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Retinal detachment (RD) is a sight-threatening condition that occurs in several retinal diseases. Microglia that reside in retina are activated after RD and play a role in the death of photoreceptor cells. The involvement of microglial pyroptosis in the early pathological process of RD is still unclear. VX-765, an inhibitor of caspase-1, may exert neuroprotective effects by targeting microglial pyroptosis in nervous system disease; however, whether it plays a role in RD is uncertain. This study detected and localized pyroptosis to specific cells by immunofluorescence co-staining and flow cytometry in rat RD models. The majority of gasdermin D N-terminal (GSDMD-N)-positive cells exhibited IBA1-positive or P2RY12-positive microglia in the early stage of RD, indicating the pyroptosis of microglia. Administration of VX-765 shifted the microglia phenotype from M1 to M2, inhibited microglial migration toward the outer nuclear layer (ONL) post-RD, and most importantly, inhibited microglial pyroptosis. The thickness of ONL increased with VX-765 administration, and the photoreceptors were more structured and orderly under hematoxylin and eosin staining and transmission electron microscopy, revealing the protective effects of VX-765 on photoreceptors. Overall, this study demonstrated that inflammation induced by pyroptosis of microglia is the early pathological process of RD. VX-765 may serve as a candidate therapeutic approach for the treatment of RD by targeting microglia., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Retinal degeneration is a collection of devastating conditions with progressive loss of vision which often lead to blindness. Research on retinal microglial cells offers great therapeutic potential in deterring the progression of degeneration. This study explored the mechanisms underlying the TREM2-mediated protective function of activated microglial cells during retinal degeneration. N-methyl-N-nitrosourea (MNU)-induced retinal degeneration was established in C57BL/6 J (WT) and Trem2 knockout (Trem2 -/- ) mice. We discovered that MNU treatment led to the concurrent processes of photoreceptor apoptosis and microglia infiltration. A significant upregulation of disease-associated microglia signature genes was observed during photoreceptor degeneration. Following MNU treatment, Trem2 -/- mice showed exacerbated photoreceptor cell death, decreased microglia migration and phagocytosis, reduced microglial PPARγ activation and CD36 expression. Pharmaceutical activation of PPARγ promoted microglial migration, ameliorated photoreceptor degeneration and restored CD36 expression in MNU-treated Trem2 -/- mice. Inhibition of CD36 activity worsened photoreceptor degeneration in MNU-treated WT mice. Our findings suggested that the protective effect of microglia during retinal degeneration was dependent on Trem2 expression and carried out via the activation of PPARγ and the consequent upregulation of CD36 expression. Our study linked TREM2 signaling with PPARγ activation, and provided a potential therapeutic target for the management of retinal degeneration., (© 2024. The Author(s).)

Inherited retinal diseases encompass a genetically diverse group of conditions caused by variants in genes critical to retinal function, including handful of ribosome-associated genes. This study focuses on the HBS1L gene, which encodes for the HBS1-like translational GTPase that is crucial for ribosomal rescue. We have reported a female child carrying biallelic HBS1L variants, manifesting with poor growth and neurodevelopmental delay. Here, we describe the ophthalmologic findings in the patient and in Hbs1ltm1a/tm1a hypomorph mice and describe the associated microscopic and molecular perturbations. The patient has impaired visual function, showing dampened amplitudes of a- and b-waves in both rod- and cone-mediated responses. Hbs1ltm1a/tm1a mice exhibited profound thinning of the entire retina, specifically of the outer photoreceptor layer, due to extensive photoreceptor cell apoptosis. Loss of Hbs1l resulted in comprehensive proteomic alterations by mass spectrometry analysis, with an increase in the levels of 169 proteins and a decrease in the levels of 480 proteins, including rhodopsin (Rho) and peripherin 2 (Prph2). Gene Ontology biological process and gene set enrichment analyses reveal that the downregulated proteins are primarily involved in phototransduction, cilium assembly and photoreceptor cell development. These findings underscore the importance of ribosomal rescue proteins in maintaining retinal health, particularly in photoreceptor cells., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

We explored the effect of inhibition of thioredoxin interacting protein (Txnip) on neuroprotection in Müller cells under high glucose. Wild-type (WT) and Txnip knockout (Txnip -/- ) mice were used to establish a streptozotocin (STZ)-induced diabetes model and a Müller cells high glucose model. We detected BDNF expression and PI3K/AKT/CREB pathway activation levels in the retina and Müller cells of each group in vivo and in vitro experiments. The Txnip -/- STZ group showed higher expression of BDNF and phosphorylation of PI3K/AKT/CREB in retina, and less retinal photoreceptor apoptosis was observed in Txnip -/- diabetic group than in WT. After using an inhibitor of PI3K signaling pathway, BDNF expression was reduced; In vitro co-cultured with Müller cells in different groups, 661 W cells showed different situations, Txnip -/- Müller cells maximum downregulated Cleaved-caspase 3 expression in 661 W, accompanied by an increase in Bcl-2/Bax ratio. These findings indicate that inhibiting endogenous Txnip in mouse Müller cells can promote their expression and secretion of BDNF, thereby reducing HG induced photoreceptor apoptosis and having important neuroprotective effects on DR. The regulation of BDNF expression by Txnip may be achieved by activating the PI3K/AKT/CREB pathway. This study suggests that regulating Txnip may be a potential target for DR treatment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yanuo Wang reports financial support was provided by National Natural Science Foundation of China [grant numbers 82201193]. Xi Shen reports financial support was provided by Shanghai Municipal Health Commission [grant numbers 202140183]. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na + - and Ca 2+ -influx, subsequent activation of voltage-gated Ca 2+ -channels (VGCC), and further Ca 2+ influx. However, a connection between excessive Ca 2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca 2+ -signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca 2+ -chelator BAPTA-AM or inhibitors of different Ca 2+ -permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca 2+ -release-activated channel (CRAC), and Na + /Ca 2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca 2+ -dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca 2+ -channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca 2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca 2+ -levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca 2+ -signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract., (© 2024. The Author(s).)

Abnormal vasculature in the retina, specifically tortuous vessels and capillary degeneration, is common in many of the most prevalent retinal degenerative diseases, currently affecting millions of people across the world. However, the formation and development of abnormal vasculature in the context of retinal degenerative diseases are still poorly understood. The FVB/N (rd1) and rd10 mice are well-studied animal models of retinal degenerative diseases, but how photoreceptor degeneration leads to vascular abnormality in the diseases remains to be elucidated. Here, we used advancements in confocal microscopy, immunohistochemistry, and image analysis software to systematically characterize the pathological vasculature in the FVB/N (rd1) and rd10 mice, known as a chronic, rapid and slower retinal degenerative model, respectively. We demonstrated that there was plexus-specific vascular degeneration in the retinal trilaminar vascular network paralleled to photoreceptor degeneration in the diseased retinas. We also quantitatively analyzed the vascular structural architecture in the wild-type and diseased retinas to provide valuable information on vascular remodeling in retinal degenerative disease., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The endoplasmic reticulum membrane protein complex (EMC) plays a critical role in the synthesis of multipass membrane proteins. Genetic studies indicated that mutations in EMC1 gene were associated with retinal degeneration diseases; however, the role of EMC1 in photoreceptor has not been confirmed. Here, we show that Emc1 ablation in the photoreceptor cells of mice recapitulated the retinitis pigmentosa phenotypes, including an attenuated scotopic electroretinogram response and the progressive degeneration of rod cells and cone cells. Histopathological examination of tissues from rod-specific Emc1 knockout mice revealed mislocalized rhodopsin and irregularly arranged cone cells at the age of 2 months. Further immunoblotting analysis revealed decreased levels of membrane proteins and endoplasmic reticulum chaperones in 1-month-old rod-specific Emc1 knockout mice retinae, and this led us to speculate that the loss of membrane proteins is the main cause of the degeneration of photoreceptors. EMC1 most likely regulated the membrane protein levels at an earlier step in the biosynthetic process before the proteins translocated into the endoplasmic reticulum. The present study demonstrates the essential roles of Emc1 in photoreceptor cells, and reveals the mechanism through which EMC1 mutations are linked to retinitis pigmentosa., (© 2023 Federation of European Biochemical Societies.)

