Molecular Aspects of Medicine 82 (2021) 101038

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Molecular Aspects of Medicine

journal homepage: www.elsevier.com/locate/mam

New insights into the role of autophagy in retinal and eye diseases
Beatriz Villarejo-Zori a, Juan Ignacio Jiménez-Loygorri a, Juan Zapata-Muñoz a, Katharina Bell b,
Patricia Boya a, *
a
Department of Cellular and Molecular Biology, Margarita Salas Center for Biological Research, CSIC, Ramiro de Maetzu, 9, 28040, Madrid, Spain
b
Singapore Eye Research Institute, Singapore National Eye Centre, Republic of Singapore

A R T I C L E I N F O A B S T R A C T

Keywords: Autophagy is a fundamental homeostatic pathway that mediates the degradation and recycling of intracellular
Autophagy components. It serves as a key quality control mechanism, especially in non-dividing cells such as neurons.
Retina Proteins, lipids, and even whole organelles are engulfed in autophagosomes and delivered to the lysosome for
Glaucoma
elimination. The retina is a light-sensitive tissue located in the back of the eye that detects and processes visual
Macular degeneration
images. Vision is a highly demanding process, making the eye one of the most metabolically active tissues in the
Retinal dystrophies
body and photoreceptors display glycolytic metabolism, even in the presence of oxygen. The retina and eye are
also exposed to other stressors that can impair their function, including genetic mutations and age-associated
changes. Autophagy, among other pathways, is therefore a key process for the preservation of retinal homeo­
stasis. Here, we review the roles of both canonical and non-canonical autophagy in normal retinal function. We
discuss the most recent studies investigating the participation of autophagy in eye diseases such as age-related
macular degeneration, glaucoma, and diabetic retinopathy and its role protecting photoreceptors in several
forms of retinal degeneration. Finally, we consider the therapeutic potential of strategies that target autophagy
pathways to treat prevalent retinal and eye diseases.

1. Introduction macroautophagy, referred to hereafter simply as “autophagy”.

Autophagy is primarily a cellular response to stress, and is classically
The word autophagy, derived from the Greek term "self-eating", re­ induced by a lack of nutrients, in particular amino acids. This process is
fers to the catabolic processes by which the cell degrades and recycles tightly regulated through signaling via the mTOR and AMPK pathways,
cellular components inside lysosomes (Galluzzi et al., 2017). There are the two main signaling routes responsible for monitoring the cell’s
three main types of autophagy, which differ according to how material nutritional status (Wong et al., 2013). Autophagy can also be induced by
destined for degradation is delivered to the lysosome (Fig. 1). In mac­ other forms of stress, including endoplasmic reticulum (ER) stress,
roautophagy, the cytoplasmic material is enveloped in a oxidative stress, hypoxia, and infections (Fig. 2). Although autophagy is
double-membrane structure that seals to form an organelle called the mainly regulated at the post-translational level, stress can also increase
autophagosome. The autophagosome subsequently fuses with the lyso­ the expression of autophagy genes (Feng et al., 2015). One of the main
some, where the cargo is degraded through the action of lysosomal regulators of these genes is transcription factor EB (TFEB). In resting
hydrolases (Fig. 1A). Chaperone-mediated autophagy (Fig. 1B), a conditions, TFEB is retained in the cytoplasm, but once phosphorylated
pathway described only in mammalian cells, selectively degrades pro­ it translocates to the nucleus where it activates the transcription of
teins expressing a specific amino acid sequence that is recognized by the lysosomal genes and autophagy-related genes (ATG) genes involved in
Hsc70 chaperone protein (Kaushik and Cuervo, 2018). In the third form the regulation of autophagy, including many lysosomal proteins (Puer­
of autophagy, known as microautophagy, the material to be degraded tollano et al., 2018). To date, more than 42 genes have been implicated
reaches the lysosome through invagination of the lysosomal or endo­ in the autophagy pathway and the list continues to grow (Mizushima,
somal membrane (Schuck, 2020). Microautophagy is less well known, 2020).
and the molecular regulators of this process are only beginning to be While autophagosome formation is a key step, regulation of auto­
described (Fig. 1C). In this review we will focus specifically on phagy is dependent on several other steps (Fig. 2). In the induction

* Corresponding author.
E-mail address: [email protected] (P. Boya).

https://doi.org/10.1016/j.mam.2021.101038
Received 8 June 2021; Received in revised form 12 September 2021; Accepted 27 September 2021
Available online 5 October 2021
0098-2997/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
B. Villarejo-Zori et al. Molecular Aspects of Medicine 82 (2021) 101038

phase, ATG1/ULK1 kinase forms a complex with ATG13, ATG101, and role of autophagy in promoting photoreceptor survival and discuss al­
FIP200 (also known as RB1CC1) in response to mTOR inhibition or terations in autophagy described in photoreceptor degeneration. The
activation of AMPK signaling. Next, the kinase activity of ULK1 triggers findings reviewed here underscore the important role of autophagy in
the formation of the phosphatidylinositol 3-kinase (PI3K) complex, maintaining proper retinal function and highlight novel therapeutic
which is comprised of VPS34, BECLIN1, VPS15, and VPS14 and favors approaches for the treatment of blindness and other diseases of the eye.
the formation of phosphatidylinositol 3-phosphate (PI3P) at phagophore
initiation sites (Mizushima, 2020). Next, two ATG7-catalyzed ubiq­ 2. The adult retina
uitin-type conjugation systems are activated for autophagosome for­
mation. The first mediates the conjugation of ATG12, ATG5, and The retina is a light-sensitive tissue in the vertebrate eye that detects
ATG16L, while the second promotes the conjugation of phosphatidyl­ and processes visual images. It senses light and creates impulses that are
ethanolamine (PE) to LC3 (light chain 3 protein) to form the transmitted to the brain via the optic nerve. Structurally, the retina
autophagosome-bound form of LC3 (the mammalian ortholog of ATG8), consists of multiple cell types arranged in layers (Fig. 3): specifically,
also called LC3-II. The continuous assembly of these protein-protein and three layers of neuronal cell bodies and two layers of synapses. Light-
protein-lipid complexes and the delivery of lipids to the autophagosome sensitive photoreceptors (rods and cones) comprise the outer nuclear
through ATG9, the only multimembrane-spanning protein in the layer (ONL) (Fig. 1). In the outer plexiform layer (OPL), these cells form
pathway, allows elongation of the autophagosome membrane (Miz­ connections with amacrine, horizontal, and bipolar cells, the nuclei of
ushima, 2020). The final stages of autophagy are controlled by mole­ which lie in the inner nuclear layer (INL). In the inner plexiform layer
cules that regulate maturation of the autophagosome, its fusion with (IPL) amacrine and bipolar cells synapse with the retinal ganglion cells
endosomes and lysosomes, and the degradation and recycling of me­ (RGCs). These cells are the only projecting neurons of the retina, and
tabolites within the lysosome (Yim and Mizushima, 2020). their axons form the optic nerve (Fig. 3). Other important cells of the
While autophagy was initially considered a non-selective process, retina are the retinal glia or Müller cells, which span the entire retina;
recent findings indicate that it is in fact highly selective. Selective astrocytes, which lie around the ganglion cell layer (GCL); and the
autophagy refers to the process whereby certain types of cargo (e.g. retinal pigment epithelium (RPE) cells, which lie immediately outside
organelles, misfolded proteins, intracellular pathogens) are specifically the neuroretina, in close contact with the photoreceptors. RPE cells
recognized and degraded (Johansen and Lamark, 2020). This process is provide trophic support to photoreceptors and mediate the recycling of
triggered by recognition of the cargo through the binding of receptors or photoreceptor outer segments (POS) (Centanin and Wittbrodt, 2014).
adapters containing LC3-interacting regions (LIRs) (Johansen and The retina has several unique features that make it an ideal window
Lamark, 2020). Selective autophagy can be classified into several types into the central nervous system (CNS), enabling the study of a range of
depending on the nature of the specific cargo. These include xenophagy neuronal processes, from development to neurodegeneration (Corro­
(degradation of pathogens), lipophagy (degradation of lipid droplets), chano et al., 2008; Mellén et al., 2009; Valenciano et al., 2008).
and aggrephagy (degradation of protein aggregates) (Johansen and Crucially, its location outside of the brain makes it the most accessible
Lamark, 2020). Similarly, organelle-specific macroautophagy can be part of the CNS. In addition, the retina can be cultivated ex vivo under
classified into ER-phagy (autophagy of the ER), peroxyphagy (auto­ semi-physiological conditions in which cell-to-cell and cell-to-matrix
phagic degradation of peroxisomes), mitophagy (autophagic degrada­ communication are maintained (Valenciano et al., 2008). Only
tion of mitochondria), and lysophagy (autophagic degradation of recently has research focused on the role of autophagy in the visual
lysosomes) (Johansen and Lamark, 2020). A recent study demonstrated system. The eye, and in particular the retina, is exposed to a variety of
that the Golgi apparatus can also be targeted for selective autophagic environmental insults and stressors, including gene mutations and
degradation (Nthiga et al., 2021). Alterations in selective autophagy can age-related changes, that lead to functional impairment (Boya et al.,
have important pathological implications. For example, the deficient 2016). The development of new therapeutic strategies for retinal dis­
removal of pathogenic protein aggregates has been linked to neurode­ eases requires a better understanding of the role of autophagy in retinal
generative and other diseases (Conway et al., 2020). homeostasis.
In this review we focus on the role of autophagy in the retina and the
pathological consequences of alterations in autophagy. We first describe 3. Autophagy in the retina
the main roles of autophagy in physiological conditions, focusing in
particular on the retinal tissue and available tools used to measure 3.1. Autophagy as a quality control mechanism
autophagy. Next, we discuss the role of autophagy in the main diseases
of the eye and retina, including age-related macular degeneration Autophagy mediates the degradation of cellular components, which
(AMD), glaucoma and diabetic retinopathy. We also address the putative are recycled to produce nutrients and building blocks to generate

Fig. 1. Main autophagy pathways described in

mammalian cells, classified according to the mode of
cargo delivery to the lysosome. A, Macroautophagy
facilitates the recycling of intracellular components,
including organelles, through the formation of an
autophagosome. B, Chaperone-mediated autophagy
enables the degradation of proteins carrying a specific
sequence (KFERQ motif) that is recognized by chap­
erones such as Hsc70. Recognition of the chaperone
by LAMP2A, the CMA receptor in the lysosomal
membrane, facilitates the unfolding and translocation
of the protein inside the lysosomal lumen. C, During
microautophagy, proteins are translocated directly
through invagination of the lysosomal membrane.
Once inside the lysosome acidic hydrolases degrade
the intracellular components and recycle the degra­
dation products, which will be exported from the
lysosome.

