Retinal Pigment Epithelium Phagocytosis Research Articles

Taurine influences the phagocytic function of retinal pigment epithelium cells in healthy and dystrophic animals

Aims/Purpose: To investigate the influence of taurine on the phagocytic function of the retinal pigment epithelium (RPE) in normal and dystrophic rats.Methods: Normal albino Sprague–Dawley (SD) and pigmented dystrophic Royal College of Surgeons (RCS‐p+) rats that suffer an inherited photoreceptor degeneration due to RPE phagocytosis impairment were used for the study. Taurine depletion was induced in SD rats (n = 8) by providing β‐alanine in drinking water (3%) from postnatal day 21 (P21). Taurine supplementation was administered to RCS‐p+ rats (n = 8) in drinking water (0.2 M) from P21. Untreated RCSp+ (n = 8) and SD (n = 8) rats served as control groups. Rats were processed at P81 (SD) and at P45 (RCS) and received an intravitreal injection (IV) of. 1.5 μL of Fluorogold (FG, 3%) diluted in saline 24 h before processing. The RPE was dissected, flat‐mounted and immunodetected with anti‐S opsin antibodies to label the RPE phagosomes which were quantified using ImageJ and size criteria.Results: The mean numbers (± SD) of phagosomes per RPE whole mount was 414 ± 118 in control SD rats, 1034 ± 528 in taurine‐depleted SD rats 88 ± 12 in untreated RCS and 119 ± 9 in taurine‐treated RCS rats. The numbers of phagosomes were significantly higher in SD rats than in RCS‐p+ rats (t‐test, p < 0.0001) documenting the impaired phagocytic function of RCS‐p+ rats. In SD rats, the number of phagosomes were significantly higher in taurine‐depleted rats evidencing that taurine depletion decreases the RPE phagocytic capacity (t‐test, p < 0.0059). Finally, in RCSp+ rats, the numbers of phagosomes were significantly higher in taurine‐treated than in non‐treated RCS rats (t‐test, p < 0.0001) indicating that taurine supplementation ameliorates the phagocytic capacity of the RPE cells in RCS‐p+ rats.Conclusions: Taurine influences the phagocytic capacity of the RPE cells in normal rats and in dystrophic RCS‐p+ rats.

A novel quantification method for retinal pigment epithelium phagocytosis using a very-long-chain polyunsaturated fatty acids-based strategy.

The vertebrate retinal pigment epithelium (RPE) lies adjacent to the photoreceptors and is responsible for the engulfment and degradation of shed photoreceptor outer segment fragments (POS) through receptor-mediated phagocytosis. Phagocytosis of POS is critical for maintaining photoreceptor function and is a key indicator of RPE functionality. Popular established methods to assess RPE phagocytosis rely mainly on quantifying POS proteins, especially their most abundant protein rhodopsin, or on fluorescent dye conjugation of bulk, unspecified POS components. While these approaches are practical and quantitative, they fail to assess the fate of POS lipids, which make up about 50% of POS by dry weight and whose processing is essential for life-long functionality of RPE and retina. We have developed a novel very-long-chain polyunsaturated fatty acids (VLC-PUFA)-based approach for evaluating RPE phagocytic activity by primary bovine and rat RPE and the human ARPE-19 cell line and validated its results using traditional methods. This new approach can be used to detect in vitro the dynamic process of phagocytosis at varying POS concentrations and incubation times and offers a robust, unbiased, and reproducible assay that will have utility in studies of POS lipid processing.

