Autophagy and phagocytosis converge for better vision

The retinal pigment epithelium (RPE) is a single layer of nonregenerating cells essential to homeostasis in the retina and the preservation of vision. While the RPE perform a number of important functions, 2 essential processes are phagocytosis, which removes the most distal tips of the photoreceptors to support disk renewal, and the visual cycle, which maintains the supply of chromophore for regeneration of photo-bleached visual pigments. We recently reported that these processes are linked by a noncanonical form of autophagy termed LC3-associated phagocytosis (LAP) in which components of the autophagy pathway are co-opted by phagocytosis to recover vitamin A in support of optimal vision. Here we summarize these findings.     

RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

Macroautophagy/autophagy is an intracellular stress survival and recycling system whereas phagocytosis internalizes material from the extracellular milieu; yet, both pathways utilize lysosomes for cargo degradation. Whereas autophagy occurs in all cells, phagocytosis is performed by cell types such as macrophages and the retinal pigment epithelial (RPE) cells of the eye where it is supported by the noncanonical autophagy process termed LC3-associated phagocytosis (LAP). Autophagy and LAP are distinct pathways that use many of the same mediators and must compete for cellular resources, suggesting that cells may regulate both processes under homeostatic and stress conditions. Our data reveal that RPE cells promote LAP through the expression of RUBCN/Rubicon (RUN domain and cysteine-rich domain containing Beclin 1-interacting protein) and suppress autophagy through the activation of EGFR (epidermal growth factor receptor). In the morning when photoreceptor outer segments (POS) phagocytosis and LAP are highest, RUBCN expression is increased. At the same time, outer segment phagocytosis activates the EGFR resulting in MTOR (mechanistic target of rapamycin [serine/threonine kinase]) stimulation, the accumulation of SQSTM1/p62, and the phosphorylation of BECN1 (Beclin 1, autophagy related) on an inhibitory residue thereby suppressing autophagy. Silencing Rubcn, preventing EGFR activity or directly inducing autophagy in RPE cells by starvation inhibits phagocytic degradation of POS. Thus, RPE cells regulate lysosomal pathways during the critical period of POS phagocytosis to support retinal homeostasis.     

Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling

In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V₀-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.     

Primary cilia in retinal pigment epithelium development and diseases

Retinal pigment epithelium (RPE) is a highly polarized epithelial monolayer lying between the photoreceptor layer and the Bruch membrane. It is essential for vision through participating in many critical activities, including phagocytosis of photoreceptor outer segments, recycling the visual cycle-related compounds, forming a barrier to control the transport of nutrients, ions, and water, and the removal of waste. Primary cilia are conservatively present in almost all the vertebrate cells and acts as a sensory organelle to control tissue development and homeostasis maintenance. Numerous studies reveal that abnormalities in RPE lead to various retinal diseases, such as age-related macular degeneration and diabetic macular oedema, but the mechanism of primary cilia in these physiological and pathological activities remains to be elucidated. Herein, we summarize the functions of primary cilia in the RPE development and the mutations of ciliary genes identified in RPE-related diseases. By highlighting the significance of primary cilia in regulating the physiological and pathological processes of RPE, we aim to provide novel insights for the treatment of RPE-related retinal diseases.     

Autophagy and heterophagy dysregulation leads to retinal pigment epithelium dysfunction and development of age-related macular degeneration

Age-related macular degeneration (AMD) is a complex, degenerative and progressive eye disease that usually does not lead to complete blindness, but can result in severe loss of central vision. Risk factors for AMD include age, genetics, diet, smoking, oxidative stress and many cardiovascular-associated risk factors. Autophagy is a cellular housekeeping process that removes damaged organelles and protein aggregates, whereas heterophagy, in the case of the retinal pigment epithelium (RPE), is the phagocytosis of exogenous photoreceptor outer segments. Numerous studies have demonstrated that both autophagy and heterophagy are highly active in the RPE. To date, there is increasing evidence that constant oxidative stress impairs autophagy and heterophagy, as well as increases protein aggregation and causes inflammasome activation leading to the pathological phenotype of AMD. This review ties together these crucial pathological topics and reflects upon autophagy as a potential therapeutic target in AMD.     

