarin gene (LRRK2) in families
with parkinsonism, frequently with dementia or a DLB presentation (97,98),
suggests the possibility that this gene may be important in some DLB/PD cases.
Understanding the pathophysiology of DLB appears important for
developing novel therapeutic strategies. Neuroprotection appears interesting
since there is evidence in DLB of significant neuronal dysfunction but no striking
cortical neuronal degeneration (see Chapter 11). Therefore, the consistent
evidence that DLB is more treatable than other neurodegenerative disorders with
available pharmacological management, make important the accurate identifi-
cation of patients with DLB and their differentiation from other common types
of dementia.

SUMMARY
An understanding of the genetics of dementia and AD has provided vital clues to
the underlying biology that leads to this disorder with the identification of APP,
PSEN1, and PSEN2. Genetic analysis, whilst useful in certain circumstances,
cannot currently be used for the majority of cases, as the APOE gene, the only
robustly identified gene for LOAD, is neither necessary nor sufficient for the
development of this disorder.

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5
Approaches to the Identification of Novel
Diagnostic and Therapeutic Targets
in Dementia

Kate E. Wilson
Institute for Ageing and Health, University of Newcastle, Newcastle
General Hospital, Newcastle upon Tyne, Tyne and Wear, U.K.
Chris Morris
Wolfson Unit of Clinical Pharmacology, Chemical Hazards and Poisons
Division–Newcastle, Health Protection Agency, and School of Neurology,
Neurobiology, and Psychiatry, The Medical School, University of Newcastle
upon Tyne, Newcastle upon Tyne, Tyne and Wear, U.K.

INTRODUCTION
With the increasing prevalence of dementia in the elderly and the recognition that
the syndrome itself is a heterogeneous disorder, there is a pressing need to
identify not only effective treatments, but also diagnostic aids that will selectively
and specifically define the different forms of dementia. Population-based studies
backed by neuropathological assessment are few; however, they show that
Alzheimer’s disease (AD), while still the major form of dementia, accounts for
about 60% of cases, with vascular dementia, dementia with Lewy bodies, frontal
lobe syndromes, and mixed pathology disorders also contributing to the overall
problem (1). Combining diagnostics and therapeutics will provide the most
significant benefits for patients, but with different syndromes making up the total
picture, it is vital to ensure that the diagnosis is correct. A major problem with
dementia diagnosis currently is the fact that gold standard diagnosis can only
come postmortem (see Chapter 11). Clinical diagnosis, while highly accurate in

137
138 Wilson and Morris

many tertiary referral centers, comes only after extensive examination over a
period of months, during which time specific treatments need to be applied. The
ability to provide a rapid and accurate diagnosis could therefore lead to
significant improvements in dementia treatment.
But how, particularly when the underlying basis for many aspects of
dementia is unknown, is it possible to identify new diagnostic targets, or new
targets for therapy? Can a technique also be applied early enough to provide
presymptomatic diagnosis? Various systems which are currently in use or are in
development allow potential novel diagnostic targets to be identified. These
methods, collectively termed functional genomics, permit the rapid and often
wholesale analysis of cells or tissues in a disease state or the effects of
pharmacological treatment, making the identification of changes possible in a
short space of time.

FUNCTIONAL GENOMICS
The various genome projects that have set out to define the molecular basis for
many major organisms, including man, and have provided the foundation for the
global analysis of cell and tissue function using functional genomics (2). By
cataloguing all the possible genes in an organism, the genome projects have
allowed the production of reagents capable of detecting all the expressed genes,
or a reference with which to identify gene products by. The former is exemplified
by the production of whole genome microarrays, now the method of choice for
determining gene expression, and the latter by the bioinformatics tools which,
when coupled to various mass spectrometry (MS) methods, can be used for high
throughput protein and peptide identification. For the identification of diagnostic
targets in dementia, the application of these methods allows the production of a
catalogue from which to choose suitable candidates for further testing.

MICROARRAY-BASED GENE EXPRESSION PROFILING


Several methods exist which allow the analysis of all the expressed genes within
a tissue or cell type, such as those based on subtractive cDNA methodology (3),
differential display-based methods (4,5), or serial analysis of gene expression (6).
Many of these methods can be used by standard laboratories with the minimum of
molecular biology equipment, though a problem with their use is in the relatively
labor-intensive nature of the methods. The advent of gene expression microarrays
has, however, largely superseded these technologies since it allows the rapid and
comprehensive analysis of gene expression, or a focused approach to the analysis
of a specific set of genes. Microarrays are high densities of specific DNA
sequences either as single stranded chemically synthesised oligonucleotides or as
cDNAs (often produced by PCR from defined clones), present as a highly ordered
array on a solid support of usually glass or plastic (7). Several thousand
individual sequences can be spotted or synthesised on an array, with each single
Novel Diagnostic and Therapeutic Targets 139

spot, often only a few microns across, representing a single mRNA species.
Microarrays are probed using a reverse hybridization procedure where the RNA
sample is labeled, normally with a fluorescent reporter molecule, and then
hybridized to the DNA sequences on the array. Since hybridization is roughly
proportional to the amount of an mRNA species present in the sample, the
presence of this highly ordered array allows each individual gene sequence to be
analyzed for the amount of mRNA present in the sample.
For any given tissue or cell, there will be a specific set of genes that are
expressed and which defines the nature of that tissue or cell. Of the 35,000–40,000
genes in the human genome, normally in any cell only a limited fraction of these
genes will be expressed, perhaps only 20–30% of the genome for any given cell
type (2). The complexities of the brain, with multiple neuronal types, vasculature,
and supporting glia, mean that the gene expression profile of any one brain area can
be extensive, and it has been suggested that perhaps half of the genome is
represented by genes specific to the brain (2). Microarrays have been designed
which are sufficient size that they represent most if not all of the possible genes in
the human genome. For the definition of brain function in health and in disease,
such arrays have the capacity to rapidly assess and identify those genes that are
expressed within a specific tissue. By extracting RNA from brain tissue and
applying it to a microarray, it is therefore possible to determine which genes are
being expressed by that tissue and also the relative amount of the particular
transcript present. Comparing healthy tissue with diseased therefore permits the
identification of which genes are present or absent and also to what extent any
particular gene is being expressed.
One potential problem of brain tissue is its highly complex nature stemming
from the numerous cell types and connections. In the various forms of dementia,
pathology is often restricted to specific anatomical locations, but also key lesions are
restricted to certain cell types (see Chapter 11). For example, neurofibrillary tangle
formation in AD may be restricted to large pyramidal neurons, sparing interneurons
(8–10). Even in areas associated with pathology, only a limited subset of the
vulnerable neurons may be affected, leaving some free of pathology. Because of this
cellular heterogeneity, it can be difficult to detect gene expression changes in a single
cell type due to dilution effects or the effects of pathology. Using laser capture
microdissection, it is, however, possible using a focused laser beam to cut through a
thin tissue section and isolate single cells of a given type, or small areas of tissue (11).
Using this methodology, specific cell groups such as neurons can be profiled
independently of any other surrounding cells, allowing a diseased neuron to be
compared with its unaffected neighbor (12). While there may be only a few
picograms of RNA in a single neuron, this is insufficient to use on a microarray as
normally an array requires microgram amounts of RNA. It is, however, possible to
use linear amplification methods to amplify endogenous RNA in a reliable and
unbiased manner from nanogram amounts of RNA for microarray analysis (13,14).
By combining methods such as laser capture microdissection with linear
amplification of RNA and microarray-based gene expression profiling, it is possible
140 Wilson and Morris

to determine gene expression profiles in isolated neurons or glia in health and disease
(15). This has been achieved to some extent in dementia with expression profiling
having been reported in AD in particular. Perhaps of significance is the report by
Blalock and colleagues who studied AD cases and also individuals with mild
cognitive impairment (MCI) in order to identify genes expressed at the earliest
stages of dementia (16). This study analysing hippocampal gene expression
identified mRNA up-regulation in several pathways involved with oligodendrocyte
differentiation and lipid metabolism, though a down-regulation of energy
metabolism pathways was evident (16). Similarly, analysis of hippocampal CA1
gene expression in AD has been used to show that there is up-regulation of some of
the inflammatory signalling pathways in AD, possibly associated with reactive
gliosis (17). This latter study also showed decreased neurotrophin and apoptotic
signalling, possibly being associated with the loss of pyramidal neurones and
neurofibrillary tangle formation (17).
The primary results of a microarray experiment represent only the
beginning of a particular gene expression study. Standard levels of analysis
identify significantly altered levels of gene expression using relatively
conservative changes, often G2 standard deviations from mean of control
expression levels, making subtle changes in gene expression difficult to detect.
Using bioinformatics approaches, however, allows the detection of specific
pathways that are affected, frequently using clustering algorithms that place
differentially expressed genes into families and pathways based on gene ontology
terms. Since the results of a microarray analysis are at best semi-quantitative, all
microarray experiments require further validation, normally by semi-quantitative
real time PCR (Q-RT-PCR). It is therefore possible to accurately determine the
levels of gene expression for a specific gene identified using the microarray, by
comparison with an appropriate housekeeping gene. Q-RT-PCR systems now
exist which make it possible to determine the levels of expression for either a few
specific genes in several samples, or to determine expression levels of a hundred
or more genes in a single sample. By this route it is a relatively straightforward
task to validate the primary targets from a microarray analysis, but also to analyze
additional components of the pathway identified as being potentially changed on
the basis of bioinformatics analysis. Microarray analysis and Q-RT-PCR
therefore can determine differential gene expression in highly selected
tissue samples.
But what of the tissue to be used in the identification of specific markers for
dementia diagnosis? One approach will be the use of suitable animal models,
most notably transgenic models showing the pathological changes associated
with dementia, which allows considerable control over agonal and pre- and post-
mortem effects. The use of transgenic models offers the opportunity to look not
just at end stage disease as is often the case with human postmortem material, but
to look at early stage pathology and even animals before the development
of pathology. One caveat of such an approach would be the complexity of the
pathology in dementia involving amyloid deposition, tangle formation, gliosis,
Novel Diagnostic and Therapeutic Targets 141

vascular changes, and white matter pathology, which is difficult to accurately


reproduce in an animal model. The use of human material is therefore perhaps
preferable, though not without difficulties in analysis. Methods for the isolation of
high-quality intact RNA from postmortem brain for gene expression profiling are
well established (18,19). A major problem with postmortem material is, however,
the uncertainties over what happens to the expression of particular genes as a
result of premortem and postmortem changes which may impact upon the
interpretation of results. It has been known for several years that RNA quality and
the expression of certain genes is heavily affected by how an individual dies (20).
Recently this has been extended by comparing gene expression in normal
individuals who died suddenly with an absence of any premortem phase, with
individuals who had various lengths of premortem illness (21). The presence of
any significant premortem coma (often seen as a reduction in the pH of tissue) is
reflected in an increase in transcripts for certain stress response genes and in the
levels of transcription factors involved in the mediation of these processes, and a
decrease in the levels of mRNAs for energy metabolism transcripts and those
involved in proteolytic events (21). Previous studies on AD where a down-
regulation of energy metabolism genes was seen may be an example of this
(16,17). Considerable care is therefore required in not only obtaining high-quality
RNA, but also in ensuring that case and control material is carefully matched for
short postmortem delay and good agonal state. This, and particularly the latter,
can be difficult to achieve particularly with dementia cases that often die in
terminal coma due to bronchopneumonia, though with access to large tissue
banks, it is possible to obtain well-matched samples.
One possibility that may avoid the problems associated with postmortem
tissue or animal models may be to use peripheral tissues, and in this instance,
lymphocytes or fibroblasts may provide the most accessible source. Given that
there is a considerable genetic influence on the development of neuro-
degenerative diseases and particularly AD (see Chapter 4), it can be argued
that the underlying genetic changes that occur in the brain will also be evident in
the periphery if that gene is expressed in a similar manner. It is also apparent that
changes in the brain impact upon the periphery as there are anatomical
connections between the central nervous system (CNS) and the periphery, and
soluble factors (cytokines, trophic molecules) can egress from the brain and have
an effect. This can be seen in changes in gene expression in lymphocytes in
response to various forms of CNS insult (22). It should therefore be possible to
investigate, for example, gene expression in lymphocytes from patients with
vascular dementia to determine if there are differential gene expression changes
compared to, for example, elderly normal individuals.
The application of microarray-based gene expression profiling to disease
tissue or animal models has the potential therefore to identify the changes that
uniquely define dementia, and moreover, different forms of dementia. These
markers, alone or in combination, provide the basis for the development of tests
to identify dementia in its earliest stages, and to provide a specific diagnosis.
142 Wilson and Morris

Cases with MCI may be one particularly fruitful source of information since, if
the individuals die early in the disease course without developing dementia, these
cases may provide information relating to the gene expression changes associated
with the primary cause of disease allowing such therapies and markers to be
developed. By using proteomic methods to identify the protein products of these
gene changes in cerebrospinal fluid (CSF), plasma, etc., or by gene expression
profiling of lymphocytes, it may be possible to develop novel tests for rapid
diagnosis. If gene expression changes are confined to the CNS, then the
development of neuroimaging ligands utilizing these targets would be one route
to improving diagnosis. Altered gene expression also provides the foundation for
potential new therapeutic strategies based on specific biochemical pathways
affected in disease, and may afford opportunities for more rational disease-
based treatment.

PROTEOMICS
Proteomics, the analysis of the protein complement of a cell or tissue, represents
the natural extension to functional genomic studies where mRNA expression is
mapped. While the sequencing of the human genome has revealed 35,000–40,000
genes, the proteome is much larger and also more dynamic in nature presenting a
unique challenge. Alternative splicing of genes results in different isoforms of a
protein while post-translational modifications also lead to multiple gene products
from a single gene. Furthermore, proteins also differ physically due to amino acid
composition, for example, strongly basic, acidic, or hydrophobic, as well as
differences in protein folding that have an effect along with protein-protein
interactions. Given the complexity of the brain, and also where comparisons
between healthy and diseased tissue are involved, the data sets generated by
proteomic techniques can be extremely large. While microarray-based gene
expression profiling can provide a rapid means to identify potential candidates for
further analysis, it can be argued that changes in gene expression are not
necessarily reflected by changes in the respective protein (23). High-throughput
proteomic techniques are therefore ideal for the direct identification of
biomarkers of disease, not only in postmortem or biopsy tissue, but also in
serum, plasma, and CSF, making the discovery of novel diagnostics and
treatment markers more effective.
Currently, no single method in a single pass is capable of accurately
identifying all proteins expressed by a cell or tissue due to the enormous variation
in the physical and chemical properties of proteins. A standard approach in
proteomic methodologies, particularly for tissue samples, is therefore to use
fractionation methods to reduce complex samples and simplify analysis. Standard
methods include centrifugation in order to separate a cell or tissue sample into,
for example, cytoplasmic and mitochondrial fractions. Alternatively, the use of
various detergents, high pH, and chaotropes can be applied to samples to extract
insoluble proteins such as membrane proteins followed by chromatographic
Novel Diagnostic and Therapeutic Targets 143

separation, which can in itself be used directly on samples. With comparative


proteomic methods, removal of certain proteins is often practiced, and this is
frequently the case when analyzing body fluids, where, for example, the plasma
may have 10,000 different proteins and peptides with concentrations ranging
from fg/ml to mg/ml. Methods to remove the most abundant proteins in a fraction
which are not thought to contribute to proteome variation are often practiced,
such as removal of albumin or immunoglobulin from plasma or serum samples.
By a combination of these techniques, it is possible to generate a series of
fractions which are sufficiently refined to allow accurate analysis. This
combination of analytical techniques, applied to even a single sample, results
in generation of very large data sets, which now means that automation and
development of high-throughput methods is becoming increasingly important in
proteomic studies, as well as tools such as protein databases for rapid data
analysis. Since an ideal biomarker of disease will reflect the underlying
mechanism or pathology of a disease, the use of automated methods is becoming
common to detect these changes, particularly where proteomic methods are being
used to identify diagnostic targets.

2D Gel Electrophoresis
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most
commonly used technique in proteomics since it is a highly effective method for
simultaneously separating complex protein mixtures (24,25). It is the only
technique currently available which allows thousands of proteins to be resolved
with one format in a single experiment and remains unchallenged as such.
Proteins are firstly separated by charge [isoelectric point (pI)] using isoelectric
focusing, followed by separation by mass in the second-dimension gel, recent
advances allowing up to 10,000 individual spots to be distinguished on a single
large format gel (26).
First-dimension isoelectric focusing separates proteins according to their pI
(the pH at which a protein has no net charge) using immobilized pH gradients
composed of thin gel strips into which ampholytes (charged carrier molecules)
are incorporated. In an electric field, proteins migrate through the gel due to the
pH gradient effect created by the ampholytes and stop migrating once their
charge is net neutral. These strips are available in a variety of formats, from wide
pH ranges covering many pH units to narrow ranges which cover just one or
two pH units. While broad range strips are ideal for initial experiments, for
example, large scale sample screening, narrow range strips have the advantage of
allowing much greater resolution. A series of gels can be overlapped to provide
greater visualization of proteins within the same range as a broad range strip yet
with much greater resolution, improving image analysis and protein identification
(24,26). Despite the advances in technology, 2D-PAGE remains unsuitable for
highly acidic or basic proteins since a lack of good ampholytes means that
focusing below pH3 and above pH11 is poor. This tends to result in reduced
144 Wilson and Morris

separation of membrane-associated proteins, which are often very acidic, due to


reduced solubility in the absence of detergents, the presence of which would
interfere with the focusing step. Additional pre-separation methods are therefore
needed to solubilize membrane proteins prior to analysis.
Following first-dimension separation by pI, proteins are separated by
molecular weight in polyacrylamide gels containing SDS, an anionic detergent
which denatures proteins, converting them into essentially linear molecules and
imparting a net negative charge. When combined with a reducing agent such as
DTT, proteins are therefore separated exclusively by mass. The resulting protein
spot map can be visualized using a variety of stains. Silver and Coomassie are
relatively simple staining techniques requiring little specialist equipment and are
therefore the most frequently used methods. Silver staining has a higher sensitivity
than traditional radiolabeling or Coomassie Brilliant Blue staining, capable of
detecting 100 pg of protein per spot (27). Recently fluorescent dyes such as
SYPRO and CyDyes have been developed with sensitivity similar to silver stains.
A major advantage of these stains is their compatibility with MS since more
traditional methods often require destaining before proteins can be identified by
MS (28–30). Traditionally one of the biggest disadvantages of 2D-PAGE was the
poor reproducibility; however, the advent of fluorescent techniques has greatly
improved this, though silver and Coomassie stains are still routinely used in many
labs and provide a simple method for building proteome databases and reference
maps for various organisms or tissues (see http://ca.expasy.org/ch2d).
A new development in 2D-PAGE ideally suited to the identification of
protein biomarkers is fluorescence difference gel electrophoresis (DIGE). This
uses different fluorescent cyanine dyes (e.g., Cy2, Cy3, Cy5) to pre-label protein
samples prior to 2D electrophoresis. Each dye, since it fluoresces at a different
wavelength, can be visualized by scanning the 2D gel at dye-specific
wavelengths, and the images overlaid to give a combined image which can
be analyzed using various software packages (31,32). This technique allows
direct comparison between samples to show either the presence or absence of a
particular protein in test compared to control sample or differences in protein
abundance, thereby reducing the effect of gel-to-gel variation and therefore
improving reproducibility (33). Since multiple samples can be separated on a
single gel, an internal standard can be incorporated into all gels within an
experiment. Using this approach, studies have demonstrated that there is little
variation between multiple operators, and differentially expressed spots can be
easily identified and analyzed (33,34). Two labeling techniques are available for
DIGE studies, minimal and saturation labeling, which in both cases have
minimal effect on the charge of the protein and add approximately 600 Da to the
mass. These CyDyes have a greater sensitivity, being capable of detecting
0.1 ng albumin, and over a greater dynamic range, around 103–104 (35). This
makes the use of the dye labeling technique ideal for applications in which there
is a limited sample, for example, where analysis of a single cell type is required
Novel Diagnostic and Therapeutic Targets 145

and so can be coupled to sample acquisition methods such as laser capture


microdissection (36,37).
Several approaches in neuroscience have used traditional 2D gel-based
approaches, and in the majority of instances have utilized prefractionation of
samples prior to analysis. For example, drug treatment has been analyzed by
2D-PAGE to identify the effects of anti-depressive drugs on the brain proteome
(38). Proteins involved in neurogenesis, such as IGF-1 and HCNP, and synaptic
plasticity, such as Rab4a and HSP10, were induced by venlafaxine or
fluoxetine, defining pathways by which these compounds can effect long-term
changes in brain structure by treatment. The pathological effects of drug-
induced seizures on brain protein expression indicate changes in heat shock and
antioxidant proteins along with synaptic proteins (39,40), which complements
work using microarray gene expression profiling on the action of anti-epileptic
drugs (41). In dementia, 2D studies using silver staining of AD and control
brains have visualized over 1500 proteins (42), though this is considerably less
than can be achieved with fluorescence-based detection methods. Analysis of
the normal human CSF proteome has produced a list of over 480 proteins using
silver-stained 2D gels in several studies, but again these represent the most
abundant proteins (43,44). Variations in Apolipoprotein E in the CSF have,
however, been identified between patients with sporadic or variant CJD (45),
and between Alzheimer’s and schizophrenia patients (46). Several proteins have
been identified as significantly different in the CSF between AD and control,
including a number of glycoproteins, again including Apolipoprotein E, but also
Apolipoproteins J and A1, raising the possibility that these protein changes
simply represent the presence of neurodegeneration. Here liquid phase IEF was
used as a pre-fractionation step prior to 2D-PAGE (47). The identification of
these possible markers for AD has not, though, been validated in any large
cohorts, and therefore additional studies are required. The use of 2D-PAGE,
however, provides a basis for other proteomic methods to build on in dementia
biomarker identification.

Isotope-Coded Affinity Tagging


While 2D-PAGE is limited in its capacity to analyze membrane proteins, isotope
coded affinity tagging (ICAT) is a new methodology in comparative proteomic
analysis that is particularly suited to membrane protein analysis as the system is
compatible with strong detergents, making it the ideal complement to DIGE (48,49).
With ICAT, samples are labeled with either an isotopically light or heavy thiol
reactive reagent which also contains a biotin affinity tag. Both samples are then
mixed together and subjected to proteolytic digestion, typically by trypsin, and the
resulting peptide mixture is then fractionated by avidin affinity chromatography
which isolates only the ICAT labelled peptides. These are then identified by liquid
chromatography (LC) and tandem MS (48–50). The eight-dalton difference between
the light and heavy reagents (containing eight hydrogen or eight deuterium atoms
146 Wilson and Morris

respectively) is detected by MS, allowing direct comparison between test and control
sample. The first generation ICAT reagents have recently been improved by
incorporating an acid-cleavable linker, allowing removal of the biotin affinity tag
prior to MS but leaving the peptide isotopically labeled. This simplifies the analysis
so that greater numbers of peptides can be identified and quantified (51). ICAT
labeling has been employed to study protein changes induced in cultures of cortical
neurons by the chemotherapeutic agent campothecin and analyzed by LC/MS,
demonstrating changes in proteins involved in transcriptional regulation, protein
synthesis, and signal transduction (52). This method is suited to the study of
relatively insoluble proteins such as membrane proteins since these can be extracted
using strong ionic detergent prior to the labeling and digestion steps, creating
peptides which are also more soluble than whole proteins (48). Given that many
ligands for positron emission tomography (PET) and single photon emission
computed tomography (SPECT) are membrane receptors, the use of methods such as
ICAT have the potential to isolate novel targets for the development of new
neuroimaging ligands in dementia.

Multidimensional Protein Identification Technology


Since the proteome of even a single cell is highly complex, for example, a typical
human cell contains many thousand different proteins and protein isoforms, direct
analysis is virtually impossible, and further complicated since several times this
number of peptides is produced by trypsin digestion prior to MS. No single
separation technique can separate all proteins and so multidimensional techniques
are often required for proteome studies. Multidimensional protein identification
technology (MudPIT) is an automated technique combining LC and MS. This is
similar to ICAT, though MudPIT tends to use online separation techniques
whereby samples flow between the different components of the system
automatically (53). Various LC techniques in sequence (affinity, size exclusion,
reversed phase) allow separation of complex samples. The flowthrough from these
columns is directly fed into an electrospray ionization (ESI) MS in which the
microcapillary tube used for injection has a diameter as small as 1–2 mm allowing
flow rates as low as 5 nl/min (53,54), greatly reducing the amount of sample
required for analysis. MudPIT is particularly useful for membrane-associated
proteins, kinases, and transcription factors which are difficult to detect using
2D-PAGE due to their low abundance (55). A similar method has been used to
characterize the serum proteome following digestion with trypsin and
fractionation using strong cation exchange and reversed phase HPLC, with
identification using ion-trap MS (56). Nearly 500 proteins were directly identified,
demonstrating the use of MudPIT methods in identifying proteomic changes with
minimal sample handling but maximal throughput. Using first-dimension anion
exchange followed by reverse phase separation, tryptic peptides from a post-
synaptic density preparation were injected directly by electrospray into a
quadrupole time of flight mass spectrometer, identifying 492 proteins associated
Novel Diagnostic and Therapeutic Targets 147

with signal transduction, the synaptic scaffold, adaptor proteins, and cell-cell
adhesion molecules (57). While some of the proteins were associated with other
cell types such as glial fibrillary acidic protein (57), the use of affinity purification
may enhance the ability to accurately define only those proteins specifically
associated with certain cell or organelle fractions (58).
Perhaps of direct relevance to dementia is the identification of markers
which indicate changes within the CNS and which are amenable to analysis by,
for instance, neuroimaging. Combining methods such as comparative 2D-PAGE,
ICAT, and MuDPIT has the potential to identify either novel pathways indicating
disease mechanisms, or biomarkers that may be present in CSF. As with methods
analysing RNA, analysis of proteins is not, however, without its difficulties. For
human postmortem material, while there is some suggestion that it is possible to
identify phosphoproteins (59), analysis of animal material postmortem suggests
that primary signalling pathways, many which rely on specific phosphorylation
events, may rapidly alter postmortem (60). This may be particularly relevant to
human studies, as similar changes appear to happen, with rapid down-regulation
of signaling pathways postmortem (Wilson, unpublished). Many of these
methods though have the potential to be used in comparative approaches and
several are now being used to provide a preliminary analysis of dementia.

FLUID ANALYSIS
One major goal in the diagnosis of any particular form of dementia would be the
availability of a simple screening tool based on key feature of the biology of the
disease. CSF and plasma testing provides an obvious route to disease marker
identification that could be applied in a routine setting (see Chapter 3). For AD,
markers such as Ab determination in plasma have shown negative results, and
in CSF have yielded conflicting results (61). Identification of elevations in
hyperphosphorylated tau in CSF in AD has improved specificity (62,63), though
the paucity of any studies with neuropathological assessment and the wide
variation of Ab and tau levels with any individual patient perhaps makes the
interpretation of these findings premature (61,64–66). Newer markers (47) have
also not been validated and therefore new markers are required. While peripheral
markers have been suggested to occur in many neurodegenerative diseases, none
have so far proved robust (61). This has been due to the commonly held view that
while there is CNS disease, there is little impact upon the periphery due to presence
of the blood-brain barrier and the very specific cell groups affected in diseases such
as AD (67). This view has recently been challenged, with the finding that there are
marked changes in lymphocyte gene expression following CNS damage (22).
Furthermore, in many psychiatric diseases, there is expression of CNS proteins on
lymphocytes, and their expression mirrors that found in the CNS (68,69). It is
therefore possible that in diseases such as AD there are changes associated with
148 Wilson and Morris

CNS disease in the peripheral circulation which are particularly amenable to


proteomic and also genomic investigation, and especially by certain MS methods.

Mass Spectrometry
MS is the preferred method for the identification of proteins by providing
structural information such as peptide mass and amino acid sequence, as well as
information on protein modifications. The data obtained can then be used to
identify a protein by searching various databases. MS measures the mass-to-
charge ratio (m/z) of gaseous ions produced by accelerating an ionized particle, in
this case the protein or peptide, through a rarefied atmosphere to a detector. Often
peptide mass fingerprint analysis is used where the isolated protein to be analyzed
is digested enzymatically, to cleave the protein at specific bonds giving a
reproducible pattern of digestion. MS is then performed on the peptide mixture,
giving masses with high accuracy the mass fingerprint of the protein. This
fingerprint is then compared to databases containing theoretical protein cleavage
data producing a list of the closest matching proteins (70). Much of this has been
achieved by matrix-assisted laser desorption/ionization (MALDI) MS which can
be used not only to identify isolated proteins, but also to directly identify peptides
from tissue samples (71). Ultra high resolution methods such as FT-NMR-MS are
now being used which may allow the detection and characterization of low-
abundance peptides in body fluids (72).

Matrix-Assisted Laser Desorption/Ionization


Body fluids are extremely complex, and it has been estimated that human plasma
may contain well over 10,000 analytes, spanning a concentration range of 10
orders of magnitude (73). Methods are required that can not only detect these
numerous different analytes at the various concentrations, but can also
distinguish these different analytes. One method that has received wide use in
this respect is surface-enhanced laser desorption/ionization (SELDI) where, like
MALDI, a laser beam is used to desorb analyte ions from a solid into the gaseous
phase for analysis by MS (74,75). While a sample such as plasma or CSF would
contain many thousands of potential peptides and would generate an extremely
complex series/smear of peaks with MALDI, SELDI utilizes a solid phase
chromatographic surface on the MALDI target plate in order to reduce the
number of peptide peaks analyzed at any one time (76). For example, the plasma
sample is applied to a target which has a cationic affinity support which binds
only peptides with high affinity for that particular support. By use of a mild
ionization procedure, SELDI produces a limited series of mass peaks for each
affinity support, and by using targets with different affinity supports, it is therefore
possible to analyze different subsets of proteins to build up a picture of the
proteins present. Using this approach it is possible to analyze a given tissue, and
Novel Diagnostic and Therapeutic Targets 149

by comparing the peak profiles of the tissue, to identify peptides which differ
between, for instance, case and control.
One major drawback, however, is the use of a relatively mild ionization
procedure which limits the eventual mass resolution of the system. Furthermore,
this also provides only a mass/charge ratio for any peak which could potentially
correspond to several, if not hundreds, of possible peptide sequences. Peptide
identification is therefore difficult unless the peptide is identified in several
different runs, and frequently the specific peptide peak has to be isolated and
identified by more traditional methods such as chromatography. Direct separation
using LC (2D LC) and fractionation of samples (77) along with high-throughput
liquid-handling robotics are now though being coupled directly to MALDI and
ESI-MS to produce more powerful systems. Here it is possible to directly analyze
the individual protein peaks in the peptide profile using TOF/TOF MS and MSn,
and the identity of the peptide can be determined rapidly. There is therefore the
prospect that proteins and peptides identified in a complex mass spectrum as
being differentially expressed between cases and controls can be directly
identified. For example, Ab has been identified in the lens of Alzheimer’s patients
suggesting that the pathological features of the disease overlap between brain and
lens (78). The identification of ovarian and prostate cancer-associated biomarkers
have also demonstrated the usefulness of direct MS-based analysis of biological
fluids for rapid discovery of markers which have extremely high specificity and
sensitivity in a clinical setting (79,80).

Protein Microarrays
Protein microarrays are gaining in popularity as miniature ligand-binding assays
which can be used for complex protein samples since they allow detection and
also quantitation of proteins. With protein arrays, a frequent approach is where
antibodies are immobilized at a high density on a solid support such as a treated
glass microscope slide. When exposed, each individual antibody captures its
target protein from the sample. By arraying hundreds or thousands of antibodies
on a single slide, this method allows large-scale and high-throughput analysis
using small sample volumes and relatively low protein concentrations (81,82).
Like gene arrays, protein arrays are probed by direct labeling of the protein
sample with fluorescent dyes (e.g., Cy3 or Cy5) and the abundance of a protein
being related to the fluorescent intensity of the particular spot on the array (82).
One problem with this approach is low sensitivity, though new approaches
include multiplexed sandwich immunoassays with ultra-high femtomolar
sensitivity (83). A major problem with this approach is the relatively limited
availability of antibodies, and in particular monoclonal antibodies, which allow
arrays to be reproduced particularly on the large scale required in proteomics.
While antibody arrays are an obvious choice for protein detection microarrays, an
alternative is the use of recombinant antibody fragments or immunoglobulin
fragments expressed on the surface of bacteriophage which provides a rapid
150 Wilson and Morris

means of producing reagents (84). The generation of large-scale protein


microarrays based on antibody fragments is now being explored and it may
soon be possible to use this high-throughput approach and with the
reproducibility that derives from recombinant techniques (85,86).
Antibody microarrays, since they are perhaps more suited to analysis of
fluid samples, are now being used in focused clinical analysis. In neurology, these
arrays have been used to analyze cord serum of children with cerebral palsy using
an antibody array capable of detecting 78 cytokines, chemokines, and growth
factors. In this instance, 12 analytes were found to be higher in cases compared to
matched controls (87). These techniques have uses as tools in differential
diagnosis of disease, both clinically and in the research setting; for example,
panels of antibodies or peptides known to be differentially expressed in various
disorders can be used to identify specific patterns of changes in CSF, plasma, or
serum, providing a rapid and easy diagnostic test, often with very high sensitivity
(83). Alternatively, with a wide enough panel of antibodies coupled to the array
or by using liquid-based multianalyte analysis (88), it may be possible to directly
analyze fluids in order to detect differential protein expression for
diagnostic purposes.

CONCLUSIONS
The diagnosis of dementia can only be achieved by the skilled clinician using
various clinical tools and personal judgement to assess the individual patient.
Currently the definitive diagnosis of dementia, particularly in the early stages, is
not possible, and reagents and tools are required which will assist the clinician in
achieving this. Using a combination of methods should, however, allow
determination of the gene and protein expression patterns of key brain regions
in health and disease which can define not only the presence of dementia, but of a
specific form of dementia such as AD. Techniques such as those described here
are already becoming routine in the search for biomarkers that can be used to
predict and diagnose disease, and to produce highly specific diagnostic tests. This
will be particularly useful in dementia diagnosis where, for example, symptoms
tend to overlap in the various different common forms, and yet accurate diagnosis
is required if the most effective treatments are to be used. However, if biomarkers
are to be used as diagnostic tools, techniques are required which are not only
sensitive and reliable but also reproducible allowing for multi-center use.
Ultimately, the technologies described here will help to unravel both the genetic
and environmental factors that predispose and precipitate the development of
dementia. These global technologies should therefore be seen, not simply as a
rapid means of identifying biological changes associated with dementia, but as a
route to more traditional methods of cell biological analysis for establishing how
dementia develops, and how new diagnostic tools can be produced.
Novel Diagnostic and Therapeutic Targets 151

ACKNOWLEDGMENTS

Supported by grants from the Alzheimer’s Research Trust, Alzheimer’s Scotland,


Alzheimer’s Society, Commission of the European Communities, GE Healthcare,
Medical Research Council, and The Health Protection Agency.

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6
Quantitative and Functional Magnetic
Resonance Imaging Techniques

Giovanni B. Frisoni and Nicola Filippini


Laboratory of Epidemiology Neuroimaging and Telemedicine (LENITEM),
IRCCS San Giovanni di Dio FBF—The National Center for Research
and Care of Alzheimer’s Disease, Brescia, Italy

IMAGE ACQUISITION: TECHNICAL FEATURES


Structural Magnetic Resonance Imaging
Hydrogen atoms are at the core of the physics of magnetic resonance imaging
(MRI) used for clinical purposes: hydrogen nuclei are made by single protons
with spinning properties that give the ability to interact with surrounding
inhomogeneous magnetic fields. Each hydrogen nucleus produces its own
magnetic field whose strength and direction can be represented by a vector known
as magnetic dipole moment (MDM). The 1.5 Tesla scanner magnet commonly
used for MRI generates a magnetic field 30,000 times greater than earth’s that
both orients the spin of hydrogen nuclei in the direction of the main magnetic
field, and at the same time leads off the “precession” phase, that is, all hydrogen
nuclei precess at the same frequency, although not at the same phase. The scanner
includes a radio wave emitter that excites hydrogen nuclei by deflecting their spin
along the x-y plane. The radio frequency pulse applied enables hydrogen nuclei
to precess at the same phase. Hydrogen nuclei revert back to their original spin
once the radio wave emission is interrupted, and give back the absorbed energy as

157
158 Frisoni and Filippini

radio waves that are picked up by an antenna. The emitted radio wave signal is
then analyzed and processed.
The signal characteristics denote some of the properties of the emitting
atoms. Clinical MRI makes use of the signal emitted by hydrogen atoms, which
are largely represented by water molecules (99.9%) and also have a strong
resonant frequency (42.577 MHz). The MRI signal gives information about
proton density, i.e., water density, and two more peculiar features are used:
T1 and T2 relaxation times. T1, also known as “spin-lattice,” denotes the time
hydrogen atoms required to revert to their initial magnetic equilibrium state
before magnetization. T2, or “spin-spin,” denotes the time hydrogen atoms
required to revert to their original orientation (dephase). T1 and T2 amplitudes
denote the difference between the MDMs of different tissue in the z-axis and in
the x-y plane, respectively, and the energy exchange of hydrogen atoms with the
surrounding molecules. In the water molecule, energy exchange is low and T1
and T2 are long, while in fatty tissue and in protein rich tissue the opposite is true.
Relaxation times of some tissues are reported in Table 1.
Repetition and echo times (TR and TE) can be modified by the scanner
operator in order to obtain greater tissue contrast based on T1 and T2: a short TR
enhances T1 differences, while a long TE enhances T2 differences.
Brain imaging acquisition time is relatively slow, ranging between 10 and
30 minutes. The bone signal is weak—black—due to low proton mobility, while
water—having long T1 and T2—will show black in T1-weighted and white in
T2-weighted images. A number of CNS (central nervous system) diseases feature
increased brain tissue water content and show as T1-hypointense and
T2-hyperintense images. Gray and white matter can be sharply differentiated
on T1-weighted sequences. Due to its higher water and lower lipid content, the
gray matter is hypointense in T1 and hyperintense in T2 images relative to
the white matter.
MRI sequences that are usually applied in clinical practice are:
T1 (anatomic sequence), where the cerebrospinal fluid (CSF) is black, the gray
matter is dark gray, and the white matter is light gray; T2 (so-called
“myelographic” or “pathological” sequence), where the CSF is white, the gray
matter is light gray, and the white matter is dark gray; fluid-attenuated inversion

Table 1 T1 and T2 Values at 1.5 T


T1 (msec) T2 (msec)

Heart 870 57
Liver 250 44
Kidney 560 58
Fat 260 84
Gray matter 920 101
White matter 790 92
Source: From Ref. 1.
Quantitative and Functional MRI Techniques 159

recovery (FLAIR), similar to T2 images, but where the CSF is suppressed and set
to black, to allow better lesion contrast in proximity to CSF spaces; and proton
density (PD), where intensity is proportional to water content. Figure 1 shows the
above sequences of a 40- and a 70-year-old healthy person.

Functional Magnetic Resonance Imaging


Functional magnetic resonance imaging (fMRI) is a tool that, by exploiting the
principles of traditional MRI, allows mapping and study of brain function, i.e.,
“looking at the brain while it works.” Effects of blood oxygen on signal decaying
rate (T2*) were first reported in MRI images by Seiji Ogawa and colleagues in
1990 (2) who noted that cortical blood vessels became more visible as blood
oxygen was lowered. They understood this to be due to the creation of local
magnetic field inhomogeneities, and thus signal losses, from deoxyhemoglobin
and termed it the Blood Oxygenation Level-Dependent (BOLD) method. Robert
Turner, at the NIH, demonstrated that with ultra-fast echo-planar imaging, he was
able to observe the time course of these oxygenation changes while an animal
breathed an oxygen-deprived nitrogen atmosphere. Shortly thereafter, Kenneth
Kwong and colleagues (3) reported seeing similar changes in humans during
breath-holding. The use for fMRI of the BOLD signal is based on the assumption
that cortical activation produces physiological and MRI parameter changes:
increased blood flow, increased oxygen consumption, increased oxyhemoglobin,
and decreased deoxyhemoglobin level, all leading to T2* changes and MRI
signal detection.

Figure 1 Axial magnetic resonance images of a 40- (left) and 70-year-old healthy person
(right). The images do not show any abnormal findings. The only minimal enlargement of
subarachnoid and frontal ventricular spaces of the older person should be appreciated.
Abbreviations: FLAIR, fluid-attenuated inversion recovery; DP, proton density.
160 Frisoni and Filippini

In active areas, blood flow can rise by 30–50%, while oxygen extraction
increases by only 5%. This leads to both a local increase of oxyhemoglobin
concentration, which has diamagnetic properties, and a reduction of deoxyhemo-
globin, which has paramagnetic properties. Changes of the oxy/deoxyhemoglobin
ratio in brain tissue is considered the physical-chemical basis of fMRI. Indeed
Pauling and Coryell showed that the presence of paramagnetic substances in the
blood could act as vascular markers acting as a natural endogenous contrast agent
(4). As such, the BOLD signal is an indirect marker of brain activity as it does not
evaluate brain activity but hemodynamic changes. Some believe that BOLD signal
changes are generated more by synaptic than neuronal body activity (5); this implies
that the region(s) apparently active during fMRI experiments can be remote from the
true site of neuronal activation. Moreover, as synaptic activity can be either
excitatory or inhibitory, the interpretation of fMRI results may be much less
straightforward than is currently believed (6). Recently Logothetis and colleagues
have analyzed the relationship between BOLD signal and local neural activity by
simultaneously acquiring electrophysiological and fMRI data from monkeys (7–9),
finding that the BOLD signal does reflect a local increase of neural activity (10) due
to both excitatory and inhibitory interneurons (11).
Presently fMRI, based on oxygen consumption, and PET, based on glucose
consumption, are the main functional techniques used to evaluate brain activity.
Although positron emission tomography (PET) and fMRI have a high spatial
resolution, the temporal resolution is limited due to the slower hemodynamic
changes related to neuronal depolarization. This is the reason for the combined
approach of PET/fMRI (high spatial resolution) with EEG/MEG (high temporal
resolution). Comparative studies between fMRI and PET have shown good
agreement, although important differences have also been highlighted (12–14).
Combined measurements of BOLD signal, CBF, cerebral metabolic rate of
oxygen (CMRO2), and oxygen extraction ratio (OER) have been performed. Feng
and colleagues have shown that CBF, CMRO2, and OER changes reached their
maximum approximately one second earlier than the BOLD signal change (15).
Non-invasive methods have been developed to improve the specificity of fMRI
data; one of the most interesting approaches is the so-called arterial spin labeling
(ASL) method, which allows simultaneous measurements of BOLD and CBF
responses providing a direct measurement of perfusion. Disadvantages of ASL
are lower signal-to-noise ratio than BOLD contrast and longer TR (16). With the
ASL method, Obata and colleagues have shown discrepancies between BOLD
and flow dynamics in primary and supplementary motor areas (17), while Uludag
and colleagues have suggested that ASL measurements of CBF change may be a
more reliable marker of neural activity than BOLD (18).
The BOLD signal is divided into three stages (Fig. 2). The “initial negative
dip” (20) appears about one second after neuronal activation and consists of a
mild signal intensity decrease under baseline. It is believed to be due to the
sudden deoxyhaemoglobin increase, subsequent to a stimulus-driven increase in
oxygen consumption, that immediately follows neuronal activation, temporarily
Quantitative and Functional MRI Techniques 161

Figure 2 Schematic representation of the common features of the fMRI BOLD response
to neuronal stimulation. Source: From Ref. 19.

uncoupled with the circulatory response. The “positive BOLD response” that
follows is a marked increase of signal intensity due to increased blood flow and
decreased oxy-/deoxyhemoglobin ratio. In this phase, the signal peak is reached.
At the very beginning of the positive bold response a sudden increase of the
BOLD signal can often be appreciated (“overshoot”), presumably due to a slow
cerebral blood volume adjustment in the face of a swift increase in cerebral
blood flow (21). The same principle applies to the start of the third stage
(“undershoot”), where the end of the task is followed by an abrupt below
threshold decrease of cerebral blood flow and a persistently high cerebral blood
volume which takes longer to return back to baseline values.
In this section two experimental designs typically used in fMRI research will
be briefly considered: block design and event related design. The former lasts
between 20 and 30 seconds, where between six and nine seconds are needed to
reach the activity peak, and between eight and 20 seconds to return to the baseline
level; the latter is variable. A BOLD response based on a typical “block design” is
divided into a “stimulation period” alternated with a “control period” (or “resting
period”).
The fMRI signal believed to be proportional to neuronal activation is the
difference between signal in the active and that in the control condition.
Activation maps are obtained through subtraction of the rest from the active
image. Well-defined functions such as motor and sensory tasks give relatively
sharp fMRI signals (22), while emotions, for example, due to slow and variable
beginning and impossibility to die off quickly, are more problematic. Despite
such methodological problems, some researchers have been able to successfully
study complex functions such as bereavement and empathy (23,24) using a
different technique that is the “event-related design.” In fact, it is difficult to
measure some cognitive states, like emotions, or some tasks not well temporally
defined, such as those analyzed in a typical “oddball paradigm,” with a block
design technique (25). The event-related design is a technique to detect
hemodynamic responses to brief stimuli or events (26). Individual, single trial
162

Table 2 Levels of Increasing Technological Sophistication of Tools to Rate Structural and Functional Imaging Findings in Patients with
Cognitive Impairment

Regional atrophy Subcortical cerebrovascular disease

Medium Technical requirements Measure Expertise Measure Expertise

Visual Routine acquisition Visual rating scales: Scheltens’ MTL 1–3 days Visual rating scales: 1–2 weeks
rating atrophy score (14) ARWMC scale (15)
Linear measures: width of the 1–3 days
temporal horn (16)
Volumetry 3D T1 acquisition manual Volumetric measures: hippocampal and 2–4 weeks Volumetric measures: 1 day
or semiautomatic post- entorhinal cortex volumes (17,18) thresholding of
processing WMHs (19)
Compu- 3D T1 acquisition Prospective whole brain assessment: brain – None –
tational software for boundary shift integral (20)
neuro- computerized post-pro-
anatomy cessing serial scans
Expertise denotes the training time needed to obtain accurate measurements.
Abbreviations: ARWMC scale, Age-Related White Matter Changes scale; MTL, medial temporal lobe; WMHs, white matter hyperintensities.
Source: From Ref. 28.
Frisoni and Filippini
Quantitative and Functional MRI Techniques 163

events are measured rather than a temporally integrated signal (27), as happens
for the block design. The main advantage of the event-related design is that
accidents such as habituation, anticipation, and strategy effects are greatly
limited, thus enhancing the possibility to draw powerful inferences.

QUANTITATIVE MAGNETIC RESONANCE IMAGING TOOLS TO


RATE REGIONAL ATROPHY AND CEREBROVASCULAR DISEASE
A number of tools of varying technological sophistication have been developed
to rate the structural changes taking place in the brains of patients with
cognitive impairment, ranging from simple subjective rating scales to
sophisticated computerized algorithms. Which tool should be used in the
clinical practice is not obvious. The aim of this section is to review those tools
that the clinical neurologist might use in his/her routine clinical practice. Part of
its contents have been published in a consensus document of the Neuroimaging
Working Group of the European Alzheimer’s Disease Consortium (EADC)
(28). The EADC is an EU-funded association of 43 centers of excellence for the
research and care of Alzheimer’s disease and related disorders of 13 European
countries (29). The tools have been categorized based on increasing levels of
technological intensity and practical usability. The features assessed by the
reviewed tools are the two main morphostructural issues of relevance in the
clinical diagnosis of cognitive disorders, i.e., regional atrophy and subcortical
cerebrovascular disease (Table 2).

Figure 3 Magnetic resonance–based visual rating of medial temporal lobe atrophy.


Score: 0 (absent), 1 (minimal), 2 (mild), 3 (moderate), and 4 (severe). The small table in
the lower right corner of the figure reports the criteria for score assignment.
164 Frisoni and Filippini

Visual Rating
Regional Atrophy: Visual Rating Scales
MRI can directly visualise the hippocampus and other critical medial temporal
lobe (MTL) structures in substantial cytoarchitectonic detail. Scheltens and
colleagues (30) have developed a subjective visual rating scale to assess MTL
atrophy on plain MRI films (the subjective MTL atrophy score). T1 weighted
sequences are used and six coronal slices (slice thickness of 5 mm) parallel to the
brainstem axis are acquired from a midsagittal scout image, the first image being
acquired directly adjacent to the brainstem. The score is assigned based on visual
rating of the width of the choroid fissure, width of the temporal horn, and height
of the hippocampal formation (Fig. 3). In 41 patients with AD and 66 non-
demented controls, the subjective MTL atrophy score showed a correct
classification of 96%, comparing favourably with volumetry (93%) (31). In a
prospective study of 31 patients with minor cognitive impairment, the subjective
MTL atrophy score improved the predictive accuracy of age and delayed recall
score for AD at follow-up (32).
Subcortical Cerebrovascular Disease: Visual Rating Scales
The European Task Force on Age-Related White Matter Changes (33) has
developed the Age-Related White Matter Changes (ARWMC) scale (34). This is
a 4-point scale which rates white matter changes separately in five areas: frontal,
parieto-occipital, temporal, infratentorial/cerebellum, and “basal ganglia”
(striatum, globus pallidus, thalamus, internal/external capsule, and insula). The
first three areas are scored as (0) no lesions (including symmetrical, well-defined
caps or bands), (1) focal lesions, (2) beginning confluence of lesions, or (3)
diffuse involvement of the entire region, with or without involvement of U fibers.

Figure 4 Visual rating scales for subcortical cerebrovascular disease: the Age-Related
White Matter Changes (ARWMC) scale. Grade 1, focal lesions; grade 2, early beginning
confluent lesions; and grade 3, confluent lesions with diffuse involvement of a lobe.
Source: From Ref. 34.
Quantitative and Functional MRI Techniques 165

The infratentorial/cerebellum and basal ganglia are scored as (0) no lesions, (1)
only one focal lesion (O5 mm), (2) more than one focal lesion, or (3) confluent
lesions. The final result of the rating are five separate scores to grade subcortical
cerebrovascular disease in the different brain regions in each hemisphere (Fig. 4).
The interrater reliability was found to be moderate (kZ0.48). Figure 4 provides
instances of three patients of increasing severity.
One should not forget to detect lacunes, which can be recognized on MRI
scans as round areas with regular contours, usually larger than 5 mm, located
more often in the basal ganglia, and featuring hyperintensity in T2 and protonic
density and hypointensity in T1 sequences.

Volumetry
Regional Atrophy: Volumetric Measures
A T1-weighted 3D-technique is employed for MRI image acquisition
magnetization prepared rapid acquisition gradient recalled echo or spoiled
gradient (MP-RAGE or SPGR). After acquisition, the digital images need to be
reconstructed on coronal, 1–2 mm-thick slices. The hippocampus is then
manually traced on all the contiguous slices where it can be appreciated
(Fig. 5). In expert hands, the reliability is high, intraclass correlation coefficients
for hippocampal measurements being 0.95 for intrarater and 0.90 for interrater
variability (35).
Subcortical Cerebrovascular Disease: Thresholding of White
Matter Hyperintensities
Quantification of the volume of white matter hyperintensities (WMHs) based on
MRI can provide an objective measure of the severity of subcortical

Figure 5 Regional atrophy assessment: hippocampal volumetry. Instance of manual


tracing of the right hippocampus (black arrow) with simultaneous view of the traced
region of interest in the coronal, sagittal, and axial planes.
166 Frisoni and Filippini

Figure 6 Volumetry through thresholding of white matter hyperintensities as marker of


subcortical cerebrovascular disease. Automatic segmentation comprises histogram
representation of the pixel intensity distribution, Gaussian modeling of the pixel
distribution separately for normal and hyperintense white matter, and identification of
the optimal intensity cutoff to separate normal from hyperintense white matter pixels.

cerebrovascular disease. A number of semi-automated methods have been


developed, most based on the notion that pixels of normal white matter can be
accurately separated from those of hyperintense white matter. A conventional
spin-echo, double-echo T2, or FLAIR sequence in the axial orientation is used for
MRI acquisition. Digital information is generally transferred for processing and
analysis to a separate workstation. Measuring involves manual tracing followed
by automatic thresholding. Manual tracing is carried out of a crudely defined
region of interest (ROI) within the white matter that completely includes all the
hyperintense white matter (Fig. 6).
Intrarater and interrater reliabilities of this method are good (36). WMH
volume has been found to be correlated with other features believed to be
indicative of subcortical cerebrovascular disease (parkinsonism and depression)
and predictive of cognitive impairment in a group of 369 cognitively intact
community-dwelling older men (37).

Computational Neuroanatomy
Recently, advances in neuroscience and neuroimaging have led to an increasing
recognition that certain neuroanatomical structures may be affected preferentially
by particular diseases. Neurodegenerative brain diseases mark the brain with a
morphological “signature”; detecting this may be useful to enhance diagnosis,
particularly in diseases lacking in other diagnostic tools. Moreover, structural
changes provide markers to track the biological progression of disease. In 1993,
Quantitative and Functional MRI Techniques 167

despite a lack of support from clinical variables, interferon beta was approved by
the Food and Drug Administration based on data from MRI (38).
Recent developments in computer science may help detect early sensitive
and specific disease signatures. The new approaches are automated, avoiding
error-prone and labour-intensive manual measurements. Second, such algorithms
can offer unprecedented precision as some can detect brain volume differences of
0.5% between images from the same subject (39).
The effort to develop such algorithms has been referred to as computational
neuroanatomy (40).
The individual algorithms can be categorized into two broad classes:
algorithms devised to detect group differences at one point in time and algorithms
devised to detect prospective changes over time. The first category may be useful
to define disease-specific signatures. The second can be applied to one or more
individuals to track natural disease progression or as modified by treatment.
While most tools have been developed to compare groups, some are being
adapted to analyze individual cases, an issue of the greatest interest for the
practicing physician.
Computational anatomy algorithms generally involve some or all of the
following steps: (1) brain extraction (brain is separated from non-brain voxels),
(2) tissue segmentation (voxels representing gray and white matter and cerebro-
spinal fluid are separated based on intensity values), (3) spatial normalization
(also called registration; the voxels of interest are matched to a template or an
earlier scan from the same individual), and (4) statistical comparison of different
subject groups or points in time. The pivotal step of all methods is registration.
Here, cross-sectional methods match images of interest to a reference stereotactic
template (a typical brain or a typical hippocampus, etc.) or vice versa, while
prospective methods match sequential images of the same patients taken at
different times.
Registration strategies differ in scope (i.e., analysis of the whole brain or
preselected regions-of-interest) and mathematical approach (accounting for
global or local variability of the brain’s size and shape). Some cross-sectional
methods that account for global variability are completely automated (such as
voxel-based morphometry based on statistical parametric mapping by Ashburner
and Friston; see Chapter 7) (41), while those that account for local variability
often require manually positioned landmarks to precisely match the image to the
template (such as cortical pattern matching) (42). Longitudinal methods use the
complexity of each individual’s brain structure to align accurately an individual’s
serial images [such as the brain-boundary shift integral (BBSI) algorithm] (43).
To perform well, all methods need high spatial resolution and clear
differentiation between tissue types. Usually, 3D high-resolution T1-weighted
MR images [spoiled gradient (SPGR) or magnetization prepared rapid
acquisition gradient recalled echo (MP-RAGE)] acquired with conventional
1.5T MR scanners and 1 mm3 voxels (ideally isotropic) across the cranium
provide sufficient detail and contrast.
168 Frisoni and Filippini

Cross-Sectional Atrophy Assessment


Voxel-based morphometry: Ashburner & Friston’s method (41)
requires that registered gray matter images are smoothed with an 8–12 mm
filter. This leads to normally distributed data and allows the use of statistical
parametric tools. The statistical approach of the A&F method (SPM) is based on
the general linear model, and identifies regions of tissue with increased or
decreased density or concentration that are significantly related to the effects
under study. Ideally, the threshold for significance should be set at p!0.05
corrected for multiple comparisons, but when there is a prior hypothesis of the
expected effect a more liberal threshold of p!0.001 uncorrected has also been
applied. However, like every statistical test, the larger the effect size and group
size, the higher the sensitivity of the method for identifying differences.
A protocol to carry out voxel-based morphometry based on SPM in patients
with neurodegenerative disorders has been developed (“optimized VBM”) which
requires images to be registered onto a “customized template,” i.e., an average
image of the cases under study (44). The optimized protocol includes (1)
generation of customized template, (2) generation of customized prior probability
maps, and (3) main VBM steps: normalization of the original MRI images,
segmentation of normalized images, cleaning of gray matter images, modulation
of gray matter images, and smoothing of modulated images. Customized prior
probability maps are computed by generating a customized gray matter template
through segmenting the original images into gray matter, white matter, and
cerebrospinal fluid, cleaning the GM, normalizing the cleaned GM images to the

Figure 7 Gray matter changes in Alzheimer’s disease assessed with Ashburner and
Friston’s voxel-based morphometry. Black arrows indicate voxels of decreased gray
matter density (p!.0001 uncorrected for multiple comparisons) in the amygdalar/hippo-
campal complex and temporal cortex in 29 mild to moderate (upper row, mean MMSE
21 G4) and three very mild (lower row, MMSE 26 and 27) AD subjects compared with 26
nondemented controls. Source: From Ref. 45.
Quantitative and Functional MRI Techniques 169

customized GM template to determine the normalization parameters, smoothing


the cleaned images, applying the normalization parameters to the smoothed
images, and averaging.
With voxel-based morphometry, hippocampal atrophy has been visualized
in Alzheimer’s disease (Fig. 7) as well as reduced volume in the posterior
cingulate gyrus and adjacent precuneus, and the temporoparietal association
cortex (45). In a small group of three very mild AD patients with Mini Mental
State Examination (MMSE) scores of 26 and 27, atrophy has been shown to affect
the amygdalar/hippocampal complex bilaterally, indicating that the technique is
sensitive also in small groups and in the earliest stages of the disease (Fig. 7).
Cortical pattern matching: This is a sensitive approach that measures
the topologic variability of the cortex (46–49). The approach consists of cortical
flattening and sulcal matching that aims to obtain an average cortical model for a
group of subjects. All MRI scans are first aligned to a standardized 3D coordinate
space. From each individual’s MRI scan, a 3D cortical surface model is extracted,
consisting of a network of discrete triangular tiles, and some sulcal/gyral
landmarks are identified on the cortical model, which is then flattened. Then, sulcal
features are co-registered to a template of sulcal curves, derived from a large group
of normal subjects, with a warping technique. The warped images are averaged,
and measures of gray matter density can be analyzed with statistical tools similar to
those used by A&F’s voxel-based morphometry. This technique can be executed
on high-end desktop machines such as the Macintosh G4, as well as Silicon
Graphics Interface or Sun workstations running UNIX. These algorithms are often
used in client-server mode, connecting to a supercomputer for very large-scale
analyzes (50).
Cortical pattern matching allows mapping of changes in cortical gray
matter density or thickness with great accuracy. Figure 8 shows the atrophic
changes found in a group of 26 mild to moderate AD patients (51).

Prospective Atrophy Assessment: Whole Brain Assessment


with the Brain Boundary Shift Integral
Serial scans within the same subject have the advantage that the wide inter-
individual variability of brain morphology is not an issue, and comparing
pre-post images of the same subject(s) carries much less error than comparing a
case to controls. Information on prospective global changes can be obtained by
rigidly matching serial scans and subtracting the superimposed images. The
difference reflects the volume of brain tissue lost or gained [e.g., BBSI (43)].
The rate of atrophy in a group of 18 AD patients was significantly greater than in
31 controls (Fig. 8: 2.78% vs. 0.24% per year with no overlap between the
groups) (39). Moreover, the rate of global cerebral volume loss was strongly
correlated with rate of cognitive change measured with the MMSE in 29 AD
patients (52).
170 Frisoni and Filippini

Figure 8 Rate of global brain atrophy computed with the BBSI method in 18 Alzheimer’s
disease patients and 31 controls. Tissue loss was 2.78% in patients and 0.24% per year in
controls. Note the lack of overlap between the groups. Source: From Ref. 39.

Information on local changes can be obtained by using non-linear


registration, which permits compression or expansion of each voxel to obtain a
precise registration (voxel compression method). The resulting deformation
fields provide a map [voxel compression map (VCM)] of the amount of
compression or expansion applied at each voxel. This reflects the amount of brain
tissue or CSF lost or gained over time in the interval between scans. Typical
patterns of change in different conditions (for example, normal aging vs.
Alzheimer’s disease) can be tapped by registering and averaging individual
VCMs and comparing the resulting averages. The method allowed the detection
of loss of brain tissue in asymptomatic individuals carrying an autosomal dominant
mutation known to cause AD more than two years before the appearance of
symptoms (53,54).
The BBSI technique might be particularly useful in detecting disease in
asymptomatic subjects at high risk of developing AD. Prospective measurements
of brain atrophy have become a very relevant issue with the advent of drugs that
might alter the natural history of AD by slowing its progression. Rates of brain
atrophy measured from serial registered MRI are being used as a surrogate
outcome of drug effectiveness in clinical trials.

