Treatment Of Retinal Degenerative Diseases Research Articles

Using Small Molecules to Reprogram RPE Cells in Regenerative Medicine for Degenerative Eye Disease.

The main purpose of regenerative medicine for degenerative eye diseases is to create cells to replace lost or damaged ones. Due to their anatomical, genetic, and epigenetic features, characteristics of origin, evolutionary inheritance, capacity for dedifferentiation, proliferation, and plasticity, mammalian and human RPE cells are of great interest as endogenous sources of new photoreceptors and other neurons for the degrading retina. Promising methods for the reprogramming of RPE cells into retinal cells include genetic methods and chemical methods under the influence of certain low-molecular-weight compounds, so-called small molecules. Depending on the goal, which can be the preservation or the replacement of lost RPE cells and cellular structures, various small molecules are used to influence certain biological processes at different levels of cellular regulation. This review discusses the potential of the chemical reprogramming of RPE cells in comparison with other somatic cells and induced pluripotent stem cells (iPSCs) into neural cells of the brain and retina. Possible mechanisms of the chemically induced reprogramming of somatic cells under the influence of small molecules are explored and compared. This review also considers other possibilities in using them in the treatment of retinal degenerative diseases based on the protection, preservation, and support of survived RPE and retinal cells.

CYLD Maintains Retinal Homeostasis by Deubiquitinating ENKD1 and Promoting the Phagocytosis of Photoreceptor Outer Segments.

Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is essential for retinal homeostasis. Dysregulation of the phagocytotic process is associated with irreversible retinal degenerative diseases. However, the molecular mechanisms underlying the phagocytic activity of RPE cells remain elusive. In an effort to uncover proteins orchestrating retinal function, the cylindromatosis (CYLD) deubiquitinase is identified as a critical regulator of photoreceptor outer segment phagocytosis. CYLD-deficient mice exhibit abnormal retinal structure and function. Mechanistically, CYLD interacts with enkurin domain containing protein 1 (ENKD1) and deubiquitinates ENKD1 at lysine residues K141 and K242. Deubiquitinated ENKD1 interacts with Ezrin, a membrane-cytoskeleton linker, and stimulates the microvillar localization of Ezrin, which is essential for the phagocytic activity of RPE cells. These findings thus reveal a crucial role for the CYLD-ENKD1-Ezrin axis in regulating retinal homeostasis and may have important implications for the prevention and treatment of retinal degenerative diseases.

Neuroprotective Effects of Astragalus Polysaccharide on Retina Cells and Ganglion Cell Projection in NMDA-Induced Retinal Injury.

Astragalus polysaccharide (APS), a water-soluble heteropolysaccharide, possesses immunomodulatory, anti-inflammatory, and cardioprotective properties. This study investigates the neuroprotective potential of APS in a model of N-Methyl-d-aspartic acid (NMDA)-induced retinal neurodegeneration, aiming to explore its potential as a treatment for retinal degenerative diseases. Retinal function was evaluated using electroretinography (ERG), optomotor reflex (OMR), and flash visual evoked potentials (FVEP). Retinal inflammatory responses were examined through immunohistochemistry, western blotting (WB), and quantitative reverse transcription PCR (qRT-PCR). To assess the integrity of visual projections, an intravitreal injection of adeno-associated virus (AAV) was employed to trace the projections of retinal ganglion cells (RGCs) to the visual centers. APS treatment conferred protection to retinal cells, as indicated by ERG and OMR assessments. And APS intervention mitigated NMDA-induced apoptosis, evidenced by a decrease in TUNEL-positive cells. Furthermore, APS treatment attenuated the NMDA-induced reduction in RGC projections to the visual centers, including the superior colliculus and lateral geniculate nucleus, as demonstrated by AAV tracing. Our findings reveal that APS shields the retina from NMDA-induced damage by inhibiting the NF-κB signaling pathway and reduces the detrimental effects of NMDA on RGC projections to the visual centers. These findings propose APS as a potential novel therapeutic agent for the treatment of retinal diseases.

GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology.

Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration-like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.

Adaptive Optics Optical Coherence Tomography Analysis of Induced Pluripotent Stem Cell-Derived Retinal Organoid Transplantation in Retinitis Pigmentosa.