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cellular structures that maintain cellular homeostasis. Furthermore, which insoluble, polyubiquitinated p62 and oxidized proteins accumu­
autophagy plays an important role in the control of cell quality, late in mice that are deficient in Atg5 or Vps34 (Morishita et al., 2013).
particularly in neurons, in which the accumulation of components such
as altered proteins or damaged organelles cannot be offset by redistri­
3.2. Autophagy and retinal metabolism
bution to daughter cells through cell division. Besides autophagy, other
proteolytic pathways such as the ubiquitin proteasome system are also
Maintenance of the neuronal excitation required for neurotrans­
key to preserve intracellular quality control (Giandomenico et al.,
mission, phototransduction, and normal cellular function means that the
2021). Crosstalk between these pathways also serves to preserve the
retina has very high energy demands (Ames and Li, 1992). The retina
intracellular proteome and deficits in one pathway often result in the
produces energy through glycolysis, even in the presence of oxygen. This
activation of other system; for example autophagy deficiency can be
process was first described by Warburg and later confirmed by others
compensated by the ubiquitin proteasome and viceversa (Ryhanen et al.,
(Ng et al., 2015; Warburg, 1956). One possible explanation for the
2009). Also, crosstalk between autophagy and other lysosomal pathways
so-called Warburg effect in the adult retina, despite its non-proliferative
such as cathepsins and calpains are also relevant as quality control
status, is that the retina has biosynthetic requirements similar to those of
pathways (Metwally et al., 2021).
neoplastic tissues due to the recycling of visual pigments in POS mem­
The key role of basal autophagy in neurons was demonstrated more
branes (Casson et al., 2013). Cellular compartmentalization provides an
than 15 years ago in mouse experiments in which Atg5 and Atg7 were
alternative explanation for the high level of glycolytic activity in pho­
selectively deleted in neuronal precursors. While deletion did not result
toreceptors: mitochondria are located in the inner segments, but are
in the perinatal lethality observed in mice with whole-body knockout of
absent from the outer segments, potentially leading to dependence on
Atg5 and Atg7, death occurred before 3 months of age due to neuro­
glycolysis in the latter region (Ng et al., 2015). In addition, recent evi­
degeneration (Hara et al., 2006; Komatsu et al., 2006) and the mice
dences show that a small fraction of pyruvate is also oxidized in
displayed degenerative changes in various brain regions, including loss
photoreceptor mitochondria and is required for visual function, photo­
of Purkinje cells, apoptosis of cerebellar granular cells, and axonal
receptor structure and viability (Grenell et al., 2019). Exactly how
swelling. Accumulation of inclusion bodies and ubiquitin-positive pro­
photoreceptors balance aerobic glycolysis and mitochondrial OXPHOS
tein aggregates was also observed in many brain regions (Hara et al.,
to regulate their survival is still unclear. However, new studies knocking
2006; Komatsu et al., 2006). Furthermore, these mice displayed signif­
down HK2 in rods in mice show that inhibition of glycolysis led to
icant retinal alterations, including increased levels of the autophagy
photoreceptor degeneration. On the one hand, metabolic reprogram­
substrate p62, ubiquitin accumulation in all retinal layers, and photo­
ming from glycolysis to mitochondrial OXPHOS may partially reduce
receptor cell death (Rodriguez-Muela et al., 2013). Crucially, these
the metabolic stress caused by deletion of HK2, on the other hand,
changes resulted in marked vision alterations as early as 5 weeks. These
excessive mitochondrial OXPHOS promotes the generation of reactive
observations suggest that basal autophagy in neuronal precursors is
oxygen species (ROS), which can finally lead to mitochondrial
critical to maintain normal retinal physiology and that decreased Atg5
dysfunction and photoreceptor degeneration (Zhang et al., 2020b).
levels result in photoreceptor neurodegeneration (Rodriguez-Muela
As demonstrated in other tissues, autophagy facilitates the supply of
et al., 2013).
metabolites that fuel anabolic reactions and sustain ATP levels, thus
Aging is linked to a general decline in the activity of proteolytic
preserving cellular functions. In the developing chick retina, pharma­
pathways (Kroemer, 2015), including macroautophagy,
cological inhibition of autophagy reduces ATP levels and alters
chaperone-mediated autophagy, and the ubiquitin-proteasome system
apoptotic cell clearance (Boya et al., 2008; Mellén et al., 2008, 2009).
(Martinez-Lopez et al., 2015). The exact reasons for this age-associated
Supporting this important metabolic role of autophagy, supplying ret­
decrease in autophagic activity are not completely understood, although
inas with methyl pyruvate (a permeable analogue of pyruvate)
it has been proposed that it is a consequence of defective lysosomal
completely reverses these engulfment defects (Mellén et al., 2008).
function (Gomez-Sintes et al., 2016). Moreover, decreases in retinal
Interestingly, methyl pyruvate also reverses the neurogenesis defects
mRNA expression of several autophagy regulators correlating with
observed in neuronal stem cells derived from autophagy-deficient ani­
decreased autophagic flux have been described in 2-year old wild type
mals (Vazquez et al., 2012). Atg5-deficient retinas also display alter­
mice, together with lipofuscin accumulation, morphological alterations
ations during embryonic development that include axonal defects and
in RPE cells and photoreceptor cell death (Rodriguez-Muela et al.,
reduced numbers of differentiated RGCs, a phenotype also found in
2013). These findings indicate that maintenance of autophagy activity in
mitophagy-deficient animals (Esteban-Martinez et al., 2017a). The se­
the eye is crucial to preserve the cellular proteome, and that decreases in
lective elimination of mitochondria via autophagy is crucial to support
autophagy caused by normal physiological aging or by the deletion of
the metabolic shift towards glycolysis that is necessary for proper retinal
autophagy genes have very similar consequences for retinal function.
neurogenesis (Esteban-Martinez et al., 2017a). These data demonstrate
The quality-control function of autophagy is also evident in the lens, in
that autophagy helps sustain a variety of metabolic functions during

Fig. 2. Macroautophagy. The ULK1/PI3K complex participates in the induction of autophagy, at which point the autophagosome membrane is formed. This
membrane becomes elongated and envelops specific cargo, subsequently closing to form the autophagosome. The next step involves fusion of the autophagosome
with the lysosome, in which the cargo is degraded by lysosomal hydrolases. Finally, the resulting products are recycled to sustain cellular functions.

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Fig. 3. The vertebrate retina. The retina is a tissue

consisting of several layers of cells. The retinal
pigment epithelium is a single layer of cells that is in
close contact with the outer segments of the photo­
receptor cells, which are classified as cones and rods.
The soma of rods and cones form the outer nuclear
layer. Photoreceptors form synapses with the bipolar
and horizontal cells that make up the outer plexiform
layer. The cell bodies of bipolar, horizontal, and
amacrine cells form the inner nuclear layer. Synapses
between retinal ganglion cells (RGCs) and bipolar and
amacrine cells form the inner plexiform layer. The
axons of RGCs form the optic nerve, which connects
the retina to the brain.

