A peripheral RPE subpopulation is identified through spatial, transcriptomic, and functional analyses, thereby contributing to the understanding of the heterogeneity of degenerative RPE cells and highlighting SERPINE3 as a protective factor with therapeutic potential for macular atrophy.
Abstract
Heterogeneous degeneration of the retinal pigment epithelium (RPE) leads to irreversible blindness in diseases associated with macular atrophy. However, the underlying mechanisms of regional RPE degeneration remain poorly understood. To address this gap, this study identified a peripheral RPE subpopulation through spatial, transcriptomic, and functional analyses, thereby contributing to the understanding of the heterogeneity of degenerative RPE cells. Specifically, omics analyses in human and macaque RPE revealed a peripheral RPE cell population with high SERPINE3 expression, while SERPINE3-GFP–knockin mice showed comparable expression patterns. SMART RNA-seq2 analysis further distinguished transcriptomic profiles between GFP+ and GFP– RPE cells. Under oxidative stress, SERPINE3 expression increased, and GFP+ cells exhibited improved survival and reentry into the cell cycle. Notably, genetic studies indicated that SERPINE3 is essential for the oxidative stress resistance of GFP+ cells. Moreover, loss of SERPINE3 resulted in regional RPE degeneration and increased microglial accumulation in aged mice. Mechanistically, proteinase screening and co-IP indicated that SERPINE3 targets caspase-1. Importantly, delivery of SERPINE3 via AAV-Serpine3 partially reduced RPE degeneration in an oxidative damage model. These findings advance the understanding of RPE heterogeneous degeneration and highlight SERPINE3 as a protective factor with therapeutic potential for macular atrophy.
Dry age-related macular degeneration (AMD) is a leading cause of blindness, characterized by progressive loss of retinal pigment epithelium (RPE) and subsequent photoreceptor degeneration. Current experimental models, including sodium iodate-induced injury, fail to fully recapitulate the chronic, age-related progression of the human disease. Although RIP3-mediated necroptosis has been strongly implicated in RPE cell death, its direct contribution to retinal degeneration in vivo remains unclear. To address this limitation, we generated two RIP3 transgenic mouse lines with distinct patterns of RIP3 overexpression. While RIP3-Tg mice exhibit systemic RIP3 overexpression, RIP3-Tg-RPE mice display additional RPE-specific overexpression beyond the levels observed in RIP3-Tg mice. We then evaluated these transgenic lines, along with wild-type controls, for age-driven retinal degeneration by using optical coherence tomography (OCT), behavior-based visual function assays, and molecular profiling of inflammation and cell death. First, RIP3-Tg mice exhibited gradual retinal thinning, progressive visual decline, and sustained upregulation of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) over 6–15 months, recapitulating the slow progression of dry-AMD. Second, RIP3-Tg-RPE mice, which exhibit further RPE-specific increases in RIP3 expression, showed markedly accelerated retinal degeneration, with significant structural and functional deficits evident as early as 2 months of age. These findings indicate that ectopic RIP3 expression in the RPE contributes to inflammatory responses and subsequent retinal degeneration. Collectively, our results highlight RIP3 as a potential contributing factor in the progression of retinal degeneration and introduce biologically relevant transgenic models that capture both slow and accelerated disease progression. These models provide a valuable platform for investigating disease mechanisms and developing therapeutic strategies targeting necroptosis in dry-AMD.
Young-Do Song, Jiyeong Kim, Jun-Ho Jang et al.· PLoS ONE· 0 citations
BACKGROUND
Dry age-related macular degeneration (AMD) is driven largely by retinal pigment epithelium (RPE) cell injury. Kinsenoside (KN) exhibits protective activity on RPE cells, but the direct effect on dry AMD and underlying molecular mechanisms remain unclear.
OBJECTIVE
This study aimed to investigate the protective effects of KN against RPE cell injury and dysfunction in dry AMD and to elucidate its regulatory mechanisms.
METHODS
An A2E- and blue light-induced ARPE-19 cell injury model and a blue light-induced AMD-like retinal injury mouse model were established. RPE cytotoxicity, apoptosis, senescence, inflammation, and melanogenesis-associated marker expression were evaluated. MAPK signaling was analyzed by Western blotting, and mechanistic analyses were performed using a JNK inhibitor, molecular docking analysis, an in vitro JNK enzymatic activity assay, exosome characterization, and exosome functional assays.