This study evaluates the transplantation of induced pluripotent stem cell (iPSC)-derived retinal organoids into patients with advanced retinitis pigmentosa using adaptive optics optical coherence tomography (AO-OCT) to monitor retinal changes over two years post transplantation. Our results confirmed successful engraftment and increased retinal thickness, with AO-OCT providing detailed visualization of cellular structures such as an outer plexiform layer-like line and highly reflective particles within rosette-like formations, indicative of photoreceptor development. Immunohistological analysis in a parallel monkey model confirmed these structures as mature, functional photoreceptor rosettes. The integration of high-resolution AO-OCT with immunohistology provides critical insights into the structural and functional outcomes of transplantation and represents a promising advancement in the treatment of retinal degenerative diseases.

Safety of intravitreally delivered AAV2 vector-mediated multi-characteristic opsin genetic construct in wild type beagle dogs.

A novel adeno-associated virus 2 (AAV2)-carried multi-characteristic opsin (MCO) (MCO-010) is undergoing several clinical trials as a novel therapeutic modality for the treatment of degenerative retinal diseases including retinitis pigmentosa and Stargardt disease. The present study aimed to determine the ocular and systemic safety of MCO-010 and the AAV2 vehicle in adult Beagle dogs following intravitreal (IVT) injection. The current safety/toxicology studies spanning 13 weeks described here utilized well-documented techniques to assess the effects of IVT injection of MCO-010 up to 2.2 × 1011 genome copies (gc) per eye, or the AAV2 capsid (vehicle control) on gross behavioral and immunogenic changes, alterations in body weights, blood biochemistry, hematology, blood coagulation, gross necropsy lesions, organ weight changes and histopathology in the dogs (n = 4 per group; two males and two females per group). Immunohistochemical and functional electroretinogram studies were also conducted to determine MCO expression in the retina and determine any retinal toxicity associated with MCO-010. There were no significant deleterious effects of the MCO-010 (or the AAV2 at the tested doses) on any of the examined parameters, including the absence of any severe ocular or systemic adverse events. However, as expected, inflammation after IVT delivery of AAV2 and MCO-010 was observed in the conjunctivae of all groups of animals, although this self-resolved within 1week post-injection. Quantitative immunohistochemical analyses of MCO-010-associated mCherry revealed successful delivery of the gene therapy within the inner retina. In summary, MCO-010 demonstrated a favorable safety profile when administered to the eyes of adult Beagle dogs of both sexes at dose levels up to 2.2 × 1011 gc per eye, with no adverse effects observed. This dose was identified as the No Observed Adverse Effect Level (i.e. NOAEL) and guided selection of safe doses for human clinical trials.

Induction of human ESC-derived and adult primary multipotent limbal stem cells into retinal pigment epithelial cells and corneal stromal stem cells

Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.

Synergistic effect of mesenchymal stem cells and silver nanoparticles in the treatment of retinal degenerative diseases

Aims/Purpose: Retinal degenerative diseases represent one of the leading causes of visual loss. Mesenchymal stem cells (MSCs) provide potential option for the treatment of several type of pathologies, including ocular diseases. Moreover, silver nanoparticles (AgNPs) are currently widely used in various fields of medicine mainly due to their antibacterial and antiangiogenic properties. Several studies showed their beneficial but also harmful effect. However, there are limited information about the synergistic effect of AgNPs with MSC‐based therapies.Methods: Chronical retinal degeneration was induced in BALB/c mice by repeated intraperitoneal application of sodium iodate (NaIO3). MSCs, AgNPs or MSCs plus AgNPs were intravitreally injected 48 h before the last application of NaIO3. Retinas were isolated after additional 7 days and tested by RT‐PCR for the expression of genes for rhodopsin (photoreceptors), Iba‐1 (microglia/macrophages), IL‐1β and galectin‐3. The presence of MSCs stained with PKH‐26 was analysed by flow cytometry.Results: It was shown that MSCs and MSCs applied with AgNPs were still present in the retina on day 7 after application. In all treated groups, the expression of the gene for galectin‐3 was decreased in contrast to untreated control. However, co‐application of MSCs with AgNPs had limited effect on downregulation of Iba‐1 and IL‐1β in contrast to MSCs or AgNPs treated group.Conclusions: The application of MSCs and AgNPs modulated immune response in chronical retinal degeneration but co‐application of MSCs with AgNPs decreased immunoregulatory effects comparted with single‐component therapy. Thus, there is a need for more investigation focusing on potential changes in MSCs applied with AgNPs.

Multidisciplinary Approaches in the Treatment of Retinal Degenerative Diseases: A Review

AbstractRetinal degenerative diseases (RDDs) cause irreversible blindness and affect ≈285 million individuals worldwide. The unavailability of treatment to stop the progression of RDDs or restore normal visual function necessitated development of novel therapeutic strategies. In recent years, multidisciplinary approaches have shown great potential for the treatment of RDDs, including intraocular delivery systems, retinal scaffolds for cell therapy, and visual prostheses with different stimulation strategies. This review focuses on the development prospects and shortcomings of various multidisciplinary treatment approaches for RDDs. The results of this review will hopefully inspire further innovation in the design of novel multidisciplinary approaches for RDD treatment and accelerate their commercialization.