QUANTITATIVE MAGNETIC RESONANCE IMAGING


FOR THE CLINICAL DIAGNOSIS
The traditional approach for imaging in the diagnostic pathway of dementia holds
that structural imaging [computed tomography (CT) or MRI] is useful to exclude
potentially treatable intracranial causes of cognitive deterioration (the so called
pseudodementias). While the increasing expertise of professionals has led to a
Quantitative and Functional MRI Techniques 171

decrease in the prevalence of pseudodementias—such that reversible causes of


cognitive impairment are recognized before the “dementia” label is attached to
the patient (55)—current guidelines still advise at least one structural imaging
exam during the course of any dementing disorder (56). The justification of this
approach is that no clinical rule can identify 100% of cases with potentially
treatable intracranial causes (57).
The enhanced anatomical detail allowed by recent MRI scanners has
permitted the delineation of atrophic patterns for different forms of dementia.
This has led to a shift from the use of imaging as an exclusion exam to an
inclusionary approach (58). Such an approach is informative in a much larger
proportion of cases than the negative approach. However, it should be highlighted
that in the diagnostic workup of patients with dementia, imaging exams fall in a
path where they have been preceded by history taking, physical, and neurological
examination, neuropsychological screening, laboratory exams, and often detailed
neuropsychological testing, such that at the time when imaging is acquired the
physician has already collected information that may have provided more or less
strong diagnostic clues. Thus, the clinically pregnant question is: what is the
diagnostic added value of imaging?
Scheltens and colleagues (58) have computed that the weighted (corrected
for number of people in the study) sensitivity and specificity for detection of mild
to moderate Alzheimer’s disease using visual assessment of medial temporal lobe

Figure 9 Post-test probability of Alzheimer’s disease with visual assessment of medial


temporal atrophy (sensitivity 85% and specificity 88%) for any given pre-test probability.
The upper curve shows the incremental diagnostic gain from a positive result
(i.e., presence of atrophy) and the lower curve shows that from a negative result (i.e.,
absence of atrophy). In the case of a pre-test probability of Alzheimer’s disease of 0.50 (the
same as tossing a coin), a positive result adds 0.27 to give a post-test probability of 0.87
and a negative result lowers the post-test probability to 0.15. Source: From Ref. 58.
172 Frisoni and Filippini

atrophy compared with controls can be calculated as 85% and 88%. The practical
implications of the results are given in Fig. 9, showing the incremental diagnostic
gain of a positive and negative visual assessment of medial temporal lobe atrophy
at any given pre-test chance (before taking MRI). For instance, in the case of a
pre-test probability of Alzheimer’s disease in a clinical setting of 0.50 (the same
as tossing a coin), a positive result adds 0.27 to give a post-test probability of 0.87
and a negative result lowers the post-test probability to 0.15.
Obviously, the higher the pre-test probability of Alzheimer’s disease, the
smaller the incremental gain obtained through neuroimaging exams. It should be
noted that to date naturalistic studies assessing the incremental value of imaging
exams—as well as studies assessing the incremental diagnostic value of any other
test used for the differential diagnosis of the dementias—are lacking. Thus,
strictly speaking, the inclusionary approach of imaging is poorly supported by
evidence. However, this fate is unfortunately shared by most radiologic and
non-radiologic diagnostic exams currently used in clinical medicine (59).

The Dementias
Alzheimer’s Disease
The preceding sections have already clearly outlined what structural MRI can
show in patients with Alzheimer’s disease, i.e., atrophy of medial temporal
structures (hippocampus, entorhinal cortex, and amygdala), of the parietotem-
poral region, and posterior cingulate. With ordinary analysis tools, i.e., the MRI
film and the physician’s own eyeballs and connected visual cortex, atrophy can
more easily be appreciated in medial temporal structures, while that in the
parietotemporal and posterior cingulate cortex is more difficult to discriminate
from age-associated changes.
Figure 10 shows how regional atrophy can be studied in a patient with AD
using assessment tools of increasing technological complexity. The anatomy of
the medial temporal lobe is such that hippocampal atrophy can be appreciated
even with CT. A CT-based marker of hippocampal atrophy (the radial width of
the temporal horn) has been shown to be sensitive and specific to AD in both
clinical and pathological series (60,61). Values greater than 5.3 mm denote
clinically significant medial temporal atrophy (45). The patient shown in
Figure 10 had a CT-based radial width of the temporal horn of 6.9 mm (above
the 99th percentile of the age-specific distribution), scored 2/4 to the right and
3/4 to the left on the MRI-based visual rating scale for medial temporal atrophy
(normal values 0 and 1), had the smallest (between right and left) hippocampal
volume of 91.5 (below the 2nd percentile of the age-specific distribution), and a
single-case analysis with voxel-based morphometry (age-adjusted at p!.05
uncorrected vs. 173 nondemented controls between 40 and 80 years of age)
showed regional gray matter loss bilaterally in the medial temporal and posterior
cingulate-precuneus regions.
Quantitative and Functional MRI Techniques 173

Figure 10 Regional atrophy in a 74-year-old patient with mild Alzheimer’s disease


(MMSEZ23/30) studied using assessment tools of increasing technological complexity.
(A) Computed tomography (CT)–based radial width of the temporal horn: 6.9 mm, above
the 99th percentile of the age-specific distribution; (B) conventional magnetic resonance
imaging (MRI)–based visual rating scale for medial temporal atrophy: 3/4 to the right and
left, normal values 0 and 1; (C) digital MRI–based hippocampal volumetry: smallest
(between right and left) normalized hippocampal volume of 91.5 (below the 2nd percentile
of the age-specific distribution); (D) digital MRI-based single-case voxel-based
morphometry: regional gray matter loss bilaterally in the medial temporal and posterior
cingulate-precuneus regions.

In a relatively large series of 55 AD patients and 42 controls, hippocampal


volumetry has shown a classification sensitivity and specificity of 94% and 90%,
respectively (62). Small hippocampal volume has been found to be predictive of
subsequent conversion to AD in 80 patients with amnestic mild cognitive
impairment (MCI) independently of neuropsychological tests, Apolipoprotein E
genotype, and cerebrovascular comorbidity (63). Of the 13 MCI patients with
hippocampal volume 2.5 SDs below the age-specific mean, six (46%) converted
to AD within the following six years, while of the 54 with hippocampal volume
between 2.5 SDs and the age-specific mean, converters were 19 (35%), and of the
13 with hippocampal volume above the mean, only two (15%) converted (63).

Frontotemporal Lobar Degeneration


This is a syndrome including—according to recent clinical criteria (64)—frontal
dementia, progressive aphasia, and semantic dementia. The frontal variant of
frontotemporal lobar degeneration (FTLD) is mainly characterized by marked
alterations of behavior and personality and by attention and executive function
impairment (65), while semantic dementia is characterized by progressive
deterioration of semantic memory with relative sparing of other cognitive func-
tions (66), and progressive aphasia is a slowly progressive impairment of speech
production (67). The most frequent etiology is nonspecific cortical degeneration,
i.e., neuronal loss associated with gliosis of the superficial cortical layers in the
frontal and temporal lobes [“dementia lacking distinctive histology” described by
174 Frisoni and Filippini

Figure 11 Frontal atrophy in a 69-year-old patient with frontotemporal lobar


degeneration—frontal dementia type of mild severity (MMSE 21/30, clinical dementia
rating 1) studied using assessment tools of increasing technological sophistication.
(A) Conventional magnetic resonance imaging (MRI)–based visual assessment: mild to
moderate enlargement of cortical sulci in the anterior frontal and temporal regions;
(B) digital MRI-based lobar volumetry: normalized frontal gray matter volumes of 142
(below the 99th percentile of the age- and gender-specific distribution—open and filled
dots denote women and men); (C) digital MRI-based single-case voxel-based
morphometry: regional gray matter loss in anterior frontal and temporal regions (anterior
cingulate, orbitomesial, dorsolateral frontal, and temporopolar areas).

Knopman and colleagues (68)]. Visual assessment of traditional structural imaging


allows the rater to appreciate mild to moderate enlargement of cortical sulci in the
anterior frontal and temporal regions (Fig. 11). This can be substantiated with a
digital analysis of frontal lobar gray matter volumes, showing marked tissue loss on
both sides, more marked to the left (Fig. 11). FDG PET also showed typical and
distinctive patterns of glucose metabolism in FTD (69,70).
Recently Miller and colleagues have suggested a different pattern of brain
atrophy to distinguish the FTLD variants (frontal dementia, progressive aphasia,
and semantic dementia) based on voxel-based morphometry analysis. Frontal and
semantic dementias seemed to be mainly characterized by common regions of
atrophy located in the ventromedial frontal cortex, the posterior orbital frontal
regions bilaterally, the insula, and the left anterior cingulate cortex. Specific
regions of atrophy in frontal but not semantic dementia affected the right
dorsolateral frontal cortex and the left premotor cortex, while semanttic dementia
patients showed atrophy in the anterior temporal cortex and the amygdala/anterior
hippocampal region bilaterally (71). In the progressive aphasia group voxel-based
morphometry showed a predominance of frontal atrophy (67), and reduced gray
matter in the left superior temporal and inferior parietal regions (71).

Dementia with Lewy Bodies


The Newcastle group has investigated the accuracy of visual assessment of medial
temporal lobe atrophy in the differential diagnosis between dementia with Lewy
Quantitative and Functional MRI Techniques 175

bodies and AD (72). Barber and colleagues (72) have shown that clinically
significant atrophy affected 100% of AD, 62% of Lewy body dementia patients,
and 4% of controls. Thus, the absence of medial temporal atrophy had 100%
specificity for dementia with Lewy bodies versus AD, while the presence of
atrophy had a sensitivity of only 38% versus controls. These data indicate that in
patients where the differential diagnosis lies between dementia with Lewy bodies
and AD, the absence of atrophy is strongly suggestive of the former. Unfortunately,
this happens in a minority (less than half) of Lewy body dementia patients.
Structural and functional imaging has allowed the elucidation of the
pathophysiology of visual hallucinations in dementia with Lewy bodies. Burton
and colleagues (73) have shown that hallucinations in dementia with Lewy
bodies are not due to occipital atrophy: unexpectedly, the occipital lobe volume
of Lewy body dementia patients with hallucinations is greater than that of
patients without hallucinations, and this holds true also in AD. Notably, despite
structural integrity, functional imaging techniques (see Chapters 7 and 8) have
shown marked occipital hypofunction in Lewy body dementia patients (74).

Other Forms of Dementia with Parkinsonism


The sections on AD, frontotemporal degeneration, and dementia with Lewy
bodies have suggested that profiles of atrophy in multiple cerebral regions can be
used to differentiate the degenerative dementias and that atrophy in a single
region is generally poorly informative.
Atrophy in a single region is poorly informative in the case of dementia in
Parkinson’s disease, (75–77) where hippocampal atrophy has been shown to be of
a degree similar to that found in AD and is therefore of no use in discriminating
between the two conditions (35).

Figure 12 The “pulvinar sign” in a patient with the new variant of Creutzfeldt–Jakob
disease. (A) T2-weighted, (B) proton density-weighted, and (C) FLAIR magnetic
resonance scans. Source: From Refs. 80, 82.
176 Frisoni and Filippini

Creutzfeldt–Jakob Disease
About 7 in 10 patients with sporadic Creutzfeldt–Jakob disease show hyperintense
lesions in the caudate and putamen and mild thalamic hyperintensities on
T2-weighted images (78,79). The low sensitivity and specificity of these findings
make them of little use in differential diagnosis. However, the new variant of
Creutzfeldt–Jakob disease (due to bovine transmission) shows mild caudate and
putaminal findings and marked thalamic hyperintensities. These are located at the
level of the pulvinar nucleus (“pulvinar sign”) and represent a moderately sensitive
(80%) but highly specific (100%) marker (Fig. 12) (80,81). The sign can be
appreciated particularly well with FLAIR sequences (82). The pulvinar sign has
been included in a suggestion for clinical criteria for the diagnosis of the disease
(81) and appears to be the diagnostically most useful instrumental sign in that the
EEG is often nonspecific and 14-3-3 protein CSF levels have low sensitivity (81).

Vascular Dementia
The clinical diagnosis of vascular dementia is a thorny issue for the lack of a
pathological standard. Consequently, although structural imaging is key to directly
appreciate the vascular lesions, its role and usefulness are still hotly debated.
NINDS-AIREN criteria (83) allow four forms of vascular dementia: (1)
multiple infarcts due to embolism or large vessel disease, (2) strategic infarcts
(due to embolism, large or small vessel disease), (3) multiple lacunes in the basal

Figure 13 Vascular dementia due to (A) multiple infarcts due to large vessel disease but
no small vessel disease; (B) multiple infarcts (1: temporal, 2: striatal) associated with small
vessel disease (3: watershed, 4: white matter lesions of the periventricular and deep
subcortical white matter); (C) multiple lacunes in the basal ganglia; and (D) extensive
lesions of the periventricular white matter associated with one deep frontal lacune in the
white matter.
Quantitative and Functional MRI Techniques 177

ganglia and white matter (generally due to small vessel disease), and (4)
extensive lesions of the periventricular white matter (due to small vessel disease).
Figure 13 shows cases of vascular dementia with multiple infarcts due to large
vessel disease but no small vessel disease, multiple infarcts associated with
small vessel disease, and extensive lesions of the periventricular white matter.
While the number, volume, and site necessary for multiple infarcts to give
vascular dementia is undetermined, strategic infarcts associated with dementia
(or—better—cognitive impairment in multiple domains) have been described in
the left angular gyrus, mono- or bilaterally in the thalamus, and bilaterally in the
globus pallidus (Fig. 14).
The diagnosis of vascular dementia due to extensive lesions of the
periventricular white matter and its differentiation from AD is particularly
difficult, although it is clinically relevant due to the poorer prognosis of the
former (85). Specific diagnostic criteria for subcortical vascular dementia have
been developed where imaging plays a major role, but these await validation (86).
Fazekas and colleagues (87,88) have shown that small punctate lesions of the
white matter (!5 mm, round, and with regular margins), when not associated
with confluent lesions, may not be due to vascular changes (87) and do not
progress over time (88). On the contrary, larger (O5 mm) lesions with irregular
margins (so called “confluent” as they appear to originate from the confluence of
smaller lesions) are due to vascular changes and progress over time (Fig. 15) (88).

Figure 14 Vascular dementia following strategic lesion. (A) Subcortical lesion in the left
angular gyrus (proton density-weighted image). (B) Unilateral (upper, FLAIR image) and
bilateral (lower, T2-weighted image) lesions following occlusion of the thalamic
paramedian artery. (C) Bilateral infarcts in the globus pallidus (T2 weighted image).
Source: From Ref. 84.
178 Frisoni and Filippini

Figure 15 Increasing severity of white matter hyperintensities on T2-weighted magnetic


resonance images. (A) Small punctate lesions of the white matter (!5 mm, round, and
with regular margins—arrows) when not associated with confluent lesions may not be due
to vascular changes and do not progress over time. On the contrary, larger (O5 mm)
lesions with irregular margins (early and late confluent—B and C) are due to vascular
changes and progress over time. Source: From Ref. 88.

Structural Imaging in Mild Cognitive Impairment


The concept of MCI is aimed at detecting patients in the transition from normality
to Alzheimer’s dementia. The contribution of structural imaging to the diagnosis
of MCI should be placed into the more general context of the search for the
signature of AD with instrumental exams.
MCI patients have a higher incidence of dementia than cognitively intact
persons of similar age (between 6% and 25% vs. 0.2–2.3% per year) (89).
Pathological and epidemiological evidence indicate that the vast majority of MCI
patients developing dementia have AD, but a relevant proportion of MCI patients
(as high as 60%) (90) remain stable and never go on to develop dementia (91). The
practical consequence of these observations is that the diagnosis of MCI is
clinically unhelpful in certain circumstances, as many MCI patients have
Alzheimer’s disease but many do not. Pathological and clinical data indicate
that some biological indicators of Alzheimer’s disease (the neurobiological
“signature” or “fingerprint,” including imaging indicators) might be used to
distinguish those MCI patients who will progress (i.e., those who already have
Alzheimer’s) from those who will not (i.e., those who do not have Alzheimer’s).
Biological indicators are hippocampal atrophy (due to early plaque and tangle
deposition in the medial temporal lobe), high concentrations of tau protein in the
CSF (due to neuronal/axonal damage following neurofibrillary tangle deposition),
and functional defects in the temporoparietal and posterior cingulate cortex (due
to deafferentation from medial temporal damage) (see Chapter 8). Indeed, when
compared to non-progressors, MCI patients who will progress to dementia
feature lower hippocampal volume measured through high-resolution structural
Quantitative and Functional MRI Techniques 179

MRI, (63,92) high levels of tau protein in the CSF, and perfusion and metabolic
defects [PET and single photon emission computed tomography (SPECT)] (93–96).
Jack et al. have tested the hypothesis that MRI-based measurements of
hippocampal volume are related to the risk of future conversion to Alzheimer’s
disease in older patients with MCI (63). Eighty consecutive patients who met
criteria for the diagnosis of MCI were recruited from the Mayo Clinic
Alzheimer’s Disease Center/Alzheimer’s Disease Patient Registry. At entry
enrolled subjects underwent an MRI examination, in order to obtain volumes of
both hippocampi, and for a period of time were also followed longitudinally, on
average 32.6 months, with approximately annual clinical/cognitive assessments.
During the period of observation 27 of the 80 MCI patients became demented,
and hippocampal atrophy at baseline was associated with crossover from MCI to
AD. The conclusions of Jack and colleagues were that MCI patients who will
progress to dementia feature lower hippocampal volume as measured through
high-resolution structural MRI.
Whether imaging or non-imaging markers are more promising for future use
in a routine clinical setting is still unknown. The accuracy of single markers varies
between 40% and 100%, (63,92–94,97,98) but studies are poorly comparable for
the doubtful reliability of current criteria when used in different memory clinics,
the heterogeneity of MCI patients enrolled, and the small study groups.
Voxel-based morphometry analysis in MCI has shown a significant agreement
in showing that patients had highly significant gray matter loss predominantly
affecting the medial temporal lobe, the hippocampal regions, the thalamus, the
cingulate gyrus, and extending also into the temporal neocortex (99–102).
A few studies have tried to include use of more than one Alzheimer’s disease
biomarker to discriminate MCI progressors from non-progressors. Okamura and
colleagues (103) studied 30 MCI patients, 22 of whom did and eight who did not
progress to cognitive impairment in the following three years, and found that a
high ratio between tau in the CSF and posterior cingulate perfusion on SPECT
could identify progressors with sensitivity of 89% and specificity of 90%. El Fakhri
and colleagues (104) studied 17 healthy controls, 56 non-demented patients with
memory problems who did not develop AD during three to five years of follow-up,
and 27 nondemented patients with memory problems who developed AD during
follow-up. Combining information coming from SPECT and structural MRI at
baseline allowed the correct classification of 94% of patients. If these early
findings can be replicated in larger and methodologically rigorous studies, the
possibility to diagnose Alzheimer’s disease before dementia has developed might
become a reality using functional and structural tools.

MICROSTRUCTURAL IMAGING: DIFFUSION AND


MAGNETIZATION TRANSFER IMAGING
In AD, the detection of gray matter loss in T1-weighted images has allowed a
better understanding of the biological basis of the clinical signs and symptoms, an
180 Frisoni and Filippini

ability to monitor disease progression, and an assessment of the effect of the


disease-modifying drugs presently under study. However, gray matter loss on
T1-weighted images has limited ability to capture the whole range of
morphostructural changes associated with the neurodegeneration of AD. First,
tissue shrinkage on T1-weighted images cannot discriminate neuronal from glial
and axonal loss as well as neuronal loss from age-associated shrinkage of healthy
neurons. Second, T1-weighted images cannot appreciate the white matter
damage that might arise in AD from neurofilament tau pathology. Techniques to
probe into the finer structure of the brain have recently been developed such as
diffusion tensor and magnetization transfer imaging that are providing
increasingly valuable information in elucidating the pathophysiology of AD.
Diffusion tensor imaging is based on the physical properties of moving
water protons. Motion is higher where protons have no constraints (e.g., in the
CSF) and lower where protons are confined within organized tissues such as the
intracellular matrix or the axonal cytoplasm. MRI can detect and quantify such
motion through the apparent diffusion coefficient (ADC) (Table 3). In the white
matter, not only is the ADC lower than in the CSF, but proton motion is highly
oriented in the direction of the axonal fiber (i.e., anisotropic), and the direction of
the motion can be quantified through the fractional anisotropy index (FA).
Axonal loss and demyelination due to Wallerian degeneration are picked up as
increased ADC and decreased FA. Gliosis of the white matter, by disrupting the

Table 3 Techniques to Probe into the Brain Microstructure with Magnetic


Resonance Imaging

Physical
phenomenon Measure Indicative of Affected by

Diffusion Mean diffusivity Apparent Axonal Cell and axonal


of water diffusion density density, glial
coefficient reaction, water
content, neurofila-
ment pathology
Diffusion Mean diffusivity Fractional Axonal den- Same as above
tensor of water in the anisotropy sity and
3 planes coherence
in a given
plane
Magneti- Transfer of magneti- Magnetization Integrity of Density of
zation zation between transfer protein macromolecules
transfer large macromol- ratio matrices
ecules and water and cell
protons mem-
branes
Source: From Ref. 28.
Quantitative and Functional MRI Techniques 181

normal axonal structure, also gives rise to decreased FA, but the ADC is normal
or decreased due to the boundaries to proton motion represented by glial cell
membranes (Table 3). Neurofilament and tau pathology is associated with normal
ADC and FA, cell membranes being intact.
Magnetization transfer imaging is based on the exchange of magnetization
between free and macromolecule bound protons and allows one to indirectly observe
semisolids, such as protein matrices and cell membranes (Table 3). Changes in
tissue structural integrity such as gliosis lead to decreased bound and increased
free protons which show as decreased magnetization transfer ratio (Table 3).
There are grounds to believe that microstructural pathology exceeding the
macrostructural changes can be appreciated through T1-weighted images present
in AD, and this is strongly correlated with cognitive performance. Of the two
pathological hallmarks of the disease, senile plaques and neurofibrillary tangles,
the latter involves the cytoskeleton and neurobiological studies have shown that it
affects axonal transport (105). Moreover, pathological studies have shown that
neurofibrillary tangle density is more closely related to cognitive performance
than senile plaques (106). Structural T1-weighted MRI studies have shown that
the correlation between regional atrophy and global cognitive impairment is
relatively weak (107).
When structural changes are assessed with magnetization transfer and
diffusion imaging, a stronger correlation emerges (107). Bozzali and colleagues
have studied 18 AD patients with MMSE between 5 and 25 and 16 elderly
controls and found that the Pearson’s r correlations with the MMSE were 0.21 for
global brain volume, 0.31 for global diffusivity of the gray matter, and 0.58 for a
magnetization transfer index of the gray matter. A composite score compounding
information on atrophy and magnetization transfer reached rZ0.65, indicating
that 42% of the MMSE variance was accounted for by these two markers (108).
Hanyu and colleagues have studied 35 AD patients with MMSE between 9 and 25
and found that the MMSE was highly correlated with the magnetization transfer
ratio in the gray matter of the hippocampus (rZ0.70, corresponding to 49% of the
explained variance). The same group had previously shown in a group of 23 AD
patients with similar range of severity (four had MMSE !10, 13 had MMSE 11–20,
and six had MMSEO21) that the MMSE had a strong correlation with both
anisotropy (rZ0.65) and magnetization transfer ratio (rZ0.64) (109). However,
the correlation with the total callosal area, a proxy of white matter atrophy, was
significantly higher, reaching rZ0.74 (56% of explained variance). Yoshiura and
colleagues have studied 34 AD patients with MMSE between 3 and 28 and found
that the mean diffusivity of the white matter in the posterior cingulate gyrus was
significantly correlated with the MMSE (rZ0.53, 28% explained variance) (110).
Twenty-five patients with MCI have so far been studied with MT in two studies.
Not unexpectedly, both have found that MT measures of MCI patients are
intermediate between those of normal controls and AD patients (111,112).
Other studies have reported high correlations between global cognitive
performance and measures of diffusion of the white matter (113) and of T of the
182 Frisoni and Filippini

gray and white matter (111), but have included controls in the correlation. This
design can be criticized in that it inflates group variance through the unlikely
assumption that the amount of variance of cognitive performance in healthy
subjects that can be accounted for by MR measures of brain structural integrity is
similar to that in Alzheimer’s patients, and that the biological underpinnings are
also similar.
Taken together, these studies suggest that microstructural pathology of the
gray and white matter might be a strong correlate of cognitive impairment in AD.
However, although one study corrected diffusion and MT measures for brain
volume, thus at least partly accounting for the effect of atrophy (111), all the
others have so far failed to partial out the effect of global or regional atrophy from
that of diffusion and MT measures. Thus, it still remains to be determined
whether microstructural change has an effect on cognitive impairment
independently of the effect of macrostructural pathology.
Individual imaging modalities have variable relationships with cognitive
impairment in AD patients. Future studies will need to combine imaging
modalities in order to obtain a multispectral image of a brain where the variance
inter-modality and among different areas of the same modality might outline a
specific disease pattern. A pioneering study (114) has obtained information on
diffusion, MT, and proton spectroscopy in a single MRI exam in order to define
profiles of microstructural and biochemical changes in individual patients with a
range of neurological diseases (MS, subcortical vascular encephalopathy, major
stroke, AD, Alexander’s disease, and haemangioma), but the specificity of
the profiles still needs to be assessed. Prospective studies will need to assess the
progression of microstructural involvement with cognitive deterioration and
compare this correlation with that of other markers used to monitor the biological
progression of the disease and as surrogate outcome measure in clinical trials (40).

FUNCTIONAL MAGNETIC RESONANCE IMAGING IN


DEMENTIAS AND MILD COGNITIVE IMPAIRMENT
fMRI studies are providing unprecedented insight into the physiological
mechanisms of the brain in a variety of conditions, from traditional neuro-
psychological task to emotional perceptions to ethical choices. In the normal
brain, fMRI allows mapping of the regions that are activated during a task with
spatial resolution presently around 1 mm at 3.0 Tesla and below 1 mm at 4–7
Tesla. Given the known functional specificity of gray matter structures, the
pattern of activation allows investigators to determine the basic functions
exploited to carry out a given task.
Neurodegenerative disorders affect the normal activation pattern in a
number of ways. As expected, in AD patients functional activation is decreased in
the areas most affected by AD pathology. Some studies of verb processing, verbal
and non-verbal learning, and working memory have found a reduced activation of
the areas activated in healthy persons to carry out the same task (115–120)
Quantitative and Functional MRI Techniques 183

(Table 4). Interestingly, medial temporal activation patterns in MCI patients


during a memory task have been found to be as much reduced as in AD patients,
possibly suggesting that the functional damage of AD neuropathology in the
medial temporal lobe is very early (116).
Some studies have provided evidence that in AD and frontotemporal
dementia reduced activation of normally active areas is accompanied by
cortical reorganization. An enlarged area of activation has been found in the
medial temporal lobe and other cortical areas with verbal and non-verbal
learning, working memory, semantic, and visuospatial tasks (Table 4)
(117–119,121–123,125,126,129,130). This finding is consistent with the
recruitment of wider networks in an attempt to preserve cognitive function. It
is conceivable that such compensatory mechanism may be functional in
the relatively early stages of the disease, while in later stages activation
decreases. Again, functional reorganization occurs in the early stages of the
disease (121).
One of the most interesting findings obtained through the use of fMRI in the
study of dementing disturbances relates to the involvement of the default network
in AD (123). In young adults, a specific set of regions consistently shows
deactivation that generalizes across a wide range of tasks and stimulus modalities,
and has been detected by using both positron emission tomography and fMRI
(131–134). These commonly deactivated regions include large sections of the
lateral parietal cortex, the medial parietal cortex (including posterior cingulate
and precuneus), and the medial frontal cortex. The function of the default network
is monitoring the environment, monitoring one’s internal state and emotions, and
various forms of undirected thought. Engaging in goal-directed, active tasks may
redirect processing resources from default activities to task-specific processes and
regions. Lustig and colleagues have studied the magnitude and dynamic temporal
properties of typically deactivated regions by using fMRI in 32 young adults, 27
older adults without dementia, and 23 older adults with mild AD. These were
imaged while performing an active semantic classification and a passive fixation
baseline task. Deactivation in lateral parietal regions was equivalent across
groups; in medial frontal regions, it was reduced by aging but was not reduced
further by AD. Of greatest interest, the posterior cingulate region showed
differences between young adults and older adults without dementia and an even
more marked difference with AD (Fig. 16). The region was initially activated by
all three groups, but the response in young adults quickly reversed, whereas AD
individuals maintained activation throughout the task.
These results can be interpreted as being related to damage of the
entorhinal/perirhinal cortex—a major source of projections to the posterior
cingulate cortex. It could be hypothesized that the deactivation of the default
network might disengage monitoring of environment, internal states and
emotions, and undirected thought, and allow the selective allocation of specific
cognitive resources to a given task. AD patients might be unable to do so, and the
(Text continues on page 187.)
184

Table 4 Functional MRI Studies with Relevance to Dementia and Alzheimer’s Disease
Reference Aim Results Test performance

Dickerson Study functional activation pattern of MCI Enlarged hippo and parahippo activation with greater
et al., 2004 during a visual encoding task clinical impairment and subsequent memory
(121) decline
Enlarged hippo and parahippo activation was
correlated with better memory performance at
baseline
Grossman Neural basis for verb processing in AD Reduced activation in AD of normally activated Poorer in AD
et al., 2003 cortical regions (post-lat temp and inf fr regions)
(115)
Lipton et al., Study working memory in 2 monozygotic Enlarged parietal and reduced prefrontal cortex Poorer in the AD
2003 (122) twins discordant for AD activation twin
Lustig et al., Study activation-deactivation pattern in a No deactivation in AD of the normally deactivated Poorer in normal
2003 (123) semantic classification task medial post par cortex aging and AD
Enlargement in normal aging and AD of normally
activated regions (dorsal front cortex)
Machulda Study activation patterns of AD and MCI Reduced activation in AD and MCI of normally Poorer in AD and
et al., 2003 patients and normals during a visual activated regions (medial temp lobe) MCI
(116) encoding task Association between activation and performance on
recognition and recall
Rombouts Study working memory in frontotemporal Reduced activation in frontotemporal dementia of Similar in
et al., 2003 dementia regions activated in AD (fr and par cortex) frontotemporal
(117) Greater cerebellar activation in frontotemporal dementia and AD
dementia
Sperling et al., Study the effect of aging and AD on Reduced activation with aging of the dorsolat prefr
2003 (118) activation patterns during a learning task cortex and enlarged activation of par cortex
Frisoni and Filippini
Reduced activation in AD of medial temp and
increased of medial par and post cing regions
Li et al., 2002 Search for a functional MRI marker of Functional synchrony of the hippocampus separates
(124) early AD AD from controls with sens and spec of 80% and
90%
Prvulovic et al., Study visuospatial processing in AD Enlargement in AD of normally activated cortical Poorer in AD
2002 (125) regions (sup temp lobule and occ-temp cortex)
Atrophy partly accounts for differences between AD
and controls
Kato et al., Study nonverbal learning in AD Reduced activation in AD of normally activated Poorer in AD
2001 (119) regions (medial temp lobe and prefr temp and par
association cortices) and enlarged activation of
visual associative cortex
Bookheimer Study activation pattern of apoE4C normal Greater magnitude and enlargement in e4C of Similar in e4C
et al., 2000 persons during encoding task and the normally activated cortical regions (hippoc, par, and e4K
Quantitative and Functional MRI Techniques

(126) predictive power of activation patterns and prefr regions). Greater activation predicted
on subsequent memory decline memory decline after 2 years
Johnson et al., Study the relationship between functional Marked effect in AD of regional cortical atrophy on Poorer in AD
2000 (127) MRI activation and cerebral atrophy activation of the left inf fr but not of the left sup
with a semantic classification task in AD temp gyri
No effect of atrophy in controls
Pihlajamaki Study verbal fluency in normal persons Verbal fluency activates the medial temp lobe
et al., 2000
(128)
Rombouts Study nonverbal learning in AD Reduced activation in AD of normally activated Poorer in AD
et al., 2000 regions (hippoc and parahippoc gyri)
(120)
(Continued)
185
186

Table 4 Functional MRI Studies with Relevance to Dementia and Alzheimer’s Disease (Continued)

Reference Aim Results Test performance

Thulborn et al., Study visuospatial attention in AD Activation in AD of silent areas (bilat dorsolat prefr Poorer in AD
2000 (129) cortex)
Reversal of the normal rightOleft activation in the
intraparietal sulcus
Saykin et al., Anatomic substrate of semantic memory Enlargement in AD of normally activated cortical Poorer in AD
1999 (130) impairment in AD regions (inf and middle fr gyrus)
Abbreviations: MCI, mild cognitive impairment; AD, Alzheimer’s disease; MRI, magnetic resonance imaging.
Frisoni and Filippini
Quantitative and Functional MRI Techniques 187

Figure 16 Effects of age and dementia on activation of the posterior cingulate area of the
default network during a semantic classification task: the region is initially activated in all
three groups, but the response in young adults quickly reverses, whereas DAT individuals
maintain activation throughout the task. (Left) Medial parietal posterior cingulate cortex.
(Right) Mean time courses of blood oxygenation level–dependent signal across active task
and passive fixation baseline conditions. Source: From Ref. 123.

activation of the default monitoring system might interfere with the activation of
task-specific regions, leading to poor performance in cognitive tasks. Better
understanding of the impairment of the default network in AD and other
dementias might help to understand the cognitive processes of patients while in
the resting state rather than performance in a challenged state—which has largely
been investigated in the last 30 years—as well as alertness and attention to outer
and inner stimuli. The neural basis of insight and awareness of own cognitive
deficits and of the surrounding environment, the development of agitation
following minor environmental stimulation in some AD patients, and the context-
dependent performance in activities of daily living might also be elucidated.
Some issues should always be kept in mind when interpreting fMRI results
in neurodegenerative conditions. First, fMRI does not allow differentiating
systems that can exert either activation or inhibition. This is a major hindrance for
tracking the cortical remodelling that likely takes place in AD and other
neurodegenerative disorders, whether excited or inhibited. Second, in normal
persons the same task gives rise to greater activation based on subjective
difficulty (cognitive tasks give more activation in persons with lower IQ, the
so-called “neural efficiency” hypothesis) (135,136). As disease specific tasks are
more difficult for patients with neurodegenerative disorders than normal persons,
this might tend to give greater activation. Unfortunately, the specific contribution
of subjective task difficulty cannot be assessed with current experimental designs.
Thus, the fMRI signal of patients with neurodegenerative conditions is the
summation of three trends: (1) lower activation due to neuronal or synaptic
damage, (2) greater activation due to compensatory recruitment, and (3) greater
activation due to subjective task difficulty. Their contribution to brain activation
remains to be investigated.
188 Frisoni and Filippini

ACKNOWLEDGMENTS

Samantha Galluzzi and Lorena Bresciani helped edit the manuscript.

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7
Perfusion Imaging with Single Photon
Emission Computed Tomography

Nadine J. Dougall and Klaus P. Ebmeier


Division of Psychiatry, Gordon Small Center for Research in Old Age
Psychiatry, University of Edinburgh, Edinburgh, U.K.

SINGLE PHOTON EMISSION COMPUTED TOMOGRAPHY


As its name suggests, single photon emission computed tomography (SPECT)
relies on the three-dimensional reconstruction of gamma emitter distributions in
the brain. Without the advantage of the special geometry of positron annihilation
into two photons travelling in opposite directions, SPECT has to use collimators
to limit the field of view of the photon detectors. This implies that a significant
amount of the radiation is absorbed by the collimator walls and does not
contribute to the reconstructed image. Consequently, the sensitivity of SPECT is
reduced compared with PET. Other differences between PET and SPECT derive
from the different radioactive half-life of the gamma and positron emitters: short
half-lives of positron-emitters make several repeat examinations within a
short period possible, while longer half-lives of gamma-emitters allow for the
examination of metabolic or pharmacological processes that require longer
periods to establish. Further, the replacement of carbon atoms of the ligand with
the positron emitter 11C can generate radio-ligands with identical pharmacologi-
cal properties, whereas substitution with the gamma-emitters 123Iodine or
99m
Technetium is likely to change the ligand’s chemical properties. Conse-
quently, the generation of new SPECT ligands has proceeded at a disappointingly
slow rate, partially because of the need to establish their pharmacological
properties after synthesis.

197
198 Dougall and Ebmeier

BLOOD FLOW TRACERS


Four SPECT tracers are now available for “cerebral blood-flow” studies. 133Xe
is an inert gas that is inhaled, equilibrates with body tissues, and, after supply is
stopped, its initial washout rate from tissue is proportional to regional cerebral
blood flow (rCBF). Its advantage is that it can be calibrated against absolute CBF,
but unfortunately, its gamma radiation is too soft (81 keV) to yield high quality
images beyond the surface of the cortex. Iodinated compounds rely on the
availability of 123I (radioactive half-life 13.2 hours; 159 keV) and a radio-
chemist, who can synthesize the radiotracer to a high standard of purity within a
reasonable time to injection. The radio-tracer N-isopropyl-[123I] beta-iodoam-
phetamine [(123I) IMP] was the first such SPECT ligand available, but currently it
is almost exclusively used in Japan. In Europe and North America, 99mTc-
substituted ligands have taken over from [123I] IMP. 99mTc has a half-life of
6 hours and generates gamma radiation of 141 keV energy, which allows for
resolutions down to 5 mm. Two ligands are commercially available, hexamethyl-
propylene amine oxime (HMPAO, Exametazime, Ceretec w) and N 0 -1,
2-ethylenediy (bis-L-cysteine) diethyl ester (ECD, bicisate, Neurolitew). The
cerebral distribution of all three tracers differs slightly, suggesting that their first
pass uptake into brain cells is due to and modified by different mechanisms. For
example, in a case control study of 90 volunteers, tracer uptake in large areas of
the parietal, occipital, and superior temporal cortices was lower in a group
imaged with 99mTc-HMPAO compared to the 99mTc-ECD group. Increases, on
the other hand, were seen in the subcortical nuclei, parts of the brain stem,
hippocampus, and small areas of the cerebellum (1). In another confirmatory
comparative study, HMPAO-SPECT images showed relatively high radioactivity
in the basal ganglia and cerebellum, whereas in the ECD-SPECT images, high
levels were observed in the medial aspect of the occipital lobe. These regions
with high radioactivity were not apparent in the rCBF-PET images (2). Because
uptake in medial temporal lobe differs between 99mTc-HMPAO and 99mTc-ECD,
specific and separate diagnostic criteria for temporal lobe pathology, such as in
dementia and temporal lobe epilepsy, are necessary (3). Moreover, different
effects of the diagnostic confounder age on perfusion can be observed, depending
on whether HMPAO or ECD is used (4).
There is some controversy about which is the best diagnostic agent, and this
will have to be decided on empirical grounds for each diagnosis separately (see
below). Here, we want to make some general points about the differences between
the first pass uptake perfusion markers 123I-IMP, 99mTc-HMPAO, and 99mTc-ECD.
99M
Tc-HMPAO is less stable after reconstitution, so that images show more
background facial uptake and retention when compared with 99mTc-ECD
images (5). Stabilization of HMPAO with methylene blue or cobalt chloride
improves image quality and reduces background activity in comparison to that
of unstabilized 99mTc-HMPAO, without reaching the quality obtained with
99m
Tc-ECD (6). On the other hand, a comparison between early and delayed
Perfusion Imaging with SPECT 199

images using both HMPAO and ECD showed that HMPAO is more stable in the
brain with no washout over time (7).
Both HMPAO and ECD underestimate perfusion in high flow areas, a
phenomenon that can be demonstrated with acetazolamide, which causes greater
increases in uptake with IMP than with the other two tracers (8,9).
Uptake of ECD depends on esterase activity, both cytosolic and membrane
bound (10), whereas HMPAO retention depends on the presence of glutathione (11).
This may, most of the time, ensure that variations in perfusion determine the
variability of tracer uptake, but it has to be understood that such variability may also
be due to factors specifically interfering with the tracer’s retention mechanism. This
will be of particular importance in pathological tissue. It appears that HMPAO
uptake in ischemic brain behaves like a rCBF tracer, i.e., is increased in areas of
luxury perfusion, while ECD and IMP do not show increased uptake, just like
regional oxygen consumption, or may even be reduced, as a marker of discrete tissue
damage (12–14). Similarly, ictal, inflammatory, and some neoplastic hyperper-
fusion is more obvious with HMPAO than with ECD SPECT (15–20). However,
relative lack of stability after reconstitution of HMPAO make ECD a logistically
more useful and effective tracer for ictal and peri-ictal studies (21,22).
Vascular lesions and hypo-perfusion in dementia appear to be detected more
easily with IMP or ECD than with HMPAO (23,24), although this may have little
impact on actual diagnostic accuracy (25). The situation may be reversed in AIDS
encephalopathy, with greater diagnostic accuracy for HMPAO than IMP (26).

ALZHEIMER’S DEMENTIA
The characteristic pattern of AD perfusion deficits in parietal and temporal lobes
is generally accepted and has been acknowledged in clinical guidelines (Figs. 1
and 2) (27). SPECT can provide rich information for the differential diagnosis of
dementia, but routine use is still controversial. Research studies have increasingly
documented modulating effects of age and gender on the typical perfusion pattern
of AD. Although confirmation of these effects is still required, the discussion here
will hopefully facilitate image interpretation and improve the usefulness of the
diagnostic changes observed with SPECT.

Effect of Age
Although presenile onset AD is recognized for its faster clinical decline and
greater symptom severity, the wider effect of age on SPECT AD diagnosis across
the whole aging spectrum has yet to be fully explored. One of the first studies by
Burns et al. (28) found age of onset correlated positively with parietal deficits and
negatively with medial temporal lobe perfusion. Jagust et al. (29) then reported
relative left frontal hypoperfusion in presenile AD compared with senile-onset
AD, and Caffarra et al. (30) tried to replicate this finding with patients matched by
illness severity, concluding that presenile AD was not associated with greater
SPECT changes than senile AD. O’Brien et al. (31) confirmed that the right
200 Dougall and Ebmeier

Figure 1 (See color insert.) Tc99m HMPAO SPECT scan of a healthy volunteer
(MMSEZ30); note that the parietal and temporal lobes are well perfused.

occipital hypoperfusion observed in their presenile patients did not distinguish


them from senile onset patients after controlling for severity and duration of
illness. However, Habert et al. (32) found a reduction in the early onset group in
bi-parietal and left temporo-occipital areas. Many possible reasons exist for the
lack of consensus of these early results, including confounding variables of age,
age of onset, illness severity, and image analysis method.
Recently, and with the benefit of enhanced imaging system resolution and
sophisticated data processing, the question of age has been revisited. A study by
Hanyu et al. (33) using 3D stereotactic surface projection (3DSSP) found that
younger AD had perfusion deficits in parietal lobe and posterior cingulate
compared with younger controls, and older AD subjects had reduced perfusion
in medial temporal and medial frontal areas, compared with older controls.
Comparing young with old AD patients directly, the younger patients had more
severe decreases in parieto-temporal and medial parietal lobes and the older group
had decreases in medial temporal, medial, and orbital frontal and medial occipital
lobes. Because posterior cingulate reductions were more severe in younger
patients, the authors conclude that the sensitivity of SPECT is reduced for
diagnosing AD in elderly cases.
Kemp et al. (34) found that 79% of a late onset AD group had medial
temporal reductions compared with posterior association cortex, and conversely,
Perfusion Imaging with SPECT 201

Figure 2 (See color insert.) Tc99m HMPAO SPECT scan of Alzheimer’s disease
(MMSEZ22), a characteristic pattern of perfusion deficits in parietal and temporal lobes.

85% of the early onset AD group had reductions in posterior association cortices
compared with medial temporal lobe. In contrast, Nitrini et al. (35) observed
bilateral parieto-temporal perfusion deficits in 23% of an early onset group and
71% of the late onset group—by combining parietal and temporal regions,
perhaps the subtle differences detected by Kemp and Hanyu were lost.
Dougall et al. (36), in a multi-center study comparing young and old AD
patients with age-matched mixed groups of healthy volunteers and depressed
subjects, found that an increase in diagnostic accuracy was achieved for younger
AD but not for the older AD group by using statistical parametric mapping (SPM)
maps in conjunction with SPECT images. Kaneko et al. (37), using 3D-SSP and
posterior cingulate reductions as a marker against other dementias, found that the
frequency of posterior cingulate hypoperfusion was greater in presenile AD than
in senile onset AD patients, thus providing further evidence of the heterogeneity
of AD across age groups.

Summary
Clinicians should be alert to possible age-related changes in the interpretation of
SPECT. Later onset AD appears to involve more medial temporal deficits and
increased image heterogeneity, and is therefore more challenging to diagnose. On
the other hand, younger onset AD has been associated with parietal and posterior
202 Dougall and Ebmeier

cingulate reductions. SPM and 3D-SSP appear to be more helpful in early than
late onset AD.

Effect of Gender
In a retrospective study of 104 SPECT scans of probable AD patients (NINCDS-
ADRDA), Nitrini et al. (35) found that disease severity, age of onset, and being
male was associated with bilateral parieto-temporal hypoperfusion. Thirty-nine
percent of females and 59% of males had this perfusion pattern. In a separate
study by Swartz et al. (38) of 63 probable and possible AD patients, both lower
MMSE scores and male gender significantly predicted reduced temporal and
parietal perfusion.
In a study by Ott et al. (39), left-right symmetric perfusion occurred more
frequently in male than female demented patients with probable AD (NINCDS-
ADRDA). Unilateral perfusion deficits in female probable AD patients were
found to be almost always on the left side; female gender and shorter disease
duration were independent predictors of unilateral left hemisphere
CBF reduction.

TECHNIQUES TO IMPROVE DIAGNOSTIC ACCURACY


In the investigation of diagnostic differences, early SPECT studies used
subjective visual inspection or quantitative region-of-interest (ROI) analysis
which is less subjective but still observer dependent. Substantial progress has
been made since then to develop objective quantitative techniques free of
observer bias and with increased sensitivity to diagnose subtle changes associated
with early dementia. Multivariate statistical techniques such as SPM, 3D-SSP,
3D fractal analysis (3D-FA), discriminant function analysis (DFA), or neural nets
(NN) have crossed over from research to clinical practice with some success.

Statistical Parametric Mapping


SPM normalizes SPECT images to standard stereotactic space, allowing for
direct statistical voxel-based comparison of scans between diagnostic groups. For
example, in a comparison with a normal comparison group, Lee et al. (40) found
reductions in left temporo-parietal cortex for mild AD and reductions in bilateral
posterior parieto-temporal cortex, contiguous parts of anterior occipital lobes,
and posterior cingulate gyri for moderate AD. A further study by Bonte et al. (41)
comparing AD with FTD found an absence of reduced perfusion in posterior
cingulate cortex to differentially diagnose FTD.
Few studies have directly compared SPM with visual assessment or ROI
analysis. In a multi-center study comparing AD patients with depressed and
healthy volunteers, Ebmeier et al. (42) reported that with SPM, between-rater
variability was reduced, with greatest improvement in diagnostic accuracy for less
experienced raters, and furthermore, normalizing to cerebellum was found to
Perfusion Imaging with SPECT 203

increase the spatial extent of reductions observed on SPMs compared with global
normalization (43). In the same multi-center study, Dougall et al. (36) found that
SPM used together with visual assessment increased specificity but not sensitivity.

3D Stereotactic Surface Projection


A multi-observer ECD-SPECT study by Honda et al. (44) directly compared
visual SPECT assessment with SPECT alongside 3D-SSP of patients with AD,
memory problems and healthy volunteers. Accuracy measured by area under the
ROC curve (AUC) was enhanced for SPECT with 3D-SSP. Accuracy was further
improved with an automated algorithm using bilateral posterior cingulate gyri in
a 3D-SSP template. Adding 3D-SSP to SPECT increased specificity, but
not sensitivity.

3D Fractal Analysis
3D fractal analysis (3D-FA) objectively measures spatial perfusion differences by
calculating an index of heterogeneity called a fractal dimension. Yoshikawa
et al. (45) used both 3D-SSP and 3D-FA to compare VaD patients with healthy
volunteers. With 3D-SSP, reduced perfusion in VaD could be divided into two
abnormal patterns: global reduction and a decrease in frontal regions only. 3D-FA
demonstrated a difference between VaD and controls and a complete discrimi-
nation from patients with moderate and severe VaD. A correlation was found in
VaD patients between the fractal dimension of image heterogeneity and cognitive
impairment measured with the MMSE (46).
Using 3D-FA, Nagao et al. (47) found differences between AD subjects and
controls, and since then Nagao et al. (48) have found differences in heterogeneity
between FTD and AD subjects.

Neural Networks
Neural networks (NN) are used to model complex nonlinear datasets where
relationships exist between SPECT perfusion patterns and diagnosis. They are
trained to recognize patterns for disease simply by presentation of a scan training
set, so that new cases can then be classified by likely diagnosis. Using this method,
Chan et al. (49) found high diagnostic accuracy measured by AUC. Dawson
et al. (50) reported a sensitivity for AD of 75% against a specificity against controls
of 69%. Page et al. (51) found NN classified diagnosis more accurately than
alternative statistical techniques and visual assessment ratings. The largest and
most recent study by Warkentin et al. (52) using NN with 133Xe SPECT obtained a
high diagnostic accuracy measured by AUC, with 86% sensitivity for AD and 90%
specificity against controls.
204 Dougall and Ebmeier

Discriminant Function Analysis


DFA derives combinations of variables that best discriminate between diagnostic
groups, and can be used to predict group membership of new cases. For example,
O’Mahony et al. (53) used DFA in a comparison of probable AD with normal
controls, determining an optimal cut-off with a sensitivity of 87% and a
specificity of 100%, then correctly categorized 93% of 15 subsequent new cases.
In another study by O’Brien and colleagues (54), optimal separation was
achieved between AD and controls with a sensitivity of 77% and specificity of
82%. Both studies were similar for MMSE score, age, and subject follow-up.

Partial Volume Correction


The lower spatial resolution of SPECT can cause partial volume effects—rCBF
in smaller brain structures is poorly estimated—further exacerbated with age-
related atrophy. An ECD study by Kanetaka et al. (55) of early AD and controls
using SPM analysis found that partial volume correction increased diagnostic
accuracy; using posterior cingulate hypoactivity, the area under the ROC curve
was much increased with both global and cerebellar mean normalization.

COHORT STUDIES
Cohort studies of consecutive patients are particularly relevant as they mimic
clinical imaging practice. To date, published cohort studies are very
heterogeneous in their diagnostic composition. For instance, cohort studies
have recruited consecutive outpatients from a memory clinic (56–59), while other
studies have recruited consecutive patients with memory problems from a nuclear
medicine department (60), a neuropsychiatry unit including neurological
disorders (61), and from neuropsychology (62) (summarized in Table 1).

VASCULAR DEMENTIA
Several studies have attempted to discriminate AD from vascular dementia
(VaD) with varying success—typically using AD criteria of temporo-parietal or
posterior deficits and diffuse multi-focal abnormalities for VaD.

Early Onset AD vs. VaD


Early studies reported promising results in spite of imaging systems with limited
resolution. Smith et al. (63) obtained a sensitivity for AD versus VaD of 73%
against a specificity of 88%, Battistin et al. (64) a sensitivity for probable AD
of 90% and a specificity against VaD of 80%, and Launes et al. (57), comparing
AD with VaD, found a sensitivity of 64% against a specificity of 85%.
(Text continues on page 209.)
Table 1 SPECT Cohort Studies Are Heterogeneous in Diagnostic Mix and Not Generalizable to the General Population
Clinical
Diagnosis definition
confirmed No. of of AD Age MMSE No. non MMSE Sensi- Speci-
First author by follow- AD in patient average mean AD in Clinical definition Age mean tivity ficity
(year) up cohort group (years) score cohort of control group average score (%) (%)
Perfusion Imaging with SPECT

Herming- No 6 Probable NR NR 27 Non-AD consecutive NR NR 71.0 96.0


haus AD cohort of MC out-
(1998) patients—other
dementia, pseudode-
mentia, or no
dementia
Launes No 36 Assumed 65 NR 62 Non-AD cohort of out- NR NR 63.9 83.9
(1991) probable patients from MC
AD (non-demented)
(Continued)
205
206

Table 1 SPECT Cohort Studies Are Heterogeneous in Diagnostic Mix and Not Generalizable to the General Population (Continued)

Clinical
Diagnosis definition
confirmed No. of of AD Age MMSE No. non MMSE Sensi- Speci-
First author by follow- AD in patient average mean AD in Clinical definition Age mean tivity ficity
(year) up cohort group (years) score cohort of control group average score (%) (%)

Lee (2001) Yes, mean 58 Probable NR NR 57 Non-AD consecutive NR NR 87.9 70.2


11.1 AD cohort of patients
months from Nuclear Medi-
cine Dept with mem-
ory impairment—PD
with dementia (12),
Picks (3), systemic
lupus (3), VaD (30),
head trauma (4),
Behcet’s disease (3)
Dougall and Ebmeier
Masterman Yes, at 51 Probable 76 17.0 31 Unlikely AD patients 73 22 74.5 54.8
(1997) 1 month AD from MC who had a
SPECT scan—VaD
(19), PD (2), FTD
(2), CJD (1), age,
associated memory
impairment (3),
others (4)
Pasquier No 127 Probable 73 17.2 115 Non-AD cohort of con- 68 22 61.4 71.3
(1997) AD secutive patients
Perfusion Imaging with SPECT

from MC—FTD
(47), VaD (21), DLB
(12), anxiety/depres-
sion (14), others (21)
Velakoulis No 9 AD (clini- 63 NR 47 Non-AD consecutive NR NR 88.9 78.7
(1998) cal cohort of neuropsy-
criteria chiatry unit patients
NR) with cognitive
impairment—FTD
(9), VaD (4), other
dementia (9), neuro-
logical diagnosis (7),
head injury (4), PD
(2), others (12)
(Continued)
207
208

Table 1 SPECT Cohort Studies Are Heterogeneous in Diagnostic Mix and Not Generalizable to the General Population (Continued)

Clinical
Diagnosis definition
confirmed No. of of AD Age MMSE No. non MMSE Sensi- Speci-
First author by follow- AD in patient average mean AD in Clinical definition Age mean tivity ficity
(year) up cohort group (years) score cohort of control group average score (%) (%)

Villa No 23 Probable 68 17.9 40 Non AD cohort of 68.4 NR 91.3 72.5


(1995) AD patients from neu-
ropsychology with
cognitive impair-
ment—VaD (12) PSP
(7) focal ventricular
lesion (12) circum-
scribed cortical
degeneration (9)
Abbreviations: AD, Alzheimer’s disease; CJD, creutzfeldt–jakob disease; DLB, dementia with lewy bodies; FTD, frontotemporal dementia; MC, memory clinic;
MMSE, mini mental state examination; NR, not reported; PD, parkinson’s disease; PSP, progressive supranuclear palsy; SPECT, single photon emission computed
tomography; VaD, vascular dementia.
Dougall and Ebmeier
Perfusion Imaging with SPECT 209

Later Onset AD vs. VaD


Weinstein et al. (65) did not find differences between older groups of AD and
VaD, concluding SPECT was not ready for routine use. In a comparison of
SPECT with MRI, Butler et al. (66) investigated late onset severe AD versus VaD
and found that SPECT correctly classified 77% of patients compared with 50%
by MRI. Late onset dementia was also examined by McKeith et al. (67), who
reported perfusion deficits for AD compared with VaD in right and left frontal,
right and left anterior, and posterior parietal areas. DeFigueiredo et al. (68) found
that using left parietal and left and right temporal areas to train a neural network,
AD was discriminated from VaD with a sensitivity of 80.0% against specificity of
86.4%; however, the AD group had lower MMSE scores than the VaD group.

AD vs. VaD—Detecting Early Dementia


Considering the differential diagnosis of early dementia, hippocampal perfusion
deficits have been reported in AD compared with VaD by Villa et al. (62), with a
sensitivity of 91.0% against a specificity of 75.0%. In a consecutive SPECT study
by Starkstein et al. (69), VaD patients had lower perfusion in frontal, superior,
and basal ganglia areas compared with AD patients (mean MMSE 19.0 and 19.9,
respectively).

AD vs. VaD with Follow-Up Confirmation of Diagnosis


A number of studies have followed up patients to confirm original diagnosis.
Bergman et al. (70) recruited patients with possible or probable AD and VaD,
followed-up at 12 months, and found a relatively poor sensitivity for AD of 55%
and a specificity of 71% against VaD. Masterman et al. (56) compared probable
AD with VaD, followed-up for 1 month, and achieved a sensitivity of 74.5% with
a relatively poor specificity of 57.9%; Pasquier et al. (59) obtained a sensitivity of
61% for probable AD against a poor specificity of 48% for VaD.
In more recent follow-up studies, Varma et al. (71) recruited consecutive
early onset probable AD and VD, finding a sensitivity of 77% for AD and a
specificity against VaD of 67%; Lee et al. (60), with a mean follow-up of
11 months in a study of consecutive patients with probable AD versus VaD and
using bilateral or unilateral temporo-parietal deficits as diagnostic criterion,
found a high sensitivity for AD 87.9%, compared with a specificity against VaD
of 76.7%.

Heterogeneity of Perfusion in AD and VaD


Using 3D-FA, Yoshikawa et al. (72) found higher heterogeneity of perfusion in
AD and VaD patients than in matched controls, AD heterogeneity was posterior
dominant, and VaD was anterior dominant—the anterior/ posterior ratio derived
by 3D-FA may thus be diagnostic of AD if !1 and of VaD if O1.
210 Dougall and Ebmeier

Systematic Review of AD vs. VaD


All extractable HMPAO-SPECT data published up to December 2002 were
combined in a systematic review and meta-analysis by Dougall et al. (73): 13 studies
with total numbers of 527 AD and 266 VaD (Table 2) gave a pooled weighted
sensitivity for AD of 71.3% (95%CI 67.5–75.2) against a pooled weighted
specificity against VaD of 75.9% (95% CI 70.8–81.1). Only two of the thirteen
studies used quantitative analysis to classify subjects; increased use of objective
multivariate statistical methods utilising whole SPECT datasets will possibly
improve the discriminative ability of SPECT.
Summary
Younger onset severely demented AD and VaD patients can be differentiated;
results for later onset dementia are not as conclusive. Early AD may have reduced
hippocampal perfusion, whereas early VaD may have reduced perfusion in
frontal and possibly basal ganglia areas.

FRONTOTEMPORAL DEMENTIA
SPECT perfusion deficits in anterior areas of the brain (frontal and anterior
temporal cortex) are associated with a frontal lobe dementing process.

Earlier FTD vs. AD


There is some evidence that FTD patients have reduced frontal, temporal, and
basal ganglia perfusion compared with controls; compared with AD they had
reduced perfusion in orbito-frontal, frontal dorso-lateral, anterior temporal, and
basal ganglia regions (74). Read et al. (75) found bilateral frontal reductions of
SPECT images in 88% of FTD compared with none of the AD, after pathological
verification of the diagnoses; unilateral left frontal hypoperfusion has also been
reported by Frisoni et al. (76).
Pasquier et al. (59) found frontal decreases in 68% of FTD patients, but these
were also present in 50% of probable AD patients; conversely, temporo-parietal
reductions were observed in 61% of patients with probable AD as opposed to 17%
in FTD. Pickut et al. (77) used discriminant function analysis (DFA) to separate
FTD from AD on the basis of frontal reductions and achieved a sensitivity of 74%
for FTD with a specificity of 81% against AD. Charpentier et al. (78), in a more
sophisticated DFA with 5 predictor ROIs and MMSE, derived a decision rule,
which correctly categorized 100% of FTD and 90% of ATD scans.
Velakoulis et al. (61) used as criterion bilateral parieto-temporal deficits
and found a sensitivity of 89% for AD and a specificity against FTD of 67%;
conversely, using frontal hypo-perfusion, the sensitivity for AD was 56% with a
specificity against FTD of 100%. In a study by Talbot et al. (79) explicitly
addressing the odds of a specific dementia diagnosis from perfusion patterns,
bilateral anterior perfusion reductions increased the odds of a patient having FTD
Table 2 HMPAO-SPECT Studies Comparing Alzheimer’s Disease and Vascular Dementia (1985–2002 Inclusive)
Diagnosis con- Age MMSE MMSE Sensi-
firmed by clini- Num- AD average mean VaD Age mean tivity
First author Year cal follow-up ber group (years) score Number group average score (%) Specificity (%)

Battistin 1990 No 19 Probable 67.7 9.0 15 VaD 71.5 16.9 89.5 80.0
Perfusion Imaging with SPECT

AD
Bergman 1997 Yes by 12 58 Mixed 75.5 21.6 17 VaD 72.9 22.4 55.0 70.6
months; then prob &
every 6–12 poss
months AD
Butler 1995 No 11 Probable 80.2 14.0 11 VaD 79.0 10.6 81.8 81.8
AD
deFigueir- 1995 No 20 Probable 72.3 14.5 22 VaD 77.0 20.8 80.0 86.4
edo AD
Launes 1991 No 36 Assumed 64.9 NR 33 VaD 68.0 NR 63.9 84.8
prob
AD
Lee 2002 Yes average 58 Probable NR NR 30 VaD NR NR 87.9 76.7
11.1 months AD
(Continued)
211
212

Table 2 HMPAO-SPECT Studies Comparing Alzheimer’s Disease and Vascular Dementia (1985–2002 Inclusive) (Continued)

Diagnosis con- Age MMSE MMSE Sensi-


firmed by clini- Num- AD average mean VaD Age mean tivity
First author Year cal follow-up ber group (years) score Number group average score (%) Specificity (%)

Masterman 1997 Yes at 1 month 51 Probable 76.0 17.0 19 VaD NR NR 74.5 57.9
AD
Pasquier 1997 No 127 Probable 72.8 17.2 21 VaD 71.9 21.6 61.4 47.6
AD
Pavics 1999 No 33 Probable 67.0 19.0 18 VaD 68.0 23.0 70.0 66.7
AD
Sloan 1995 No 43 AD (DSM- 70.1 18.8 25 VaD 76.0 20.2 74.4 92.0
III-R)
Smith 1988 No 26 AD(DSM- 64.6 NR 25 VaD 67.8 NR 73.0 88.0
III-R)
Varma 2002 Yes, 6 monthly 22 Probable 62.8 18.0 18 VaD 66.4 21.0 77.3 66.6
for 1–3years AD
Villa 1995 No 23 Probable 67.9 17.9 12 VaD 68.9 NR 91.0 75.0
AD
Abbreviations: AD, Alzheimer’s disease; MMSE, mini mental state examination; NR, not reported; SPECT, single photon emission computed tomography; VaD,
vascular dementia.
Dougall and Ebmeier
Perfusion Imaging with SPECT 213

as opposed to AD (likelihood ratioZ15.9) and decreased the odds of a patient


having AD as opposed to FTD (likelihood ratioZ0.1).