Microglia preserve visual function in the aging retina by supporting retinal pigment epithelial health

BackgroundIncreased age is a risk factor for the development and progression of retinal diseases including age-related macular degeneration (AMD). Understanding the changes that occur in the eye due to aging is important in enhancing our understanding of AMD pathogenesis and the development of novel AMD therapies. Microglia, the resident brain and retinal immune cells are associated with both maintaining homeostasis and protection of neurons and loss of microglia homeostasis could be a significant player in age related neurodegeneration. One important characteristic of retinal aging is the migration of microglia from the inner to outer retina where they reside in the subretinal space (SRS) in contact with the retinal pigment epithelial (RPE) cells. The role of aged subretinal microglia is unknown. Here, we depleted microglia in aged C57/BL6 mice fed for 6 weeks with a chow containing PLX5622, a small molecule inhibitor of colony-stimulating factor-1 receptor (Csf1r) required for microglial survival.ResultsThe subretinal P2RY12 + microglia in aged mice displayed a highly amoeboid and activated morphology and were filled with autofluorescence droplets reminiscent of lipofuscin. TEM indicates that subretinal microglia actively phagocytize shed photoreceptor outer segments, one of the main functions of retinal pigmented epithelial cells. PLX5622 treatment depleted up to 90% of the retinal microglia and was associated with significant loss in visual function. Mice on the microglia depletion diet showed reduced contrast sensitivity and significantly lower electroretinogram for the c-wave, a measurement of RPE functionality, compared to age-matched controls. The loss of c-wave coincided with a loss of RPE cells and increased RPE swelling in the absence of microglia.ConclusionsWe conclude that microglia preserve visual function in aged mice and support RPE cell function, by phagocytosing shed photoreceptor outer segments and lipids, therefore compensating for the known age-related decline of RPE phagocytosis.

Loss of Prom1 impairs autophagy and promotes epithelial-mesenchymal transition in mouse retinal pigment epithelial cells.

Mutations in the Prominin-1 (Prom1) gene disrupt photoreceptor disk morphogenesis, leading to macular dystrophies. We have shown that human retinal pigment epithelial (RPE) homeostasis is under the control of Prom1-dependent autophagy, demonstrating that Prom1 plays different roles in the photoreceptors and RPE. It is unclear if retinal and macular degeneration caused by the loss of Prom1 function is a cell-autonomous feature of the RPE or a generalized disease of photoreceptor degeneration. In this study, we investigated whether Prom1 is required for mouse RPE (mRPE) autophagy and phagocytosis, which are cellular processes essential for photoreceptor survival. We found that Prom1-KO decreases autophagy flux, activates mTORC1, and concomitantly decreases transcription factor EB (TFEB) and Cathepsin-D activities in mRPE cells. In addition, Prom1-KO reduces the clearance of bovine photoreceptor outer segments in mRPE cells due to increased mTORC1 and reduced TFEB activities. Dysfunction of Prom1-dependent autophagy correlates with both a decrease in ZO-1 and E-cadherin and a concomitant increase in Vimentin, SNAI1, and ZEB1 levels, consistent with induction of epithelial-mesenchymal transition (EMT) in Prom1-KO mRPE cells. Our results demonstrate that Prom1-mTORC1-TFEB signaling is a central driver of cell-autonomous mRPE homeostasis. We show that Prom1-KO in mRPE leads to RPE defects similar to that seen in atrophic age-related macular degeneration and opens new avenues of investigation targeting Prom1 in retinal degenerative diseases.

Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis.

Phagocytosis is one of the key functions of retinal pigment epithelium (RPE) cells, which maintain photoreceptor health by removing photoreceptor outer segments (POSs) that are regularly shed. A deficiency in RPE function to phagocytose POSs may lead to vision loss in inherited retinal diseases and eventually to age-related macular degeneration (AMD) with geographic atrophy. Significant progress has been made in the field of cell replacement therapy for AMD using stem-cell-derived RPE. To test their function, RPE cells are incubated with purified bovine POSs for the demonstration of efficient binding, internalization, and digestion of POSs. Here, we present an image-based method to measure phagocytosis activity by using POSs labeled with a pH-sensitive fluorescent dye, which has low fluorescence at neutral pH outside of the cell and high fluorescence at low pH inside the phagosome. Further, we introduce a unique flow-cytometry-based method for the characterization of POSs by measuring specific markers for POSs such as rhodopsin and opsin. Using this method, we demonstrated a comparable quality of several bovine POS isolation batches and a reliable assessment of POS quality on RPE phagocytosis assay performance when subjected to different stress conditions. This work provides new tools to characterize POSs and insight into RPE phagocytosis assay development for the functional evaluation of RPE cells in the field of cell replacement therapy.