Modifying treatment of degenerative retinal diseases. Part 2. Conditioning therapy techniques and the problem of maximizing retinal plasticity

In the first part of the review [ROJ, 2023; 16 (2): 160–2], we discussed the common and specific features of adaptive and non-adaptive retinal plasticity characteristic of glaucoma, age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and retinopathy of prematurity. The presented part of the review discusses the issues of axon regeneration of retinal ganglion cells and analyzes therapeutic approaches aimed at maximizing the plasticity and stimulating the reparative potential of the retina. The protective effects of "conditioning" stimuli in the modifying treatment of retinal diseases are considered. Some of the present-day visual rehabilitation strategies based on visual perception training and visual fixation training using biofeedback systems are reported.

Therapeutic Extracellular Vesicles from Tonsil-Derived Mesenchymal Stem Cells for the Treatment of Retinal Degenerative Disease.

Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic. To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa. T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dose-dependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat. Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.

Modifying treatment of degenerative retinal diseases. Part 1. Adaptive and non-adaptive retinal plasticity

Retinal structural plasticity is manifested in multiple damages of the retina. In many cases, the response to these damages is identical at both the cellular and molecular levels, involves similar sets of cellular signals, and is associated with a change in the structure of the retina and remodeling of the neural connections. The review discusses the common and specific features of adaptive and non-adaptive retinal plasticity, which characterize glaucoma, age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and retinopathy of prematurity. Given the common features of neurodegeneration and retinal plasticity in brain and retinal diseases, similar therapeutic strategies can be used in many cases to preserve the structure connectivity and retinal function, which stop or slow down the clinical evolution of the disease by either suppressing primary events or enhancing compensatory and regenerative mechanisms in the nervous tissue. Part 2 of the review will present neuroplasticity-based modifying therapy methods for retinal degenerative diseases.

Affinity-controlled release of rod-derived cone viability factor enhances cone photoreceptor survival.

Retinitis pigmentosa (RP) is a group of genetic diseases that results in rod photoreceptor cell degeneration, which subsequently leads to cone photoreceptor cell death, impaired vision and eventual blindness. Rod-derived cone viability factor (RdCVF) is a protein which has two isoforms: a short form (RdCVF) and a long form (RdCVFL) which act on cone photoreceptors in the retina. RdCVFL protects photoreceptors by reducing hyperoxia in the retina; however, sustained delivery of RdCVFL remains challenging. We developed an affinity-controlled release strategy for RdCVFL. An injectable physical blend of hyaluronan and methylcellulose (HAMC) was covalently modified with a peptide binding partner of the Src homology 3 (SH3) domain. This domain was expressed as a fusion protein with RdCVFL, thereby enabling its controlled release from HAMC-binding peptide. Sustained release of RdCVFL was demonstrated for the first time as RdCVFL-SH3 from HAMC-binding peptide for 7 d in vitro. To assess bioactivity, chick retinal dissociates were harvested and treated with the affinity-released recombinant protein from the HAMC-binding peptide vehicle. After 6 d in culture, cone cell viability was greater when cultured with released RdCVFL-SH3 relative to controls. We utilized computational fluid dynamics to model release of RdCVFL-SH3 from our delivery vehicle in the vitreous of the human eye. We demonstrate that our delivery vehicle can prolong the bioavailability of RdCVFL-SH3 in the retina, potentially enhancing its therapeutic effects. Our affinity-based system constitutes a versatile delivery platform for ultimate intraocular injection in the treatment of retinal degenerative diseases. STATEMENT OF SIGNIFICANCE: Retinitis pigmentosa (RP) is the leading cause of inherited blindness in the world. Rod-derived cone viability factor (RdCVF), a novel protein paracrine factor, is effective in preclinical models of RP. To extend its therapeutic effects, we developed an affinity-controlled release strategy for the long form of RdCVF, RdCVFL. We expressed RdCVFL as a fusion protein with an Src homology 3 domain (SH3). We then utilized a hydrogel composed of hyaluronan and methylcellulose (HAMC) and modified it with SH3 binding peptides to investigate its release in vitro. Furthermore, we designed a mathematical model of the human eye to investigate delivery of the protein from the delivery vehicle. This work paves the way for future investigation of controlled release RdCVF.