KRT8 phosphorylation regulates the epithelial-mesenchymal transition in retinal pigment epithelial cells through autophagy modulation

Proliferative vitreoretinopathy (PVR) is a severe ocular disease which results in complex retinal detachment and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is considered to be critical in the pathogenesis of PVR. In this study, we focused on the potential impact of keratin 8 (KRT8) phosphorylation and autophagy on TGF-β2–induced EMT of RPE cells and explored the relationship between them. Using immunofluorescence and Western blot analysis, the co-localization of KRT8 and autophagy marker, as well as the abundance of phosphorylated KRT8 (p-KRT8) expression, was observed within subretinal and epiretinal membranes from PVR patients. Moreover, during TGF-β2–induced EMT process, we found that p-KRT8 was enhanced in RPE cells, which accompanied by an increase in autophagic flux. Inhibition of autophagy with pharmacological inhibitors or specific siRNAs was associated with a reduction in cell migration and the synthesis of several EMT markers. In the meantime, we demonstrated that p-KRT8 was correlated with the autophagy progression during the EMT of RPE cells. Knockdown the expression or mutagenesis of the critical phosphorylated site of KRT8 would induce autophagy impairment, through affecting the fusion of autophagosomes and lysosomes. Therefore, this study may provide a new insight into the pathogenesis of PVR and suggests the potential therapeutic value of p-KRT8 in the prevention and treatment of PVR.     

Impacts of two point mutations of RPE65 from Leber's congenital amaurosis on the stability, subcellular localization and isomerohydrolase activity of RPE65

RPE65, a membrane-associated protein in the retinal pigment epithelium, is the isomerohydrolase essential for regenerating 11-cis retinal, the chromophore for visual pigments. RPE65 mutations are associated with inherited retinal dystrophies. Here we report that single point mutations of RPE65, Y144D and P363T, identified in patients with Leber's congenital amaurosis (LCA), significantly decreased the stability of RPE65. Moreover, these mutations altered subcellular localization of RPE65 and abolished its isomerohydrolase activity. These observations suggest that the decreased protein stability and altered subcellular localization of RPE65 may represent a mechanism for these mutations to lead to vision loss in LCA patients.     

Cholesterol-mediated activation of acid sphingomyelinase disrupts autophagy in the retinal pigment epithelium

Autophagy is an essential mechanism for clearing damaged organelles and proteins within the cell. As with neurodegenerative diseases, dysfunctional autophagy could contribute to blinding diseases such as macular degeneration. However, precisely how inefficient autophagy promotes retinal damage is unclear. In this study, we investigate innate mechanisms that modulate autophagy in the retinal pigment epithelium (RPE), a key site of insult in macular degeneration. High-speed live imaging of polarized adult primary RPE cells and data from a mouse model of early-onset macular degeneration identify a mechanism by which lipofuscin bisretinoids, visual cycle metabolites that progressively accumulate in the RPE, disrupt autophagy. We demonstrate that bisretinoids trap cholesterol and bis(monoacylglycero)phosphate, an acid sphingomyelinase (ASMase) cofactor, within the RPE. ASMase activation increases cellular ceramide, which promotes tubulin acetylation on stabilized microtubules. Live-imaging data show that autophagosome traffic and autophagic flux are inhibited in RPE with acetylated microtubules. Drugs that remove excess cholesterol or inhibit ASMase reverse this cascade of events and restore autophagosome motility and autophagic flux in the RPE. Because accumulation of lipofuscin bisretinoids and abnormal cholesterol homeostasis are implicated in macular degeneration, our studies suggest that ASMase could be a potential therapeutic target to ensure the efficient autophagy that maintains RPE health.     

Deletion of autophagy inducer RB1CC1 results in degeneration of the retinal pigment epithelium

Autophagy regulates cellular homeostasis and response to environmental stress. Within the retinal pigment epithelium (RPE) of the eye, the level of autophagy can change with both age and disease. The purpose of this study is to determine the relationship between reduced autophagy and age-related degeneration of the RPE. The gene encoding RB1CC1/FIP200 (RB1-inducible coiled-coil 1), a protein essential for induction of autophagy, was selectively knocked out in the RPE by crossing Best1-Cre mice with mice in which the Rb1cc1 gene was flanked with Lox-P sites (Rb1cc1^flox/flox). Ex vivo and in vivo analyses, including western blot, immunohistochemistry, transmission electron microscopy, fundus photography, optical coherence tomography, fluorescein angiography, and electroretinography were performed to assess the structure and function of the retina as a function of age. Deletion of Rb1cc1 resulted in multiple autophagy defects within the RPE including decreased conversion of LC3-I to LC3-II, accumulation of autophagy-targeted precursors, and increased numbers of mitochondria. Age-dependent degeneration of the RPE occurred, with formation of atrophic patches, subretinal migration of activated microglial cells, subRPE deposition of inflammatory and oxidatively damaged proteins, subretinal drusenoid deposits, and occasional foci of choroidal neovascularization. There was secondary loss of photoreceptors overlying the degenerated RPE and reduction in the electroretinogram. These observations are consistent with a critical role of autophagy in the maintenance of normal homeostasis in the aging RPE, and indicate that disruption of autophagy leads to retinal phenotypes associated with age-related degeneration.     

