Phagocytosis Of Photoreceptor Outer Segments Research Articles

Retinal atrophy, inflammation, phagocytic and metabolic disruptions develop in the MerTK-cleavage-resistant mouse model.

In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of photoreceptors (PRs) and vision. Among those, RPE cells phagocytose under a circadian rhythm photoreceptor outer segment (POS) tips that are constantly subjected to light rays and oxidative attacks. The MerTK tyrosine kinase receptor is a key element of this phagocytic machinery required for POS internalization. Recently, we showed that MerTK is subjected to the cleavage of its extracellular domain to finely control its function. In addition, monocytes in retinal blood vessels can migrate inside the inner retina and differentiate into macrophages expressing MerTK, but their role in this context has not been studied yet. We thus investigated the ocular phenotype of MerTK cleavage-resistant (MerTKCR) mice to understand the relevance of this characteristic on retinal homeostasis at the RPE and macrophage levels. MerTKCR retinae appear to develop and function normally, as observed in retinal sections, by electroretinogram recordings and optokinetic behavioral tests. Monitoring of MerTKCR and control mice between the ages of 3 and 18 months showed the development of large degenerative areas in the central retina as early as 4 months when followed monthly by optical coherence tomography (OCT) plus fundus photography (FP)/autofluorescence (AF) detection but not by OCT alone. The degenerative areas were associated with AF, which seems to be due to infiltrated macrophages, as observed by OCT and histology. MerTKCR RPE primary cultures phagocytosed less POS in vitro, while in vivo, the circadian rhythm of POS phagocytosis was deregulated. Mitochondrial function and energy production were reduced in freshly dissected RPE/choroid tissues at all ages, thus showing a metabolic impairment not present in macrophages. RPE anomalies were detected by electron microscopy, including phagosomes retained in the apical area and vacuoles. Altogether, this new mouse model displays a novel phenotype that could prove useful to understanding the interplay between RPE and PRs in inflammatory retinal degenerations and highlights new roles for MerTK in the regulation of the energetic metabolism and the maintenance of the immune privilege in the retina.

Live imaging of photoreceptor outer segment phagocytosis in zebrafish

Retina depends on retinal pigment epithelium (RPE) for its survival. To maintain normal functionality, photoreceptors renew 10% of their outer segments every day and this process requires efficient phagocytosis of the aged photoreceptor fragments by the RPE. Phagocytosis is a dynamic process with strong circadian regulation, but detailed understanding on the regulation of the process and differences between photoreceptor types is presently inadequate. Previous studies have been carried out mainly with in vitro models and cell cultures, and therefore, the dynamics of the phagocytosis process in the context of a living animal has remained elusive. Here, we present an approach that utilizes transgenic zebrafish lines with fluorescently tagged photoreceptor and RPE cells for in vivo imaging of their interactive functions in a living animal.Zebrafish is one of the most accessible animal models to study retinal physiology in vivo and currently the only vertebrate model that can be used to image retinal tissue dynamics in real time. By utilizing already existing low‐pigmented zebrafish lines and microinjecting plasmids coding for photoreceptor and RPE cell membrane targeted tags, we have created low‐pigmented fish with fluorescent RPE cells and the outer segments of the different photoreceptor types. Here, we first show with histological sections how the plasmids are expressed in the correct photoreceptor types and RPE cells in larval zebrafish eyes. Then, we present our in vivo imaging approach where we anesthetise and mount the zebrafish to a confocal microscope. Our results using time‐lapse imaging demonstrate how we can follow the interaction between photoreceptors and RPE in live animals in resolution that enables detecting the movement of photoreceptor outer segment fragments in the subretinal space. Taken together, our study introduces a new approach to investigate the dynamics of phagocytosis of different photoreceptor types by the RPE in real time in a living animal.

