Photoreceptor Cell Survival Research Articles

All-in-one AAV-mediated Nrl gene inactivation rescues retinal degeneration in Pde6a mice.

Retinitis pigmentosa (RP) is a complex group of inherited retinal diseases characterized by progressive death of photoreceptor cells and eventual blindness. Pde6a, which encodes a cGMP-specific phosphodiesterase, is a crucial pathogenic gene for autosomal recessive RP (RP43); there is no effective therapy for this form of RP. The compact CRISPR/SaCas9 system, which can be packaged into a single adeno-associated virus, holds promise for simplifying effective gene therapy. Here, we demonstrated that all-in-one AAV-SaCas9-mediated Nrl gene inactivation can efficiently prevent retinal degeneration in a RP mouse model with Pde6anmf363/nmf363 mutation. We screened single guide RNAs (sgRNAs) capable of efficiently editing mouse Nrl gene in N2a cells and then achieved effective gene editing by using a single AAV to co-deliver SaCas9 and an optimal Nrl-sg2 into the mouse retina. Excitingly, in vivo inactivation of Nrl improved photoreceptor cell survival and rescued retinal function in treated Pde6a deficient mice. Thus, we showed that a practical, gene-independent method, AAV-SaCas9-mediated Nrl inactivation, holds promise for future therapeutic applications in patients with RP.

HSPB4/CRYAA Protect Photoreceptors during Retinal Detachment in Part through FAIM2 Regulation.

Our previous study discussed crystallin family induction in an experimental rat model of retinal detachment. Therefore, we attempted to evaluate the role of α-crystallin in photoreceptor survival in an experimental model of retinal detachment, as well as its association with the intrinsically neuroprotective protein Fas-apoptotic inhibitory molecule 2 (FAIM2). Separation of retina and RPE was induced in rat and mouse eyes by subretinal injection of hyaluronic acid. Retinas were subsequently analyzed for the presence αA-crystallin (HSPB4) and αB-crystallin (HSPB5) proteins using immunohistochemistry and immunoblotting. Photoreceptor death was analyzed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining and cell counts. The 661W cells subjected to FasL were used as a cell model of photoreceptor degeneration to assess the mechanisms of the protective effect of αA-crystallin and its dependence on its phosphorylation on T148. We further evaluated the interaction between FAIM2 and αA-crystallin using a co-immunoprecipitation assay. Our results showed that α-crystallin protein levels were rapidly induced in response to retinal detachment, with αA-crystallin playing a particularly important role in protecting photoreceptors during retinal detachment. Our data also show that the photoreceptor intrinsically neuroprotective protein FAIM2 is induced and interacts with α-crystallins following retinal detachment. Mechanistically, our work also demonstrated that the phosphorylation of αA-crystallin is important for the interaction of αA-crystallin with FAIM2 and their neuroprotective effect. Thus, αA-crystallin is involved in the regulation of photoreceptor survival during retinal detachment, playing a key role in the stabilization of FAIM2, serving as an important modulator of photoreceptor cell survival under chronic stress conditions.

Preservation of vision by transpalpebral electrical stimulation in mice with inherited retinal degeneration.

The potential neuroprotective and regenerative properties of electrical stimulation (ES) were studied in rhodopsin knockout mice (Rho -/- ), a murine model of inherited retinal degeneration. The study focused on assessing the impact of varying ES frequencies on visual functions and photoreceptor cell survival in Rho -/- mice. To elucidate the impact of electrical stimulation on cone survival, Rho -/- mice received either sham or transpalpebral ES using biphasic ramp or rectangular waveforms at 100µA amplitude, starting at sixweeks of age. The treatment duration spanned from one to three weeks. The optimal treatment frequency of ES sessions was determined by applying ES every one, two, or three days in three separate groups of Rho -/- mice. The sham group received daily treatments without the application of ES. Our study revealed significant improvement of visual function in Rho -/- mice following daily or every-other-day noninvasive transpalpebral ES, as evidenced by electroretinogram and optomotor response-based visual behavior assays. Concurrently, assessment of outer nuclear thickness and immunohistochemistry for the cone photoreceptor cell marker PNA demonstrated pronounced increases in the survival of rods and cones and improvement in the morphology of the inner and outer segments. This study underscores the protective effect of non-invasive ES in rhodopsin knockout-induced retinal degenerative disorders, providing a foundation for developing targeted therapeutic interventions for retinitis pigmentosa.