neuronal differentiation. Further studies are needed to better under­ 3.3. LC3-associated phagocytosis and visual function
stand the interaction between metabolism and autophagy during later
stages of neuronal differentiation as well as during normal retinal Vision begins with the absorption of light by light-sensitive photo­
function. In the adult retina, Zhou et al. demonstrated that starvation receptors in the retina. This induces conformational changes in chro­
induced AMPK phosphorylation in cone cells. Specific Atg5 deletion in mophores present in these cells, triggering the phototransduction
cones results in impaired cone function as measured by electroretino­ cascade. To sustain vision, the chromophore 11-cis-retinal must be
gram at 10 months of age (Zhou et al., 2015b). Cones from 7-week-old recycled back into its original conformation via a process known as the
Atg5ΔCone mice showed increased accumulation of mitochondria and visual cycle. This involves tight regulation of the interaction between
p62 in photoreceptor inner segments as compared with control animals. photoreceptors and the RPE, where POS, which are damaged by the
Furthermore, cone-specific elimination of Atg5 resulted in increased impact of millions of photons per day, are degraded and visual pigment
susceptibility to light-induced damage (Zhou et al., 2015b). Atg5 dele­ recycled each morning. Deletion of Atg5 in the cells of the RPE has
tion in cones thus renders these cells sensitive to both age- and revealed the importance of a non-canonical autophagy pathway for vi­
light-induced degeneration. sual pigment and POS recycling (Boya and Codogno, 2013; Kim et al.,
Aerobic glycolysis is thought to be the most important mechanism to 2013). In this pathway, known as LC3-associated phagocytosis (LAP),
fulfil the metabolic demands of photoreceptors, whereas OXPHOS ap­ LC3 and Atg5 promote the degradation of extracellular cargo such as
pears to be the main energy source for RGC dendrites in the vascularized apoptotic cells, bacteria and, in the case of the RPE, POS (Heckmann and
retina. Metabolically, RGCs can be divided into 3 components: the soma, Green, 2019). LAP in the RPE is not autophagy in sensu stricto, as the
dendrites, and axon. Mitochondrial trafficking in the axon suggests that material to be degraded is not derived from the cell itself. This material
OXPHOS is the main energy source, although glycolysis may play an enters the cell via a phagosome, and the conjugation machinery adds
additional role, especially in thinner axons (Casson et al., 2021). The LC3 to the phagosome membrane, generating what is known as a
importance of OXPHOS for the metabolic status of RGCs was recently LAPosome. LAP thus occurs without the formation of a
demonstrated in various glaucoma models treated with nicotinamide double-membrane and independently of the initiation complex, as evi­
(Casson et al., 2021; Tribble et al., 2021). Mitochondrial quality control, denced by the recruitment of LC3 to the phagosome membrane in the
and hence mitophagy, thus play an important role in RGCs. Further absence of FIP200, ATG13, and ULK1 proteins. Essential LAP regulators
supporting this view, deletion of UCP2 (uncoupling protein 2) in RGCs include Rubicon (which is not essential for autophagy) and NOX2, which
exerts a neuroprotective effect, which is driven by increases in mito­ are required during LAP for localization of PI3P at the phagosomal
chondrial quality control and mitophagy (Hass and Barnstable, 2019). membrane and the production of reactive oxygen species (ROS),
Whether this is also linked to a metabolic role after mitochondria respectively (Martinez et al., 2015). Other studies have shown that
degradation or not remains to be elucidated. The induction of autophagy RUBCN expression peaks in the morning during maximal POS degra­
and mitophagy thus appears to play an important role in retinal cells. dation (Muniz-Feliciano et al., 2017). Thus the pathway for delivery of
However, given the complex structure of the retina and the cells within apoptotic cells and pathogens to the lysosome (Mehta et al., 2014) also
the retina, more detailed analyses of the links between autophagy and plays a central role in recycling POS and sustaining the visual cycle in
the metabolic status of retinal cells will need to be performed. the RPE (Kim et al., 2013).

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3.4. Circadian rhythm and autophagy SQSTM1/p62 can also be affected by proteasome inhibitors (Klionsky
et al., 2021). This method is well described in vitro, and can also be
In 1977 Remé et al. first reported a 24-h cyclic increase in the applied to ex vivo samples such as retinal explants incubated in the
number of autophagosomes in rat rod photoreceptors immediately after absence or presence of lysosomal inhibitors (Mellén et al., 2008, 2009).
light exposure (Remé et al., 1985; Remé and Sulser, 1977; Reme et al., Another key advantage of the retina in the context of research is that
1999). More recent data indicate that autophagosome levels increase in whole retinas can be cultured in defined media. Controlling the exact
photoreceptors due to translocation of transducin and arrestin from amount of metabolites and growth factors in culture conditions allows
outer to inner photoreceptor segments, and vice versa, following a fine-tuning of metabolic substrates and determination of the conse­
bimodal pattern after dark-to-light and light-to-dark transitions. This quences for autophagic flux (Esteban-Martinez et al., 2015, 2017b;
pattern is abolished when animals are kept in darkness, and mice defi­ Gomez-Sintes et al., 2017). More importantly, autophagic flux can be
cient for transducin (Gnat1− /− ) and arrestin (Sag− /− ) do not display determined in vivo in animals, as the blood retinal barrier (BRB) is
light and dark-induced LC3-II peaks, respectively (Yao et al., 2014). permeable to protease inhibitors (Esteban-Martinez and Boya, 2015).
Xenopus laevis also shows an increase in autophagosome formation in On the other hand, the study of autophagy flux by flow cytometry using
photoreceptors after light exposure, an effect that is lost following reporter proteins has been described (Shvets et al., 2008). EGFP-LC3 is a
CRISPR-mediated deletion of GNAT1 or RPE65, indicating that photo­ substrate for autophagic degradation, total fluorescence intensity of
transduction and chromophore availability, respectively, are essential EGFP-LC3 can be used to indicate levels of autophagy in living
for diurnal induction of autophagy (Wen et al., 2019). mammalian cells. When autophagy is induced, the decrease in total
Streptozotocin-treated T1D mice and BBZDR/wor T2D rats, both of cellular fluorescence can be precisely quantified as the GFP is quenched
which are models of diabetic retinopathy, show dysregulated diurnal inside the acidic environment of the lysosome. Flux can also be directly
regulation of autophagy in the neuroretina and retinal vessels, an effect associated with an increase of detectable puncta (Klionsky et al., 2021).
that is exacerbated in older animals (Qi et al., 2020). The recent generation of autophagy and mitophagy reporter animals
In the RPE, engulfment and recycling of visual pigments can occur has enabled in vivo assessment of autophagy and mitophagy in the retina
via non-canonical LAP (Kim et al., 2013). However, this process simul­ (McWilliams et al., 2019). The use of these animals has revealed dif­
taneously triggers the formation of double membrane vesicles, corre­ ferential activation of each of these processes in distinct retinal cell types
sponding to canonical autophagy, following the same bimodal pattern during development, in adult mice (McWilliams et al., 2019), and after
(dark-to-light and light-dark) as photoreceptors (Yao et al., 2014). in vivo optic nerve crush (Rosignol et al., 2020). An increase in RGC
Importantly, in contrast to what happens in photoreceptors, the increase mitophagy after optic nerve damage had been previously hypothesized,
of LC3-II levels during “dark hours” is independent of light exposure, but was very difficult to detect and quantify. Cells isolated from reporter
suggesting concomitant circadian regulation of autophagy (Yao et al., mice are also a very good source of primary RGCs (Rosignol et al., 2020).
2014). All in all, autophagy appears to play an important role in regu­ Using primary RGCs isolated from the mito-QC mouse, we demonstrated
lating the levels of phototransduction proteins in photoreceptors and that induction of mitophagy exerts a neuroprotective effect in RGCs
supporting visual function. In line with this view, transducin accumu­ exposed to oxidative stress (Rosignol et al., 2020).
lation is described in Atg5ΔRod mice (Zhou et al., 2015a). These data also In conclusion, the development of new tools and methods has been
underscore the essential role of canonical autophagy in POS component pivotal in furthering our knowledge of the basic functions of autophagy
post-processing and maintenance of overall photoreceptor and RPE in the retina both in vitro and in vivo (Boya et al., 2016). In the following
homeostasis throughout the 24-h cycle, which appears to be altered in sections of this review, we will discuss the main findings supporting a
neurodegenerative diseases of the retina such as diabetic retinopathy (Qi role of autophagy dysregulation in the onset and progression of the most
et al., 2020). common diseases of the retina.

4. Methods to monitor autophagy in the retina 5. Autophagy in glaucoma and optic neuropathies

Autophagosome formation is the key regulatory step of autophagy, Glaucoma and optic neuropathies are the two main groups of dis­
and can be directly assessed by monitoring levels of the autophagosomal eases that lead to vision loss and blindness due to irreversible damage of
binding protein MAP-LC3 (microtubule-associated protein LC3) (Klion­ the optic nerve. The optic nerve is made up by the axons of RGCs, which
sky et al., 2021). Autophagosome formation involves ATG7-mediated represent the 3rd retinal neuron involved in transporting light signals to
lipidation of ATG8 proteins (LC3 in mammalian cells) by covalent the brain and the innermost cellular retinal layer. Both glaucoma and
attachment of phosphatidyl ethanolamine to the autophagosomal other optic neuropathies lead to RGCs death, resulting in visual
membrane. This lipidation process can be detected by Western blot impairment or even blindness. However, although RGCs are the main
because lipidated LC3 (LC3-II) migrates faster through the gel than non- target for these diseases, other cells, such as retinal glia, likely contribute
conjugated LC3, and is visualized as a lower molecular-weight band, to the pathogenesis of the diseases as well (Liao et al., 2017). This is also
allowing differentiation of free from autophagosome-bound forms of represented in various disease models primarily aiming to mimic
LC3 (Kabeya et al., 2000). different mechanisms of RGC death but also show different aspects of
While LC3 levels provide an indication of autophagosome number at neuroinflammation (Soto and Howell, 2014).
a given time point, they do not constitute a direct read-out of autophagy
activity per se, as autophagosome number can also be increased by 5.1. Autophagy in glaucoma
lysosomal inhibition (Boya et al., 2005; Gonzalez-Polo et al., 2005).
This, together with the fact that autophagy is a highly dynamic process Glaucoma is characterized by progressive, irreversible, painless loss
(10 min between autophagosome formation and fusion with the lyso­ of RGCs, leading to visual field defects and blindness. Because this
some), means it is essential to determine the rate of autophagosome condition generally progresses slowly, it tends not to be detected until
turnover by assessing autophagic flux. This can be achieved by later stages. Glaucoma is one of the leading causes of blindness world­
comparing autophagosome levels (measured by Western blot) in control wide. Although it is difficult to calculate its global prevalence, studies
conditions with those observed after lysosomal inactivation. In addition, estimate that at least 111.8 million people will be affected by the year
it is probably prudent to monitor the turnover of LC3-II together with an 2040 (Tham et al., 2014). The two main risk factors for glaucoma are
autophagosome substrate, due to the fact that LC3 might be coupled to increased intraocular pressure (IOP) and ageing (Tham et al., 2014).
endosomal and other membranes and not just autophagosomes, and the However, the pathophysiology of glaucoma still remains unclear. Ge­
levels of well-characterized autophagosome substrates such as netic studies indicate a link between glaucoma and autophagy-related