Stem Cells As Potential Regenerative Treatment For Retinal Degenerative Diseases And Diabetic Retinopathy

Stem Cells As Potential Regenerative Treatment For Retinal Degenerative Diseases And Diabetic Retinopathy

Targeting Necroptosis: A Novel Therapeutic Option for Retinal Degenerative Diseases.

The discovery of the necroptosis, a form of regulated necrosis that is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like pseudokinase (MLKL), represents a major breakthrough that has dramatically altered the conception of necrosis - traditionally thought of as uncontrolled cell death - in various human diseases. Retinal cell death is a leading cause of blindness and has been identified in most retinal diseases, e.g., age-related macular degeneration, glaucoma, retinal detachment, retinitis pigmentosa, etc. Increasing evidence demonstrates that retinal degenerative diseases also share a common mechanism in necroptosis. Exacerbated necroptotic cell death hinders the treatment for retinal degenerative diseases. In this review, we highlight recent advances in identifying retinal necroptosis, summarize the underlying mechanisms of necroptosis in retinal degenerative diseases, and discuss potential anti-necroptosis strategies, such as selective inhibitors and chemical agents, for treating retinal degenerative diseases.

Retinal Degeneration: Molecular Mechanisms and Therapeutic Strategies.

Retinal degenerative diseases are the main retinal diseases that threaten vision. Most retinal degenerative diseases are inherited diseases, including autosomal recessive inheritance, autosomal dominant inheritance, X-linked inheritance, and mitochondrial inheritance; therefore, emerging gene therapy strategies may provide an alternative method of treatment. Currently, three viral vectors are usually used in gene therapy studies: adenovirus, lentivirus, and adeno-associated virus. Other gene therapies have their own advantages, such as DNA nanoparticles, antisense oligonucleotides, and gene editing therapies. In addition, retinal degenerative diseases are often accompanied by abnormalities of retinal cells, including photoreceptor and retinal pigment epithelial cells. At present, stem cell transplantation is a promising new treatment for retinal degenerative diseases. Common sources of stem cells include retinal progenitor cells, induced pluripotent stem cells, embryonic stem cells, and mesenchymal stem cells. In addition, retina explant cultures in vitro can be used as an effective platform for screening new therapies for retinal degenerative diseases. Drugs that actually reach the retinal layer are more controlled, more consistent, and less invasive when using retinal explants. Furthermore, studies have shown that the imbalance of the gut microbiota is closely related to the occurrence and development of diabetic retinopathy. Therefore, the progression of diabetic retinopathy may be restrained by adjusting the imbalance of the gut microbiota. The purpose of this review is to discuss and summarize the molecular mechanisms and potential therapeutic strategies of retinal degenerative diseases.

Optogenetic Therapy for Visual Restoration.

Optogenetics is a recent breakthrough in neuroscience, and one of the most promising applications is the treatment of retinal degenerative diseases. Multiple clinical trials are currently ongoing, less than a decade after the first attempt at visual restoration using optogenetics. Optogenetic therapy has great value in providing hope for visual restoration in late-stage retinal degeneration, regardless of the genotype. This alternative gene therapy consists of multiple elements including the choice of target retinal cells, optogenetic tools, and gene delivery systems. Currently, there are various options for each element, all of which have been developed as a product of technological success. In particular, the performance of optogenetic tools in terms of light and wavelength sensitivity have been improved by engineering microbial opsins and applying human opsins. To provide better post-treatment vision, the optimal choice of optogenetic tools and effective gene delivery to retinal cells is necessary. In this review, we provide an overview of the advancements in optogenetic therapy for visual restoration, focusing on available options for optogenetic tools and gene delivery methods.

Hydrogen sulfide protects retinal pigment epithelium cells against ferroptosis through the AMPK- and p62-dependent non-canonical NRF2-KEAP1 pathway

Oxidative stress-induced ferroptosis of retinal pigment epithelium (RPE) cells contributes to retinal degenerative diseases. The antioxidant molecule hydrogen sulfide (H2S) regulates oxidative stress response, but its effect on the ferroptosis of RPE cells is unclear. In this study, sodium hydrosulfide (NaHS) was used as an exogenous H2S donor to intervene tert-butyl hydroperoxide (t-BHP)-induced ferroptosis of APRE-19 cells. We found that NaHS pretreatment attenuates t-BHP-induced oxidative stress and ferroptosis. Analysis of mRNA-sequencing coupled with FerrDb database identified nuclear factor erythroid-2-related factor 2 (NRF2) as a primary target for the cytoprotective role of H2S. NRF2 inhibitor ML385 reverses the effects of H2S on ferroptosis. Biochemical analysis revealed that H2S stabilizes NRF2. H2S decreases the interaction between NRF2 and KEAP1, but enhances the interaction between KEAP1 and p62. These results suggest that H2S activates the non-canonical NRF2-KEAP1 pathway. Further study demonstrated that H2S stimulates AMPK to interact and phosphorylate p62. Additionally, inhibiting AMPK or knocking down p62 blocks the effects of H2S. We speculate that targeting the non-canonical NRF2-KEAP1 pathway by H2S-based drug may benefit the treatment of retinal degenerative diseases.