MIR96 Has Good Potential to Differentiate Human Bone Marrow-Derived Mesenchymal Stem Cells into Photoreceptor-Like Cells.

MicroRNAs play an important role in the development and function of neuron cells. Among these, the miRNA known as MIR96 is abundantly expressed in mammalian retina and significantly affects differentiation, maturation, and survival of human photoreceptor cells. In this study, a mimic to miRNA-96 was transfected into human bone marrowderived mesenchymal stem cells to explore the biological functions of MIR96 at differentiation processing. A mimic to miRNA-96 and a competitive control were transfected into human bone marrow-derived mesenchymal stem cells using Lipofectamine. After 24 and 48 hours, we evaluated changes in expression levels of genes associated with neural progenitor and photoreceptor differentiation (OTX2, NRL, protein kinase C, SLC1A1, and recoverin) by real-time polymerase chain reaction. In addition, we measured expression of mRNA and protein of the CRX gene (neuroretinal progenitor cell marker) and the RHO gene (terminal differentiation marker) using real-time polymerase chain reaction and immunocytochemistry, respectively. Real-time polymerase chain reaction results showed increased levels of RHO and recoverin mRNA after 24 hours in transfected cells. In addition, mRNA levels of OTX2, CRX, NRL, RHO, recoverin, and protein kinase C increased after 48 hours in transfected cells. Immunocytochemistry results confirmed these findings by demonstrating RHO and CRX at both 24 and 48 hours in transfected cells. Control of the expression of MIR96 can be a good strategy to promote cell differentiation and can be used in cell therapy for retinal degeneration. Our results showed that human bone marrow-derived mesenchymal stem cells can differentiate into photoreceptor cells after transfection with MIR96. These results support therapeutic use of MIR96 in retinal degeneration and suggest human bone marrowderived mesenchymal stem cells as a promising tool for interventions.

The temporal progression of retinal degeneration and early-stage idebenone treatment in the Pde6brd1/rd1 mouse model of retinal dystrophy

Photoreceptor cell death, primarily through apoptosis, related to retinal disorders like retinitis pigmentosa (RP), would result in vision loss. The pathological processes and crucial mutant conditions preceding photoreceptor cell demise are not well understood. This study aims to conduct an in-depth examination of early-stage changes in the widely utilized Pde6brd1/rd1 (rd1) mouse model, which has Pde6b gene mutations representing autosomal recessive RP disorder. We investigated the morphology and ultrastructure of retinal cells, including second-order neurons, during the initial phase of disease progression. Our findings revealed that mitochondrial alterations in rod photoreceptors were present as a predeath mutant state as early as postnatal day 3 (P3). The bipolar and horizontal cells from the rd1 mouse retina exhibited significant morphological changes in response to loss of photoreceptor cells, indicating that second-order neurons rely on these cells for their structures. Subsequent oral administration of idebenone, a mitochondria-protective agent, enhanced retinal function and promoted both photoreceptor cell survival and inner retinal second-order synaptogenesis in rd1 mice at P14. Our findings offer a mechanistic framework, suggesting that mitochondrial damage acts as an early driver for photoreceptor cell death in retinal degeneration.

The many faces of SIRT6 in the retina and retinal pigment epithelium.