Recent FTD Studies


Pagani et al. (80) compared FTD, AD, and normal controls in a 3D standardized
volume of interest analysis. They found fronto-temporal, anterior cingulate, and
caudate nucleus reductions for FTD compared with normal controls. Pagani et al.
(81) expanded this study using cluster analysis; a joint feature vector for the
separation of scans by diagnosis yielded correct classification of 98% of FTD
against controls and 94% of AD against FTD.
With an anterior-posterior perfusion ratio, Sjogren et al. (82) achieved a
sensitivity of 86% for FTD and specificity against early onset AD of 96% and late
onset AD of 80%. Nagao et al. (48) used DFA-derived anterior and anterior-
posterior fractal dimensions that separated FTD from AD and controls with a
sensitivity of 86% and specificity of 84%.
While hypoperfusion in the posterior cingulate has been found to be
specific to AD, Bonte et al. (41) found that the absence of impaired perfusion
favors FTD.
Varrone et al. (83) used SPM and found that relatively lower perfusion was
observed in right medial frontal, anterior cingulate, and temporal areas as well as
orbitofrontal and ventrolateral prefrontal cortex in FTD compared with AD.
Comparing AD with FTD, a perfusion decrease in bilateral superior parietal
cortex was observed, more extensive on the left than the right with involvement
of superior occipital and temporo-occipital regions.

Systematic Review of AD vs. FTD


A meta-analysis of AD from FTD up to December 2002 and, using temporo-
parietal reductions as a diagnostic marker, seven eligible studies with a total of
119 FTD subjects compared with 265 AD subjects were identified by Dougall
et al. (Table 3) (73). The pooled weighted sensitivity was 71.5% against a
specificity of 78.2% with a diagnostic odds ratio of 10.6 (95% CI 6.2 to 17.9).
Using an additional marker such as fronto-temporal reductions may further
improve diagnostic accuracy.

Summary
In the differential diagnosis of FTD from AD, the presence of frontal
hypoperfusion is highly specific for FTD even when observed in combination
with bilateral temporoparietal deficits. Anterior-posterior ratios appear more
useful in younger age groups. Whereas posterior cingulate reductions have been
reported as diagnostic for AD, conversely some evidence exists that anterior
cingulate reductions are diagnostic of FTD.
214

Table 3 HMPAO-SPECT Studies Classifying Alzheimer’s Disease from Frontotemporal Dementia (1985–2002 Inclusive)
Diagnosis
confirmed by Clinical defi- Age MMSE MMSE Sensi-
First clinical nition of AD average mean FTD Age mean tivity
author Year follow-up Number group (years) score Number group average score (%) Specificity (%)

Launes 1991 No 36 Assumed 64.9 NR 5 FTD 66 NR 63.9 80.0


prob AD
Pasquier 1997 No 127 Probable AD 72.8 17.2 47 FTD 68 20.3 61.4 83.0
Pickut 1997 No 19 Probable AD 70 14.8 21 FTD 70 15.4 74.0 81.0
Sjogren 2000 No 52 Probable AD 67.4 NR 16 FTD 62 NR 88.5 87.5
Testa 1988 No 26 Probable AD NR NR 14 FTD NR NR 84.6 71.4
Varma 2002 Yes, 6 monthly 22 Probable AD 62.8 18 21 FTD 63 21 77.3 66.6
for 1–3 years
Vela- 1998 No 9 AD (clinical 63 NR 9 FTD 56 NR 88.9 67.0
koulis criteria
NR)
Abbreviations: AD, alzheimer’s disease; FTD, frontotemporal dementia; MMSE, mini mental state examination; NR, not reported; SPECT, single photon emission
computed tomography.
Dougall and Ebmeier
Perfusion Imaging with SPECT 215

DEMENTIA WITH LEWY BODIES


SPECT studies comparing dementia with Lewy bodies (DLB) with AD are rare.
An early study by Varma et al. (84) reported posterior cortical reductions in both
DLB and AD; both Pasquier et al. (85) and Talbot et al. (79) have concluded that
classification is difficult. Ceravolo et al. (86) and Donnemiller et al. (87) found
evidence exists for reductions in occipital lobe perfusion in DLB compared with
AD and, conversely, reduced temporoparietal regions in AD relative to DLB.
Ishii (88) found reduced perfusion in occipital areas, as well as relatively
preserved medial temporal lobes in DLB compared with AD. These authors also
reported that DLB and AD patients both had reduced parieto-temporal and
posterior cingulate perfusion when compared with a group of normal controls.
Lobotesis et al. (89) used left occipital and right temporal reductions in
perfusion to differentiate between DLB and AD and achieved a sensitivity of 64%
against a specificity of 69.6% (Table 4). Colloby (90) re-analysed Lobotesis’ data
with SPM and found relative reductions in DLB relative to AD in right parietal,
bilateral central parietal cortex, the precuneus, and the medial occipital gyri—and
for DLB relative to normal controls, reductions in central and inferior parietal,
left medial occipital, superior frontal regions, precuneus, and cingulate.

Summary
Occipital and posterior cingulate reductions and perhaps also the absence of
medial temporal reductions are probable SPECT markers of DLB. Voxel-based
techniques such as SPM may be more sensitive in earlier detection compared
with ROI analysis.

MILD COGNITIVE IMPAIRMENT


Individuals presenting with cognitive impairment who do not meet clinical
criteria for a dementing illness may qualify for a diagnosis of mild cognitive
impairment (MCI). MCI is typically characterized by a decline from a previous
level of memory functioning, which exceeds that expected by normal aging, with
otherwise good functioning.
Predicting with SPECT whether individuals will make the transition from
MCI to early AD or not may increase its value for planning appropriate early
treatment interventions (91). Selected studies with accuracy measures
investigating MCI, memory problems, as well as depression are summarized
in Table 5.
One of the first published longitudinal studies by Celsis et al. (92) followed
up subjects recruited from a memory clinic with a diagnosis of age related
cognitive decline (ARCD) over a mean of two years. 133Xe SPECT was used at
baseline and during follow-up and compared with a normal comparison group
(Text continues on page 220.)
216

Table 4 HMPAO-SPECT Studies Classifying Alzheimer’s Disease from Dementia with Lewy Bodies (1985–2002 Inclusive)
Diagnosis Age Clinical
confirmed Clinical defi- aver- MMSE definition MMSE Sensi-
First by clinical nition of AD age mean of control Age mean tivity
author Year follow-up Number patient group (years) score Number group average score (%) Specificity (%)

Lee 2001 Yes, mean 58 Probable AD NR NR 12 Parkin- NR NR 87.9 41.7


11.1 sons
months disease
with
deme-
ntia
Pasquiera 1997 No 127 Probable AD 72.8 17.2 12 DLB 72.3 22.7 61.4 75
Pasquiera 1997 No 156 Mixed probable 70.6 20.8 12 DLB 72.3 22.7 57.1 75
(n=127) and
Possible AD
(n=29)
Lobotesis 2001 NR 50 Definite (n=2) 81.6 17.3 23 DLB 79.4 16.0 64 69.6
and prob
(n=21) and
poss (n=27)
AD
a
Same study.
Abbreviations: AD, Alzheimer’s disease; DLB, dementia with Lewy bodies; MMSE, mini mental state examination; NR, not reported; SPECT, single photon
emission computed tomography.
Dougall and Ebmeier
Table 5 Summary of Selected SPECT Studies Investigating Alzheimer’s Disease, Mild Cognitive Impairment, and Memory Problems
Clinical
Diagnosis con- definition
First firmed by of AD Age MMSE MMSE Sensi- Speci-
author clinical follow- Num- patient average mean Num- Definition of control Age aver- mean tivity ficity
(year) up ber group (years) score ber group age score (%) (%)

Encinas Every 21 Progressed 75.3 NR 21 Stable MCI 71.6 NR 79.0 76.0


(2003) 6 months for to prob-
1–3 years able AD
Perfusion Imaging with SPECT

from
MCI
Huang Average of 82 Progressive 63.4 25.7 20 Stable MCI 59.6 26.9 Area under ROC
(2003) 26.4 months MCI curveZ0.75
Okamura Mean follow-up 22 Progression 71.9 25.6 8 Stable MCI 72.1 26.6 88.5 90.0
(2002) 3.1 years from
MCI to
AD on
follow-
up
Knapp NR 30 Probable 73.6 18.1 50 MCI 65.0 27.3 70.0 70.0
(1996) AD
Johnsona Yes, all subjects 56 Probable 77.4 18.0 27 Questionable AD 72.4 NR 80.4 77.8
(1998) AD (CDRZ0.5)
Johnsona Yes, all subjects 56 Probable 77.4 18.0 18 Converters to AD on 72.6 NR 80.4 94.4
(1998) AD mean follow-up
16.7 m (nZ18)
(Continued)
217
218

Table 5 Summary of Selected SPECT Studies Investigating Alzheimer’s Disease, Mild Cognitive Impairment, and Memory Problems
(Continued)

Clinical
Diagnosis con- definition
First firmed by of AD Age MMSE MMSE Sensi- Speci-
author clinical follow- Num- patient average mean Num- Definition of control Age aver- mean tivity ficity
(year) up ber group (years) score ber group age score (%) (%)

Greene Every 31 Probable 69.9 23.5 24 Controls with 65.4 NR 80.6 66.7
(1996) 6 months for AD memory problems
2 years
Hashi- NR 12 Probable 57.7 NR 18 Controls with 58.7 NR 100.0 100.0
kawa AD headaches or
(1995) dizziness
Mattman NR 128 73 Probable 70.0 NR 48 Controls with 65.0 NR 78.9 64.6
(1997) and 55 memory problems
possible
AD
Dougall and Ebmeier
Mielke NR 20 Probable 68.8 20.9 13 Controls with 59.5 28.8 80.0 65.0
(1994) AD memory problems
Muller NR 116 AD (DSM- 66.0 19.9 20 Controls with 56.0 28.8 48.0 75.0
(1999) III-R) memory problems
Perfusion Imaging with SPECT

Launes NR 36 Assumed 64.9 NR 62 Controls with NR NR 63.9 83.8


(1991) probable memory problems
AD including
psychiatric
disorder
(depression or
anxiety nZ21)
a
Johnson 1998 same study.
Abbreviations: AD, Alzheimer’s disease; CDR, clinical dementia rating; MCI, mild cognitive impairment; MMSE, mini mental state examination; NR, not reported;
ROC, receiver operating characteristic, SPECT, single photon emission computed tomography.
219
220 Dougall and Ebmeier

and a probable AD group with mild to moderate severity. ARCD subjects had
pronounced asymmetrically decreased temporo-parietal perfusion at baseline
compared with controls that declined further on follow up.
A large HMPAO-SPECT study by Johnson et al. (93) investigated differences
between groups of normal cognition, questionable AD, converters to AD from
questionable AD by two years follow-up, and probable AD (NINCDS-ADRDA)
for preclinical predictors of developing AD. Singular value decomposition was
used to reduce the SPECT data set to 20 vector scores, which were analyzed in
turn by DFA. Probable AD could be differentiated from questionable AD with a
sensitivity of 80% and specificity of 78%, and from converters to AD (mean
16.7 months) with a sensitivity of 80% against a specificity of 94%.
Using patients as their own controls, Kogure et al. (94) observed the
changes of ECD-SPECT scans in 32 patients with MCI as they developed AD
(mean follow-up 15 months). At baseline, MCI patients had bilateral reductions
in posterior cingulate gyri and precunei compared with healthy volunteers—
using SPM for analysis. Further decreases on follow-up and after conversion to
AD were observed in left hippocampus and left parahippocampal gyrus.
Selective perfusion decreases in posterior cingulate gyrus and precuneus in MCI
as measured with sophisticated techniques of analysis may be a marker
predicting future AD.
More recently, a follow-up study by Tanaka et al. (95) retrospectively
examined non-demented subjects with memory loss of whom half converted to
AD within two years. SPECT patterns at initial presentation suggested that both
converters and non-converters had medial temporal and posterior cingulate
reductions in perfusion (using SPM), with additional reductions in parietal and
anterior cingulate in the individuals who later developed AD. SPECT could be
useful in predicting conversion to AD, but typical AD perfusion deficits were also
observed in subjects who did not convert to AD.
Posterior cingulate perfusion was again abnormal in an HMPAO study by
Huang et al. (96) of 54 consecutively recruited MCI subjects, 17 of whom
progressed to AD within two years (DSM-IV criteria). Activity in left posterior
cingulate cortex was found to be decreased in those who progressed compared to
those who retained a diagnosis of stable MCI. In a further SPM analysis of
baseline HMPAO-SPECT images, Huang et al. (97) found bilateral prefrontal
perfusion increases and parietal perfusion decreases (left more than the right) in
the progressive group compared to the stable group and controls; the authors did
not find any differences between groups in posterior cingulate—in contrast to
their previous study.
An attempt to combine 123I-IMP-SPECT and CSF tau protein levels to give
higher predictive accuracy was reported by Okamura et al. (98). Perfusion in
posterior cingulate was found to be lower in both progressive MCI and probable
AD compared with cognitively normal subjects and the stable MCI group. In
addition, CSF tau levels were higher in the progressive MCI and AD groups
compared with normal subjects and stable MCI. Combining CSF and CBF data in
Perfusion Imaging with SPECT 221

a ratio [CSF (tau level)/CBF (posterior cingulate)], and using an optimum cut-off
value, the authors achieved a sensitivity of 89% for progressive MCI and a
specificity of 90% against stable MCI.
Encinas et al. (99) found that medial and anterior temporal perfusion
deficits were of no value in predicting conversion to dementia since both stable
and progressive MCI groups showed these deficits; this is in agreement with
Tanaka et al. (95), who also found medial temporal deficits to be of no predictive
value. In contrast, right and left pre-frontal deficits discriminated stable MCI from
AD converters with accuracy measured by area under ROC curve of 75–78%.
El Fakhri et al. (100) extended the study of Johnson, this time comparing,
both independently and in combination, MRI volume with HMPAO-SPECT
perfusion estimates. Using DFA, both baseline SPECT and MRI were highly
significant predictors of conversion to AD; they discriminated between
questionable AD and converters to AD. Assessed by area under the ROC curve
(AUC), MRI had relatively higher sensitivity and lower specificity than SPECT
and vice versa. Taking MRI and SPECT in combination increased accuracy
further—providing evidence that the two methods contribute independent
information. For SPECT, Johnson reported that the best areas to discriminate
between questionable AD and converters were: amygdala followed by superior
temporal sulcus, basal forebrain, caudal anterior cingulate, hippocampus, and
rostral anterior cingulate. In this study, the classification rules were derived from
the study subjects themselves—testing these classification rules in a new group of
patients would provide further insight as to the usefulness of the methodology.
Not all published SPECT studies have positive findings. A 3-year
prospective study by McKelvey et al. (101) followed up MCI subjects at 9–12
monthly intervals and found that half progressed to probable AD. Of the MCI
subjects, 36% were judged to have normal SPECT scans by visual assessment
and 64% abnormal scans; 52% of those with abnormal scans progressed to
dementia compared with 55% of those with normal scans. Since SPECT
abnormalities did not predict cognitive decline (MMSE score) or conversion to
dementia, McKelvey concluded that SPECT was not useful in predicting
conversion—at least with visual inspection of images.

Summary
SPECT evidence demonstrates that the longitudinal spectrum of decline from
normal cognitive function to MCI, and possible further decline to early AD is
most likely associated with reductions in cingulate gyrus, in particular posterior
cingulate, possible precuneus, hippocampal, and amygdala involvement as well
as temporo-parietal cortex. It would appear that visual inspection of images does
not discriminate between MCI and healthy volunteers. Regions of interest
analysis is not as powerful or statistically robust as the technique of SPM for
detecting subtle changes in small structures prone to partial volume effects.
222 Dougall and Ebmeier

CONCLUSION
Clinical criteria such as NINCDS-ADRDA for “probable AD” against the
neuropathological gold standard have been estimated to have an average sensitivity
of 81% and specificity of 70% (102). Comparing SPECT against clinical criteria
in a meta-analysis finds it to be a test with relatively higher specificity than
sensitivity. Therefore SPECT can be a useful adjunct to clinical criteria in the
differential diagnosis of dementia. Diagnostic accuracy can be improved with
SPECT for those demented patients not conforming with clinical criteria or who
have possible co-morbid symptoms of vascular disease.
Recent research indicates that sophisticated multi-variate statistical
processing of image data such as SPM enhances the diagnostic power of
SPECT. Using such methods could help improve prognostic accuracy—for
example, in patients presenting with memory problems or MCI. Visual inspection
does not appear to be sufficient to discriminate between dementia sub-types in the
early stages. While SPM is readily available and has been successfully
implemented in clinical practice, subtle abnormalities associated with early
disease are more likely to be identified with SPM or 3D-SSP (94,95).
To date most SPECT studies have been concerned with defining accurate
markers for dementia sub-types that could be readily adopted in clinical use.
Future research should investigate the value of SPECT in combination with other
predictor variables, such as biological and cognitive markers, to optimize
prediction and, by implication, therapeutic power.

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8
FDG PET: Imaging Cerebral Glucose
Metabolism with Positron
Emission Tomography

Karl Herholz
Wolfson Molecular Imaging Centre, University of Manchester,
Manchester, U.K., and Department of Neurology,
University of Cologne, Cologne, Germany

POSITRON EMISSION TOMOGRAPHY


Around 1975, positron emission tomography (PET) was used for the first time to
image brain function (1). It remained primarily a research tool for the next 20 years,
until its cost-effectiveness in oncology was proven and widely accepted (2). Since
then, PET has become widely available in major hospitals, and therefore now also
has the perspective for clinical use in neurology (3).
PET is based on the detection of coincident 511 keV gamma rays that
originate from positron electron pair annihilation. At the core of PET scanners are
scintillation detectors associated with coincidence electronics to detect the paired
511 keV gamma rays. Detectors made of scintillation crystals such as bismuth
germanate (BGO), sodium iodide, cesium fluoride, barium fluoride, gadolinium,
or lutetium oxyorthosilicate are typically arranged as hexagonal, octagonal, or
circular rings. The width of the detector ring is larger in whole body scanners than
in dedicated brain scanners, and both can be used for brain studies. Some older
scanners had axial fields of view (FOV) of less than 12 cm, but modern scanners
have an axial FOV of 12 to 15 cm at a nearly isotropic spatial resolution of 2.2 to
6 mm that allows scanning of the entire brain without repositioning. Image

229
230 Herholz

reconstruction from the recorded coincident events by filtered backprojection or


iterative procedures needs to take into account corrections for gamma ray
attenuation, scatter, and isotope decay.
Positron emitting isotopes typically have nuclear masses that are smaller
than those of stable isotopes, and most of them are very short lived. Compared to
other tracers that are commonly used in nuclear medicine, such as 99mTc (see
Chapter 7), higher doses (typically 185 to 740 MBq) can be applied at rather low
effective biological radiation doses (4). In addition, the efficiency of PET
scanners is substantially higher than that of single-photon emission computed
tomography (SPECT) scanners, resulting typically in high-count PET images
with excellent signal-to-noise ratio.
The physical half-lives of oxygen-15 (15O, 2 minutes) and carbon-11 (11C,
20 minutes) require an on-site cyclotron for tracer preparation, and therefore the
availability of these tracers usually is limited to highly specialized research
laboratories. Tracers labeled by fluorine-18 (18F, 109 minutes), however, can be
reasonably well stored and transported over several hours and therefore
increasingly become available by regional suppliers. The most widely used
tracer, not only for brain studies but also for cancer studies, is 18F-2-fluoro-2-
deoxy-D-glucose (FDG) (5), which is usually synthesized in a radiochemistry
laboratory by a stereospecific procedure based on nucleophilic substitution (6).

MEASUREMENT OF LOCAL CEREBRAL GLUCOSE METABOLISM


Glucose is the main substrate for energy supply of the brain. Measurement of
local cerebral metabolic rates of glucose (lCMRglc) by PET are based on the fact
that FDG is transported into tissue and phosphorylated to FDG phosphate, like
glucose, but does not undergo significant further metabolism. It accumulates in
the brain in proportion to lCMRglc, and after the first 10 to 20 minutes after i.v.
bolus injection the distribution of FDG in brain approximates lCMRglc. Thus,
images of FDG distribution recorded over 20 to 40 minutes within a time window
of 20 to 90 minutes after tracer injection are suited to represent lCMRglc.
Quantification of lCMRglc can be achieved by comparing measured cerebral
activity with the plasma input function, obtained by multiple arterial or
arterialized venous blood samples (7,8). FDG uptake of the brain is reduced in
hyperglycemia, which leads to poor signal-to-noise and problems with
measurement quantification. Thus, clinical FDG PET studies should be done
under fasting conditions at normoglycemia (9).
In normal subjects, typical resting state gray matter CMRglc values are in
the range of 40 to 60 mmol glucose/100 g/minute, and they are around 15 mmol
glucose/100 g/minute in white matter. There are regional differences, with
highest values in striatum and parietal cortex close to the parieto-occipital sulcus.
Some phylogenetically old brain structures such as the mesial temporal cortex
and cerebellum have metabolic rates below the gray matter average but are still
higher than normal white matter. There is probably a slight decline of lCMRglc
FDG PET 231

with age, most prominently seen in frontal cortex (10), but this has not been
confirmed in all studies.
Local brain glucose metabolism depends on brain function. During
functional activation lCMRglc increases, and it is decreased by sedation and
during slow-wave sleep (11). Thus, a valid measurement of lCMRglc must
include adherence to a defined functional state. Usually this is resting state
characterized by lying comfortably in the scanner in supine position in a quiet
examination room with dimmed lights. In many laboratories patients are asked to
close their eyes before beginning the examination and to keep them closed. It is
important to make subjects familiar with the surroundings prior to examination to
avoid any unnecessary level of anxiety and restlessness.

MAIN FINDINGS IN ALZHEIMER’S DISEASE


A consistent finding that has been noted since the earliest PET studies in
Alzheimer’s disease (AD) is hypometabolism affecting the temporal and parietal
association cortex (12–15), with the angular gyrus usually being the center of the
metabolic impairment (Fig. 1). Frontolateral association cortex is also frequently
involved to a variable degree (16–19). Primary motor, somatosensory, and visual
cortical areas are relatively spared. This pattern corresponds in general to the
clinical symptoms, with impairment of memory and associative thinking,
including higher-order sensory processing and planning of action, but with
relative preservation of primary motor and sensory function. These changes differ
from those of normal aging, which leads to predominantly mesial frontal
metabolic decline and may cause some apparent dorsal parietal and
frontotemporal (perisylvian) metabolic reduction due to partial volume effects
caused by atrophy (10,20–23).
More recent studies that used voxel-based comparisons to normal reference
data clearly showed that the posterior cingulate cortex (PCC) and the precuneus
are also impaired early in AD (24). The histochemical correlate of reduced FDG
is a pronounced decline in cytochrome oxidase activity in AD relative to controls,
whereas adjacent motor cortex does not show such differences (25). Metabolic
impairment of the posterior cingulate gyrus is not directly obvious by inspection
of FDG PET scans because metabolism in that area is typically above the normal
cortical average level (26). When impairment develops, it decreases to the level
of surrounding cortex but is not a visually apparent hypometabolic lesion. Thus,
this potential diagnostic sign is easily missed by standard visual interpretation of
FDG scans.
Automatic detection of abnormal metabolism on individual PET scans
requires appropriate reference data sets, spatial normalization of scans, statistical
algorithms to compare the voxels in scan data with normal reference data, and
suitable display of the results (Fig. 2). Signorini et al. (27) demonstrated that this
can be achieved by adapting the statistical parametric mapping software package
that was developed at the Wellcome Institute, London, U.K., originally for
232 Herholz

Figure 1 (See color insert.) Typical findings with FDG PET in Alzheimer’s disease.

analysis of activation studies (28). Some commercial software packages provide


similar approaches, but users should take care to check the validity of normal
reference data, statistics, and normalization procedures. Special non-linear
warping procedures for spatial normalization and surface renderings that provide
a quick overview of abnormalities have been developed for diagnosis of AD by
FDG PET scans by Minoshima et al. (29,30). This software package, 3D-SSP or
NEUROSTAT, has since been used successfully to identify metabolic alterations
in dementia and MCI by several groups (31–33).
Even more advanced approaches go beyond detection of abnormal voxels
and aim at automatic recognition of the typical anatomical distribution of
metabolic abnormalities in AD. Discriminant functions derived by multiple
regression of regional data achieved 87% correct classification of AD patients
versus controls (34), and a neural network classifier arrived at 90% accuracy (35).
The sum of abnormal t-values in regions that are typically hypometabolic in AD
FDG PET 233

Figure 2 (See color insert.) Automatic detection of abnormal metabolism, including


correction for age (PMOD Technologies Ltd., Adliswil, Switzerland).

has been used as an indicator with 93% accuracy (23). The same accuracy was
achieved even without image reconstruction by a special pattern extraction
technique from PET sinograms (36). Several discrimination functions combined
with principal component analysis or partial least-squares have been tested for
discrimination between AD and FTD in a sample of 48 patients with autopsy-
confirmed diagnosis and achieved accuracies between 80% and 90% (37).
Use of FDG PET to diagnose AD rests on the typical distribution of these
functional changes in brain. Impairment of local FDG uptake by itself, however,
is not specific for AD pathology and could potentially be caused by many other
disorders, e.g., ischemic lesions, when they just happen to affect those areas that
are typically affected in AD. Correspondingly, a high sensitivity in the order of
90% to 95% for FDG PET to detect AD has been documented in several studies,
but specificity for discrimination from other neurodegenerative disorders is lower
and in the order of 65–75% (38). Patients with Parkinson’s disease (PD) may
show a very similar metabolic impairment (39,40), even in absence of major
234 Herholz

cognitive deficits (41). These changes may even be reversible with successful
electrical stimulation of the subthalamic nucleus (42). With appropriate clinical
information, the “pseudo-AD” metabolic pattern should not be a major problem
because it is seen only in patients with long-standing PD who also have the
clinical motor symptoms of PD. Thus FDG PET scans always need to be
interpreted in the context of clinical history and symptoms. They also cannot
replace structural imaging by computed tomography (CT) or magnetic resonance
imaging (MRI), which are needed to recognize non-degenerative lesions such as
infarcts, tumors, hematomas, or hydrocephalus that may also lead to focal or
generalized cortical hypometabolism (43).
Patients with late-onset AD may show less difference between typically
affected and non-affected brain regions than usually seen in early-onset AD, which
could potentially lead to reduced diagnostic accuracy with FDG PET (44–46). This
could reflect the fact that at older age multifactor damage to the brain is likely to
accumulate, and, actually, also in neuropathological studies the proportion of
unclassifiable dementia is highest in the oldest old (see Chapter 11). Thus, in very
old multimorbid patients, FDG PET is probably of little diagnostic use, which is in
accord with general clinical wisdom.
Atrophy of hippocampal and parahippocampal structures is a main finding of
structural imaging in AD (see Chapter 6). Therefore one would expect also major
functional changes of lCMRglc in this brain area, but this has not generally been the
case (47). It is difficult to identify hippocampal metabolic impairment on FDG PET
scans because it exhibits lower resting metabolism than neocortex and pathological
changes are not obvious by visual image analysis. However, by coregistration of
MRI and standardized placement regions of interest onto the hippocampus in FDG
PET scans to increase spatial accuracy, a reduction, especially of entorhinal
metabolism, has indeed been observed in mild cognitive impairment (MCI) and AD
(48). In normal controls, lCMRglc in the neocortex is correlated with entorhinal
cortex lCMRglc across both hemispheres, whereas in AD patients these functional
metabolic correlations are largely lost (49). Metabolic impairment in the
parahippocampal gyrus had also been noted in a previous study during activation
using a simple memory task (50). As would be expected, hypometabolism in that
region is associated with memory impairment (51).
Atrophy may cause an apparent reduction in local FDG uptake due to
increased partial volume effects, even if local glucose metabolic rate in remaining
cortex was constant. Algorithms to correct for that effect have been applied in
AD (52,53) and concluded that partial volume effects contribute to apparent
hypometabolism but also that in spite of that local glucose metabolism remains
reduced in temporoparietal cortex even after correction. In contrast, the negative
correlation between frontal and perisylvian apparent glucose metabolism and age
seen in normal subjects could largely be resolved by partial volume correction (54).
Partial volume correction is not usually applied to clinical data because it requires
accurate coregistration with high-quality MRI, is very sensitive to slight mismatch
potentially producing severe artifacts, increases image noise, and is not needed for
FDG PET 235

diagnostic purposes because metabolic impairment and atrophy generally go in


parallel in neurodegenerative diseases. Readers of clinical PET images, however,
need to keep in mind that atrophy in otherwise healthy individuals, which most
frequently is most pronounced in frontal and upper parietal cortex as well as adjacent
to the major fissures (interhemispheric and Sylvian), may cause an apparent
reduction of FDG uptake in those areas that should not be confused with AD.
Both local cerebral blood flow (CBF) and local cerebral glucose
metabolism are coupled to neuronal function and are reduced in neuro-
degenerative disease (55). Thus, regional reductions of CBF similar to the
reductions of CMRglc can be observed with CBF imaging methods in AD and
other dementias. In clinical practice, especially SPECT is widely used for that
purpose (see Chapter 7) because it is less expensive and more widely available
than PET. In direct comparisons of the two techniques (56,57), SPECT always
showed inferior diagnostic discrimination which limits its power to detect early
cases. O-15 water PET has also been used to measure CBF in AD, but in a clinical
study its diagnostic power turned out to be much lower than in FDG studies (58).
Several receptor ligands with high blood-brain transfer rates provide information
about local CBF when early uptake is measured by appropriate kinetic modeling,
in addition to measurements of receptor binding potential (see Chapter 9). This
property of 11C-dihydrotetrabenazine, a vesicular monoamine transporter ligand,
has been used to demonstrate that it may also serve for detection of AD in addition
to its ability to detect degeneration of aminergic pathways (59).

LONGITUDINAL STUDIES AND TRIALS


Longitudinal studies have shown that the severity and extent of metabolic impairment
in temporal and parietal cortex increases as dementia progresses, and frontal
involvement becomes more prominent (60,61). The decline of metabolism is in the
order of 16% to 19% over three years in association cortices, which contrasts with an
absence of significant decline in normal control subjects (62). Our own longitudinal
data indicate that the total cortical metabolic impairment in AD increases at a constant
rate to reach the maximum observed impairment after eight to nine years. Metabolic
asymmetries and associated predominance of language or visuospatial impairment
tends to persist during progression (63,64). Metabolic rates in basal ganglia and
thalamus remain stable and are unrelated to progression (62). Thus in late dementia,
there is typically a pattern of severe hypometabolism in temporoparietal and frontal
association cortices, with a relative sparing of primary cortical areas.
In a few clinical trials, the effects of nootropic drugs on cerebral glucose
metabolism in AD have been explored. An increase of CMRglc has been
demonstrated with piracetam (a putative enhancer of cerebral metabolism) (65),
propentofylline (an adenosine uptake blocker) (66), and metrifonate (a long-
lasting cholinesterase inhibitor) (67); the latter two studies showed an associated
improvement in certain cognitive measures. Reductions of CMRglc were found
with physostigmine (68), however, in spite of improved attention. Studies with
236 Herholz

other centrally acting cholinergic drugs generally did not show a main treatment
effect on cerebral glucose metabolism, but when subjects were divided into
responders and non-responders the former showed a metabolic increase that was
mostly located in frontal cortex and associated areas (69–71). These trials studied
the effect of drugs on CMRglc but did not address whether drugs might slow
disease progression. To perform such a trial, it has been estimated that 24 patients
with Alzheimer’s disease per active treatment and placebo group would be
needed to detect a significant 33% treatment response with p!0.05 and 80%
power in a one-year, double-blind, placebo-controlled treatment study (72).

EARLY DIAGNOSIS OF ALZHEIMER’S DISEASE


The current main issue for diagnosis of AD with PET is early diagnosis when
patients present with a mild cognitive deficit, but before clinical dementia arises.
This may be of particular importance for subjects with a high premorbid cognitive
level, who can experience a substantial decline of cognitive function before
reaching the lower normal limit of standard neuropsychological tests. There are
many indications that this will be possible with FDG PET. Even at an
asymptomatic stage, impairment of cortical glucose metabolism has been
observed in subjects at high risk for AD due to family history of AD and
Apolipoprotein E (ApoE) 34 homozygosity (73,74) and this abnormality is seen
decades before the likely onset of dementia (75). In middle-aged and elderly
asymptomatic ApoE 34-positive subjects, temporoparietal and posterior cingulate
lCMRglc declines by about 2% per year (76). The potential for primary
intervention studies employing FDG PET as an outcome measure in samples of 50
and 115 cognitively normal 34 heterozygotes has been documented (77). After
onset of manifest dementia, 34-positive patients still had more severe metabolic
deficits in some studies (78,79). Mesial temporal hypometabolism is often seen in
patients with severe memory impairment, including MCI and other amnesic
disorders (48,80–82). Depending on subject selection, it may also have prognostic
impact. A longitudinal study of cognitively normal subjects indicated that
cognitive decline to MCI within three years, follow-up is related to metabolic
reductions in entorhinal cortex at entry, independent of 34 status (83). Progression
to dementia usually is associated with additional metabolic impairment in
temporoparietal and posterior cingulate cortex (82,84).
Nondemented patients with MCI may already show the metabolic impair-
ment of association cortices that is characteristic of AD. MCI patient groups when
compared to normal controls typically show significantly impaired metabolism
(24,33). In our multicenter database (85), about one-third of patients clinically
diagnosed as having MCI have significant metabolic impairment of association
cortex. Severe impairment was observed in one subject with a presenilin-1
mutation (A431V) who clinically still was at the MCI stage of AD (86).
Data are accumulating that the presence of the AD metabolic pattern in MCI
predicts conversion to clinical dementia of Alzheimer type, and therefore indicates
FDG PET 237

“incipient AD.” The predictive power of posterior cingulate metabolic impairment


was documented by Minoshima and colleagues as early as 1997 (24). We studied
31 patients with cognitive deficits, mostly limited to the memory domain, with
Mini-Mental State Examination (MMSE) scores of 24 or higher and not yet
fulfilling the criteria of probable AD. They were therefore diagnosed as “possible
AD,” and most of these patients would have fulfilled the criteria of MCI (which
were not yet used by us at that time). We found that 60–70% of those patients who
already had moderate or severe metabolic impairment of association cortices in
FDG PET declined on MMSE by three points or more within two years (mostly
leading to clinical dementia), whereas only 10–20% of patients without such
metabolic impairment had that decline (87). Similar conversion rates (seven of 10
within three years in subjects with abnormal metabolism, three of 10 with normal
lCMRglc) were reported by Berent et al. (88). In a three-year follow-up study of 20
MCI patients, Arnaiz et al. (89) observed nine converters. The two variables that
most effectively predicted future development of AD were lCMRglc from the left
temporoparietal area and performance on the block design. These combined
measures gave an optimal 90% correct classification rate, whereas only lCMRglc
or neuropsychology alone gave 75% and 65% correct classification, respectively.
In a one-year study of 22 patients, eight converters had reduced lCMRglc in PCC
(compared to normal controls and non-converters) and temporoparietal
cortex (32). After one year, additional bilateral reduction of lCMRglc in prefrontal
areas along with a further progression of the abnormalities in the parietal and
posterior cingulate cortex was observed in converters, whereas non-converters had
unchanged metabolism. Among 17 patients followed for 18 months, seven
converters had significantly lower lCMRglc in right temporoparietal cortex than
non-converters (90). In a recent study, Anchisi et al. (91) have demonstrated that by
neuropsychological testing alone one can identify subjects who are likely not to
progress to dementia because their memory deficit is relatively mild, thus
providing a high negative predictive value with regard to progression. However,
prediction based on neuropsychological testing is less reliable for MCI patients
with severe memory impairment. In these patients, FDG PET adds significant
information by separating those who will progress within the next from those who
will remain stable.
Few studies so far compared FDG PET with other biomarkers. The
combination with ApoE 34 has been addressed by Mosconi et al. (92) in 37
patients who were followed over one year. There were eight converters, and
inferior parietal cortex lCMRglc predicted conversion with 84% accuracy,
compared to 62% prediction accuracy of 34 status. PET prediction accuracy was
best (94%) within the 34-positive group. In a series of 30 patients who were
followed over 16 months, the positive and negative predictive values of FDG
PET for progression to AD were 85% and 94%, respectively, whereas
corresponding values for the ApoE4 genotype were 53% and 77% only (93).
By combination of the two indicators, predictive values increased to 100% in
subgroups of patients with concurrent genetic and metabolic findings. When
238 Herholz

comparing phosphorylated tau protein in CSF with FDG PET in MCI, Fellgiebl
et al. found similar findings with both tests (94). Seven of 16 patients had
increased phosphorylated tau levels, and six of them also had AD-typical FDG
PET findings.

CLINICAL DIAGNOSTIC USE OF 18F-2-FLUORO-2-DEOXY-


D-GLUCOSE POSITRON EMISSION TOMOGRAPHY
IN ALZHEIMER’S DISEASE
FDG PET is an expensive procedure, but with its broader use mainly in oncology,
costs for the tracer and scanning have been declining from initial levels of several
thousand dollars and are now in the order of US $1000 or even lower in some
places. There have been model calculations indicating that it could be cost
efficient under certain circumstances (95), but studies do not yet fulfill generally
accepted standards. Although the promise of FDG PET in clinical diagnosis was
recognized by the Quality Standards Subcommittee of the American Academy of
Neurology (96) and other professional organizations (97,98), its routine use had
not been recommended until 2004 in most countries. Meanwhile, the US
Medicare and Medicaid services refund FDG PET studies in dementia if certain
conditions are met, including difficulty of clinical diagnosis because of atypical
cause or presentation and FDG PET (99). Similar regulations are active in
Switzerland, whereas many other countries do not yet have obligatory refunding
regulations.

DEMENTIA WITH LEWY BODIES


Patients with dementia with Lewy bodies (DLB) often clinically have fluctuating
levels of attention and consciousness, visual hallucinations, and may develop the
motor features of Parkinson’s disease (100,101). Reduced FDG uptake is found
to be very similar to AD, but also in primary visual cortex which is usually spared
in AD (Fig. 3) (102–105). The impairment of glucose metabolism in visual cortex
may well be the correlate of the impairment of visual processing and visual
hallucinations. The diagnostic reliability of the finding is not yet clear and could
potentially be confounded by reduced occipital FDG uptake secondary to vision
impairment. Another characteristic finding in DLB is the reduction of F-18-
DOPA uptake in the putamen that has been described in DLB (106,107) but is
absent in AD (108).

FRONTOTEMPORAL DEMENTIA
Frontotemporal dementia (FTD) is characterized clinically by leading changes in
personality and behavior, such as apathy or disinhibition, whereas memory
impairment may be absent or less prominent (109). In practice, FTD is readily
identified on FDG PET scans by a distinct frontal or frontotemporal metabolic
FDG PET 239

Figure 3 (See color insert.) Asymmetric frontotemporal atrophy and metabolic impair-
ment in frontotemporal dementia.

impairment that typically is quite asymmetrically centered in frontolateral cortex


and the anterior pole of the temporal lobe from where it may extend to other
association areas (110–113). Mesial frontal metabolic impairment can be found
in nearly every case of FTD (114). Apathy was found to be associated with a
prevalent dorsolateral and frontal medial hypometabolism, whereas disinhibition
demonstrated a more severe hypometabolism in limbic structures (115).
Frequently there is also unilateral focal atrophy of the frontal and temporal
lobe corresponding to the metabolic deficit.
Automatic methods have been explored in order to distinguish between
FTD and AD. An analysis using multivariate methods such as principal
components analysis and partial least squares regression achieved over 90%
accuracy in a sample of 48 patients (116). Preliminary results of a prospective
study indicated that FDG PET may be more accurate than clinical judgment in
predicting histopathological diagnosis (117). This discrimination is of primary
interest in dementia patients with presenile onset, whereas there may be
substantial overlap of the FTD pattern with frontotemporal impairment and the
AD pattern with predominant temporoparietal impairment, especially in senile
dementia patients (118).
240 Herholz

Semantic dementia is usually regarded as a variant of FTD with similar


histopathological features and tau protein deposition (119). Its main clinical
symptoms are the progressive inability to comprehend common concepts, often
associated with fluent aphasia, but there are less emotional disturbances and
repetitive, compulsive behaviors than in FTD (120). Metabolic impairment is
similar to FTD (121) but appears to be more focused on the left temporal rather
than the frontal lobes (122).
Primary progressive (non-fluent) aphasia is another related syndrome
associated with left frontal and temporal hypometabolism (123–127) that may
also affect additional brain areas to a lesser degree, suggesting that it is not a
strictly focal impairment. A similar condition also seems to exist for the right
hemisphere, clinically consisting of progressive prosopagnosia (128). Some cases
seem to be histopathologically related to FTD, whereas others may represent a
posterior variant of AD (129).
FTD can be differentiated from corticobasal degeneration with pre-
dominant parietal metabolic reduction (130), although histopathological features
may overlap (131). Frontal metabolic impairment is also part of many other
diseases and conditions, including progressive supranuclear palsy (in combination
with midbrain impairment) (132), spinocerebellar atrophy (133), and cocaine
abuse (134). Moderate frontal dysfunction and hypometabolism is also observed in
many psychiatric disorders, and therefore cannot be regarded as a specific
diagnostic feature.

VASCULAR DEMENTIA
Diagnosis of vascular dementia (VaD) is easily made in the case of multiple
cortical infarcts multi-infarct dementia, but may be a difficult issue in cases with
severe microvascular changes but without major cortical infarcts. There is
currently no consensus about clinical criteria, and correspondence between
existing criteria (e.g., ICD-10, DSM-IV, NINDS-AIREN, CAMDEX) is poor
(135,136). Several studies have suggested that a diffuse global reduction of
cerebral glucose metabolism is a typical finding in VaD (Fig. 4), and that the degree
of that reduction in association cortex is similar to that seen in AD (137,138). In
cases without gross cortical infarcts, the effects of WML are generalized and
frontal hypometabolism correlates with memory and global impairment, cognitive
as well as executive function. The effects of subcortical cerebrovascular disease
appear to converge on the frontal lobes but are diffuse, complex, and of modest
magnitude (139). Thus, the contrast between metabolic impairment in association
areas and preserved metabolism in primary areas, basal ganglia, and cerebellum,
which is typical for AD but not for VaD, seems to provide some distinction with
FDG PET between these two types of dementia (137). Only large white matter
lesions may cause moderate focal reductions of overlying cortical blood flow and
metabolism (140), and it therefore is likely that presence of the typical metabolic
AD pattern in demented patients with multiple deep white matter lesions does
FDG PET 241

Figure 4 (See color insert.) Global reduction of cerebral glucose metabolism in vascular
dementia.

indicate that this is predominant AD rather than VaD (141). However, a definitive
distinction between VaD and AD or the relative importance of vascular and
Alzheimer-type lesions in patients who have signs of both often remains uncertain,
even after autopsy and histopathological examination (see Chapter 11).
VaD may also be caused by strategic infarcts in structures that are essential
for integrating higher cognitive functions, especially the thalamus. Even small
thalamic lesions may lead to considerable deactivation of cortex, as demonstrated
by FDG PET (142–144).

CREUTZFELDT–JAKOB DISEASE
Clinically, this disease is characterized by rapidly progressive dementia, often
accompanied by insomnia, myoclonus, and other extrapyramidal disorders. In all
cases reported so far, cerebral glucose metabolism was severely reduced in a
multifocal fashion (145–149).

SUMMARY
Imaging cerebral glucose metabolism with PET has a proven potential for early
diagnosis of AD at the clinical stage of MCI. The typical regional pattern of
metabolic impairment differs between major neurodegenerative diseases that
may cause dementia. Thus, FDG PET also has the potential to improve early and
242 Herholz

differential diagnosis, and it may be used to monitor disease progression and


treatment effects. Because of its widespread use in oncology it is now available in
most major hospitals. Due to its high cost it can currently not be recommended as
a routine diagnostic tool, but PET continues playing a major role in clinical and
scientific studies and as a supplementary diagnostic test in selected cases.

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9
Imaging of Neurotransmitter Systems
in Dementia

Steen G. Hasselbalch
Neurobiology Research Unit and Memory Disorders Research Group,
Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark
Gitte M. Knudsen
Neurobiology Research Unit, Copenhagen University Hospital,
Rigshospitalet, Copenhagen, Denmark

INTRODUCTION
Chemical neurotransmission as a tool for cell communication in the human brain
relies on the interaction of neurotransmitters acting on their target receptors. At
the moment, over 100 neurotransmitters with 300 corresponding receptors have
been identified. Malfunction of some of these neurotransmitter systems in
degenerative disorders may provide part of the pathophysiological basis for the
varying symptomatology found in these disorders.
Animal models, peripheral measures of receptors, neurotransmitters and their
metabolites, and clinical effects of neuropharmacological treatment are crucial to
improving our understanding of the underlying pharmacology involved in aging and
cognitive dysfunction. As opposed to these indirect approaches, molecular imaging
of the neurotransmitter systems with positron emission tomography (PET) or single
photon emission computed tomography (SPECT) is able to characterize the
functional state of human central receptor systems in vivo. PET is a powerful
technique for investigating human brain physiology and pathophysiology. When
used with appropriate radioligands, PET can reveal the distribution of
neuroreceptors in the living human brain, and their interactions with neurotrans-
mitters or administered drugs. PET radioligands suitable for these purposes give
large signals that are quantifiable in terms of robust measures of regional receptor

253
254 Hasselbalch and Knudsen

concentration. They should also be selective and sensitive to occupancy of receptors


by administered psychoactive drugs, and some of them are even sensitive to
competition with an endogenously released neurotransmitter. Though some
receptors have been imaged successfully with PET, many others lack effective
radioligands. An overview of published data on some radiopharmaceuticals that
with reasonable success have been applied in humans is given in Table 1.

Table 1 Examples of Ligands Used for Neuroreceptor Imaging in Humans


Transmitter involved Target Examples of ligands

Monoamines Vesicular monoamine (C)11C-DTBZ


transporter 2
11
Monoamine oxidase A C-clorgyline
11
Monoamine oxidase B C-L-deprenyl
18
Dopamine Dopa synthesis F-dopa
11
Dopamine reuptake site C-CFT
123
I-PE2I/11C-PE2I
11
D1 C-NNC112
11
C-SCH23390
[11C]-NNC 756
11
D2-like C-raclopride
123
I-IBZM
11
Serotonin Serotonin synthesis C-5-hydroxytryptophan
11
Serotonin reuptake site C-DASB
11
5HT1A C-WAY-100635
11
5HT2A C-MDL100907
18
F-altanserin
123
I-R91150
123
Acetylcholine Vesicular acetylcholine I-IBVM
transporter
11
Cholinesterase activity C-MP4A
11
C-PMP
11
Muscarinic C-NMPB
11
C-TRB
18
M2 selective F-FP-TZTP
11
Nicotinic C-nicotine
a4b2 6-18Fluoro-A-85380
11
Opioid Mu C-carfentanyl
11
Mu/delta/kappa C-diprenorphine
11
Histamine H1 C-pyrilanine
11
C-doxepine
11
GABA a1 C-flumazenil
123
I-iomazenil
11
a5 C-Ro154513
11
Glutamate NMDA C-MK801
11
Microglia activation t3-benzodiazepine C-PK 11195
Imaging of Neurotransmitter Systems in Dementia 255

Molecular imaging is likely to provide a better insight into the malfunction


of the different transmitter systems, and at some point it may also lend itself to
detection of gene expression and monitoring of gene therapy, thereby holding a
promising potential for future research areas with the ultimate goal to better
understand and treat the underlying pathophysiology in different neuro-
degenerative disorders.
The interaction between the functioning receptor systems render it likely
that the affection of one transmitter system is associated with malfunction of
other receptor systems. Very little is, however, known about such interactions
and their impact for the clinical picture of different neurodegenerative
disorders.
In the following, a review of neurotransmitter dysfunction in the most
prevalent dementia disorders will be given. We focus on Alzheimer’s disease
(AD), but will briefly touch upon dementia with Lewy bodies (DLB) and
frontotemporal dementia (FTD). Neurotransmitter imaging shows great potential
in diagnosing Parkinson’s disease (PD) and related disorders. Although several of
these movement disorders are associated with cognitive dysfunction, they fall
outside the scope of this chapter. In the daily clinical practice, differential
diagnosis between primary neurodegenerative diseases and cognitive dysfunction
due to cerebrovascular disease is often difficult, and clinical application of
neurotransmitter imaging will also briefly be touched upon.

NORMAL AGING
Although the amount of receptors varies considerably between individuals (1) it
is well established that a number of different receptor systems decline with age—
frequently at a rate that varies between the different brain areas. This has been
demonstrated both in postmortem human and in brain imaging studies.
Postmortem studies have shown a loss of dopamine transporters amounting
to 5–7% per decade in striatum (2) and that dopaminergic D1 and D2 receptors
in striatum also decline with age (3). These findings have been corroborated
in imaging studies where van Dyck et al. found a 6.6% decrease per decade in
striatal dopamine transporters (Fig. 1) (4), and postsynaptically, dopamine D1 and
D2 receptors in the same areas seem to decrease at the same rate (5,6).
For the serotonergic system, postmortem studies showed evidence for a
decrease in 5-HT2A receptor binding with age (7). A gender-specific effect of age
on 5-HT1A receptor density was initially reported with an inverse relationship
between 5-HT1A receptor density and age in men, but not in women (8). PET
data from a large sample of healthy control subjects have, however, not been
able to confirm this finding, and the density of 5-HT1A receptors seemed to be
remarkably constant within the age span from 23 to 54 years (9). Neocortical
5-HT2A receptor binding, by contrast, was found to decline by 6% per decade in a
large group of healthy controls aged between 21 and 79 years (Fig. 2) (10),
corroborating previous findings in a smaller sample (11).
256 Hasselbalch and Knudsen

Figure 1 (See color insert.) Loss of dopamine transporters with age. Dopamine trans-
porters measured using SPECT and [123I]-PE2I in a 21-year-old healthy male (left) and in a
70-year-old healthy male (right). Source: Courtesy of the Neurobiology Research Unit,
Copenhagen, Denmark.

For the glutamatergic NMDA receptor complex a similar decrease has


been reported in postmortem frontal cortex (12,13). Since no appropriate gluta-
matergic PET tracers are available yet, imaging data cannot add any further
information.

2
BP1

0
0 10 20 30 40 50 60 70 80 90
Age (years)

Figure 2 The correlation between age and the averaged binding potential of 5-HT2A
receptors for all cortical brain regions (measured with PET and [18F]-altanserin in healthy
controls). The regression coefficient corresponds to an average decrease of 5-HT2A
receptors of about 6% per decade. Source: From Ref. 10.
Imaging of Neurotransmitter Systems in Dementia 257

Postmortem data have shown a significant decrease in M1 and M2


receptors as well as in nicotinic receptors in the cerebral cortex (14,15), whereas
one PET study using 18FP-TZTP as a selective ligand for the M2 receptor found a
higher binding throughout the brain in older controls as compared to younger
controls (16). One possible explanation for this could be a lower concentration of
acetylcholine in the synapse in older controls. Data have been less consistent for
the presynaptic marker choline acetyltransferase (ChAT) that catalyzes the
biogenesis of acetylcholine (17,18). Thus in general, it seems that several—but
not all—receptor systems decline with age, but the rate of decline varies between
systems. Further, within one system it may also differ between different brain
regions, which necessitates a close matching between control and patient groups.
Event though most imaging studies so far have not taken into account that brain
atrophy associated with age invariably will be associated with the so-called
partial volume effect and thereby a tendency for underestimation of the receptor
binding in a given brain region, the decrease in receptor binding with age is not
explained fully by these effects.

ALZHEIMER’S DISEASE
The earliest and most prominent symptom of AD is problems with episodic
memory due to failure of learning and retention of new material. Therefore
neurotransmitter systems involved in memory processes, such as the cholinergic
system, have generally been studied in more detail than other systems, although
lack of specific ligands for this system have hampered in vivo imaging data.
However, behavioural and neuropsychiatric symptoms are frequently occurring in
the course of AD, and even extrapyramidal symptoms are encountered in the
disease, sometimes rendering a firm diagnosis difficult. One of the purposes of past
and recent neurotransmitter imaging studies has been to aid clinicians in improving
diagnostic accuracy by revealing disease-specific neurotransmitter changes in the
most prevalent neurodegenerative disorders, including AD. Another purpose has
been to better understand the pathophysiology behind certain symptoms with the
ultimate goal of improving treatment. In the following, only receptor systems
considered of importance for AD are included. A summary of these main findings
is given in Table 2. Data based both on postmortem studies and in vivo emission
tomography studies will be reviewed and the potential impact of disturbances
within the receptor systems discussed.

Cholinergic Transmitter System


The most prominent and specific neurotransmitter deficit in AD is found in the
cholinergic system. According to current concepts, cholinergic neurons are
involved in specific forms of attention, thereby serving a modulatory function in
cognition by optimizing cortical information processing (47). Cholinergic
receptors comprise transmitter-gated ion channels [nicotinic acetylcholine
258

Table 2 Single Photon Emission Computed Tomography and Positron Emission Tomography Neuroreceptor Studies in Alzheimer’s Disease
Number of Number of
Receptor system controls patients Ligand Results Author

Cholinergic
Muscarinic receptors 11 14 [123I]-QNB Y Weinberger 1991 (19)
4 8 [123I]-QNB 4 (Y) Wyper 1993 (20)
5 5 [123I]-IDEX Y Claus 1997 (21)
18 18 [11C]-NMPB Y Yoshida 1998 (22)
18 6 [11C]-NMPB 4 Zubieta 2001 (23)
Cholinergic
Nicotinic receptors 3 8 [11C]-nicotine Y (high affinity) Nordberg 1995 (24)
Cholinergic
Acetylcholin-esterase 8 5 [11C]-MP4A Y Iyo 1997 (25)
4 4 [11C]-MP4A Y Herholz 2000 (26)
14 28 [11C]-MP4A Y Shinotoh 2000 (27)
26 14 [11C]-PMP Y Kuhl DE 1999 (28)
GABAergic
GABAA receptors (central 5 6 [123I]-iomazenil Y Soricelli 1996 (29)
benzodiazepine receptors) 4 8 [123I]-iomazenil Y Fukuchi 1997 (30)
6 13 [11C]-flumazenil 4 Meyer 1995 (31)
5 5 [11C]-flumazenil 4 Ohyama 1999 (32)
Hasselbalch and Knudsen
Serotonergic
5-HT2A receptors 37 9 [11C]-setoperone Y Blin 1993 (33)
10 9 [18F]-altanserin Y Meltzer 1999 (34)
26 9 [123I]-R91150 Y Versijpt 2003 (35)
Glutamatergic
NMDA receptors 5 5 [11C]-MK 801 ([) Brown 1997 (36)
Microglia marker (peripheral
benzodiazepine receptors)
8 8 [11C]-PK11195 4 Groom 1995 (37)
15 8 [11C]-PK11195 [ Cagnin 2001 (38)
9 10 [123I]-PK11195 [ Versijpt 2003 (39)
Dopaminergic
D2 (striatum) 9 15 [123I]-IBZM Y Pizzolato 1996 (40)
10 10 [11C]-raclopride 4 Kemppainen 2000 (41)
5 10 [11C]-raclopride Y Tanaka 2003 (42)
D2(D3) (hippocampus) 11 14 [11C]-FLB 457 Y Kemppainen 2003 (43)
Dopaminergic
D1 (striatum) 8 10 [11C]-NNC 756 Y Kemppainen 2000 (41)
Imaging of Neurotransmitter Systems in Dementia

Dopaminergic
DAT 12 15 [18F]-dopa 4 Tyrrell 1990 (44)
15 12 [11C]-betaCFT Y Rinne 1998 (45)
33 34 [123I]-FP-CIT 4 O’Brien 2004 (46)
259
260 Hasselbalch and Knudsen

receptors (nAChR)] and G protein-coupled muscarinic acetylcholine receptors


(mAChR), which are located both presynaptically and postsynaptically. nAChRs
belong to a superfamily of homologous receptors including GABA, glycine, and
5-HT3 receptors. Each nAChR consists of five membrane spanning subunits
arranged around an aqueous channel in the center. So far, nine different types
have been identified and cloned (48). In vitro studies of human autopsy brain
tissue suggest heterogeneity regarding nAChRs which can be rationalized into
three different types of binding sites: super-high, high, and low affinity sites
corresponding to the a3, a4, and a7 subunits (15,49,50). The nAChRs are
expressed in low density in the human brain, as compared to the muscarinic
receptors. The distribution of nAChRs is relatively homogenous and not
restricted to defined cholinergic pathways, although it is more dense in some
brain regions including thalamus, cortex, and basal ganglia. Nicotinic receptors
are distributed to influence many neurotransmitter systems at more than one
location, and the broad, but sparse, cholinergic innervation throughout the brain
enables nAChRs to become important modulators of neuronal excitability (51).
An abundant amount of research has provided evidence that the nAChRs are
involved in several important functional processes including cognition, learning,
memory, and arousal (52). Substantial evidence also indicates the involvement of
the nicotinic cholinergic system in the pathology of AD (53). Drugs targeting
these sites may not only have a positive effect on cognitive function, but also have
additional therapeutic benefits in terms of restoring the hypoactivity in the
excitatory amino acid pyramidal system and even slowing the emergence of
AD pathology.
Nicotinic Receptors
A consistent and severe loss of nicotinic receptors in AD was reported in early
postmortem studies (54,55). The major loss in cortical nAChRs in AD appears to
occur in the a4-b2 nAChR subtype (56), and a study on nAChR expression in the
frontal and temporal cortex of AD patients has demonstrated that both the numbers
of a4- and a7-immunoreactive neurons and the quantitative amount, in particular
of the a4 protein, are markedly decreased in AD (57).
Until recently only one tracer ligand was available for PET studies, namely
11
C-nicotine (58). 11C-nicotine imaging suffers from high nonspecific binding
and rapid metabolism; but even so, 11C-nicotine binding seems to agree well with
the distribution of nAChR observed in vitro in human postmortem brain tissue
(24,59,60). Significant reductions in 11C-nicotine binding were found in AD
patients in frontal and temporal cortices and in hippocampus when compared to
age-matched controls, and the changes were associated with cognitive function
(24,61). 11C-nicotine-PET has also been used to evaluate the effect of cholinergic
drug treatment: after three months of treatment with the AChE inhibitor tacrine,
the binding of 11C-nicotine was increased in temporal cortex in AD patients (61)
suggesting a restoration of nAChR with treatment, which may explain part of the
therapeutic benefit of AChE inhibitors (62). Recent reports point towards the a7
Imaging of Neurotransmitter Systems in Dementia 261

subtype as a target for specific amyloid binding (63), but at present there are no
suitable tracers available for in vivo imaging of this subtype. Recently,
radiohalogenated analogs of 3-(2(S)-azetidinylmethoxy)pyridine (A-85380)
were used successfully for the in vivo visualization of alpha4beta2 nicotinic
receptors in the human brain with PET/SPECT (64), but so far, data for 6-18F-
fluoro-A-85380 PET imaging in AD are still missing.
Muscarinic Receptors
Although some biopsy and autopsy studies point to nAChR as the receptor
subtype most affected in AD with relative sparing of the mAChR subtype (55,65),
a selective loss of the subtype M2 compared to M1 has been shown (66,67).
Recent studies suggesting a role for muscarinic agonists in regulating the
production of Ab-amyloid raise the possibility that selective M1 agonists could be
useful in treating not only the symptoms, but also the underlying cause(s) of AD.
Unfortunately, due to several methodological problems, imaging of mAChR has
so far revealed contradictory results, and has not been able to demonstrate the
selective loss of M2 as compared to M1 receptors obtained from postmortem
studies. Using non-selective M1/M2 mACh receptor tracers, PET and SPECT
studies have rather consistently shown a reduction in mAChR in AD (19–22).
Thus, using 11C-NMPB and PET, Yoshida et al. found significant reductions in
mAChR binding in the parietal cortex of AD patients as compared to controls.
However, taking some of several methodological problems into account, Zubieta
et al. could not demonstrate a reduction in mAChR in six mild to moderate AD
patients (23). In healthy APOE 34-positive subjects, a higher M2 receptor binding
as assessed with 18F-TZTP PET has been demonstrated as compared to APOE 34-
negative subjects, presumably because of a lower synaptic concentration of
acetylcholine in these individuals (16). So far, no studies have been conducted
with selective muscarinic tracers in AD, but future studies should reveal mAChR
subtypes are differentially affected in vivo in AD.
Choline Acetyltransferase and Acetylcholinesterase
Marked reductions in the enzymes ChAT and acetylcholinesterase (AChE) and
cholinergic neuronal loss have been reported in postmortem studies as well as in
biopsies from AD brains, and this cholinergic dysfunction correlates with the
cognitive function (65,68,69). Currently, there are no tracers to map the key
cholinergic enzyme ChAT, but AChE tracers are in use. Pappata et al. used a
labeled inhibitor of AChE (11C-physostigmine) in normal subjects to demonstrate
tracer distribution corresponding to that found in postmortem studies (70). The
PET tracer 11C-MP4A serves as an in vivo AChE substrate so that the local rate of
radiotracer hydrolysis can be followed by PET. Based on this methodology,
AChE levels were reported to be decreased by 30–40%, especially in
temporoparietal regions in 1997 by Iyo et al. (25), and this finding was later
confirmed by Kuhl and coworkers using 11C-PMP in patients with mild to
moderate AD (28). These data agreed with earlier postmortem data with regard to
262 Hasselbalch and Knudsen

normal relative cerebral distributions, absence of large age-effect in normal


aging, and deficits in AD (see above). AChE activity has, however, been found to
be more globally reduced in the brain in contrast to the more selective reductions
in CMRGlc and CBF in the temporoparietal association areas (Fig. 3) (26).
Further, Kuhl et al. demonstrated a 50% displacement of the radiolabeled ligand
following acute treatment with physostigmine, suggesting that this tracer can be
used to assess the efficacy of AChE inhibitors which currently are the most
widely used drugs in AD (28). Using the same method as Iyo et al. Shinotoh et al.
found significantly reduced AChE activity in neocortex, hippocampus, and
amygdala in 28 AD patients (71). In seven of these patients where PET scans
were repeated after two years, a progressive loss of AChE activity was
found (27). Thus, PET studies using AChE tracers might be used to evaluate and
monitor the concentration of AChE in AD patients, thereby assessing drug
efficacy objectively. In particular, the objective demonstration of a lack of
efficacy of AChE inhibitors in some patients and in different disease severity
states could be useful knowledge to guide the clinician.
Elucidation of whether AChE acting drugs applied in AD exert their effect
by an inhibitory activity on AChE or by other complex actions may be
investigated by following the pharmacokinetics of the drug in the target organ by
PET. [11C]N-methyl-labeled tacrine (MTHA) has for example been shown to
cross the blood-brain barrier easily in healthy human volunteers and to be highly
concentrated in the brain, though the regional brain distribution of [11C]MTHA
does not correspond to that of in vivo AChE concentrations (72).