RESULTS
KN treatment significantly attenuated cytotoxicity, apoptosis, cellular senescence, inflammatory responses, and melanogenesis-associated marker alterations in AMD-like ARPE-19 cells. In vivo, KN reduced drusen-like deposition, improved retinal structural integrity, and partially restored outer nuclear layer thickness while suppressing senescence and inflammation. Mechanistically, KN preferentially attenuated pathological JNK activation under the examined conditions and reduced JNK enzymatic activity in vitro. Pharmacological JNK inhibition phenocopied the protective effects of KN with no additive benefit, indicating pathway convergence. Exosome inhibition, transfer, and depletion experiments supported the contribution of exosome-associated signaling to KN-mediated cytoprotection.
CONCLUSION
These findings suggest that KN alleviates RPE cell injury under AMD-like stress, at least partly through exosome-associated modulation of JNK/MAPK signaling.
Yue Bai, Maosong Xie, Yihua Yao et al.· International Immunopharmaco...· 0 citations
PRPH2 mutations cause inherited retinal dystrophies (IRDs), but how photoreceptor outer segment (OS) disruption reshapes the surrounding retina remains unclear. Using a heterozygous Prph2C213Y/+ mouse model generated by CRISPR/Cas9, we characterized age-related retinal pathology and responses of retinal pigment epithelium (RPE) and Müller glia. Independent age- and sex-matched cohorts were examined at 1, 3, and 6 months by electroretinography, optical coherence tomography, and fundus autofluorescence. Mutant mice showed rod dysfunction from 1 month, RPE dysfunction from 3 months, and cone dysfunction by 6 months, accompanied by progressive outer retinal thinning and hyperautofluorescent deposits. Histological and ultrastructural analyses revealed OS disorganization, shortened RPE microvilli, RPE monolayer remodeling, increased RPE autofluorescence, and reactive Müller gliosis. Single-cell spatial transcriptomics of wild-type and mutant retinas at 6 months resolved nine cell populations and identified RPE cells and Müller glia as prominently perturbed non-photoreceptor populations. RPE cells showed an epithelial-mesenchymal transition-related remodeling state linked to a candidate Nfib-Fstl1 module, whereas Müller glia showed activation of activator protein 1 (AP-1) regulons, including Fos, Fosl2, and Junb, with predicted targets Osmr, A2m, and Stat3. Cell-cell communication analyses indicated coordinated changes in neuroprotective, inflammatory, and matrix-related signaling from RPE cells and Müller glia toward photoreceptors. These findings indicate that PRPH2-associated retinal dystrophy is a multicellular process in which OS disruption drives coordinated RPE and Müller glial remodeling with potentially protective or pro-degenerative effects, and nominate the RPE Nfib-Fstl1 program, Müller glial AP-1 responses with predicted STAT3 involvement, and support-cell-derived growth factor signaling as candidate mutation-independent therapeutic targets.
Haoxin Guo, Linfei Wei, Binghan Chen et al.· Neurobiology of Disease· 0 citations
Usher syndrome type 1, caused by pathogenic variants in the USH1C gene, leads to congenital deafness and progressive retinal degeneration resulting in vision loss. While auditory deficits can be compensated by cochlea implants and hearing aids, no treatment exists to prevent retinal degeneration. Here, we generated retinal organoids from induced pluripotent stem cells of two USH1C patients to elucidate the cellular and molecular mechanisms driving ocular pathogenesis. Single-cell RNA sequencing of healthy and USH1C retinal organoids identified differential expression of genes related to phototransduction in photoreceptors, as well as alterations in cell adhesion and canonical Wnt signaling in Müller glia cells. Analysis of intercellular communication revealed an overall reduced signaling efficiency, particularly affecting Müller glia-mediated retinal adhesion processes. Morphological characterization of organoids confirmed transcriptome changes by showing degeneration of the outer limiting membrane and loss of adherens junction architecture. Moreover, photoreceptors revealed increased levels of apoptosis, as well as morphological and functional changes related to phototransduction. Our results demonstrate that disruption of Müller glia signaling contributes to an overall loss of retinal integrity, providing novel insights into USH1C pathogenesis and offering targets for therapeutic interventions.