Sirtuin 6 (SIRT6) is a member of the mammalian sirtuin family of NAD+-dependent protein deacylases, homologues of the yeast silent information regulator 2 (Sir2). SIRT6 has remarkably diverse functions and plays a key role in a variety of biological processes for maintaining cellular and organismal homeostasis. In this review, our primary aim is to summarize recent progress in understanding SIRT6's functions in the retina and retinal pigment epithelium (RPE), with the hope of further drawing interests in SIRT6 to increase efforts in exploring the therapeutic potential of this unique protein in the vision field. Before describing SIRT6's role in the eye, we first discuss SIRT6's general functions in a wide range of biological contexts. SIRT6 plays an important role in gene silencing, metabolism, DNA repair, antioxidant defense, inflammation, aging and longevity, early development, and stress response. In addition, recent studies have revealed SIRT6's role in macrophage polarization and mitochondrial homeostasis. Despite being initially understudied in the context of the eye, recent efforts have begun to elucidate the critical functions of SIRT6 in the retina and RPE. In the retina, SIRT6 is essential for adult retinal function, regulates energy metabolism by suppressing glycolysis that affects photoreceptor cell survival, protects retinal ganglion cells from oxidative stress, and plays a role in Müller cells during early neurodegenerative events in diabetic retinopathy. In the RPE, SIRT6 activates autophagy in culture and protects against oxidative stress in mice. Taken together, this review demonstrates that better understanding of SIRT6's functions and their mechanisms, both in and out of the context of the eye, holds great promise for the development of SIRT6-targeted strategies for prevention and treatment of blinding eye diseases.

The effects of ush2a gene knockout on vesicle transport in photoreceptors

USH2A (Usher syndrome type 2A) gene mutations are the predominant cause of Usher syndrome type 2, characterized by sensorineural hearing loss and retinitis pigmentosa (RP), and also significant contributors to non-syndromic RP. To date, there is a lack of definitive therapeutic interventions to mitigate the associated disorders caused by USH2A mutations, and the precise pathogenic mechanisms underlying their onset remain unclear. In the present study, we utilized the ush2a knockout zebrafish model to investigate the pathological mechanisms of RP. In late-stage ush2a-/- zebrafish, the outer segments of rods displayed shortened length and decreased number. Anomalous vesicle accumulation was observed at the junction between the inner and outer segments, accompanied by reduced expression and structural damage of actin filaments in the photoreceptor cells. Furthermore, we discovered that Rab8 expression was downregulated and exhibited aberrant localization in ush2a-/- zebrafish. Additionally, we identified an interaction between USH2A and Rab8. Therefore, the knockout of ush2a may potentially affect vesicle transport through the regulation of Rab8, providing a novel target for maintaining the survival of photoreceptor cells. These findings also contribute to our understanding of the potential molecular pathogenesis underlying RP caused by USH2A gene mutations.

Human Neural Progenitors Expressing GDNF Enhance Retinal Protection in a Rodent Model of Retinal Degeneration.

Stem cell therapy for retinal degenerative diseases has been extensively tested in preclinical and clinical studies. However, preclinical studies performed in animal models at the early stage of disease do not optimally translate to patients that present to the clinic at a later stage of disease. As the retina degenerates, inflammation and oxidative stress increase and trophic factor support declines. Testing stem cell therapies in animal models at a clinically relevant stage is critical for translation to the clinic. Human neural progenitor cells (hNPC) and hNPC engineered to stably express GDNF (hNPCGDNF) were subretinally injected into the Royal College of Surgeon (RCS) rats, a well-established model for retinal degeneration, at early and later stages of the disease. hNPCGDNF treatment at the early stage of retinal degeneration provided enhanced visual function compared to hNPC alone. Treatment with both cell types resulted in preserved retinal morphology compared to controls. hNPCGDNF treatment led to significantly broader photoreceptor protection than hNPC treatment at both early and later times of intervention. The phagocytic role of hNPC appears to support RPE cell functions and the secreted GDNF offers neuroprotection and enables the extended survival of photoreceptor cells in transplanted animal eyes. Donor cells in the RCS rat retina survived with only limited proliferation, and hNPCGDNF produced GDNF in vivo. Cell treatment led to significant changes in various pathways related to cell survival, antioxidative stress, phagocytosis, and autophagy. A combined stem cell and trophic factor therapy holds great promise for treating retinal degenerative diseases including retinitis pigmentosa and age-related macular degeneration.