Retinal detachment (RD) is a neurodegenerative blinding disease caused by plethora of clinical conditions. RD is characterized by the physical separation of retina from the underlying retinal pigment epithelium (RPE), eventually leading to photoreceptor cell death, inflammation, and vision loss. Albeit the activation of complement plays a critical role in the pathogenesis of RD, the retinal cellular source for complement production remains elusive. Here, using C3 tdTomato reporter mice we show that retinal injury upregulates C3 expression, specifically in Müller cells. Activation of the complement cascade results in the generation of proinflammatory cleaved products, C3a and C5a, that bind C3aR and C5aR1, respectively. Our flow cytometry data show that retinal injury significantly upregulated C3aR and C5aR1 in microglia and resulted in the infiltration of peripheral immune cells. Loss of C3, C5, C3aR or C5aR1 reduced photoreceptor cell death and infiltration of microglia and peripheral immune cells into the sub-retinal space. These results indicate that C3/C3aR and C5/C5aR1 play a crucial role in eliciting photoreceptor degeneration and inflammatory responses in RD.

Ceramide (Cer) plays an essential role in photoreceptor cell death in the retina. On the one hand, Cer accumulation emerges as a common feature during retina neurodegeneration, leading to the death of photoreceptors. On the other hand, Cer deficiency has also recently been associated with retinal dysfunction and degeneration. Although more and more evidence supports the importance of maintaining Cer homeostasis in the retina, mechanistic explanations of the observed phenotypes, especially in the context of Cer deficiency, are still lacking. An enhanced understanding of Cer's role in the retina will help us explore the underlying molecular basis for clinical phenotypes of retinal dystrophies and provide us with potential therapeutic targets., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Retinitis pigmentosa (RP) causes blindness in 1 out of 3000-4000 individuals worldwide. Understanding the disease mechanism underlying the death of photoreceptors in RP patient is crucial for the discovery and development of therapies to prevent and stop the progression of retinal degeneration. Despite having provided valuable insight into RP pathology, several shortcomings of animal models warrant the need for a better modeling system. This review discusses the current use of patient-derived induced pluripotent stem cells (iPSCs) to model RP and its advantages over animal models. Further improvement to enhance the representativeness of iPSC RP models is also discussed., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Pathologies of the retina are clinically visualized in vivo with OCT and ex vivo with immunohistochemistry. Although both techniques provide valuable information on prognosis and disease state, a comprehensive method for fully elucidating molecular constituents present in locations of interest is desirable. The purpose of this work was to use multimodal imaging technologies to localize the vast number of molecular species observed with matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) in aged and diseased retinal tissues. Herein, MALDI IMS was utilized to observe molecular species that reside in photoreceptor cells and also a basal laminar deposit from two human donor eyes. The molecular species observed to accumulate in these discrete regions can be further identified and studied to attempt to gain a greater understanding of biological processes occurring in debilitating eye diseases such as age-related macular degeneration (AMD)., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The cone-rod homeobox (CRX) protein is a key transcription factor essential for photoreceptor function and survival. Mutations in human CRX gene are linked to a wide spectrum of blinding diseases ranging from mild macular dystrophy to severe Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP). These diseases are still incurable and mostly inherited in an autosomal dominant form. Dysfunctional mutant CRX protein interferes with the function of wild-type CRX protein, demonstrating the dominant negative effect. At present, gene augmentation is the most promising treatment strategy for hereditary diseases. This study aims to review the pathogenic mechanisms of various CRX mutations and propose two therapeutic strategies to rescue sick photoreceptors in CRX-associated retinopathies, namely, Tet-On-hCRX system and adeno-associated virus (AAV)-mediated gene augmentation. The outcome of proposed studies will guide future translational research and suggest guidelines for therapy evaluation in terms of treatment safety and efficacy., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Pathological deterioration of mitochondrial function is increasingly linked with multiple degenerative illnesses as a mediator of a wide range of neurologic and age-related chronic diseases, including those of genetic origin. Several of these diseases are rare, typically defined in the United States as an illness affecting fewer than 200,000 people in the U.S. population, or about one in 1600 individuals. Vision impairment due to mitochondrial dysfunction in the eye is a prominent feature evident in numerous primary mitochondrial diseases and is common to the pathophysiology of many of the familiar ophthalmic disorders, including age-related macular degeneration, diabetic retinopathy, glaucoma and retinopathy of prematurity - a collection of syndromes, diseases and disorders with significant unmet medical needs. Focusing on metabolic mitochondrial pathway mechanisms, including the possible roles of cuproptosis and ferroptosis in retinal mitochondrial dysfunction, we shed light on the potential of α-lipoyl-L-carnitine in treating eye diseases. α-Lipoyl-L-carnitine is a bioavailable mitochondria-targeting lipoic acid prodrug that has shown potential in protecting against retinal degeneration and photoreceptor cell loss in ophthalmic indications., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Several pathogenic variants have been reported in the IMPG1 gene associated with the inherited retinal disorders vitelliform macular dystrophy (VMD) and retinitis pigmentosa (RP). IMPG1 and its paralog IMPG2 encode for two proteoglycans, SPACR and SPACRCAN, respectively, which are the main components of the interphotoreceptor matrix (IPM), the extracellular matrix surrounding the photoreceptor cells. To determine the role of SPACR in the pathological mechanisms leading to RP and VMD, we generated a knockout mouse model lacking Impg1 , the mouse ortholog. Impg1 -deficient mice show abnormal accumulation of autofluorescent deposits visible by fundus imaging and spectral-domain optical coherence tomography (SD-OCT) and attenuated electroretinogram responses from 9 months of age. Furthermore, SD-OCT of Impg1 -/- mice shows a degeneration of the photoreceptor layer, and transmission electron microscopy shows a disruption of the IPM and the retinal pigment epithelial cells. The decrease in the concentration of the chromophore 11- cis -retinal supports this loss of photoreceptors. In conclusion, our results demonstrate the essential role of SPACR in maintaining photoreceptors. Impg1 -/- mice provide a novel model for mechanistic investigations and the development of therapies for VMD and RP caused by IMPG1 pathogenic variants., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Retinitis pigmentosa (RP) is genetically heterogeneous retinopathy caused by photoreceptor cell death and retinal pigment epithelial atrophy that eventually results in blindness in bilateral eyes. Various photoreceptor cell death types and pathological phenotypic changes that have been disclosed in RP demand in-depth research of its pathogenic mechanism that may account for inter-patient heterogeneous responses to mainstream drug treatment. As the primary method for studying the genetic characteristics of RP, molecular biology has been widely used in disease diagnosis and clinical trials. Current technology iterations, such as gene therapy, stem cell therapy, and optogenetics, are advancing towards precise diagnosis and clinical applications. Specifically, technologies, such as effective delivery vectors, CRISPR/Cas9 technology, and iPSC-based cell transplantation, hasten the pace of personalized precision medicine in RP. The combination of conventional therapy and state-of-the-art medication is promising in revolutionizing RP treatment strategies. This article provides an overview of the latest research on the pathogenesis, diagnosis, and treatment of retinitis pigmentosa, aiming for a convenient reference of what has been achieved so far.