Neuroprotective effects of syngeneic bone marrow‐derived mononuclear stem cell transplantation and intraperitoneal minocycline in the treatment of inherited photoreceptor retinal degeneration

Inherited retinal degenerations are diseases characterized by photoreceptor loss secondary to genetic defects and Retinitis Pigmentosa (RP) is the most frequent inherited retinal degeneration. This disease has a prevalence of around 1 in 4000 individuals and is caused by mutations in more than 300 genes. RP is characterized by progressive loss of photoreceptors (fist rods following by cones), activation of microglial cells and gliosis increases of macroglial cell.The Royal college of Surgeon (RCS) rat is a well characterized animal models of RP which suffer a mutation of the MERTK gene, that impedes the normal photoreceptor outer segment phagocytosis by RPE cells thus triggers the simultaneous degeneration of rods and cones. Recent studies have shown that microglial activation starts simultaneously with the beginning of photoreceptor death in the RCS retinas, suggesting that microglial cells play an important role in the retinal degeneration progression.Therapies for inherited photoreceptor degeneration aim to prevent or delay death of the photoreceptors, or in specific circumstances try to replace them.The Bone marrow derived‐mononuclear cells (BM‐MNCs) are a mononuclear CD34+ subpopulation of bone marrow‐derived cells that can be separated from the other cellular fractions of the bone marrow aspirate using a Ficoll density gradient‐based protocol. These cells have shown promising results in different animal models of neuronal degeneration, including inherited photoreceptor degenerations such us: improve the morphology of the photoreceptors outer segment, decrease synaptic degeneration in the outer plexiform layer and reduce glial fibrillary acidic protein expression in Müller cells.On the other hands minocycline is a broad‐spectrum tetracycline antibiotic, of which it has been demonstrated has antimicrobial, anti‐inflammatory, antiapoptotic, neuroprotective, and microglial inhibition effects in central nervous system diseases.In RCS rat retina the neuroprotective effect of minocycline against photoreceptor apoptosis is can be explained by mainly two different mechanisms: (1) a direct antiapoptotic effect on photoreceptors and/or (2) an effect through its anti‐inflammatory properties reducing microglial cells activation and migration.The question arises whether the combination of intravitreal BM‐MNCs transplantation with minocycline, substances that increase survival presumably through different mechanisms, could further increase photoreceptor survival and therefore be used for the treatment of retinal degenerations.Our results showed that intravitreal transplantation of BM‐MNCs increase photoreceptor survival, improved the morphology of the photoreceptors outer segment, and reduce retinal thinning (in vivo). On the other hands intraperitoneally minocycline treatment, and reduced activation and migration of microglia and produced a significant rescue of photoreceptors. Finally, our result show that combination both treatments had higher neuroprotective effects in the retina, increasing photoreceptor survival, than each of the treatments alone.In conclusion in RCS rat animal model of photoreceptor degeneration, minocycline reduces microglial activation and migration, and intravitreal transplantation of BM‐MNCs and minocycline increase photoreceptor survival. The combination of both treatments shows greater neuroprotective effects than their isolated effects, and therefore could be indicated for treatment of the early stages of photoreceptor degenerations.

Oxidative stress and docosahexaenoic acid injury lead to increased necroptosis and ferroptosis in retinal pigment epithelium

Age-related macular degeneration (AMD) is a complex disease caused by different genetic and environmental risk factors leading to loss of cells in the central part of the retina. Oxidative stress appears to be an important environmental risk factor that contributes to both the initiation and progression of AMD. Retinal pigment epithelium (RPE) plays an important role in regulating oxidative stress in the retina and is one of the main retinal cell types affected in AMD. A main function of RPE is to phagocytose photoreceptor outer segments (POS) which are rich in the polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA), making this cell type potentially more susceptible to oxidative stress-induced lipid peroxidation which can lead to cell death. RPE is known to undergo necrotic cell death in response to oxidative stress. The aim of this study was to determine if DHA in POS can increase oxidative damage to RPE. It was found that RPE undergo increased lipid peroxidation and decreased cell viability when stressed with hydrogen peroxide in combination with DHA or POS. H2O2-induced oxidative stress was found to cause both ferroptosis and necroptosis. However, the ferroptosis regulator acyl-CoA synthetase long-chain family member 4 (ACSL4) was found to be downregulated in RPE exposed to H2O2 and this effect was exacerbated when the RPE cells were simultaneously treated with DHA. Together, these results show a response of RPE when stressed which will likely be overwhelmed under disease conditions such as AMD resulting in cell death.

Oxidative and Endoplasmic Reticulum Stress Represent Novel Therapeutic Targets for Choroideremia.