Müller cell vulnerability in aging human retina: Implications on photoreceptor cell survival

Müller glial cells (MC) support various metabolic functions of the retinal neurons, and maintain the homeostasis. Oxidative stress is intensified with aging, and in human retina, MC and photoreceptors undergo lipid peroxidation and protein nitration. Information on how MC respond to oxidative stress is vital to understand the fate of aging retinal neurons. This study examined age-related changes in MC of donor human retina (age: 35–98 years; N = 18 donors). Ultrastructural and immunohistochemical observations indicate that MC undergo gliosis and increased lipid peroxidation, and show osmotic changes with advanced aging (>80 years). Photoreceptor cells also undergo oxidative-nitrosative stress with aging, and their synapses also show clear osmotic swelling. MC respond to oxidative stress via proliferation of smooth endoplasmic reticulum in their processes, and increased expression of aquaporin-4 in endfeet and outer retina. In advanced aged retinas (81–98 years), they showed mitochondrial disorganisation, accumulation of lipids and autophagosomes, lipofuscin granules and axonal remnants in phagolysosomes in their inner processes, suggesting a reduced phagocytotic potential in them with aging. Glutamine synthetase expression does not alter until advanced aging, when the retinas show its increased expression in endfeet and Henle fiber layer. It is evident that MC are vulnerable with normal aging and this could be a reason for photoreceptor cell abnormalities reported with aging of the human retina.

Effect on cell survival and cytoophidium assembly of the adRP-10-related IMPDH1 missense mutation Asp226Asn.

Introduction: Inosine monophosphate dehydrogenase 1 (IMPDH1) is a critical enzyme in the retina, essential for the correct functioning of photoreceptor cells. Mutations in IMPDH1 have been linked to autosomal dominant retinitis pigmentosa subtype 10 (adRP-10), a genetic eye disorder. Some of these mutations such as the Asp226Asn (D226N) lead to the assembly of large filamentous structures termed cytoophidia. D226N also gives IMPDH1 resistance to feedback inhibition by GDP/GTP. This study aims to emulate the adRP-10 condition with a long-term expression of IMPDH1-D226N in vitro and explore cytoophidium assembly and cell survival. We also assessed whether the introduction of an additional mutation (Y12C) to disrupt the cytoophidium has an attenuating effect on the toxicity caused by the D226N mutation. Results: Expression of IMPDH1-D226N in HEp-2 cells resulted in cytoophidium assembly in ∼70% of the cells, but the presence of the Y12C mutation disrupted the filaments. Long-term cell survival was significantly affected by the presence of the D226N mutation, with a decrease of ∼40% in the cells expressing IMPDH1-D226N when compared to IMPDH1-WT; however, survival was significantly recovered in IMPDH1-Y12C/D226N, with only a ∼10% decrease when compared to IMPDH1-WT. On the other hand, the IMPDH1 expression level in the D226N-positive cells was <30% of that of the IMPDH1-WT-positive cells and only slightly higher in the Y12C/D226N, suggesting that although cell survival in Y12C/D226N was recovered, higher expression levels of the mutated IMPDH1 were not tolerated by the cells in the long term. Conclusion: The IMPDH1-D226N effect on photoreceptor cell survival may be the result of a sum of problems: nucleotide unbalance plus a toxic long-life cytoophidium, supported by the observation that by introducing Y12C in IMPDH1 the cytoophidium was disrupted and cell survival significantly recovered, but not the sensibility to GDP/GTP regulation since higher expression levels of IMPDH1-D226N were not tolerated.

Drosophila Phosphatase of Regenerating Liver Is Critical for Photoreceptor Cell Polarity and Survival during Retinal Development.