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures.

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

Biallelic CLCN2 mutations cause retinal degeneration by impairing retinal pigment epithelium phagocytosis and chloride channel function

CLCN2 encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association between Clcn2and the retina is well-established in mice, as loss-of-function of CLC-2can cause retinopathy in mice; however, the ocular phenotypes caused by CLCN2 mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms of CLCN2 variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsense CLCN2 mutation was followed up for > 6years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro, mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying the CLCN2 mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of the CLCN2 mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelic CLCN2 variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of human CLCN2-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.

Phagocytosis in the retina promotes local insulin production in the eye.

The retina is highly metabolically active, relying on glucose uptake and aerobic glycolysis. Situated in close contact to photoreceptors, a key function of cells in the retinal pigment epithelium (RPE) is phagocytosis of damaged photoreceptor outer segments (POS). Here we identify RPE as a local source of insulin in the eye that is stimulated by POS phagocytosis. We show that Ins2 messenger RNA and insulin protein are produced by RPE cells and that this production correlates with RPE phagocytosis of POS. Genetic deletion of phagocytic receptors ('loss of function') reduces Ins2, whereas increasing the levels of the phagocytic receptor MerTK ('gain of function') increases Ins2 production in male mice. Contrary to pancreas-derived systemic insulin, RPE-derived local insulin is stimulated during starvation, which also increases RPE phagocytosis. Global or RPE-specific Ins2 gene deletion decreases retinal glucose uptake in starved male mice, dysregulates retinal physiology, causes defects in phototransduction and exacerbates photoreceptor loss in a mouse model of retinitis pigmentosa. Collectively, these data identify RPE cells as a phagocytosis-induced local source of insulin in the retina, with the potential to influence retinal physiology and disease.

Evaluation of CRISPR/Cas9 exon-skipping vector for choroideremia using human induced pluripotent stem cell-derived RPE.

Exon-skipping is a powerful genetic tool, especially when delivering genes using an AAV-mediated full-length gene supplementation strategy is difficult owing to large length of genes. Here, we used engineered human induced pluripotent stem cells and artificial intelligence to evaluate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9-based exon-skipping vectors targeting genes of the retinal pigment epithelium (RPE). The model system was choroideremia; this is an X-linked inherited retinal disease caused by mutation of the CHM gene. We explored whether artificial intelligence detected differentiation of human OTX2, PAX6 and MITF (hOPM) cells, in which OTX2, PAX6 and MITF expression was induced by doxycycline treatment, into RPE. Plasmid encoding CHM exon-skipping modules targeting the splice donor sites of exons 6 were constructed. A clonal hOPM cell line with a frameshift mutation in exon 6 was generated and differentiated into RPE. CHM exon 6-skipping was induced, and the effects of skipping on phagocytic activity, cell death and prenylation of Rab small GTPase (RAB) were evaluated using flow cytometry, an in vitro prenylation assay and western blotting. Artificial intelligence-based evaluation of RPE differentiation was successful. Retinal pigment epithelium cells with a frameshift mutation in exon 6 showed increased cell death, reduced phagocytic activity and increased cytosolic unprenylated RABs only when oxidative stress was in play. The latter two phenotypes were partially rescued by exon 6-skipping of CHM. CHM exon 6-skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress.

Does taurine have a role in the phagocytic function of the retinal pigment epithelium?