Nicole Wenck, Mark Zorin, Qiang Wang et al.· Cellular and Molecular Life...· 0 citations
Age-related macular degeneration (AMD) remains a leading cause of irreversible blindness worldwide, characterized by the progressive breakdown of the outer blood-retinal barrier, accumulation of protein and lipid deposits in the subretinal space, and the consequential degeneration of macular photoreceptors. Geographic atrophy (GA) is one of the blinding end points of AMD. While current pharmaceutical interventions can slow lesion expansion or counter choroidal neovascularization, they fail to address the most significant clinical unmet need: regeneration of damaged retinal tissue to restore vision. Regenerative medicine via stem cell transplantation offers a definitive curative approach by replacing the structural and immune framework of the outer retina. This review synthesizes current advancements in human embryonic and pluripotent stem cells platforms engineered for outer retinal reconstruction. We evaluate the differentiation, culturing, and quality validation required to generate clinical-grade, polarized RPE monolayers and homeostatic microglia-like cells. Structurally, single-cell suspensions are contrasted against bioengineered patches, scaffold-free cell sheets, and advanced cell strips, analyzing how graft configuration dictates post-transplantation integration and visual recovery while balancing procedural adverse events like cell reflux and epiretinal membrane formation. Furthermore, this review addresses the microenvironmental challenges of transplanting allogeneic constructs into an inflamed, senescent host niche. We examine CRISPR-Cas9 genome editing paradigms, including cytosine base and prime editing, designed to rectify cell-intrinsic genetic vulnerabilities such as the complement factor H (CFH) risk variant. We highlight translational applications, such as knocking out the Class II transactivator to eliminate major histocompatibility complex class II (MHC-II) expression, which successfully circumvents host T-cell immune surveillance and prevents graft rejection in non-human primates. Additionally, we analyze how engineering an inhibitor-resistant colony-stimulating factor 1 receptor (CSF1R) point mutation enables exogenously administered microglia to robustly outcompete and replace maladaptive, pro-inflammatory host microglia under selective small-molecule pressure. Finally, we discuss future directions, emphasizing multi-lineage bilayered co-transplantation models that combine genome-edited RPE patches with homeostatic microglia or retinal organoids to achieve durable synaptic repair, alongside automated artificial intelligence manufacturing pipelines. Together, these combined structural, molecular, and immune-modulating strategies represent the next clinical frontier in restoring clear central vision and achieving permanent neurovascular rescue in advanced macular degeneration.
Nuntachai Surawatsatien, Rajvir Mukesh Solanky, Robert P. van de Werken et al.· American journal of ophthalm...· 0 citations
Proliferative diabetic retinopathy (PDR) is a leading cause of irreversible blindness worldwide, characterized by pathological neovascularization and progressive retinal fibrosis. Although anti-VEGF therapy effectively suppresses neovascularization, it does not address fibrovascular membranes (FVMs) formation and may paradoxically accelerate membrane contraction, increasing the risk of tractional retinal detachment. The lack of effective anti-fibrotic strategies highlights the importance to elucidate the key cellular mechanisms underlying retinal fibrosis in PDR. We performed single-cell RNA sequencing (scRNA-seq) on primary retinal tissues from PDR organ donors and non-diabetic controls, generating a single-cell transcriptomic atlas of the human PDR retina. Critical findings were validated by immunofluorescence, in vivo and in vitro functional assays. Stromal-specific Sox15 conditional knockout mice in a two-stage laser-induced fibrosis model and siRNA-mediated knockdown in fibroblasts, and intravitreal administration of the EDNRB antagonist were performed. scRNA-seq of primary human PDR retinas generated the first comprehensive single-cell transcriptomic atlas of human PDR, revealing profound alterations in cellular composition including microglial expansion and Müller glia reprogramming as central neuroinflammatory features of PDR, and selective loss of metallothionein enriched rods subpopulation and S-cones. Notably, We identified a stromal cell population that was enriched in PDR retinas, localized in FVMs, and characterized by high SOX15 expression. Cell-cell communication analysis revealed that EDN1-EDNRB signaling between these stromal cells and Müller glia represented the most prominent intercellular interaction in PDR. Mechanistically, SOX15 regulates EDN1 transcription through binding to its promoter, driving EDN1 expression that activates EDNRB-expressing retinal macroglia to promote reactive gliosis and retinal fibrosis. Stromal-specific Sox15 conditional knockout significantly attenuated fibrotic lesion formation in vivo, and PDR-derived fibrocytes with elevated SOX15 exhibited enhanced fibroblast differentiation and EDN1 secretion, providing translational validation in human disease. Intravitreal EDNRB blockade with BQ-788 significantly reduced fibrotic area in a murine fibrosis model. This study identifies key pathological features of the human PDR retina, including pervasive neuroinflammation and selective loss of photoreceptor subpopulations, and delineates the SOX15–EDN1-EDNRB axis as a novel mechanism driving retinal fibrosis. Targeting this axis may represent a promising anti-fibrotic strategy for advanced PDR.
F. Tang, Wenjing He, Zhijie Niu et al.· Journal of Translational Med...· 0 citations