Figure 3 (See color insert.) Acetylcholinesterase activity in the central nervous system
evaluated with C11-MP4A. Source: From Ref. 26.
Imaging of Neurotransmitter Systems in Dementia 263

Central Benzodiazepine Receptor Imaging


The central benzodiazepine receptor (BZR) is part of the major inhibitory
neurotransmitter system g-aminobutyric acid (GABAA) receptor complex.
GABAA is widely expressed in cortical neurons, and is considered a marker
for neuronal integrity and viability.
Although one study found a modest 22% reduction in frontal cortex (73),
most postmortem studies have found GABAA receptor subtypes relatively
preserved in the hippocampus in AD patients in different disease stages, despite
marked loss of neurons in this area (74,75). On the other hand, SPECT studies
using [I-123]labeled iomazenil have shown reduced cortical BZR in temporo-
parietal areas (76,77), and iomazenil may even be a more sensitive marker for
cortical dysfunction than the cerebral blood flow tracer [Tc-99m]HMPAO (30,78).
In contrast, a PET and [C-11]-flumazenil study by Meyer et al. showed relatively
preserved benzodiazepine binding sites in AD (31). In a later PET-flumazenil
study it was also found that the BZR reduction was less prominent than the CBF
suppression as measured by [15-O]H2O (32). The apparent discrepancy between
the PET and SPECT results of BZRs may be caused by differences in the two
tracers, and at this time it is not clear whether the moderate reductions in BZR
density is more sensitive or specific than conventional cerebral blood flow imaging
in the differential diagnosis between different degenerative disorders.

Serotonergic Transmitter System


Animal and human studies suggest that serotonin (5-HT) is linked to many functions,
such as cognition, body temperature, hormonal release, mood, aggression, feeding,
and sleep. Modulation for cholinergic neuronal activity by 5-HT may play a role in
higher cognitive processes such as memory and learning. 5-HT1A receptor
antagonism appears to enhance activation and signaling through neuronal circuits
known to be involved in cognitive processes (79). There is also substantial evidence
from postmortem studies on human brain, clinical pharmacology, animals studies,
and recently PET studies in humans to implicate this transmitter in several major
neuropsychiatric disorders, including depression (80). This observation is of
contextual interest because depression is commonly seen in AD patients and may
further interfere with cognitive function. The relationship between dementia and
depression in elderly people is incompletely understood and probably complex. A
large fraction of elderly depressives have evidence of significant cognitive
dysfunction, which may be reversible (“pseudodementia”). On the other hand,
depression may be the initial sign of a neurodegenerative disease and is known to
constitute a nearly five-fold increased risk for later development of dementia (81),
and depression complicates AD in about one-fifth of patients (82).
In postmortem frontal cortical brain samples it has been found that
serotonin levels correlate negatively and 5-HT1A receptor density correlates
positively with the rate of cognitive decline in patients with AD (83). By contrast,
264 Hasselbalch and Knudsen

in younger individuals with major depressive disorder, a PET study has revealed
a more widespread reduction in 5-HT1A receptor binding (80).
The 5-HT2A receptors are highly expressed in neocortex but less so in limbic
cortex and basal ganglia (84). 5-HT2A receptors are also associated with cholinergic
nerve terminals in the cerebral cortex and hippocampus. In a postmortem study of
hippocampus receptor binding, the largest decrease was found for 5-HT2 binding,
with N-methyl-D-aspartate (including glutamate, phencyclidine, and glycine
binding sites), quisqualate, kainic acid, adenosine A1, benzodiazepine, 5-HT1,
muscarinic cholinergic, beta-adrenergic, neurotensin, and opioid receptors showing
parallel but less pronounced reductions in binding (85). Bowen et al. also showed
that 5-HT1 and 5-HT2 receptors (7,86) and serotonin reuptake sites (87) were
decreased in postmortem AD studies with 5-HT2A receptors preferentially affected
over 5-HT1A receptors (86,88). Using 18F-setoperone and PET in moderately to
severely demented AD patients, Blin et al. demonstrated large reductions in 5-HT2A
receptor binding in frontal, temporal, parietal, and occipital cortices (33). This
finding was corroborated in a more recent study by Meltzer et al. (34). They studied
5-HT2A receptor binding using 18F-altanserin in patients with depression and in
patients with mild to moderate AD, including three with concurrent depression, and
compared these results to age-matched controls after controlling for partial volume
effects. No differences were, however, found between AD patients with and without
depression, but the limited sample size prevented any examination of relations
between neuropsychiatric symptoms and 5-HT2A receptor binding. Likewise, using
SPECT in nine AD patients, Versijpt et al. found a generally decreased 5-HT2A
receptor density in the orbitofrontal, prefrontal, lateral frontal, cingulate,
sensorimotor, parietal inferior, and occipital regions (35). In very early AD (mild
cognitive impairment of the amnestic type), reduction in 5-HT2A density has been
demonstrated, suggesting that the 5-HT2A receptors are affected early in the course
of the disease (Hasselbalch et al., unpublished data).
The 5-HT4 receptor is highly expressed in cortex and hippocampus and
seems to enhance memory formation through its action on cholinergic neurons
(89). One postmortem study found significant reductions of 5-HT4 receptors in
hippocampus (67%), and smaller reductions in the neocortex in postmortem AD
brains (90), whereas another study did not find 5-HT4 receptor density affected in
the frontal and temporal cortex in AD (91).
A more widespread decrease in 5-HT transporter sites was also found in
brain tissue from AD patients with depressive symptoms as compared to AD
patients without depressive symptoms (92).
In conclusion, both postmortem and imaging studies have consistently
found evidence for serotonergic dysfunction in AD with 5-HT2A receptors most
prominently affected. Although there is substantial evidence for a serotonergic
dysfunction in AD, no PET studies have so far addressed the relationship between
on one hand the serotonergic system, and on the other hand neuropsychiatric and
behavioral symptoms in AD. In particular, the question of to what extent deficits
Imaging of Neurotransmitter Systems in Dementia 265

in serotonergic function are linked to coexisting depression in AD needs to be


clarified in larger human studies.

Peripheral Benzodiazepine Receptor Imaging


It is well established that glial cells play an important role during injury and
neurodegenerative processes in the central nervous system. There is no global
glial proliferation in normal aging, but reactive gliosis has been described in
specific areas of the limbic system and neocortex in non-demented elderly
persons. In contrast to the relatively moderate overall glial changes in normal
aging, the close association of activated astrocytes and microglial cells with
neuritic plaques and cells undergoing neurofibrillary degeneration in AD, the
expression of receptors for complement by glial cells, and the release of soluble
cytokines strongly suggest that inflammatory processes may play an important
part in the complex pathophysiological interactions that occur in AD (93).
Visualization of the extent of microgliosis in age-associated neurodegenerative
disorders may provide new tools to detect and monitor the disease process. In an
early report on the peripheral BZR ligand PK11195, it was concluded that the
peripheral BZR binding sites associated with microgliosis and cellular
inflammation in AD at postmortem are undetectable by PET using 11C-PK-
11195 in patients with mild to moderate dementia (37). A more recent paper did,
however, possibly due to improvements in quantification methods and in scanner
sensitivity, show an increased binding in the entorhinal cortex, temporoparietal,
and cingulate (94). Further evidence for increased microgliosis in AD was
obtained recently by a SPECT study using 123I-iodo-PK11195, where significant
increases were found in frontal and mesotemporal cortex in AD (39). Whether
11
C-PK11195 turns out to be specific enough for the diagnosis of early AD and
whether it holds potential as a prognostic tool still needs to be clarified.

Dopaminergic Transmitter System


Dopamine is well known as a neurotransmitter with an important regulatory role
for motor and limbic functions. It may, however, also be involved in cognition
and attentional processes, and since it is considered a key neuroregulator in
behavioral adaptation (95), alterations in the dopaminergic transmission could,
therefore, be associated with cognitive disturbances.
Postmortem receptor studies have generally failed to show any changes in
D1, D2, or D3 receptors in AD (96–98). In patients with combined AD and Lewy
body pathology or with significant extrapyramidal symptoms, however, D1
receptors are upregulated, whereas D2 receptors are downregulated (96,98). In a
PET study of mild to moderate AD patients with only mild extrapyramidal
symptoms, Kemppainen et al. found a selective loss of D1 receptors in striatum,
whereas D2 receptor density was unchanged (41). Also, in AD patients without
overt extrapyramidal symptoms, SPECT scanning showed a significant reduction
in striatal D2-like receptors as compared to healthy controls (40), suggesting that
266 Hasselbalch and Knudsen

the dopamine system may be affected in AD patients without clinical signs of


motor abnormalities. The role of dopamine in neuropsychiatric symptoms in AD
has also been explored. Using 11C-raclopride and PET, Tanaka et al. found that
striatal D2 receptor density correlated inversely with behavioral symptoms in
severely affected AD patients (42). Further, in AD patients with psychotic
symptoms, D3 receptors have, in postmortem tissue, been found to be
upregulated as compared to AD patients without psychosis (98). Finally, an
implication of the dopamine system in cognitive function was suggested by
Kemppainen et al., who found a bilateral reduction in hippocampal D2/D3
receptor density in AD patients which correlated positively with memory
performance (43).
In a postmortem study by the same group, Kemppainen and co-workers
found a 30–50% reduction in the number of dopamine transporters in the striatum
of patients dying of, or with, AD (2), with the putamen being more severely
affected as compared to normal aging, where dopamine transporter density in the
caudate nucleus seemed to decline more rapidly with age. By contrast, in a PET
study, Rinne et al. found a less pronounced decline (20%) in putamen and caudate
dopamine transporter density in AD patients (45), even though the same tracer
was used. Interestingly, the reduction in [11C]beta-CFT uptake correlated with
the severity of the extrapyramidal symptoms of the patients. In another PET study
of presynaptic dopaminergic function (striatal dopa synthesis measured by
18
F-dopa), no significant differences were found in AD patients with rigidity as
compared to healthy controls (44).
Thus, part of the apparently contradicting data obtained from postmortem
studies and the few PET studies may be explained by the clinical classification of
patients and differences in the severity of extrapyramidal and behavioral
symptoms in the AD groups. Further, at the moment it is not clear whether
significant dopaminergic dysfunction exists in early “pure AD,” and if so, whether
the profile of this dysfunction differs from that of DLB.

LEWY BODY DEMENTIA


Core symptoms of DLB consist of a mixed cortical/subcortical dementia,
extrapyramidal symptoms, a fluctuating course with episodes of disturbed
consciousness, and recurrent detailed visual hallucinations (99). Because
differential diagnosis between DLB and AD can be difficult, neuroimaging may
be of importance by increasing the diagnostic accuracy of these disorders. Due to
the nature of the symptoms in DLB, several studies have looked for dysfunction in
the dopaminergic system, but also profound changes in the cholinergic system
have been demonstrated.
Neurochemically, the decline of cholinergic (ChAT) activity has been
shown to be greater than that of AD (100) and to occur early in the course of the
disease (101). As in AD, loss of nAChR occurs in parietal cortex, but in contrast
Imaging of Neurotransmitter Systems in Dementia 267

to AD, loss of nAChR is also evident in putamen, probably reflecting loss of


nicotinic receptors from dopaminergic neuronal projections to striatum as found
in PD (102).
In a postmortem study comparing AD, DLB, and PD, Piggott et al. found
dopamine transporter activity reduced by 57% in DLB, by 75% in PD, and
normal in AD when compared to controls (97). Further, D2 receptor density was
more reduced in striatum in DLB than in PD, whereas D1 and D3 receptor density
was unchanged. Similarly, in AD patients additional Lewy body pathology has
been associated with decreases in D2 and D3 receptor density (97). Some of these
findings have been corroborated in SPECT and PET studies. Using the dopamine
transporter ligand 123I-FP-CIT and SPECT, significantly lower uptake in striatum
has been found in DLB and PD when compared to AD and to controls (103,104).
Using the same tracer, similar findings were obtained by O’Brien et al. (46), but
they also demonstrated a more uniform tracer distribution in striatum in DLB as
compared to PD (Fig. 4). Likewise, in a PET study Hu et al. found pronounced

Figure 4 (See color insert.) Dopamine transporter loss visualized with FP-CIT SPECT
in the differential diagnosis of dementia with Lewy bodies (DLB). Reductions in FP-CIT
binding occurred in the caudate and anterior and posterior putamens in subjects with DLB
compared with subjects with Alzheimer’s disease and controls. Transporter loss in DLBs
was of similar magnitude to that seen in Parkinson’s disease (PD), while the greatest loss in
all three areas was seen in those who had PD and dementia. In Parkinson’s disease, there
was a greater selective reduction in putamen uptake compared with DLB and PD and
dementia. Source: From Ref. 46.
268 Hasselbalch and Knudsen

reductions in striatal 18F-dopa uptake in moderately demented DLB patients


when compared to moderate AD (105). Using a tracer that binds to the
presynaptic monoamine vesicular transporter, Gilman et al. found striatal mean
binding potential decreased by 45% to 67% in a group of DLB patients, as
compared to both controls and AD patients (106). Interestingly, in a few AD
patients binding values were below the range of the controls, and in one of these
patients autopsy showed neuropathological features of both DLB and AD,
although the patient had no extrapyramidal symptoms (Fig. 4). In line with this,
the PET study by Rinne et al. suggests that decreases in striatal dopamine
transporter density correlate with the severity of extrapyramidal symptoms (45),
perhaps due to concomitant Lewy body pathology in AD patients. Studying
postsynaptic D2 receptor density in striatum with IBZM-SPECT, similar
reductions were found in DLB and AD when compared to controls; although
the reduction was somewhat more pronounced in DLB, a considerable overlap
between AD and DLB was found (107). These few neuroimaging studies suggest
that presynaptic dopamine imaging might prove useful in the differential
diagnosis between AD and DLB, and that imaging may be sensitive to Lewy
body pathology already in the very early stages of the disease.

FRONTOTEMPORAL DEMENTIA
With regard to neuroreceptor imaging, FTD is the least studied progressive
neurodegenerative disorder. Furthermore, FTD comprise a group of disorders
with different neuropathological findings, and therefore neurochemical post-
mortem studies might reflect this heterogeneity.
In a postmortem study of 16 FTD patients (10 Pick pathology patients, and
six patients with unspecific changes), cholinergic activity (measured as ChAT
activity and M1 receptors) was not significantly reduced in cortical areas in FTD
as compared to controls (108). This data supports the empirical clinical finding of
no effect of cholinergic drugs in FTD. By contrast, in the same study both 5-HT1A
and 5-HT2A receptors were reduced in temporal and frontal cortices in FTD as
compared to controls. Interestingly, serotonergic receptors were also lost in AD,
but only in temporal and parietal areas. Thus, there might be regional differences
in the serotonergic dysfunction in FTD and AD, but whether imaging of
serotonergic receptors might be helpful in the differential diagnosis remains to be
settled, since no imaging studies have explored this possibility.
Since extrapyramidal symptoms are relatively common in FTD, the
expected dopaminergic dysfunction has indeed been supported by one post-
mortem study in which neuronal loss in the substantia nigra was found (109).
In familial FTD with parkinsonism due to a mutation in chromosome 17, one
SPECT study (110) and one PET study (111) have found evidence for a reduced
dopamine transporter binding. In the latter study, striatal D2 receptors were
preserved much in line with what is found in PD. In a 11C-CFT PET study,
dopamine transporter binding was reduced by 18% and 14% in putamen and in
Imaging of Neurotransmitter Systems in Dementia 269

Figure 5 (See color insert.) Differences in cortical GABAA receptor density between
patients with leukoaraiosis and dementia (A) and patients with leukoaraiosis without
dementia (B), indicating that severe white matter ischemic lesions may cause cortico-
subcortical disruption, which may augment cognitive dysfunction. Source: From Ref. 116.

caudate, and the reductions correlated with extrapyramidal symptoms (112).


In the differential diagnosis between AD, DLB, and FTD, these findings suggest
that dopamine transporter imaging might not be very helpful, since reductions
have been found in all three disorders, and seem to be related to extrapyramidal
symptoms, rather than to a specific neurodegenerative disorder.

COGNITIVE DYSFUNCTION DUE TO


CEREBROVASCULAR DISEASE
The concept of vascular dementia is currently changing since it is increasingly
understood that cerebrovascular disease leading to cognitive dysfunction cannot
be categorized under one disease, but results from different pathophysiological
processes. Structural imaging, especially magnetic resonance imaging (MRI), has
increased our understanding of cerebrovascular disease and cognitive dysfunc-
tion considerably over the last decades. With regard to neurotransmitter imaging,
research has focused on neuronal dysfunction due to ischemic stroke (113),
270 Hasselbalch and Knudsen

where markers of neuronal integrity, such as GABAA ligands, combined with


measurements of cerebral blood flow, oxygen metabolism, and oxygen extraction
may determine the extent of tissue damage (114). Few studies have investigated
more global cortical or subcortical neurotransmitter changes (outside ischemia
injured tissue) in cerebrovascular disease. One study found that cortical 5-HT2
receptor binding positively correlated with cognitive performance in post-stroke
patients, suggesting that alterations in the serotonergic system following stroke
may affect cognition (115). Ihara et al. demonstrated significant differences in
cortical GABAA receptor between patients with leukoaraiosis and dementia and
patients with leukoaraiosis without dementia, indicating that severe white matter
ischemic lesions may cause cortico-subcortical disruption, which may augment
cognitive dysfunction (Fig. 5) (116).
In conclusion, few studies have addressed the relationship between
cognitive dysfunction and neurotransmitter changes in cerebrovascular disease
in vivo. In particular, it would be interesting to look further into the
pathophysiological background for the specific symptomatology found in
subcortical vascular dementia.

CONCLUSIONS
Among the primary degenerative disorders, AD is the most extensively
investigated when it comes to neuroreceptor studies. In postmortem studies,
marked reductions in some of the receptor systems, in particular 5-HT2 receptors,
nicotinic a4-b2, and a preferential loss of M2 as compared to M1 muscarinic
receptors have been found. Studies of muscarinic and dopaminergic receptors
have shown conflicting results. Some of these findings have been partially
replicated in in vivo imaging studies: a decrease in 5-HT2, nicotinic, and possibly
muscarinic receptors.
In DLB cholinergic and dopaminergic dysfunctions have been established,
and in FTD reductions in serotonergic receptors and dopamine reuptake are among
the neurochemical changes. Some of these results have been replicated in vivo in
neuroimaging studies, and imaging of neurotransmitters may prove useful in the
differential diagnosis between the primary neurodegenerative disorders.
Generally, the development of more selective radiotracers that are
accurately quantifying receptor binding would greatly enhance our knowledge
on in vivo changes in the transmitter systems in dementia disorders. Neurotrans-
mission imaging has an enormous potential for increasing our understanding of the
complex relationship between neurochemical processes and cognitive and
neuropsychiatric symptoms in dementia.

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10
Development and Application
of b-Amyloid Imaging Agents
in Alzheimer’s Disease

William E. Klunk, Robert D. Nebes, and Nicholas Tsopelas


Department of Psychiatry, University of Pittsburgh, Pittsburgh,
Pennsylvania, U.S.A.
Brian J. Lopresti and Julie C. Price
Department of Radiology, University of Pittsburgh, Pittsburgh,
Pennsylvania, U.S.A.
Steven T. DeKosky
Departments of Psychiatry and Neurology, University of Pittsburgh,
Pittsburgh, Pennsylvania, U.S.A.
Chester A. Mathis
Departments of Radiology, Pharmacology, and Pharmaceutical Sciences,
University of Pittsburgh, Pittsburgh, Pennsylvania, U.S.A.

INTRODUCTION
The ability to localize and quantify amyloid deposition in the living brain can
advance the study and management of Alzheimer’s disease (AD) in several
important ways. This chapter describes some recent progress in the development
and application of amyloid imaging agents. Sensitive in vivo detection of amyloid
deposition could aid in early, perhaps even pre-clinical, diagnosis. Longitudinal

279
280 Klunk et al.

studies of amyloid deposition could shed new light onto the controversial “amyloid
cascade hypothesis.” The ability to assess amyloid deposition pre- and post-
treatment with anti-amyloid therapies could significantly facilitate the develop-
ment of these promising experimental treatments. Surrogate marker questions
must await clinical trials in which amyloid imaging is performed before and after
treatment with anti-amyloid therapies. While the results of initial studies are
promising, they must be followed by larger studies, employing a wider range of
disease severity, incorporating longitudinal studies, and examining amyloid
imaging agent retention in dementias other than AD.

Neuropathology of Alzheimer’s Disease


The definitive diagnosis of AD relies on the demonstration of amyloid plaques
and neurofibrillary tangles (NFT) at autopsy (1) (see Chapter 11). Amyloid
plaques are composed of 40–42 amino acid amyloid-beta (Ab) peptides (2), while
NFT are composed mainly of a hyperphosphorylated form of the microtubule-
associated protein, tau (3). Frequently, a-synuclein deposits in the form of Lewy
bodies or threads also are present making the Lewy body variant of AD a “triple
amyloidosis.” Plaques occur earliest in neocortex, where they are relatively
evenly distributed (2), and tangles appear first in limbic areas, such as the
transentorhinal cortex, and progress in a predictable pattern of regional
distribution to the neocortex (3). Arnold et al. (4) mapped the distribution of
NFT and neuritic plaques (amyloid plaques surrounded by dystrophic neurites) in
the brains of patients with AD. Compared to NFT, neuritic plaques were generally
more evenly distributed throughout the cortex, with the exceptions of notably
fewer neuritic plaques in limbic periallocortex and allocortex (the areas with
greatest NFT density such as the hippocampus). This is supported by the findings
of Price and Morris (5), who found little amyloid pathology in the hippocampus of
amyloid-positive controls or very mild AD patients. Thus, while limbic areas have
early and severe tangle pathology, the medial temporal lobe has relatively little
neuritic plaque pathology early in the disease. The cerebellum is notably free
of neuritic plaques in AD, although diffuse amyloid deposits that do not label with
fibrillar dyes, such as Congo red, are commonly observed (6,7).
The following facts support the use of amyloid plaque imaging for the
study of AD. First, while small degrees of AD-like pathology can be found in
cognitively normal individuals over the age of 75, large deposits of these
pathological entities or deposits prior to that age appear specific to AD (5).
Second, although the time course of amyloid deposition in AD has not been
elucidated, evidence gained through postmortem study of Down syndrome (DS)
(a condition in which Ab amyloid deposition is always present by age 40 and
dementia is very common) suggests that amyloid deposition begins over a decade
prior to the clinical symptoms of dementia (8).
b-Amyloid Imaging Agents 281

The Central Role of Ab in the Pathophysiology


of Alzheimer’s Disease
A growing consensus points to Ab deposition as a central event in the
pathogenesis of AD. The single, most important piece of evidence for this
“amyloid cascade hypothesis” of AD is the demonstration that mutations in the
Ab precursor protein (APP) gene on chromosome 21 cause early onset AD (9).
While there are a very small number of families affected with this form of AD, the
disease is phenotypically indistinguishable from the more common sporadic form
of AD, save only for its early age of onset. Further genetic support for the
amyloid cascade hypothesis comes from the finding that the most common form
of autosomal dominant AD is caused by mutations in the presenilin-1 gene on
chromosome 14, which codes for a protein that is strongly suggested to be an
essential component of the “gamma-secretase” enzyme complex (10).

Ab Deposition as a Therapeutic Target in Alzheimer’s Disease


It is not surprising that the metabolism of Ab has become an important
therapeutic target in AD research. A corollary of the amyloid cascade hypothesis
is that prevention of Ab accumulation in oligomers or plaques should prevent
AD. Approaches to “anti-amyloid” therapy have focused both on decreasing
production and increasing clearance of Ab. Attempts to decrease Ab production
involve inhibition of two distinct “secretase” enzymes responsible for cleavage of
Ab from its much larger precursor protein (11). The b-secretase or b-amyloid
cleaving enzyme cleaves the N-terminus of Ab, and the g-secretase enzyme
complex cleaves the C-terminus (12). Studies with transgenic mice that deposit
Ab plaques in their brains have shown that g-secretase inhibitors can prevent
amyloid deposition (13). Human studies have begun with g-secretase inhibitors,
although few details have yet been released.
A second “anti-amyloid” approach makes use of immunotherapy against
Ab. It is generally regarded that this approach lowers Ab levels by augmenting
clearance of Ab. The first iteration of the immunotherapeutic approach involved
active immunization with fibrillar Ab itself. This approach had marked anti-
amyloid effects in amyloid-depositing transgenic mice (14). Much interest was
given to the first human trials of this approach, sometimes referred to as the
“Alzheimer’s vaccine.” Unfortunately, this trial was suspended due to a 6%
incidence of a serious adverse event of meningoencephalitis (15). Follow-up of
the initially treated cases continues, and a preliminary report on a subset
of patients suggests that successful immunization against Ab slows cognitive
decline (16). Analysis of the full cohort showed a modest effect on slowing
cognitive decline that appeared to be related to the level of antibody response to
the immunizations (17). Proof-of-concept evidence supporting the removal of
amyloid deposits from the human brain emerged from three autopsy studies from
the Ab-immunization trials (18–20). While all of these cases showed evidence of
significant amyloid clearance, it is interesting that this was typically a very focal
282 Klunk et al.

finding. This focality underscores the need to have an in vivo method capable of
measuring amyloid burden in the entire brain when assessing the effects
of immunotherapy. The promising findings from the Ab-immunization trial have
prompted more intense interest in further refinements of the immunotherapeutic
anti-amyloid approach such as passive immunization with anti-Ab antibodies
(21,22) that should avoid many untoward effects of active immunization
including meningoencephalitis. These passive immunization trials have now
entered early-phase human studies.

Need for In Vivo Quantitation of Ab


The advent of anti-amyloid therapies clearly demonstrates one of the most
pressing needs for a technology that would allow non-invasive, in vivo
quantitation of amyloid deposition in human brain. For example, even though
the autopsy cases reported from the vaccine study appeared to show decreased
amyloid load, some have pointed out that we can not be sure that these were not
just anomalous cases since no measures of pre-treatment amyloid load were
available. In addition, the authors of the preliminary study that suggested
cognitive slowing due to Ab immunization stated, “We do not know whether
brain Ab-amyloid load was reduced in our study patients; in vivo imaging
techniques will be required to answer this question” (16). These anti-amyloid
therapies are among the most promising in the current pharmaceutical industry
pipeline, and the ability to quantify amyloid load before treatment and then
follow the effects of treatment is critical to the efficient development of this class
of drugs. The success of these or any preventive therapy for AD will require an
accurate means of early, and ideally pre-symptomatic, detection of those with the
disease or at high risk for the disease. The significant overlap of symptoms among
various forms of dementia can make diagnosis challenging, and clinicians would
benefit from a method to detect the neuropathology of AD in vivo. Amyloid
imaging could thus become an important diagnostic tool to clinicians. Finally, the
ability to follow the natural history of amyloid deposition beginning in
pre-symptomatic stages in subjects at very high risk for AD (e.g., carriers of
mutations causing early onset familial AD) should yield important pathophysio-
logical insights regarding the accuracy of the amyloid cascade hypothesis of AD.

DEVELOPMENT OF AMYLOID IMAGING TECHNOLOGIES


Preclinical Development
A number of groups have worked to develop radiolabeled amyloid-specific
imaging agents, but early efforts were limited by poor brain entry, high levels of
non-specific binding, or low levels of specific binding in brain regions known to
contain high concentrations of Ab (23). Most groups used postmortem tissue dyes
known to bind amyloid as the starting point. We reported that Chrysamine G
(CG), a lipophilic analog of the amyloid dye Congo red, possessed high affinity
b-Amyloid Imaging Agents 283

for Ab fibrils and plaques (24), but the penetration of an I-125-labeled CG


derivative into normal rat brain was low (25). Similar results were reported using
a Tc-99 m-labeled CG derivative (26). Other more chemically distant analogs of
CG comprised of styrylbenzene salicylic acids, such as X-34, provided similar
results of high in vitro binding affinity to aggregated Ab but low brain penetration
in normal rodent brain (23,27–29). Further modification of the structure by
removing the carboxylic acid groups to provide Methoxy-X04 resulted in
improved brain entry, but not to the degree typically necessary to provide a good
non-invasive positron emission tomography (PET) imaging agent (27). To
overcome the low brain penetration of the styrylbenzenes, we developed neutral
carbon-11-labeled derivatives of another amyloid dye, thioflavin-T. Modification
of the amyloid-binding histologic dye, thioflavin-T, led to the finding that neutral
benzothiazole-anilines (or BTAs) bound to amyloid with high affinity and
crossed the blood-brain barrier (BBB) very well (30). The basic properties of the
prototypical benzothiazole amyloid binding agent, termed BTA-1, and related
derivatives have been described in detail (23,31–33). These studies showed that
these compounds could bind to amyloid with low nanomolar affinity, enter brain
in amounts sufficient for imaging with PET, and clear rapidly from normal brain
tissue. At the low nanomolar concentrations typically used in PET studies, the
binding of BTA-1 to postmortem human brain was shown to be a good indication
of Ab amyloid deposition, but did not appear to detect the presence of NFT (34).
These postmortem data suggested benzothiazole derivatives could be good
in vivo PET amyloid imaging agents. A structure-activity study of a series of
benzothiazoles suggested that a hydroxylated BTA-1 derivative had brain
clearance properties typical of many useful PET radiotracers (32). Therefore, this
hydroxybenzothiazole was chosen as the lead compound for the first human trial
of benzothiazole amyloid imaging agents performed in Uppsala, Sweden (35).
The compound, {N-methyl-[11C]}2-(4 0 -methylaminophenyl)-6-hydroxyben-
zothiazole, was given the Uppsala University PET Center code of “Pittsburgh
compound-B” or PIB. Pre-clinical studies showed that PIB bound to AD brain
with a Kd of 1–2 nM entered the brain rapidly (w7%ID/g two minutes post IV
injection in mice), and cleared rapidly from normal mouse brain and baboon
brain (32). Using real-time in vivo multiphoton microscopy, BTA amyloid-
imaging agents have been shown to label individual amyloid plaques in
transgenic mouse models of AD within three minutes following IV injection and
clear rapidly from normal brain parenchyma (36).
Kung and co-workers developed similar radioiodinated thioflavin-T
derivatives as potential Ab imaging agents for single photon emission computed
tomography (SPECT), and several of these compounds (e.g., IMPY) show
promise for this purpose with high binding affinities for aggregated Ab and good
penetration in mouse brain (37–39). This same group, working in collaboration
with researchers from the University of Toronto, has reported C-11-labeled
stilbene derivatives for amyloid imaging in human studies (see below) (40).
284 Klunk et al.

Barrio and co-workers reported the synthesis of a lipophilic F-18-


radiolabeled tracer for PET imaging of plaques and NFTs in the brains of AD
patients (41,42). This tracer is a fluorinated derivative of a non-specific cellular
membrane dye, 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene
(DDNP). This agent is capable of crossing the BBB and entering brain tissue,
and the nature and degree of its specific binding to b-amyloid and NFTs has been
partially characterized (43). [F-18]FDDNP was used in one of the early human
amyloid imaging studies (see below).
Several groups have taken a large biomolecule approach to amyloid
imaging using either antibodies to Ab (44–47) or labeled Ab itself (48–51). All of
these approaches have met with the problem of achieving substantial penetration
of the BBB (23).
In addition to these PET and SPECT radiotracer approaches, several groups
have sought to image amyloid using magnetic resonance imaging (MRI) or
spectroscopy (MRS). These earliest of these approaches utilized gadolinium-
labeled putrescine modified Ab (52) or monocrystalline iron oxide nanoparticle
(MION)-labeled Ab (53). The MION approach required mannitol infusion to
temporarily open the BBB to allow passage of the large modified Ab probe. The
gadolinium-labeled Ab approach suffered from similar brain uptake difficulties.
More recently, another group has reported using an fluorine-labeled derivative of
X-34 with fluorine-MRS (54). Interestingly, Jack et al. (55) have shown that it is
possible to use MRI to resolve individual plaques in living transgenic mice
without MRI contrast agents. Unfortunately, current MRI technical limitations do
not allow either the fluorine-MRS or the no-contrast approach to be applied to
human studies. It is possible that refinements of MRI technology will allow
human MRI amyloid imaging studies in the future, and this would be
advantageous given the widespread availability of MRI.

Human Amyloid Imaging Studies


Radiolabeled Monoclonal Antibody Fragments
The first attempt to non-invasively image brain amyloid deposits in probable AD
patients was made using 10H3, a monoclonal antibody fragment targeting the
Ab1-28 residues, which was labeled with technetium-99 m for SPECT imaging
(45). While this agent showed specific binding to senile plaques and
cerebrovascular amyloid in postmortem tissue sections (46), only non-specific
retention in scalp was observed in human subjects in a manner that did not
distinguish AD and control subjects. A major limitation of this approach is the
inability of such large molecules to readily enter brain and achieve concentrations
sufficient for detection by standard molecular imaging techniques. It may be
possible to achieve detectable brain concentrations of this and other antibody
fragments over a protracted period of observation of days or weeks, although the
relatively short biologic half-life of this injected antibody fragment (two to
b-Amyloid Imaging Agents 285

three hours) and the half-life of the radionuclide would render such an approach
impractical for in vivo investigations in human subjects.
[18F]FDDNP
The second agent for the in vivo visualization of amyloid deposits was a
radiofluorinated derivative of the solvent and viscosity sensitive fluorophore
2-{1-[6-(dimethylamino)-2-naphthyl]ethylidene}malononitrile (DDNP), termed
[18F]FDDNP. In vitro characterization of FDDNP demonstrated that it bound to
amyloid-beta fibrils with high affinity and fluorescently stained both plaques and
NFTs at high concentrations (43,56). Human studies in AD patients (nZ9) and
controls (nZ7) showed slightly greater retention of [18F]FDDNP in frontal,
parietal, temporal, and occipital cortices at steady-state (60–120 minutes post
injection), though this increased cortical retention was shown to exceed the
reference region (pons) by only a margin of 10–15%. The area of highest
retention at equilibrium was a region encompassing hippocampus/amygdala/
entorhinal (h-a-e) cortex region, which was shown to exceed retention in the pons
by w30% (57). Interestingly, autopsy studies (4) showed that neuritic plaques are
more densely concentrated in neocortex, while the mesial temporal lobe
structures, including the h-a-e cortex region, contain the fewest neuritic plaques.
NFTs, in contrast, are densely concentrated in the mesial temporal lobe where
[18F]FDDNP retention is greatest (3).
Initial analyses of [18F]FDDNP PET data were performed using a novel
kinetic analysis method, termed the relative residence time (RRT), which relates
specific radiotracer binding to the negative net difference between the reciprocal
of the tissue clearance constants (k2) for the reference and target tissues. The
authors demonstrated significantly longer h-a-e RRT values for AD patients as
compared to controls, and that the value of the RRT parameter was significantly
correlated with Mini-Mental State Examination (MMSE) (58). A concern with
this method of analysis is that it is sensitive to both peak and steady-state levels of
[18F]FDDNP in tissue, which could potentially result in aberrations of the
outcome measure by blood flow and transport phenomenon. For instance, brain
areas with nearly identical equilibrium levels of tracer, such as temporal cortex
and occipital cortex, produced RRT estimates that differed by more than a factor
of 15. Additional analyses of [18F]FDDNP have been reported in a preliminary
form comparing 13 AD subjects (age: 76G8 years; MMSE: 18G6) and 10
controls (age: 63G10 years; MMSE: all scored 30). The method used a standard
uptake value (SUV) computed from 60–120 minutes tissue radioactivity
concentrations that were normalized to a reference region devoid of specific
binding (SUVR), such as cerebellum. These analyses have shown significant
differences (p!0.001) in the retention of [18F]FDDNP between control and AD
subjects in regions such as the medial temporal, parietal, and prefrontal cortices,
although the magnitude of the signal in the AD subjects did not exceed the control
value by more than 15% in any region (59). The applicability of conventional
quantitative analysis methodologies, such as compartmental modeling and
286 Klunk et al.

graphical analyses, to dynamic [18F]FDDNP PET data remains to be


demonstrated. These studies are necessary to fully understand the nature of
[18F]FDDNP retention and to assess the overall sensitivity and specificity of the
compound for the detection of AD pathology in vivo.
[11C]SB-13
The properties of a stilbene derivative, 4-N-methylamino-4 0 -hydroxystilbene or
SB-13, were recently reported (60). In vitro, [3H]SB-13 was shown to bind
specifically and with high affinity (KdZ2.4G0.2 nM) to cortical homogenates
prepared from postmortem brain tissue samples of four patients with a
pathological confirmation of AD. In contrast, homogenates of cerebellar and
white matter tissues prepared from both AD or control elderly brain tissue did not
represent significant sources of [3H]SB-13 specific binding. In vitro competition
binding studies demonstrated comparable potencies in the low nanomolar range
(KiZ6.9 nM) for BTA-1 displacement of [3H]SB-13. FDDNP exhibited less
competition with [3H]SB-13 (KiZ294 nM) as compared to BTA-1. This
suggested that benzothiazoles, such as BTA-1 and PIB, share a binding site on
the Ab peptide with SB-13, while FDDNP does not (61). [3H]SB-13 was shown
to label Ab plaques in sections of human AD cortex, but not in control brain.
These favorable in vitro properties supported the continued investigation of
SB-13 as a potential molecular imaging probe for the non-invasive assessment
of brain amyloid deposition in human subjects.
SB-13 was labeled with carbon-11 for in vivo investigations in five female
AD subjects and six healthy controls (HCs) using PET (60). These same subjects
were imaged with PIB, in order to provide a basis of comparison for [11C]SB-13.
Venous blood samples were drawn throughout the first 70 minutes of PET data
acquisition to characterize the metabolism of [11C]SB-13 and to provide an
approximation of the arterial input function for quantitative data analysis. To
estimate non-specific binding of [11C]SB-13, the cerebellum was selected as a
reference region as it has been shown to contain a relative paucity of fibrillar
amyloid deposits in AD and can be assumed to be nearly devoid of [11C]SB-13
specific binding. Following the injection of [11C]SB-13, cerebellar time-activity
data showed similar retention characteristics between AD patients and controls.
Clearance of cerebellar radioactivity after injection of [11C]SB-13 was somewhat
slower than that observed for PIB in the same subjects, with [11C]SB-13
exhibiting a w3:1 ratio in the peak to 90 minutes radioactivity concentrations as
compared to w8:1 for PIB. Increased retention of [11C]SB-13 was observed in
AD subjects compared to controls in cortical areas known to contain significant
amyloid deposits in AD, such as the frontal cortex (FRC). Across the four cortical
areas included in this investigation (frontal, temporal, parietal, occipital), SUVs
were determined from 40–120 minutes post-injection emission data and AD
cortical averages were shown to exceed control averages by a ratio of 1.44–1.75,
with the greatest distinction observed in left FRC. In the same subjects, the ratio
of AD to control SUV values for PIB ranged from 1.96 to 2.52, which was also
b-Amyloid Imaging Agents 287

maximal in the left FRC. It is likely that the improved distinction between AD
and control subjects using PIB is a result of more rapid clearance of non-specific
binding. While the pattern of retention of [11C]SB-13 appears to mirror that of
PIB, the latter provides a greater dynamic range and improved distinction of AD
subjects from controls.
Pittsburgh Compound-B
The first human studies with PIB were presented in preliminary form in 2002 (35)
and were followed by a full report in 2004 (62). The initial study included 16
probable AD patients (65.9G11 years; 12.3G4 years education), six elderly age-
matched controls (69.0G7 years; 12.7G4 years education), and three 21-year-old
controls (young controls). The young controls were included in the study because of
the near certainty that these young subjects would represent true plaque-negative
controls. Dementia severity in the probable AD subject group, as evaluated by the
MMSE, varied from mild (MMSEZ18) to very mild/questionable (MMSEZ29)
with a mean MMSE of 24.9G3.4. Subjects were administered w300 MBq (8 mCi)
of PIB, and dynamic PET data were acquired for 60 minutes. In this study, neither
MRI images nor arterial blood samples were obtained for anatomical co-registration
and input function determination, and analyses of these studies were limited to semi-
quantitative SUV measures of PIB uptake. Nevertheless, striking differences in PIB
retention were observed between control and AD subjects in brain areas known to
contain significant amyloid deposits in AD [e.g., FRC, and parietal cortex (PAR)].
The six elderly control subjects showed a brain entry and clearance pattern that was
indistinguishable from that of the three young control subjects. This permitted young
and elderly control groups to be combined to form a unified (HC) group for
comparison to the AD patients. As a group, the control subjects showed rapid entry
and clearance of PIB from all cortical and subcortical gray matter areas, including
the cerebellar cortex (Fig. 1). Cerebellum, an area generally lacking fibrillar amyloid
plaques in AD, showed nearly identical uptake and clearance of PIB in the
cerebellum of HC and AD groups (Fig. 1A). Subcortical white matter showed
relatively lower entry and slower clearance in both HC subjects and AD patients
compared to cortical and subcortical gray matter areas (Fig. 1B). In contrast, the AD
patients showed a markedly enhanced retention of PIB compared to HC subjects in
areas of the brain known to contain high levels of amyloid deposits in AD (Figs. 1C,
D, and E), such as parietal and frontal cortices (2,4).
The regional distribution of PIB retention was clearly different in AD
patients compared to the HC subjects (Fig. 2). PIB accumulation in AD patients
as a group was most prominent in cortical association areas and lower in white
matter areas, a pattern consistent with that described in postmortem studies of
amyloid deposition in AD brain (2). PIB images from HC subjects showed little
or no PIB retention in cortical areas, leaving the subcortical white matter regions
highest in relative terms. In absolute terms, the accumulation of PIB in white
matter was essentially the same in AD patients and HC subjects (Fig. 1B).
A series of axial and sagittal SUV images provides a three-dimensional sense of
288 Klunk et al.

(A) (B) (C)


5 5 5
A Subcortical B Frontal C
Cerebellum
White Matter Cortex
4 4 4
AD AD AD
HC HC HC
3 3 3
SUV

SUV

SUV
2 2 2

1 1 1

0 0 0
0 20 40 60 0 20 40 60 0 20 40 60
Time (min) Time (min) Time (min)

(D) (E)
5 5
Fr Par D Fr Par E
SWM Cb SWM Cb
4 4
Older Control AD
3 3
SUV

SUV

2 2

1 1

0 0
0 20 40 60 0 20 40 60
Time (min) Time (min)

Figure 1 Pittsburgh compound-B (PIB) is differentially retained only in amyloid-laden


cortical areas of the Alzheimer’s disease (AD) brain. Standardized uptake values (SUV)
demonstrating brain entry and clearance of PIB. (A–C) represent averaged SUV values for
all HC subjects (open symbols; nZ9) and all AD patients (filled symbols; nZ15) in
cerebellum, subcortical white matter, and frontal cortex. (D) and (E) show brain entry and
clearance in cerebellum (triangles), subcortical white matter (diamonds), frontal cortex
(squares), and parietal cortex (circles) for an older control subject (D) and an AD patient
(E). Error bars represent one standard deviation (SD) and are too small to be seen in some
of the HC subject data in (A–C). Asterisks indicate a significant difference between AD
and HC values (p!0.006). Shaded areas highlight the 40–60 minute time period used for
the summed SUV data displayed in Figures 2–4. Source: From Ref. 62.

the regional distribution of PIB retention (Fig. 3). The marked difference between
PIB retention in the AD patient and the HC subject is apparent throughout most of
the forebrain. FRC was widely affected in the AD patient, but intense PIB
retention also was observed in temporal and parietal cortices, part of the occipital
cortex, and in the striatum. Lateral temporal cortex (LTC) appeared to have
greater PIB accumulation than mesial temporal areas. Consistent with previous
reports of extensive amyloid deposition in the striatum of virtually all AD
patients (63–66), the striatum was found to have significantly higher PIB
retention in AD patients than in HC subjects. Cerebellar cortex (Fig. 3) showed
little PIB retention and was similar in AD patients and HC subjects. In general,
the observed pattern of PIB retention in AD subjects was found to be consistent
b-Amyloid Imaging Agents 289

Figure 2 (See color insert.) Pittsburgh compound-B (PIB) standard uptake value (SUV)
images demonstrate a marked difference between PIB retention in Alzheimer’s disease
(AD) patients and HC subjects. PET images of a 67-year-old HC subject (left) and a
79-year-old AD patient [AD6; Mini-Mental State ExaminationZ21; (right)]. (Top) PIB
SUV images summed over 40–60 minutes; (bottom): fluorodeoxyglucose (FDG) rCMRglc
images (mmol/min/100 ml). The left column shows lack of PIB retention in the entire gray
matter of the HC subject (top left) and normal FDG uptake (bottom left). Nonspecific PIB
retention is seen in the white matter (top left). The right column shows high PIB retention
in the frontal and temporoparietal cortices of the AD patient (top right) and a typical
pattern of FDG hypometabolism present in the temporoparietal cortex [arrows; (bottom
right)] along with preserved metabolic rate in the frontal cortex. PIB and FDG scans were
obtained within three days of each other. Source: From Ref. 62.

with the pattern of amyloid plaque deposition described in postmortem studies of


AD brain (2,4). PIB retention typically predominated in FRC, but it should be
noted that FRC did not always show the highest PIB retention in a given subject
and mean levels of frontal PIB retention exceed parietal levels by less than 10%.
Three patients diagnosed as probable AD had high MMSE scores (28–29)
and showed no significant deterioration over the two-to-four years follow-up
period (i.e., MMSE remained 28–29). These atypical subjects had levels of PIB
retention in cortical regions typical of controls (open triangles in Fig. 4), though
they were retained in the AD group for all analyses. Other mild AD patients with
similar clinical profiles showed typical AD-like changes in PIB retention and
rCMRglc. It was unclear whether PIB was simply insensitive to the amount of
amyloid deposits in the brains of these three atypical AD patients with MMSE
scores of 28–29 or whether PIB imaging had correctly identified subjects without
290 Klunk et al.

Figure 3 (See color insert.) Serial planes demonstrate the topography of Pittsburgh
compound-B (PIB) retention. Axial (top two rows) and sagittal (bottom two rows) PIB
standard uptake value (SUV) images of the subjects shown in Figure 2. The HC subject
data is shown in rows (1) and (3). The AD patient data is shown in rows (2) and (4). The
reference region, the cerebellum, can best be appreciated in the images at the far right. The
cerebellar peduncles (white matter) show some nonspecific retention, but the cerebellar
cortex shows negligible retention. Scale bar indicates relative levels of PIB SUV values.
Source: From Ref. 62.

amyloid deposits in whom the clinical diagnosis of AD was incorrect and would
not be confirmed by postmortem evaluation. In the elderly control group, the
oldest subject (76 years old) consistently showed the highest cortical PIB
retention and the lowest cortical rCMRglc (boxed circles in Fig. 4). This subject
had not expressed any subjective memory complaints and performed within the
normal range on the neuropsychological test battery except for difficulty copying
a complex cube. This type of case, which could be described as an asymptomatic
amyloid-positive case, highlights the issue of specificity versus early detection.
One possibility could be that a high PIB signal was obtained in the absence of
amyloid deposits (i.e., a false-positive). If this finding does represent the true
b-Amyloid Imaging Agents 291

Cortical Areas White Matter


(A) 2.5 (B) 2.5

2 2

1.5 1.5
SUV

SUV
1 1

0.5 0.5

0 *** *** ** 0
Temporal
Parietal
Frontal

Pons

SWM
Cb

Cb
(C) 80 (D) 80
*
rCMRglc (µmol/min/100mL)
rCMRglc (µmol/min/100mL)

60 60

40 40

20 20

0 0

Figure 4 Differences in Pittsburgh compound-B (PIB) retention between Alzheimer’s


disease (AD) patients and HC subjects can be quantified and are statistically significant.
Accumulation of PIB and rCMRglc in selected regions. Average PIB standard uptake
values (SUV) were summed over 40–60 minutes in cortical areas (A) or white matter areas
(B) and compared to cerebellum. Values for fluorodeoxyglucose (FDG) uptake were
calculated with the Gjedde-Patlak method in cortical areas (C) or white matter areas (D)
and compared to cerebellum. The mean and one SD are indicated with the error bars beside
the individual points. HC subjects (circles, nZ9): filled circles represent the three young
HC subjects; boxed circle represents the outlier in the HC group (oldest subject). AD
patients [triangles, nZ15 (SUV) or nZ16 (FDG)]: open triangles represent the three
outliers in the AD group. AD mean and SD values include all 15 (SUV) or 16 (FDG) AD
subjects; p!0.01 (*); p!0.002 (**); p!0.0002 (***). Source: From Ref. 62.

presence of amyloid in an asymptomatic individual, the question becomes


whether substantial amyloid deposition can be found as part of the normal aging
process in subjects who will never develop AD (67) or is increased amyloid
deposition always a sign of pre-clinical AD (68–70). The ability to longitudinally
292 Klunk et al.

follow PIB retention as an in vivo measure of amyloid deposition provides a new


tool through which we may be able to answer this question in a manner that
postmortem studies can not.

Pittsburgh Compound-B Methodology Development


Since the initial report detailing the PIB proof-of-concept studies, additional
studies conducted at the University of Pittsburgh have sought to extend these
studies to include in vitro analyses of PIB binding kinetics, fully quantitative
imaging, and a third group of subjects with a diagnosis of mild cognitive
impairment (MCI). Significant extensions of the original imaging paradigm
included: (1) MRI image acquisition (for PET image co-registration, region-of-
interest definition, and partial volume correction), (2) the determination of the
arterial input function, and (3) the collection of 90 minutes of PET emission data.
The ultimate goal of these studies was to apply quantitative imaging techniques,
such as compartmental modeling, to identify a suitable method for the analysis of
PIB PET data that could be used to validate the utility of PIB across the spectrum
of AD. A second goal of these fully quantitative studies was to provide a basis for
which to evaluate the performance of simplified methods of analysis applied to
human PIB studies in AD, MCI, and control subjects, such as the substitution of
reference tissue data in place of an arterial input function.

Fully Quantitative Analyses


The initial quantitative studies conducted at the University of Pittsburgh included
five subjects with mild to moderate AD (5M; age: 68G10 years; MMSE
18–26), five control subjects (3F, 2M; age: 59G16 years; MMSE 28–30), and
five subjects characterized as MCI (5M; age 71G10 years; MMSE 23–29). All
AD subjects met NINDS-ADRDA criteria for Probable AD and DSM-IV criteria
for Dementia of the Alzheimer’s type (71,72). MRI was performed on all
subjects, and a volumetric spoiled gradient recalled sequence optimized for
contrast between gray matter, white matter, and cerebrospinal fluid was
obtained. Ninety minutes of PET data were acquired following the injection of
370–555 MBq (10–15 mCi) of PIB. Arterial blood samples were withdrawn from
a catheter placed in the radial artery for the purposes of input function
determination. Radiolabeled metabolites in plasma were determined using a
combination of high-performance liquid chromatography and a liquid-liquid
extraction technique. Details of the methods used in these studies are described
elsewhere (73).
PET emission data were analysed using a variety of techniques routinely
employed for the analysis of PET receptor studies using reversibly binding
radioligands. These techniques included spectral analysis, a data driven
method that does not assume an underlying model configuration (74,75),
conventional 2- and 3- compartment pharmacokinetic models (76), and regression
methods such as the Logan graphical analysis (77). These models of radioligand
b-Amyloid Imaging Agents 293

binding assume reversible in vivo kinetics and provide outcome measures, such as
the binding potential (BP, unitless), distribution volume, and the distribution volume
ratio (DVR), which are related to Bmax, the concentration of free receptors, and the
ligand dissociation constant Kd.
Determination of the PIB tissue-to-plasma ratios for a variety of cortical and
subcortical regions showed that a constant ratio (plateau) was achieved after
approximately 20 minutes in the cerebellar regions of both AD and control subjects.
Tissue-to-plasma ratios in regions known to contain significant amyloid deposits in
AD, such as the posterior cingulate gyrus (PCG), also achieved a constant ratio that
was greater than threefold higher than that observed for controls, though the time to
plateau was considerably delayed (45–50 minutes). Furthermore, the specific
binding of [3H]PIB to human AD brain tissue homogenates was shown to be clearly
reversible with an off-rate (koff) of 0.0027/min, or a clearance half-life of
252 minutes (73). These observations were consistent with reversible radioligand
binding and supported the use of models of reversible radioligand binding.
Compartmental modeling of PIB data showed that PIB kinetics were best
described by a model that allowed for influx and efflux from two tissue
compartments (2T-4k) for all subject groups and regions, including cerebellum.
Nonspecific PIB retention was found to be similar across subjects, as no
significant group differences in the cerebellar 2T-4k DV were observed. In
cortical regions, the 2T-4k DVR value showed greater PIB retention in PCG,
PAR, LTC, and FRC relative to control subjects.
The Logan graphical analysis, a regression method for describing
reversible radioligand binding, was applied to regional and cerebellar PIB data
over the 35–90 minutes post-injection interval. Linear regression of the
graphical variables yields slope values that are equivalent to the 2T-4k
radiotracer DV (77). Good agreement between compartmental and Logan DVR
values (e.g., PCG: rZ0.89, slopeZ0.91) was observed, although the
Logan results were considerably less variable. Statistically significant differences
(p!0.05) in Logan DVR values were observed for AD subject relative to
controls in cortical areas after correction for multiple comparisons. The Logan
DVR measure also demonstrated favorable intrasubject variability, as
assessed in five subjects (2AD, 1 MCI, 2C) who returned for a repeat PIB PET
scan 8–20 days later. Test-retest variability averaged 8.4G5.4% across 11
cortical and subcortical regions-of-interest, and 6.1G1.5% across the five areas
with the highest PIB retention in AD: PCG, PAR, FRC, LTC, and caudate.
The intersubject variability was greatest in regions dominated by non-specific
PIB retention, such as the subcortical white matter and pons.
In general, mean MCI Logan DVR values fell between control and AD
means in brain areas that showed high PIB retention in AD. Figure 5 shows that
individual MCI subjects do not fall as a group into an area that is intermediate
between the control and AD ranges, but, instead, MCI subjects are either
indistinguishable from controls, or indistinguishable from AD subjects. This was
equally true for SUV and compartmental analyses (data not shown).
294 Klunk et al.

Figure 5 Scatter-plot showing the distribution of individual subject Logan distribution


volume ratio values. Control (triangles, nZ5), mild cognitive impairment (MCI) (circles,
nZ5) and Alzheimer’s disease (AD) subject (squares, nZ5) data in the five brain regions
showing the highest Pittsburgh compound-B (PIB) retention in AD subjects are shown. The
two MCI subjects that had relatively stable clinical courses (MCI-2 and MCI-5) are shown
with filled circles. Note that MCI-2 and MCI-5 are indistinguishable from controls in all five
brain areas while the other MCI subjects are essentially indistinguishable from the AD
subjects. The AD subject with the highest PIB retention in the posterior cingulate gyrus
(filled squares) also had the highest retention in the other brain areas. Source: From Ref. 62.

Figure 6 shows examples of parametric Logan DVR images that were


generated for a control (C-5), a “control-like” MCI subject (MCI-2), an “AD-like”
MCI subject (MCI-4), and the AD subject with the highest PIB retention (AD-1).
The parametric images show greatest PIB retention in cortical areas of the AD
brain and the “AD-like” MCI brain consistent with known patterns of amyloid
deposition (2,4,78), with comparatively minimal retention in the control and
MCI-2 subjects.
Overall, this initial quantitative assessment of PIB binding yielded differences
between AD subjects and controls that were consistent with the distribution of
amyloid deposition over the AD disease spectrum. The quantitative results were
found to be entirely consistent with the data obtained in the proof-of-concept study
(see above), and supported the use of arterial blood sampling and 90 minutes of data
acquisition. Most importantly, the study demonstrated that conventional modeling
approaches that assume reversible binding kinetics (i.e., the Logan graphical
method) can be employed in the analysis of PIB data and produce results that agree
with SUV-based analyses. The Logan graphical analysis and the DVR were
identified as the preferred method and outcome measure, due to its favorable level of
inter- and intra-subject variability across regions, high correlation with compart-
mental 2T-4k outcome measures, and ease of implementation. The data support the
b-Amyloid Imaging Agents 295

Figure 6 (See color insert.) Examples of Pittsburgh compound-B (PIB) Logan


distribution volume ratio (DVR) images. Shown are a control (C-5), a “control-like”
mild cognitive impairment (MCI) (MCI-2), an “Alzheimer’s disease (AD)-like” MCI
(MCI-4), and the AD subject with highest PIB retention. Images were generated using
arterial plasma data and a 90-minute analysis. The DVR images reflect greater PIB binding
in many cortical areas of the AD subject and MCI-4, with background levels in the control
and MCI-2 subjects. Similar DVR values were measured in the cerebellum across all
subject groups.

feasibility of performing multiple fully-quantitative PET studies in AD subjects, and


provide a basis to validate simplified methods of analysis for routine use across the
AD disease spectrum.

Simplified Analysis Methods


Ongoing studies at the University of Pittsburgh seek to extend the quantitative
PIB studies of Price et al. (73) to include an evaluation of simplified methods
for PIB PET imaging, with the ultimate goal of identifying a methodology that
can be simply and reliably applied across the AD disease spectrum. PIB PET
scans were conducted in 24 subjects (6 AD, 10 MCI, 8 controls) with arterial
blood sampling. Repeat scans were performed in eight subjects (3 AD, 1 MCI,
4 controls) within 28 days. Data were analyzed over 60 and 90 minutes using the
Logan analysis and (1) metabolite-corrected input functions based on arterial
plasma, (2) input functions based on carotid artery time-activity data with a
population average metabolite correction applied, and (3) cerebellar reference
tissue. Data also were analyzed using the simplified reference tissue method and
a single-scan method based on late-scan ratios of SUV.
All simplified methods of analysis effectively discerned regional
differences between AD and control subjects in amyloid-laden cortical regions
(p!0.001), although the performance of the simplified methods varied in terms
296 Klunk et al.

of bias, test-retest variability, and Cohen’s effect size (AD vs. control) (79).
Carotid volume-of-interest-based methods showed the lowest bias of any method
relative to the metabolite-corrected arterial methods. Although the test-retest
variability was the poorest of all the simplified methods examined, the carotid-
based methods demonstrated acceptable test-retest variability (G7.1% across 11
regions). Cerebellar input methods showed negative biases relative to the
metabolite-corrected arterial methods that were greater in subjects with higher
levels of PIB retention, although these cerebellar reference-based
methods showed the lowest test-retest variability of any method (G4.5% across
11 regions) and a large effect size [w6.5 for PCG]. The single-scan SUV-based
methods showed the largest effect sizes for AD and control group differences (6.9
for PCG) and performed very well in terms of inter-subject and test-retest
variability (G5.0%), although the SUV measures were positively biased relative
to the arterial methods. While the results of the comparison of simplified methods
of PIB analysis are preliminary, they suggest that several simplified methods of
analysis may be applicable to human PIB PET data, some requiring only a
20 minutes PET scanning session.