The breakdown of all-trans-retinal (atRAL) clearance is closely associated with photoreceptor cell death in dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1), but its mechanisms remain elusive. Here, we demonstrate that activation of gasdermin E (GSDME) but not gasdermin D promotes atRAL-induced photoreceptor damage by activating pyroptosis and aggravating apoptosis through a mitochondria-mediated caspase-3-dependent signaling pathway. Activation of c-Jun N-terminal kinase was identified as one of the major causes of mitochondrial membrane rupture in atRAL-loaded photoreceptor cells, resulting in the release of cytochrome c from mitochondria to the cytosol, where it stimulated caspase-3 activation required for cleavage of GSDME. Aggregation of the N-terminal fragment of GSDME in the mitochondria revealed that GSDME was likely to penetrate mitochondrial membranes in photoreceptor cells after atRAL exposure. ABC (subfamily A, member 4) and all-trans-retinol dehydrogenase 8 are two key proteins responsible for clearing atRAL in the retina. Abca4 -/- Rdh8 -/- mice exhibit serious defects in atRAL clearance upon light exposure and serve as an acute model for dry AMD and STGD1. We found that N-terminal fragment of GSDME was distinctly localized in the photoreceptor outer nuclear layer of light-exposed Abca4 -/- Rdh8 -/- mice. Of note, degeneration and caspase-3 activation in photoreceptors were significantly alleviated in Abca4 -/- Rdh8 -/- Gsdme -/- mice after exposure to light. The results of this study indicate that GSDME is a common causative factor of photoreceptor pyroptosis and apoptosis arising from atRAL overload, suggesting that repressing GSDME may represent a potential treatment of photoreceptor atrophy in dry AMD and STGD1., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetic retinopathy is a frequent complication of longstanding diabetes, which comprises a complex interplay of microvascular abnormalities and neurodegeneration. Zebrafish harboring a homozygous mutation in the pancreatic transcription factor pdx1 display a diabetic phenotype with survival into adulthood, and are therefore uniquely suitable among zebrafish models for studying pathologies associated with persistent diabetic conditions. We have previously shown that, starting at three months of age, pdx1 mutants exhibit not only vascular but also neuro-retinal pathologies manifesting as photoreceptor dysfunction and loss, similar to human diabetic retinopathy. Here, we further characterize injury and regenerative responses and examine the effects on progenitor cell populations. Consistent with a negative impact of hyperglycemia on neurogenesis, stem cells of the ciliary marginal zone show an exacerbation of aging-related proliferative decline. In contrast to the robust Müller glial cell proliferation seen following acute retinal injury, the pdx1 mutant shows replenishment of both rod and cone photoreceptors from slow-cycling, neurod-expressing progenitors which first accumulate in the inner nuclear layer. Overall, we demonstrate a diabetic retinopathy model which shows pathological features of the human disease evolving alongside an ongoing restorative process that replaces lost photoreceptors, at the same time suggesting an unappreciated phenotypic continuum between multipotent and photoreceptor-committed progenitors.

Retinitis pigmentosa (RP) is a major cause of blindness that is difficult to diagnose and treat. PKM2, a subtype of pyruvate kinase, is strongly associated with oxidative stress and is expressed in photoreceptors. We investigated whether PKM2 reduces photoreceptor cell apoptosis and evaluated possible antiapoptotic mechanisms in RP. We established RP models by exposing 661W cells to blue light and modulated PKM2 activity using a PKM2 inhibitor. We measured the apoptosis rates using calcein-acetoxymethyl ester/propidium iodide double staining and Cell Counting Kit-8, the oxidative stress levels using a reactive oxygen species assay, and the changes in protein expression by western blotting. Photodamage increased PKM2 expression, cellular oxidative stress, and apoptosis of 661W cells. PKM2 inhibition significantly reduced the levels of apoptosis and oxidative stress induced by photodamage. Our data suggest that PKM2 is a potential disease marker and therapeutic target for RP., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Peiwen Zhu et al.)

Usher syndrome type I (USH1) is characterized by deafness, vestibular areflexia, and progressive retinal degeneration. The protein-truncating p.Arg245* founder variant of PCDH15 (USH1F) has an ~2% carrier frequency amongst Ashkenazi Jews accounts for ~60% of their USH1 cases. Here, longitudinal phenotyping in 13 USH1F individuals revealed progressive retinal degeneration, leading to severe vision loss with macular atrophy by the sixth decade. Half of the affected individuals were legally blind by their mid-50s. The mouse Pcdh15 R250X variant is equivalent to human p.Arg245*. Homozygous Pcdh15 R250X mice also have visual deficits and aberrant light-dependent translocation of the phototransduction cascade proteins, arrestin, and transducin. Retinal pigment epithelium (RPE)-specific retinoid cycle proteins, RPE65 and CRALBP, were also reduced in Pcdh15 R250X mice, indicating a dual role for protocadherin-15 in photoreceptors and RPE. Exogenous 9- cis retinal improved ERG amplitudes in Pcdh15 R250X mice, suggesting a basis for a clinical trial of FDA-approved retinoids to preserve vision in USH1F patients., Competing Interests: SS, WZ, SR, AG, TD, RH, CB, AG, TR, SR, TF, ZA No competing interests declared, JS Julie M. Schultz is affiliated with GeneDx, Inc. The author has no financial interests to declare.

Age-related macular degeneration (AMD) is a common blinding disease in the western world that is linked to the loss of fenestration in the choriocapillaris that sustains the retinal pigment epithelium and photoreceptors in the back of the eye. Changes in ocular and systemic zinc concentrations have been associated with AMD; therefore, we hypothesized that these changes might be directly involved in fenestrae formation. To test this hypothesis, an endothelial cell (bEND.5) model for fenestrae formation was treated with different concentrations of zinc sulfate (ZnSO 4 ) solution for up to 20 h. Fenestrae were visualized by staining for Plasmalemmal Vesicle Associated Protein-1 (PV-1), the protein that forms the diaphragms of the fenestrated endothelium. Size and distribution were monitored by transmission electron microscopy (TEM). We found that zinc induced the redistribution of PV-1 into areas called sieve plates containing ~70-nm uniform size and typical morphology fenestrae. As AMD is associated with reduced zinc concentrations in the serum and in ocular tissues, and dietary zinc supplementation is recommended to slow disease progression, we propose here that the elevation of zinc concentration may restore choriocapillaris fenestration resulting in improved nutrient flow and clearance of waste material in the retina.