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy, affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, with no approved therapy. CHM is caused by mutations in the CHM gene, which encodes the ubiquitously expressed Rab escort protein 1 (REP1). REP1 is involved in prenylation, a post-translational modification of Rab proteins, and plays an essential role in intracellular trafficking. In this study, we examined oxidative and endoplasmic reticulum (ER) stress pathways in chmru848 zebrafish and CHMY42X patient fibroblasts, and screened a number of neuroprotectants for their ability to reduce stress. The expression of the oxidative stress markers txn, cat and sod3a, and the ER stress markers bip, atf4 and atf6, were dysregulated in chmru848 fish. The expression of SOD2 was also reduced in CHMY42X fibroblasts, along with reduced BIP and increased CHOP expression. The lack of REP1 is associated with defects in vesicular trafficking, photoreceptor outer segment phagocytosis and melanosome transport, leading to increased levels of stress within the retina and RPE. Drugs targeting oxidative and ER stress pathways represent novel therapeutic avenues.

Optineurin tunes outside-in signaling to regulate lysosome biogenesis and phagocytic clearance in the retina

Balancing the competing demands of phagolysosomal degradation and autophagy is a significant challenge for phagocytic tissues. Yet how this plasticity is accomplished in health and disease is poorly understood. In the retina, circadian phagocytosis and degradation of photoreceptor outer segments by the postmitotic retinal pigment epithelium (RPE) are essential for healthy vision. Disrupted autophagy due to mechanistic target of rapamycin (mTOR) overactivation in the RPE is associated with blinding macular degenerations; however, outer segment degradation is unaffected in these diseases, indicating that distinct mechanisms regulate these clearance mechanisms. Here, using advanced imaging and mouse models, we identify optineurin as a key regulator that tunes phagocytosis and lysosomal capacity to meet circadian demands and helps prioritize outer segment clearance by the RPE in macular degenerations. High-resolution live-cell imaging implicates optineurin in scissioning outer segment tips prior to engulfment, analogous to microglial trogocytosis of neuronal processes. Optineurin is essential for recruiting light chain 3 (LC3), which anchors outer segment phagosomes to microtubules and facilitates phagosome maturation and fusion with lysosomes. This dynamically activates transcription factor EB (TFEB) to induce lysosome biogenesis in an mTOR-independent, transient receptor potential-mucolipin 1 (TRPML1)-dependent manner. RNA-seq analyses show that expression of TFEB target genes temporally tracks with optineurin recruitment and that lysosomal and autophagy genes are controlled by distinct transcriptional programs in the RPE. The unconventional plasma membrane-to-nucleus signaling mediated by optineurin ensures outer segment degradation under conditions of impaired autophagy in macular degeneration models. Independent regulation of these criticalclearance mechanisms would help safeguard the metabolic fitness of the RPE throughout the organismal lifespan.

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients' fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

Comparison of Fucoidans from Saccharina latissima Regarding Age-Related Macular Degeneration Relevant Pathomechanisms in Retinal Pigment Epithelium.

Fucoidans from brown algae are described as anti-inflammatory, antioxidative, and antiangiogenic. We tested two Saccharina latissima fucoidans (SL-FRO and SL-NOR) regarding their potential biological effects against age-related macular degeneration (AMD). Primary porcine retinal pigment epithelium (RPE), human RPE cell line ARPE-19, and human uveal melanoma cell line OMM-1 were used. Cell survival was assessed in tetrazolium assay (MTT). Oxidative stress assays were induced with erastin or H2O2. Supernatants were harvested to assess secreted vascular endothelial growth factor A (VEGF-A) in ELISA. Barrier function was assessed by measurement of trans-epithelial electrical resistance (TEER). Protectin (CD59) and retinal pigment epithelium-specific 65 kDa protein (RPE65) were evaluated in western blot. Polymorphonuclear elastase and complement inhibition assays were performed. Phagocytosis of photoreceptor outer segments was tested in a fluorescence assay. Secretion and expression of proinflammatory cytokines were assessed with ELISA and real-time PCR. Fucoidans were chemically analyzed. Neither toxic nor antioxidative effects were detected in ARPE-19 or OMM-1. Interleukin 8 gene expression was slightly reduced by SL-NOR but induced by SL-FRO in RPE. VEGF secretion was reduced in ARPE-19 by SL-FRO and in RPE by both fucoidans. Polyinosinic:polycytidylic acid induced interleukin 6 and interleukin 8 secretion was reduced by both fucoidans in RPE. CD59 expression was positively influenced by fucoidans, and they exhibited a complement and elastase inhibitory effect in cell-free assay. RPE65 expression was reduced by SL-NOR in RPE. Barrier function of RPE was transiently reduced. Phagocytosis ability was slightly reduced by both fucoidans in primary RPE but not in ARPE-19. Fucoidans from Saccharina latissima, especially SL-FRO, are promising agents against AMD, as they reduce angiogenic cytokines and show anti-inflammatory and complement inhibiting properties; however, potential effects on gene expression and RPE functions need to be considered for further research.