AbstractPurpose: To study the role of taurine in the morphology and phagocytic function of the retinal pigment epithelium (RPE) in normal and dystrophic rats.Methods: Albino Sprague–Dawley (SD) rats and pigmented dystrophic Royal College of Surgeons (RCS‐p+) rats that suffer a severe form of inherited photoreceptor degeneration due to RPE phagocytosis impairment, were used for this study. SD rats received β‐alanine in drinking water (3%) for 2 months from P21 to induce taurine depletion and RCS‐p + rats received taurine supplementation in drinking water (0.2 M) for 24 days from P21. Groups of untreated RCS and SD rats were used as control. SD rats were processed 2 months after the start of treatment, while RCS rats were processed at P45. Twenty‐four hours before processing, the animals received an intravitreal injection of 1.5 μl of 3% fluorogold (FG) diluted in saline 24 to label the RPE cells. Animals were processed and the RPE was dissected flat‐mounted and observed using a confocal microscope (Leica SP8).Results: In control SD animals, Fluorogold (FG) accumulates homogeneously in the cytoplasm of the RPE cells. Taurine‐depleted SD animals showed less FG accumulation in the cytoplasm in all cells and, and some cells had FG accumulation only in part of the cytoplasm or no FG accumulation at all. Untreated RCS‐p + animals showed an inverted accumulation of FG because it appeared mainly in the membrane of the RPE but not in the cytoplasm. Finally, taurine‐treated RCS‐p + animals showed FG accumulation in the cytoplasm of the RPE cells, although the accumulation of FG was smaller than that found in SD rats.Conclusions: Taurine influences the phagocytic capacity of the RPE cells. Taurine depletion impairs the phagocytic capacity of the RPE cells in healthy retinas, and taurine supplementation improves the phagocytic function of RPE cells in dystrophic RCS‐p + rats.

Subretinal application of cell cultures: Surgical methods and its transition to the use in human

AbstractThe progression of age‐related macular degeneration (AMD) results in irreversible and untreatable impairment of central vision. The formation of the drusen and subsequent atrophy of in the retinal pigmented epithelium (RPE) with development of neovascular membranes remain the typical features of AMD pathogenesis. As the result of the disorganization of the RPE the death photoreceptor cell develops, which is associated with altered RPE phagocytosis, increased RPE permeability, formation of choroidal neovascularization, and involvement of resident and monocytic microglial cells. Nevertheless, the exact cause of the RPE degeneration is still not fully understood. Up to date there is no therapeutic approach, which could reverse the course of AMD progression. Injection of anti‐vascular endothelial growth factors (VEGFs), laser‐photo coagulation and photodynamic therapy of the neovascularisations are being widely used to treat the complications of exudative AMD and therefore to prevent visual loss. Since few decades, the concept of substitutional cell‐based regenerative treatment has been investigated by many groups. It could offer in the near future an alternative approach to restore impaired vision in AMD. Th studies have shown that subretinal implantation of RPE cells seeded on a biodegradable scaffold enhances the integration of the donor cells compared to the injection of cell‐suspension as method of delivery. This could improve the differentiation of donor RPE cells into the host retina and help with the proper cellularization of the RPE monolayer. However, the implantation techniques including surgical procedure with exposion of subretinal space and the development and use of delivery instruments remains challenging. Experimental studies with subretinal RPE transplantation are focused on development of optimal surgical techniques, which could be easily transferred to the application in humans. The cultivation and reproducible viability of hRPEs for transplantation purposes on biodegradable scaffolds for human transplantation shall correspond and be adapted to the instrumentation used during the surgery. Hence, the advances in the methodology and the results of improved approaches shall be investigated further.

Systemic taurine treatment affords functional and morphological neuroprotection of photoreceptors and restores retinal pigment epithelium function in RCS rats