Obesity-associated type 2 diabetes (T2D) is on the rise in the United States due to the obesity epidemic, and 60% of T2D patients develop diabetic retinopathy (DR) in their lifetime. Chronic inflammation is a hallmark of obesity and T2D and a well-accepted major contributor to DR, and retinal photoreceptors are a major source of intraocular inflammation and directly contribute to vascular abnormalities in diabetes. However, how diabetic insults cause photoreceptor inflammation is not well known. In this study, we used a high-fat diet (HFD)-induced T2D mouse model and cultured photoreceptors treated with palmitic acid (PA) to decipher major players that mediate high-fat-induced photoreceptor inflammation. We found that PA-elicited microRNA-150 (miR-150) decreases with a consistent upregulation of ETS-domain transcription factor 1 (Elk1), a downstream target of miR-150, in PA-elicited photoreceptor inflammation. We compared wild-type (WT) and miR-150 null (miR-150 -/- ) mice fed with an HFD and found that deletion of miR-150 exacerbated HFD-induced photoreceptor inflammation in conjunction with upregulated ELK1. We further delineated the critical cellular localization of phosphorylated ELK1 at serine 383 (pELK1 S383 ) and found that decreased miR-150 exacerbated the T2D-induced inflammation in photoreceptors by upregulating ELK1 and pELK1 S383 , and knockdown of ELK1 alleviated PA-elicited photoreceptor inflammation., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Photoreceptor degeneration caused by genetic defects leads to retinitis pigmentosa, a rare disease typically diagnosed in adolescents and young adults. In most cases, rod loss occurs first, followed by cone loss as well as altered function in cells connected to photoreceptors directly or indirectly. There remains a gap in our understanding of retinal cellular responses to photoreceptor abnormalities. Here, we utilized single-cell transcriptomics to investigate cellular responses in each major retinal cell type in retinitis pigmentosa model (P23H) mice vs. wild-type littermate mice. We found a significant decrease in the expression of genes associated with phototransduction, the inner/outer segment, photoreceptor cell cilium, and photoreceptor development in both rod and cone clusters, in line with the structural changes seen with immunohistochemistry. Accompanying this loss was a significant decrease in the expression of genes involved in metabolic pathways and energy production in both rods and cones. We found that in the Müller glia/astrocyte cluster, there was a significant increase in gene expression in pathways involving photoreceptor maintenance, while concomitant decreases were observed in rods and cones. Additionally, the expression of genes involved in mitochondrial localization and transport was increased in the Müller glia/astrocyte cluster. The Müller glial compensatory increase in the expression of genes downregulated in photoreceptors suggests that Müller glia adapt their transcriptome to support photoreceptors and could be thought of as general therapeutic targets to protect against retinal degeneration., (© 2021. The Author(s).)

This study tested the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca 2+ -sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy. Nondiabetic and 2-month diabetic C57Bl/6J and calpain1 knockout (Capn1 -/- ) mice were studied; subgroups were treated with a calpain inhibitor (CI). Ca 2+ content was measured in photoreceptors using Fura-2. Retinal calpain expression was studied by quantitative RT-PCR and immunohistochemistry. Superoxide and expression of inflammatory proteins were measured using published methods. Proteomic analysis was conducted on photoreceptors isolated from untreated diabetic mice or treated daily with CI for 2 months. Cytosolic Ca 2+ content was increased twofold in photoreceptors of diabetic mice as compared with nondiabetic mice. Capn1 expression increased fivefold in photoreceptor outer segments of diabetic mice. Pharmacologic inhibition or genetic deletion of Capn1 significantly suppressed diabetes-induced oxidative stress and expression of proinflammatory proteins in retina. Proteomics identified a protein (WW domain-containing oxidoreductase [WWOX]) whose expression was significantly increased in photoreceptors from mice diabetic for 2 months and was inhibited with CI. Knockdown of Wwox using specific siRNA in vitro inhibited increase in superoxide caused by the high glucose. These results suggest that reducing Ca 2+ accumulation, suppressing calpain activation, and/or reducing Wwox up-regulation are novel targets for treating early diabetic retinopathy., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Progressive photoreceptor death occurs in blinding diseases such as retinitis pigmentosa. Myeloid differentiation primary response protein 88 (MyD88) is a central adaptor protein for innate immune system Toll-like receptors (TLR) and induces cytokine secretion during retinal disease. We recently demonstrated that inhibiting MyD88 in mouse models of retinal degeneration led to increased photoreceptor survival, which was associated with altered cytokines and increased neuroprotective microglia. However, the identity of additional molecular changes associated with MyD88 inhibitor-induced neuroprotection is not known. In this study, we used isobaric tags for relative and absolute quantification (iTRAQ) labelling followed by LC-MS/MS for quantitative proteomic analysis on the rd10 mouse model of retinal degeneration to identify protein pathways changed by MyD88 inhibition. Quantitative proteomics using iTRAQ LC-MS/MS is a high-throughput method ideal for providing insight into molecular pathways during disease and experimental treatments. Forty-two proteins were differentially expressed in retinas from mice treated with MyD88 inhibitor compared with control. Notably, increased expression of multiple crystallins and chaperones that respond to cellular stress and have anti-apoptotic properties was identified in the MyD88-inhibited mice. These data suggest that inhibiting MyD88 enhances chaperone-mediated retinal protection pathways. Therefore, this study provides insight into molecular events contributing to photoreceptor protection from modulating inflammation., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10 ), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5 -/- zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5 -/- mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.

We developed a model of retinal degeneration in rabbits based on exposure to light with a wavelength of 405 nm. This model allows reproducing structural and functional disorders in the central parts of the retina, including primarily degeneration of the outer layers of the retina (retinal pigment epithelium and layer of photoreceptor cells), and is designed to study the mechanisms of formation, progression and effectiveness of new drugs and methods of treatment of degenerative diseases of the retina.

The BBSome is a protein complex consisting of BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and BBS18 that associates with intraflagellar transport complexes and specializes in ciliary trafficking. In primary cilia, ciliary entry requires the fully assembled BBSome as well as the small GTPase, ARL6 (BBS3). Retinal photoreceptors possess specialized cilia. In light of key structural and functional differences between primary and specialized cilia, we examined the principles of BBSome recruitment to photoreceptor cilia. We performed sucrose gradient fractionation using retinal lysates of Bbs2-/-, Bbs7-/-, Bbs8-/- and Bbs3-/- mice to determine the status of BBSome assembly, then determined localization of BBSome components using immunohistochemistry. Surprisingly, we found that a subcomplex of the BBSome containing at least BBS1, BBS5, BBS8 and BBS9 is recruited to cilia in the absence of BBS2 or BBS7. In contrast, a BBSome subcomplex consisting of BBS1, BBS2, BBS5, BBS7 and BBS9 is found in Bbs8-/- retinas and is denied ciliary entry in photoreceptor cells. In addition, the BBSome remains fully assembled in Bbs3-/- retinas and can be recruited to photoreceptor cilia in the absence of BBS3. We compared phenotypic severity of their retinal degeneration phenotypes. These findings demonstrate that unlike primary cilia, photoreceptor cilia admit a partially assembled BBSome meeting specific requirements. In addition, the recruitment of the BBSome to photoreceptor cilia does not require BBS3. These findings indicate that the ciliary entry of the BBSome is subjected to cell-specific regulation, particularly in cells with highly adapted forms of cilia such as photoreceptors., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Vision loss is among the characteristic symptoms of neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative lysosomal storage disorder. Here, we performed an in-depth analysis of retinal degeneration at the molecular and cellular levels in mice lacking the lysosomal aspartyl protease cathepsin D, an animal model of congenital CLN10 disease. We observed an early-onset accumulation of storage material as indicated by elevated levels of saposin D and subunit C of the mitochondrial ATP synthase. The accumulation of storage material was accompanied by reactive astrogliosis and microgliosis, elevated expression of the autophagy marker sequestosome 1/p62 and a dysregulated expression of several lysosomal proteins. The number of cone photoreceptor cells was reduced as early as at postnatal day 5. At the end stage of the disease, the outer nuclear layer was almost atrophied, and all cones were lost. A significant loss of rod and cone bipolar cells, amacrine cells and ganglion cells was found at advanced stages of the disease. Results demonstrate that cathepsin D deficiency results in an early-onset and rapidly progressing retinal dystrophy that involves all retinal cell types. Data of the present study will serve as a reference for studies aimed at developing treatments for retinal degeneration in CLN10 disease.