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genes (Wiggs, 2015). Of the genes that have been linked to glaucoma in model (Russo et al., 2011). Although there is strong evidence that
genome-wide association studies (GWAS), both Optn (Swarup and autophagy induction has neuroprotective effects in the context of
Sayyad, 2018) and Tbk1 (Sears et al., 2019) are implicated in autophagy glaucoma, more detailed research is required to understand timing and
regulation, as well as mitochondrial function (Choquet et al., 2020; Lang dosing of autophagy modulators.
et al., 2017; Youngblood et al., 2019). Levels of autophagy in the brain In conclusion, autophagy plays an important role in glaucoma by
and retina decline with increasing age (Lipinski et al., 2010; Rodri­ regulating both IOP and the response to RGC damage caused in this
guez-Muela et al., 2013). This is thought to contribute to the patho­ disease. A better understanding of the role of autophagy within the
genesis of various age-related degenerative diseases, including different RGC compartments and the relevance of age-related changes in
glaucoma (Boya, 2017; Wong et al., 2019). We recently demonstrated an autophagy will be necessary to fully realize the potential of autophagy as
age-related increase in RGC vulnerability in the context of autophagy a therapeutic target in glaucoma.
deficiency in young (3 months old) and aged (13 months old)
AMBRA1+/gt heterozygote mice subjected to optic nerve crush (Bell 5.2. Autophagy in optic neuropathies
et al., 2020). AMBRA1 (autophagy/beclin-1 regulator 1) participates in
the initiation of the autophagy process (Fimia et al., 2007) but also is an Optic neuropathies, unlike glaucoma, generally present with acute or
important mitophagy receptor that can act in both a subacute symptom onset and focal rather than diffuse damage to the
PINK1/PARKIN-dependent and -independent manner (Di Rita et al., optic nerve or optic nerve head. The main causes of optic neuropathies
2018; Strappazzon et al., 2015; Van Humbeeck et al., 2011). We are demyelinating processes such as multiple sclerosis, ischemic pro­
observed no differences in the rates of RGC death in young wildtype cesses, and inflammatory and infectious lesions (Abel et al., 2019;
(WT) or AMBRA1+/gt mice. However, aged old AMBRA1+/gt showed Augstburger et al., 2020; Kale, 2016). Optic neuropathies can be he­
significantly greater RGC loss after ONC than their WT littermates (Bell reditary, such as the mitochondrial disorder Leber’s hereditary optic
et al., 2020). This could be explained by a decrease in the oxidative atrophy (LHON) (Yu-Wai-Man et al., 2002) and dominant optic atrophy
stress response and a failure to increase Bnip3 levels and (DOA) (Lenaers et al., 2012), or caused by toxins (Grzybowski et al.,
Bnip3l-mediated autophagy after injury. 2015) such as alcohol, tobacco, and ethambutol (which is used to treat
Age-related dysregulation of autophagy in the retina, as well as the tuberculosis). The list of causes continues to grow (Behbehani, 2007).
trabecular meshwork, has also been detected in an elevated IOP model Given the possible contributions of autophagy and mitophagy to optic
of glaucoma (Nettesheim et al., 2020). Because increased IOP is a key neuropathies and the therapeutic potential of strategies that modulate
risk factor for glaucoma, the trabecular meshwork, which serves as the autophagy, studies have sought to tease apart the roles of autophagy in
main outflow pathway for aqueous humour, is especially important for LHON and DOA. Interestingly, genetic mutations in mitochondrial
maintaining normal IOP. Dysregulation of autophagy in the trabecular proteins have been described in both LHON and DOA, and although
meshwork occurs during ageing and has also been associated with these mutations are not limited to RGCs or even the eye, the neuronal
elevated IOP (Hirt et al., 2018). The importance of autophagy was damage in most patients with disease-specific mutations is very specif­
recently demonstrated in the context of TGF-β-induced trabecular ically limited to RGCs (Heiduschka et al., 2010; Kirches, 2011). Muta­
meshwork fibrosis (Nettesheim et al., 2020). While this review focuses tions of OPA1 account for the majority of DOA cases, although mutations
on retinal cells, readers may be interested in the comprehensive review in OPA3 and OPA7 have also been identified (Lenaers et al., 2012). OPA1
by Hirt and Liton on this extensive topic (Hirt and Liton, 2017). is a well-conserved gene; the translated protein is located in the inner
In general, cells induce autophagy as a response to stress in order to mitochondrial membrane and is essential for mitochondrial fusion and
ensure cell survival. This is also the case for injured RGCs. Induction of mitochondrial quality control (Gao and Hu, 2021). Splice variants,
autophagy in the early phases after injury has been demonstrated in frame shifts, nonsense mutations, deletions, and duplications have all
different mouse models of glaucoma, including damage induced by been detected in patients carrying OPA1 mutations (Lenaers et al.,
axonal injury (Rodriguez-Muela et al., 2012), in response to IOP eleva­ 2012). Although evidence indicates that DOA mutations increase auto­
tion by occlusion of the episcleral vein, and after retinal phagy and mitophagy, the effect of these mutations on autophagy and,
ischemia-reperfusion (Park et al., 2012). There is strong evidence sug­ specifically, mitophagy can vary, particularly in cases of mutations that
gesting that autophagy induction exerts a neuroprotective effect on cause haploinsufficiency (Alavi and Fuhrmann, 2013; Kane et al., 2017;
damaged RGCs in the context of glaucoma, although other findings Moulis et al., 2017). Analysis of a nonsense OPA1 mutation in a het­
contradict this view. Activation of autophagy by pharmacologic or ge­ erozygous OPA1 mouse model revealed increased numbers of auto­
netic manipulation improves RGC survival in various in vivo experi­ phagosomal structures in RGCs on electron microscopy analysis (White
mental models of glaucoma. Using an optic nerve transection model, we et al., 2009). Increased mitophagy, measured by analyzing colocaliza­
demonstrated a 40% increase in RGC survival 10 days after axotomy in tion of LC3-positive puncta with TOM20-stained mitochondria or by
mice treated with rapamycin (Rodriguez-Muela et al., 2012). Rapamy­ counting the number of LC3-positive puncta in cells co-stained with
cin, which was first discovered as an antifungal agent produced by lysotracker and mitotracker, has been described in fibroblasts from DOA
Streptomyces hygroscopicus, inhibits mTOR (mechanistic target of patients with OPA1 deletion and dominant negative mutations (Kane
rapamycin) and therefore induces autophagy not only in peripheral et al., 2017; Liao et al., 2017). A more detailed analysis of the effects on
organs but also in the CNS, including the retina and brain (Dehay et al., autophagy and mitophagy of mutations in the GTPase and the
2010; Rodriguez-Muela et al., 2012). By comparison, in Atg4b− /− mice coiled-coil domains of OPA1 revealed mitochondrial depletion in axons
RGC loss after ONT was 28% greater than in WT controls, and specific of RGCs expressing these mutations and accumulation of
deletion of Atg5 in RGCs resulted in similar outcomes (33% decrease in mito-autophagosomes in the axonal hillock (Zaninello et al., 2020). The
RGC survival after ONT) (Rodriguez-Muela et al., 2012). In a rat model increase in autophagy activation in this part of the cell could be
of IOP induced by laser, Kitaoka et al. demonstrated neuroprotection of explained by increased AMPK activation caused by OPA1 mutations. The
the optic nerve axons following treatment with rapamycin, and an in­ authors demonstrated that inhibition of autophagy restored axonal
crease in axonal degeneration following treatment with 3-methylade­ mitochondrial content in these cells. Moreover, inhibition of autophagy
nine (3-MA) (Kitaoka et al., 2013). Another study in which 3-MA was in the respective OPA1 mutant mouse models attenuated the loss of
used to inhibit autophagy in a glaucoma model induced by episcleral vision.
vein cauterization reported neuroprotective effects (Park et al., 2012), Mutations affecting mitochondrial proteins have also been found in
although the studies differed in terms of the timing of 3-MA adminis­ LHON, in all cases leading to amino acid changes in subunits of complex
tration. The neuroprotective effect of autophagy induction with rapa­ I of the mitochondrial respiratory chain. The most common mutations
mycin was further demonstrated in a mouse ischemia/reperfusion affect the proteins ND1 (13%), ND4 (69%), and ND6 (14%)