The aim of our work was to study whether taurine administration has neuroprotective effects in dystrophic Royal College of Surgeons (RCS) rats, suffering retinal degeneration secondary to impaired retinal pigment epithelium phagocytosis caused by a MERTK mutation. Dystrophic RCS-p + female rats (n = 36) were divided into a non-treated group (n = 16) and a treated group (n = 20) that received taurine (0.2 M) in drinking water from postnatal day (P)21 to P45, when they were processed. Retinal function was assessed with electroretinogram. Retinal morphology was assessed in cross-sections using immunohistochemical techniques to label photoreceptors, retinal microglial and macroglial cells, active zones of conventional and ribbon synaptic connections, and oxidative stress. Retinal pigment epithelium function was examined using intraocular fluorogold injections. Our results document that taurine treatment increases taurine plasma levels and photoreceptor survival in dystrophic rats. The number of photoreceptor nuclei rows at P45 was 3–5 and 6–11 in untreated and treated animals, respectively. Electroretinograms showed increases of 70% in the rod response, 400% in the a-wave amplitude, 30% in the b-wave amplitude and 75% in the photopic b-wave response in treated animals. Treated animals also showed decreased numbers of microglial cells in the outer retinal layers, decreased glial fibrillary acidic protein (GFAP) expression in Müller cells, decreased oxidative stress in the outer and inner nuclear layers and improved maintenance of synaptic connections. Treated animals showed increased FG phagocytosis in the retinal pigment epithelium cells. In conclusion, systemic taurine treatment decreases photoreceptor degeneration and increases electroretinographic responses in dystrophic RCS rats and these effects may be mediated through various neuroprotective mechanisms.

Targeting Phospholipase D Pharmacologically Prevents Phagocytic Function Loss of Retinal Pigment Epithelium Cells Exposed to High Glucose Levels.

We previously described the participation of canonical phospholipase D isoforms (PLD1 and PLD2) in the inflammatory response of retinal pigment epithelium (RPE) cells exposed to high glucose concentrations (HG). Here, we studied the role of the PLD pathway in RPE phagocytic function. For this purpose, ARPE-19 cells were exposed to HG (33 mM) or to normal glucose concentration (NG, 5.5 mM) and phagocytosis was measured using pHrodo™ green bioparticles® or photoreceptor outer segments (POS). HG exposure for 48 and 72 h reduced phagocytic function of ARPE-19 cells, and this loss of function was prevented when cells were treated with 5 μM of PLD1 (VU0359595 or PLD1i) or PLD2 (VU0285655-1 or PLD2i) selective inhibitors. Furthermore, PLD1i and PLD2i did not affect RPE phagocytosis under physiological conditions and prevented oxidative stress induced by HG. In addition, we demonstrated PLD1 and PLD2 expression in ABC cells, a novel human RPE cell line. Under physiological conditions, PLD1i and PLD2i did not affect ABC cell viability, and partial silencing of both PLDs did not affect ABC cell POS phagocytosis. In conclusion, PLD1i and PLD2i prevent the loss of phagocytic function of RPE cells exposed to HG without affecting RPE function or viability under non-inflammatory conditions.

Spatiotemporal control of actomyosin contractility by MRCKβ signaling drives phagocytosis

Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKβ/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKβ/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKβ is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKβ signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKβ is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKβ signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK.

Differences in Diurnal Rhythm of Rod Outer Segment Renewal between 129T2/SvEmsJ and C57BL/6J Mice.

In all mammalian species tested to date, rod photoreceptor outer segment renewal is a circadian process synchronized by light with a burst of outer segment fragment (POS) shedding and POS phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning at light onset. Recent reports show that RPE phagocytosis also increases shortly after dark onset in C57BL/6 (C57) mice. Genetic differences between C57 mice and 129T2/SvEmsJ (129) mice may affect regulation of outer segment renewal. Here, we used quantitative methods to directly compare outer segment renewal in C57 and 129 mouse retina. Quantification of rhodopsin-positive phagosomes in the RPE showed that in 129 mice, rod POS phagocytosis after light onset was significantly increased compared to C57 mice, but that 129 mice did not show a second peak after dark onset. Cone POS phagosome content of RPE cells did not differ by mouse strain with higher phagosome numbers after light than after dark. We further quantified externalization of the “eat me” signal phosphatidylserine by outer segment tips, which precedes POS phagocytosis. Live imaging of retina ex vivo showed that rod outer segments extended PS exposure in both strains but that frequency of outer segments with exposed PS after light onset was lower in C57 than in 129 retina. Taken together, 129 mice lacked a burst of rod outer segment renewal after dark onset. The increases in rod outer segment renewal after light and after dark onset in C57 mice were attenuated compared to the peak after light onset in 129 mice, suggesting an impairment in rhythmicity in C57 mice.