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.     

Autophagy,exosomes and drusen formation in age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of loss of vision in developed countries. AMD is characterized by a progressive degeneration of the macula of the retina, usually bilateral, leading to a severe decrease in central vision. An early sign of AMD is the appearance of drusen, which are extracellular deposits that accumulate on Bruch’s membrane below the retinal pigment epithelium (RPE). Drusen are a risk factor for developing AMD. Some of the protein components of drusen are known, yet we know little about the processes that lead to formation of drusen. We have previously reported increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we used in vitro modeling of increased mtDNA damage. Under conditions of increased mtDNA damage, autophagy markers and exosome markers were upregulated. In addition, we found autophagy markers and exosome markers in the region of Bruch’s membrane in the retinas of old mice. Furthermore, we found that drusen in AMD donor eyes contain markers for autophagy and for exosomes. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients.     

Oxidative stress in retinal pigment epithelium cells increases exosome secretion and promotes angiogenesis in endothelial cells

The retinal pigment epithelium (RPE), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species (ROS). As the RPE, because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell‐to‐cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors (VEGFR) were analysed by both flow cytometry and Western blot. RT‐PCR and qPCR were conducted to assess mRNA content of VEGFRs in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA. Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes.     

Effects of white light‐emitting diode (LED) exposure on retinal pigment epithelium in vivo

Ageing and alteration of the functions of the retinal pigment epithelium (RPE) are at the origin of lost of vision seen in age‐related macular degeneration (AMD). The RPE is known to be vulnerable to high‐energy blue light. The white light‐emitting diodes (LED) commercially available have relatively high content of blue light, a feature that suggest that they could be deleterious for this retinal cell layer. The aim of our study was to investigate the effects of “white LED” exposure on RPE. For this, commercially available white LEDs were used for exposure experiments on Wistar rats. Immunohistochemical stain on RPE flat mount, transmission electron microscopy and Western blot were used to exam the RPE. LED‐induced RPE damage was evaluated by studying oxidative stress, stress response pathways and cell death pathways as well as the integrity of the outer blood–retinal barrier (BRB). We show that white LED light caused structural alterations leading to the disruption of the outer blood–retinal barrier. We observed an increase in oxidized molecules, disturbance of basal autophagy and cell death by necrosis. We conclude that white LEDs induced strong damages in rat RPE characterized by the breakdown of the BRB and the induction of necrotic cell death.     

A mutation in the silver gene leads to defects in melanosome biogenesis and alterations in the visual system in the zebrafish mutant fading vision

Forward genetic screens have been instrumental in defining molecular components of visual function. The zebrafish mutant fading vision (fdv) has been identified in such a screen due to defects in vision accompanied by hypopigmentation in the retinal pigment epithelium (RPE) and body melanocytes. The RPE forms the outer most layer of the retina, and its function is essential for vision. In fdv mutant larvae, the outer segments of photoreceptors are strongly reduced in length or absent due to defects in RPE cells. Ultrastructural analysis of RPE cells reveals dramatic cellular changes such as an absence of microvilli and vesicular inclusions. The retinoid profile is altered as judged by biochemical analysis, arguing for a partial block in visual pigment regeneration. Surprisingly, homozygous fdv vision mutants survive to adulthood and show, despite a persistence of the hypopigmentation, a partial recovery of retinal morphology. By positional cloning and subsequent morpholino knock-down, we identified a mutation in the silver gene as the molecular defect underlying the fdv phenotype. The Silver protein is required for intralumenal fibril formation in melanosomes by amylogenic cleavage. Our data reveal an unexpected link between melanosome biogenesis and the visual system, undetectable in cell culture.     