The retinal pigment epithelium displays electrical excitability and lateral signal spreading

BackgroundThe non-neuronal retinal pigment epithelium (RPE) functions in intimate association with retinal photoreceptors, performing a multitude of tasks critical for maintaining retinal homeostasis and collaborating with retinal glial cells to provide metabolic support and ionic buffering. Accordingly, the RPE has recently been shown to display dynamic properties mediated by an array of ion channels usually more characteristic of astrocytes and excitable cells. The recent discovery of canonical voltage-activated Na+ channels in the RPE and their importance for phagocytosis of photoreceptor outer segments raises a question about their electrogenic function. Here, we performed a detailed electrophysiological analysis related to the functioning of these channels in human embryonic stem cell (hESC)-derived RPE.ResultsOur studies examining the electrical properties of the hESC-RPE revealed that its membrane mainly displays passive properties in a broad voltage range, with the exception of depolarization-induced spikes caused by voltage-activated Na+ current (INa). Spike amplitude depended on the availability of INa and spike kinetics on the membrane time constant, and the spikes could be largely suppressed by TTX. Membrane resistance fluctuated rapidly and strongly, repeatedly changing over the course of recordings and causing closely correlated fluctuations in resting membrane potential. In a minority of cells, we found delayed secondary INa-like inward currents characterized by comparatively small amplitudes and slow kinetics, which produced secondary depolarizing spikes. Up to three consecutive delayed inward current waves were detected. These currents could be rapidly and reversibly augmented by applying L-type Ca2+ channel blocker nifedipine to diminish influx of calcium and thus increase gap junctional conductance.ConclusionsThis work shows, for the first time, that INa and INa-mediated voltage spikes can spread laterally through gap junctions in the monolayer of cells that are traditionally considered non-excitable. Our findings support a potential role of the RPE that goes beyond giving homeostatic support to the retina.

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.

Aging induces cell loss and a decline in phagosome processing in the mouse retinal pigment epithelium

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss and dysfunction in the retinal pigment epithelium (RPE) with age is known to contribute to disease development. The aim of this study was to investigate how the C57BL/6J mouse RPE changes with age. RPE structure was found to change with age and eccentricity, with cell size increasing, nuclei lost, and tight junctions altered in the peripheral retina. Phagocytosis of photoreceptor outer segments (POS) by the RPE was investigated using gene expression analysis and histology. RNA-Seq transcriptomic gene profiling of the RPE showed a downregulation of genes involved in phagosome processing and histological analysis showed a decline in phagosome-lysosome association in the aged tissue. In addition, failures in the autophagy pathway that modulates intracellular waste degradation were observed in the aged RPE tissue. These findings highlight that RPE cell loss and slowing of POS processing contribute to RPE dysfunction with age and may predispose the aging eye to AMD development.

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.

Inhibition of the LRRC8A channel promotes microglia/macrophage phagocytosis and improves outcomes after intracerebral hemorrhagic stroke.