Acquired and inherited retinal disorders are responsible for vision loss in an increasing proportion of individuals worldwide. Photoreceptor (PR) death is central to the vision loss individuals experience in these various retinal diseases. Unfortunately, there is a lack of treatment options to prevent PR loss, so an urgent unmet need exists for therapies that improve PR survival and ultimately, vision. The retina is one of the most energy demanding tissues in the body, and this is driven in large part by the metabolic needs of PRs. Recent studies suggest that disruption of nutrient availability and regulation of cell metabolism may be a unifying mechanism in PR death. Understanding retinal cell metabolism and how it is altered in disease has been identified as a priority area of research. The focus of this review is on the recent advances in the understanding of PR metabolism and how it is critical to reduction-oxidation (redox) balance, the outer retinal metabolic ecosystem, and retinal disease. The importance of these metabolic processes is just beginning to be realized and unraveling the metabolic and redox pathways integral to PR health may identify novel targets for neuroprotective strategies that prevent blindness in the heterogenous group of retinal disorders.

Channelrhodopsin (ChR)-based optogenetics is one promising approach to restore vision in photoreceptor degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Currently, a large number of ChRs from different alga species as well as engineered variants are available. They vary with their light response properties like peak sensitive wavelength (λ max ), current amplitude, and kinetics. Therefore, it is important to choose an appropriate ChR for practical applications, such as vision restoration. Here we describe a standard laboratory protocol for characterizing properties of ChRs in in vitro in human embryonic kidney (HEK) cells. Based on such characterization, we also discuss the criteria for selecting optimal ChRs for optogenetic vision restoration.

The aim of this study was to analyze photoreceptor alterations occurring in eyes with intermediate age-related macular degeneration (AMD) and to investigate their associations with choriocapillaris (CC) flow. In this retrospective case-control study, we collected data from 35 eyes with intermediate AMD from 35 patients who had swept source optical coherence tomography structural and angiography imaging obtained. A control group of 35 eyes from 35 healthy subjects was included for comparison. Our main outcome measure for comparison between groups was the normalized reflectivity of en face image segmented at the ellipsoid zone (EZ) level, which was calculated to quantify the photoreceptor damage. OCTA metrics to quantify CC flow signal were also computed. These metrics were measured in a circle centered on the fovea and with a diameter of 5 mm. In intermediate AMD eyes, the macular area occupied by drusen was identified. Therefore, the EZ reflectivity and CC flow signal were separately measured in regions without drusen ("drusen-free" region). Measurements were generated using previously published algorithms. Mean ± SD age was 74.1 ± 6.8 years in the intermediate AMD group and 72.1 ± 6.0 years in the control group (p = 0.206). The normalized EZ reflectivity was 0.76 ± 0.10 in the intermediate AMD group and 0.85 ± 0.08 in the control group (p < .0001). In the "drusen-free" region, the normalized EZ reflectivity was 0.77 ± 0.10 (p < .0001  vs. healthy controls) and was positively correlated with the CC flow signal density (ρ = - 0.340 and p = 0.020). In conclusion, eyes with intermediate AMD exhibit a diffuse reduced EZ normalized reflectivity, and this reduction is correlated with CC flow signal in the regions without drusen. This study supports the concept of the damage of the unit comprised of photoreceptor, CC, and intervening tissues as an early event in AMD.

Background: Inflammation and particularly interleukin-1β (IL-1β), a pro-inflammatory cytokine highly secreted by activated immune cells during early AMD pathological events, contribute significantly to retinal neurodegeneration. Here, we identify specific cell types that generate IL-1β and harbor the IL-1 receptor (IL-1R) and pharmacologically validate IL-1β's contribution to neuro-retinal degeneration using the IL-1R allosteric modulator composed of the amino acid sequence rytvela (as well as the orthosteric antagonist, Kineret) in a model of blue light-induced retinal degeneration., Methods: Mice were exposed to blue light for 6 h and sacrificed 3 days later. Mice were intraperitoneally injected with rytvela, Kineret, or vehicle twice daily for 3 days. The inflammatory markers F4/80, NLRP3, caspase-1, and IL-1β were assessed in the retinas. Single-cell RNA sequencing was used to determine the cell-specific expression patterns of retinal Il1b and Il1r1. Macrophage-induced photoreceptor death was assessed ex vivo using retinal explants co-cultured with LPS-activated bone marrow-derived macrophages. Photoreceptor cell death was evaluated by the TUNEL assay. Retinal function was assessed by flash electroretinography., Results: Blue light markedly increased the mononuclear phagocyte recruitment and levels of inflammatory markers associated with photoreceptor death. Co-localization of NLRP3, caspase-1, and IL-1β with F4/80 + mononuclear phagocytes was clearly detected in the subretinal space, suggesting that these inflammatory cells are the main source of IL-1β. Single-cell RNA sequencing confirmed the immune-specific expression of Il1b and notably perivascular macrophages in light-challenged mice, while Il1r1 expression was found primarily in astrocytes, bipolar, and vascular cells. Retinal explants co-cultured with LPS/ATP-activated bone marrow-derived macrophages displayed a high number of TUNEL-positive photoreceptors, which was abrogated by rytvela treatment. IL-1R antagonism significantly mitigated the inflammatory response triggered in vivo by blue light exposure, and rytvela was superior to Kineret in preserving photoreceptor density and retinal function., Conclusion: These findings substantiate the importance of IL-1β in neuro-retinal degeneration and revealed specific sources of Il1b from perivascular MPs, with its receptor Ilr1 being separately expressed on surrounding neuro-vascular and astroglial cells. They also validate the efficacy of rytvela-induced IL-1R modulation in suppressing detrimental inflammatory responses and preserving photoreceptor density and function in these conditions, reinforcing the rationale for clinical translation.

Inherited retinal degenerative disorders such as retinitis pigmentosa and Usher syndrome are characterized by progressive death of photoreceptor cells. To restore vision to patients blinded by these diseases, a stem cell-based photoreceptor cell replacement strategy will likely be required. Although retinal stem cell differentiation protocols suitable for generating photoreceptor cells exist, they often yield a rather heterogenous mixture of cell types. To enrich the donor cell population for one or a few cell types, scientists have traditionally relied upon the use of antibody-based selection approaches. However, these strategies are quite labor intensive and require animal derived reagents and equipment that are not well suited to current good manufacturing practices (cGMP). The purpose of this study was to develop and evaluate a microfluidic cell sorting device capable of exploiting the physical and mechanical differences between retinal cell types to enrich specific donor cell populations such as Retinal Pigment Epithelial (RPE) cells and photoreceptor cells. Using this device, we were able to separate a mixture of RPE and iPSC-derived photoreceptor precursor cell lines into two substantially enriched fractions. The enrichment factor of the RPE fraction was 2 and that of the photoreceptor precursor cell fraction was 2.7. Similarly, when human retina, obtained from 3 independent donors, was dissociated and passed through the sorting device, the heterogeneous mixture could be reliably sorted into RPE and photoreceptor cell rich fractions. In summary, microfluidic cell sorting is a promising approach for antibody free enrichment of retinal cell populations., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In patients with retinitis pigmentosa (RP), color fundus photography and fundus autofluorescence (FAF) have been used to estimate the disease progression. To understand the origin and the diagnostic interpretation of the fundus color and FAF, we performed in vivo imaging of fundus color and FAF together with histological analyses of the retinal degeneration process using the RP model mice, rd10. FAF partly represented the accumulation of microglia in the photoreceptor outer segments. Fundus whitening suggested the presence of apoptotic cells, which spatiotemporally preceded increase in FAF. We observed two patterns of FAF localization, arcuate and diffuse, each indicating different pattern of apoptosis, wavy and diffuse, respectively. Diffuse pattern of apoptosis was suppressed in dark-raised rd10 mice, in which outer nuclear layer (ONL) loss was significantly suppressed. The occupancy of FAF correlated with the thinning rate of the ONL. Fractalkine, a microglia chemotactic factor, was detected in apoptotic photoreceptors, suggesting chemokine-induced recruitment of microglia into the ONL, which paralleled with accelerated ONL loss and increased FAF occupancy. Thus, we propose that the degree of photoreceptor apoptosis and the rate of ONL thinning in RP patients might be read from the fundus color and the FAF.