Pattern of PIB Retention in Alzheimer’s Disease, Mild Cognitive


Impairment, and Controls
The finding of Price et al. (73) that MCI subjects may have as much amyloid as
AD patients must be interpreted with caution due to the small number of subjects
(nZ5), but this is a finding with potentially important implications. At some point
in the development of AD pathology, there must be a stage at which amyloid
levels are midway between typical controls and typical AD patients. It would be
expected that anti-amyloid therapeutic approaches (e.g., passive immunization,
secretase inhibitors, etc.) would be optimally effective at these earliest stages of
amyloid buildup. These findings suggest that, at the very least, MCI patients do
not uniformly represent a homogeneous group that is at the very beginning or
middle stages of amyloid deposition. Although the numbers are small, these
findings hint that a significant percentage of MCI subjects will have amyloid
pathology nearly as well established as mild-moderate AD subjects. This finding
of AD-like levels of PIB retention in MCI is not particularly surprising in light of
the previous postmortem reports of AD-like levels of amyloid deposition in some
MCI subjects and in some elderly subjects with normal cognition (67–69,80).
It should be noted that the findings of Price et al. (73) in MCI patients do not
appear to be due to an unusually severe cohort of subjects since the least impaired
MCI subject (MCI-4; MMSEZ27) actually showed the greatest PIB retention.
In addition, the fact that 40% of the MCI subjects in the Price et al. study had
no evidence of amyloid deposition compared to controls (73) is entirely consis-
tent with the fact that 40–50% of clinically diagnosed MCI subjects do not
develop AD over a 10-year period of follow-up and may revert to a diagnosis of
“normal” (81,82).
b-Amyloid Imaging Agents 297

FUTURE APPLICATIONS OF AMYLOID IMAGING


The studies described above suggest that amyloid load can be accurately and
reproducibly measured in living human brain. As with all new imaging
technologies, the challenge that lies ahead will be to discover the unique
advantages of this technology as well as its limitations. Amyloid imaging will not
serve all purposes in neuroimaging studies of AD, but will likely find a niche
alongside the current standards, FDG PET, and structural MRI.

Improved and Earlier Diagnosis


Most imaging techniques have been used with the intent of identifying preclinical
pathology and developing a predictive diagnostic method (i.e., an antecedent
biomarker). Amyloid imaging holds potential as an antecedent biomarker if
detectable amyloid deposition occurs before the onset of clinical symptoms.
Evidence for pre-clinical amyloid deposition comes from postmortem histologi-
cal studies in AD and cognitively normal controls (69) and from the “Nun Study”
(66), in which preliminary data suggests that the prevalence of amyloid in
clinically unimpaired elderly may be on the order of 25–40%. Perhaps the
strongest indication of the time course of preclinical amyloid deposition comes
from postmortem studies of DS patients. Having three copies of the APP gene on
chromosome 21, nearly all DS patients develop amyloid pathology by their 30s or
40s and a large percentage later show clinical dementia in their 40s and 50s.
Although the time course of amyloid deposition in AD is not known, evidence
gained through postmortem study of DS suggests that amyloid deposition begins
over a decade prior to the clinical symptoms of dementia (8,83).
The question for amyloid imaging is when in this possible 10-year
prodromal phase can preclinical amyloid deposition be detected? It is possible
that detectable fibrillar amyloid deposits would occur only late in the preclinical
phase or even only after the onset of clinical symptoms. Existing data from the
application of PIB imaging to MCI subjects certainly shows that amyloid
deposition is easily detectable at this very early clinical stage and is already well-
established (73). Early PIB studies (62,73) also suggest that increased PIB
retention can be detected in cognitively normal control subjects. Natural history
studies are underway in subjects who carry early-onset, autosomal dominant
familial AD gene mutations in efforts to define when in the course of the illness
amyloid deposition can be detected. Similar PIB PET studies in other populations
with predictable amyloid deposition, such as DS, could be similarly useful.
In addition to improved early diagnosis, amyloid imaging could play a role
in the differential diagnosis of dementias other than AD or when an AD
component is suspected. In this context, we might think of defining dementia
syndromes as “dementia with b-amyloidosis” and “dementia without b-amyloi-
dosis.” Dementia with b-amyloidosis would presumably be equivalent to the
presence of AD (alone or in combination with another dementing disorder).
A large variety of dementias could make up the group of dementias without
298 Klunk et al.

b-amyloidosis. In particular, the finding of a negative PIB PET study could be


very useful in defining pure frontotemporal dementia. One might be able to
identify cases of “pure” dementia with Lewy bodies (DLB) and “pure” vascular
dementia (VaD), although they may be relatively rare as neuropathological
studies indicate that many patients with DLB and VaD also have amyloid
deposits.

Prediction of Mild Cognitive Impairment Conversion


to Alzheimer’s Disease
A closely related application of imaging technology in AD research is the
prediction of which MCI patients will progress to a diagnosis of AD. On average,
patients diagnosed with MCI progress to AD at a rate of about 10–15% per year,
but nearly half do not develop AD even after follow-up periods as long as five
years (81,84). It would be very useful to accurately distinguish these two types of
MCI patients, particularly for inclusion in clinical trials of anti-amyloid drugs. In
the only group of MCI subjects thus far reported (nZ5), PIB imaging has been
able to distinguish MCI patients with AD-like retention patterns from patients
diagnosed with MCI but who have control-like PIB retention. While this is
promising, it is too early to tell if this distinction will accurately predict
conversion to AD, and if so, how many years before the clinical diagnosis of AD
can an AD-like pattern of PIB retention be detected.

Natural History of Amyloid Deposition


The use of amyloid imaging as an antecedent biomarker and for prediction of
conversion from MCI to AD can be thought of in the wider context of the natural
history of amyloid deposition. To optimally achieve preclinical diagnosis or
prediction of MCI progression to clinical AD, we should ideally understand the
entire process of amyloid deposition. This understanding will require a coupling
of detailed cognitive assessment with the amyloid imaging studies. Correlations
between mild though specific cognitive deficits and early focal amyloid
deposition can then be made. These natural history studies are critical to fully
understand the still controversial role of amyloid deposition in the pathogenesis
of AD. For example, deposition of Ab protein in the brain has been postulated to
be the cause of AD (9). However, the finding of postmortem amyloid plaque
deposition in the brains of individuals in whom the absence of clinical AD was
well documented prior to their death (85) has been interpreted to both support
(68) and refute (86) the amyloid cascade hypothesis. This wide range of
interpretations exists partly because of the lack of in vivo longitudinal data on the
natural history of amyloid deposition. Such data could clarify whether
cognitively normal individuals with brain amyloid deposition are destined to
develop clinical dementia.
b-Amyloid Imaging Agents 299

In ongoing amyloid imaging studies, we are assessing the in vivo


prevalence of brain amyloid deposition in cognitively “normal” elderly subjects
who do not meet clinical criteria for MCI or AD. With increasing age, the
cognitive performance of many individuals will decline while still not meeting
criteria for MCI or AD (i.e., cognitive decline below the threshold of clinical
impairment). We prefer to refer to these subjects as “clinically unimpaired.”
Although these elderly subjects do not meet clinical diagnostic criteria for
cognitive impairment, previous studies have shown that a high percentage of
these clinically unimpaired elderly will have cognitive performance below that of
young subjects with equal education (Fig. 7A) (87). We refer to these subjects as
“low-normal performers” (Fig. 7A). We have recently initiated studies aimed to
assess whether in vivo evidence of amyloid deposition is associated with
cognitive performance in the lower end of this “normal” range (i.e., being a “low-
normal performer” Fig. 7A). In these studies, we will assess whether evidence of
amyloid deposition is associated with greater decrements in cognitive
performance which could ultimately lead to conversion to MCI or AD over
time (Fig. 7B).
There are at least three reasons to relate cognition and amyloid deposition
in clinically unimpaired elderly: (1) definition of the prevalence of amyloid
deposition in clinically unimpaired elderly, (2) determination of whether the
greater variability of cognitive performance in the clinically unimpaired elderly
(compared to the young) is explained by the presence or absence of amyloid
deposition, and (3) assessment of whether clinically unimpaired individuals with
substantial amyloid deposition will invariably progress to clinical dementia.
Studies have attempted to address the first two questions with postmortem
assessment of amyloid deposition, but there are obvious weaknesses to this
approach. First, it is difficult to acquire cognitive testing close to the time of
death—when the amyloid assessment is made. The time period between cognitive
testing and death can vary by over a year in some studies. Even cognitive testing
occurring very close to the time of death is problematic since any cognitive
impairment present could be due to effects of the disease that led to the individual’s
death. Second, there is an additional selection bias added by the decision to agree to
autopsy. This means that not all of the cognitively assessed group will be
represented in the amyloid assessment. Finally, many of these studies involve
cognitive testing that was not performed for the specific purposes of the study, but
was retrospectively gathered to the best degree possible. Much of the cognitive
data available comes from cognitive screening tests such as the MMSE (58) and
not measures that examine the specific components of cognition thought to be
particularly affected by aging (e.g., information processing speed, working
memory, inhibitory efficiency). Even when longitudinal data on cognitive
performance are available [e.g., (88,89)], it is not possible to link an age-related
decrease in cognitive performance to an increase in amyloid load (i.e., to establish
causality) because amyloid load could only be determined at one time point—after
death. All of these factors have hindered attempts to determine the role that
300 Klunk et al.

Figure 7 Schematic of the distribution of cognitive performance measures in young


subjects, “clinically unimpaired” elderly, and clinically affected individuals who meet
criteria for mild cognitive impairment (MCI) or Alzheimer’s disease (AD). (A) Clinically
unimpaired elderly show increased variability compared to young subjects, skewed towards
poorer performance, but not reaching clinical criteria for MCI or AD. (B) We hypothesize
that much of the poorer performance of the elderly is explained by a subset who have
amyloid deposition and that this “amyloid-positive” subset contains those individuals who
may convert to MCI or AD over time (i.e., amyloid-negative subjects will become amyloid-
positive before converting to MCI or AD).

amyloid deposition may play in the various cognitive decrements found in the
“normal” old. The availability of amyloid imaging techniques that can measure the
amyloid load at multiple time points in living individuals who are not close to death
is thus a major advantage.

Natural History in Early-Onset, Autosomal Dominant Familial


Alzheimer’s Disease
Discussion of the three issues described above regarding the natural history of
amyloid deposition presumes that, in a person destined to demonstrate clinical
symptoms of AD, amyloid deposition precedes these clinical symptoms and can
b-Amyloid Imaging Agents 301

be detected by in vivo amyloid imaging. This can be a difficult assumption to


demonstrate in normal aging studies due to the difficulty in identifying with
certainty people who will develop clinical AD. Although tragic, the predictable
clinical outcome of individuals who carry an early-onset autosomal dominant
familial AD mutation offers an opportunity to efficiently gain insights into the
natural history of amyloid deposition in general. In addition, the clinical time
course can also be reasonably predicted by the family history. Therefore, studies
can be designed with relatively few subjects that can be known with certainty to
be on a predictable course toward the development of clinical AD. The imaging
studies can be timed to occur at points before pathological changes are expected
and be continued through the onset of clinical symptoms. Studies such as these
have previously been performed using volumetric MRI (90).
Knowledge of this natural history has implications not only for early onset
familial AD, but for typical late-onset sporadic AD as well. Information from
early-onset familial AD studies could help address the natural history issue
regarding the validity of the amyloid cascade hypothesis. If possible, definition of
a particular distribution or amount of amyloid deposition which heralds clinical
decline in early-onset familial AD could add important information to the design
of studies aimed to determine whether non-demented individuals with substantial
amyloid deposition (with or without an early-onset familial AD mutation)
invariably progress to clinical dementia. This issue is key to the amyloid cascade
hypothesis and to health care policy decisions regarding the need to treat
asymptomatic individuals with evidence of amyloid deposition when and if
effective anti-amyloid therapies are successfully developed. This in turn could
lead to more effective treatments, not only for those with early onset familial AD,
but also for those destined to develop sporadic AD.

Amyloid Imaging in Clinical Trials of Anti-amyloid Drugs


One of the most important uses of amyloid imaging technology may be in the
facilitation of new drug development. While amyloid imaging may or may not
have a general role to play in the development of the so-called “disease
modifying” therapies, it is relatively easy to envision its role in the evaluation of
drugs aimed specifically at amyloid production (e.g., secretase inhibitors) or
amyloid clearance (e.g., plaque breakers and immunotherapies). For the optimal
evaluation of these anti-amyloid therapies, it will first be essential to define
homogeneous trial populations that have some identifiable baseline level of
amyloid deposition. In other words, it would make little sense to use anti-amyloid
drugs in subjects who have no brain amyloid deposits to begin with. Furthermore,
since effects on amyloid deposition are not likely to be an all-or-none
phenomenon, it will be essential to quantitatively and precisely define the
baseline status of amyloid deposition for the purposes of comparison to post-
treatment outcomes. Early studies suggest that amyloid imaging with PIB is a
302 Klunk et al.

very stable and reproducible measure over short time periods with test-retest
variability of only 5–10% (73). Therefore, reductions in amyloid load of 20% or
more should be easily measurable, once corrected for the increase in amyloid
load over the time period of the study determined from natural history studies.
One must consider whether imaging large fibrillar Ab deposits is the
appropriate method for assessing the efficacy of anti-amyloid therapies. Evidence
is accumulating to suggest that small, soluble oligomers may be the toxic species
of Ab in the human brain (91,92). Some would even suggest that large insoluble
aggregates of Ab are beneficial in that they serve to “detoxify” these soluble
oligomers. This concept appears to have some support from in vitro studies of
other amyloid-related pathologies (e.g., prion protein accumulator in yeast) in
which large intracellular aggregates of the protein in discrete cellular organelles
appear to be related to cell survival (93). However, plaques are extracellular
aggregates and are very different from the carefully managed intracellular
“detoxification” programs seen in cellular models of prion disease that use both
the cell membrane and organelle membrane to control the toxic waste. Plaques
are more akin to an unmanaged dumping of barrels of toxic waste into a land fill.
Even when the soluble/free toxin is removed, the plaque serves as a source from
which more toxin can leach into the surrounding extracellular environment. To
keep the potential danger of the insoluble Ab deposits in perspective, we must
remember that in AD brain, soluble forms of Ab make up less than 1% of total
brain Ab (94). In addition, it is almost certain that soluble and insoluble Ab pools
are in equilibrium. This is supported by the fact that it is extremely difficult to
separate soluble and insoluble Ab in the laboratory, because even after
ultracentrifugation and removal of soluble Ab, resuspension of the “insoluble”
Ab results in the appearance of more soluble Ab (unpublished observation).
Other support for this equilibrium comes from the observation that immunization
of amyloid-depositing transgenic mice in a manner that produces antibodies
specific for oligomeric Ab leads to marked reduction of not only oligomeric
forms of Ab, but also results in clearance of thioflavin-S positive plaque forms of
Ab as well (95,96). This suggests that any meaningful anti-amyloid therapy will
need to have a significant impact on insoluble brain Ab deposits for there to be a
long-lasting lowering of soluble Ab. Such an effect should be detectable with
agents that bind to fibrillar amyloid.

SUMMARY
In vivo amyloid imaging technology has largely overcome the hurdle of
achieving sufficient brain entry while retaining adequate affinity for the Ab target.
Brain clearance and the resulting specific labeling of Ab deposits also appears
sufficient in some of the existing tracers, but future radiopharmaceutical
development may result in tracers with even better signal-to-noise ratio and
sensitivity. Existing tracers all appear to target aggregated b-sheet forms of Ab,
but further work is necessary to precisely define the subtypes of Ab deposits that
b-Amyloid Imaging Agents 303

are labeled by each tracer. The early proof-of-concept phase has produced
convincing evidence that amyloid load can be quantitatively assessed in living
humans. Future challenges lie in the application of amyloid imaging to increase
the understanding of the natural history of amyloid deposition and its relationship
to cognitive change. Perhaps the most important impact of amyloid imaging
technology will be in the facilitation of the development of anti-amyloid
therapies, early (even preclinical) identification of cognitively normal individuals
with amyloid deposition who would be good candidates for anti-amyloid therapy,
and individual patient follow-up to monitor the success of anti-amyloid
treatment regimens.

ACKNOWLEDGMENTS

This work was supported by grants from The National Institutes of Health (R01
AG018402, P50 AG005133, K02 AG001039, R01 AG020226, R01 MH070729,
K01 MH001976), The Alzheimer’s Association (TLL-01-3381), The U.S.
Department of Energy (DE-FD02-03 ER63590), and GE Healthcare, Inc. GE
Healthcare entered into a license agreement with the University of Pittsburgh
based on some of the technology described in this manuscript. Drs. Klunk and
Mathis are co-inventors of PIB and, as such, have a financial interest in this
license agreement.

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11
A View on Early Diagnosis of Dementias
from Neuropathology

Kurt A. Jellinger
Institute of Clinical Neurobiology, Vienna, Austria

INTRODUCTION
With an increasingly elderly population the incidence of dementing disorders is
rapidly increasing in frequency and represents a major scientific, humanitarian,
and socioeconomic problem. Due to recent progress in genetic, molecular
biological, imaging, and neuropsychological techniques, and detection of disease
specific biological markers, the diagnosis and classification of these processes
have increased in accuracy. However, despite the establishment of diagnostic
guidelines and consensus criteria, a definite diagnosis still depends on neuro-
pathological examination of the brain at autopsy and, rarely, by biopsy. This is
particularly true for early stages of such processes, in which the validity of
clinical and neuroimaging criteria is limited. Studies of the brains of elderly
individuals without cognitive changes and in those with Alzheimer’s disease (AD)
and other dementias have provided a major input for progress in our under-
standing of brain aging and the lesion thresholds for mild cognitive impairment
(MCI) and initial stages of dementia (1–16).
The quality and pattern of brain lesions in aging and AD, their spreading
along well-defined anatomical pathways, and the consecutive biochemical
changes have been described and correlations with cognitive function and clinical
course have been performed. Recent insights into the cell biology of protein
misfolding and its consequences for neuronal (dys)function offer hope for a better
understanding of the underlying molecular processes (17–21). However, many

311
312 Jellinger

questions concerning the pathology of dementia, its relations to clinical


phenotypes, and the lesion threshold for cognitive impairment remain
unanswered. Additional difficulties arise from the multimorbidity of aged
individuals and frequent concurrence of age-related brain lesions with other
pathologies that may interact in “unmasking” or promoting mental impairment.
The majority of demented patients die in advanced stages of the disorder,
and only in a few instances is postmortem examination possible in early stages
when death occurs under acute and unforeseen circumstances, e.g., due to fatal
trauma, suicide, or intercurrent diseases.
The present chapter aims to give a brief update of the neuropathology of
major dementia disorders and an outline of the current pathological guidelines
for their diagnosis. Regarding the possibilities of early diagnosis, the available
morphological data in cognitively normal aged subjects and the lesion threshold
between physiological aging and MCI as a pre- or subclinical stage of AD and
other dementias (22–28) are reviewed. The few available and partly contradicting
results of comparative biopsy and autopsy studies in the same patients have
provided some opportunities to follow the progression of AD. Early
morphological changes in other diseases have hardly been described and only
can be suggested from their full-blown pathology. The relations between disease
morphology and various biomarkers will be discussed, and a conclusion will
critically summarize the current possibilities for early diagnosis of dementing
disorders from the view of neuropathology.

CLASSIFICATION OF DEMENTIA DISORDERS


Dementia has recently been redefined as the differential manifestation of
deteriorating brain functions over time as a part of aging due to cell deaths in the
brain caused by neurodegeneration or any other disease (29). Currently,
dementing disorders are classified according to their principal underlying
molecular biological and genetic changes (Table 1). Here only the morphology of
the major types of dementia will be reviewed.

BASIC PATHOLOGY OF MAJOR DEMENTING DISORDERS


Alzheimer’s Disease
The morphological features of AD include extracellular deposition of amyloid
b peptide (Ab) in plaques and cerebral vasculature [cerebral amyloid angiopathy
(CAA)] and accumulation of microtubule-associated hyperphosphorylated
tau-protein forming paired helical filaments (PHF) in neurons [neurofibrillary
tangles (NFT)], dendrites [neuropil threads (NT)] , and around plaques [neuritic
plaques (NP)]. Although most of these changes are nonspecific, the diagnosis
of AD depends on their semiquantitative assessment. Other lesions are loss of
synapses, synaptic proteins, and neurons causing cerebral volume loss (atrophy)
Early Diagnosis of Dementias from Neuropathology 313

Table 1 Classification of Dementing Disorders According to Hitherto Known Cause


Degenerative dementias (biomolecular types)
Proteinopathies
Tauopathies (secondary)
Alzheimer’s disease (AD) (3C4-R tau-tripletCb-amyloid deposition)
Tauopathies (primary)
Progressive supranuclear palsy (PSP) (4-R tau doubletCExon 10)
Corticobasal degeneration (similar to PSP)
Tangle-predominant dementia (NFTs, no/little amyloid)
Argyrophilic grain disease (4-R tau with/without AD lesions)
Frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17T)
(mutations Exon 9, 10, 12, 13 of tau gene)
Pick disease (3-R tau without exon 10)
Familial progressive subcortical gliosis (tau mutation on chromosome 17q)
Multisystem tauopathy with presenile dementia (4-R tau)
a-Synucleinopathies
Dementia with lewy bodies (DLB)
Parkinson disease with dementia (PDD)
LB-variant of AD (LBV/AD) (DLBCAD)
Ubiquitinopathies
Familial frontotemporal dementia (FTD) with Ub-positive cell inclusions (FTD-U)
FTD with motoneuron disease (FTD-MND)
Lobar atrophies
Frontotemporal lobar degeneration (FTLD) without tau pathology
Polyglutamic disorders
Huntington disease (CA6-trinucleotid repeat disorder)
Prion disease (transmissible spongiform encephalopathies)
Creutzfeldt–Jakob disease (sporadic)
Creutzfeldt–Jakob disease (familial)
Creutzfeldt–Jakob disease (iatrogenic)
Creutzfeldt–Jakob disease (new variant)
Gerstmann-Sträussler-Scheinker disease
Fetal insomnia
Vascular-ischemic dementias (VID)
Vascular cognitive impairment (VCI)
Multiinfarct encephalopathy (MIE)
Subcortical atherosclerotic encephalopathy (SAE)
Strategic infarct dementia (SID)
Postanoxic-hypoxic encephalopathy
“Mixed dementias”
Combination ADCVID
Combination ADCother pathologies
Infectious-inflammatory disorders
Viral
HIV dementia
Herpes encephalitis (residual)
(Continued)
314 Jellinger

Table 1 Classification of Dementing Disorders According to Hitherto Known


Cause (Continued)
Progressive multifocal leukoencephalopathy (PML)
Neurosyphilis (paretic dementia)
Demyelinating disorders
Multiple sclerosis
Subacute sclerosing panencephalopathy (SSPE)
Leukodystrophies
Neoplasia
Primary and secondary brain tumors
(Para-) neoplastic processes
Internal hydrocephalus
Obstructive vs. non-obstructive
Normal-pressure hydrocephalus
Head injuries
Sequelae of brain trauma
Subdural hematoma
Boxer’s encephalopathy (dementia pugilistica)
Toxic injuries
Alcohol
Drugs
Metals, other chemicals
Nutritional and metabolic disorders
Vitamine deficiencies
Systemic metabolic disease (endocrine, kidney, liver, etc.)
Lysosomal/peroxisomal disorders (gangliosidoses, leukodystrophies)
Mitochondrial encephalopathies (MELAS, etc.)

and progressive disruption/disconnection of neuronal circuits as major substrates


of dementia (30–33), microglial activation (inflammatory cascades) (34,35),
astroglial proliferation, granulovacuolar degeneration, and Lewy bodies (LBs)
(Table 2). Neuron loss, particularly affecting glutamatergic neurons, is prominent
in the neocortex and hippocampus, areas of importance in memory and cognition,
as well as in some subcortical regions, e.g., in the cholinergic and GABAergic
systems (36).
Amyloid or Tau Pathology—Chicken or Egg?
It is generally accepted that b-amyloid precursor protein (APP) functioning,
metabolism, and intraneuronal transport are key factors in the pathogenesis of AD
(37). Ab is considered central to the pathogenesis of AD (38–42). Ab derived from
the proteolysis of a large transmembrane glycoprotein precursor, the APP, is
released as soluble peptides of 39 to 46 residues, with Ab 40 as the most abundantly
produced isoform. The balance between biogenesis versus catabolism of Ab may
be an important factor in the pathogenesis of AD. The balance is disturbed between
overproduction of Ab through enhancement of b- and g-secretase activity, which
Early Diagnosis of Dementias from Neuropathology 315

Table 2 Histopathology of Alzheimer’s Disease—Diagnostic Markers

appears to be constantly expressed in normal tissues, or an altered conformation of


substrate found in some inherited mutations of APP, and failure to adequately
degrade or clear Ab from its intracellular and extracellular compartment in the
brain (43). It has been proposed that the Ab pathology seen in AD is a continuous
process from an initial abnormal Ab intracellular accumulation to the well-
established extracellular Ab aggregation, culminating in the formation of amyloid
plaques and dystrophic neurites (44). Water-soluble Ab (wsAb) is present in
cerebral cortex of subjects at risk of AD as well as in normal elderly subjects as a
mixture of three major species: 1-42, py3-42, and py11-42. The three wsAb species
are not detectable in the brains of young people, free of immunohistochemically
detectable APs. In the brains of Down syndrome (DS) and APP-mutant tg mice,
wsAb appears a long time before amyloid deposition, indicating that it represents
the first form of Ab aggregation and accumulation. In the normal brain, wsAb is
bound to apolipoprotein E (ApoE) which favours its degradation by proteases.
The composition of wsAb, in terms of the ratio between the full-length 1-42 and
the py3-42 peptides, correlates with the severity of clinical and pathological
phenotype in familial early onset AD. Water-soluble Ab is the native counterpart
of the Ab small aggregates (soluble oligomers) that show, in vitro, an early and
high neuronal toxicity (45). They localize to axons and axon terminals with a
higher density in AD than in non-demented brains and might be related to synaptic
dysfunction in AD (46). The central questions in AD focus on whether cerebral and
cerebrovascular Ab accumulation is (1) a final neurotoxic pathway, common to all
forms of AD; (2) a toxic by-product of an independent primary metabolic lesion
that, by itself, is also neurotoxic; or (3) an inert by-product of an independent
primary neurotoxic reaction (47). In late onset AD, there are compelling reasons
that a failure of degradation or clearance of Ab from the brain underlies its
316 Jellinger

accumulation. Clearance of aggregated Ab is a complex process, for which


microglia, macrophages, and bulk flow across the blood-brain barrier (BBB) are
important. Ab42 can form fibrils more rapidly and at lower concentrations than
Ab40 (48). The most abundant form of Ab42 fibril exhibits a nodular structure with
a 100-nm periodicity. This length is very similar (1) to the length of protofibril
bundles that are the dominant feature at earlier stages in the aggregation process,
(2) to the period of helical structures that have been observed in the core of fibrils,
and (3) to the distance between regularly spaced, structurally weak fibril points.
Taken together, these data are consistent with the existence of an approximately
similar to 100-nm long basic protofibril unit that is a key fibril building block
(49). Cleavage of APP by a- and b-secretase is determined by dynamic
interactions with lipid rafts (50), that are also associated with the active
g-secretase complex (51). Hence, lipid rafts play an important role in APP
processing, in the generation, aggregation, and degradation of Ab (52), and are
a common component of human extracellular amyloid fibrils (53). A fraction
of these Ab molecules never leave the membrane lipid bilayer after they
are generated, but instead exert their toxic effects by competing with and
compromising the functions of intramembranous segments of membrane-bound
proteins that serve many critical functions. Based on the presence of shared
amino acid sequences it was speculated that accumulations of intramembranous
Ab might affect the functions of APP itself and the assembly of the PSEN1,
Aph1, Pen 2, Nicastrin complex (54).
Clouds and fine diffuse plaques (DP), initial stages of Ab deposition with
accumulation of nonfibrillary Ab42, are followed by deposition of Ab40 (55) that
is inserted into lipid layers (56), and oligomerization of both (57), later evolving
into primitive plaques containing fibrillary Ab, tau-positive dystrophic neurites,
activated glia, and subsequent development of NFTs (58). Ab42 plays a more
important role in the pathogenesis of AD since its aggregative ability and
neurotoxicity are considerably greater than those of Ab40 (59). Increased
concentrations of Ab lead to the formation of insoluble Ab plaques (60).
Evidence that synaptically released Ab accumulates as extracellular deposits in
the hippocampus of transgenic (tg) mice suggests that these are dynamic
structures and a perforant path lesion alters the equilibrium between Ab
production/deposition towards clearance as a consequence of blocked APP
transport from the entorhinal cortex to terminal fields in the hippocampus
(61,62). Intracellular Ab42 increases with aging and accumulates in multi-
vesicular bodies within presynaptic and postsynaptic compartments (63,64) in the
endoplasmic reticulum (ER), mitochondria, and Nissl bodies (65), and a positive
feedback mechanism between Ab production and intracellular APP levels has
been described (66); deposition of Ab fibrils promotes cell-surface accumulation
of APP (67).
Brain-derived neurotrophic factor (BDNF) induces APP expression in vivo,
but a significant reduction of BDNF in AD brain suggests that cellular APP
expression is mainly modulated by other factors (68). There is growing evidence
Early Diagnosis of Dementias from Neuropathology 317

that reduced neurotrophic support is a significant factor in the pathogenesis of


AD and other neurodegenerative diseases (69). ER-localized Ab is degraded in the
cytosol (70). Intracellular accumulation of Ab42, detected in human brain (71–73)
and in APP and presenilin-1 (PSEN1) double-tg mice (74–76) and rats (77), is
associated with abnormal synaptic morphology before Ab pathology, and synaptic
alterations predominate in tg mice where amyloid is primarily targeted (78–80).
Recently, Ab peptides, whether added to cultures or produced by neurons, have
been shown to decrease the number and activity of synaptic glutamate receptors
(81,82). This confirms the critical role of intraneuronal A42 for synaptic plasticity
and neuronal loss and may provide alternative explications for the early
pathogenesis of AD, other than the conventional extracellular Ab deposition
hypothesis (83–85). Co-localization of intraneuronal phosphorylated APP and tau
has been observed in very early AD (86). Important synaptic loss may take place
even in the absence of amyloid plaques (87,88), implying that the major causative
synaptotoxic insult in AD occurs during the early steps of amyloid processing
and fibrillar aggregation. Another possible way of plaque-independent
neurotoxicity might be membrane permeabilization or disruption (89,90).
In AD, endosome abnormalities are among the earliest neuropathologic features
to develop and have now been closely linked to genetic risk factors for AD,
including APP triplication in Trisomy 21 (DS) and APOE 34 and 32 genotype in
sporadic AD. Recent findings on endosome regulation and developmental and
late-onset neurodegenerative disease disorders are beginning to reveal how
endocytic pathway impairment may lead to neuronal dysfunction and cell death
in these disorders and may also promote amyloidogenesis in AD (91).
Animal Models
The identification of genes and pathways implicated in the pathogenesis of AD
led to the production of tg models of Ab amyloidosis, which recapitulate many of
the neuropathological features of AD, including progressive accumulation of
SPs, synaptic loss, and gliosis, associated with learning and memory deficits
(92,93). The molecular basis of the disease phenotype in the majority of these tg
animals is probably enhanced production of Ab species. Furthermore, the
mechanisms by which additional factors implicated in Ab metabolism and AD,
such as ApoE, cholesterol, and inflammatory mediators, have been explored (92).
In some triple-tg models, ultrastructural changes, such as dystrophic neurites and
behavioural changes, precede Ab deposition, which in turn precedes tangle
formation (94–96), while tg mice expressing the b-cleaved C-terminal APP show
no alterations of hippocampal neuronal and synaptic bouton numbers (97), but
dynamin-1 depletion in hippocampal neurons, a potential mechanism for early
cognitive loss without a major decline in synapse number (98). However,
tg animals overexpressing Ab lacking tau pathology and showing little or no
neuronal damage (99) are an incomplete model of AD (100). In APP/PSEN1
double-tg mice, Ab pathology does not correlate with synaptic and cognitive
deficits, hippocampal cell loss, or central cholinergic function, which occur only
318 Jellinger

at a later age as amyloid burden increases, suggesting that Ab levels are not
a marker of memory decline that is related to disease progression (101–103),
whereas in the APPSLPS1KI mice massive CA1/2 neuronal loss correlates with
strong accumulation of intraneuronal Ab42 (104), and APP/tau double-tg mice
show accelerated Ab deposition, neurofibrillary degeneration, and neuronal loss
suggesting a reciprocal interaction between Ab and tau (105). In triple-tg
(3xTgAD) mice harboring PSEN1, APP, and tau (P301L) antigens and
progressively developing SPs and NFTs, synaptic dysfunction manifests in an
age-related manner before plaque and tangle pathology (96), suggesting that
plaque or tangle pathology contribute to cognitive dysfunction at later time
points. APP(SL) PSEN1 tg mice develop age-related synaptophysin-IR
presynaptic boutons within the hippocampus, which supports their role in
neurodegeneration (106). Clearance of the intraneuronal AD pathology by
immunotherapy rescues the early cognitive deficits on a hippocampal-dependent
task, strongly implicating intraneuronal Ab in the onset of cognitive dysfunction
(107). This AD model and APP(SL) PS1 K1 mice show massive CA1/2 neuronal
loss with intraneuronal and N-terminal truncated Ab42 accumulation (104). The
introduction of Ab into tau-tg mice has led to enhanced tau pathology with no
change in Ab deposition, further supporting the amyloid cascade hypothesis
(92,95,108). Tangle formation in tg mice expressing mutant human tau is
accompanied by extensive axonal and neuronal damage (109).

Ab Neurotoxicity
Amyloid proteins appear as a subgroup of misfolded proteins, where misfolding
leads to subsequent aggregation. This aggregation may be a generic property of
polypeptide chains possibly linked to their common peptide backbone that does
not depend on specific amino acid sequences. And, in fact, many proteins can,
in vitro, form amyloid-like aggregates, while in vivo, only 20 amyloid proteins
have been so far identified. Although misfolding and aggregation are quite well
studied in vitro, the last step of amyloid deposition, i.e., anchorage to the
extracellular matrix, cannot be so easily approached. Proteoglycans and serum
amyloid P component have nevertheless been identified as key elements involved
in extracellular deposition of amyloid (110).
Mounting data suggest that soluble Ab oligomers (protofibrils), intraneur-
onal and spherical aggregates of Ab42 (amylospheroids), rather than Ab fibrils,
may be the primary toxic species and the principal cause of synaptotoxicity
(111–118). Ab cytotoxicity is mediated by p38 inducing oxidative stress (119).
Soluble Ab oligomers ultrastructurally localize to cell processes and might be
related to synaptic dysfunction in AD brain (46). These soluble neurotoxins
[known as “amyloid b-derived diffusible ligands” (ADDLs) (120,121) and
protofibrils] seem likely to account for the imperfect correlation between
insoluble fibrillar amyloid deposits and AD progression. ADDLs are known to be
potent inhibitors of hippocampal long-term potentiation in vivo, which is
Early Diagnosis of Dementias from Neuropathology 319

a paradigm for synaptic plasticity, and have been linked to synapse loss and
reversible memory failure in tg mouse AD models (111), and binding occurs
predominantly at synapses (117). If such oligomers were to build up in human
brain, their neurological impact could provide the missing link that accounts for
the poor correlation between AD dementia and amyloid plaques. Oligomers in
AD reach levels up to 70-fold over control brains. Brain-derived and synthetic
oligomers exhibit the same striking patterns of attachment to cultured
hippocampal neurons, binding on dendrite surfaces in small clusters with
ligand-like specificity. Binding assays using solubilized membranes show
oligomers to be high-affinity ligands for a small number of low abundance
proteins. Current results confirm the prediction that soluble oligomeric Ab
ligands are intrinsic to AD pathology (121). Recent experiments have detected
the presence of ADDLs in AD-afflicted brain tissue and in tg models of AD. The
presence of high affinity ADDL binding proteins in hippocampus and frontal
cortex parallels the regional specificity of AD pathology and suggests
involvement of a toxin receptor-mediated mechanism. The properties of
ADDLs and their presence in AD-afflicted brain are consistent with their
putative role even in the earliest stages of AD (116). The strong correlation
between soluble Ab brain concentrations and severity of neural circuitry
dysfunction (122) and early aggregates of nondisease-associated proteins are
potent cytotoxics, whereas their final fibrillar aggregates (indistinguishable from
AD fibrils) are not (123). The presence of misfolded proteins in the ER triggers
a cellular stress response called the unfolded protein response (UPR) that may
protect the cell against the toxic buildup of misfolded proteins. Phosphorylated
(activated) pancreatic ER kinase (p-PERK), which is activated during the UPR,
was observed in neurons in AD patients, but not in nondemented controls and did
not co-localize with AT8-positive tangles. These data show that the UPR is
activated in AD, and the increased occurrence of p-PERK in cytologically
normal-appearing neurons suggests a role for the UPR early in AD neuro-
degeneration. Although the initial participation of the UPR in AD pathogenesis
might be neuroprotective, sustained activation of the UPR in AD might initiate or
mediate neurodegeneration (124).
On the other hand, recent data suggest that synapse-associated Ab is
prominent in regions relatively unaffected by AD lesions, and that amyloid
accumulation in surviving terminals is accompanied by gliosis and alteration in
the postsynaptic structure (125). In accordance, single systemic administration of
an antibody against Ab produces rapid improvement in behavioural performance
of APP-tg mice without affecting total amyloid levels, obviously because of
blockage of a diffusible Ab species (126). The binding of Ab to membrane lipids
facilitates Ab fibrillation which in turn disturbs the structure and function of
neuronal membranes (127). Fibrillary Ab deposition has been shown to be more
detrimental to neuronal circuitry than previously thought (128). Ab toxicity
mediated by the formation of APP complexes (129) but not by secreted Ab (130)
and mitochondrial dysfunction (131,132) induce oxidative stress-mediated
320 Jellinger

(133–136), apoptotic neuronal death in vitro and in vivo (137). Ab further


induces impairment of endothelial function causing vascular disorders and
disorders of the BBB (138). An increased influx of circulatory Ab into the CNS
across the BBB may result in accumulation of soluble neurotoxic Ab in brain
interstitial fluid and/or its subsequent aggregation and deposition in the CNS
(139). All these changes were previously suggested to be caspase-independent
(140), but recent studies indicate that Ab could function as a key suicide molecule
through caspase-3 in AD (141). Ab further induces activation of the p38, JNK
pathways, and NF-kB in control cybrids and offers protection against the
neurotoxic effects of Ab. Expression of AD mitochondrial genes in cybrids
activates stress-related signaling pathways and reduces viability. This AD
phenotype is produced by endogenously generated Ab and can be replicated by
exogenous Ab acting through AGEs (142). Proteins are modified by oxidation,
glycoxidation, and lipoxidation, an early event in MCI (143), supporting an
important role for lipid-peroxidation-derived protein modifications in AD
pathogenesis (144,145). On the other hand, in tg animal models of AD, brain
oxidative damage precedes Ab formation (146). Both extracellular Ab deposits
and intraneuronal Ab42 production triggered by sustained increase of cytosolic
calcium concentration resulting from Ab ion channel-like structures in the
cellular lipid-bilayer, allowing calcium uptake (147,148), may activate caspases
leading to cleavage of nuclear and cytoskeletal proteins, induce neuronal death,
and underlie disruptions of neuronal signaling in the early stages of AD. Thus, Ab
channels may provide a direct pathway for calcium-dependent Ab toxicity in AD,
which can be prevented by zinc, an Ab-channel blocker and by the removal of
extracellular calcium (147) or a g-secretase inhibitor (148–150).

Mechanisms of Cell Death in AD


Aging renders the brain vulnerable to Ab neurotoxicity (151), and DNA
replication precedes neuronal death in AD (152). Recent evidence suggests that
two intrinsic pathways, mitochondrial dysfunction and ER stress, are central in
the execution of apoptosis in AD, mediated by organelle dysfunction (153). In
particular, the cleavage of cytoskeletal components, which clearly alters cell
morphology, may either activate death signals or disrupt survival signals
necessary to suppress cell death (154). Accumulation of both Ab42 and p53 was
found in some neurons with degenerative morphology in both tg mice and human
AD cases. Thus, the intracellular Ab42/p53 pathway may be directly relevant to
neuronal loss in AD. Although neurotoxicity of extracellular Ab is well known
and both synaptic and mitochondrial dysfunction by intracellular Ab42 has been
suggested, intracellular Ab42 may cause p53-dependent neuronal apoptosis
through activation of the p53 promoter, thus demonstrating an alternative
pathogenesis in AD (155). Furthermore, Ab induces cell death by an apoptotic
process due to activation of the caspase-3 apoptotic cascade regulated
by calcineurin dephosphorylation. Calcineurin inhibitors were also effective by
Early Diagnosis of Dementias from Neuropathology 321

preventing loss of mitochondrial membrane potential induced by Ab, not


allowing cytochrome c release from mitochondria and subsequently caspase-3
activation. Thus calcineurin activation and BAD dephosphorylation are are
upstream in premitochondrial signaling events leading to caspase-3 activation in
Ab-treated cells (156). The detection of active caspases and the accumulation of
cleaved substrates, such as fodrin, actin, and APP, in postmortem AD brain tissue
support the hypothesis that apoptosis-like mechanisms may contribute to neuronal
loss in AD (157–159), and studies indicate that caspase-mediated cleavage of
critical proteins contributes to neuronal degeneration in AD (160,161). Although
an in vivo model suggests that a relation between apoptosis and AD may exist,
apoptosis, at least in human postmortem AD brains, is rare (162–164). A complex
cascade involving immune reactive cells, cytokines, chemokines,
and neurotransmitters may contribute to Ab-induced toxicity and may be related
to the delay of Ab-induced neuronal cell death in vivo (85,142,165). It has
been suggested that the presence of a disordered BBB and the presence of
immunoglobuline in neurons (166) is associated with microglial activation and
neuroinflammation, this being subsequent to AP formation (35,167). Misfolded
Ab, via a disturbance of signaling pathways, may lead to cycles of synapse loss
and aberrant sprouting (168). However, the mechanisms by which the toxic form
of Ab acts remain to be elucidated. Ab leads to internalization of NMDA
receptors, reducing their availability at synapses (169). Paradoxical effects occur
when Ab forms complexes with metal ions (iron, zinc, or copper), where
Ab-metal complexes are capable of being neurotoxic or neuroprotective
(170,171). Amorphous plaques in the cerebral cortex of AD patients and
nondemented subjects are associated with glial Ab (111,172–175), which may be
related to glial clearance of non-fibrillary Ab, whereas expression of the b-site
APP-cleaving enzyme (BACE1) by reactive astrocytes may contribute to
developing AD (176). ApoE promotes Ab aggregation as well as senile plaque
(SP) and astrocyte colocalization and degradation of deposited Ab (177). Ab42
accumulation and the selective lysis of Ab-burdened neurons and astrocytes make
a major contribution to the formation of SPs (178,179). Astrocytes can protect
neurons from Ab toxicity, but when they interact with Ab, neuronal damage is
enhanced and the expression of synaptic vesicle proteins is decreased (180).
Cortical synaptic integration in vivo is disrupted by amyloid plaques (APs) (181).
The levels of both Ab40 and Ab42 are elevated early in dementia and are
correlated with the severity of cortical AD changes and with cognitive decline
(182–186), this being confirmed in a Drosophila model (187). Elevation of Ab in
the frontal cortex demonstrated in vivo supports the importance of Ab in early
AD (188,189).
However, in support of a physiological role for Ab was the demonstration
that its production is important for neuronal viability and modulation of synaptic
functions, protection against oxidative stress, and surveillance against toxins and
pathogens (190,191), and blockade of endogenous rodent Ab production
enhances spontaneous neuronal activity and synaptic plasticity. For this
322 Jellinger

regulatory feedback loop that is broken in AD, resulting in unchecked


accumulation of Ab and neurotoxicity, two possible scenarios were proposed:
either neurons might fail to be depressed by Ab, leading to a gradual build-up of
neuronal activity and further Ab secretion, or the machinery for Ab production
becomes independent from neuronal activity (41). Different causes may underlie
the familial and sporadic forms of AD. These studies have challenged our
traditional perception of Ab in the pathological processes. Originally thought
of as a toxic waste product, it has now been revealed as an endogenous regulator
of synaptic structure/plasticity, and neuronal activity (192). Amyloid may also be
beneficial, in this case, following neuronal stress or disease. Although
controversial, a protective function for Ab is supported by the available literature
(66) and also explains why many aged individuals, despite the presence of high
numbers of SPs, show little or no cognitive decline (193).
Tau Pathology and AD
Cytoskeletal lesions, resulting from accumulation of phosphorylated (p)-tau
protein (194), include NFTs in the neuronal cell soma, pretangles, defined as non-
fibrillar accumulations of tau and considered to be premature NFTs (195,196),
and NTs, which account for 85–90% of cortical tau pathology in aging and early
AD (197). The vast majority of NTs occurs in dendrites, while a small proportion
is present in axons (198). In early stages, NTs occur occasionally in the cerebral
cortex, corresponding to a dendritic tree that is arborized from a tau-positive
neuron (199). There is an argument that NFTs in the cell soma and NTs in the cell
processes are formed simultaneously (196); however, it has been argued that
more than half of the NTs arise from neurons without NFTs (200). P-tau
accumulates simultaneously in the cell soma and processes in early stages of AD
and in nondemented aged subjects. NFTs and NTs contain either 3-repeat tau,
4-repeat tau, or both (201,202). Sections CA 2–4 of the Amon’s horn show
predominantly 4-R NFTs containing the pSer422 epitope, while pSer262 may
detect the process of transformation from p-NFT to intracellular NFT and
extracellular NFT consisting predominantly of 3-R tau (202a). This shift in 3-R to
4-R tau may lead to NFT formation. Such isoform differences may depend on the
nature of individual neurons bearing NFTs and NTs, though the process of aging
and AD is not likely to be tau isoform-specific (199). The most established and
the most compelling cause of dysfunctional tau in AD is its abnormal
hyperphosphorylation. It not only results in the loss of tau function in promoting
assembly and stabilizing microtubules but also in a gain of a toxic function
whereby the pathological tau sequesters normal tau and mitogen-activated
proteins (MAP1A or MAP1B) and causes inhibition and disruption of
microtubules (203). In addition changes in phosphorylation state, tau undergoes
multiple truncations and shifts in conformation as it transforms from an unfolded
monomer to the structured polymer characteristic of NFT (204). Truncations at
both the amino- and carboxy-termini directly influence the conformation into
which the molecule folds, and hence the ability of tau to polymerize into fibrils.
Early Diagnosis of Dementias from Neuropathology 323

Certain of these truncations may be due to cleavage by caspases as part of the


apoptotic cascade (205). Although to date no mutations or changes of the splicing
regulation of the tau gene and the relative expression of tau isoforms have been
found in sporadic cases of the disease, differential expression of tau isoforms in
temporal cortex might underlie the susceptibility of certain brain areas to NFT
formation (206,207).
Alzheimer-specific epitopes of tau representing lipid peroxidation-induced
conformations support the idea that oxidative stress is involved in NFT formation in
AD brain (208). Creation of the Tau-C3 epitope specific for tau cleavage of aspartic
acid 421 appears to occur relatively early in the disease state, at the same time as the
initial Alz50 folding event that heralds the appearance of filamentous tau in NFTs,
NTs, and the dystrophic neurites surrounding APs (209). Quantification of tau-
positive regional NFT density showed that the AD-associated phosphorylation
process progresses from the C-terminus to the N-terminus of the amino acid
sequence, and correlation of the Gallyas (silver) stained NFT density was more
significant in the limbic cortices than in neocortices with a heterogenous pattern,
suggesting that stereotypical phosphorylation occurs in the limbic structures (210).
Recent biochemical studies on brains from elderly people at Braak stages I
to III showed a steep rise in the levels of tau and possibly Ab42 in the enterlinal
cortex (EC) at approximately 75 years of age. The levels of insoluble tau
increased as the Braak stage increased from I to II, but had a tendency to remain
stable between stages II and III. Ab42 showed a small increase with increasing
SPs, while Ab40 increased continuously with advancing Braak stage. There was
no significant correlation between the levels of insoluble tau and Ab in the EC.
Even if Ab did not accumulate to a significant extent, substantial accumulation of
insoluble tau occurred. These data clearly indicate that accumulation of tau and
Ab occur independently in the EC (211).
In AD, phosphorylated tau proteins are considered a control mediator of
disease pathogenesis. On the other hand, it has been proposed that tau
phophorylation represents a compensatory response mounted by neurons against
oxidative stress and serves a protective function. In a new tau-tg mouse in which
the overexpression of mutant human tau can be regulated by doxycycline, turning
off tau expression after deposits have formed halts neuronal loss and reverses
memory deficits. But surprisingly, NFTs continue to accumulate, suggesting that
they are not responsible for neurodegeneration (212). These findings provide
compelling evidence that perikaryal tau inclusions alone may not cause disease
and do not directly serve a role in neuronal loss or cognitive impairment (213). This
is consistent with a recent report that neurofibrillary pathology was not correlated
with neuronal death in a human tau-tg model displaying neurodegeneration (214).
The study by SantaCruz et al. (212) also provides the exciting prospective that
recovery of cognitive function is possible even after significant progression of
neurodegeneration. These findings have implications in the understanding of AD
and other tauopathies. This novel concept, which can also be applied to protein
aggregates in other neurodegenerative diseases, opens a new window of
324 Jellinger

knowledge with broad implications for the understanding of mechanisms


underlying disease pathophysiology (215).

Spreading Patterns of AD Pathologies


The cytoskeletal lesions that are associated with conformational changes of
tau-protein (216,217) show a distinct and predictable spreading pattern from the
(trans)entorhinal cortex in the mediobasal temporal lobe via the hippocampus to
neocortical association areas and, later, to subcortical nuclei (218,219). This
pattern correlates with early memory disorders due to synaptic dysfunction and
deafferentiation of the hippocampus by dissection of the GABAergic “perforant
pathway” (220,221), followed by disturbances of higher cortical functions due to
disorganization of cortico-cortical circuitry (222). However, due to considerable
overlap particulary in intermediate (limbic) stages and deviations from the
stereotypic expansion pattern, this model is of limited value (223–226). Different
populations of neurons prone to NFT formation are lost at different rates. In the
prefrontal cortex non-phosphorylated NF protein-enriched neurons represent
a vulnerable subpopulation. Their preferential involvement suggests that neurons
providing specific cortico-cortical connections between association areas predict
cognitive impairment in AD (227). Involvement of the primary motor and
association cortices, affected only in late stages of AD, provide significant
morphologic markers for dementia (219,228).
The extension of tau pathology is different from the phases of Ab deposition
(Fig. 1A and B). Diffusion of soluble Ab in the extracellular space is involved in
the spread of Ab pathology and can lead to neurodegeneration (230). Amyloidosis
usually begins in the neocortex and later progresses to allocortical regions
expanding anterogradely into regions that receive neuronal projections from
already affected brain areas (229,231). In contrast to the usually precise pathway
of tau pathology, Ab pathology is more diffusely and less predictably distributed.
Both lesions start before symptoms become apparent (10,229), and clinically
manifest AD is considered to be a late stage of these processes. While AP variants
are present in both early and late AD, disease progression is associated with both a
shift to a higher proportion of fibrillar plaques that induce local neuritic alterations,
and transformation of cytoskeletal proteins within associated neuronal processes
(232). Increased Ab correlates with cognitive decline in early AD, and demented
individuals usually show higher Ab loads than nondemented ones (185,233), but
the individual variability and independence of equilibrium distribution of Ab40
and Ab42 may explain the inconsistent and weak correlations between disease
progression, neuronal loss, and Ab phases (229,234–236), while tau pathology is
strongly correlated with disease progression and the degree of dementia
(6,22,237–239). However, while large numbers of NFTs exist in neocortical
projection neurons late into the course of AD, it is not possible to assess
whether these neurons are dead or functional and may respond to therapeutic
strategies (240).
Early Diagnosis of Dementias from Neuropathology 325

Figure 1 (See color insert.) Spreading pattern of (A) cytoskeletal/tau pathology and
(B) of Ab deposition. Source: From Refs. 218, 229.

Interaction Between Ab and Tau


Although it is now widely believed that an increase in the production of Ab is
central to the pathogenesis of AD, little is known about the relationship between
APP/Ab and tau changes (150,241,242). In the rare familial forms of AD,
pathogenic mutations have been identified in both the gene encoding the precursor
of the Ab peptide, APP gene itself, and in the presenilin genes which encode part of
the APP-protease complex, supporting the “amyloid cascase hypothesis” (48,243)
which claims that Ab causes the imbalance between Ab generation and clearance
326 Jellinger

as the basis of AD or enhances the tau pathology. For the more frequent sporadic
form of AD, the pathogenic trigger has not been unambiguously identified.
Whether Ab is again the main cause remains to be determined, although tau-tg
mice and tissue-culture models provide insight into the biochemical mechanisms
of tau aggregation and nerve cell degeneration (244,245). Chronic activation of
microglia, via the secretion of cytokines and reactive molecules, may exacerbate
plaque pathology and enhance the hyperphosphorylation of tau and the subsequent
development of NFTs. Suppression of microglial activity in AD brain has been
considered as a potential treatment of AD and may slow disease progression (246).
Recent results clearly indicate that Ab25-35, the peptide region to which the
cytotoxic properties of Ab can be assigned, interacts with the peptide region of tau
protein involved in microtubule binding; intracellular binding of Ab oligomeres to
soluble tau may promote tau phosphorylation. This interaction produces the
aggregation of tau peptide and the concomitant disassembly of Ab25-35, offering an
explanation for the lack of co-localization of NFTs and SPs in AD, and suggesting
the possibility that tau protein may have a protective action by preventing Ab from
adopting the cytotoxic, aggregated form (247).
Recent data indicate nonoverlapping but synergistic actions of both
pathologies in sporadic AD (248,249). APP dysfunction/mismetabolism inducing
Ab accumulation in the brain is believed to be the primary influence driving AD
pathology due to a failure of an autoregulation feedback reducing neuronal
activity and could contribute to cognitive decline in early AD and to disease
progression (41,250). Tau colocalizes with Ab42 and is induced by Ab42 in vitro.
Recent evidence suggests that plaques, NFTs, and caspases share a common
pathway (251). Caspases cleavage of APP and tau has been demonstrated in AD
(158,252,253). Ab accumulation triggers caspase activation which, in turn, leads
to caspase-cleavage of tau which is an early event that precedes hyperpho-
sphorylation in the evolution of AD tangle pathology (252,254,255). Caspase-
cleaved tau (Dtau) may initiate or accelerate the development of tangle pathology
(164,253). Its accumulation may represent a common pathway associated with
abnormal intracellular accumulation of tau or a-synuclein (AS) and may be less
dependent on the extracellular accumulation of Ab in non-AD dementias (256).
Dtau occurs early in the development of tangle pathology within AD brains and in a
tg mouse AD model, suggesting that caspase cleavage of tau plays an important
role in the development of NFT pathology. Alterations in tau phosphorylation and
cleavage by caspases have been reported in neuronal apoptosis. The presence of
activated caspase-6 in pre-tangles suggests that it occurs early and supports
previous studies demonstrating caspase activation in MCI but not in AD subjects.
While caspase-6-cleaved tau was found in NPs, NTs, and NFTs, active caspase-6
localized primarily to neurites (161). This is consistent with the hypothesis that
apoptosic-like mechanisms can damage synapses, axons, and dendrites, without
causing overt neuronal death. These results also lend support to the hypothesis that
the activation of apoptosis-like mechanisms may be involved in AD pathogenesis
(160,251). Caspase inhibition prevents tau cleavage without reversing changes
Early Diagnosis of Dementias from Neuropathology 327

in tau phosphorylation linked to apoptosis. The microtubule depolymerizing


agent, colchicine, induces tau dephosphorylation and caspase-independent tau
cleavage and degradation. Both phenomena are blocked by inhibiting protein
phosphatase 2A (PP2A) by okadaic acid (257). Thus, caspase cleavage of tau
provides a mechanistic link between the development of amyloid and tangle
pathologies in AD (251). However, others suggest that tau is essential for Ab-
induced neurotoxicity (258), and precedes the occurrence of Ab deposits
(259,260). Elevated tau inhibits the axonal transport of APP, suggesting a possible
link between the two key proteins in AD (261,262), and is likely to be independent
of amyloidosis or APP dysfunction. Both APP and tau are implicated in axonal
transport. Dysregulation of APP and tau metabolism by abnormalities in APP,
PSEN, ApoE, and tau can cause impairment of fast axonal transport, leading to
axonal depletion of critical components and neurodegeneration (263,264). The
suggestion that both processes evolve systematically in parallel and that APP
amplifies tau pathology (248) is supported by findings that Ab induces PHF-like
tau filaments in tissue culture (265), and a reciprocal interaction between Ab and
tau in vivo is supported by tg mouse findings (265a).
AD lesions in the primary visual cortex (Brodmann area 17), which is
affected only late in AD, were scored into 4 grades. At grade 1 only deposits of Ab
were noticed. Grade 2 showed congo red positive deposits and processes con-
taining Ub and cathepsin D immunoreactivity around plaque cores. At grade 3,
NPs and NTs were present, and NFTs at grade 4. The density of all lesions
drastically increased at grade 4 (266). This sequence is believed to be compatible
with a cascade of events beginning with deposition of Ab and ending with NFT
pathology (267) but awaits further confirmation. The spatial relationship in early
AD stages indicating that SPs lie in the terminal fields of NFT-bearing neurons
suggests that NFTs either antecede plaques or, less likely, are independently
formed (268). An explanation for the current results of independent accumulation
of tau and Ab in the human EC (211) is that the amyloid cascade hypothesis is
valid in the neocortex but not in the hippocampus or the EC. This cannot explain
accelerated alterations in both regions of the brain affected by familial AD
(FAD). Another possibility would be an additional effect(s) of mutant APP and
PSEN 1/2. In addition to increased production of Ab42, this may lead to
premature NFT formation and degeneration in the hippocampus and entorhinal
cortex and could have a significant role for enhanced neuronal degeneration in the
hippocampus and EC in FAD. There is evidence that the massive neurodegenera-
tion at an early age in FAD patients could be a consequence of an increased
vulnerability of neurons by mitochondrial abnormalities resulting in activation of
different apoptotic pathways as a consequence of elevated oxidative stress.
A hypothetical sequence of the pathogenic steps linking sporadic AD, FAD, Ab
production, mitochondrial dysfunction with caspase pathway activation, and
neuronal loss has been proposed (269). There is a correlation of the duration
of dementia with the degree of NFT formation and synapse loss (31), but not
with any Ab measures. The accumulation of Ab is markedly increased in AD
328 Jellinger

brain independent of disease duration, even in cases of short duration (270).


Others suggest a sequence of events whereby the effect of Ab deposition on
cognitive impairment is mediated by NFTs (271). NF triplet and a-internexin
immunoreactive neurites were localized to plaques densely packed with Ab
fibrils in preclinical AD cases, indicating that certain plaques may cause
structural injury or impediment of local axonal transport. However, a-internexin,
and not NF triplet, ring-like reactive neurites were present in end-stage AD cases,
indicating the relatively late involvement of neurons that selectively contain
a-internexin. These results implicate the expression of specific intermediate
filament proteins in a distinct hierarchy of differential neuronal vulnerability to
AD (272).
Although the relationship between SP, NFTs, and neuronal/synapse loss
remains to be elucidated, there is a cascade of reactions (273), and most clinico-
pathological studies have shown that both lesions, if present in sufficient
amounts, particularly in the neocortex, are considered the best markers for
dementia, and that all clinically defined AD cases have huge amounts of amyl-
oid deposits and widespread tau pathology (7,228,274). On the other hand,
widely distributed diffuse and neuritic plaques in allocortex are frequently
present in both nondemented old subjects and those with MCI. Thus, they often
do not permit a distinction between questionable and definite dementia, although
preclinical (CERAD) “possible” AD or “pathological aging” often has more
neuritic pathology mainly in hippocampus than “normal” aging with no or single
Ab deposits and no to moderate NFTs in hippocampus and frontal cortex (7,238).
They also differ in both PHF-tau and Ab biochemistry (219,248). However,
some highly functioning seniors may show severe Ab deposition (275–277).
Breakdown of white matter is seen in normal aging but is exagerated in AD due to
Ab deposition and toxicity resulting in “disconnection” of widely distributed
neuronal networks (278).