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(Yu-Wai-Man et al., 2002). Less is known about alterations in autophagy example, ARPE-19 cells cultured in high concentrations of glucose
and mitophagy in LHON. However, in vitro findings suggest that display upregulation of autophagy, which may constitute a pro-survival
mitophagy is decreased in cells harboring mutations (Sharma et al., mechanism. Autophagy blockade increases oxidative stress and the ac­
2019). Two studies using cybrid cell lines reported decreased autophagy tivity of IL-1β and NLRP3 (a NOD-like receptor responsible for pro­
in cells carrying the LHON-specific ND4 and ND1 mutations, leading to cessing pro-IL-1β to produce the active form of IL-1β) (Shi et al., 2015).
an accumulation of dysfunctional mitochondria, and showed that in­ Recent findings suggest that this pro-survival effect may be mediated by
duction of autophagy with rapamycin promoted the clearance of the selective elimination of mitochondria by thioredoxin-interacting
damaged mitochondria (Dai et al., 2014; Sharma et al., 2019). The less protein (TXNIP) via PINK1/PARKIN-mediated mitophagy (Huang
common ND5 mutation is also proposed to decrease levels of mitophagy et al., 2018; Su et al., 2020). A similar effect is observed in Müller cells,
(Zhang et al., 2018). In summary, further studies are needed to fully in which TXNIP increases the expression of HMGB1, a nuclear protein
understand the role of autophagy in both these diseases, and more that is transported from the nucleus in conditions of oxidative stress and
detailed analysis of the various mutations involved will be necessary to interacts with Beclin-1 to enable autophagy. Activation of autophagy
pave the way towards more personalized therapeutic approaches. may therefore represent a survival mechanism, degrading mitochondria
Little is known about the role of autophagy in optic neuropathies damaged by ROS (Devi et al., 2012). Further studies will be needed to
other than LHON and DOA. One important form of toxic optic neurop­ confirm this hypothesis as other evidence points to a detrimental effect
athy is that induced by ethambutol, a drug frequently used for the of TXNIP-mediated mitophagy in the RPE in high glucose conditions
treatment of tuberculosis. In addition to RGC death, this drug can lead to (Devi et al., 2019). These dual effects of autophagy and mitophagy in DR
autophagy dysregulation by blocking autophagic flux and neutralizing may be associated with disease progression, as observed in a recent
lysosomal pH, ultimately resulting in RGC apoptosis (Huang et al., 2015; study in hyperglycemic mice (Hombrebueno et al., 2019).
Yamada et al., 2016).
7. Diseases associated to photoreceptor degeneration
6. Autophagy in diabetic retinopathy
Several blindness-associated diseases are caused by the progressive
Diabetes is a chronic disease that results in hyperglycemia, which dysfunction and death of retinal photoreceptors. These are highly
occurs when the pancreas does not produce enough insulin or when the specialized neurons capable of detecting light stimuli and converting
body cannot effectively use the insulin it produces. Diabetic retinopathy them into electrical impulses. There are two types of photoreceptors:
is a complication of diabetes caused by the long-term accumulation of cones, which are responsible for vision at high light intensity (photopic
damage to small blood vessels in the retina. DR is an important cause of vision); and rods, which support vision at low light intensity (scotopic or
blindness, accounting for 2.6% of all cases of blindness worldwide night vision) (Lamb et al., 2007).
(Bourne et al., 2013). Blood vessel damage can result in ischemia and Phototransduction subjects photoreceptors to constant stress, and
neovascularization, eventually leading to blindness. Chronic hypergly­ the need to continuously regenerate POS imposes a high metabolic de­
cemia has multiple effects on retinal cells. It can induce ER-stress, mand. Damage caused by light and by oxidation of cell membranes,
mitochondrial and oxidative damage resulting in the death of neurons including those of the POS, can result in photoreceptor death. Photo­
and pericytes, loss of synapses and dendrites, and alterations in synaptic receptor damage can also be caused by calcium-activated enzyme
activity (Oshitari et al., 2011; Ozcan et al., 2006). In addition, hyper­ signaling, cyclic nucleotide signaling, and cathepsin-mediated lyso­
glycemia, results in the formation of advanced-glycation end products somal cell death (Murakami et al., 2013; Rodriguez-Muela et al., 2015;
(AGEs) and induces ocular tissue dysfunction and its contribution to the Wang et al., 2018). Moreover, a wide-range of mutations, generally
onset and development of eye disorders (Bejarano and Taylor, 2019). those that affect the phototransduction cascade or cause protein mis­
Furthermore, activation of microglia and Müller cells induces the pro­ folding or mislocalization, can induce photoreceptor death. The role of
duction of vascular endothelial growth factor (VEGF), which promotes autophagy in photoreceptor survival remains a topic of debate, and
neovascularization, contributing to vision loss. although most studies indicate a neuroprotective effect, some authors
Pericyte loss is considered an initiating event in DR, as pericytes have proposed that autophagy inhibition may be beneficial under
protect the endothelial cells in the retinal capillaries and are also certain conditions. In this section, we will discuss the opposing roles of
responsible for maintaining integrity of the BRB. One hallmark of dia­ autophagy in photoreceptor survival.
betes is post-translational modification, including glycosylation and
oxidation, of plasma lipoproteins, including low-density lipoproteins 7.1. Photoreceptor degeneration in autophagy-deficient animals
(LDLs). Pericyte loss results in increased BRB permeability, favoring the
extravasation of LDLs into the retina. Pericytes treated with sub-lethal In recent years several laboratories have investigated the conse­
doses of LDL show an increased autophagy that preserves cell quences of deleting autophagy genes in retinal photoreceptors. The first
viability. However, at high LDL doses this pro-survival autophagy mouse with photoreceptor-specific deficiencies in autophagy, the
response is no longer observed, and autophagy inhibition by deletion of Atg7ΔRod mouse, was generated by crossing Atg7flox/flox mice with rod-
the autophagy regulator Beclin-1 rescues pericytes from LDL-induced specific LMOP-Cre mice (Chen et al., 2013). These animals displayed
cell death. This dual effect of ER-stress-induced autophagy has also no detectable phenotype in normal conditions. However, exposure of
been described in a rat model of diabetic retinopathy (Fu et al., 2016). Atg7ΔRod mice to intense illumination resulted in retinal degeneration
How autophagy levels are regulated in pericytes in human DR remains (Chen et al., 2013). Deletion of Atg5 in rods by crossing
unknown. A recent study reported that retinal ischemia alters blood flow rhodopsin-iCre-75 transgenic mice with Atg5flox/flox mice resulted in
and neurovascular coupling by damaging intercellular communication reduced ONL thickness at 5 months of age and rod photoreceptor
between pericytes. Pericytes connect to one another and transfer or­ degeneration, which was also observed when these mice were raised in
ganelles including vesicles and mitochondria via processes that resemble darkness (Zhou et al., 2015a). Although there are differences between
tunneling nanotubes (Alarcon-Martinez et al., 2020). How this inter­ these two studies, deletion of the essential autophagy genes Atg5 and
cellular communication impacts pericyte function in DR remains to be Atg7 in rods results in a clear detrimental phenotype in conditions of
determined. light stress.
While DR is largely considered a vascular disease, recent evidence Decreases in autophagy in cones have been achieved by crossing
indicates that abnormalities in the neural retina and the adjacent RPE Atg5flox/flox mice with HRGP-Cre transgenic mice, in which Cre recom­
contribute to the pathogenesis of DR (Tonade and Kern, 2020) and that binase is controlled by a fragment of the promoters of the human red
autophagy could represent a prosurvival response in those cells. For (OPN1LW) and green (OPN1MW2) opsin genes (Zhou et al., 2015b). The