Purpose: Photoreceptor precursor cells (PRPs) differentiated from human embryonic stem cells can serve as a source for cell replacement therapy aimed at vision restoration in patients suffering from degenerative diseases of the outer retina, such as retinitis pigmentosa and AMD. In this work, we studied the electrophysiologic maturation of PRPs throughout the differentiation process., Methods: Human embryonic stem cells were differentiated into PRPs and whole-cell recordings were performed for electrophysiologic characterization at days 0, 30, 60, and 90 along with quantitative PCR analysis to characterize the expression level of various ion channels, which shape the electrophysiologic response. Finally, to characterize the electrically induced calcium currents, we employed calcium imaging (rhod4) to visualize intracellular calcium dynamics in response to electrical activation., Results: Our results revealed an early and steady presence (approximately 100% of responsive cells) of the delayed potassium rectifier current. In contrast, the percentage of cells exhibiting voltage-gated sodium currents increased with maturation (from 0% to almost 90% of responsive cells at 90 days). Moreover, calcium imaging revealed the presence of voltage-gated calcium currents, which play a major role in vision formation. These results were further supported by quantitative PCR analysis, which revealed a significant and continuous (3- to 50-fold) increase in the expression of various voltage-gated channels concomitantly with the increase in the expression of the photoreceptor marker CRX., Conclusions: These results can shed light on the electrophysiologic maturation of neurons in general and PRP in particular and can form the basis for devising and optimizing cell replacement-based vision restoration strategies.

Purpose: To study if human embryonic stem cell-derived photoreceptors could survive and function without the support of retinal pigment epithelium (RPE) after transplantation into Royal College of Surgeons rats, a rat model of retinal degeneration caused by RPE dysfunction., Methods: CSC14 human embryonic stem cells were differentiated into primordial eye structures called retinal organoids. Retinal organoids were analyzed by quantitative PCR and immunofluorescence and compared with human fetal retina. Retinal organoid sheets (30-70 day of differentiation) were transplanted into immunodeficient RCS rats, aged 44 to 56 days. The development of transplant organoids in vivo in relation to the host was examined by optical coherence tomography. Visual function was assessed by optokinetic testing, electroretinogram, and superior colliculus electrophysiologic recording. Cryostat sections were analyzed for various retinal, synaptic, and donor markers., Results: Retinal organoids showed similar gene expression to human fetal retina transplanted rats demonstrated significant improvement in visual function compared with RCS nonsurgery and sham surgery controls by ERGs at 2 months after surgery (but not later), optokinetic testing (up to 6 months after surgery) and electrophysiologic superior colliculus recordings (6-8 months after surgery). The transplanted organoids survived more than 7 months; developed photoreceptors with inner and outer segments, and other retinal cells; and were well-integrated within the host., Conclusions: This study, to our knowledge, is the first to show that transplanted photoreceptors survive and function even with host's dysfunctional RPE. Our findings suggest that transplantation of organoid sheets from stem cells may be a promising approach/therapeutic for blinding diseases.

Rod-cone degenerations, for example, retinitis pigmentosa are leading causes of blindness worldwide. Despite slow disease progression in humans, vision loss is inevitable; therefore, development of vision restoration strategies is crucial. Among others, promising approaches include optogenetics and prosthetic implants, which aim to bypass lost photoreceptors (PRs). Naturally, the efficacy of these therapeutic strategies will depend on inner retinal structural and functional preservation. The present study shows that in photoinducible I307N rhodopsin mice (Translational Vision Research Model 4 [Tvrm4]), a 12k lux light exposure eliminates PRs in the central retina in 1 week, but interneurons and their synapses are maintained for as long as 9 weeks postinduction. Despite bipolar cell dendritic retraction and moderate loss of horizontal cells, the survival rate of various cell types is very high. Significant preservation of conventional synapses and gap junctions in the inner plexiform layer is also observed. We found the number of synaptic ribbons to gradually decline and their ultrastructure to become transiently abnormal, although based on our findings intrinsic retinal architecture is maintained despite complete loss of PRs. Unlike common rodent models of PR degeneration, where the disease phenotype often interferes with retinal development, in Tvrm4 mice, the degenerative process can be induced after retinal development is complete. This time course more closely mimics the timing of disease onset in affected patients. Stability of the inner retina found in these mutants 2 months after PR degeneration suggests moderate, stereotyped remodeling in the early stages of the human disease and represents a promising finding for prompt approaches of vision restoration., (© 2019 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