Guidelines for the Neuropathological Diagnosis of AD


Current criteria for the neuropathological diagnosis of AD are based on
(semi)quantitative assessment of plaques and tangles, considered to be
histological hallmarks of the disease (Table 3). Several guidelines have been
suggested:
1. National Institute of Aging (NIA) criteria emphasizing neocortical SPs
per unit corrected for age which could include diffuse as well as
neuritic types (279).
2. Criteria based on the semiquantitative assessment of plaques and NFTs
in neocortex and hippocampus (280).
3. The Consortium to Establish a Registry for Alzheimer’s Disease
(CERAD) criteria, using semiquantitative NP counts with adjustment
for age together with clinical history (dementia) to give the level of
likelihood of AD (281).
Early Diagnosis of Dementias from Neuropathology 329

Table 3 Currently Used Consensus Criteria for the Pathological Diagnosis of AD


Criteria according to Khachaturian (1985) (279)
Senile (SP) or neuritic plaques (NP) in the presence of neurofibrillary degeneration (NFT) in
neocortex (any region)
Age ! 50 a: 2–5/mm2
Age 50–60 a: R8/mm2
Age 66–75 a: O10/mm2
Age O 75 a: O15/mm2
Criteria according to Tierney et al. (1988) (280) Lesion/x25 field
A1: one/several SPCNFT in hippocampus (without neocortex)
A2: one/several SPCNFT in neocortexChippocampus
A3: one/several SPCNFT in neocortex (without hippocampus)
CERAD criteria (Mirra et al., 1993) (281)
Method
Semiquantitative assessment of neuritic plaque density, graded by “cartoon” comparison
as sparse, moderate, and frequent. Sampling of multiple cortical areas and midbrain
Generation of “age-related plaque score”
Definite AD: “C” age-adapted plaque scorea, clinical dementia
Probable AD: “B” age-adapted plaque score, clinical dementia presence/absence of
other lesions causing or related to dementia
Potential disadvantages: Neurites in plaques do not have to display tail immunoreactivity
Braak & Braak scheme (1991) (218)
Topographic pattern of neurofibrillary tangle spreading (stage I—VI)
NIA-Reagan Institute Criteria (1997) (282)
Probability statements based upon topographic “staging” of NFT and SP
All lesions considered (amyloid deposits, neuritic plaques, neuropil threads, and NFT)
“Age-related plaque score” and topographic staging of NFT combined with clinical
information
Probabilistic approach for diagnosis of dementia
Low probability: CERAD “sparse” and Braak stage I/II
Intermediate probability: CERAD “moderate” and Braak stage III/IV
High probability: CERAD “frequent” and Braak stage V/VI
Disadvantage: Other possible combinations of CERAD and Braak scores not considered.
Uncertainty over application when no clinical details
Specific recommendations—routine diagnosis
Semi-quantitative methodologies (i.e., the CERAD approach) should be used to assess
neuritic plaques and neurofibrillary tangles
Examination of the hippocampus and the neocortex for the presence of neurofibrillary tangles
is essential
a
Age-adapted plaque score:
Frequency of plaques

Age at death None Few Moderate Numerous

!50 a 0 C C C
50–75 a 0 B C C
O75 a 0 A B C

Abbreviations: 0, no evidence; A, uncertain evidence of AD; B, suggestive of AD; C, indicative of AD.


Source: From Refs. 218, 279–282.
330 Jellinger

4. Topographic staging of neuritic (neurofibrillary) changes, distinguish-


ing six different stages—entorhinal (1 & 2), hippocampal (3 & 4), and
neocortical (5 & 6) (218);
5. The Washington University quantitative criteria for diagnosis of AD
(5,6,283). These criteria are a modification of the 1985 NIA consensus
criteria (279). The main modifications are: 1) a modified Bielschowsky
silver stain that better visualizes the whole range of SPs, including the
diffuse variety (284); and 2) a counting protocol that evaluates the total
number of SPs in 10 contiguous 1 mm2 microscopic fields in each brain
region. This strategy was designed to assess total average plaque
distribution across a 10 mm2 expanse of cortex, thus precluding
a diagnosis of AD based on only one to three selected fields. The
counting protocol differs from the 1985 NIA consensus quantitative
method where SP density in only one single microscopic field has to
meet or exceed criteria, and from the CERAD semiquantitative
counting strategy in which only NPs are evaluated and maximal plaque
counts in any three microscopic fields are averaged and compared
to cartoons that depict mild, moderate, and severe plaque numbers in
one field.
6. The guidelines of the NIA and the Ronald and Nancy Reagan Institute
of the Alzheimer’s Association (RI) for making the postmortem
diagnosis of AD, combining the CERAD and the Braak scores
(285,282). Assessment by NIA-RI guidelines leads to a probability
statement for a low (CERAD 0-A, Braak stages 1–2), intermediate
(CERAD B, Braak 3–4), or high likelihood (CERAD C, Braak 5–6)
that dementia is due to AD. These categories apply only to individuals
with dementia, but the underlying guiding principle was that any
degree of Alzheimer changes is abnormal.
These algorithms that only consider the classical “plaque and tangle”
phenotype of AD have some weaknesses and do not recognize other dementias
and AD subtypes, e.g., the “plaque predominant” type with abundant APs, no or
very little neuritic AD pathology restricted to the hippocampus (Braak stages 4 or
less), and abnormal p-tau in neocortical pyramidal neurons but lacking overt
tangle formation, accounting for 3.5–8% of demented subjects over age 85 years
(286–288), the “tangle-predominant type” with NFT pathology in the limbic
areas, absence of NPs and no or very little amyloidosis, accounting for 5–7%
of oldest-olds with female predominance (288,289), and the Lewy body variant
of AD (LBV/AD), displaying cortical and subcortical LBs with severe AD
pathology (290). Although the interlaboratory comparison of neuropathological
assessment of AD when using standardized criteria showed reasonable interrater
agreement (291–294), it is important to recognize that pathological diagnosis
merely represents the association of a pattern of pathological changes with
a clinical phenotype. Therefore, it should be acknowledged that, although Ab
Early Diagnosis of Dementias from Neuropathology 331

detection and semiquantification have some diagnostic utility, the simple


presence of APs, as with proteinaceous accumulations in essentially all
neurodegenerative diseases, does not presume aetiology. The major morpho-
logical lesions differentiating AD from other neurodegenerative dementing
disorders are given in Table 4.
The evaluation of the NIA-RI criteria demonstrated fairly good correlations
with clinical dementia and good agreement with pathological methods, and their
easy and rapid use in AD and nondemented subjects, but much less reliability
for other dementing disorders. Comparison of these criteria with clinical scores
identified almost all cases with severe dementia, but often failed in mild to
moderate dementias. However, use of these criteria would result in many of CDR
0.5 and some CDR 1 cases being classified as “low probability” AD despite
convincing clinical and psychometric data that indicate that they have
recognizable progressive dementia, distinct from cognitive changes associated
with nondemented aging (25). This suggests that NFT-based neuropathologic
criteria will be weighted toward diagnosing AD in its moderate and severe stages,
because neocortical neurofibrillary pathology—although present—is relatively
modest in early-stage AD (i.e., very mild and mild DAT). Most nondemented
cases were assigned to the low or intermediate categories, but several studies in
those with no or only MCI showed a wide range of AD-related pathology, and
even the combined use of all current criteria often cannot distinguish between
questionable and definite dementia (Table 5). Although the sensitivity and
specificity of the above algorithm is suggested to be 90%, 40–50% of the brains
of patients with the clinical diagnosis of AD show “pure” AD pathology
(Table 6). Thus, their predictive value may be reduced to 38–44% (299). It should
be borne in mind that all additional pathologies may interact in inducing
cognitive impairment. Therefore, the reliability and clinical relevance of the
current diagnostic criteria need better qualification and validation.

Dementia with Lewy Bodies vs. Parkinson’s Disease with Dementia


Dementia with Lewy bodies (DLB), a relatively new term for a progressive
dementia syndrome, is associated clinically with the core neuropsychiatric
features of fluctuating cognition and visual hallucinations with parkinsonian
features. It represents the second most frequent cause of dementia in the elderly
after AD, accounting for 7–30% with means of 15–25% in several autopsy series
(300–302). An early-onset case of an atypical form of DLB developing
neurological signs at 13 years of age was reported recently (303). DLB is
characterized by a variable burden of a-synucleinopathy and AD pathology
(300,304). Morphological hallmarks are LB and Lewy neurites (LN) in the
brainstem, basal ganglia, limbic cortex, and neocortex, which are scored
semiquantitatively according to the severity and anatomical distribution,
separating brainstem predominant (i.e., PD), limbic (or transitional), and
neocortical types (Table 7a). Case validation is compromised by the lack of
332

Table 4 Morphological Differential Diagnosis of Alzheimer’s Disease


Vascu- MIX
Major mor- Plaque Tangle lar type
phological AD only demen- demen- demen- LBV/ Diffuse Pick’s Prion
lesions disease AD tia tia tia AD LBD FTD disease PSP CBD MSA disease

Brain atro- CCC/CC CC C G CC CC G C C C G C CC


phy, dif-
fuse
Brain atro- CC K K C C K K CCC CCC C/CC CC G G
phy, focal
Amyloid CCC CCC K/G G/C CCC CCC C/CC G/C G/C G C G/C C
plaques
Neuritic CCC G G K CCC CCC K K G CC C/CC K K
plaques
C tangles
limbic/
temporal
Neocortex CCC K K/C K CCC CCC K K G C G/C K K
Tangles sub- C/CCC K CCC K G/C C/CC K K K CCC CCC K K
cortical
Lewy bodies G C K K K CCC CCC K K K K K K
cortical
Lewy bodies G G K K K CCC CCC K K G/C K K K
subcortical
Jellinger
Pick bodies K K K K K K K K CCC K K K K
Spongy G K K K K C C C C K K K CC/CCC
changes
Ballooned K K K K K K K/C C C C/CC CCC K K
neurons
GCIs K K K K K K K K K K K CCC K
Glial tau C G K C K K C K G/C CC CC C K
inclusions
Glial plaques K K K K K K K K K K CCC K K
Vascular G K K CCC C K K K K K K K K
lesions
subcorti-
cal
Vascular G K K G/C CCC K K K K K K K K
lesions
large/
cortical
Prion (anti- K K K K K K K K K K K K CCC
Early Diagnosis of Dementias from Neuropathology

PrP)
immuno-
histo-
chemistry
Key: K, lacking; G, rare; C, occasional; CC, moderate; CCC, severe.
Abbreviations: AD, Alzheimer’s disease; CBD, corticobasal degeneration; DLB, dementia with Lewy bodies; FTD, frontotemporal dementia; GCIs, glial
cytoplasmic inclusions; LBV/AD, Lewy body variant of Alzheimer’s disease; MSA, multiple system atrophy; PSP, progressive supranuclear palsy.
333
334 Jellinger

Table 5 Likelihood of Dementia (in Percent) Due to Alzheimer’s Disease According to


NIA-R-Institute Criteria in Various Autopsy Series

Low Interm. High Mean age


Disorder CERAD/Braak A/0-II B/III-IV C/V/VI (years)

Cochran et al. (1998) (295)


Demented (nZ17) 47 41 12 ?
Nondemented (nZ40) 72.5 22.5 5 ?
Newell et al. (1999) (23)
AD (nZ33) 0 3 97 83
DLB (nZ15) 48 26 26 81
PSP (nZ12) 75 17 8 68
Controls (nZ17) 76 24 0 77
Harding et al. (1999) (296)
AD (nZ31/22-no LB) (CDR 1–3) 26/13 20/27 54/60 77
DLB, neocort. (nZ11) 73 18 9 76
PD (nZ7) (CDR 0–0.5) 83 17 0 79
Controls (nZ18) (CDR 0–0.5) 83 17 0 79
Davis et al. (1999) (297)
Controls (nZ57, MMSE 27–29) 88 – 12 84
McKee et al. (2002) (298)
AD (nZ12) (CDR 1–3) 0 17 83 81
Cogn. normal (nZ23) (CDR 0) 62 38 0 83
Jellinger (2003 f) (277)
AD (nZ100) (MMSE 0–17) 0 24 76 85
DLB (nZ36) (MMSE 0–20) 25 33 42 77
PSP (nZ10) 70 20 10 72
PD dem. (nZ20, MMSE 0–20) 25 50 25 83
PD nondem. (nZ17, MMSE O20) 70 30 0 72
Controls (nZ20, MMSE 28–30) 100 0 0 81
Abbreviations: AD, Alzheimer’s disease; DLB, dementia with Lewy bodies; MMSE, Mini-Mental
State Examination; PSP, progressive supra nuclear palsy; PD, Pick’s disease.
Source: From Refs. 23, 277, 295–298.

defined neuropathological criteria for DLB and the presence of LBs in many
cases at autopsy with non-DLB clinical presentations. The distribution of cortical
LBs in DLB does not follow the spread of NFTs (196), while the spreading
pattern of AS pathology with onset in the lower brainstem and progression to the
midbrain, dorsal forebrain, amygdala, limbic cortex, and final extension to the
neocortex is similar to that in sporadic PD (306). The late stages 5 and 6 of LB
pathology (involvement of sensory association and prefrontal, primary sensory,
and motor areas) suggest transition between PD and DLB (275,307,308). DLB
was classified into the limbic type and neocortical type according to the degree
of Lewy pathology, including LBs and LNs, and, on the other hand, into the
pure form, common form, and AD form according to the degree of Alzheimer
Early Diagnosis of Dementias from Neuropathology 335

Table 6 Morphological Diagnosis in Consecutive Vienna Autopsy Series (1985–2004)


(A) of Demented Individuals, 541 Males, 899 Females, Age 50–103 (Mean 83.3G
6.0) Years; and (B) of Patients with Clinical Diagnosis of Probable or Possible
Alzheimer’s Disease (Mean Age 81.3G6.0 Years)

(A) (B)

Morphological diagnosis n % n %

“Pure” AD (CERAD pos., Braak V-VI) 623 41.5 432 52.0


Alzheimer type path, (plaque, limbic, NFT/SD) 107 7.1 58 7.0
(26/53/28, 12/22/24)
ADCCVD (lacunar state, old/acute infarcts old, 236 15.7 167 20.1
AH-sclerosis (129/66/37/4, 97/18/41/11)
ADCcerebral hemorrhage (CAA) 44 2.9 17 2.0
Lewy body variant AD/Diff, LB disease (33/33, 22/7) 66 4.4 29 3.5
ADCParkinson pathol., PD, Incid, LBD, SN lesions 73 4.8 43 5.2
(51/14/8, 21/14/8)
MIX type dem, (ADCMIE, CSAE, CSID) (39/22/7, 68 4.6 20 2.4
12/7/1)
ADCother pathol. (tumors, MS, MSA, etc.) 39 2.6 13 1.6
Alzheimer pathology total 1256 83.7 779 93.9
Vascular dementia (MIE, SAE, SID) (61/79/22, 5/6/6) 162 10.8 17 2.0
Other disorders (Huntington disease, FTD, CJD, others) 67 4.5 28 3.4
Nothing abnormal beyond age 15 1.0 6 0.7
Non-Alzheimer pathologies 244 16.3 51 6.1
Total 1500 100.0 830 100.0
Abbreviations: AD, Alzheimer’s disease; AH, Amon’s horn; CVD, cerebrovascular disease; CAA,
cerebral amyloid angiopathy; CJD, Creutzfeldt–Jakob disease; FTD, frontotemporal dementia; LBD,
Lewy body disease; MIE, multi-infarct encephalopathy; NFT, neurofibrillary tangles; SAE,
subcortical atherosclerotic encephalopathy; SID, strategic infarct dementia; SD, senile dementia;
SN, substantia nigra.

pathology including NFT and amyloid deposits by Braak staging (218). These
combined subtypes were lined up on a spectrum not only with Lewy pathology
but also with other DLB-related lesions including Alzheimer pathology, neuronal
loss in the SN, spongiform changes in the transentorhinal cortex, and LNs in the
CA2-3 region. There were some similarities in the extent of Lewy pathology
between PD and DLB, although Lewy pathology of PD was below the lowest
stage of Lewy pathology. In contrast, AD did not meet the stages of DLB Lewy
pathology, and there were also no similarities in other DLB-related pathologies
between AD and DLB. In addition, LBs of AD showed characteristics different
from those of DLB in the coexistence of LBs with NFTs. These findings suggest
that DLB has pathological continuity with PD, but can be pathologically
differentiated from AD. While this would suggest that DLB exists as a discrete
pathological entity, as with AD and PD (309), others showed no (310) or only
336 Jellinger

Table 7a Consensus Pathological Guidelines for Scoring Cortical Lewy


Body Deposition

Brodmann
Cortical region area Anatomy Score

Entorhinal cortex 29 Medial flank of collateral sulcus 0 1 2


Cingulate gyrus 24 Whole gyral cortex 0 1 2
Mid-frontal cortex 8/9 Lateral flank of superior frontal 0 1 2
sulcus
Mid-temporal cortex 21 Inferior surface of superior 0 1 2
temporal sulcus
Inferior parietal 40 Lateral flank of parietal sulcus 0 1 2
lobule
For each region Lewy bodies are counted from the depth of the sulcus to the lip. Counts are not made
over the crest of the gyri except for the cingulate gyrus. Lewy bodies are predominantly located in
deeper cortical layers (layers 5 and 6). In each region a count of up to five Lewy bodies in the cortical
ribbon gives a score of 1 in the table. Counts greater than five score as 2. The sum of the five areas is
used to derive the category of cortical spread (maximum score 10). Cortical Lewy body score: 0–2,
brainstem-predominant; 3–6, limbic or “transitional”; 7–10, neocortical.
Source: From Ref. 305.

little difference between Parkinson’s disease with dementia (PDD) and DLB
(310a), in particular, more frequent involvement of the amygdala-limbic system,
and neocortex in DLB (308).
LB pathology may progress in a systematic fashion through the brain
regardless of the clinical phenotype. Except for some deviations in the severity and
distribution of lesions in substantia nigra pars compacta (SNc) and a more frequent
involvement of the CA2-3 region of hippocampus in DLB, these similarities
suggest morphological and pathogenetic relations between both disorders. DLB
is frequently associated with AD pathology of variable intensity and extent.
A subgroup with diffuse Ab deposition and neuritic AD lesions absent or restricted
to the hippocampus is referred to as “pure” DLB, while those showing significant
neuritic pathology that fits with the diagnosis of definite AD (281) have been
classified as LBV/AD (290,311). They occupy higher Braak stages of AD
pathology than age-matched controls, but lower stages than pure AD (290,312).
Among 96 autopsy cases, 62% were classified as “pure” DLB and 37% had severe
additional AD pathology (Braak stages 5 and 6). LBV/AD was present in 33% each
of the limbic and neocortical subtypes (276,313). “Pure” DLB shows no significant
differences in neocortical synapse density and synaptophysin immunoreactivity
compared to controls, while severe synapse loss comparable to AD is seen in LBV/
AD (314). Although neuronal loss in the cholinergic basal forebrain is consistently
found in DLB, PDD, and AD (1), early and more widespread cholinergic decline
differentiates DLB from AD (300), while LBV/AD shows an increase of cortical
M1 muscarinic cholinergic receptors compared to both AD and controls (315).
Early Diagnosis of Dementias from Neuropathology 337

Selective neuronal loss in the presubiculum in DLB (296), decreased expression of


BDNF in hippocampal neurons and increased activated microglia producing
neurotrophic cytokines, eg. interleukin-6 (IL-6), in PD and DLB brain may be
related to functional changes affecting cognitive function (316,317). The clinical
diagnostic accuracy was higher in DLB cases with low (75%) compared to high
(39%) Braak stages (318). Unlike AD, neurochemical markers [synaptophysin and
choline acetyl transferase (ChAT)] often do not correlate with cognitive decline in
DLB (319) that showed a significant inverse association between NFT burden and
psychosis (320). Microvascular disease due to hypotension related to carotid sinus
syndrome may be a (reversible) substrate of cognitive impairment in DLB (321).
Patients with AD alone or with LB pathology usually have more severe memory
impairment than those with DLB alone that is associated with more severe
executive dysfunctions. AD with LB pathology has the most rapid rate of cognitive
decline (322). However, there is neither correlation between LB density in any
brain area among DLB patients with cognitive changes or parkinsonism, nor
between LB density and neuritic Braak stages or frequency of NPs, nor between
LBs in cortex and SN (323), and no correlation betweeen the distribution pattern of
NFTs, AP, and AS-positive structures, and ChAT activity (324).
Whereas cortical LB densities, in general, cannot separate DLB from PDD,
the severity and duration of dementia appears to be related to both increasing
parahippocampal LB densities and NP grade. Both LB and AD pathology
contribute to dementia severity, but there may be considerable overlap between
demented and nondemented patients. LBs appear to be a major determinant of
dementia severity in DLB cases with milder AD pathology (Braak 3–4), but not in
those with severe AD pathology (Braak 5–6). Advanced AD pathology may
facilitate LB formation and, reciprocally, neocortical LBs may promote secondary
AD pathology. It was concluded that the same neuronal circuits within the
hippocampal formation are involved by both pathologies in AD and DLB and may
contribute to cognitive impairment in both diseases (325). The relatively more
severe executive impairment in DLB than in PDD may relate to the loss of
frontohippocampal projections in DLB (326). Despite the morphological
similarity of a-synucleinopathy in different types of LBD, indicating its
coexistence with both PD and AD, their pathogenic relationship remains to be
clarified (300,301,327). DLB and PDD could be so similar at a biological level
that a categorical distinction is difficult or inappropriate (328–330).
Recent biochemical studies of tau, Ab, and AS deposition showed an
overlap of all three pathologies in sporadic AD with similar progression from the
limbic system to the neocortex. Tau pathology and Ab deposits were
biochemically identical in both AD and DLB, but, corroborating neuropatholo-
gical findings, tau pathology was usually less severe in sporadic DLB than in AD,
the mean Braak scores being 4.1 versus 5.1 in AD, and the tau stages according to
Delacourte et al. (248) were 7.3 versus 9.3 in DLB, respectively. These studies
suggest that Ab may stimulate progression of cortical synucleinopathy in
sporadic DLB, while “pure” DLB without essential AD lesions was found only
338 Jellinger

in a single familial PD case (331). The presence of AS-positive lesions in 7–71%


of sporadic and familial AD in the amygdala, even in the absence of subcortical
LBs (332–336), associated with the APOE 34 and 32 allele (337), and
involvement of other brain areas (275,308,338,339), the co-localization of tau
and AS epitopes in LBs (340) as well as clinical, biochemical and morphological
overlaps between PD, DLB, and AD suggest that the process of LB formation is
triggered, at least in part, by AD pathology (307,341). This collision of two
processes may occur within the same region or within a single cell in the human
brain (336,341) and in tg mice (342), though in an early-onset case of DLB no
co-localization of tau and AS was found (303). The upregulation of the PD-
associated protein DJ-1 in tau neuronal inclusions in Pick’s disease (PiD),
progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and AD
and in glial inclusions in PSP, CBD, and multiple system atrophy (MSA) also
suggests a common reaction, but human LBs and LB-like inclusions in AS-tg
mice were negative (343). In vitro, AS promotes tau aggregation (fibrillation) and
vice versa (344), suggesting a synergy between tau and AS (345). Furthermore,
the presence of Ab deposits in the cerebral cortex was often associated with
extensive AS lesions and higher levels of insoluble AS. This suggests that
Ab enhances the development of cortical AS lesions in LB disorders (346).
This evidence challenges the view of DLB as a distinct entity and suggests that
in neurodegeneration certain neurons display fibrillary aggregates that are
typical of two or more different disease processes (double or triple amyloidosis).
It is unclear whether there is a common underlying pathological mechanism
or if these lesions represent a common final pathology leading to
neuronal degeneration.
Guidelines for the Diagnosis of DLB
The pathological guidelines, intended to provide a method of scoring the severity
and distribution of LBs in the cerebral cortex (300,305), do not provide
diagnostic criteria. Since they were formulated prior to the introduction of
specific AS immunohistochemistry for the detection of LBs (347), the scores for
LBs per region are based on ubiquitin (Ub) or conventional H & E staining.
Inclusion of this method into the CERAD protocol (281) has not been achieved.
The LB scoring system is based on five cortical areas, chosen on the basis of their
frequent involvement. A simplified protocol by excluding the frontal region has
been proposed because of the common finding of occasional LBs in this region in
PD (312) (Table 7b). Another screening algorithm suggested that semiquanti-
tative LB density thresholds in the parahippocampus may distinguish PDD and
demented from nondemented DLB cases independent from other patholo-
gies (348). Within pathological studies of patients with clinically diagnosed DLB
and PDD, there is heterogeneity in terms of Alzheimer’s and LB pathology.
There do not seem to be obvious neuropathological differences between DLB and
PDD, so a descriptive clinicopathological approach to their classification will
probably be most productive, with specificity both about clinical terms (DLB or
Early Diagnosis of Dementias from Neuropathology 339

Table 7b Modified Criteria of Dementia with Lewy Bodies, Cases Without Brainstem
Lewy Bodies Excluded

Maximum number of Lewy bodies per field Rating score for each region

0 0
1–5 1
6C 2

Region sampled

Temporal Cingulate Transentorhinal cortex


Category cortex cortex C hippocampus Total

Brainstem predominant 0 0–1 0–1 0–2


Limbic (transitional) 1 1–2 1 3–4
Neocortical 1–2 2 2 5–6
Source: From Ref. 312.

PDD, largely determined by the temporal order of symptoms) and also about
pathological findings (LBD, AD). The latter “categories” will need to be further
illustrated by details of lesion density and distribution (330). Thus, a practical
consensus for the diagnosis of DLB at present is not available.

Frontotemporal Dementias and Related Tauopathies


The heterogeneous group of frontotemporal dementias (FTD) or—neuropatho-
logically more correct—frontotemporal lobar degeneration (FTLD), previously
subsumed as PiD or Pick complex (349), represents the third most frequent cause
of dementia (10–20%) (350,351). Clinico-pathological correlates in FTD have
recently been summarized (352,353), and with awareness of the unique clinical
manifestation of FTD, accurate antemortem diagnosis appears feasible (354).
The temporal variant of FTD follows a characteristic cognitive and behavioral
progression that suggests early spread from one anterior temporal lobe to the
other. Later symptoms implicate ventromedial frontal, insular, and inferoposter-
ior temporal regions, but their precise anatomic correlates await confirmation
(355). Morphologically it is featured by focal atrophy of the frontal and temporal
lobes with neuronal loss, microvacuolar (spongiform) changes, and gliosis, with
or without tau or Ub pathology (356). Neurodegenerative mechanisms in FTD
may be assisted or precipitated by the loss of astrocytic support (357).
Constantinidis (358) distinguished type A, classic PiD with Pick bodies (Pib)
and cells, and type B, with frontal and parietal atrophy, ballooned neurons, but no
Pibs (359). Most cases of type B are now considered as CBD or FTD and
parkinsonism related to chromosome 17 (FTDP-17) (353). Phenotype C without
Pib or tau pathology is now classified as dementia lacking distinctive
340 Jellinger

histopathology (DLDH) (360). Updated classifications of FTD suggest the


following diagnoses (361,362):

1. Tau-positive lesions with predominantly insoluble 3-repeat tau:


(i) PiD
(ii) FTDP-17
2. Tau-positive lesions with predominantly insoluble 4-repeat tau:
(i) CBD
(ii) PSP
(iii) FTDP-17
3. Tau-positive inclusions and insoluble 3- and 4-repeat tau:
(i) NFT dementia
(ii) FTDP-17
4. Frontotemporal neuronal loss and gliosis without tau/ubiquitin-positive
inclusions, no insoluble and reduced soluble tau:
(i) DLDH
5. Ub-positive, tau-negative inclusions without detectable tau, with or
without motoneuron disease (MND), but MND-type inclusions:
(i) FTD with MND that may be closely linked to ALS with
dementia (363)
(ii) FTD with MND-type inclusions without clinical MND present-
ing as semantic dementia (364)
(iii) Motoneuron disease inclusion dementia (MNDID) related to
chromosome areas 9 and 11
Ub intraneuronal inclusions and dendrites have also been reported in
familial FTD (365,366), in FTD-17 (367), in familial and non-familial cases
of FTD-MND (368), and a few sporadic FTD cases in the absence of MND
(369–371), while neuronal intranuclear inclusions distinguish familial FTD-
MND type from sporadic cases (372). There is a close relationship between CBD,
PSP, and FTD (373,374), but, despite the identical composition of tau isoforms,
different proteolytic processing of abnormal tau distinguishes PSP from CBD
(375). Tau mutations have not been identified in sporadic FTD cases (376) and in
some FTD families showing genetic linkage to the tau locus (377).
Specific subtypes of FTD that deserve description in detail are:

Pick’s Disease
PiD is a rare variant FTD that accounts for 1–2% of all elderly dementia cases; its
classical type shows frontotemporal lobe and limbic atrophy with neuronal loss,
spongiosis, and gliosis including microglial activation, achromatic (Pick) cells,
and intraneuronal globose inclusions (Pib) in hippocampus, in particular in
dentate granule neurons, in cerebral cortex, and in selected brainstem nuclei.
Ultrastructurally, they are composed of straight and long-period twisted
filaments, made up of 3-repeat tau doublets (60 and 64 kDa) and a minor
Early Diagnosis of Dementias from Neuropathology 341

68 kDa band (332,378). Tau-positive glial inclusions, mainly in the white matter,
NFTs, and a network of dystrophic neurites differentiate PiD from FTD with
tau-negative astrocytes (379). In sporadic PiD cases, in addition to 3- repeat
tau deposits, isolated filaments formed from 3- and 4-repeat tau isoforms, and tau
phosphorylation-dependent and exon 10-specific epitopes are found. Thus,
accumulation of Pibs with 3- and 4-repeat tau pathology distinguish PiD from
other tauopathies (380) along with a lack of any association with the tau H2
haplotype (381). A novel presenilin 1 mutation with familial Pick-type tauopathy
without tau gene mutation and without Ab plaques (382), a familial FTD with
Pick-like pathology associated with Q336R mutation of the tau gene (383),
another phenotype with a missense mutation of S305N (384), and a family with
the R406W mutation and pathology consistent with NFTD (362) have been
reported recently.

Frontotemporal Dementia Without Specific Lesions


FTD, without disease-specific lesions or DLDH, is a sporadic or familial “tau-
less” tauopathy with no insoluble or fibrillary tau inclusions, and reduction of
soluble tau but normal tau mRNA levels (385). Similar brain pathology was
seen in a US family without known genetic background (386) and in a pedigree
known as hereditary dysphasia disinhibition dementia (HDDD2) with linkage
to chromosome 17q21–22, but no tau mutation. Reduction of soluble tau in
most brains with DLDH and in some HDDD2 brains differs from previous FTD
cases containing substantial mounts of insoluble tau (376,385). The phenotypic
heterogenity of HDDD2 parallels that of other hereditary FTDs caused by tau
gene mutation (387,388). No tau mutations have been identified in sporadic FTD
cases (376) and in FTD families showing genetic linkage to the tau locus (377).

Familial Tauopathies
Several genetically distinct groups of inherited FTD have been identified:
(1) FTDP-17; (2) FTD-MND linked to chromosome 3; and (3) FTD linked to
chromosome 3 (FTD-3). Tau mutations have been found in 25% of familial cases
of dominantly inherited FTD, but only in 4% of sporadic cases (389). Tau on
chromosome 17 is the only gene where mutations have been identified, while Ub
and tau inclusions have been found in the frontal cortex of patients from a Danish
family with FTD-MND that shows more diffuse cortical involvement than other
forms of FTD (390,391). Autosomal-dominantly inherited disorders linked to
chromosome 17q21–22 show diverse but overlapping phenotypes (392), with tau
pathology in neurons and glia, but no Ab deposits or other disease-specific brain
lesions (393). Two classes of tau mutations have been found in 10–40% of
familial FTD cases—those directly affecting the microtubule-binding sites of tau
and those that alter tau splicing (378,387). The majority of the currently known
tau mutations are located in exons 9 to 13. P301L mutation was detected in 11%
of familial FTD cases. The H1 haplotype was not overrepresented, but the P301L
342 Jellinger

mutation appeared in the background of the H2 tau haplotype. Single nuclear


polymorphisms in intron 9 and deletion in other introns upstream from exon
10 were increased in FTD cases with an increase in exon 10-containing tau
transcripts (388). Thus, sequence variations in regulatory regions may lead to
tau dysfunction and neurodegeneration (394). The aggregation of tau protein
produces a significant accumulation of intracellular Ab42 in cortical neurons,
which, however, does not reach the critical concentrations needed for AP
formation (395). A San Francisco family of 17q-linked FTD-amyotrophic lateral
sclerosis (ALS) with AS and tau inclusions (4-R/ON isoform) but without tau
mutations is different from other familial forms of FTD and ALS (396).
A study of 55 autopsy cases of FTD revealed tau pathology in 14, and tau-
positive PiD inclusions in four, while 60% showed no tau pathology in the brain,
except for rare NFTs. The 33 non-tau cases showed variable loss of soluble tau
protein, broadly comparable with the extent of neuronal loss (397). In other series
there was a predominance of FTLD-MND (398) and non-tauopathies with FTLD
with Ub-only neuronal changes (FTLD-U) (370,399).
Neurochemical changes in FTD contrast with those of AD: a much lower
cholinergic deficit and more serotonergic disturbance is linked with impulsivity,
irritability, and affective change which are common features of FTD (400,401).

Consensus criteria for the postmortem diagnosis of FTDs: A recent


pathological classification of FTDs suggests (402):

1. 3-repeat tauopathies—PiD.
2. 4-repeat tauopathies—CBD.
PSP.
Argyrophilic grain disease (AGD).
3. 3- and 4-repeat tauopathies; tangle-predominant dementia.
4. DLDH.
5. Diseases with motor neuron disease-type inclusions (DMNDI).
Another classification distinguishes pathological subtypes of FTD (352):
† with tau-immunopositive inclusions (with and without Pib)—CBD
and AGD
† with Ub-immunopositive inclusions—FTD-MND/FTLD-U
† lacking distinctive histology (DLDH).
FTLD-U or FTLD-MND should be considered in the differential diagnosis
of progressive FTD with an akinetic-rigid syndrome and PSP (399). An
additional, recently described form is neuronal intermediate filament inclusion
disease (NIFID) (372,403), representing a neuropathologically distinct, clinically
heterogenous variant of FTD that may include parkinsonism or MND and is
distinct from other FTDs (405). Genetic alterations can give rise to phentoypes
more or less similar to any of the above. Tau mice recapitulate the key phenotypic
hallmarks of human tauopathies (404).
Early Diagnosis of Dementias from Neuropathology 343

Other Tauopathies
Hippocampal Sclerosis Dementia (with Tauopathy)
In a subset of elderly individuals, hippocampal sclerosis (HS) is the only
remarkable morphological finding (406–412). Its frequency in autopsy series of
elderly demented ranged from 0.4 to 26% (413), while it is almost never seen in
nondemented oldest-olds (414,415). HS shows a wide range of severity and
distribution, with damage of the hippocampus and subiculum ranging from
neuronal loss and gliosis to frank infarction. It is occasionally accompanied by
multiple small infarcts in other brain regions or leukoencephalopathy or both,
while only rarely such brains show additional AD lesions (410,413). The cause of
HS has been suggested to be due to occult hypoxic-ischemic episodes (408,409)
or as a sequela of limbic encephalitis (416). Classical HS dementia (HSD),
clinically being more similar to FTD than to AD (417), is morphologically
characterized by severe loss of neurons and gliosis in the hippocampal CA1
region and subiculum. The EC, temporal pole, inferior frontal neocortex, and
frontal pole may also be affected. Frequent tau-positive neurons and/or glial cells
in neocortex, basal ganglia, thalamus and/or limbic regions, resembling to FDTP-
17 and a mixture of 3- and 4-repeat tau isoforms, suggested the term “HSD with
taupathy” (HSDT) (413). “Pure” HSD (406), where no other cause of dementia
could be identified, shows morphological similarities to DLDH or ubiquitinated
neuronal inclusions, similar to those of MNDID (363,418), but with brain levels
of soluble and insoluble tau being normal, suggesting that “pure HSD” may
represent FTD and, in particular, its MNDID variant (419). In addition, several
cases had argyrophilic grains (413) that are also seen in argyrophilic grain disease
(AGD). Selective degeneration of the CA2 sector of the hippocampus is rare, but
when detected, it is associated with 4-repeat tauopathies, particularly AGD (420).
Argyrophilic Grain Disease
This neurodegenerative disorder of the elderly is seen in about 5% of demented
patients and in subjects progressing from MCI to dementia (421–423). It may or
may not be associated with a cognitive decline in the presence of only moderate
amounts of AD-related pathology (424) and may be associated with anxiety,
restlessness or depression (422) or may present with FTD (425). Morphologically
AGD displays abnormal argyrophilic grains (AG), coiled bodies, and “pre-
tangles” mainly in the limbic system (medial temporal lobe, amygdala) and
insular cortex containing 4-repeat tau deposits with isoforms of 64 and 69 kDa
(380,426–428). It shows genetic differences to other tauopathies (422,429). AGD
is frequently associated with AD-related changes, low Ab-load, and mild to
moderate NFT pathology. A recent comparative morphological study showed
that demented AGD cases showed lower stages of AD-related pathology, but
higher ones than non-demented AGD patients. AGD associated dementia was
associated with Braak stages 2–4 and Ab phases 2–3, while those stages were not
associated with dementia in the absence of AGD (424). Another recent study
344 Jellinger

showed that the distribution of AGs follows a stereotypic regional pattern: in


stage I, they are restricted to the ambient gyrus; in stage II they appear in the
anterior and medial temporal lobe, subiculum, and entorhinal cortex; in stage III
abundant AGs involve the septum, insular cortex, and anterior cingulate cortex,
accompanying spongy degeneration of the ambient gyrus. 97% of AGD stage III
cases without other pathology showed CDROZ0.5 (430). These data suggest
that AGD is a clinically relevant neurodegenerative age-related tauopathy that
independently contributes to the development of dementia by lowering the
threshold for cognitive deficits in the presence of moderate degrees of AD-
pathology (424,430). Since patients with AGD-associated dementia exhibit
significant morphological and genetic differences from patients with “pure” AD,
(431) they should be excluded from the AD group in future studies, although their
clinical and morphological distinction may be very difficult, and recent studies
using 4-repeat tau-specific immunohistochemistry detected AGD in 26% of
neuropathologically confirmed AD cases (427).

Vascular-Ischemic Dementia—Vascular
Cognitive Impairment
Vascular dementia (VaD) is a heterogeneous group due to lesions caused by
different pathophysiological mechanisms and with different combinations of
brain pathologies. It is therefore necessary to identify the various types of
vascular brain lesions in order to correlate correlation with clinical symptoms
and for diagnostic purposes to search for risk factors and therapeutic strategies.
Dementias related to cerebrovascular disease (CVD) and ischemic brain
damage were previously considered to be the second most common type of
dementia (432–436) in the Western world after AD and DLB, with a possible
incidence of 8–15% (415). Clinical diagnostic criteria for VaD–ICD-10 [WHO
93 (437)] exists: DSM-IV (438), the SCADDTC (439), and NINDS-AIREN
criteria (440), supported by the Hachinski Ischemic score (HIS) (441,442),
show variable sensitivity (average 50%) and specificity (range 64–98%)
(443–446) and may exclude a number of subjects with VaD (447). NINDS-
AIREN neuroimaging criteria have been found not to distinguish stroke
patients with and without dementia (448). In an interobserver study, use of the
operational definitions for the NINDS-AIREN criteria improved agreement but
only for already experienced observers (449), whereas in another study none of
the currently used clinical criteria identified the same group of incidentally
demented subjects (450). On the other hand, integration of neuropsychological
and neuroimaging data was suggested to be sufficient for the diagnosis
of subcortical VaD (451). The classification may be based on: 1) primary
vascular etiology, 2) primary type of ischemic brain lesions, 3) primary
location of the brain lesions, and 4) primary clinical syndrome. Subcortical
ischemic VaD is an example of such a subgroup. Vascular cognitive disorder
(VCD) has been recently limited to cases without dementia, i.e., vascular
Early Diagnosis of Dementias from Neuropathology 345

cognitive impairment–no dementia (VCI-ND), but there are no universally


accepted criteria for VCI (452–454). Other subgroups include post-stroke
dementia (PSD) and mixed AD plus CVD (455). The Mayo Clinic criteria
(temporal relationship between stroke and dementia or worsening of cognition,
or bilateral infarction in specific regions) had 75% sensitivity and 81%
specificity for autopsy-proven VaD (444). While the validity and liability of
clinical diagnostic criteria is a matter of discussion (452), generally accepted
and validated neuropathological criteria for VaD have not been established
so far.
Morphological lesions associated with vascular cognitive disorder include
focal and multifocal CVD (415,433,456–463):
1. Multifocal lesions with large territorial or borderline infarcts,
microinfarcts and lacunar lesions in subcortical regions, cortical and
cortico-subcortical infarcts, cortical pseudolaminar necrosis, granular
cortical atrophy, and white matter lesions (WMLs) (Binswanger type
of subcortical arteriosclerotic leukoencephalopathy), resulting from
chronic ischemia, hypoperfusion, and other mechanisms (464–467),
and multiple postischemic lesions.
2. Focal disease with strategically placed infarcts, i.e., in functionally
important brain areas and neuronal circuits (see below).
Another classification distinguishes large and small vessel disease:
1. Large vessel dementia (LVD): classical multi-infarct encephalopathy/
large vessel disease—rather rare.
2. Small vessel dementia (SVD): microangiopathic (small vessel infarct)
dementia with lacunes, strategic infarcts mainly in subcortical areas,
WMLs (468–473).
3. Other types of VaD. Although cause-relationships between CVD and
dementia evade strict classification, brain lesions associated with
cognitive impairment are summarized in Tables 8 and 9.
Silent and symptomatic infarcts are related to an increased rate of dementia
(474), and the presence of an APOE E4 and E2 allele is associated with greater
progression of cognitive decline (475). Impairment of cognitive function is
less related to the volume of brain destruction (476) than to the location of
cerebrovascular lesions (CVLs), in particular to involvement of the hippocampus,
entorhinal area, thalamus, basal forebrain, and interruption of corticothalamic and
other important neuronal circuits (409,460,461,477–480). Cognitive impairment
has been found in 20–41% of patients with ischemic stroke (473,481–483) and
40% with recurrent transient ischemic attacks (484). It is also associated
with asymptomatic high-grade stenosis of the left internal carotid artery (485),
and common carotid artery media thickness (486), although many patients with
dementia identified after stroke already had dementia before (487). Older age,
prestroke cognitive decline, stroke recurrence, hypoxic-ischaemic disorders,
346 Jellinger

Table 8 Major Morphological Types of Vascular Dementia


Classical multi-infarct encephalopathy (MIE)
Multiple large (sub/territorial) infarcts in cortex and white matter/basal ganglia in
territories of large cerebral arteries, MCA, MCA C PCA; involving the left or both
hemispheres
Strategic infarct dementia (SID)
Small or medium-sized infarcts/ischemic scars in functionally important brain regions:
thalamus, hippocampus (PCA), basal forebrain angular gyrus (ACA), bilaterally or
dominant hemispheres
Microangiopathic (small vessel infarct) dementia (SMVA)
Subcortical arteriosclerotic leukoencephalopathy Binswanger (SAE)
Multiple small infarcts in basal ganglia C white matter with preservation of cortex
Multilacunar state
Multiple microinfarcts (scars up to 1.5 cm Ø); basal ganglia, hemisphereal white
matter, pontine basis
Multiple cortico-subcortical microinfarctions (mixed encephalopathies)
Granular cortical atrophy
Multiple small scars within border zones ACA/MCA in one/both hemispheres
Subcortical microvascular leukoencephalopathy
(acquired/genetically determined)
Gliosis or hippocampal sclerosis
Inflammatory angiopathy and other mechanisms
Abbreviations: ACA, anterior cerebral artery; MCA, middle cerebral artery; PCA, posterior cerebral
artery.
Source: From Refs. 440, 456, 463.

left-side infarcts, strategic infarcts, and white matter lesions appear to be the main
predictive factors of PSD (488). During a four-year follow-up the incidence of
PSD increased gradually, shifting from an initial AD-type picture to a VaD-type
later (489). Many patients with lacunar infarcts have a good functional outcome
at five years. For older patients, for patients with an initial severe stroke, and with
additional vascular risk factors, however, the prognosis is more severe, with an
increased risk for mortality, stroke recurrence, and physical and cognitive decline
(490). Dementia showed a correlation with widespread small ischemic lesions
throughout the CNS, mainly lacunes, microinfarcts, and hippocampal injury, and
much less with larger infarcts, many brains showing more than one type of CVLs,
although in cognitively normal aged controls similar lesions were present
(297,415,460,461,491). The size of WMLs in the elderly may progress with time
and may relate to clinical symptoms (492–494) and thus can be regarded as a risk
factor for cognitive impairment (495), whereas others may show territorial
infarcts and older age as predictors of cognitive impairment in the first year after
stroke (496). They impair frontal functions regardless of their location (497),
are associated with cortical more than entorhinal and hippocampal atrophy (498),
and increase the risk of dementia (499), particularly in patients with lacunar
Early Diagnosis of Dementias from Neuropathology 347

Table 9 Pathophysiological Classification of Vascular Dementia


Multifocal/diffuse disease
Large vessel dementia (LVD)
Multiple infarct dementia (MID)
Multiple large artery/borderline infarcts, cortical and subcortical, with perifocal
incomplete infarcts, especially in white matter
Small vessel dementia (SVD)
Subcortical infarct dementia
Multiple small lacunar infarcts with perifocal lesions in white matter
“Granular atrophy” of cortex (multifocal cortical microinfarcts)
Lacunar state
Binswanger subcortical leukoencephalopathy (BSLE)
Hereditary angiopathies — CADASIL (cerebral autosomal dominant arteriopathy
with subcortical infarcts and leukoencephalopathy)
Cortical plus subcortical infarct dementia
Multiple, restricted small infarcts due to:
Hypertensive and arteriolosclerotic angiopathy
Amyloid angiopathy, with/without hemorrhages
Collagen or inflammatory vascular disease (angiitis, PCNSA, FMD)
Hereditary forms
Hypoperfusive, hypoxic-ischemic dementia (HHD)
Incomplete white matter infarcts
Anti-PL related ischemia
Diffuse hypoxic-ischemic encephalopathy (cortical lacunar necrosis, post cardiac
arrest, hypotension)
Venous infarct dementia
Large hemorrhagic, congestive symmetric infarcts due to thrombosis of the sagittal
sinus or the great vein of Galen
Hemorrhagic dementia
Subdural hemorrhage
Subarachnoid hemorrhage
Intracerebral hemorrhage
Focal disease/strategic infarct dementia (SID)
Few infarcts restricted to functional important regions
Mesial temporal (including hippocampal) infarcts/ischemia/sclerosis
Caudate and thalamic infarcts (especially DM nucleus, bilateral damage)
Fronto-cingulate infarcts (basal forebrain, ACA territory)
Angular gyrus infarct (dominant cerebral hemisphere—ACA and MCA territories)
White matter key areas
Abbreviations: Anti-PL, anti-phospholipid; PCNSA, primary angiitis/arteritis of the central nervous system;
FMD, fibromuscular dysplasia; ACA, anterior cerebral artery; DM, dorsomedial; MCA, middle cerebral
artery.
Source: From Ref. 470.
348 Jellinger

infarcts (500,501), but WMLs and lacunes may be independently associated with
cognitive dysfunction (502). Other studies showed that subcortical vascular
disease on computed tomography (CT) is frequent in older patients with MCI,
but does not appear to be associated with the severity of cognitive deficits (503).
Postmortem detection of WMLs by MRI was less sensitive than pathology (504).
These and recent neuroimaging data indicate that subcortical lacunes, WMLs,
and multiple disseminated microinfarcts resulting from small vessel disease
that damage structures of the prefrontal-subcortical circuits (505) are the most
common pathological features of VCI/VaD, whereas large infarcts are less
frequent (506).

Guidelines for the Morphological Diagnosis


of Vascuar-Ischemic Dementia
While most of the currently used clinical guidelines for diagnosis of vascular-
ischemic dementia (VID)/VCI show variable sensitivity and specificity, and the
recent Mayo Clinic criteria (444) await validation by further clinicopathological
studies, at present, no generally accepted and validated neuropathological criteria
are available. Cognitive impairment appears to correlate with widespread
ischemic and/or vascular lesions throughout the brain with particular involve-
ment of functionally important areas and neuronal loops. “Pure” VaD/VCI should
only be diagnosed in the absence of AD lesions beyond age-related levels or other
concomitant pathologies (462). However, the postmortem diagnosis of VaD/VCI
currently is a subjective one and a matter of discussion.

Mixed Dementias
Mixed type dementia (MD) is characterized by combined pathologies of both AD
and VaD or other dementing disorders, but the distinction between these two
pathological diseases is controversial (356,434,460,507–509). Recent emphasis on
co-morbidity of AD and CVD (510–512), the link between AD and atherosclerosis
(513), cognitive impairment associated with amyloid angiopathy (514,515),
significant cerebral microvascular pathology (516,517), and deficient clearance
of Ab across the BBB in AD (518,519) all indicate that vascular disorder is an
important feature of the chronic neurodegeneration in AD. Therefore, neurovas-
cular dysfunction could have a major role in the pathogenesis of AD (140,520).
ApoE E4 and E2 with its potential amyloidogenic role may be responsible for some
of the microvascular changes found in AD (521). Many patients with dementia
have radiological and neuropathological features of AD and VaD, with the
classical NFTs and Ab plaques of AD together with the cerebral infarcts of VaD.
A close relationship between AD and VaD has been suggested since the age-related
changes in cerebral blood vessels that are the basis of CVD and VaD may also be
responsible for the failure of elimination of Ab from the brain in AD (522).
Neuronal overexpression of human APP renders the brain more vulnerable to
ischemic injury and describes the factors that are involved in increased neuronal
Early Diagnosis of Dementias from Neuropathology 349

susceptibility to ischemic stroke (523). Criteria for the clinical diagnosis of mixed
dementia are variable (439,440,524–528), and it has been questioned whether
mixed dementia really exists as a separate entity (529,530). Generally accepted and
validated histopathological criteria for the diagnosis of MD are currently not
available, and its true frequency is unknown. Its prevalence rate in autopsy series
showed a wide range from 2 to 56% in retrospective studies and 2.9 to 54.2% in
prospective studies with means around 15% (13,460,461,477,531). Complicating
the studies, many persons exhibit neuropathologic changes similar to AD, VaD, or
mixed dementia, but do not meet clinical criteria for dementia (2,532).
Patients with AD frequently have other concomitant pathological lesions
(533) and strokes are common and increase with age (534). Elderly people with
silent brain infarcts have an increased risk of dementia and a steeper decline in
cognitive function than those without such lesions (535). The severity of cognitive
impairment was correlated to the total volume of infarcts with impact on lesions in
limbic and medial association areas, frontal cortex, and white matter (536). In the
Nun study, patients with autopsy-confirmed AD and CVLs had a higher prevalence
of dementia than those without infarcts (24). The risk of being demented was
20-fold higher in subjects with AD and lacunar infarcts, but much lower and non-
significant when a large territorial infarct was present (525,527,537). Other
studies, however, showed lower Mini-Mental State Examination (MMSE) scores
in AD patients without than in those with concomitant CVLs, the latter having
a significantly more frequent history of strokes (Table 10). The presence of
cerebral infarcts in AD increases significantly with age, but has little influence on
the clinical features, and cannot be predicted from common vascular risk factors.
In spite of a trend, there are no major differences in neurodegenerative lesion load
(Ab and NFT) between AD and AD with cerebral infarcts groups, except when
cerebral infarcts are located in the temporal lobe (including hippocampus) where
AD pathology was frequently lower, suggesting that this location may be
important in the pathophysiology of mixed vascular and AD dementia (538).
In a population-based study in the UK among 209 autopsies of elderly subjects,
48% being demented, 78% with CVD, and 70% AD pathology, the proportion of
multiple vascular pathology was higher in the demented group (539), in which only
21% showed “pure” AD (471). In a health maintenance organization dementia
registry, only 36% of patients had AD and no other findings, while 45% had
pathologically definite AD plus coexistent CVLs, and 22% had AD plus DLB
features (540). Comparison of 186 AD cases and 13 individuals with no cognitive
impairment (NCI) suggested that neocortical core plaques were more related to
dementia severity than the density of total SPs and NPs, while diffuse SPs were not.
In those patients with infarcts, hemorrhages, or PD, NFT and SP densities were
lower despite the presence of dementia (6). Among 333 autopsied men in the
Honolulu-Asian Aging Study (120 demented, 115 marginal, 96 normal cognition),
24% of all dementias were linked with CVLs, and dementia frequency more than
doubled with coexistent CVLs (45% vs. 20%). Findings suggest CVLs are
associated with a marked excess of dementia in cases with low neuritic plaque
350 Jellinger

Table 10 Relation Between Cognitive State and Vascular Lesions in


Alzheimer’s Disease

Age at Braak stage Final History of


Disorder n (M/F) death (mean) MMSE (n) stroke (%)

“Pure” AD 400 (159/241) 80.3G8.9 5.2 1.1 (77)a 10.0


AD with lacu- 133 (37/96) 83.2G6.2 5.0 4.9 (19) 21.0b
nar state
AD with old 37 (12/25) 84.6G6.7 4.8 7.0 (5) 33.0b
infarcts
(!10 mL)
AD with hip- 16 (9/7) 86.1G6.7c 4.7 5.0 (5) NG
pocampal
sclerosis
MIX (AD 33 (7/26) 82.7G5.9 4.8 7.3 (4) 95.0b
C CVD)
infarcts
O10 mL
a
p!0.01 vs. other groups.
b
p!0.01 vs. other groups.
c
p!0.05 vs. “pure” AD.
Abbreviations: AD, Alzheimer’s disease; CVD, cerebrovascular disease; MMSE, Mini-Mental State
Examination; M/F, male/female; NG, not given.

frequency. Prevention of CVLs may be critically important in preserving late-life


cognitive function (541). A consecutive autopsy series of 1500 demented aged
subjects showed AD-type pathology in 83.7%, but “pure” or atypical AD only in
48.6%, while 23% had additional CVLs, 9.2% a-synucleinopathy, and 2.6% other
pathologies. Other disorders were seen in 16%, almost two thirds of them classified
as “pure” VaD. Similar results were seen in 830 cases of the same autopsy cohort
with the clinical diagnosis of probable or possible AD (Table 6). These and other
studies indicate that most elderly patients with or without cognitive impairment
have mixed disease.
There appears to be a close relationship between AD, atherosclerosis (542),
and vascular pathology (460,510,512,543). Amyloid deposits in the vessel walls
in aging and AD may be linked to consecutive CVLs and development of
dementia (507,544–546). Soluble Ab has a toxic effect on vascular endothelium
via NOS phosphorylation, Ca2C leakage, and other mechanisms causing vascular
dysfunction (139), and CAA may be an independent risk factor for vascular
cognitive impairment (471,514). CAA and hypertension are considered
important contributors to mixed dementia (547), while severe CAA, more
extensive than in AD and causing cortical infarctions, was seen in VaD (548).
Persons with hypertension have increased NFTs and brain atrophy at
autopsy (549,550), and large vessel CVD, or atherosclerosis, is strongly
Early Diagnosis of Dementias from Neuropathology 351

associated with the presence of plaques (542). In autopsy series of demented


patients, 18–80% had AD with CVLs (460,551–553). In a recent study the
prevalence of vascular pathology in AD was significantly higher than in age-
matched controls (48.0% vs. 32.8%, p!0.01), in particular the presence of severe
CVLs (old and recent infarcts and hemorrhages). The brain weight and severity of
dementia did not correspond to the degree of vascular pathology, but higher
Braak scores contributed to cognitive impairment (554). Another study of
a consecutive series of autopsy-proven AD cases and age-matched controls
revealed a higher frequency of CVLs and CAA in AD [57.4% vs. 33.2% and
94.5% vs. 33.3%, respectively (552)]. Concomitant cerebral infarction and, in
particular, strategic lesions may have synergistic effects, lowering the threshold
of AD pathology required for the development of dementia, and thus may
aggravate cognitive impairment (525,527,533,555–558). In patients with CVD,
the densities of plaques and neuritic AD lesions were significantly lower than in
“pure” AD for every given level of cognitive deficit (6,546).
The contribution of small concomitant infarcts to cognitive decline in AD is
unclear. In a cohort of longitudinally followed autopsy cases, significantly higher
age and lower Braak stages were seen in patients with small infarcts of !10 mL
volume compared to those with “pure” AD, but small infarcts had no impact on
cognitive decline (559). These data were at variance with others, suggesting
a contribution of CVLs to cognitive decline in AD even with volumes of ! 1 mL
(508,536) but were confirmed by another autopsy study (526). This study
indicated that coexistent small CVLs do not significantly influence the rate of
progression of dementia in AD, although a concomitant vascular pathology may
modulate both cognitive and non-cognitive features (560). In AD with minor
CVLs, the majority of lesions were lacunes in basal ganglia and/or white matter
and multiple microinfarcts, while in MD large lobar infarcts or multiple infarcts
in the left or both hemispheres were more frequent, suggesting different
pathogenic mechanisms between VaD, AD with small CVLs, and MD (460,461).
The relevance of WMLs is a matter of discussion. WMLs in AD are significantly
correlated with cortical atrophy and cognitive impairment (561), and together
with cortical microinfarcts may contribute to the progression of cognitive
deficits, but they do not contribute to the progression of AD (562). No
interactions between WMLs and AD were found (563), and the neuropatholo-
gical evaluation of focal cortical and white matter gliosis had no clinical validity
(564). On the other hand, regional WML volumetry may be helpful in correlating
subcortical pathology and cognitive impairment (565). In patients with
subclinical AD and little functional brain reserve, additional cerebral infarcts
independently contribute to cognitive decline, but do not interact with AD
pathology to increase the likelihood of dementia beyond their additive
effect (557). On the other hand, CVD has been suggested to be the most
important cause in the elderly for the conversion of low-grade AD to dementia
either by itself or acting as a catalyst (433). In full-blown AD with small CVLs,
frequently observed by modern neuroimaging methods, cognitive decline is
352 Jellinger

mainly related to the severity and extent of AD pathology (Table 10). Hence, the
combination of two or more pathologic processes may influence the severity
of cognitive deficits and represents a major diagnostic challenge.
Guidelines for the Diagnosis of Mixed Dementia
At present, there are no generally accepted and validated clinical or neuro-
pathological guidelines for the diagnosis of MD. Criteria for AD and VaD are of
limited value for the diagnosis of MD, and more distinct criteria for this diagnostic
category are necessary.

Other Dementias
The pathological findings and diagnostic criteria for other types of dementia will
not be discussed here (292,566).

EARLY PATHOLOGY OF AD AND CLINICAL CORRELATES


Longitudinal clinico-pathological studies have enhanced our understanding of
early AD changes and their relationship to cognitive function (566a). Here we
review (1) the most important studies comparing neuropathology of normal brain
aging to AD, (2) morphological differences between MCI/early AD and AD
dementia, (3) the role of hippocampus and the cholinergic system in early AD, (4)
early involvement of the olfactory system, (5) comparison between biopsy and
later autopsy findings, and (6) the value of biopsy demonstration of CAA.

Neuropathology in Cognitively Normal Aged Subjects (and AD)


Universally accepted neuropathologic criteria for differentiating AD from
healthy brain aging do not exist. Older studies in small autopsy samples reported
numerous SPs in 9–100% of nondemented elderly, but none of them met
pathological criteria of AD (567). These and other data suggest that (1) Ab and
diffuse SPs in the absence of neuritic pathology either appear to be part of normal
aging with no clinical significance or may represent an early stage of AD, and (2)
Ab deposits appear to be necessary but not a sufficient condition for inducing
neuritic components and thus may not be, per se, markers of incipient AD
(7,568,569). AD and controls could be distinguished from each other by mean NP
and NFT counts, but sufficient overlap caused difficulty in diagnosing any
individual case. Abundant diffuse SPs and NPs in the neocortex were seen only in
AD cases (570), while in the neocortex of cognitively normal subjects, plaques
are either absent or present as diffuse plaques only in scattered patches (571).
Thus, both plaque burden and plaque morphology appear to be useful in
distinguishing the earliest detectable form of AD from nondementing aging. In
contrast, the slow accumulation of NFTs restricted to entorhinal cortex and
related medial temporal lobe structures occurs as a function of age and appears
not to reliably distinguish AD from aging (14,238).
Early Diagnosis of Dementias from Neuropathology 353

Other studies of nondemented elderly individuals reveal at least a few NFT


and SPs in ento- and perirhinal cortex, and the CA1 of hippocampus but with less
involvement of the neocortex. The distribution of NFTs follows a similar pattern
to that of AD, while the number of SPs was not related to the severity of NFTs
(15). Only 17% of cognitively normal individuals (mean age 83.9 years) had no
or few SPs and few hippocampal and entorhinal NFTs. Sixty-nine percent
showed variable numbers of NPs in neocortex, amygdala, and EC. Forty-eight
percent met Khachaturian criteria for AD, 25.4–27% CERAD, 11% intermediate
NIA-RI criteria, and almost 9% Braak stages 5 or 6. Only NFTs in hippocampal
CA1 region correlated with higher Braak stages and autopsy age, suggesting that
they are associated with normal brain aging. Although many subjects had AD-
like changes, they showed no differences in mental performance, suggesting
compensatory mechanisms (298,572,573).
Among 39 nondemented elderly (mean age 85 years) only one brain was
without NFTs, 56% had Braak stages 1 or 2, 28% stage 3, and 13% stage 4 or
higher. Eighty-two percent and 95% had less than moderate numbers of cored SPs
or NPs, 49% had moderate to frequent DPs, with 23% having a typical Braak
staging in one region discordant from the scheme. 18% were CERAD positive for
AD, 49% Khachaturian positive, and 13% had LBs, but only one with numerous
neocortical LBs. Small, old infarcts were seen in 46%, with 23% having more than
one, most of those being subcortical, rarely cortical, and none had a large infarct.
Thus, the majority of cognitively normal individuals had minimal neuritic tau
pathology (Braak stage !4 and NPs !6/100 field) in most affected neocortical
regions (574). These and other studies, e.g., the Nun study, demonstrate a broad
spectrum of neuropathology in the elderly cognitively normal. Several studies
have indicated that the densities of total neocortical SPs (neuritic and diffuse),
although correlating moderately and not as robustly as neocortical NFTs with
dementia severity, constitute the best marker to differentiate AD, even in the
earliest symptomatic stage (CDR 0.5), from nondemented controls (5,6,238).
Neither neocortical nor hippocampal NFTs discriminated nearly as well between
the nondemented and demented AD groups (282).