PEDF Deletion Induces Senescence and Defects in Phagocytosis in the RPE.

The retinal pigment epithelium (RPE) expresses the Serpinf1 gene to produce pigment epithelium-derived factor (PEDF), a retinoprotective protein that is downregulated with cell senescence, aging and retinal degenerations. We determined the expression of senescence-associated genes in the RPE of 3-month-old mice that lack the Serpinf1 gene and found that Serpinf1 deletion induced H2ax for histone H2AX protein, Cdkn1a for p21 protein, and Glb1 gene for β-galactosidase. Senescence-associated β-galactosidase activity increased in the Serpinf1 null RPE when compared with wild-type RPE. We evaluated the subcellular morphology of the RPE and found that ablation of Serpinf1 increased the volume of the nuclei and the nucleoli number of RPE cells, implying chromatin reorganization. Given that the RPE phagocytic function declines with aging, we assessed the expression of the Pnpla2 gene, which is required for the degradation of photoreceptor outer segments by the RPE. We found that both the Pnpla2 gene and its protein PEDF-R declined with the Serpinf1 gene ablation. Moreover, we determined the levels of phagocytosed rhodopsin and lipids in the RPE of the Serpinf1 null mice. The RPE of the Serpinf1 null mice accumulated rhodopsin and lipids compared to littermate controls, implying an association of PEDF deficiency with RPE phagocytosis dysfunction. Our findings establish PEDF loss as a cause of senescence-like changes in the RPE, highlighting PEDF as both a retinoprotective and a regulatory protein of aging-like changes associated with defective degradation of the photoreceptor outer segment in the RPE.

Arap1 loss causes retinal pigment epithelium phagocytic dysfunction and subsequent photoreceptor death.

ABSTRACTRetinitis pigmentosa (RP), a retinal degenerative disease, is the leading cause of heritable blindness. Previously, we described that Arap1−/− mice develop a similar pattern of photoreceptor degeneration. Arap1 is an Arf-directed GTPase-activating protein shown to modulate actin cytoskeletal dynamics. Curiously, Arap1 expression was detected in Müller glia and retinal pigment epithelium (RPE), but not the photoreceptors themselves. In this study, we generated conditional knockout mice for Müller glia/RPE, Müller glia and RPE via targeting Rlbp1, Glast and Vmd2 promoters, respectively, to drive Cre recombinase expression to knock out Arap1. Vmd2-Cre Arap1tm1c/tm1c and Rlbp1-Cre Arap1tm1c/tm1c mice, but not Glast-Cre Arap1tm1c/tm1c mice, recapitulated the phenotype originally observed in germline Arap1−/− mice. Mass spectrometry analysis of human ARAP1 co-immunoprecipitation identified candidate binding partners of ARAP1, revealing potential interactants involved in phagocytosis, cytoskeletal composition, intracellular trafficking and endocytosis. Quantification of outer segment phagocytosis in vivo demonstrated a clear phagocytic defect in Arap1−/− mice compared to Arap1+/+ controls. We conclude that Arap1 expression in RPE is necessary for photoreceptor survival due to its indispensable function in RPE phagocytosis. This article has an associated First Person interview with the first author of the paper.

Microglia Prevent the Loss of Visual Function in the Aging Retina by Maintaining the Health of Retinal Pigment Epithelial Cells