Background: Subretinal fluid (SRF) can be associated with choroidal nevus and can cause progressive change in the morphology of overlying photoreceptors., Methods: A retrospective observational study was performed using optical coherence tomography to assess nevus and SRF features, as well as photoreceptor morphology over time., Results: There were 232 choroidal nevi that presented with or developed SRF. Photoreceptor morphology at presentation was classified as normal (n=60, 26%), shaggy (elongated) (n=73, 31%), retracted (stalactite appearance) (n=76, 33%), or absent (n=23, 10%). There was a progression in photoreceptor morphology with increasing SRF chronicity (p=0.003). For nevus presenting with normal photoreceptors and later developed SRF (n=60), photoreceptors became shaggy in 29 (48%), retracted in 24 (40%), and absent in 7 (12%) after 15, 19 and 22 months, respectively. For nevus presenting with SRF and shaggy photoreceptors (n=73), progression to retracted photoreceptors occurred in 31 (42%) after a mean of 22 months; for nevus with SRF and retracted photoreceptors (n=76), progression to absent photoreceptors occurred in 19 (25%) after a mean of 34 months; and for nevus with absent photoreceptors (n=23), photoreceptor morphology showed no change after mean follow-up of 33 months. Risk of nevus growth to melanoma was not associated with photoreceptor morphology at presentation (p = 0.19)., Conclusion: In eyes with choroidal nevus and SRF, there is a longitudinal evolution in photoreceptor morphology from normal to shaggy to retracted then absent with increasing SRF chronicity. SRF chronicity, as indicated by photoreceptor morphology on presentation, did not correlate with nevus growth to melanoma., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Photoreceptor cells are first-order retinal neurons that directly contribute to the formation of vision. Photoreceptor degeneration is the primary cause of vision impairment during the course of retinopathies such as retinitis pigmentosa and age-related macular degeneration, for which photoreceptor-targeted therapies are currently unavailable. Shihu Yeguang Pill (SYP), a classic formula in traditional Chinese medicine, has a long histology of clinical application for the treatment of a wide range of retinopathies in China. However, whether SYP is pharmacological effective at protecting photoreceptor cells is unclear. The current study thus directly addressed the pharmacological implications of SYP in photoreceptor degeneration in a mouse model characterized by bright light-induced retinal degeneration. Non-invasive full-retinal assessment was carried out to evaluate the effect of SYP on the retinal structure and function through optical coherence tomography and electroretinography, respectively. In addition, photoreceptor apoptosis, second-order neuron impairment and reactive changes in retinal microglial and müller cells, hallmark pathologies associated with photoreceptor degeneration, were assessed using immunohistochemistry and real-time PCR analyses. The results showed that SYP treatment attenuated bright light-induced impairment of the retinal structure and function. Moreover, SYP treatment suppressed photoreceptor apoptosis, alleviated the impairment of bipolar and horizontal cells and mitigated the reactive changes of müller and microglial cells in the bright light-exposed retinas. Real-time PCR analyses showed that dysregulated expression of pro-apoptotic c-fos and c-jun and anti-apoptotic bcl-2 as well as proinflammatory TNF-α in the bright light-exposed retinas was partially normalized as a result of SYP treatment. In summary, the work here demonstrates for the first time that SYP treatment protects the retinas from developing bright light-induced photoreceptor degeneration and associated alterations in second-order neurons and glial cells. The findings here thus provide experimental evidence to better support the mechanism-guided clinical application of SYP in the treatment of related retinal degenerative diseases., Competing Interests: Declaration of Competing Interest The authors declare no commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Photoreceptors possess ribbon synapses distinct from the conventional synapses in the brain. Little is known about the function of the BBSome, a complex integral in ciliary and intracellular trafficking, in ribbon synaptic formation. We performed immunohistochemistry using retinas from Bardet-Biedl Syndrome (BBS) mouse models and found that BBS mutant animals have significantly fewer ribbon synapses in the outer plexiform layer and increased ectopic synapses in the outer nuclear layer compared to controls. Many ectopic synapses in BBS mutant retinas are associated with horizontal cell axonal processes that aberrantly intrude into the outer nuclear layer. To determine whether this horizontal cell phenotype is a consequence of retinal degeneration, we examined this phenotype in mice with photoreceptor-specific inactivation of the BBSome induced by Cre recombinase driven by the rhodopsin promoter. At three months of age, despite retinal degeneration, Bbs8 floxed/floxed ; Rho-Cre + mice lack the aberrant intrusion of horizontal cell processes. At 6 months, some horizontal cell processes intrude into the outer nuclear layer in Bbs8 floxed/floxed ; Rho-Cre + mice, but the phenotype does not recapitulate the phenotypic severity observed in young congenital BBS mutant mice. Therefore, the lack of BBSome function negatively impacts retinal synaptogenesis, and causes horizontal cell defects in a potentially cell-autonomous fashion.

Purpose: To evaluate the effect of elongated photoreceptor outer segment length on the visual prognosis of patients with chronic central serous chorioretinopathy after treatment using half-dose and half-fluence photodynamic therapy (reduced PDT)., Methods: The study included 36 eyes of 36 patients with chronic central serous chorioretinopathy who underwent reduced PDT and were followed up for at least 1 year. Spectral domain optical coherence tomography measurement was conducted at baseline and 12 months after reduced PDT. Thereafter, the association between the best-corrected visual acuity (BCVA) at 12 months after reduced PDT and 7 baseline variables (age, symptom duration, BCVA, outer nuclear layer thickness, elongated photoreceptor outer segment length, height of subretinal detachment, and subfoveal choroidal thickness) was evaluated. Multivariate analyses using the model selection with the corrected Akaike Information Criterion index were performed., Results: The optimal model for BCVA at 12 months only included baseline BCVA (coefficient = 0.90, P < 0.0001) and baseline elongated photoreceptor outer segment length (coefficient = -0.0016, P = 0.034), but not outer nuclear layer thickness., Conclusion: Elongated photoreceptor outer segment length was significantly associated with BCVA prognosis in patients with chronic central serous chorioretinopathy after reduced PDT and can be useful for predicting residual photoreceptor function during the active phase of chronic central serous chorioretinopathy.

Diabetic macular edema (DME) and retina vein occlusion (RVO) are macular diseases in which central photoreceptors are affected due to pathological accumulation of fluid. Optical coherence tomography allows to visually assess and evaluate photoreceptor integrity, whose alteration has been observed as an important biomarker of both diseases. However, the manual quantification of this layered structure is challenging, tedious and time-consuming. In this paper we introduce a deep learning approach for automatically segmenting and characterising photoreceptor alteration. The photoreceptor layer is segmented using an ensemble of four different convolutional neural networks. En-face representations of the layer thickness are produced to characterize the photoreceptors. The pixel-wise standard deviation of the score maps produced by the individual models is also taken to indicate areas of photoreceptor abnormality or ambiguous results. Experimental results showed that our ensemble is able to produce results in pair with a human expert, outperforming each of its constitutive models. No statistically significant differences were observed between mean thickness estimates obtained from automated and manually generated annotations. Therefore, our model is able to reliable quantify photoreceptors, which can be used to improve prognosis and managment of macular diseases.

Age-related macular degeneration (AMD) is a complex eye disease underlined by the death of photoreceptors and degeneration of retinal pigment epithelium (RPE) and choriocapillaris (CC). The mechanism(s) responsible for massive and progressive retinal degeneration is not completely known. Senescence, a state of permanent inhibition of cell growth, may be induced by many factors important for AMD pathogenesis and results in senescence-associated secretory phenotype (SASP) that releases growth factors, cytokines, chemokines, proteases and other molecules inducing inflammation and other AMD-related effects. These effects can be induced in the affected cell and neighboring cells, leading to progression of AMD phenotype. Senescent cells also release reactive oxygen species that increase SASP propagation. Many other pathways of senescence-related AMD pathogenesis, including autophagy, the cGAS-STING signaling, degeneration of CC by membrane attack complex, can be considered. A2E, a fluorophore present in lipofuscin, amyloid-beta peptide and humanin, a mitochondria-derived peptide, may link AMD with senescence. Further studies on senescence in AMD pathogenesis to check the possibility of opening a perspective of the use of drugs killing senescent cells (senolytics) and terminating SASP bystander effects (senostatics) might be beneficial for AMD that at present is an incurable disease.