Promoting microglial/macrophage (M/Mφ) phagocytosis accelerates hematoma clearance and improves the prognosis of intracerebral hemorrhagic stroke (ICH). Cation channels such as Piezo1 modulate bacterial clearance by regulating M/Mφ. Whether LRRC8A, an anion channel, affects M/Mφ erythrophagocytosis and functional recovery after ICH was investigated here. We found that LRRC8A is highly expressed on M/Mφ in the perihematomal region of ICH mice. Conditional knockout of Lrrc8a in M/Mφ or treatment with an LRRC8A channel blocker accelerated hematoma clearance, reduced neuronal death, and improved functional recovery after ICH. Mechanistically, the LRRC8A channel inhibition promoted M/Mφ phagocytosis by activating AMP-activated protein kinase (AMPK), thereby inducing nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2) and increasing Cd36 transcription. Our findings illuminate the regulation of M/Mφ phagocytosis by the LRRC8A channel via the AMPK-Nrf2-CD36 pathway after ICH, suggesting that LRRC8A is a potential target for hematoma clearance in ICH treatment.

Neuroprotective effects of intraocular bone marrow‐derived mononuclear cell transplants in inherited retinal degenerations

AbstractInherited retinal degenerations are diseases characterized by photoreceptor loss secondary to genetic defects. At present, only a minority are susceptible of genetic treatment and therefore are a frequent cause of blindness in infancy and adulthood.Retinitis Pigmentosa (RP) is the most frequent inherited retinal degeneration. This disease has a prevalence of around 1 in 4000 individuals and is caused by mutations in more than 300 genes. RP is characterized by progressive loss of photoreceptors, rods and then cones that may die secondarily to rod loss.The rats P23H and Royal college of Surgeon (RCS) are two well characterized animal models of RP because they bear genetic defects observed in RP patients. The P23H rat suffers an autosomal dominant mutation of the rhodopsin gene that causes protein misfolding and endoplasmic reticulum stress in rods, which die first followed by cones. The RCS rat has a mutation of MERTK gene, that impedes the normal photoreceptor outer segment phagocytosis by RPE cells thus causing simultaneous rod and cone degeneration.Therapies for inherited photoreceptor degeneration aim to prevent or delay death of the photoreceptors, or in specific circumstances try to replace them.The Bone marrow derived‐mononuclear cells (BM‐MNCs) are a mononuclear CD34+ subpopulation of bone marrow‐derived cells that can be separated from the other cellular fractions of the bone marrow aspirate using a Ficoll density gradient‐based protocol. The BM‐MNCs is a mixture of specific bone marrow cell subtypes in which is possible to find cells: Lin+, Lin−, CD34+, c‐Kit+, CD133+ or Sca‐1+ HSCs, endothelial precursor cells, and Mesenchymal Stem Cells.BM‐MNCs transplantation has shown promising results in different animal models of neuronal degeneration, including inherited photoreceptor degenerations.In our studies we have performed a syngeneic intravitreal or subretinal transplantation of BM‐MNCs in a P23H‐1 and RCS rats, and we have documented that both increase photoreceptor survival. They also improve the morphology of the photoreceptors outer segment, decrease synaptic degeneration in the outer plexiform layer and reduce glial fibrillary acidic protein (GFAP) expression in Müller cells. However, the transplanted cells did not penetrate the retina, only survived for up to 15 days, and did not modify microglial cell activation and migration or the electroretinographic responses. Their beneficial effects are maybe due to a paracrine trophic effect achieved through release of neurotrophic, anti‐angiogenic and/or immunomodulatory factors.In conclusion, the intra ocular transplantation BM‐MNCs shows photoreceptor neuroprotective actions in inherited retinal degenerations. These cells may have the potential to slow down photoreceptor loss, and therefore could be indicated for treatment of the early stages of photoreceptor degenerations.

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.

Regulation of the rhythmic diversity of daily photoreceptor outer segment phagocytosis in vivo.