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resulting mice show a decrease in cone number at 2 months of age that pathways, including the ubiquitin-proteasome pathway and the UPR,
correlates with reduced cone function on electroretinogram at 10 are essential for the elimination of toxic protein aggregates.
months of age. Moreover, an increased accumulation of mitochondria is The results of a study performed using two different models of retinal
observed in the inner segment of cones of 7-week-old Atg5ΔCone mice degeneration suggest that upregulation of autophagy using trichostatin
compared with control animals. Interestingly, these accumulations of A (TSA) may delay cone death (Samardzija et al., 2021). In that study,
mitochondria are age-dependent, indicating that autophagy down­ secondary cone cell death was associated with exacerbated histone
regulation results in a phenotype characterized by progressive cone deacetylate (HDAC) activity. Inhibition of HDAC activity using a single
degeneration. Furthermore, cone-specific elimination of Atg5 results in intravitreal injection of trichostatin A (TSA), administered at an age at
increased susceptibility to light-induced damage as well as a decrease in which most rods have degenerated, delayed cone death in both rd10 and
cone POS length and enlargement of inner segments, with accumulation rd1 mice. Transcriptional changes associated with cone survival
of mitochondria (Zhou et al., 2015b). More recent studies have included regulation of distinct pro-survival mechanisms, including
corroborated the importance of autophagic flux in sustaining cone autophagy and both MAPK and PI3K/Akt signaling (Samardzija et al.,
function. For example, Vps34 (also called Class III PI3K) has an impor­ 2021). HIF-1 signaling and autophagy are also among the neuro­
tant role in cones. Vps34 selectively phosphorylates PI to PI(3)P, but protective pathways proposed to be upregulated by the HDAC6 inhibitor
does not phosphorylate other PIPs. The PI(3)P lipid generated by Vps34 tubastatin A in both atp6v0e1− /− zebrafish and rd10 mice (Sundar­
plays an important role in endocytic membrane trafficking, canonical amurthi et al., 2020).
autophagy, and cell survival (Stenmark et al., 2002). Specific deletion of Changes in autophagic flux can also occur as a consequence of
Vps34 in cones also results in impaired visual function (Rajala et al., lysosomal alterations in RP. Rodriguez-Muela et al. found that intra­
2020) and photoreceptor degeneration is observed as early as eye cellular calcium accumulation led to calpain activation in rd10 mice
opening in mice deficient in Rabgef1, a protein implicated in endoly­ before peak rod cell death (Rodriguez-Muela et al., 2015). Moreover, the
sosomal function (Hargrove-Grimes et al., 2020). Rabgef1-deficient authors found that calpain and cathepsin inhibitors attenuated photo­
mice also display accumulations of autophagosomes and p62 in photo­ receptor cell death in these mice in vitro, ex vivo, and in vivo, suggesting
receptor inner segments consistent with compromised autophagy that calpain-mediated lysosomal membrane permeabilization underlies
(Hargrove-Grimes et al., 2020). the lysosomal dysfunction and autophagy downregulation associated
with photoreceptor cell death in RP (Rodriguez-Muela et al., 2015).
7.2. Autophagy in retinal dystrophies Given that lysosome damage is often accompanied by reduced auto­
phagic flux, candidate neuroprotective strategies for these pathologies
Retinal dystrophies are a group of diseases that lead to progressive should include restoration of lysosomal activity. One such example is
loss of vision due to the death of retinal cells. Photoreceptor death is a trehalose-dependent TFEB upregulation, which reverses lysosomal
feature of most of these diseases (Olivares-González et al., 2021). The damage and ameliorates vision loss in several models of retinal diseases
most common retinal dystrophy is retinitis pigmentosa (RP), a group of (Abokyi et al., 2020; Lotfi et al., 2018; Naso et al., 2020; Rodri­
degenerative retinal diseases characterized by rod and cone degenera­ guez-Muela et al., 2015).
tion (Olivares-González et al., 2021). Clinically, this disease is charac­ In general terms, proper autophagy can be considered essential to
terized by pigment accumulation and progressive blindness, and there is maintain vision. Deficient autophagy affects both the structure and
currently no cure. RP can be caused by mutations in over 80 different function of rods. While autophagy is essential for recycling visual cycle
genes that alter photoreceptor homeostasis, ultimately leading to cell proteins such as rhodopsin and transducin, overactivation of autophagy
death (Olivares-González et al., 2021). RP-associated gene mutations can be counterproductive in certain cases, exacerbating photoreceptor
encode enzymes (and their regulators) involved in the photo­ degeneration. As we mentioned above, misfolded proteins are typically
transduction cascade among others (Athanasiou et al., 2013). degraded by one of two mechanisms. The first involves translocation of
Several genetic mouse models of these diseases have been used to the misfolded protein out of the ER and shuttling to the proteasome. The
identify the pathological changes associated with photoreceptor second involves the induction of autophagy. In such cases, activation of
degeneration and to search for putative strategies to delay degeneration the proteasome in the retina is triggered by autophagy blockade. This
and preserve visual function. For example, in their 2009 study, Punzo activation is necessary for the elimination of misfolded proteins and it
and coworkers performed microarray analyses at peak cone cell death in can compensate for the degeneration produced by an overactivation of
four different RP models, and demonstrated upregulation of several autophagy (Yao et al., 2018). Autophagy is also necessary to maintain
genes involved in the insulin/mTOR pathway (Punzo et al., 2009). They photopic vision. It should be noted that in most models in which rod
found that insulin administration attenuated photoreceptor cell death, autophagy is specifically altered, cone neurodegeneration is also
suggesting that cone starvation contributes to cell death in RP (Punzo observed. Therefore, homeostatic autophagy is necessary to maintain
et al., 2009). Overactivation of autophagy has also been described in a proper photoreceptor function and sustain vision.
mice deficient in UXT, a ubiquitously expressed prefoldin-like chap­
erone that regulates mTOR activity (Pan et al., 2020). These mice 7.3. Autophagy in models of light-induced retinal damage and retinal
display RP-like features including apoptotic photoreceptor degeneration detachment
and vision loss.
Recent studies have also demonstrated increased autophagy activa­ Prolonged exposure to intense visible light can lead to photoreceptor
tion in P23H rhodopsin (RHO) mice. Interestingly, Yao and coworkers cell damage. The consequences of light-induced retinal damage vary
found that photoreceptor structure and function was improved in these greatly, depending on the type of light and duration of exposure, as well
mice by reducing autophagic flux either pharmacologically or by rod- as the genetic background of the animal model studied (Organisciak and
specific deletion of the autophagy-activating gene Atg5 (Yao et al., Vaughan, 2010). Data from Drosophila studies support the view that
2018), suggesting that a balance between autophagy and the protea­ autophagy helps protect against light-induced damage by eliminating
some pathway is essential to maintain photoreceptor homeostasis. rhodopsin-arrestin complexes that form during phototransduction
Sizova et al. reported improved visual function in P23H mice 4 weeks (Midorikawa et al., 2010; Wang et al., 2009b). Beclin1 hemizygous mice
after rapamycin treatment (Sizova et al., 2014). However, the beneficial are more sensitive to light-induced retinal damage and show reduced
effects of rapamycin treatment appeared to be linked to the activation of retinal thickness and alterations in RPE cells (Chen et al., 2013). A
the unfolded protein response (UPR) and not to upregulation of auto­ similar phenotype has been described in mice with rod-specific deletion
phagy. These data suggest that multiple pathways participate in pre­ of Atg7 (Chen et al., 2013). These findings suggest that autophagy pri­
serving photoreceptor integrity and that proper functioning of these marily triggers a cytoprotective response in the light-challenged retina

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(Chen et al., 2016). However, the results of other studies indicate that 8.1. Importance of autophagy in age-related macular degeneration
the induction of autophagy in response to light-induced retinal damage
can also have detrimental effects. Kunchithapautham reported that in­ The RPE is the first component of the retina affected in AMD. It
jection of rapamycin for 10 days in a mouse model of light-induced consists of a monolayer of polarized epithelial cells containing melanin
retinal damage protected against rod cell death, although cone func­ granules. In the apical compartment, microvilli form a meshwork with
tion was diminished (Kunchithapautham et al., 2011), and a recent POS, the distal tips of which are shed, phagocytosed, and recycled daily
study found that inhibition of ER stress-induced autophagy protected by the RPE in the aforementioned visual cycle. The basal membrane of
against light-induced retinal damage in mice (Song et al., 2020). In the RPE is part of Bruch’s membrane, and forms part of the BRB, helping
conclusion, these data demonstrate that the outcomes of autophagy in­ isolate the retina from the choroidal bloodstream. In addition to facili­
duction in response to light stress can vary significantly, possibly tating chromophore recycling, the RPE also supports glycolysis-driven
depending on photoreceptor cell type. Further studies are necessary to photoreceptor metabolism by providing glucose and other essential
understand the contributions of other extrinsic and intrinsic factors to nutrients (Fisher and Ferrington, 2018). Metabolically-demanding POS
photoreceptor survival. processing, high levels of production of OXPHOS-derived ATP, constant
Retinal detachment is an eye disorder in which the retina is sepa­ exposure to photo-oxidative stress, and choroidal blood flow all combine
rated from the RPE, resulting in photoreceptor cell death and blindness. to create an environment of chronic oxidative stress in the RPE (Datta
However, vision restoration can be achieved if the detachment is et al., 2017; Fisher and Ferrington, 2018). Furthermore, oxidized visual
repaired within 1 week. Research has thus sought to identify therapeutic pigments cannot be fully degraded by lysosomal enzymes and accu­
strategies that prolong photoreceptor survival. One such mechanism is mulate in the form of the end product bis-retinoid (Sparrow et al., 2012).
autophagy (Chinskey et al., 2014), where its downregulation decreases Within increasing age, the progressive build-up of oxidized bis-retinoid
photoreceptor survival following retinal detachment (Besirli et al., leads to the accumulation of lipofuscin inside lysosomes, altering lyso­
2011). Moreover, autophagy appears to be essential to degrade material somal pH and inhibiting the degradation of both endogenous and
produced by the retraction of photoreceptors during the process of exogenous material (Bergmann et al., 2004). The RPE is a post-mitotic
retinal detachment (Xiao et al., 2021). Rapamycin has been proposed to tissue that generates and accumulates damaged intracellular compo­
protect against photoreceptor damage in retinal detachment (Ding et al., nents throughout its lifetime. Maintenance of RPE homeostasis therefore
2019): it prevents necroptosis by inhibiting ROS production and requires a tightly-regulated intracellular housekeeping system, which
apoptosis inducing factor (AIF) release, and promotes photoreceptor cell includes autophagy (Zhang et al., 2020c).
survival by increasing autophagy (Ding et al., 2019). Retinal
detachment-induced hypoxia also activates autophagy in photorecep­ 8.2. Autophagy-related phenotypes in age-related macular degeneration
tors via a HIF-1α-mediated mechanism, and silencing of HIF-1α de­ patients
creases autophagy and increases cell death (Shelby et al., 2015).
Together, these findings point to a clear pro-survival role of autophagy In their study of donor eyes, Wang et al. detected markers of auto­
in photoreceptor cells during retinal detachment. phagy (ATG5) and exosomes (CD63, CD81, LAMP2) in drusen from the
eyes of AMD donors (Wang et al., 2009a). By contrast, they detected
8. Age-related macular degeneration only small ATG5-positive foci inside small deposits in the sub-RPE space
in the eyes of age-matched healthy donors without previous eye dis­
AMD is a progressive bilateral neurodegenerative retinopathy that eases. Mitter and coworkers described an age-associated increase in the
affects the macula, the cone-rich central region of the retina responsible number of autophagosomes in the RPE and neuroretina of healthy do­
for high-resolution color vision. Its complex etiology includes genetics, nors that was not observed in early and late AMD donors, in which
diet, smoking, and high blood pressure, among other environmental decreased ATG7 and ATG9 immunoreactivity was detected (Mitter et al.,
factors, although age is considered the primary risk factor (Mitchell 2014). Another study reported that levels of LC3-II and p62 were
et al., 2018). Variants in proteins involved in lipid metabolism, extra­ increased and decreased, respectively, in the RPE of AMD patients,
cellular matrix remodeling, angiogenesis, and complement activation suggesting induction of autophagy (Ye et al., 2016). Primary RPE cells
have also been implicated in AMD (Fritsche et al., 2016; Lim et al., from AMD patients also contain enlarged autophagosomes and show
2012). It is estimated that around 25.3% of people aged over 60 years impaired autophagy induction upon nutrient starvation, and reduced
present early or intermediate AMD, while 2.4% present late AMD, and autophagic flux, evaluated in presence of protease inhibitors (Golesta­
these numbers are expected to increase in the coming years due to neh et al., 2017). AMD donor cells contain swollen lysosomes, a sign of
population ageing (Li et al., 2020). Late AMD can be further classified as lysosomal stress, as well as cytoplasmic glycogen and lipid granules
“dry” or “wet”. The wet or exudative form of the disease affects only (Golestaneh et al., 2017). Zhang and coworkers reported marked
around 10% of patients and is driven by abnormal choroidal neo­ metabolic alterations in AMD patient-derived RPE cultures, including
vascularization into the macular subretinal space and neuroretina, overactivation of mTORC1 (the main inhibitor of canonical autophagy)
where newly-formed leaky vessels cause macular edema, neuro­ and decreased glycerophospholipid metabolism (Zhang et al., 2020a).
degeneration and, ultimately, central vision loss. The dry form of the Decreased autophagy-mediated phagocytosis of POS has also been re­
disease is characterized by the build-up of extracellular deposits, called ported in a human cell model of AMD (Inana et al., 2018).
drusen, between the RPE and Bruch’s membrane. Drusen are composed Single-nucleotide polymorphisms (SNPs) in core autophagy regulators
of a mixture of apolipoproteins, oxidized proteins, complement com­ (mTOR, ATG5, ULK1, MAP1LC3A, SQSTM1) have been identified in
ponents, and trace elements, among other components, and their for­ patients with wet AMD, in some cases correlating with enhanced or
mation remains poorly understood (Bergen et al., 2019). Dry AMD can attenuated responses to anti-VEGF treatment (Paterno et al., 2020).
eventually progress to geographic atrophy leading to loss of chorioca­ Overall, these data point to an induction of autophagy upon AMD onset
pillaris, RPE cells and, subsequently, photoreceptors (Fleckenstein et al., that cannot be completed due to decreased lysosomal function, leading
2018). Anti-VEGF immunotherapy has shown promising results, either to an intracellular accumulation cell debris (Golestaneh et al., 2017;
stabilizing or improving visual acuity in wet AMD patients (Mitchell Mitter et al., 2014; Ye et al., 2016). The available evidence suggests that
et al., 2018). However, to date there is no curative treatment for either autophagy plays an essential role in the development of AMD and that
dry AMD or geographic atrophy. blockade of autophagic flux could be a key contributor to the patho­
genesis of AMD.