Purpose: To explore the expression and role of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) in retinal degeneration., Methods: The retinal degeneration of BALB/c mice was induced by light exposure. BV2 cells were activated by LPS stimulation. Retinas or BV2 cells were pretreated with LOX-1 neutralizing antibody or Polyinosinic acid (PolyI) (the inhibitor of LOX-1) before light damage (LD) or LPS stimulation. LOX-1, TNF-α, IL-1β, CCL2 and NF-κB expression were detected in retinas or BV2 cells by real-time RT-PCR, western blot or ELISA. Histological analyses of retinas were performed. Photoreceptor cell death was assessed by TUNEL assay in retinas or by flow cytometry in 661W cells cultured in microglia-conditioned medium., Results: Photoreceptor cell death and elevated expression of LOX-1 were induced by LD in retinas of BALB/c mice. LOX-1 neutralizing antibody or PolyI pretreatment significantly reduced the elevated expression of LOX-1, TNF-α, IL-1β, CCL2 and p-NF-κB caused by LD in retinas. Inhibition of LOX-1 by LOX-1 neutralizing antibody or PolyI significantly reduced photoreceptor cell death induced by LD in retinas. Elevated levels of TNF-α, IL-1β and CCL2 caused by LPS were down-regulated by inhibition of LOX-1 in BV2 cells. Inhibition of LOX-1 reduces microglial neurotoxicity on photoreceptors., Conclusions: LOX-1 expression is increased in light induced retinal degeneration, what's more, inhibition of LOX-1 prevents inflammation and photoreceptor cell death in retinal degeneration and reduces microglial neurotoxicity on photoreceptors. Therefore, LOX-1 can be used as a potential therapeutic target for such retinal degeneration diseases., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Combined administration of N-Methyl-D-Aspartate (NMDA) and kainic acid (KA) on the inner retina was studied as a model of excitotoxicity. The right eye of C57BL6J mice was injected with 1 µL of PBS containing NMDA 30 mM and KA 10 mM. Only PBS was injected in the left eye. One week after intraocular injection, electroretinogram recordings and immunohistochemistry were performed on both eyes. Retinal ganglion cell (RGC) projections were studied by fluorescent-cholerotoxin anterograde labeling. A clear decrease of the retinal "b" wave amplitude, both in scotopic and photopic conditions, was observed in the eyes injected with NMDA/KA. No significant effect on the "a" wave amplitude was observed, indicating the preservation of photoreceptors. Immunocytochemical labeling showed no effects on the outer nuclear layer, but a significant thinning on the inner retinal layers, thus indicating that NMDA and KA induce a deleterious effect on bipolar, amacrine and ganglion cells. Anterograde tracing of the visual pathway after NMDA and KA injection showed the absence of RGC projections to the contralateral superior colliculus and lateral geniculate nucleus. We conclude that glutamate receptor agonists, NMDA and KA, induce a deleterious effect of the inner retina when injected together into the vitreous chamber., Competing Interests: The authors declare no financial conflicts of interest.

Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661 W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.

Activation of the inflammasome is involved in the progression of retinal degenerative diseases, in particular, in the pathogenesis of Age-Related Macular Degeneration (AMD), with NLRP3 activation the focus of many investigations. In this study, we used genetic and pharmacological approaches to explore the role of the inflammasome in a mouse model of retinal degeneration. We identify that Casp1/11 -/- mice have better-preserved retinal function, reduced inflammation and increased photoreceptor survivability. While Nlrp3 -/- mice display some level of preservation of retinal function compared to controls, pharmacological inhibition of NLRP3 did not protect against photoreceptor cell death. Further, Aim2 -/- , Nlrc4 -/- , Asc -/- , and Casp11 -/- mice show no substantial retinal protection. We propose that CASP-1-associated photoreceptor cell death occurs largely independently of NLRP3 and other established inflammasome sensor proteins, or that inhibition of a single sensor is not sufficient to repress the inflammatory cascade. Therapeutic targeting of CASP-1 may offer a more promising avenue to delay the progression of retinal degenerations.

Purpose: Diabetic retinopathy (DR) is a leading cause of vision impairment and blindness worldwide in the working-age population, and the incidence is rising. Until now it has been difficult to define initiating events and disease progression at the molecular level, as available diabetic rodent models do not present the full spectrum of neural and vascular pathologies. Zebrafish harboring a homozygous mutation in the pancreatic transcription factor pdx1 were previously shown to display a diabetic phenotype from larval stages through adulthood. In this study, pdx1 mutants were examined for retinal vascular and neuronal pathology to demonstrate suitability of these fish for modeling DR., Methods: Vessel morphology was examined in pdx1 mutant and control fish expressing the fli1a:EGFP transgene. We further characterized vascular and retinal phenotypes in mutants and controls using immunohistochemistry, histology, and electron microscopy. Retinal function was assessed using electroretinography., Results: Pdx1 mutants exhibit clear vascular phenotypes at 2 months of age, and disease progression, including arterial vasculopenia, capillary tortuosity, and hypersprouting, could be detected at stages extending over more than 1 year. Neural-retinal pathologies are consistent with photoreceptor dysfunction and loss, but do not progress to blindness., Conclusions: This study highlights pdx1 mutant zebrafish as a valuable complement to rodent and other mammalian models of DR, in particular for research into the mechanistic interplay of diabetes with vascular and neuroretinal disease. They are furthermore suited for molecular studies to identify new targets for treatment of early as well as late DR.

Background/aims: It is well established that oxidative stress and inflammation are common pathogenic features of retinal degenerative diseases. ITH12674 is a novel compound that induces the transcription factor Nrf2; in so doing, the molecule exhibits anti-inflammatory, and antioxidant properties, and affords neuroprotection in rat cortical neurons subjected to oxidative stress. We here tested the hypothesis that ITH12674 could slow the retinal degeneration that causes blindness in rd10 mice, a model of retinitis pigmentosa., Methods: Animals were intraperitoneally treated with 1 or 10 mg/Kg ITH12674 or placebo from P16 to P30. At P30, retinal functionality and visual acuity were analyzed by electroretinography and optomotor test. By immunohistochemistry we quantified the photoreceptor rows and analyzed their morphology and connectivity. Oxidative stress and inflammatory state was studied by Western blot, and microglia reactivity was monitored by flow cytometry. The blood-brain barrier permeation of ITH12674 was evaluated using a PAMPA-BBB assay., Results: In rd10 mice treated with 10 mg/Kg of the compound, the following changes were observed (with respect to placebo): (i) a decrease of vision loss with higher scotopic a- and b-waves; (ii) increased visual acuity; (iii) preservation of cone photoreceptors morphology, as well as their synaptic connectivity; (iv) reduced expression of TNF-α and NF-κB; (v) increased expression of p38 MAPK and Atg12-Atg5 complex; and (vi) decreased CD11c, MHC class II and CD169 positive cell populations., Conclusion: These data support the view that a Nrf2 inducer compound may arise as a new therapeutic strategy to combat retinal neurodegeneration. At present, we are chemically optimising compound ITH12674 with the focus on improving its neuroprotective potential in retinal neurodegenerative diseases., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Purpose: To determine whether the size of the foveal avascular zone (FAZ) is significantly correlated with the best-corrected visual acuity (BCVA) and to examine the relationship between the size and microstructural changes of the photoreceptors in eyes with a branch retinal vein occlusion., Methods: The medical records of 69 eyes of patients (mean age, 64.6 ± 11.7 years) with a branch retinal vein occlusion were reviewed after the resolution of macular edema. All the patients underwent optical coherence tomography angiography for measurement of the FAZ area and spectral domain optical coherence tomography for determination of microstructural changes of the photoreceptors at the fovea., Results: The superficial and deep FAZ areas in eyes with a branch retinal vein occlusion were 0.39 ± 0.36 mm and 0.63 ± 0.18 mm, respectively, and both were significantly larger than those observed in the fellow eyes (both, P < 0.001). The superficial FAZ area correlated with the posttreatment BCVA (r = 0.285, P = 0.027) but not with any parameters regarding the microstructures of the photoreceptors. Multivariate linear regression analysis showed that the pretreatment BCVA (β = 0.519, P < 0.001) and integrity of the external limiting membrane (β = -0.373, P = 0.001) were independent factors that significantly correlated with the posttreatment BCVA., Conclusion: There was no significant correlation between the FAZ area and microstructural parameters. However, the integrity of the external limiting membrane was significantly correlated with the posttreatment BCVA in eyes with a branch retinal vein occlusion.