Brain Morphology in MCI Compared to Cognitively Normal


Controls and AD
MCI is the term commonly used for a prominent memory impairment and relative
sparing of other cognitive domains that is intermediate between CNS aging
and very early dementia, particularly AD, and is a high-risk predementia state
(26,503,574–582). Its clinical representation and morphological basis are hetero-
genous and include patients with both early AD and significant CVD, in whom
memory and executive performance impairment predict likelihood of developing
dementia (583–588).
Few NFTs in anterior olfactory nucleus and parahippocampal gyrus were
seen in young nondemented individuals, with more NFTs in these areas and in
354 Jellinger

the hippocampal area CA1 in older ones (73–89 years), while MCI cases had
more NFTs in the same areas. Severely demented patients had large numbers of
NFTs in neocortex, while only part of the nondemented group showed a few
primitive plaques, and all the MCI to mildly demented groups had large numbers
of primitive or mature SPs. Unlike NFTs they were not considered a feature of
aging up to 80 years (14).
In another cohort, all nondemented individuals had NFTs in (para)-
hippocampal areas, their mean density showing an exponential increase with age,
even in the absence of SPs. A group of nondemented elderly showed widely
distributed DPs and NPs in the neocortex and limbic areas; those with NPs were
suggested to represent preclinical AD, CDR 0.5 in this particular study being the
threshold of very mild dementia. NFTs in EC, suggested to be the earliest stages
of AD, closely paralled cell loss in very mild demented subjects, but NFT
formation was not associated with age-related cell loss, which was not found in
nondemented subjects (237,238,589).
In another study, no clear differences in the frequence of NFTs between
cognitively normal persons and CERAD “possible” AD cases were seen. Cogniti-
vely normal individuals had Braak stages 1 or 2 while “possible” AD cases had
Braak stages 1–3 (590).
In a further study, diffuse SPs were seen in all cases with moderate dementia
(mean age 60 years), in 65% with mild dementia, and in 58% with questionable or
no dementia. SP density increased as a function of dementia severity and was
correlated with NP and NFT formation (4). Diffuse SPs may not be part of normal
aging but instead represent presymptomatic AD. The high density of DPs in
controls just at the threshold of detectable dementia is consistent with the
hypothesis that Ab deposition is an initial event in development of AD (237).
This was confirmed in a 74-year-old female with advanced AD and
her nondemented 47-year-old daughter, both dying from homicidal strangula-
tion (591). While the mother showed fully-developed AD pathology, the
daughter’s brain revealed only perineuronal deposition of diffuse Ab in cerebral
cortex and abnormalities of the endosomal lysosomal system, without NFTs
or glial changes, suggesting that amyloid deposition and endosomal-lysosomal
changes are early events in late-onset AD that precede the onset of dementia.
No genetic information about this AD family was available.
Elderly cognitively normal individuals had no or only few neocortical SPs
or NFTs in limbic areas (Braak stages 0–2). While there was no significant
relation between age at death and density of limbic or neocortical NFTs, the rate
of cognitive change was correlated with NFT burden in the neocortex and to
a lesser degree in limbic areas. NFT density decreased in order from the EC
through amygdala, subiculum, and CA1, to infratemporal region. In patients who
became demented, more severe NFT and SPs were seen (3).
These and data from the Vienna Prospective Dementia Study (mean age
81.7G8.6 years) confirm the significant negative correlation between neuropsy-
chological status and Braak stages in patients without other pathologies (509).
Early Diagnosis of Dementias from Neuropathology 355

Neocortical Braak stages 5 and 6 were mainly, but not exclusively, seen in
severely demented individuals, while limbic stages (2–4) were associated with
a wide range between the cognitively normal to overt dementia (Fig. 2). Similar
correlations have been observed in both younger (219,223,274,298) and
older subjects (224,592,593), indicating a “gray” transitional zone of mild to
moderate dementia showing a wide range of AD lesions (Fig. 3). These data
support a continuum in which AD is infrequent in healthy, cognitively stable
seniors, and preclinical pathology precedes cognitive impairment. All MCI
patients had a tau pathology, but not necessarily Ab pathology, whereas not all
patients with tau pathology did not have MCI (219).
Among 11 MCI cases (mean age 89 years; CDR 0.5) five patients showed
frequent NFTs and NTs in limbic regions and moderate DPs with sparse NPs and
NFTs in neocortex, but were insufficient for the diagnosis of definite AD, while
the others showed extensive tau pathology in the medial temporal lobe suggesting
very early AD (594).
In the Religious Order Study (ROS) fewer NFTs and NTs in the
parahippocampus were seen in cognitively unimpaired subjects compared to
MCI and AD (10,295). NFT density within peri- and entorhinal cortex correlated
with measures of episodic memory. NTs were not correlated with any cognitive
ability. The NT burden increased from normal to MCI and then decreased in AD.
Major findings were (1) that granulovacuolar lesions and NFTs correlated with
measurements of episodic memory; (2) NTs precede NFTs that in turn precede

Figure 2 (See color insert.) Relationship between Mini-Mental State Examination


(MMSE) and Braak neuritic Alzheimer’s disease stages in 207 consecutive autopsies of
aged individuals (mean age at death 81.4G8.6 years).
356 Jellinger

Figure 3 Column scatter plot of Braak stages versus Clinical Dementia Rating (CDR)
scores in 100 oldest-old people (mean age at death 92.5G2.5 years).

NPs; and (3) conformational tau changes emerged as a result of in situ reactivity
to different antibodies, reflecting its structural status in NFTs that correlated with
memory deficits (216). The authors presented a model for PHF degeneration
beginning with NT formation, followed by NFT development and ending with
appearance of NPs. Nondemented subjects showed the hierarchical pattern of
NFTs, while the distribution of NPs varied among individuals. MCI cases had
higher NFT densities than the cognitively normal in the medial temporal lobe,
this being related to performance on memory tests, while amyloid SPs
showed no correlation (11). It seems that the progression from Braak stages I
and II to stage III, which may or may not be associated with signs and symptoms
of mild AD or MCI, takes decades to occur and is presumably critical for
developing AD.
The transition to clinical dementia (CDR R1) was associated with PHF-tau
pathology in areas A9/10, A22, A23, and A39. A9/10 represents a prefrontal
cortical network that provides “executive control” over complex goal-directed
behaviors, while the other areas provide afferents to other frontal systems and
have been implicated in early AD (595). Executive MCI in the absence of
dementia has been suggested to be associated with decrease of soluble Ab in
the frontal cortex (596) and with frontal and anterior cingulate tau and Ab load
that may be early features of the frontal variant of AD (597).
Among 162 older Catholic clergy (mean age 75.8 years; 31 MCI, 57
cognitively unimpaired, and 53 clinical AD), nearly all brains had some AD
pathology. Its average level in MCI was significantly higher than in controls and
significantly lower than in AD. The occurrence of infarcts and LBs showed no
difference between MCI and the two other groups. This suggests that patients
with MCI show a level of Alzheimer type pathology that is significantly different
Early Diagnosis of Dementias from Neuropathology 357

from both AD and cognitively unimpaired individuals. LBs were usually not
related to MCI (16), although MCI can evolve into DLB in some cases (598).
Similar findings were reported recently from the ROS: among 180 participants
(60 normal, 37 MCI, and 88 demented), nearly all had some AD pathology,
cerebral infarctions were present in 35.3%, and 15.6% had DLB. Persons with
MCI were intermittent in terms of Braak stage, CERAD, and NIA-RI
neuropathologic criteria for AD compared to the other two groups, and the
relationship between cognition and AD pathology did not significantly differ
between the three groups which also showed intermediate levels of cerebral
infarction, while only 8% had LB pathology (583).
In the Nun study, Braak stages 1 and 2 showed the greatest variation in
cognitive profile, while in other reports stages 3 and 4 seemed to be the most
diverse in terms of cognitive deterioration. Although stages 1 and 2 have been
described as “clinically silent” (218), some subjects in these stages already had an
identifiable cognitive impairment. Only cognitively unimpaired subjects were
classified as either Braak stage 0 or 2–3, while those with MCI showed all Braak
stages (24), but others found no or little pathological differences between persons
with MCI and cognitively normal individuals (25), and between MCI and
AD (216). Of the cognitively unimpaired individuals, 87% showed allocortical
NFTs, only 37% displayed neocortical NPs, 19% had hippocampal NPs, and none
exhibited neocortical NFTs, though the latter were not detected in 10% of AD
brains or in nearly 50% of mild AD. Although NP and NFT density increased
with dementia severity, significant differences emerged for NFTs alone. The
increase in NPs and NFTs, in patients with even mild AD compared to normal
controls, suggests that both lesions are associated with the earliest symptoms of
AD (566a,599). Hence, NFT and NP pathology may constitute pathological
substrates for memory loss in AD but also in normal aging and MCI.
However, demonstration of a distinct sequence of Ab deposition suggests
that nondemented individuals with Ab or NPs may also represent early stages
of AD (229). An asymptomatic prodromal stage appears to be associated with
increasing Ab in the brain. The initial symptoms of MCI may develop when
a threshold level of neuronal and synaptic loss is reached in the EC and
hippocampus concurrent with NFT formation and gliosis. Subsequent disease
progression and conversion to clinical AD are associated with progressive
neuronal and synaptic loss, and NFT formation on a background of elevated
Ab that reaches a “ceiling” early in the disease. NFT formation and synapse loss
continue throughout the disease course (270). Dementia conversion rates increase
as cognitive impairment increases, and the likelihood of subsequent conversion
of MCI to dementia is usually related to the severity of local AD pathology (24),
particularly in the ventromedial temporal lobe (566a).
In general, neuropathology data do not permit extrapolation in terms of
prediction of progressive cognitive decline in individual cases. However,
respondents with a final MMSE score in the “medium MMSE” group, analogous
to but not identical with clinical MCI, show indices of AD pathology which
358 Jellinger

appear intermediate between high and low performing groups: namely a paucity
of neocortical tau pathology but with more advanced temporal lobe pathology
than high performing patients.

The Role of Hippocampus in Early AD


The earliest clinical phase of AD, characterized by impairment of episodic
memory and delayed recall, begins with subtle hippocampal synaptic dysfunction
prior to frank neuronal degeneration, but their causes are unclear. Ab deposition
is associated with decreased hippocampal glucose metabolism and spatial
memory impairment in APP/PSEN1 mice (600). Parahippocampal tau pathology
was significantly less severe in cognitively normal individuals compared to MCI
and/or full-blown AD, and the NFT density was significantly correlated with
memory deficits but no other cognitive abilites. This indicates that tau pathology
in the ventromedial temporal lobe develops prior to the onset of clinical dementia
and is associated with cognitive impairment restricted to dysfunction of episodic
memory. Substantial involvement of the hippocampus is often a key step for the
transition from MCI to dementia, probably irrespective of the patient’s age (601).
Progression of the disease, especially beyond the boundaries of the limbic
region, is associated with marked cognitive decline (602), and neuron number
and volume measures significantly decline with increasing duration of AD.
Tauopathy of the hippocampal formation in humans appears to be an age-related
process that can be compensated for by some individuals and appears also to be
independent of AD, but amplified by APP dysfunction (603).
Numerous primitive plaques and hippocampal alterations correlate with
age-associated memory impairment, whereas substantial NFT formation in the
neocortical temporal association areas is a prerequisite for the development of
AD. No correlation was observed between SP densities and severity of dementia.
Very old demented AD subjects had high NFT densities in the anterior CA1 field,
but not in the entorhinal and anterior temporal cortices (2), while other authors
considered the extent of NFT pathology in perforant pathway neurons to be a key
determinant of dementia in the very old (592). Oldest-old AD patients showed
decreased entorhinal PHF-tau lesions compared to younger ones, suggesting that
it is not simply correlated with aging and that AD pathology may differ in the
oldest age group (604). Thomas et al. (605) found a correlation between memory
impairment, but not global cognitive impairment, and amyloid load particularly
for Ab42 and in the EC in AD and VaD but not in DLB. They conclude that the
anatomic location of amyloid deposition is an important factor-specific item of
memory impairment in AD and VaD.
Although the mesolimbic cortex and hippocampus are thought to be central
in the integration of emotional responses, behavior, and intellect, neuritic SPs and
NFTs in these areas do not appear to be a reliable predictor of dementia, as
20–100% of mentally intact elderly show neuritic lesions in these regions and
cannot be clearly distinguished from demented persons (7,14). Early NFTs in
Early Diagnosis of Dementias from Neuropathology 359

layer II of the EC are present in normal aging (7,14,15,218,567,569,606). In


nondemented elderly, Alz-50 immunoreactive neurons were found most
frequently in the CA2 sector of hippocampus and only occasionally in layer II
of the EC (607). In both nondemented elderly and late onset AD NFTs are
frequently found in the CA1, and rarely in CA2 hippocampal area; their numbers
increase with age in CA1, but not until the age of 90 years in the CA2 area (608).
A heterogenous pathology of the hippocampus appears to be related to
degeneration of the perforant pathway: NFT formation is restricted to CA2
related to the non-perforating route, while degeneration of the perforant pathway
precedes NFT formation in pre-CA1 and presubiculum (609).
Aged cognitively unimpaired controls showed no changes in neuronal
numbers in entorhinal and superior temporal cortices (589,610); MCI had
a significant (32%) cell loss in EC, but none in superior temporal cortex, while
in AD cell loss was 66% and 52%, respectively, and in entorhinal layer II was
60–90% (12). Others reported a substantial decrease (35%) in EC, 50% in layer
II, and 46% in the CA1 in very mild AD (CDR 0.5), and a greater cell loss in CA1
in the severe AD group (589). MCI cases in layer II of the EC showed 65% of
neurons compared to cognitively normal individuals; mild to moderate AD had
45% with 25% atrophy of layer II. Both lesions correlated with performance on
MMSE, indicating that cell loss and atrophy of entorhinal layer II occur in elderly
subjects with MCI before the onset of dementia (8). Memory impairment can
occur in the presence of intact numbers of hippocampal neurons in non-AD
dementias, while nerve cell loss in hippocampus might be characteristic of AD
(611). Stereological studies gave evidence for persistence of viable neurons in the
entorhinal layer II and the CA1 hippocampal field even in advanced stages of AD
(612), and no significant loss of neurons in any other subdivisions of the
hippocampus was seen in preclinical AD (613). There is a stage-dependent and
sector-specific neuronal loss in AD hippocampus, with reduction of pyramidal
cells in CA1 by 33–51%, in Braak stages 4 and 5 compared to stage I, while in the
subiculum a 22% cell loss was seen only in stage 5 (614). Neuronal loss was
higher than the numbers of NFTs with a relation of 10:1 (234), while there was no
correlation with SP numbers. In stratum lacunosum moleculare, a subfield of
the hippocampus regulating declarative memory function, elevated growth
protein was positively correlated with the severity of AD, suggesting aberrant
neuroplasticity (615), while hippocampal neurogenesis is apparently increased in
AD (616). Functional magnetic resonance imaging (fMRI) studies suggest
a phase of increased hippocampal activation in MCI, i.e., early in the course of
prodromal AD, followed by a subsequent decrease as the disease progresses
(617). In addition to increased glial fibrillary acidic protein (GFAP) expression
due to astrogliosis, accumulation of specific GFAP isoforms have been
observed in degenerating hippocampal neurons in AD brain, indicating probably
a protective mechanism that remains to be elucidated (618).
Stereological studies fo the hippocampus showed significantly more severe
loss of CA1 and CA2 neurons in AD compared with vascular ischemic dementia,
360 Jellinger

without significant difference in neuron size. The number of CA1 neurons


correlated with MRI-derived hippocampal volume and memory score, indicating
that severity of cell loss shows correlations with structure and function across
causative subtypes which have distinct pathologic correlates (619).
Analysis of AD-related pathology in 12 oldest-old individuals including
assessment of total NFT, neuron numbers, and amyloid volume in EC, CA fields,
and dentate gyrus showed that the progression of NFT numbers and amyloid
volume across the different CDR groups was slower compared to younger cases.
Although patients with mild and moderate dementia showed significantly lower
mean neuron numbers compared to CDR 0–0.5 cases, there was a marked overlap
in individual values among CDR groups. A modest proportion of the variability
in CDR scores was explained by NFT numbers in the CA2 field (18.1%) and the
dentate gyrus (17.3%), while neither neuron numbers nor total amyloid volume in
the areas studied significantly predicted cognitive status. This indicates that the
occurrence and progression of AD-related pathologic changes are not an
unavoidable consequence of aging. The data also suggests that dementia in
extreme age is dependent on damage to hippocampal subdivisions that normally
show minimal pathology rather than on severe NFT formation and neuronal loss
in the CA1 field and EC (620), while synaptic loss is an early event in AD (620a).
Alterations in the g-aminobutyric acid (GABA) neurotransmitter and
receptor systems contribute to vulnerability of hippocampal pyramidal neurons in
AD. In control subjects (Braak I/II), the intensity of neuronal GABA receptor R1
protein (GBR1) immunoreactivity (IR) differed among hippocampal fields, being
most prominent in the CA4 and CA3/2 fields, mild in the CA1 field, and very
light in the dentate gyrus. AD cases with moderate NFT pathology (Braak III/IV)
showed increased GBR1-IR, particularly in the CA4 and CA3 fields. In the CA1
field of the majority of AD cases, the numbers of GBR1-IR neurons were
significantly reduced, despite the presence of neurons in this region. These data
indicate that GBR1 expression changes with the progression of NFT in AD.
At onset of hippocampal pathology, increased or stable expression of GBR1
could contribute to neuronal resistance to the disease process. Advanced
hippocampal pathology appears to be associated with decreased neuronal GBR1
staining in the CA1 region, which precedes neuronal cell death. Thus, changes in
hippocampal GBR1 may reflect alterations in the balance between excitatory and
inhibitory neurotransmitter systems, which likely contributes to dysfunction of
hippocampal circuitry in AD (621).
Synapsin I, a synaptic vesicle-associated protein participating in synapse
formation, regulation of the synthesis of other synaptic vesicle proteins, and
promotion of neurotransmitter release, is significantly decreased in the stratum
radiatum of the CA1 subfield and the molecular layer of the dentate gyrus in AD
brains, but was not reduced in the stratum pyramidale, where pretangles were
observed (622). This indicates differential synaptic alterations in AD.
Chromogranin B and secretoneurin, which are soluble constituents of large
dense-core vesicles, were significantly reduced in the entorhinal and orbitofrontal
Early Diagnosis of Dementias from Neuropathology 361

cortex in AD, while chromogranin A was less reduced. This altered availability
may be responsible for impaired transmission and reduced function of dense core
vesicles (623,624). Loss of synaptophysin mRNA in NFT-bearing neurons in the
hippocampus (625) and defects in expression of genes related to synaptic vesicle
trafficking in frontal cortex of AD occur as well (626).
Caspase 3, a marker of apoptosis, is activated in the parahippocampal gyrus
in subjects with MCI and low CERAD/intermediate Braak scores; caspase-like
immunoreactiviy has a tangle-like appearance and co-evolved with PHF
pathology in neurons, suggesting activation of apoptosis and of caspase-cleaved
APP occurs very early in the medial temporal lobe in aging and AD (627,628).
Mitochondrial defects in AD include COX-deficient hippocampal neurons that do
not appear to be prone to apoptosis or directly participate in the overproduction of
tau or Ab. They are likely to contribute to the overall CNS dysfunction and
possibly cause neurodegeneration via mechanisms other than apoptosis (629).
Microarray analyses of hippocampal gene expression revealed a major
transcriptional response comprising thousands of genes correlated with AD
markers. There was up-regulation of many transcription factor/signalling genes
regulating proliferation and differentiation, tumor suppressors, glial growth
factors, protein kinase A modulators of adhesion, apoptosis, lipid metabolism,
initial inflammation processes, and down-regulation of protein folding/transport,
some energy metabolism, and signaling pathways. These findings suggest a new
model of AD pathogenesis in which a genomically orchestrated up-regulation of
tumor suppressor-mediated differentiation and involution processes induces the
spread of pathology along myelinated axons (630).

The Cholinergic System in MCI and Early AD


The cholinergic neurons of the basal forebrain system provide a topographically
organized input to all regions of the allo- and neocortex that is important for
cortical activation. They not only influence their target neurons, but are also
influenced by target-derived neurotrophic factors, such as nerve growth factor
(NGF), which modulate cholinergic transmitter production and cellular structure
(631). Depriving the cholinergic neurones of NGF-producing target cells in
experimental animals causes pronounced cell atrophy. While neither aged
animals nor AD patients show decline in cortical NGF production, both show
evidence of disturbed retrograde transport of NGF (632). Precursor of NGF
(proNGF) levels increase during the preclinical stage of AD—possibly an early
biomarker for the onset of AD—and its accumulation in cerebral cortex
correlates with loss of cognitive performance (633). Depending upon the cellular
context these changes may result in increased pro-apoptotic signaling, cell
survival, or a defect in retrograde transport mechanisms, the latter not being
confirmed. Alterations in NGF and its receptors within the cholinotrophic basal
forebrain system in early AD suggest that NGF-mediated cell signalling is
required for the long-term survival of these neurons. Neurochemical and
362 Jellinger

behavioral studies support the possibility that aging reveals the vulnerability of
an abnormally regulated cortical cholinergic input system. Its decline and the
decline in cognitive functions are further accelerated as a result of interactions
with APP processing (634). Extracellular Ab aggregation may affect cholinergic
terminations prior to progression onto other neurotransmitter systems (635), and
there may be relations between calbindin-D-28k, FADD expression, and
phosphorylation of tau within the basal forebrain (636), but the interaction
between cholinergic dysfunction and Ab are not fully clarified (637,638).
Cognitively normal subjects and early stage AD (average Braak stage !2)
in plaque-containing cases showed a significant decrease of ChAT activity
(71–79.5%) compared to plaque-free cases, and in inferior temporal cortex,
ChAT had an inverse correlation with Ab concentration (639), suggesting
a preclinical onset of the cholinergic deficit in AD (640). NFTs and AT8
immunoreactive neurons in the basal forebrain were seen even in cognitively
unimpaired subjects, but the percentage of tau-positive basal forebrain neurons
was greater in MCI cases and showed a significant correlation with memory
scores (27). These results and reduction of cortical AChE activity (640a) indicate
that the cholinergic deficit occurs at an early stage of AD before the onset of
clinical symptoms. Whereas both the subicular cholinergic fibers and basal
forebrain neurons were lost in AD, no cholinergic deficit was found in the temporal
cortex in “pure” VaD (641), and subicular cholinergic fibers are unimpaired in
Binswanger’s disease (642).
Changes in some cholinergic basal forebrain markers, e.g., the high affinity
TrkA receptor, but not others [e.g., cortical ChAT activity, the number of ChAT
and vesicular acetylcholine (ACh) transporter-immunoreactive neurons], suggest
specific phenotypical changes, but not the frontal neuronal degeneration that
occurs early in cognitive decline. APOE E4 and E2 genotype, a predictor of
cholinergic deficits (643), is accompanied by lower metabolic activity in the
basal forebrain neurons in AD patients and also in aged controls as indicated by
the size of the Golgi apparatus and may represent a risk factor for cognitive
impairment (644). Others saw no correlation between APOE E4 and E2 and
cholinergic decline (ChAT activity) in AD, while the presence of two E4 alleles
was an important determinant of both NP and NFT accumulation in the
neocortex. By contrast, a strong relationship between APOE E4 and E2 alleles
and decreased neocortical NP counts suggests a putative protective role for E2 in
AD (645). However, the fact that cholinergic neuron numbers and brain ChAT
activity are not altered in MCI suggests that compensatory mechanisms occur in
the remaining cholinergic perikarya that normalize cortical ChAT levels.
Reductions of cortical TrkA in prodromal AD may be one of the earliest signs
of subtle neurodegeneration in the basal forebrain system leading to the inability
of this system to utilize NGF and may have important consequences for
cholinergic basal forebrain function during the transition from MCI to AD (646).
The accumulation of proNGF in the basal forebrain cholinergic target zones and
the lack of retrograde signals are crucial for nuclear transcription of genes
Early Diagnosis of Dementias from Neuropathology 363

mediating cell survival. Increases in cell cycle proteins may be a response to the
lack of cell survival signals, suggesting that basal forebrain neurons undergo
degenerative events during the prodromal stages of AD (647). Progressive loss of
cortical acetylcholinesterase (AChE) activity, indicating a progressive dysfunc-
tion of the ascending cholinergic system, correlated with cognitive decline (648).
A significant reduction in the number of basal forebrain p75 neurotrophin
receptor-immunoreactive neurons was seen in MCI (38%) and mild AD
(43%). Its correlation with performance in the MMSE, some tests of working
memory and attention, and other data lend support to the hypothesis that MCI is a
preclinical stage of AD (649).
While strong correlations have been found between the density of NFTs in
the hippocampus and neocortex with the degree of cholinergic pathology and
dementia (650,651), many elderly subjects with MCI or early/very mild AD do
not display deficits in ChAT activity (297,652), or show an upregulation in
hippocampus and frontal cortex (9,647). A significant elevation of hippocampal
ChAT in MCI was found selectively in the limbic (3/4) Braak stages which may
reflect a compensatory response to the progressive denervation of the
hippocampus by lost EC input (653). Hippocampal upregulation was no longer
present in mild AD cases compared to cognitively unimpaired subjects, while
severe AD showed markedly depleted ChAT levels. Cognitively normal
individuals, MCI and mild-moderate AD, showed a positive correlation between
hippocampal ChAT activity and progression of NP pathology in this area. In MCI
and mild AD, ChAT activity was normal in inferior frontal, superior temporal,
and anterior cingulate cortex (9). This suggests that earliest cognitive deficits,
e.g., short-memory loss in MCI, do not involve cholinergic deficits, but more
likely relate to disrupted entorhinal-hippocampus connectivity. Increased medial
temporal NFT correlates strongly and better than hippocampal ChAT activity
with impairment of episodic memory (654). Increased cholinergic activity during
the early stages of AD may contribute to synaptic scaling, but the mechanisms of
these changes are unknown (655). No changes in cortical or hippocampal levels
of NGF were found in the brains of MCI individuals, and they did not correlate
with an increase in ChAT activity in these regions, suggesting that brain NGF
levels appear sufficient to support cholinergic plasticity (656). In vivo studies in
mild to moderate AD showed significant reduction of ACh levels in amygdala
and neocortex, whereas ACh activity and glucose metabolism appeared
preserved or even increased in the basal forebrain. This suggests that changes
in the cholinergic system are an early and leading event in AD rather than the
consequence of neurodegeneration of basal nuclei (657). Cortical AChE activity
is more robustly associated with functions of attention and working memory
compared to performance on primary memory tests in AD (658).
While early-onset AD patients show a wide range of expression levels of
the hippocampal cholinergic neurostimulating peptide (HCNP) precursor protein
mRNA, in late-onset AD it is progressively decreased in hippocampus but not in
the dentate gyrus (651). Since it stimulates the production of ChAT, this low
364 Jellinger

expression in CA 1 may explain the downregulation of cholinergic neurons which


show a progressive loss of synapsis and synaptic proteins in hippocampus and
cortex even in early stages of AD (659).

Early Olfactory Involvement in AD and Other


Neurodegenerating Diseases
Olfactory dysfunction (anosmia) is a common feature in old age and in AD
(660–662), PD (663–668), DLB, and other neurodegenerative disorders
(669,670), but not in vascular parkinsonism, PSP, and CBD (671). Anosmia is
more frequent in DLB than in AD (672) and very common in LBV/AD (673). It
also occurs in tg mice overexpressing human tau protein (674). NFTs and SPs in
the olfactory system in AD have been reported previously (675–679), while LBs
have been described in PD (306,680,681).
Recent studies indicate that the involvement of the olfactory bulb and tract is
an early event in AD. Autopsy examination in elderly subjects showed
degenerative olfactory changes in more than 84%, i.e., in all cases of definite
AD and in 2/19 cognitively normal controls. NFTs and NTs appeared in the
olfactory system as early as in the EC, and there was a statistical association
between olfactory and olfactory system neocortical neuritic changes with respect
to their frequency and severity (682), while others observed olfactory tau
pathology in one-third of AD cases with Braak stage 2. They demonstrated that tau
pathology in the olfactory system correlates with Braak stage and APOE 34 and 32
(683). These data suggest that olfactory dysfunction and APOE 34 and 32 are
associated with a high risk of cognitive decline (660). Tau pathology in the
olfactory bulb was common in AD and DLB, but minimal or absent in PSP, CBD,
and controls, while AS-positive LBs were detected in most cases of DLB but absent
in AD, PSP, and CBD. The severity of tau pathology also correlated with cortical
and amygdala LBs, suggesting a possible synergistic effect between tau and AS,
the main component of LBs, in the olfactory system in both pathological processes.
In a personal consecutive autopsy study of 240 aged brains, tau pathology in the
olfactory system was seen in all cases of definite AD (Braak stages 5 and 6), in 88%
of Braak stage 4, 46% of Braak stage 3, and in 30% of MCI cases (Braak stage 2),
while all NCI controls (Braak 0 and 1) were negative (684). Recently, hyposmia in
PD was explained by increase of dopaminergic cells in the olfactory bulb (685).

Comparison Between Biopsy and Autopsy Findings in AD


Brain biopsy has an uncertain role in the diagnosis of dementia, although it may be
of some value, when a treatable disease cannot be excluded by other means (686).
Comparison of biopsy and autopsy findings in the same individuals provide
opportunities to follow the disease process. Among four patients meeting the
clinical criteria for AD who showed progressive deterioration from the time of
biopsy to autopsy (nine to 11 years), three cases showed a striking increase in the
density of SPs and NFTs in frontal cortex, and a significant increase of microglial
Early Diagnosis of Dementias from Neuropathology 365

activation in one. Severity of CAA varied significantly among patients (687).


These findings contrasted with those of two other groups. Biopsies performed in
five AD patients were compared with autopsies after AD duration of one to
seven years (688). In all brains, density and nuclear volume of pyramidal neurons
were significantly less than in controls and both measures fell significantly further
from biopsy to autopsy, while NFT density either did not change or decreased. SP
type in four brains was similar, and no increase in Ab SPs was seen. Three brains
showed no consistent changes in SP densities, while in one SP density increased
only in layers I–III. In one brain density of SPs decreased at autopsy, while
density of NFTs remained unchanged. Comparing autopsy findings in four
patients with advanced AD (MMSE 2–20) who underwent biopsy one year after
onset of symptoms and one to 47 years before death, despite marked decline in
mental state, there was no consistent change in SP and NFT densities in the left
frontal cortex. In one patient each, total SP count dropped and increased, while
two cases showed little changes. NFT density in three brains dropped
considerably and only in one case increased (689). These studies did not
demonstrate an increase in Ab density from biopsy to autopsy, which is in
keeping with data indicating that amyloid burden does not correlate with duration
of AD and that a dynamic balance exists between Ab deposition and resolution
(270,690).
In frontal cortical biopsy samples of autopsy-proven AD mean levels of
ChAT activity were decreased to 36% of age-matched controls and were further,
but not significantly, decreased in autopsied cases (691). The biopsy AD group
showed a significant loss of synapses (35%), and a further loss in the autopsy AD
group (692). These and comparable findings (693) indicated a continous decline
of both ChAT activity and synapse density through the course of disease. Both
changes correlated with the dementia score, cortical ChAT activity being
considerably weaker than cortical synapse density.

Is Bioptic Demonstration of CAA a Dagnostic Tool for AD?


CAA is defined as deposition of congophilic material (Ab) in the walls of
meningeal and cerebral vessels (694–696). It is common in the brains of elderly
demented and nondemented individuals; its incidence and severity increase with
age (460,697–700), and it is associated with cerebral hemorrhages, infarcts, and
WMLs (460,694,698,699,701–703). The prevalence of CAA in AD is 70–100%
(460,696,704,705). In CAA, Ab40 affects vessel walls more frequently and more
severely than Ab42, whereas in SPs, Ab42 is predominant (696,706–708).
For the origin of Ab in CAA three different mechanisms have been
proposed (698): (1) derivation of Ab from blood and/or cerebrospinal fluid (CSF)
(709); (2) production by smooth muscle cells (SMC) within vessel walls and/or
pericytes (710,711); or (3) derivation from the neuropil, i.e., SPs and NPs, in the
course of its perivascular drainage (694,712–715). Ab is present as globular
deposits on the capillary wall and as thin layers in the pericapillary basement
366 Jellinger

membrane, with dyshoric Ab deposits in the adjacent neuropil (229,694,696,


714). This CapCAA is probably related to AD-associated Ab deposition in the
brain parenchyma, supporting the concept of its derivation from the neuropil,
suggesting that transport across the BBB regulates brain Ab (518).
The clinical diagnosis of “probable” CAA can be made in patients over age
60 years with multiple hemorrhages confined to lobar brain areas showing no
other causes of hemorrhage. Over 50% of the patients develop new petechial
hemorrhages within one to two years (716). Detection of small hemorrhages on
MRI may be an efficient surrogate endpoint for pilot studies (717). Validation of
the Boston criteria (Table 11) pathologically confirmed all 13 individuals
diagnosed clinically with probable CAA (718). The frequency of cerebral
infarctions and hemorrhages increased with the severity of CAA that was
associated with the APOE 34 and 32 genotype (719). In brain biopsies CAA was
seen in 13.1% of cases with cerebral infarcts, but only 3.7% Ab in blood vessels
of controls. CAA mainly involved large cortical vessels, while Ab deposits in
meningeal vessels were not different between infarct and control groups (701).
This indicates that CAA, but not AP formation, is a risk factor for cerebral

Table 11 Boston Criteria for Diagnosis of Cerebral Amyloid Angiopathy–Related


Hemorrhage
Definite CAA
Full postmortem examination demonstrating:
Lobar, cortical, or corticosubcortical hemorrhage
Severe CAA with vasculopathy
Absence of other diagnostic lesion
Probable CAA with supporting pathology
Clinical data and pathologic tissue (evacuated hematoma or cortical biopsy)
demonstrating:
Lobar, cortical, or corticosubcortical hemorrhage
Some degree of CAA in specimen
Absence of other diagnostic lesion
Probable CAA
Clinical data and MRI or CT demonstrating:
Multiple hemorrhages restricted to lobar, cortical, or corticosubcortical regions
(cerebellar hemorrhage allowed)
Age R55 years
Absence of other cause of hemorrhage
Possible CAA
Clinical data and MRI or CT demonstrating:
Single lobar, cortical, or corticosubcortical hemorrhage
AgeR55 years
Absence of other cause of hemorrhage
Abbreviations: CAA, cerebral amyloid angiopathy; CT, computed tomography; MRI, magnetic
resonance imaging.
Source: From Ref. 718.
Early Diagnosis of Dementias from Neuropathology 367

infarction being significantly more common in elderly patients with cerebral


infarction than in age-matched controls. While in nondemented subjects, the
presence of CAA had no effect on cognitive ability, subjects with AD plus CAA
had a lower test score than AD patients without CAA (720).
Recent studies reported both positive (229) and negative correlations
between CAA and AD pathology (704,705). Neuritic AD pathology and
CapCAA are highly correlated with only low correlations with general (non-
capillary) CAA (696). Ab42 deposits in capillaries, more precisely in the glia
limitans, correlated highly with both Ab42 plaques and morphological AD
criteria, while only a low correlation with CAA was observed. Ab40 deposits
were significantly less frequent in both capillaries and plaques, and showed a low
correlation with morphological AD criteria. These data suggest different
pathomechanisms for both types of CAA, and a close relationship between
CapCAA and AD pathology (721). Others observed different types of amyloid
(Ab42 and immunoglobulin-l light chain amyloid) in CAA with spontaneous
intracranial hemorrhage (722). Based on these findings, the demonstration of
CAA in meningeal and superficial cortical vessels in brain biopsies may not
indicate the presence of significant AD pathology and, therefore, may not be
a reliable tool for diagnosing AD. This is illustrated by mildly to severely
demented aged individuals who, at autopsy, showed severe general CAA, but no
or only few cortical amyloid plaques and neuritic Braak stages 2 to 4, not fitting
the morphological criteria of AD (722a,723), while other patients die showing
clinical and pathological AD with virtually no Ab within blood vessels (724).
However, CAA, due to the effects of Ab on cerebrovascular functions, may be an
independent risk factor for vascular cognitive impairment (471,514).

EARLY MORPHOLOGICAL CHANGES IN DEMENTIA


DISORDERS AND RELATION TO BIOMARKERS
Progress in our understanding of the molecular pathology and development of
AD and its relationship to brain aging have provided clinically meaningful
advances in the development of biomarkers, which may provide reasonable
evidence for association with key mechanisms of pathogenesis and neurodegen-
eration. Emerging methods that are based on biochemical and imaging markers
of disease specific pathology hold the potential to provide effective measures of
early diagnosis, biological activity, disease outcome and markers of surrogate
endpoints for their clinical use in diagnostic and preclinical purposes.
Considerable progress has been made in the evaluation of biomarkers for AD.
However, the nature of the majority of reported findings are still preliminary and
most of them retrospective, and have been compared against a clinical rather than
“gold standard” pathological diagnosis. Because clinical criteria lack specificity,
particularly early in the disease course, quoted sensitivities and specificities for
putative biomarkers are difficult to interpret.
368 Jellinger

Alzheimer’s Disease
According to the guidelines of an international consensus group, a biomarker for
AD should detect a manifestation of the fundamental neuropathology and be
validated in morphologically confirmed cases. Its sensitivity for detecting AD and
its specificity in differentiating AD and other dementias should exceed 80%.
Ideally, a biomarker should also be reliable, reproducible, non-invasive, simple
to perform, and inexpensive. Of particular interest is its ability to detect
the disease at the earliest possible stage, i.e., before significant irreversible disease
progression has occurred. Biomarkers are potentially useful to study disease
pathogenesis in vivo and to study presymptomatic AD (725).
The relationship of functional neuroimaging to early morphological changes
are reviewed in the respective chapters (see Chapters 6 and 8) (726). They appear
superior to cognitive testing for early diagnosis of AD (727), but combined
evaluation of neuropsychological, structural, and functional imaging offers greater
predictive value (580). High resolution magnetic resonance imaging (MRI)
showed that the volume of frontal grey matter was strongly associated with age,
less with occipital grey and white matter, while the reduction in hippocampal
volume (727a) was best modelled as a cubic regression model. Common changes
in brain morphology as observed by cerebral MRI are associated with diminished
cognitive functions in elderly individuals (728). Cerebral atrophy seems to closely
parallel the distribution of neuritic pathology (and less the distribution of SPs).
Significant correlations were observed between antemortem hippocampal
volumes and both dementia severity and the density of hippocampal
neurofibrillary tangles at autopsy. Total cerebral volumes, in contrast, were
significantly correlated with the density of hippocampal SPs. This data suggests
that hippocampal volume assessed in living subjects with probable AD is both a
good marker of dementia severity and of the AD disease process (729). Voxel-
based morphometry and automated brain volumetry showed a high discrimination
accuracy between AD and controls, and could open up new possibilities for
early diagnosis of AD (730,731). MRI shows differences in Braak stages and
hippocampal volume between normal individuals and mild AD (601,732) with
increased changes related to the duration of disease and may predict AD pathology
(733). Early predictors of onset of AD in nondemented elderly subjects and of
progression of amnestic MCI to AD include increased annual atrophy rate in
the medial temporal lobe (734), inward deformation of the left hippocampal
surface in a zone corresponding to the CA1 subfield (735), atrophy of the
hippocampus (736), in particular of the EC (737), whole brain MRI spectroscopy
(738), and reduced hippocampal metabolism (739–741). Findings from the
Nun study indicated that both delayed recall measure and neuritic AD pathology
in the CA1 region reflect associated hippocampal atrophy (742,743). Memory
complaints in patients without any cognitive impairment were associated with
smaller left hippocampal volumes and more depressive symptoms (744).
As a result of neuron loss in the right amygdala-hippocampus complex it is
Early Diagnosis of Dementias from Neuropathology 369

correlated with declarative memory, in the left temporal-parietal region with


performance in naming and praxia (745). Dementia severity is also reflected by
reduced metabolism in posterior and frontal association areas in AD (746).
Morphological analysis in AD revealed severe atrophy in medial temporal
lobe and in inferior temporal, superior, and middle frontal cortex. It was
significantly greater for the fusiform gyrus, while the inferior frontal lobes were
entirely spared. Atrophy of other cortical regions was less marked and not related
to disease duration (747). Comparative volumetry revealed a strong relationship
between neuron number and both hippocampal and brain volume in brains with
and without AD (748). MRI volume measurements over time are valid biomarkers
of AD progress (749–751). Higher atrophy rates in the EC than in the hippocampus
are consistent with the view that AD pathology begins in the EC region (752,753)
and are a good predictor of conversion from MCI to AD (754,755). Grey matter
loss in the medial temporal lobe characterizes MCI, while in the parietal and
cingulate cortex it may be a feature of AD (756). Increased activation of the medial
temporal lobe, reflecting a compensatory response to accumulating AD pathology,
may serve as a marker for impending clinical decline (757). Reduced medial
temporal lobe N-acetylaspartate, together with reduced hippocampal gray
matter values, may be early indicators for dementia-related pathology (758).
Diffusion tensor MRI, helpful in quantifying MCI pathology to distinguish it from
aged controls (759), can also detect differential vulnerability of anterior white
matter with minimal acceleration in AD (760), while others show a similar
distribution of WMLs in VaD, AD, and healthy aging, the frontal lesions
correlating with MMSE scores (565). The pattern of hippocampus and corpus
callosum atrophy in relation to dementia severity gives evidence for early
neocortical degeneration in AD (761), and differs from VaD (762).
A novel amyloid-imaging PET tracer (see Chapter 10) showed marked
consistence with the pattern of Ab plaques in AD brain (188), and a combination
of two imaging agents (IMPY and SB13); targeting APs may be useful to
quantitate AP burden in the living AD patient, but not in tg mouse brains (763)
while positron emission tomography (PET) imaging NFTs is under discussion
(764,765). Increased [11C](R)-PK11195 binding in limbic and temporoparietal
cortex in mild AD suggests early microglial activation (766), while regional
cerebral blood flow patterns suggest that AD is a heterogenous disease (767).
A promising source of biomarkers is CSF which is in direct contact with the
extracellular space of the CNS, where biochemical changes in the brain could
potentially be reflected (see Chapter 3). However, CSF examination is not a “liquid
biopsy.” A growing body of literature has shown that total tau (t-tau),
phosphorylated tau (p-tau), and Ab42 in CSF and, in particular, combined
measurement of tau and Ab42 have reasonable specificity and sensitivity when
differentiating AD from normal aging, to detect incipient AD and candidates for
conversion of MCI to AD (768–781). Longitudinal CSF and MRI biomarkers may
improve the diagnosis of MCI (782). One main criticism is that only three studies
have compared CSF biomarkers and postmortem confirmation of diagnosis.
370 Jellinger

Large numbers of NPs were strongly associated with lower CSF Ab42 levels
that may reflect processes implicated in amyloid pathology (783). Studies in 106
patients with dementia and four NCI persons confirmed the association between
elevated CSF p-tau premortem and the pathological hallmarks of AD, indicating
that elevated CSF p-tau levels strongly support a diagnosis and may be helpful in
distinguishing AD from other dementing disorders (784). A reduced ratio of Ab42
to total Ab in CSF of AD patients was consistent with previous clinical reports, but
because of the broad range of Ab values provided no meaningful additional
diagnostic information when tau was elevated. Meta-analysis of CSF levels in a
large number of AD cases (only 23% confirmed at autopsy) and controls showed
significant decrease of Ab42 and an increase of tau (785). A small autopsy cohort
showed a significant decrease of postmortem CSF melatonin in aged individuals
with early AD changes in the temporal cortex, suggesting that this may also be an
early event in the development of AD (786). In view of diverging data between
in vitro and postmortem CSF, this study awaits confirmation. Although CSF tau
and Ab42 may not be specific for AD, their evaluation combined with
neuroimaging may improve diagnostic specifity (787). Quantitative proteomics
of CSF from AD patients compared to age-matched cotrols, as well as from other
neurodegenerative diseases, will allow us to generate a roster of proteins that may
serve as specific biomarker panels for AD and other geriatric dementias (788).
With regard to plasma Ab40 and Ab42 levels, these may increase with
age but are not specific for MCI or sporadic AD (789). Reports show these to
have been elevated before and during the early stages of AD though then decline
thereafter (790). These findings may be associated with APOE E4 (791) and may
be a risk factor for microvascular damage (791a), although there are no reports of
any neuropathological confirmation of this data. The same findings are evident for
APP in platelets and other peripheral marker for sporadic AD (792). Morover,
plasma Ab42 levels are highly influenced by concomitant medications (793).
Salivary AChE activity may prove to be a useful marker of central
cholinergic activity (794). It is important that any future studies on biomarkers
are long-term prospective investigations with the aim of confirming the diagnosis
pathologically (795).

PDD and DLB


Early morphological changes have been described and related to in vivo markers
in PD and DLB. However, except for rare patients who died in early or preclinical
stages of the disorder, the morphology of initial changes, beginning in the lower
brainstem and olfactory system, with upward progression to the nigrostriatal
dopaminergic system (306,307), is not available, and information depends on
functional neuroimaging (652,796,797) and monitoring dopaminergic degener-
ation (798–802).
In DLB, determination of the loss of dopamine transporter as marker of
striatonigral degeneration (803,804) and use of presynaptic and postsynaptic
Early Diagnosis of Dementias from Neuropathology 371

ligands may distinguish it from AD (805,806). A sensitivity of 83% and specificity


of 100% have been reported for the association of an abnormal scan with an
autopsy diagnosis in DLB (329). Regional correlation of dopaminergic functions
in the striatum, using PET analysis, may be useful in distinguishing DLB from PD
(807). Neuroimaging studies and their correlation with neuropathology may also
be helpful in supporting the clinical diagnosis (808). Voxel-based morphometry
(VBM) showing preservation of the medial temporal lobe, hippocampus,
and amygdala in DLB was considered a potential antemortem marker in
distinguishing it from AD (809). PD cases showed gray matter loss in prefrontal
and limbic/paralimbic areas (810), and in PDD this extended to temporal and
subcortical areas, with occipital atrophy in PDD being the only difference between
the two groups, while no volumetric differences were observed between PDD and
DLB (811). Diffuse glucose hypometabolism in the entire cortex including
occipital cortex, only sparing the primary sensory-motor cortex, also seems to be a
typical pattern for DLB distinct from AD (812–814). In vivo diffusion MRI
showing selective involvement of parietal, frontal, and occipital lobe as well as
subcortical abnormalities indicating nigrostriatal involvement in DLB and PD
may be useful in the diagnosis of DLB (815).
Another possibility would be histological examination of biopsies of the
olfactory epithelium (OE) showing prominent expression of AS and dystrophic
neurites in patients with PD, DLB, multiple system atrophy (MSA), and AD
(669). Further studies should validate the diagnostic sensitivity of olfactory
mucosa biopsies.
No clinically useful tests have been reported using plasma and CSF
analysis for the diagnosis of both PD and DLB (816). Some studies indicated that
CSF p-tau may be lower in DLB than in AD, both showing decreased levels of
Ab (347,817,818), while others showed decreased Ab42 in AD and a slight
increase in DLB, but follow-up CSF analyses are of limited value for the
differentiation of AD and DLB. This suggests that more specific markers have to
be established for the differentiation and follow-up of these diseases (819). While
the native form of AS was previously not found in CSF (820), recent studies
demonstrated increased soluble AS in the blood and brain from familial PD with
SNCA locus triplication (821).

Frontotemporal Dementias
Based on the severity of gross atrophy in FTD, four progressive stages were
identified (822): stage 1: initial mild atrophy in the orbital and superior medial
frontal regions, and hippocampus; stage 2: atrophy of the other anterior frontal
regions, temporal cortices, and basal ganglia; stage 3: involvement of all
remaining tissues on coronal slices; stage 4: marked atrophy in all brain areas.
Severe atrophy of the frontal lobe, amygdala, and hippocampus in stage 2 suggests
that they are among the earliest affected in FTD (823). Comparison of PID, DLDH,
and FTD-MND found no differences in these subtypes, suggesting that disease
372 Jellinger

stage rather than FTD subtype determines the pattern and extent of neuronal
degeneration (824). This scheme provides the framework to determinate
correlates relating to disease progression, e.g., with neuroimaging data (825–828).
FTD revealed elevated CSF tau (though lower than in AD), high APOE E4
and E2 frequency, and decreased Ab42 levels (829). However, these values are in
the intermediate range between FTD, AD, and other dementias, and are not useful
for the diagnosis of FTD (830,831), and others showed significant reduction of t-tau
levels in CSF (832). CSF t-tau was not increased in FTD patients with tau mutations
(833), while p-tau 231 and p-tau 181 yielded excellent distinction between AD and
FTD (834). It could be useful to develop techniques to study tau-isoform
patterns in CSF, because they are disease specific when measured in brain
homogenates (835). Unfortunately, none of these CSF studies was confirmed by
postmortem studies, and a sensitive and a specific biomarker enabling differen-
tiation of FTD from other conditions is still lacking.
Vascular Dementias
Previous neuroimaging studies in vascular cognitive impairment (VCI) have been
partly contradictory (452,836). Independent correlates of post stroke dementia are
the combination of infarct feature (volume of infarcts in middle cerebral artery,
higher frequency of left-sided infarcts), extent of WMLs, medial temporal atrophy,
and host factors (836). The volume of functional tissue loss may be more important
than of total tissue because it also includes the effect of deafferented cortex
(837,838). Absence of CVLs on CT and MRI is evidence against a vascular
etiology of dementia, which is in agreement with neuropathological studies
(460,461,477). Although the hippocampus is less affected by subcortical CVLs,
dementia due to microvascular pathology showed significant hippocampal
neuronal loss (478,839), and hippocampal atrophy may increase the development
of post-stroke dementia (840). To date, however, it has not been possible to
determine the specific amount of WML necessary to cause cognitive impairment,
but it has been found that even relatively mild WMH can have deleterious effects
on cognition (472). A standardized assessment of subcortical CVD on CT films
can be combined into a unique score that is in good agreement with
neuropathology. This supports the validity of the CT-based visual rating scale
as a valid tool to detect subcortical vascular changes in elderly persons (841).
MRI studies confirmed that infarcts located in strategic areas have a role in
cognitive impairment, and their quantification correlates with neuropathological
findings (842). There is a substantial overlap between cases classified as AD by
NINCDS/ADRDA, and VaD by modified SCADDTC criteria. The substantial
contribution of vascular disease would be missed without inclusion of MRI.
Treatment of risk factors for VaD could have an important impact on the
incidence of dementia (843).
Studies of cerebral blood flow (CBF) and metabolism, in general, failed to
identify diagnostic features of VaD and to provide a robust, easily applicable
diagnostic technique because of the heterogenous pathology underlying dementia.
Early Diagnosis of Dementias from Neuropathology 373

WMLs usually have an independent correlation with VaD, while hyperperfusions


and fronto-subcortical atrophy are substrates of mild cognitive deficits after stroke
(844). They are often correlated with atrophy of the corpus callosum (845),
supporting the role of disruption of white matter connections in the pathogenesis of
cognitive impairment (846). WMLs are associated with reduced rCBV in the
cerebral cortex, particularly in individuals with extensive hypertension (847).
Neurochemical markers, due to a wide range of levels, appear inconclusive
for the diagnosis of VaD and its distinction from other dementing disorders (848).
The diagnosis of VaD is difficult in epidemiologic studies because post-stroke
dementia can be due to AD and evidence of VaD can be found in the MRI
of dementia cases without clinical strokes. Whether the clinical progression is
related to AD pathology or vascular disease is difficult to establish (450). Similar
difficulties arise for the in vivo diagnosis of mixed type dementia.

CORRELATION OF CLINICAL AND PATHOLOGICAL CRITERIA


While recent studies clearly point to the importance of AD pathology in limbic
areas for memory impairment, involvement of the neocortex is significantly
correlated to the progress of dementia. According to most clinico-pathological
studies, all demented patients had dystrophic neurites both free in the neuropil or
surrounding SPs. The number of NFTs and, much less so, that of NTs usually
correlated with the clinical severity of dementia, while even widespread Ab
deposition may be necessary but by itself is often insufficient for the development
of dementia (298). Although there may be considerable overlap with isocortical
Braak stages in cognitively normal individuals and MCI subjects (10,24,297), the
only brain region in which NFT count is suggested to provide a significant marker
for dementia is the association isocortex (223,650). Only up to 20% of aged
cognitively normal subjects had no or only few AD changes, while all the others
showed numerous AD lesions including 9% with isocortical Braak stags 5 or 6,
indicating that some patients tolerate the insults of AD pathology, probably due
to neurocognitive reserve or compensation (24,297). In other cohorts, 7–50% of
individuals with no impairment or MCI met one or more of the morphological
AD criteria or had tau pathology in the perforant path target zone and loss of
entorhinal neurons, while only 10–20% were free of considerable AD-related
lesions (10,24,238,599). About 29% of cases pathologically defined by CERAD
criteria as AD do not have clinical dementia, whereas more than half of
octogenarians without dementia meet CERAD criteria for pathologically
confirmed AD (849). Therefore re-evaluation of the diagnostic criteria are
necessary and require further development for reliable screening.

EFFECTS OF CONCURRENT PATHOLOGIES


Comorbidity in aged individuals may influence the clinical and morphological
features of AD, and it is well established that AD patients frequently have other
374 Jellinger

lesions (533). Among the most frequent coexisting pathologies are CVLs with an
incidence of 18–80% (460,461,463,539,551,553,554), followed by LB pathology
in 12–20% (275,540). Cortical LBs have been observed in 7–71% (334,335,850),
and AS pathology in the amygdala and other brain areas in 15–60% of sporadic
AD cases (275,332,338,339). The pathogenesis and clinico-pathological impact
remains to be determined. While the APOE genotype usually shows no differences
between AD and vascular disease, cerebral infarcts in regions typically destroyed
by AD (e.g., limbic areas and the thalamus), and cortical LBs which frequently
occur in both AD and DLB, all may exacerbate the clinical manifestations of AD
and thus contribute to the heterogeneity of the disease (299,533).

NEUROPATHOLOGY OF AD AND CLINICAL RELEVANCE


AD is a heterogeneous disorder with variable clinical and pathological
phenotypes. Current guidelines for the neuropathological diagnosis of AD are
based on age-adapted (semi)quantitative assessment of plaques, either amyloid or
dystrophic (279), neuritic plaques (CERAD), topographic spreading of neuritic
AD pathology (218), and a combination of Braak and CERAD staging with
probabilistic statement that dementia may be associated with AD. Khachaturian
criteria may not be useful to differentiate between diffuse and neuritic plaques,
and between AD cases and cognitively normal individuals. Neither CERAD nor
NIA-RI guidelines discriminate AD from pathological aging in the absence of
clinical findings. Braak staging does not correlate well with psychometric data in
clincally nondemented subjects (297,851). Multivariate analysis of aged individuals
revealed significant correlations between cognitive function assessed by the MMSE
and Blessed test, and both the CERAD criteria and Braak staging, but much weaker
correlations for both Tierney and Khachaturian criteria (289,291). High Braak stages
and CERAD criteria identified 54% and 62% of demented individuals respectively,
and eliminated 84% and 70% of non-demented individuals.
The major clinico-pathological correlates of AD can be summarized as
follows:
1. Early, often preclinical involvement of the allocortex causes deafferen-
tation of the hippocampus due to involvement of the (trans)-entorhinal
region by alterations of synaptic function and neuronal degeneration due
to cytoskeletal PHF changes and Ab deposition clinically presenting as
early cognition, olfactory, and amnestic deficits without overt dementia.
2. Spreading of AD pathology to the isocortex with hierarchic involvement
of isocortical association areas cause the following syndromes:
(i) temporal isocortex induces degeneration and disruption of
cortico-cortical connections to prefrontal and parietal areas and
causes visuospatial, speech, and auditory disorders.
(ii) frontal cortex: high NFT- and Ab-load in the frontal variant of AD
with isolated executive impairment (597).
Early Diagnosis of Dementias from Neuropathology 375

(iii) orbito-frontal and cingulate pathology often causes agitation.


(iv) major NFT load in areas 7, 24, and CA1 induces spatial-
temporal disorientation.
(v) occipital cortex induces rare visual variant of AD (852) recently
referred to as progressive posterior cortical dysfunction involving
selective visuospatial deficits (853).
(vi) higher levels of AD pathology are related to lower levels of
implicit memory and conceptual but not to perceptual, proceeding
resources (854).
(vii) progressive degeneration of intracortical, cortico-cortical con-
nections causes global cortico-cortical discontinuation with loss
of higher cortical functions and dementia.
3. Late spreading of AD pathology to subcortical areas cause degeneration
of cortico-subcortical neuronal systems and induces primary sensory
deafferentation with affective, extrapyramidal, and vegetative signs
or symptoms.

CONCLUSIONS
In general, there is a good correlation between tau pathology and cognitive state as
well as between the progress of both neuroimaging and the clinical manifestations
of disease. These morphological markers (tau pathology, amyloidosis, and
synaptic proteins as well as biochemical markers, i.e., ChAT and related mRNAs)
usually identify cases with moderate to severe dementia. However, due to variable
overlaps these changes often fail to distinguish between cognitively intact aged
subjects from those with MCI/preclinical or mild AD. In particular these latter
groups show a wide variety in the intensity and pattern of AD-related lesions.
Although they often differ from “normal” aging, only a small proportion of
cognitively intact aged individuals are free of AD pathology, while up to 50% may
show various AD-related alterations or even definitive AD pathology. Additional
difficulties arise from frequent coexistence with other pathologies that have a
synergistic effect and may act by contributing to cognitive impairment.
In view of these difficulties, further studies are needed to increase the
diagnostic validity of current diagnostic criteria of AD and its distinction from
“normal” aging and from other disorders causing dementia.

ABBREVIATIONS
3-R 3-repeat
ACh acetylcholine
AChE acetylcholinesterase
AD Alzheimer’s disease
ADDL amyloid-ß derived diffusable ligand
ADRDA Alzheimer’s Disease and Related Disorders Association
376 Jellinger

AGD argyrophilic grain disease


AP amyloid plaque
APP amyloid precursor protein
AS a-synuclein
Aß amyloid ß peptide
BBB blood-brain barrier
BDNF brain-derived neurotrophic factor
CAA cerebral amyloid angiopathy
CBD corticobasal degeneration
CDR Clinical Dementia Rating Scale
CERAD Consortium to Establish a Registry for Alzheimer’s Disease
ChAT choline acetyl transferase
CN cognitively normal
CVD cerebrovascular disease
CVL cerebrovascular lesion
DLB dementia with Lewy bodies
DLDH dementia lacking distinctive histopathology
DP diffuse plaque
EC entorhinal cortex
ER endoplasmic reticulum
FAD familial AD
FTD frontotemporal dementia
FTDP-17 frontotemporal dementia and parkinsonism related to chromo-
some 17
FTLD frontotemporal lobe degeneration
FTLD-U FTLD with Ub-only neuronal changes
GABA g-aminobutyric acid
GBR1 GABA receptor R1 protein
GFAP glial fibrillary acidic protein
HHD hypoperfusive hypoxic-ischemic dementia
HSD hippocampal sclerosis dementia
HIS Hachinski Ischemic Score
ICD International Classification of Diseases
kDA kilodalton
LB Lewy body
LBD Lewy body disease
LBV/AD Lewy body variant of Alzheimer’s disease
LVD large vessel dementia
MAP mitogen-activated protein
MBP myelin basic protein
MCI mild cognitive impairment
MD mixed type dementia
MID multiple-infarct dementia
MMSE Mini-Mental State Examination
Early Diagnosis of Dementias from Neuropathology 377

MND motoneuron disease


MNDID motor neuron disease inclusion dementia
MRI magnetic resonance imaging
NAA N-acetyl aspartate
NCDS Neurological and Communicative Disorders and Stroke
NF neurofilament
NFT neurofibrillary tangle
NGF nerve growth factor
NIA National Institute for Aging
NINDS-AIREN National Institutes of Neurological Diseases and Stroke
NP neuritic plaque
NT neuropil thread
OE olfactory epithelium
OS olfactory system
PD Parkinson’s disease
PDD Parkinson’s disease with dementia
p-PERK phosphorylated pancreatic ER kinase
PHF paired helical filaments
Pib Pick body
PIB Pittsburgh compound-B
PiD Pick’s disease
PSEN1 presenilin-1
PSD post-stroke dementia
PSP progressive supranuclear palsy
p-tau phosphorylated tau protein
rCBF Regional cerebral blood flow
RI Ronald and Nancy Reagan Institute of the Alzheimer’s
Association
ROS Religious Order Study
SCAA severe CAA
SCADDTC State of California Alzheimer’s Disease Diagnostic and
Treatment Center
SID strategic infarct dementia
SNc substantia nigra pars compacta
SP senile plaque
SVD small vessel disease
Tg transgenic
t-tau total tau protein
Ub ubiquitin
UPR unfolded protein response
VaD vascular dementia
VID vascular-ischemic dementia
VBM voxel based morphometry
378 Jellinger

VCI vascular cognitive impairment


WML white matter lesion

ACKNOWLEDGMENTS

The study was supported by the Society for the Support of Research in
Neurological Sciences, Vienna, Austria. Special thanks are due to Mr. E. Mitter-
Ferstl, PhD, for secretarial and computer work.