Outer segment phagocytosis (OSP) is a highly-regulated, biological process wherein photoreceptor outer segment (OS) tips are cyclically phagocytosed by the adjacent retinal pigment epithelium (RPE) cells. Often an overlooked retinal process, rhythmic OSP ensures the maintenance of healthy photoreceptors and vision. Daily, the photoreceptors renew OS at their base and the most distal, and likely oldest, OS tips, are phagocytosed by the RPE, preventing the accumulation of photo-oxidative compounds by breaking down phagocytosed OS tips and recycling useful components to the photoreceptors. Light changes often coincide with an escalation of OSP and within hours the phagosomes formed in each RPE cell are resolved. In the last two decades, individual molecular regulators were elucidated. Some of the molecular machinery used by RPE cells for OSP is highly similar to mechanisms used by other phagocytic cells for the clearance of apoptotic cells. Consequently, in the RPE, many molecular regulators of retinal phagocytosis have been elucidated. However, there is still a knowledge gap regarding the key regulators of physiological OSP in vivo between endogenous photoreceptors and the RPE. Understanding the regulation of OSP is of significant clinical interest as age-related macular degeneration (AMD) and inherited retinal diseases (IRD) are linked with altered OSP. Here, we review the in vivo timing of OSP peaks in selected species and focus on the reported in vivo environmental and molecular regulators of OSP.

Mitophagy initiates retrograde mitochondrial-nuclear signaling to guide retinal pigment cell heterogeneity

ABSTRACT Age-related macular degeneration (AMD), the leading cause of blindness among the elderly, is without treatment for early disease. Degenerative retinal pigment epithelial (RPE) cell heterogeneity is a well-recognized but understudied pathogenic factor. Due to the daily phagocytosis of photoreceptor outer segments, unique photo-oxidative stress, and high metabolism for maintaining vision, the RPE has robust macroautophagy/autophagy, and mitochondrial and antioxidant networks. However, the autophagy subtype, mitophagy, in the RPE and AMD is understudied. Here, we found decreased PINK1 (PTEN induced kinase 1) in perifoveal RPE of early AMD eyes. PINK1-deficient RPE have impaired mitophagy and mitochondrial function that triggers death-resistant epithelial-mesenchymal transition (EMT). This reprogramming is mediated by novel retrograde mitochondrial-nuclear signaling (RMNS) through superoxide, NFE2L2 (NFE2 like bZIP transcription factor 2), TXNRD1 (thioredoxin reductase 1), and phosphoinositide 3-kinase (PI3K)-AKT (AKT serine/threonine kinase) that induced canonical transcription factors ZEB1 (zinc finger E-box binding homeobox 1) and SNAI1 (Snail family transcriptional repressor 1) and an EMT transcriptome. NFE2L2 deficiency disrupted RMNS that paradoxically normalized morphology but decreased function and viability. Thus, RPE heterogeneity is defined by the interaction of two cytoprotective pathways that is triggered by mitophagy function. By neutralizing the consequences of impaired mitophagy, an antioxidant dendrimer tropic for the RPE and mitochondria, EMT (a recognized AMD alteration) was abrogated to offer potential therapy for early AMD, a stage without treatment. Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; AMD: age-related macular degeneration; CCCP: cyanide m-chlorophenyl hydrazone; CDH1: cadherin 1; DAVID: Database for Annotation, Visualization and Integrated Discovery; DHE: dihydroethidium; D-NAC: N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSEA: Gene Set Enrichment Analysis; HSPD1: heat shock protein family D (Hsp60) member 1; IVT: intravitreal; KD: knockdown; LMNA, lamin A/C; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NAC: N-acetyl-l-cysteine; NQO1: NAD(P)H quinone dehydrogenase 1; NFE2L2: NFE2 like bZIP transcription factor 2; O2 −: superoxide anion; OCR: oxygen consumption rate; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; RMNS: retrograde mitochondrial-nuclear signaling; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SNAI1: snail family transcriptional repressor 1; TJP1: tight junction protein 1; TPP-D-NAC: triphenyl phosphinium and N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; Trig: trigonelline; TXNRD1: thioredoxin reductase 1; VIM: vimentin; WT: wild-type; ZEB1: zinc finger E-box binding homeobox 1

Tissue-specific modifier alleles determine Mertk loss-of-function traits.