Ginkgo biloba Extract Attenuates Light-Induced Photoreceptor Degeneration by Modulating CAV-1-Redoxosome Signaling.

The clinical potential of Ginkgo biloba extract (GBE) in the prevention and/or treatment of retinal degenerative diseases has been widely explored; however, the underlying molecular mechanism is poorly understood. Photoreceptor degeneration is the hallmark of retinal degenerative diseases and leads to vision impairment or loss. In this study, the effect of GBE against white light (WL) illumination-induced photoreceptor degeneration was investigated, as well as its underlying mechanism. To evaluate the in vitro activity of GBE, analysis of cell viability, cell apoptosis, oxidative stress, NOX (NADH oxidase) activity and mitochondrial membrane potential (MMP), as well as Western blotting and transcriptome sequencing and analysis, were conducted. To evaluate the in vivo activity of GBE, HE staining, electroretinography (ERG), Terminal-deoxynucleoitidyl transferase (TdT)-mediated nick end labeling (TUNEL) assay and immunofluorescence analysis were conducted. Our study showed that GBE treatment significantly attenuated WL illumination-induced oxidative damage in photoreceptor 661W cells—a finding that was also verified in C57BL/6J mice. Further molecular study revealed that WL illumination downregulated caveolin-1 (CAV-1) expression, interrupted CAV-1-NOX2 interaction, re-located NOX2 from the cell membrane to the cytoplasm and induced the formation of redoxosomes, which led to cell death. However, these cytotoxic events were significantly alleviated by GBE treatment. Interestingly, CAV-1 overexpression showed a consistent protective effect with GBE, while CAV-1 silencing impacted the protective effect of GBE against WL illumination-induced oxidative damage in in vitro and in vivo models. Thus, GBE was identified to prevent photoreceptor cell death due to CAV-1-dependent redoxosome activation, oxidative stress and mitochondrial dysfunction resulting from WL illumination. Overall, our study reveals the protective effect of GBE on photoreceptors against WL illumination-induced oxidative damage in in vitro and in vivo models, which effect is mediated through the modulation of CAV-1-redoxosome signaling. Our findings contribute to better understanding the therapeutic effect of GBE in preventing photoreceptor degeneration in retinal degenerative diseases, and GBE may become a novel therapeutic agent that is effective in reducing the morbidity of these diseases.

Somatostatin signalling promotes the differentiation of rod photoreceptors in human pluripotent stem cell-derived retinal organoid.

ObjectivesStem cell‐derived photoreceptor replacement therapy is a promising strategy for the treatment of retinal degenerative disease. The development of 3D retinal organoids has permitted the production of photoreceptors. However, there is no strategy to enrich a specific photoreceptor subtype due to inadequate knowledge of the molecular mechanism underlying the photoreceptor fate determination. Hence, our aim is to explore the uncharacterized function of somatostatin signalling in human pluripotent stem cell‐derived photoreceptor differentiation.Materials and Methods3D retinal organoids were achieved from human embryonic stem cell. The published single‐cell RNA‐sequencing datasets of human retinal development were utilized to further investigate the transcriptional regulation of photoreceptor differentiation. The assays of immunofluorescence staining, lentivirus transfection, real‐time quantitative polymerase chain reaction and western blotting were performed.ResultsWe identified that the somatostatin receptor 2 (SSTR2)‐mediated signalling was essential for rod photoreceptor differentiation at the precursor stage. The addition of the cognate ligand somatostatin in human 3D retinal organoids promoted rod photoreceptor differentiation and inhibited cone photoreceptor production. Furthermore, we found that the genesis of rod photoreceptors was modulated by endogenous somatostatin specifically secreted by developing retinal ganglion cells.ConclusionsOur study identified SSTR2 signalling as a novel extrinsic regulator for rod photoreceptor fate determination in photoreceptor precursors, which expands the repertoire of functional signalling pathways in photoreceptor development and sheds light on the optimization of the photoreceptor enrichment strategy.