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12
Guideline and Perspective

Karl Herholz
Wolfson Molecular Imaging Centre, University of Manchester, Manchester,
U.K., and Department of Neurology, University of Cologne,
Cologne, Germany
Daniela Perani
Departments of Neuroscience and Nuclear Medicine, Vita-Salute
San Raffaele University and San Raffaele Scientific Institute, Milan, Italy
Chris Morris
Wolfson Unit of Clinical Pharmacology, Chemical Hazards and Poisons
Division–Newcastle, Health Protection Agency, and School of Neurology,
Neurobiology, and Psychiatry, The Medical School, University of Newcastle
upon Tyne, Newcastle upon Tyne, Tyne and Wear, U.K.

INTRODUCTION
Early diagnosis of dementia has many aspects. There is no, and probably will not
be any, diagnostic measure that can be expected to suit all diagnostic situations
equally well and therefore a differentiated approach is necessary. Three gross
scenarios can be distinguished: (1) diagnosis of mild dementia that is clinically
manifest, (2) diagnosis of a dementing disease in a subject who has cognitive
impairment but is not yet demented, and (3) detection of a dementing disease in
an asymptomatic individual.
In addition to these diagnostic aspects, new methods may play an important
role in clinical trials, e.g., by defining efficient inclusion criteria particularly at a
presymptomatic stage, or by providing pathophysiological insights into treatment
effects that have not been available in the past. In this context, imaging and
biomarkers are expected to play an increasing role. A list of criteria for such
markers to be clinically acceptable has been defined by a joint working group of

429
430 Herholz et al.

the Ronald and Nancy Reagan Research Institute of the Alzheimer’s Association
and the National Institute on Aging (1):
1. There should be at least two independent studies that specify the
biomarker’s sensitivity, specificity, and positive and negative
predictive values
2. Sensitivity and specificity should be no less than 80%; positive
predictive value should approach 90%
3. The studies should be well powered, conducted by investigators with
expertise to conduct such studies, and the results published in peer-
reviewed journals
4. The studies should specify type of control subjects, including normal
subjects and those with a dementing illness but not Alzheimer’s
disease (AD)
5. Once a marker is accepted, follow-up data should be collected and
disseminated to monitor its accuracy and diagnostic value.
Some imaging techniques now appear to fulfill or come close to these
requirements, although the request for 90% positive prediction obviously
depends on the selection of subjects. Even excellent biomarkers providing more
than 95% specificity in selected samples would fall short of that goal in the
context of population screening. It can be achieved only in subjects who have
been selected on the grounds that their clinical symptoms already include
dementia or at least a severe memory deficit.

DIAGNOSIS OF MILD MANIFEST DEMENTIA


Quite frequently under standard health care in most countries, even clinically
manifest dementia is diagnosed only at a late stage of the illness, or sometimes
not at all. There are several possible reasons for this including a reluctance to seek
medical help on the patient’s part, and to make a specific diagnosis of dementia
on the part of the physician. Patients may not feel impaired to a degree where they
would ask for treatment or they may be simply unaware of their deficits and their
severity as a consequence of dementia. But even when recognizing the deficits
and possibly complaining about them, they may not expect effective treatment
and may be afraid of being diagnosed with dementia and therefore avoid
medical advice. Such fears are probably not entirely unfounded since a
diagnosis of dementia may mean a loss of self-esteem and personal and financial
independence, and may lead to social isolation and perhaps even discrimination.
Only with adequate and unconditional support from friends, relatives, caregivers,
and social institutions can an early diagnosis of dementia help in maintaining
self-esteem and smooth the transition to dependency, thereby improving the
situation of the affected individual rather than deteriorating it. Thus, early
diagnosis touches many ethical issues, and it is therefore mandatory that seeking
early diagnosis needs to be a decision of the affected individual rather than a
Guideline and Perspective 431

requirement from other perhaps (but not necessarily always) well-meaning


people. The situation changes as soon as dementia is severe enough that patients
endanger other people or themselves by hazardous actions or neglect, and social
and legal actions are then required for protection—yet, it is at this stage that we
have left the arena of early diagnosis that is treated in this book.
Physicians and nurses may also be reluctant to diagnose dementia at an
early stage, perhaps because they may feel that such a diagnosis is not in their
patient’s interest. Since there is currently no effective treatment that prevents
progression of disease, the quest for an early diagnosis may be seen as an
unwarranted action that incurs costs to cover diagnostic procedures but provides
little benefit. Health care systems may explicitly or implicitly discourage early
diagnosis of dementia, most likely for economic reasons. Standard diagnostic
criteria are also imprecise and do not correspond with each other entirely at the
very mild stages of dementia, causing objective difficulties in obtaining a reliable
diagnosis. Nevertheless, N. Foster provides many good reasons in chapter 1 why,
even in the current situation, obtaining an early diagnosis is important and
worthwhile, and the urgency of that will of course increase enormously once drug
treatments become available that are either highly effective in ameliorating
symptoms, or may even delay or prevent progression of dementia. There is
already limited albeit controversial evidence for this with currently used drugs;
therefore, even small advances in treatment possibilities will cause an enormous
demand for better diagnosis to avoid under, or inappropriate treatment.
Diagnosis of early dementia has two components: revealing the clinical
syndrome of dementia and distinguishing between dementing diseases, such
as AD, frontotemporal dementia, dementia with Lewy bodies, and vascular
dementia (VaD). Obviously, the distinction between diseases is more demanding
than detecting the dementia syndrome. Many studies that deal with issues of
sensitivity and specificity only address the discrimination of one dementia type
(most frequently AD, of course) from normal individuals. Thus, they often are
validated against the clinical standard of comprehensive assessment using
neuropsychological test batteries and exclusion of other diseases by clinical
examination, computed tomography (CT) or magnetic resonance imaging (MRI),
and standard laboratory tests. Thus, it is difficult to judge whether there is any
additional benefit to be had by using new tests beyond those that are the clinical
standard, especially when implemented optimally as part of a multidisciplinary
dementia or memory clinic.
Comprehensive neuropsychological assessment is a demanding procedure
with respect to expertise of the examiner, time needed, and patient cooperation.
However, several compact cognitive batteries have been designed which show
considerable promise for dementia screening in the earliest phase of AD (2–5).
A wide range of cognitive functions appear to decline in persons who are
diagnosed with AD, including memory, attention, language, visuospatial skill,
perceptual speed, and executive functioning. There is sufficient evidence to
recommend specific tests or cut-off scores. Daniela Perani makes the case for
432 Herholz et al.

using certain neuropsychological tests for the detection of the earliest stages of
dementia, though these may only be able to detect individuals with certain types
of dementia amnestic mild cognitive impairment (MCI). While requiring
longitudinal validation, the application of such tests should in the future provide
much simplified diagnosis. Thus, in a clinical setting, the degree of impairment
can be assessed neuropsychologically, but fulfillment of different dementia
criteria is ultimately determined through clinical judgement using information
from these tests within a framework that includes other tools. New biomarkers
probably could not reasonably aim at replacing the detection of dementia by
clinical judgement, but should always be seen as part of that process.
Claims for the more ambitious goal of an early distinction between
dementing diseases are difficult to verify because the appropriate gold standard
for this is histopathological postmortem examination and, as pointed out by
Jellinger (see Chapter 11), these standards are lacking at the earliest stages of
dementing diseases. Thus, only longitudinal studies that cover the five to 10 years
that may pass between onset of dementia and death with definitive pathological
diagnosis could have a chance to achieve this. The lack of such studies, though,
demonstrates an urgent scientific need for carefully designed longitudinal studies
that include modern diagnostic procedures that assess specific pathophysiological
processes. Particularly in very elderly individuals with dementia, they should also
address the issue of how various pathophysiological processes interact, such as
cytoskleletal and synaptic changes, deposition of amyloid, tau protein, and
a-synuclein with vascular pathology, and especially during the progression of
dementia. Such studies are costly and difficult to organize, but the payoff is likely
to be enormous.
Somewhat less firm evidence can be gathered when comparing new
diagnostic procedures against each other and against standard clinical criteria.
From the correspondence of diagnostic classifications, we can at least get an
upper estimate of their accuracy and, by considering their pathophysiological
rationale and closeness to purported core pathophysiological events, an idea of
their potential. Most of the studies that are available today fall into this category
and guide our ideas about their relative merits and perspectives.
Qualitative judgment of atrophy on CT and MRI is already in common use
as part of clinical assessment. Progress is to be expected by using age-adjusted
quantitative measures or, at least, standardised performance and reading of scans,
as described by Frisoni and Filippini (see Chapter 6), but the specificity of using
simple measures of atrophy will probably remain low. New techniques, e.g.,
diffusion-weighted imaging and magnetisation transfer techniques, could be
added relatively easily to the standard clinical test batteries in use and show some
promise in improving diagnostic specificity. Molecular laboratory blood tests
could be integrated easily, but none with high predictive power is currently
available. Cerebrospinal fluid (CSF) testing is less likely to be adopted widely due
to its invasiveness. Functional imaging by single photon emission computed
tomography (SPECT) (see Chapter 7) and 18F-2-fluoro-2-deoxy-D-glucose
Guideline and Perspective 433

(FDG) positron emission tomography (PET) (see Chapter 8) can be a component


of the clinical workup where the necessary technical equipment, expertise in
performing scans and reading results, and financial resources are available, but
more standardisation and proof of efficacy is required to allow implementation as
a standard procedure. Molecular tracers, especially for amyloid imaging, may
potentially make a major contribution once the remaining open issues about their
diagnostic specificity, costs, and availability have been solved. Genetic testing has
a clear role in familial disease (see Chapter 4), but its efficacy is still too low in
sporadic disease to be of much clinical value.

NONDEMENTED SUBJECTS WITH COGNITIVE IMPAIRMENT


Subjects with progressive cognitive impairment who are not demented, which
essentially means that cognitive impairment is not severe enough to impair
activities of daily living, are at increased risk of becoming demented. This would
be the primary target population for a drug or other intervention that could
prevent dementia. The degree of risk at which that intervention was effective
would depend on its efficiency, cost, and side effects. A drug with moderate
efficiency but significant side effects or high cost probably should be reserved for
individuals at very high risk, whereas a cheap drug without significant side effects
could be given to a much more broadly defined at risk population.
In this context, a major difficulty exists with diagnostic classification.
There is a widely accepted operational definition for MCI based on clinical and
neuropsychological examination (6), and it is well documented that individuals
with predominant severe memory impairment (amnesic MCI) are at particular
risk of developing AD and this risk increases steeply with age. However, when
applying the criteria on a population basis, most studies found that specificity is
low and up to 50% of individuals fulfilling the criteria do not develop AD within
the next few years (7). For example, in the PAQUID study, while MCI was
found to be a good predictor of dementia with a conversion rate of
approximately 8%, over 40% of those diagnosed with MCI did not convert to
dementia over a five-year period. In contrast, conversion rates of MCI to
dementia (either AD or VaD) of 44% were found in a clinic-based study with
half of the MCI patients remaining cognitively impaired but dementia free and
with a very small (!5%) proportion being free of dementia at follow up. The
suggestion has been made that the discrepancy for these results may be due to
selection bias in clinic-based studies, participants having more severe cognitive
impairment and therefore being more likely to present and also to progress to
dementia. In population based studies, MCI may not simply represent prodromal
AD but may be due to a variety of factors including late onset depression and be
of a milder nature.
Neuropsychological tools for early detection of dementia that are sensitive
enough to differentiate among incipient dementia, normal aging, and other brain
disorders that can mimic dementia, in particular depression (8) have been
434 Herholz et al.

proposed. The evaluation of memory is the basis for the differential diagnosis of a
dementia in its early stages and cognitive changes related to normal aging. The
availability of proper normative data, however, represents a current challenge
that requires a solution. In the last few years, specific criteria for the diagnosis
of memory deficits in elderly have been proposed. The criteria for “age-
associated memory deficit” and for the nosographic picture of MCI underline
that, in the two conditions, the diagnosis is based on very different psychometric
criteria of memory performance. In MCI an impairment on tests of episodic
memory is generally the most predictive measure (6), but also tests of many
different cognitive capacities have been shown to be predictive of a future
diagnosis of AD among memory-impaired subjects (9–11).
The results from a general practice study of cognitive impairment have
suggested guidelines for the detection of MCI (12). The role of the general
practitioner in the diagnosis of MCI and the potential feasibility of general
practice screening is particularly relevant. It may be possible for the family
practitioner to verify cognitive complaints and to screen for MCI with a high
degree of accuracy using a brief test battery derived from empirical observations
in population studies. Neuropsychological tests with the highest predictive value
for dementia conversion and suitable for use in general practice comprise three
tests (delayed auditory verbal recall, verbal fluency, and visuospatial
construction), giving a specificity of 99% and sensitivity of 73%. In addition,
MCI detection should not be limited to cognitive performance alone. Proxy
observations of behavioral change and information relating to loss of ability to
perform activities of daily living should also be used to improve sensitivity and
also to provide information needed in patient management. However, the
assessment of specific domains of complex instrumental activities of daily living
that might be impaired in MCI need to be determined.
There is a consensus that cognitive and functional abilities need to be
considered in the evaluation of MCI. Individual slopes of decline in both
functional and cognitive performance may be better measures than deficits
assessed according to age-specific norms. However, a true consensus can only be
achieved after longitudinal studies establish the age-specific levels of cognitive
functioning, as well as normal rates of cognitive decline over specific time
periods.
Thus, there is a substantial need for improvement in detection by means of
more reliable diagnostic methods. We are also lacking appropriate diagnostic
terms to label individuals who present clinically as MCI with additional evidence
that this is early AD. Even when the histopathological signs of AD are found at
postmortem examination, standard diagnostic criteria require evidence of
dementia during life to diagnose AD since the presence of some plaques and
tangles is quite common in cognitively intact elderly subjects and the correlation
between these changes and cognitive status is not very close. Quite obviously, the
issue of whether those individuals with histopathological signs of AD but no
dementia would eventually have become demented cannot be clarified based on
Guideline and Perspective 435

postmortem research because it cannot be done longitudinally. We can, however,


longitudinally follow subjects who are asymptomatic or who present with MCI
and show significant amyloid deposition based on PET images, to clarify whether
this is indeed an indicator of incipient AD (see Chapter 10).
If a reliable predictor of AD was found, the situation would become
somewhat similar to how it is in Huntington’s disease today. Before onset of
chorea in that disorder, there is a stage when unspecific motor symptoms and
psychiatric symptoms may be present. Prediction of manifestation of this
autosomal dominant disease with complete penetrance can be made on the basis
of genetic testing, and the degree of caudate atrophy provides additional clues to
predict time until onset quite accurately (13). Thus, an optimum time window for
entry into prevention trials with development of clinical symptoms as outcome
parameter can be defined in order to achieve high trial power.
Since AD is a multifactorial disorder, it is unlikely that the same degree of
predictive accuracy as in Huntington’s disease can be achieved. A scientifically
appropriate approach to the problem of quantifying risk is studying dementia-free
survival, this probably being best achieved by using a proportional hazards
model. This approach has been used to determine the predictive utility of APOE
genotyping for AD in MCI patients (14) demonstrating that its predictive power
is higher at age 70 to 85 than below age 70. Dementia-free survival curves have
also been plotted for different degrees of hippocampal atrophy on MRI by Jack
et al. (15), demonstrating that the risk is more significant than that associated with
age or APOE status. The application of novel statistical methods based on
Bayesian theory has been applied in conjunction with APOE genotype to at risk
families with AD, though even here margins of error are perhaps larger than
are needed for application in a clinical setting. Methods such as these may
though in future be applied to give the higher sensitivity and specificity required
for clinical use. There is therefore a need to perform larger studies with
statistical analyses including other biomarkers to obtain a more complete and
reliable picture.
Since for practical purposes medicine requests diagnostic categories, we
should find a consensus to define a new diagnostic category for cognitively
impaired individuals at very high risk to develop AD within the next two years.
As a suggestion for a suitable term we would like to put forward MCI-AD as an
abbreviation for “MCI, at high risk for AD.” This group would be a prime target
for clinical trials of neuroprotective agents. Some authors suggest that this is
already possible by careful patient selection based on careful clinical and
extended neuropsychological examination (16), but that involves a substantial
degree of subjective clinical judgement that should be overcome by better
objective tests. Memory loss alone rarely predicts AD, whereas multiple mild
deficits do, especially if reported or confirmed by informants (17,18). Deficits not
only in memory but also in problem solving, slowed psychomotor performance,
and depressive features are further clinical predictors of dementia in old age (19).
To achieve reasonable specificity, it is also important to exclude reversible
436 Herholz et al.

conditions that can cause memory impairment by appropriate medical tests


(see Chapter 1) (20,21). Visser and colleagues achieved 80% specificity and a
positive predictive value of 77% for AD within five years using a combination of
neuropsychological testing, assessment of hippocampal atrophy, and APOE
genotyping in an MCI sample drawn from a memory clinic (22). Some studies
indicate that combining targeted neuropsychology testing with MRI volumetry
(23) or with PET (24), combining MRI with SPECT (25), or CSF testing with
SPECT (26) may reach a prediction accuracy very close to 90%. Hampel and
Burger (Chapter 3) show that newer in vitro tests for MCI may provide high
sensitivity for detecting those individuals which progress to AD on the basis of
reductions in CSF b-amyloid (Ab) and elevations in phosphorylated tau.
Similarly, Klunk and colleagues (Chapter 10) demonstrate the possibility of
detecting MCI-AD using Ab-specific PET imaging agents where Ab plays a
significant part in the etiology of the dementia. With a combination of tests it may
therefore be possible in the near future be able to identify individuals with MCI
who are at high risk of AD (MCI-AD) who are candidates for early therapeutic
intervention, and trials of such combination tests are currently underway. These
very promising results, however, still have to be confirmed in larger studies
before current practice guidelines that rely on clinical judgement and
neuropsychological testing (27) should be revised substantially.

ASYMPTOMATIC SUBJECTS
On a population basis, early diagnosis may mean screening of asymptomatic
individuals who may be at increased risk of AD because they are old or have a
relative with AD and carry a common risk factor such as the APOE epsilon 4
allele. In the current situation without proven interventions that could
prevent onset of dementia, there is usually little reason to perform such screening
other than for the purpose of epidemiologic studies, but individuals may still be
worried and seek advice (28).
Genetic counselling is usually recommended for subjects who have
multiple (typically three or more) family members who developed early onset
AD, especially when age of onset was at 55 years or younger. Up to 70% of such
families may have mutations in the amyloid precursor protein (APP), presenilin-1
(PSEN1), or presenilin-2 (PSEN2) genes that can be identified by comprehensive
genetic screening (29). Yet, even in such cases, genetic testing may not provide a
conclusive answer in some families (30). In those rare families where mutations in
APP, PSEN1, or PSEN2 are identified, or in other families where there is a clear
indication of genetically defined autosomal dominant dementia, there are good
reasons to consider predictive testing in at risk family members since, in the vast
majority of cases, the presence of a mutation indicates a high likelihood of
developing dementia (see Chapter 4). This, however, brings with it certain ethical
issues particularly since there are currently no disease slowing therapies. As with
other neurodegenerative disorders with a clearly defined genetic basis, prior to
Guideline and Perspective 437

any genetic testing, support in the form of counselling should be provided to


family members prior to any specific testing, and regimes and guidelines
recommended for Huntington’s are strongly recommended. Individuals are then
free to make any life choices and undertake testing if they wish. If an individual
agrees to undergo testing or not, there is also a need to provide support and
counselling for a prolonged period after the initial stages of counselling, and this
is particularly the case where a mutation is found, where systems should be in
place for monitoring and supporting the individual (32). There is now a consensus
in most countries that genetic testing should be offered for adults asking for it only
on the background of professional genetic counselling that addresses and deals
with the psychological and social implications (31).
The presence of the APOE E4 allele is currently the strongest and most
consistent indicator of genetic risk for “sporadic” AD. The remaining lifetime
risk at age 65 of developing AD for APOE E4 homozygotes is about four times,
and for heterozygotes about twice that of non-carriers (where it is 4.6% in men
and 9.3% in women) (30). With advancing age, however, the influence of APOE
E4 status gradually weakens, and for individuals living beyond 90 years of age
where the prevalence of dementia is higher than 30%, dementia is largely
independent of APOE E4 status. Some of the new techniques presented in this
book might provide additional evidence to adjust that risk individually, though
one should keep in mind that providing an individual prognosis before age 65
will be fraught with a significant number of false positive predictions because at
low baseline prevalence, even 90% specificity of a test (currently most in vivo
tests provide less than that and there is little that achieves much beyond that)
implies a low positive predictive value, and many people will not live long
enough to develop dementia. Thus, there is a very real danger of worrying and
thus reducing the quality of life of a significant number of healthy individuals.
This in our opinion should lead to a policy of not providing any new diagnostic
procedures to individuals who are at a risk that is so low as to imply that testing
would lead to more false than correct positive findings. Most recommendations
of professional organizations agree that the predictive value of APOE testing or
other diagnostic procedures at an asymptomatic stage in subjects who do not
have a clear family history of early-onset AD is too low to justify the burdens of
testing (32,33).

MONITORING TREATMENT
The traditional approach for assessment of treatment outcome involves clinical
endpoints that are directly related to symptoms of the disease under investigation,
and this is the approach required by drug licensing agencies, such as the United
States Food and Drug Administration, for approval of drugs. For dementia, these
endpoints are usually based on clinical ratings and neuropsychological test scores
to demonstrate an improvement of symptoms (34). It is much more difficult to
prove a reduction in progression of the underlying disease by clinical trials, but
438 Herholz et al.

that could perhaps be achieved by the use of a biomarker that is closely linked to
disease pathophysiology (35,36).
In a recent trial with Ab immunization that was stopped because some
patients developed meningoencephalitis, a decline in CSF tau was observed in
antibody responders compared to placebo controls (37). In a trial of the cholesterol-
lowering drug simvastatin, CSF alpha-sAPP and CSF beta-sAPP were
significantly reduced, but the CSF levels of tau, phosphorylated tau (p-tau),
and Ab (42) and the plasma levels of Ab (42) were unchanged after 12 weeks of
treatment (38). Thus, molecular analysis of CSF appears useful in order to obtain
some insight into possible mechanisms of drug action, but because of the invasive
nature of CSF sampling and a lack of a clear association with clinical benefits, it is
unlikely to be useful as a surrogate marker to determine drug efficacy.
Imaging proposals have been put forward for measurement of atrophy
progression by MRI (39), although it is still difficult to achieve consistency of
measurements across scanners and centres. Nutrition and hydration status may
also have an influence on brain volume (40), but they are unlikely to influence
specifically local mesial temporal atrophy that is typical of AD. Feasibility of
quantitative PET analysis has been demonstrated in multi-center studies (41,42).
Tools for efficient quantitative regional image analysis have been developed for
multi-center trials that are now underway (43).
Measurement of the progression of hippocampal atrophy by MRI appears
to have some face validity as a marker of degenerative disease progression and is
beginning to be used in drug trials (44). Analysis of the recent immunization trial,
however, has suggested that under certain circumstances there may be a
dissociation between clinical scores and progression of atrophy, e.g., if volume
changes were due to amyloid removal and associated cerebral fluid shifts (45).
Measurement of functional changes in the brain in therapeutic trials have
been accomplished with SPECT (46–48) and PET (49) (see Chapter 8 for further
citations), and also more recently with magnetic resonance spectroscopy (MRS)
(44,50) and functional magnetic resonance imaging (fMRI) (51). In these studies,
there was generally a good correlation between clinical response and an increase
in regional blood flow and glucose metabolism. Such data suggests that
functional imaging techniques have the potential to be used as a surrogate
endpoint in clinical trials although a number of limitations such as the technical
variability across centers and a clear definition of reproducible functional
conditions during studies still applies (35).
Molecular tracers also permit measurement of specific drug effects. The
degree of acetylcholinesterase (AChE) inhibition in the brain of AD patients has
been measured by PET (52,53), demonstrating that cognitive effects are related to
local drug activity which varies among patients and may not be very well
predicted by preclinical studies (54). Amyloid imaging tracers are likely to
provide the most direct in vivo evidence of whether new drugs are actually able to
reduce Ab deposition in the brain and whether that is related to clinical benefits
(see Chapter 10). In addition, drug companies are increasingly likely to study
Guideline and Perspective 439

human regional pharmacokinetics and receptor binding using PET and SPECT at
the preclinical and early clinical phase of drug development (55).

CONCLUSION
A large number of new tools have been developed in recent years for the
improved early diagnosis of AD. New neuropsychological screening tools
facilitate diagnosis of MCI as a condition with substantially increased risk for
developing AD. Functional imaging methods and MRI volumetry have
demonstrated usefulness in identifying subgroups of MCI patients with a very
high likelihood of developing AD within a few years. Molecular CSF tests and
imaging techniques even hold the promise for the early distinction of AD from
other disorders that lead to dementia prior to its onset, and new molecular
techniques for identifying new diagnostic targets may provide improved
methods of detection. Genetic counselling is recommended in early-onset
familial AD, where specific monogenetic mutations may play a causative role.
In the future we should see integration of these new possibilities into longi-
tudinal and therapeutic intervention studies that should lead to more effective
development of drugs to slow progression or even prevent development of
dementia.

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Index

2D-PAGE. See two-dimensional Age effects


polyacrylamide gel Alzheimer’s disease, 195–198, 230
electrophoresis amyloid deposition, 296
3D-FA. See three-dimensional fractal neurotransmitter systems, 251–253
analysis PIB retention, 286
3D-SSP. See three-dimensional Age related cognitive decline (ARCD),
stereotactic surface projection 212
3NT. See 3-nitrotyrosine Age-Related White Matter Changes
5-HT. See serotonin (ARWMC) scale, 160–161
14-3-3 protein, 87 Aggressive behavior, 53
Alpha-secretase, 111
AADL. See Advanced Activities of Daily Alpha-synuclein, 124–125
Living scale Alzheimer’s disease (AD)
Aß. See beta-amyloid 2D-PAGE analysis, 141
Aß40. See amyloid ß peptide, 40 apraxia, 42
Aß42. See amyloid ß peptide, 42 biomarkers, 71–105
Acetylcholine, 253 biopsy/autopsy findings
See also cholinergic system comparison, 360
Acetylcholinesterase (AChE), cerebral amyloid angiopathy, 360–362
260–261 cholinergic system, 356–359
ACT. See antichymotrypsin cohort studies, 200–217
Activities of Daily Living (ADL) scale, depression, 51–52
45 diagnosis, 6, 20–21, 33, 57, 232–234
AD. See Alzheimer’s disease early olfactory involvement, 359
ADAS. See Alzheimer’s Disease early pathology and clinical
Assessment Scale correlates, 347–362
ADDLs. See amyloid ß-derived early symptom recognition, 6, 232–234
diffusable ligands FDG PET imaging, 227–234, 237
ADL. See Activities of Daily fluid analysis, 143
Living scale functional MRI, 178–183
Advanced Activities of Daily Living gene expression profiling, 136
(AADL) scale, 45 genetics, 107, 108, 113–120
AGD. See argyrophilic grain disease hippocampus, 353–356

443
444 Index

[Alzheimer’s disease (AD)] Amyloid precursor protein (APP), 75,


language impairments, 42 108–112, 310–317
MCI conversion, 47 Anatomic MRI sequence, 154
microstructural imaging, 175–178 Animal models, 136–137, 313–314
morphological changes and relation to Anosmia, 359
biomarkers, 362–368 Anosognosia, 3, 33
multiple domain cognitive deficits, 15 Anti-amyloid drugs, 297–298
natural history, 297 Antibodies, 145–146
neuropathology, 276 Antichymotrypsin (ACT), 81–82
neuropsychological assessment, Anti-depressive drugs, 141
47–48 Apolipoprotein E (Apo E)
neuroreceptor studies, 254–256 2D-PAGE analysis, 141
neurotransmitter systems, 253–262 AD/AD prediction, 115–119, 432–433
pathology, 308–329, 347–362 biomarkers, 80
pathophysiology, 277 MCI and early AD, 357
PIB patterns of retention, 292–293 PET comparison, 233–234
presentation and course, 8 APP. See amyloid precursor protein
quantitative MRI tools, 168–169 Apraxia, 42
short-term memory, 39 ARCD. See age related cognitive decline
SPECT, 195–200 Argyrophilic grain disease (AGD), 339
therapeutic targets, 277–278 Arterial spin labelling (ASL), 156
vascular pathology, 22–23 ARWMC. See Age-Related White Matter
visual MTA assessment, 167–168 Changes scale
visuospatial impairments, 42–43 ASL. See arterial spin labelling
voxel-based morphometry, 165 Asymmetric atrophy, 235
Alzheimer’s Disease Assessment Scale Asymptomatic subjects, 432–433
(ADAS), 37–38 Attention assessment, 41
Amyloid ß. See beta-amyloid Autopsy, 360
Amyloid ß-derived diffusable ligands See also postmortem examinations
(ADDLs), 314–315 Autosomal dominant AD, 48, 297
Amyloid ß peptide, 40 (Aß40) 72–75
Amyloid ß peptide, 42 (Aß42) 72–75, BACE1/BACE2. See beta-site APP
87–88, 89 cleaving enzyme
Amyloid cascade theory, 112, 119–120 Basic Activities of Daily Living (BADL)
Amyloid deposition scale, 45
AD, 276 Batteries, neuropsychological tests,
animal models, 313–314 36–38
drug trials, 298 BBB. See blood–brain barrier
natural history, 294–296 BBSI. See brain boundary shift integral
therapeutic target in AD, 277–278 technique
Amyloid imaging agents Bedford Alzheimer Assessment
AD, 275–305 Nursing Scale, 45
development of technologies, Behavioral disturbance, 16, 43–44,
278–293 49–50, 57–58
drug trials, 297–298, 434 Benzodiazepine receptor
future applications, 293–298 (BZR), 261–262
studies, 280–288 Benzothiazole-anilines (BTAs), 279
Index 445

Beta-amyloid (Aß) 11C-nicotine, 259


AD, 277, 310–323 C1q, 83–84
cerebrospinal fluid, 72–75, 87–88 CAA. See cerebral amyloid angiopathy
genetics, 110, 112–113, 119–120 CADL. See Communicative Activities
therapeutic target in AD, 277–278 of Daily Living scale
in vivo quantitation, 278 California Verbal Learning
Beta-amyloid antibodies, 87–88 Test (CVLT), 40
Beta-amyloid imaging Cambridge Examination for Mental
agents, 275–305 Disorders of the Elderly
Beta-secretase, 111–112 (CAMDEX), 36–37
b-secreted APP (b-APP), 75 Cancellation tests, 41
Beta-site APP cleaving enzyme Caspase cleavage, 321–322
(BACE1/BACE2), 111–112 Category Cued Recall (CCR) test, 41
Beta-synuclein, 125–126 CBF. See cerebral blood flow
Bioinformatics, 136 CCR. See Category Cued Recall test
Biomarkers CDR. See Clinical Dementia
AD, 363–365 Rating Scale
blood and cerebrospinal fluid, 71–105 Cell death, 316–317
FTD, 367 Central benzodiazepine receptor
morphological changes, 362–368 imaging, 261–262
PDD and DLB, 366 Central nervous system (CNS), 261
VaD, 367–368 Centrifugation methods, 138
Blessed–Roth test, 35 CERAD. See Consortium to Establish a
Block design, fMRI, 157 Registry for Alzheimer’s Disease
Blood biomarkers, 71–105 Cerebellar cortex, 283, 285, 286
Blood–brain barrier (BBB), 100, 261, Cerebral amyloid angiopathy (CAA),
279, 311, 315, 361 346, 360–362
Blood flow tracers, 194–197 Cerebral blood flow (CBF), 231
Blood Oxygenation Level Dependent Cerebral cortex impairments, 23
(BOLD) signal, 155–157 Cerebral glucose metabolism, 225–247
Braak staging, 330–332 Cerebrospinal fluid (CSF), 71–105, 365
Brain biochemistry, 135, 156 See also spinal fluid examination
Brain biopsy, 19, 360 Cerebrovascular disease (CVD),
Brain boundary shift integral (BBSI) 265–266, 339–340
technique, 165–166 Cerebrovascular lesions (CVLs),
Brain imaging 344–347
ß-amyloid in AD, 275–305 CG. See Chrysamine G
cerebral glucose metabolism, 225–247 ChAT. See choline acetyltransferase
neurotransmitter systems, 249–273 Cholesterol metabolism, 85–86
new research approaches, 57 Choline acetyltransferase (ChAT)
treatment decisions, 23–24 Alzheimer’s disease, 260–261
See also functional brain imaging; MCI and early AD, 357–359
magnetic resonance imaging; normal aging, 253
structural brain imaging Cholinergic system
Broca’s aphasia, 5 Alzheimer’s disease, 258–261
BTAs. See benzothiazole-anilines DLB, 262–263
BZR. See benzodiazepine receptor MCI and early AD, 356–359
446 Index

Chromosome, 17, 337 Compartmental modelling, PIB, 289


Chrysamine G (CG), 278–279 Computational neuroanatomy, 162–164
CIND. See cognitive impairment, not Concurrent validity, neuropsychological
demented tests, 32
CJD. See Creutzfeldt–Jakob disease Consortium to Establish a Registry for
Classification of dementia disorders, Alzheimer’s Disease (CERAD),
308–310 37, 325
Clinical criteria, correlation with Construct validity, neuropsychological
pathological criteria, 368–369 tests, 32
Clinical Dementia Rating Scale (CDR), Controls, PIB, 283, 290, 292–293
33–35 Cortex
Clinical diagnosis GABAA receptor, 265–266
FDG PET in AD, 234 Pittsburgh compound-B, 283–287
neuropsychological assessment, 33 receptor binding, 257–258
See also diagnosis; diagnostic criteria Cortical pattern matching, 165–166
Clinical Insight Rating Scale, 44 Corticobasal degeneration, 15
Clinical issues, 1–30 Counselling, 432
crossroads and decision C-reactive protein (CRP), 83
points, 13–24 Creutzfeldt–Jakob disease (CJD), 19, 77,
dementia evaluation, 2–13 172, 237
Clinical trials, 24, 166, 231–232, Cross-sectional methods, 163–165
297–298 CRP. See C-reactive protein
Clock drawing test, 35–36, 51 [11C]SB-13, 282–283
CNS. See central nervous system CSF. See cerebrospinal fluid
Cognitive impairment CVD. See cerebrovascular disease
AD prediction, 47 CVLT. See California Verbal Learning
amyloid deposition, 295–296 Test
cerebrovascular disease, 265–266 Cytoskeletal lesions, 318–320
determination of cause, 6–11
development relative to Dardarin gene, 126
motor symptoms, 22 Decision points in diagnosis,
prodromal dementia identification, 3, 5, 13–24
14–15 Default network, functional MRI studies,
psychiatric illness, 16–17 179, 183
recognizing, 3–4 Delayed recall tests, 39–40
single and multiple domain, 15 Delirium, 17
VaD, 341 Dementia, causes of
Cognitive impairment, not demented clinical evaluation, 6–11
(CIND), 5–6, 14–15, 428–432 rapidly progressive dementia, 18–20
Cognitive profiles, 56 Dementia lacking distinctive
Cohort studies, 200–217 histopathology (DLDH), 336
Collateral sources, 4 Dementia with Lewy bodies (DLB)
Combined imaging methods, 156, 178 amyloid imaging, 294
Communicative Activities of Daily biomarkers, 73, 77, 79
Living (CADL) scale, 45–46 differential diagnosis, 21, 51, 329–333
Comorbidity, 17–18, 369 FDG PET imaging, 234
Comparative proteomic methods, 139 genetics, 124–126
Index 447

[Dementia with Lewy bodies (DLB)] [Differential diagnosis]


morphological changes and relation to AD and normal aging, 429
biomarkers, 366 DLB, 21, 51, 329–333
neurotransmitter systems, 262–264 FTD, 56–57
presentation and course, 8 FTLD, 49–51
quantitative MRI tools, 170–171 neuropsychological
SPECT, 210, 212, 213 assessment, 48–52
Dementia-free survival, 431 normal aging, 47–48
DemTect, 36 VaD, 48–49
Deoxyhemoglobin, 156 Diffusion tensor imaging, 176–178
Depression, 4, 16–17, 44, DIGE. See fluorescence difference gel
51–53, 257–258 electrophoresis
DFA. See discriminant function Digit span test, 39
analysis Discriminant function analysis (DFA),
Diagnosis 200, 228–229
AD versus VaD with follow-up Disease progression, 52–53
confirmation, 206 DLB. See dementia with Lewy bodies
amyloid imaging, 293–294 DLDH. See dementia lacking
crossroads and decision distinctive histopathology
points, 13–24 Dopaminergic transmitter system
guidelines and perspectives, 425–438 AD, 253, 256
asymptomatic subjects, 432–433 DLB, 263, 264
cognitive impairment, not demented, normal aging, 251, 252
428–432 Double Memory Test (DMT), 40–41
diagnosis of mild manifest dementia, Down syndrome (DS), 276, 293
426–428 DS. See Down syndrome
importance, 2 Dye labelling techniques, 140
new technologies, 12–14
novel diagnostic targets, 133–152 EADC. See European Alzheimer’s
quantitative MRI tools, 166–175 Disease Consortium
SPECT, 198 Early diagnosis
See also clinical diagnosis; AD, 232–234
differential diagnosis; amyloid imaging, 293–294
early diagnosis; neuropsychological assessment, 46–48
evaluation of dementia Early onset dementia
Diagnostic criteria AD versus VaD, 200, 206
AD neuropathology, 323–329 amyloid ß 277
DLB neuropathology, 333–334 autosomal dominant familial AD, 297
MCI, 429–430 FTD versus AD, 207, 210
usefulness, 7 genetic analysis, 113–114
Diagnostic technology, misuses, 12–13 ECD. See diethyl ester
Diagnostic validity, neuropsychological Electroencephalography (EEG), 19, 156
tests, 32 Emotions, and event-related fMRI, 157,
Diethyl ester (ECD), 194–195 159
Differential diagnosis Encoding specificity, 40
AD, 20–21, 33, 47, 57 Endosome abnormalities, 312–313
AD and depression, 51–52 Episodic memory, 39, 48
448 Index

Ethical issues, 426, 432 fMRI. See functional magnetic


European Alzheimer’s Disease resonance imaging
Consortium (EADC), 159 Focal CVD, 340
Evaluation of dementia Folate, 84–85
confirming presence of Follow-up confirmation, AD versus VaD
dementia, 4–6 diagnosis, 206
determining cause of, 6–11 Fractionation methods, 138–139
rationale for early recognition and Free-recall test, 39
evaluation, 11–12 Freed and Cued Selective Reminding
recognizing cognitive impairment, 3–4 Test (FCSRT), 40
role of evaluation, 2–3 Frontotemporal dementia (FTD)
See also diagnosis behavioral/personality disturbance, 16,
Event-related design, fMRI, 157, 159 57–58
Evidence-based medicine, 13 differential diagnosis, 20–21, 56–57
Executive functions, 41 early onset, 207, 210
Extrapyramidal symptoms, 264–265 early symptom recognition, 6
FDG PET imaging, 234–236
18
genetics, 121–123
F-2-fluoro-2-deoxy-D-glucose. HMPAO-SPECT classification
See FDG studies, 211
Familial Alzheimer’s disease (FAD), morphological changes and
108–110, 297, 322–323 relation to biomarkers, 367
See also genetics neuropathology, 334–338
Familial tauopathies, 336–338 neurotransmitter systems, 263–265
Family histories, 114 Parkinson’s disease, 22
FCSRT. See Freed and Cued Selective presentation and course, 8, 56
Reminding Test recent studies, 210
18
[ F]FDDNP, amyloid imaging, 281–282 single domain cognitive deficits, 15
FDG (18F-2-fluoro-2-deoxy-D-glucose), SPECT perfusion, 207
225–247 Frontotemporal lobar degeneration
Fibroblast analysis, 137 (FTLD), 49–51, 169–170
First dimension isoelectric FTD. See frontotemporal dementia
focusing, 139 FTLD. See frontotemporal lobar
FLAIR. See fluid attenuated inversion degeneration
recovery Functional brain imaging, 15
Floor effects, neuropsychological Functional genomics, 134
tests, 52 Functional impairment, 4–5, 44–46
Fluid analysis, 143–146 Functional magnetic resonance imaging
mass spectrometry, 144 (fMRI), 155–159, 178–183,
matrix laser assisted desorption 363–364
ionization, 144–145 Future trends
protein microarrays, 145–146 neuropsychological assessment, 54–58
Fluid attenuated inversion prevalence of dementia, 1
recovery (FLAIR), 154–155
Fluorescence difference gel GABA receptor R1 protein
electrophoresis (DIGE), 140 (GBR1), 355–356
Fluorescent staining, 140 GABAA, 265–266
Index 449

Gamma-secretase, 111, 112 Huntington’s disease, 430


GBR1. See GABA receptor R1 protein Hydrogen atoms, magnetic resonance
GDS. See Global Deterioration Scale imaging, 153
Gender effects Hyperphosphorylated tau, 318–319
Alzheimer’s disease, 198 Hypertension, 346
neurotransmitter systems, 251
Gene expression profiling, 134–138, 356 IADL. See Instrumental Activities of
Gene–gene interaction, 120 Daily Living scale
General practitioners, 429–430 ICAT. See isotope coded
Genetics, 13, 107–131, 134–138, affinity tagging
356, 432 IDE. See insulin degrading enzyme
Geriatric major depression. See major IL-6. See interleukin-6
depression Imaging. See brain imaging
Global Deterioration Scale (GDS), 33 Immunization, 120, 277–278
Glutamatergic NMDA receptor Instrumental Activities of
complex, 252 Daily Living (IADL) scale, 45
Insulin degrading enzyme
Hallucinations, 171 (IDE), 118, 119
HDDD2. See hereditary dysphasia Intelligence quotient (IQ), 33
disinhibition dementia Intelligence tests, 33–34
Hearing loss, 4 Interleukin-6 (IL-6), 82–83
Hemorrhages, 361 Interpersonal Adjectives Scales, 58
Hereditary dysphasia disinhibition Intravenous immuoglobulin
dementia (HDDD2), 336 preparations (IVIG), 88
Heterogeneity of perfusion, 206 Intronic mutations, TAU, 121, 122
Hexamethyl-propylene amine oxime IQ. See intelligence quotient
(HMPAO) Isoelectric focusing, 139
AD and VaD comparison studies, Isotope coded affinity tagging (ICAT),
208–209 141–142
AD/DLB classification studies, 213 Isprostanes, 81
AD/FTD classification IVIG. See intravenous immuoglobulin
studies, 211 preparations
blood flow tracers, 194–197
Hierarchic Dementia Scale, 54
Hippocampal sclerosis Laboratory tests, 7, 9–10, 14
dementia (HSD), 338 Lacunes, 161
Hippocampus Language impairment
receptor binding, 257–258 AD, 42
role in early AD, 353–356 FTLD, 50
volume in MCI, 174–175 Primary Progressive Aphasia, 50–51
HMPAO. See hexamethyl-propylene psychometric assessment, 42
amine oxime semantic dementia, 51
Hold–Don’t Hold deterioration Large vessel dementia (LVD), 340
quotient, 33–34 Laser capture microdissection,
Homocysteine, 84–85 135–136
HSD. See hippocampal sclerosis Late onset dementia, 206
dementia LBs. See Lewy bodies
450 Index

LBV/AD. See Lewy body Mass spectrometry (MS), 144


variant of AD Matrix assisted laser desorption
Lewy bodies (LBs), 124–125, 330–334, ionization (MALDI), 144–145
359, 369 Mattis Dementia Rating Scale, 35
See also dementia with Lewy bodies MCI. See mild cognitive impairment
Lewy body variant of AD (LBV/AD), MCI-AD. See mild cognitive
326 impairment at risk for AD
Linear amplification methods, 135–136 MD. See major depression
Lipid rafts, 311 Medical history, 4, 7, 33
Lipoprotein receptor-related protein Medication, 54
(LRP), 119 Medication-induced cognitive
LOAD cases, 117–119 impairment, 17–18
Local cerebral glucose Membrane protein analysis, 141–142
metabolism, 226–227 Memory impairment
Logan graphical analysis, 289–291 dementia evaluation, 5
Long-term memory, 39–41 differential diagnoses, 47, 50, 429
Longitudinal studies, 231–232, 276, neurotransmitter systems, 253
427–428 psychometric assessment, 38–41
LRP. See lipoprotein receptor-related SPECT studies, 214–215
protein Mental status assessment, 5
LVD. See large vessel dementia Metabolic impairment, 225–247
Lymphocyte analysis, 137 Microarray based gene
expression profiling, 134–138
mAChR. See muscarinic receptors Microstructural imaging, 175–178
Magnetic dipole moment Mild cognitive impairment (MCI)
(MDM), 153–155 amyloid deposition, 295
Magnetic resonance imaging (MRI), APOE E4 allele, 115–116
153–191 biomarkers, 72, 73, 76–77
amyloid imaging, 280–288 brain morphology, 349–353
computational neuroanatomy, cholinergic system, 356–359
162–164 conversion to AD, 294, 429
cortical pattern matching, 165–166 differential diagnosis, 33
diffusion tensor imaging, 176–178 early diagnosis of AD, 232–234
functional MRI, 155–159, 178–183, limitations of current criteria, 55
363–364 microarray based gene expression
magnetization transfer imaging, profiling, 138
177–178 multiple domain cognitive deficits, 15
microstructural imaging, 175–178 neuropsychological assessment, 47
structural MRI, 153–155 Pittsburgh compound-B, 289–290,
visual rating scales, 160–161 292–293
volumetry, 161–162 presentation and course, 8
voxel-based morphometry, 164–165 prodromal dementia
Magnetization transfer imaging, identification, 14–15
177–178 recognizing, 5–6
Major depression (MD), 77–78 SPECT, 212, 214–217
MALDI. See matrix assisted structural brain imaging, 174–175,
laser desorption ionization 178–183
Index 451

Mild cognitive impairment at risk for Natural history, 294–297


AD (MCI-AD), 431–432 Nerve growth factor (NGF), 356–357
Mild manifest dementia, 426–428 Neural networks (NN), 199
Mini Mental Status Examination Neurodegenerative diseases, 17–18, 359
(MMSE), 32, 34, 281, 283, 285 Neurofibrillary degeneration, 122
Missense mutations, tau protein Neurofibrillary tangles (NFTs)
gene, 122 AD, 177, 276
Mixed dementias, 22–23, 343–347 early AD, 353–356
See also multiple pathologies MCI, 349–353
MMSE. See Mini Mental Status normal aging, 348
Examination tau pathology, 318, 322–323
MoCA. See Montreal Cognitive Neurological examination, 7
Assessment Neuron loss, 310
Molecular imaging, 251 Neuropathology, 307–423
Monitoring treatment, 433–434 AD, 276, 308–329, 347–362, 369–370
Monoclonal antibody fragments, biomarkers and early morphological
280–283 changes, 362–368
Montreal Cognitive Assessment clinical/pathological criteria
(MoCA), 36 correlation, 368–369
Morphological changes DLB versus PDD, 329–333
AD, 363–365 FTD, 334–338
FTD, 367 mixed dementias, 343–347
PDD and DLB, 366 Neuropil threads (NTs), 318, 351
relation to biomarkers, 362–368 Neuropsychiatric Inventory (NPI), 43
VaD, 367–368 Neuropsychological assessment, 31–69
Morphological differential advanced tests and future research
diagnosis, 327–328, 330, 341 approaches, 54–58
Motor symptoms, 22 attention, 41
MP-RAGE technique, 161 batteries, 36–38
MRI. See magnetic resonance clinical assessment, 33
imaging differential diagnosis, 48–52
MS. See mass spectrometry early screening/diagnosis, 46–48, 427,
99MTc, 194–197 429
Multidimensional protein identification evaluation of disease progression,
technology (MudPIT), 142–143 52–53
Multifocal CVD, 340 executive functions, 41
Multiinfarct dementia, 55 intelligence tests, 33–34
Multiple domain cognitive deficits, 15 language, 42
Multiple pathologies, 9–10 memory, 38–41
See also mixed dementias monitoring treatment effects, 54
Muscarinic receptors (mAChR), 258, praxis, 42
259–260 psychological and behavioral
Myelographic MRI sequence, 154 disturbances, 43–44
psychometric adequacy, 32–33
nAChR. See nicotinic receptors screening tests, 34–36
National Institute of Aging severe dementia, 53–54
(NIA), 325–326 visuospatial abilities, 42–43
452 Index

Neuroreceptor studies, 254–256 Peptide mass fingerprint analysis, 144


See also receptor binding Perfusion SPECT, 193–224
Neurotransmitter systems, 249–273 Peripheral benzodiazepine
New diagnostic technologies, 12–14 receptor imaging, 262
NFT. See neurofibrillary tangles Peripheral tissue analysis, 137
NGF. See nerve growth factor Personality alterations, 57–58
NIA. See National Institute of Aging PET. See positron emission
Nicotinic receptors (nAChR), tomography
258–259, 263 Phosphorylated tau (p-tau), 78–80, 89,
3-Nitrotyrosine (3NT), 86–87 318–319
NN. See neural networks Physical abnormalities, 7, 9
Normal aging PIB. See Pittsburgh compound-B
differential diagnosis, 47–48 Pick’s disease (PiD), 336
neuropathology, 347–348 Pittsburgh compound-B (PIB)
neurotransmitter systems, 251–253 amyloid imaging, 279, 282,
Normative data, 32–33 283–293
NPI. See Neuropsychiatric Inventory methodology development, 288–292
NTs. See neuropil threads modelling, 289
patterns of retention in AD, MCI, and
Occam’s Razor, 9 controls, 292–293
Olfactory dysfunction, 359 Plaque-predominant AD, 326
Oligomers, 112, 314 Plasma
Oxyhemoglobin, 155–156 amyloid ß 74–75
Oxysterols, 85–86 biomarkers, 365
matrix assisted laser desorption
P-tau. See phosphorylated tau ionization, 144
PAL test, 52 PLAU. See urokinase-plasminogen
Parkinson’s disease (PD), 229–230, 251, activator
263, 264 Positron emission tomography (PET),
Parkinson’s disease with 225–226
dementia (PDD) amyloid imaging, 279–280, 288–292
differential diagnosis, 329–333 benzodiazepine receptor imaging,
distinguishing causes, 21–22 261–262
11
Lewy bodies, 124 C-nicotine, 259
morphological changes and cerebral glucose metabolism,
relation to biomarkers, 366 225–247
presentation and course, 8 combined methods, 156
quantitative MRI tools, 171 early morphological changes in AD,
Partial volume correction, 200 364
Pathological criteria, 368–369 FDG, 225–247
Pathological MRI sequence, 154 neuroreceptor studies in AD,
Pathophysiology 254–256
Alzheimer’s disease, 277 neurotransmitter systems, 249–250
VaD, 342 PIB, 288–292
PD. See Parkinson’s disease Postmortem examinations
PDD. See Parkinson’s disease with amyloid deposition, 295–296
dementia causes of dementia, 10
Index 453

[Postmortem examinations] Relaxation times, body tissue, 154


early stage dementia, 308 Reliability, neuropsychological
MudPIT technique, 143 tests, 32
neurotransmitter systems, 251, RNA, 135–137
253, 256 RRT. See relative residence time
RNA quality, 137
Preclinical development, 278–280 Schizophrenia, 16
Predictive testing, 114 Screening, 4, 34
Presenilins, 109–115 Seizures, 18
Prevalence of dementia, SELDI. See surface-enhanced laser
future trends, 1 desorption/ionization
Primary progressive (non-fluent) Semantic dementia, 51, 236
aphasia, 50–51, 236 Semi-quantitative real-time PCR
Prodromal dementia, 5–6, 14–15 (Q-RT-PCR), 136
Prospective methods Senile plaques (SPs)
atrophy assessment, 165–166 AD, 177
combined imaging modalities, 178 MCI, 349–350
computational neuroanatomy, 163 normal aging, 347–348
Protein microarrays, 145–146
Sensitivity, neuropsychological
Proteomics, 138–143
tests, 32
2D-PAGE technique, 139–141
Serotonergic transmitter system
isotope coded affinity tagging,
Alzheimer’s disease, 256–258
141–142
FTD, 264
MudPIT technique, 142–143
normal aging, 251
Proton density MRI sequence, 155
Serotonin (5-HT), 256–258
Pseudodementias, 167
Seven minute test, 36
Psychiatric disorder, 16–17, 43–44
Severe dementia, 53–54
Pulvinar sign, CJD, 172
Severe Impairment Battery (SIB), 54
Q-RT-PCR. See semi-quantitative Short Mental Status Test, 34
real-time PCR Short-term memory (STM), 38–39
Quantitative analyses, Pittsburgh SIB. See Severe Impairment Battery
compound-B, 288–291 sIL-6RC. See soluble interleukin-6-
Quantitative MRI, 159–175 receptor-complex
Silver staining, 140
Radiolabeled monoclonal antibody Simplified analysis method, 291–292
fragments, 280–283 Single domain cognitive deficits, 15
Radioligands, 249–250 Single photon emission computed
Rapidly progressive dementia, 18–20 tomography (SPECT), 193–195
Receptor binding amyloid imaging, 279–280
cholinergic transmitter system, benzodiazepine receptor imaging,
258–261 261–262
normal aging, 251–253 blood flow tracers, 194–197
serotonergic transmitter cohort studies, 200–217
system, 257–258 neuroreceptor studies, 254–256
See also neuroreceptor studies neurotransmitter systems, 249
Regional atrophy, 158, 159–166 perfusion, 193–224
Relative residence time (RRT), 281 PET comparison, 231
454 Index

Small vessel dementia (SVD), 340 Tau proteins, 76–80, 89


Soluble interleukin-6-receptor-complex Test-retest variability,
(sIL-6RC), 82–83 PIB analysis, 292
Specificity Theory of mind, 58
encoding, 40 Therapeutic targets, amyloid ß
neuropsychological tests, 32 deposition, 277–278
SPECT. See single photon emission Three-dimensional fractal analysis
computed tomography (3D-FA), 199
SPGR technique, 161 Three-dimensional stereotactic surface
Spin-lattice relaxation times, 154 projection (3D-SSP), 199
Spin-spin relaxation times, 154 Thresholding, white matter
Spinal fluid examination, 18–19 hyperintensities, 161–162
See also cerebrospinal fluid Tissue-to-plasma ratios, PIB, 289
SPM. See statistical parameter mapping TMT. See Trail Making Test
Staining techniques, 140 Total tau (t-tau), 76–78, 89
Statistical parameter mapping (SPM), Trail Making Test (TMT), 41
198–199 Transgenic models, 136–137,
STM. See short-term memory 313–314
Striatum, 284–285 Transmitters. See neurotransmitter
Stroke, 5, 10 systems
Stroop test, 41 Treatment
Structural brain imaging, 7, 9, 11, 21,
identification of novel therapeutic
153–155
targets, 133–152
Subcortical cerebrovascular
monitoring, 433–434
disease, 158, 159–166
selection and monitoring, 23–24
Subcortical dementia, 55
See also medication
Subjective MTL atrophy score, 160
Triggers, dementia evaluations, 3
Substance abuse, 16
Two-dimensional polyacrylamide
Surface-enhanced laser
gel electrophoresis (2D-PAGE),
desorption/ionization
139–141
(SELDI), 144
SVD. See small vessel dementia
Symptoms, differentiation across Ubiquitin (Ub), FTD, 335
syndromes, 6 Unfolded protein response (UPR),
Synaptic alterations, 356 315
Systematic review Urokinase-plasminogen activator
AD versus FTD, 210 (PLAU), 118–119
AD versus VaD, 207
VaD. See vascular dementia
T-tau. See total tau VaD–VCI. See vascular-ischemic
T1/T2 relaxation times, 154 dementia–vascular cognitive
Tangle-predominant AD, 326 impairment
TAU gene, 121–123 Validity, neuropsychological
Tau pathology tests, 32
AD, 318–323 Vascular dementia (VaD)
DLB versus PDD, 333 amyloid imaging, 294
hippocampus, 353 cohort studies, 200, 206–209
olfactory bulb, 359 differential diagnosis, 48–49
Index 455

[Vascular dementia (VaD)] Voxel-based morphometry, 164–165, 175


FDG PET imaging, 236–237 Voxel compression maps, 166
morphological changes and relation
to biomarkers, 367–368 WAIS. See Weschler Adult Intelligence
pathophysiological Scale
classification, 342 Washington University diagnostic
presentation and course, 8 criteria, 325
quantitative MRI tools, 172–174 Water-soluble Aß (wsAß), 311
recognizing, 55 WCST. See Wisconsin Card Sorting Test
Vascular-ischemic dementia–vascular Weschler Adult Intelligence Scale
cognitive impairment (WAIS), 33–34
(VaD–VCI), 339–343 White matter hyperintensities
Vascular pathology, 22–3 (WMHs), 161–162
Verbal fluency tests, 41 White matter lesions (WMLs), 343,
Visual problems, 4 346–347, 368
Visual rating scales, Wisconsin Card Sorting
MRI findings, 160–161 Test (WCST), 41
Visuospatial abilities, 39, 42–43 WMHs. See white matter
Vitamin B group, 84 hyperintensities
Voiced complaints, 6 Working memory, 38–39
Volumetric measures, MRI findings,
161–162 Xe, 194
natively unfolded αSN monomers

oxidative stress, ligands, pH or ∆T


posttranslational modifications ligands
conformational changes posttranslational modifications
molecular crowding conformational changes

spherical protofibrils partially folded intermediates

fibrils chain and annular


protofibrils

vesicle disruption 'toxic' soluble normal functions


(neurotransmitters, lysosomes) αSN protein (chaperone, DA transmission,
golgi fragmentation complexes proteasome functions)

inclusions
('protective sinks' & 'delayed-toxicity')

fibrillar αSN non-fibrillar accumulation


(Lewy bodies) αSN aggregates of Ub+ –proteins

Figure 4.5 Molecular pathophysiology of a-synuclein (aSN) monomers converting into


partially folded, toxic, and/or aggregated forms. (See page 128.)

Figure 7.1 Tc99m HMPAO SPECT scan of a healthy volunteer (MMSEZ30).


(See page 200.)
Figure 7.2 Tc99m HMPAO SPECT scan of Alzheimer’s disease (MMSEZ22).
(See page 201.)

Figure 8.1 Typical


findings with FDG PET
in Alzheimer’s disease.
(See page 232.)
Figure 8.2 Automatic detection of abnormal metabolism, including correction for age.
(See page 233.)

Figure 8.3 Asymmetric frontotemporal atrophy and metabolic impairment in


frontotemporal dementia. (See page 239.)
Figure 8.4 Global reduction of cerebral glucose metabolism in vascular dementia.
(See page 241.)

Figure 9.1 Loss of dopamine transporters with age. (See page 256.)
Figure 9.3 Acetylcholinesterase activity in the central nervous system evaluated with
C11-MP4A. (See page 262.)

Figure 9.4 Dopamine transporter loss visualized with FP-CIT SPECT in the differential diagnosis
of dementia with Lewy bodies (DLB). (See page 267.)
Figure 9.5 Differences in cortical GABAA receptor density between patients with leuko-
araiosis and dementia (A) and patients with leukoaraiosis without dementia (B). (See page 269.)

Figure 10.2 Pittsburgh


compound-B (PIB) stan-
dard uptake value (SUV)
images demonstrate a
marked difference between
PIB retention in Alzhei-
mer’s disease (AD)
patients and HC subjects.
(See page 289.)
Figure 10.3 Serial planes demonstrate the topography of Pittsburgh compound-B (PIB)
retention. (See page 290.)

Figure 10.6 Examples of Pittsburgh compound-B (PIB) Logan distribution volume ratio
(DVR) images. (See page 295.)
Figure 11.1 Spreading pattern of (A) cytoskeletal/tau pathology and (B) of Ab
deposition. (See page 325.)

Figure 11.2 Relationship


between Mini-Mental State
Examination (MMSE) and
Braak neuritic Alzheimer’s
disease stages in 207 consecu-
tive autopsies of aged individ-
uals (mean age at death 81.4G
8.6 years). (See page 355.)

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