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8.3. Role of autophagy in experimental models of age-related macular followed by secondary photoreceptor degeneration and vision loss (Yao
degeneration et al., 2015). TSC1 is a negative upstream regulator of mTORC1 that
promotes autophagy, and Tsc1ΔRPE animals lacking TSC1 in the RPE
The etiology of AMD is complex, and is influenced by both genetic present features of both wet and dry AMD, including fundus auto­
and environmental factors. Given that only primates present a macula fluorescence and atrophy of the choroid and RPE, ultimately resulting in
per se, there is no small animal model that fully recapitulates the disease progressive retinal degeneration (Go et al., 2020; Huang et al., 2019).
(Soundara Pandi et al., 2021). Because AMD is characterized by primary Mice lacking the autophagy regulators Atg5/Atg7 in the RPE also present
RPE degeneration, the main in vitro model used is the ARPE-19 cell line, age-dependent increases in levels of p62-positive aggregates, oxidative
derived from the RPE of a 19-year-old male donor. ARPE-19 cells express stress markers, RPE atrophy, and retinal degeneration (Zhang et al.,
characteristic RPE markers, including RPE65 and CRALBP, and can 2017). These changes have been attributed to alterations in the
eventually form polarized monolayers and become pigmented if non-canonical function of Atg5 in LAP, leading to inadequate recycling
cultured in confluence for long periods of time in defined medium (Dunn of retinoids, accumulation of POS, and RPE atrophy, and can be reversed
et al., 1996). 3-MA-induced inhibition of autophagy in ARPE-19 cells by supplementing mice with 9-cis-RAL to fuel phototransduction and
leads to increased accumulation of endogenous lipofuscin, while inhi­ visual function. While retinal morphology and rod/cone numbers
bition with bafilomycin A1 leads to the accumulation of the oxidative remain normal throughout the life, Atg5ΔRPE mice present reduced visual
stress-derived 4-HNE-protein adduct (Mitter et al., 2014; Piippo et al., function upon aging (Kim et al., 2013). However, in another study of
2014). Moreover, POS exposure increases autofluorescence in this cell Atg7ΔRPE mice, no retinal degeneration was detected and the authors
line, an effect exacerbated by autophagy inhibitors (3-MA, leupeptin, concluded that autophagy was not involved in A2E (a component of
chloroquine, NH4Cl) and attenuated by autophagy induction with lipofuscin) accumulation or maintenance of the visual cycle (Perusek
rapamycin (Lei et al., 2017; Mitter et al., 2014). et al., 2015). It is unclear whether these conflicting findings are due to
Sodium iodate (SI) is an inorganic salt that selectively induces differences between studies in the protocols used for Cre-mediated Atg7
geographic atrophy-like RPE degeneration, leading to subsequent deletion or in the readouts used, and further studies will be required to
photoreceptor cell death (Sorsby, 1941). It induces ROS generation, RPE resolve these discrepancies. Furthermore, the involvement of Atg5 and
atrophy, and cell death, and is commonly used as a pharmacological Atg7 in both canonical autophagy and LAP also complicates the inter­
model to simulate AMD-like damage both in vitro and in vivo (Chowers pretation of these results, making it difficult to determine which
et al., 2017). SI basifies acidic compartments, leads to autofluorescence pathway is the main contributor to the resulting phenotype (Heckmann
accumulation, and partially blocks autophagy in ARPE-19 cells in vitro and Green, 2019).
(Lin et al., 2018). Furthermore, SI-induced ROS generation is required to LAMP2-deficient mice also exhibit some features of dry AMD,
trigger autophagy and blocking autophagic flux with 3-MA or Baf-A1 including increased fundus autofluorescence and atrophy of Bruch’s
results in decreased cell viability (Chan et al., 2019). membrane and the RPE, most likely caused by defective POS processing
Supporting these findings, mice lacking the antioxidant enzyme (Notomi et al., 2019). Deficits at any of the multiple stages of the
SOD2 in the retina display a phenotype similar to that found in humans autophagy process thus lead to RPE dysfunction and retinal degenera­
with AMD: increased autophagy in the early stages of degeneration (1 tion, highlighting the importance of this process in maintaining cellular
month) and blockade of autophagic flux at later stages (6 months) homeostasis. As previously mentioned, AMD patients also present
(Mitter et al., 2014). Mice lacking the antioxidant master regulator impaired autophagy, lysosomal cargo accumulation, and
NFE2L2 or the mitochondrial biogenesis inducer PGC-1α also present autophagy-related proteins in drusen (Golestaneh et al., 2017; Mitter
visual dysfunction associated with primary RPE degeneration. Both KO et al., 2014; Ye et al., 2016). These evidences suggest autophagy as one
mice show damaged mitochondria, mitophagy induction, and accumu­ of the main drivers of AMD pathology, and should therefore be
lation of LC3- and p62-positive autophagosomes in the RPE. This considered both as a cause and as a putative therapeutic target in the
phenotype is further exacerbated in NFE2L2/PGC-1α double KO mice, search for new curative treatments.
which present higher levels of the aforementioned markers, as well as
accumulation of autofluorescent material inside the RPE and extracel­ 9. Conclusions and further challenges
lular drusenoid deposits (Felszeghy et al., 2019; Sridevi Gurubaran
et al., 2020; Zhao et al., 2011). Therefore, a healthy mitochondrial pool The studies reviewed here highlight the significant role of autophagy
and antioxidant response are required to maintain RPE homeostasis, and in sustaining the function of both the neural retina and the RPE, and its
lack of any or both systems leads to AMD-like degeneration concomitant involvement in some of the most prevalent diseases of these structures
with dysregulated autophagy in the RPE. Smoking is one of the main (Fig. 4). Key roles of autophagy proteins in the retina include quality
environmental factors contributing to the development of AMD, and control functions, elimination of toxic aggregates, and facilitation of
exposure of WT mice to cigarette smoke leads to increases in the levels of POS degradation and visual pigment recycling to sustain photoreceptor
ubiquitinated proteins and p62, as well as LC3-II accumulation; function. However, overactivation of autophagy in a cell type-
although autophagic flux was not evaluated and there is a concomitant dependent manner may have detrimental consequences in the context
increase on p62 mRNA levels (Wang et al., 2014). This cigarette of light-induced photoreceptor damage. In RGCs autophagy has been
smoke-mediated induction of autophagy is diminished in implicated in axonal homeostasis and exerts a protective function,
NFE2L2-deficient mice (Wang et al., 2014). These data suggest a pro­ possibly by minimizing ROS levels and sustaining mitochondrial func­
tective role of autophagy upon induction of AMD-like oxidative cellular tion. In certain conditions, blockade of autophagy in a specific subtype
stress in animal and cell models of AMD. of RGCs has been shown to ameliorate the pathogenic phenotype and
attenuate vision loss. Finally, in the RPE autophagy is essential to pre­
8.4. Alterations in the RPE of autophagy-deficient mice serve degradative capacity, provide metabolic support, and ensure
quality control. Thus, it is clear from the literature that alterations in
Selective knock-out of genes in the RPE can be achieved using Cre- autophagy and lysosomal pathways are implicated in many if not all
VMD2 mice, in which the BEST1 promoter is exclusively expressed in diseases of the eye. Moreover, the decline in lysosomal activity associ­
the RPE and testis (Le et al., 2008). RPE-specific deletion of the ca­ ated with age exacerbates alterations in autophagy, potentially aggra­
nonical autophagy initiator RB1CC1 (also known as FIP200) results in vating related conditions. Future studies will undoubtedly help further
primary RPE degeneration, morphological abnormalities, accumulation our understanding of the potential of therapeutic strategies that target
of autofluorescent material, oxidized proteins, and intracellular debris, autophagy and the lysosomal pathway in diseases of the retina and eye.
microglial infiltration, and disruption of Bruch’s membrane upon aging, However, many challenges lie ahead.