Establishing apicobasal polarity, involving intricate interactions among polarity regulators, is key for epithelial cell function. Though phosphatase of regenerating liver (PRL) proteins are implicated in diverse biological processes, including cancer, their developmental role remains unclear. In this study, we explore the role of Drosophila PRL (dPRL) in photoreceptor cell development. We reveal that dPRL, requiring a C-terminal prenylation motif, is highly enriched in the apical membrane of developing photoreceptor cells. Moreover, dPRL knockdown during retinal development results in adult Drosophila retinal degeneration, caused by hid-induced apoptosis. dPRL depletion also mislocalizes cell adhesion and polarity proteins like Armadillo, Crumbs, and DaPKC and relocates the basolateral protein, alpha subunit of Na+/K+-ATPase, to the presumed apical membrane. Importantly, this polarity disruption is not secondary to apoptosis, as suppressing hid expression does not rescue the polarity defect in dPRL-depleted photoreceptor cells. These findings underscore dPRL's crucial role in photoreceptor cell polarity and emphasize PRL's importance in establishing epithelial polarity and maintaining cell survival during retinal development, offering new insights into PRL's role in normal epithelium.

Mechanisms of Arrestin-Mediated Signaling.

Arrestins were first discovered as proteins that selectively bind active phosphorylated GPCRs and suppress (arrest) their G protein-mediated signaling. Nonvisual arrestins are also recognized as signaling proteins regulating a variety of cellular pathways. Arrestins are highly flexible; they can assume many different conformations. In their receptor-bound conformation, arrestins have higher affinity for a subset of binding partners. This explains how receptor activation regulates certain branches of arrestin-dependent signaling via arrestin recruitment to GPCRs. However, free arrestins are also active molecular entities that regulate other signaling pathways and localize signaling proteins to particular subcellular compartments. Recent findings suggest that the two visuals, arrestin-1 and arrestin-4, which are expressed in photoreceptor cells, not only regulate signaling via binding to photopigments but also interact with several nonreceptor partners, critically affecting the health and survival of photoreceptor cells. Detailed in this overview are GPCR-dependent and independent modes of arrestin-mediated regulation of cellular signaling. © 2023 Wiley Periodicals LLC.

The endoplasmic reticulum membrane protein complex subunit Emc6 is essential for rhodopsin localization and photoreceptor cell survival

The endoplasmic reticulum (ER) membrane protein complex (EMC) is responsible for monitoring the biogenesis and synthetic quality of membrane proteins with tail-anchored or multiple transmembrane domains. The EMC subunit EMC6 is one of the core members of EMC and forms an enclosed hydrophilic vestibule in cooperation with EMC3. Despite studies demonstrating that deletion of EMC3 led to rhodopsin mislocalization in rod photoreceptors of mice, the precise mechanism leading to the failure of rhodopsin trafficking remains unclear. Here, we generated the first rod photoreceptor-specific knockout of Emc6 (RKO) and cone photoreceptor-specific knockout of Emc6 (CKO) mouse models. Deficiency of Emc6 in rod photoreceptors led to progressive shortening of outer segments (OS), impaired visual function, mislocalization and reduced expression of rhodopsin, and increased gliosis in rod photoreceptors. In addition, CKO mice displayed the progressive death of cone photoreceptors and abnormal localization of cone opsin protein. Subsequently, proteomics analysis of the RKO mouse retina illustrated that several cilium-related proteins, particularly anoctamin-2 (ANO2) and transmembrane protein 67 (TMEM67), were significantly down-regulated prior to OS degeneration. Detrimental rod photoreceptor cilia and mislocalized membrane disc proteins were evident in RKO mice. Our data revealed that in addition to monitoring the synthesis of rhodopsin-dominated membrane disc proteins, EMC6 also impacted rod photoreceptors' ciliogenesis by regulating the synthesis of membrane proteins associated with cilia, contributing to the mislocalization of membrane disc proteins.

Gene augmentation of LCA5-associated Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme.

Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy, we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localized at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient in lebercilin exhibited early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, adeno-associated virus-based LCA5 gene augmentation partially restored the bulge region, preserved OS axoneme structure and membrane disc formation, and resulted in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.

Mutations in BCOR, a co-repressor of CRX/OTX2, are associated with early-onset retinal degeneration.