Activated monocytes resist elimination by retinal pigment epithelium and downregulate their OTX2 expression via TNF‐α

Orthodenticle homeobox 2 (OTX2) controls essential, homeostatic retinal pigment epithelial (RPE) genes in the adult. Using cocultures of human CD14⁺ blood monocytes (Mos) and primary porcine RPE cells and a fully humanized system using human‐induced pluripotent stem cell‐derived RPE cells, we show that activated Mos markedly inhibit RPEOTX2 expression and resist elimination in contact with the immunosuppressive RPE. Mechanistically, we demonstrate that TNF‐α, secreted from activated Mos, mediates the downregulation of OTX2 and essential RPE genes of the visual cycle among others. Our data show how subretinal, chronic inflammation and in particular TNF‐α can affect RPE function, which might contribute to the visual dysfunctions in diseases such as age‐related macular degeneration (AMD) where subretinal macrophages are observed. Our findings provide important mechanistic insights into the regulation of OTX2 under inflammatory conditions. Therapeutic restoration of OTX2 expression might help revive RPE and visual function in retinal diseases such as AMD.     

Hyperbaric oxygen therapy suppresses hypoxia and reoxygenation injury to retinal pigment epithelial cells through activating peroxisome proliferator activator receptor-alpha signalling

Retinal ischemia followed by reperfusion (IR) is a common cause of many ocular disorders, such as age-related macular degeneration (AMD), which leads to blindness in the elderly population, and proper therapies remain unavailable. Retinal pigment epithelial (RPE) cell death is a hallmark of AMD. Hyperbaric oxygen (HBO) therapy can improve IR tissue survival by inducing ischemic preconditioning responses. We conducted an in vitro study to examine the effects of HBO preconditioning on oxygen–glucose deprivation (OGD)-induced IR-injured RPE cells. RPE cells were treated with HBO (100% O₂ at 3 atmospheres absolute for 90 min) once a day for three consecutive days before retinal IR onset. Compared with normal cells, the IR-injured RPE cells had lower cell viability, lower peroxisome proliferator activator receptor-alpha (PPAR-α) expression, more severe oxidation status, higher blood-retinal barrier disruption and more elevated apoptosis and autophagy rates. HBO preconditioning increased PPAR-α expression, improved cell viability, decreased oxidative stress, blood-retinal barrier disruption and cellular apoptosis and autophagy. A specific PPAR-α antagonist, GW6471, antagonized all the protective effects of HBO preconditioning in IR-injured RPE cells. Combining these observations, HBO therapy can reverse OGD-induced RPE cell injury by activating PPAR-α signalling.     

Di-retinoid-pyridinium-ethanolamine (A2E) Accumulation and the Maintenance of the Visual Cycle Are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium

Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7^flox/flox;VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7-deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7-deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7^flox/flox;VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E accumulation. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4^−/− and Abca4^+/+ backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation.     

Identification of conserved histidines and glutamic acid as key residues for isomerohydrolase activity of RPE65, an enzyme of the visual cycle in the retinal pigment epithelium

We have recently reported that RPE65 from the retinal pigment epithelium is the isomerohydrolase, a critical enzyme in the visual cycle for regeneration of 11-cis retinal, the chromophore for visual pigments. Here, we demonstrated that mutation of any one of the absolutely conserved four histidine and one glutamic acid residues to alanine in RPE65 abolished its isomerohydrolase activity. Substitution of the conserved glutamic acid with glutamine also resulted in loss of the activity. Moreover, these mutations significantly reduced protein stability of RPE65. These results indicate that these conserved residues are essential for the isomerohydrolase activity of RPE65 and its stability.     

Palmitoyl transferase activity of lecithin retinol acyl transferase

Lecithin retinol acyl transferase (LRAT) has the essential role of catalyzing the transfer of an acyl group from the sn-1 position of lecithin to vitamin A to generate all-trans-retinyl esters (tREs). In vitro studies had shown previously that LRAT also can exchange palmitoyl groups between RPE65, a tRE binding protein essential for vision, and tREs. This exchange is likely to be of regulatory significance in the operation of the visual cycle. In the current study, the substrate specificity of LRAT is explored with palmitoylated amino acids and dipeptides as RPE65 surrogates. Both O- and S-substituted palmitoylated analogues are excellent substrates for tLRAT, a readily expressed and readily purified form of LRAT. Using vitamin A as the palmitoyl acceptor, tREs are readily formed. The cognate of these reactions occurs in crude retinal pigment epithelial (RPE) membranes as well. RPE membranes containing LRAT transfer palmitoyl groups from radiolabeled [1-(14)C]-l-alpha-dipalmitoyl diphosphatidylcholine (DPPC) to RPE65. Palmitoyl transfer is abolished by preincubation with a specific LRAT antagonist both in membranes and with purified tLRAT. These experiments are consistent with an expanded role for LRAT function as a protein palmitoyl transferase.