Even in healthy individuals, aging leads to the progressive deterioration of visual function that coincides with increasing cellular dysfunction in retinal neurons and retinal pigment epithelial (RPE) cells. Interestingly, aging also coincides with the migration of microglia from the inner to the outer retina where they reside in the subretinal space between photoreceptor outer segments and the apical surface of RPE. Healthy young microglia act as the immune sentinels of the retina, and thus are protective and maintain retinal homeostasis. However, age alters cellular functions and the role of aging subretinal microglia is not known. Here we investigated whether aged microglia residing in the subretinal space are either preventing or promoting loss of visual function.Microglia were depleted in 33‐month‐old C57/BL6 mice fed for 6 weeks with a chow containing PLX5622, a small molecule inhibitor of colony‐stimulating factor‐1 receptor (Csf1r) which is expressed by microglia and is required for their proliferation, differentiation, and survival. As a negative control, age‐matched mice received a control diet. Following microglia depletion, the optomotor reflex (OMR) was used to assess visual acuity and contrast sensitivity and electroretinogram (ERG) assessed retinal function. Morphological analysis was determined by immunohistochemical staining and TEM.To distinguish microglia from infiltrating macrophages, retinas from old mice were stained with P2RY12, a microglia‐specific marker. The vast majority of cells residing between the photoreceptor outer segments and RPE layer in old mice were P2RY12+ microglia. These cells displayed a highly amoeboid and activated morphology and were filled with autofluorescence droplets similar to lipofuscin. PLX5622 treatment depleted up to 90% of the microglia. Microglia preserved visual function, and in the absence of microglia, there was a significant loss of contrast sensitivity and ERG c‐wave, as compared with untreated controls. Loss of microglia coincided with a loss of RPE cells and increased RPE swelling indicating that microglia support RPE in aged animals. TEM indicates that subretinal microglia actively phagocytize shed photoreceptor outer segments, thereby compensating for the known age‐related decline of RPE phagocytosis.We conclude that microglia in aging mice migrate to the subretinal space of the outer retina to support and maintain RPE cell functions that preserves vision.

Mitochondria dynamics in the aged mice eye and the role in the RPE phagocytosis

Aging is a predominant risk factor for various eye diseases. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, and its etiology remains unclear. Fragmented and dysfunctional mitochondria are associated with age-related diseases. The retinal pigment epithelium (RPE), a polarized cell layer that functions in visual pigment recycling and degeneration, is suspected as the primary region site of AMD. In the present study, we investigated the relationship between mitochondrial dysfunction and RPE aging. Compared to young mice, aged pigmented mice (C57BL/6J, 12-month-old) exhibit decreased visual function without retinal thinning. Consistently, the rhodopsin expression level decreased in the outer segment of aged mice. Moreover, the cell volume of the RPE increased in aged animals. Interestingly, the expression of mitochondria dynamics-related proteins, including Drp1, was altered in the RPE-choroid complex but not in the neural retina after aging. Electron microscopy revealed that mitochondrial size decreased and cristae width increased in aged RPE. The photoreceptor outer segment (POS) treatment of ARPE-19 cells causes Drp1 activation. Furthermore, pharmacological suppression of mitochondrial fission improved the phagocytosis of the POS. These findings indicate that mitochondrial dysfunction and fission in RPE impede phagocytosis and cause retardation of the visual cycle, which can be one of the age-related defects in the retina that may contribute to the onset of AMD.

Lack of the antioxidant enzyme methionine sulfoxide reductase A in mice impairs RPE phagocytosis and causes photoreceptor cone dysfunction

Methionine sulfoxide reductase A (MsrA) is a widely expressed antioxidant enzyme that counteracts oxidative protein damage and contributes to protein regulation by reversing oxidation of protein methionine residues. In retinal pigment epithelial (RPE) cells in culture, MsrA overexpression increases phagocytic capacity by supporting mitochondrial ATP production. Here, we show elevated retinal protein carbonylation indicative of oxidation, decreased RPE mitochondrial membrane potential, and attenuated RPE phagocytosis in msra−/− mice. Moreover, electroretinogram recordings reveal decreased light responses specifically of cone photoreceptors despite normal expression and localization of cone opsins. Impairment in msra−/− cone-driven responses is similar from 6 weeks to 13 months of age. These functional changes match dramatic decreases in lectin-labeled cone sheaths and reduction in cone arrestin in msra−/− mice. Strikingly, cone defects in light response and in lectin-labeled cone sheath are completely prevented by dark rearing. Together, our data show that msra−/− mice provide a novel small animal model of preventable cone-specific photoreceptor dysfunction that may have future utility in analysis of cone dystrophy disease mechanisms and testing therapeutic approaches aiming to alleviate cone defects.