Many transcription factors regulating the production, survival, and function of photoreceptor cells have been identified, but little is known about transcriptional co-regulators in retinal health and disease. Here, we show that BCL6 co-repressor (BCOR), a Polycomb repressive complex 1 factor mutated in various cancers, is involved in photoreceptor degenerative diseases. Using proteomics and transcription assays, we report that BCOR interacts with the transcription factors CRX and OTX2 and reduces their ability to activate the promoters of photoreceptor-specific genes. CUT&RUN sequencing further shows that BCOR shares genome-wide binding profiles with CRX/OTX2, consistent with a general co-repression activity. We also identify missense mutations in human BCOR in five families that have no evidence of cancer but present severe early-onset X-linked retinal degeneration. Last, we show that the human BCOR mutants cause degeneration when expressed in the mouse retina and have enhanced repressive activity on OTX2. These results uncover a role for BCOR in photoreceptors in both health and disease.

Complement C3 deficiency alleviates alkylation-induced retinal degeneration in mice

BackgroundIt has been found that the extensive use of anticancer drugs containing DNA-alkylating agents not only target cancer cells but also cause retinal inflammation through toxic intermediates. Complement C3 (C3) is a core component of the complement activation pathway, and dysregulation of the complement pathway is involved in several retinal degenerative diseases. However, whether C3 plays a critical role in alkylation-induced retinal degeneration is unclear.MethodsFollowing treatment with the alkylating agent methyl methane sulfonate (MMS), the C3 mRNA and protein level was measured, DNA damage and photoreceptor cell death were assessed in both wild-type (WT) C57BL/6J and C3 knockout (KO) mice.ResultsWe determined that complement pathway is activated following MMS treatment, and C3 knockout (KO) increased the rate of photoreceptor cell survival and preserved visual function. The mRNA levels of nuclear erythroid-related factor 2 (Nrf2) and related genes were higher after MMS application in C3 KO mice.ConclusionIn summary, our study found that C3 KO promotes photoreceptor cell survival and activates the Nrf2 signaling pathway in the context of alkylation-induced retinal degeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40662-022-00292-4.

Mice Lacking the Systemic Vitamin A Receptor RBPR2 Show Decreased Ocular Retinoids and Loss of Visual Function

Simple SummaryThis work represents an initial evaluation of the second RBP4-vitamin A receptor RBPR2 in a mammalian model. We provide evidence that the membrane localized RBPR2 protein, under variable conditions of dietary vitamin A intake, plays an important role for dietary vitamin A transport to the eye for ocular retinoid homeostasis and visual function. These findings are of general interest, as disturbances in blood and ocular vitamin A homeostasis are linked to retinal degenerative diseases, which are blinding diseases. The animal model described here could also serve as an in vivo tool to study mechanisms related to retinal cell degeneration that are associated with vitamin A deficiency.The systemic transport of dietary vitamin A/all-trans retinol bound to RBP4 into peripheral tissues for storage is an essential physiological process that continuously provides visual chromophore precursors to the retina under fasting conditions. This mechanism is critical for phototransduction, photoreceptor cell maintenance and survival, and in the support of visual function. While the membrane receptor STRA6 facilitates the blood transport of lipophilic vitamin A into the eye, it is not expressed in most peripheral organs, which are proposed to express a second membrane receptor for the uptake of vitamin A from circulating RBP4. The discovery of a novel vitamin A receptor, RBPR2, which is expressed in the liver and intestine, but not in the eye, alluded to this long-sort non-ocular membrane receptor for systemic RBP4-ROL uptake and transport. We have previously shown in zebrafish that the retinol-binding protein receptor 2 (Rbpr2) plays an important role in the transport of yolk vitamin A to the eye. Mutant rbpr2 zebrafish lines manifested in decreased ocular retinoid concentrations and retinal phenotypes. To investigate a physiological role for the second vitamin A receptor, RBPR2, in mammals and to analyze the metabolic basis of systemic vitamin A transport for retinoid homeostasis, we established a whole-body Rbpr2 knockout mouse (Rbpr2−/−) model. These mice were viable on both vitamin A-sufficient and -deficient diets. Rbpr2−/− mice that were fed a vitamin A-sufficient diet displayed lower ocular retinoid levels, decreased opsins, and manifested in decrease visual function, as measured by electroretinography. Interestingly, when Rbpr2−/− mice were fed a vitamin A-deficient diet, they additionally showed shorter photoreceptor outer segment phenotypes, altogether manifesting in a significant loss of visual function. Thus, under conditions replicating vitamin A sufficiency and deficiency, our analyses revealed that RBPR2-mediated systemic vitamin A transport is a regulated process that is important for vitamin A delivery to the eye when RBP4-bound ROL is the only transport pathway in the fasting condition or under vitamin A deficiency conditions.