10
B. Villarejo-Zori et al. Molecular Aspects of Medicine 82 (2021) 101038

Fig. 4. Main roles of autophagy in retinal cells and in the most prevalent retinal diseases.

According to the WHO blindness is one of the most debilitating observed in Atg5-deficient retinas, underscoring the crucial role of
disabilities, resulting in significant impairment of social activity and chaperone-mediated autophagy in the retina (Rodriguez-Muela et al.,
changes in personality. Globally, cataracts account for the majority of 2013). Exploiting these compensatory changes when other pathways are
cases of blindness in adults aged 50 years and older. However, in 2020 downregulated, either due to mutations or aging, thus constitutes an
other less tractable and irreversible diseases such as glaucoma, diabetic interesting therapeutic avenue. Finally, boosting lysosomal activity is
retinopathy, and AMD collectively accounted for more than 19 million another potential means of simultaneously potentiating macro­
cases of moderate or severe vision impairment in adults aged 50 years autophagy and chaperone-mediated autophagy, and may be the best
and older, making these diseases important targets for prevention and option in cases of generalized lysosomal damage (e.g. caused by exces­
treatment. The fact that the molecular bases of these diseases are not sive ROS production or calcium-dependent calpain activation in pho­
completely understood and the lack of good mouse models hinders the toreceptors) (Rodriguez-Muela et al., 2015). Moreover, therapies that
development of effective treatments. Therefore, identifying both the increase levels of the transcription factor TFEB could prove promising
causes of these diseases and understanding how the retina responds to for the treatment of retinal diseases and other neurodegenerative con­
stress is crucial to facilitate the development of novel, effective therapies ditions (Cortes and La Spada, 2019).
for eye diseases. Finally, there is an urgent need for disease models that better
Another challenge is the complexity of the tissue itself. Retinal dis­ reproduce the alterations observed in human patients with retinal and
eases can primarily affect different cell types that compose the retina, eye diseases. Retinal organoids are a particularly promising tool to
and thus how these respond under pathological conditions may vary improve ophthalmic research (Kruczek and Swaroop, 2020). In
considerably. For example, in the context of autophagy, how a photo­ conclusion, while our knowledge of autophagy in the eye and retina has
receptor deals with toxic waste products in its outer segment, which is increased greatly in the last decade, research must continue apace to
completely devoid of degradative organelles such as lysosomes, is facilitate the development of autophagy-targeting therapies to treat
unique. RGCs are compartmentalized into the soma, axon, and den­ retinal diseases.
drites, each of which are separately metabolically regulated (Casson
et al., 2021). Removal of damaged mitochondria from the soma along Acknowledgements
the long axons of RGCs also poses a complex problem that does not arise
in other retinal cells. Because autophagy plays an important role in Research in the P.B. lab is supported by funding (PGC2018-098557-
removing damaged organelles and proteins in these different cell com­ B-I00) from Spain’s Ministerio Ciencia e Innovación, Agencia Estatal de
partments (Stavoe and Holzbaur, 2019), it is crucial to understand how Investigación (AEI), the Fondo Europeo de Desarrollo Regional
this process is specifically regulated in each compartment. Autophagy (FEDER), the European Union’s Horizon 2020 research and innovation
also provides the building blocks for synapses, and therefore contributes program (grant agreement No 765912), the Fundación Tatiana Pérez de
to synaptic plasticity and neuronal survival (Nikoletopoulou et al., Guzmán el Bueno Proyectos en Neurociencia 2018, and Redes de Bio­
2017). These specific challenges, and the manner in which they are Medicina de la Comunidad de Madrid (BMD-3813). JIJL and JZPM are
resolved in different cell types, can strongly impact the cell’s response to recipients of FPI (Ministerio Ciencia e Innovación) and FPU (Ministerio
stress. More research will be essential to understand how the autophagy de Universidades) fellowships, respectively. We thank O. Howard for
process is specifically regulated in different cell types of the eye. English-language editing.
It is worth also mentioning that autophagy occurs in coordination
with other degradative pathways such as chaperone-mediated auto­ Abbreviations
phagy and the ubiquitin-proteasome system (Koga et al., 2011). For
example, in the retina, chaperone-mediated autophagy compensates for 3-MA 3-methyladenine
the age-associated decreases in (macro)-autophagy, at least for some AMD Age-related macular degeneration
time (Rodriguez-Muela et al., 2013). Similar compensatory effects are AIF Apoptosis-inducing factor

11
B. Villarejo-Zori et al. Molecular Aspects of Medicine 82 (2021) 101038

AMBRA1 Autophagy/beclin-1 regulator 1 Causes of vision loss worldwide, 1990-2010: a systematic analysis. The Lancet.
Global health 1 (6), e339–349.
ARPE-19 Adult retinal pigment epithelial cell line-19
Boya, P., 2017. Why autophagy is good for retinal ganglion cells? Eye (London, England)
Baf-A1 Bafilomycin A1 31 (2), 185–190.
BRB Blood retinal barrier Boya, P., Codogno, P., 2013. Recycling in sight. Nature 501 (7465), 40–42.
DR Diabetic retinopathy Boya, P., Esteban-Martinez, L., Serrano-Puebla, A., Gomez-Sintes, R., Villarejo-Zori, B.,
2016. Autophagy in the eye: development, degeneration, and aging. Prog. Retin. Eye
DOA Dominant optic atrophy Res. 55, 206–245.
GWAS Genome-wide association studies Boya, P., Gonzalez-Polo, R.A., Casares, N., Perfettini, J., Dessen, P., Larochette, N.,
HDAC Histone deacetylate Metivier, D., Meley, D., Souquere, S., Yoshimori, T., Pierron, G., Codogno, P.,
Kroemer, G., 2005. Inhibition of macroautophagy triggers apoptosis. Mol. Cell Biol.
IOP Intraocular pressure 25 (3), 1025–1040.
INL Inner nuclear layer Boya, P., Mellen, M.A., de la Rosa, E.J., 2008. How autophagy is related to programmed
IPL Inner plexiform layer cell death during the development of the nervous system. Biochem. Soc. Trans. 36
(Pt 5), 813–817.
LHON Leber’s hereditary optic atrophy Casson, R.J., Chidlow, G., Crowston, J.G., Williams, P.A., Wood, J.P.M., 2021. Retinal
LC3 Light chain 3 protein energy metabolism in health and glaucoma. Prog. Retin. Eye Res. 81, 100881.
LDLs Low density lipoproteins Casson, R.J., Chidlow, G., Han, G., Wood, J.P., 2013. An explanation for the Warburg
effect in the adult mammalian retina. Clin. Exp. Ophthalmol. 41 (5), 517.
NLRP3 a NOD-like receptor responsible for the processing of pro-IL- Centanin, L., Wittbrodt, J., 2014. Retinal neurogenesis. Development 141 (2), 241–244.
1β to the active form of IL-1β Chan, C.M., Huang, D.Y., Sekar, P., Hsu, S.H., Lin, W.W., 2019. Reactive oxygen species-
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Sci. 26 (1), 40.
OPL Outer plexiform layer Chen, Y., Perusek, L., Maeda, A., 2016. Autophagy in light-induced retinal damage. Exp.
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PE Phosphatidylethanolamine Chen, Y., Sawada, O., Kohno, H., Le, Y.Z., Subauste, C., Maeda, T., Maeda, A., 2013.
Autophagy protects the retina from light-induced degeneration. J. Biol. Chem. 288
PI3P Phosphatidylinositol 3-phosphate (11), 7506–7518.
PI3K Phosphatidylinositol 3-kinase Chinskey, N.D., Besirli, C.G., Zacks, D.N., 2014. Retinal cell death and current strategies
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Choquet, H., Wiggs, J.L., Khawaja, A.P., 2020. Clinical implications of recent advances in
RGCs Retinal ganglion cell primary open-angle glaucoma genetics. Eye (London, England) 34 (1), 29–39.
RPE Retinal-pigmented epithelial Chowers, G., Cohen, M., Marks-Ohana, D., Stika, S., Eijzenberg, A., Banin, E.,
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RGCL RGC layer Conway, O., Akpinar, H.A., Rogov, V.V., Kirkin, V., 2020. Selective autophagy receptors
SI Sodium iodate in neuronal health and disease. J. Mol. Biol. 432 (8), 2483–2509.
TXNIPT hioredoxin-interacting protein Corrochano, S., Barhoum, R., Boya, P., Arroba, A.I., Rodriguez-Muela, N., Gomez-
Vicente, V., Bosch, F., de Pablo, F., de la Villa, P., de la Rosa, E.J., 2008. Attenuation
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Cortes, C.J., La Spada, A.R., 2019. TFEB dysregulation as a driver of autophagy
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