Knockout (KO) mouse models play critical roles in elucidating biological processes behind disease-associated or disease-resistant traits. As a presumed consequence of gene KO, mice display certain phenotypes. Based on insight into the molecular role of said gene in a biological process, it is inferred that the particular biological process causally underlies the trait. This approach has been crucial towards understanding the basis of pathological and/or advantageous traits associated with Mertk KO mice. Mertk KO mice suffer from severe, early-onset retinal degeneration. MERTK, expressed in retinal pigment epithelia, is a receptor tyrosine kinase with a critical role in phagocytosis of apoptotic cells or cellular debris. Therefore, early-onset, severe retinal degeneration was described to be a direct consequence of failed MERTK-mediated phagocytosis of photoreceptor outer segments by retinal pigment epithelia. Here, we report that the loss of Mertk alone is not sufficient for retinal degeneration. The widely used Mertk KO mouse carries multiple coincidental changes in its genome that affect the expression of a number of genes, including the Mertk paralog Tyro3. Retinal degeneration manifests only when the function of Tyro3 is concomitantly lost. Furthermore, Mertk KO mice display improved anti-tumor immunity. MERTK is expressed in macrophages. Therefore, enhanced anti-tumor immunity was inferred to result from the failure of macrophages to dispose of cancer cell corpses, resulting in a pro-inflammatory tumor microenvironment. The resistance against two syngeneic mouse tumor models observed in Mertk KO mice is not, however, phenocopied by the loss of Mertk alone. Neither Tyro3 nor macrophage phagocytosis by alternate genetic redundancy accounts for the absence of anti-tumor immunity. Collectively, our results indicate that context-dependent epistasis of independent modifier alleles determines Mertk KO traits.

Aberrant Retinal Pigment Epithelial Cells Derived from Induced Pluripotent Stem Cells of a Retinitis Pigmentosa Patient with the PRPF6 Mutation.

Pre-mRNA processing factors (PRPFs) are vital components of the spliceosome and are involved in the physiological process necessary for pre-mRNA splicing to mature mRNA. As an important member, PRPF6 mutation resulting in autosomal dominant retinitis pigmentosa (adRP) is not common. Recently, we reported the establishment of an induced pluripotent stem cells (iPSCs; CSUASOi004-A) model by reprogramming the peripheral blood mononuclear cells of a PRPF6-related adRP patient, which could recapitulate a consistent disease-specific genotype. In this study, a disease model of retinal pigment epithelial (RPE) cells was generated from the iPSCs of this patient to further investigate the underlying molecular and pathological mechanisms. The results showed the irregular morphology, disorganized apical microvilli and reduced expressions of RPE-specific genes in the patient’s iPSC-derived RPE cells. In addition, RPE cells carrying the PRPF6 mutation displayed a decrease in the phagocytosis of fluorescein isothiocyanate-labeled photoreceptor outer segments and exhibited impaired cell polarity and barrier function. This study will benefit the understanding of PRPF6-related RPE cells and future cell therapy.

Microcarrier-Based Culture of Human Pluripotent Stem-Cell-Derived Retinal Pigmented Epithelium.

Dry age-related macular degeneration (AMD) is estimated to impact nearly 300 million individuals globally by 2040. While no treatment options are currently available, multiple clinical trials investigating retinal pigmented epithelial cells derived from human pluripotent stem cells (hPSC-RPE) as a cellular replacement therapeutic are currently underway. It has been estimated that a production capacity of >109 RPE cells annually would be required to treat the afflicted population, but current manufacturing protocols are limited, being labor-intensive and time-consuming. Microcarrier technology has enabled high-density propagation of many adherent mammalian cell types via monolayer culture on surfaces of uM-diameter matrix spheres; however, few studies have explored microcarrier-based culture of RPE cells. Here, we provide an approach to the growth, maturation, and differentiation of hPSC-RPE cells on Cytodex 1 (C1) and Cytodex 3 (C3) microcarriers. We demonstrate that hPSC-RPE cells adhere to microcarriers coated with Matrigel, vitronectin or collagen, and mature in vitro to exhibit characteristic epithelial cell morphology and pigmentation. Microcarrier-grown hPSC-RPE cells (mcRPE) are viable; metabolically active; express RPE signature genes including BEST1, RPE65, TYRP1, and PMEL17; secrete the trophic factors PEDF and VEGF; and demonstrate phagocytosis of photoreceptor outer segments. Furthermore, we show that undifferentiated hESCs also adhere to Matrigel-coated microcarriers and are amenable to directed RPE differentiation. The capacity to support hPSC-RPE cell cultures using microcarriers enables efficient large-scale production of therapeutic RPE cells sufficient to meet the treatment demands of a large AMD patient population.