Lysophosphatidylcholine acyltransferase 1 controls mitochondrial reactive oxygen species generation and survival of retinal photoreceptor cells

Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice bearing a frameshift mutation in the gene encoding lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration in mice; however, the mechanism by which this mutation affects degeneration is unclear. In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 not only decreased saturated PC production but also affected membrane lipid composition, presumably by altering saturated fatty acyl-CoA availability. Furthermore, we demonstrated that Lpcat1 deletion led to increased mitochondrial reactive oxygen species levels in photoreceptor cells, but not in other retinal cells, and did not affect the OS structure or trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC plays critical roles in photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration–related retinal diseases.

Long-Term Ultrastructural Outcomes of Autologous Transplantation of Retinal Pigment Epithelium-Partial Thickness Choroid (RPE-PTC) Sheet in Rabbits.

To observe the ultrastructural outcomes of autologous transplantation of retinal pigment epithelium-partial-thickness choroidal (RPE-PTC) sheets in rabbits after 6 months. Eighteen pigmented rabbits were used in this study. Among them, nine rabbits were used for autologous transplantation of RPE-PTC sheets. Tissue sections were observed under a transmission electron microscope for one, three, and six months after transplantation, respectively. One, three, and six months after the autologous transplantation of RPE-PTC sheets, the inner and outer segments of photoreceptor cells were arranged regularly, and the connection between the inner and outer segments was normal. The inner structure of the RPE cells and tight junctions among them remained normal. Phagocytosis of outer segment of photoreceptor cells could also be observed in RPE cells. The structure of the Bruch's membrane appeared loose, rather than being dense as normal, and it was undulated after one and three months, while it became dense after six months. The graft and the bed were healed well, the boundary was unclear, and the graft was vascularized after one, three, and six months, respectively. Our findings revealed that the RPE-PTC sheets could quickly rebuild blood vessels, thereby maintaining the normal physiological functions of RPE cells, as well as the survival and functional status of photoreceptor cells for a long-time.

Automated evaluation of retinal pigment epithelium disease area in eyes with age-related macular degeneration

The retinal pigment epithelium (RPE) is essential for the survival and function of retinal photoreceptor cells. RPE dysfunction causes various retinal diseases including age-related macular degeneration (AMD). Clinical studies on ES/iPS cell-derived RPE transplantation for RPE dysfunction-triggered diseases are currently underway. Quantification of the diseased RPE area is important to evaluate disease progression or the therapeutic effect of RPE transplantation. However, there are no standard protocols. To address this issue, we developed a 2-step software that enables objective and efficient quantification of RPE-disease area changes by analyzing the early-phase hyperfluorescent area in fluorescein angiography (FA) images. We extracted the Abnormal region. This extraction was based on deep learning-based discrimination. We scored the binarized extracted area using an automated program. Our program’s performance for the same eye from the serial image captures was within 3.1 ± 7.8% error. In progressive AMD, the trend was consistent with human assessment, even when FA images from two different visits were compared. This method was applicable to quantifying RPE-disease area changes over time, evaluating iPSC-RPE transplantation images, and a disease other than AMD. Our program may contribute to the assessment of the clinical course of RPE-disease areas in routine clinics and reduce the workload of researchers.