bims-mideyd 2025-09-07 papers

Stem Cell Reports. 2025 Aug 19. pii: S2213-6711(25)00215-2. [Epub ahead of print] 102611

Single-cell transcriptome and surfaceome profiling of the adult human retinal pigment epithelium.

Farhad Farjood, Swapna Nandakumar, Taylor Bertucci, Thomas Kiehl, Steve Lotz, Yue Wang, Jacob Black, Skanda Sai, Jade Kozak, Brigitte L Arduini, Sally Temple, Nathan C Boles, Jeffrey H Stern.

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells beneath the neural retina that supports photoreceptor cell function essential for vision. Our study explores the diversity of adult human RPE subpopulations and associated implications for retinal biology. Employing cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), we identified distinct RPE cell subpopulations characterized by unique single-cell transcriptomic and surface protein signatures. Immunohistochemical analysis using CITE-seq markers demonstrated that different RPE subpopulations had previously unappreciated spatial patterns. Enrichment by CITE-seq surface marker selection revealed that different RPE subpopulations have distinct functions. By comparing native RPE cells isolated from the adult RPE layer to cultured RPE cells, we demonstrated that most RPE subpopulations were preserved during culture, a finding with relevance to an RPE cell product currently in clinical trial for treatment of non-exudative age-related macular degeneration. These findings deepen understanding of human RPE biology and provide valuable insights to optimize RPE-cell-based therapy.

Keywords: AMD; CITE-seq; RPE; age-related macular degeneration; retinal pigment epithelium

DOI: https://doi.org/10.1016/j.stemcr.2025.102611

Invest Ophthalmol Vis Sci. 2025 Sep 02. 66(12): 11

Lack of Pnpla2 Accelerates Progression of AMD-Like Features in Mice.

Juan Yang, Alexandra Bernardo-Colón, S Patricia Becerra.

Purpose: Lipid accumulation in the retinal pigment epithelium (RPE) contributes to cellular stress and progression of age-related macular degeneration (AMD). However, the regulation of lipid homeostasis in AMD development is not fully elucidated. The study investigates the effects of Pnpla2 deletion, a gene involved in lipid regulation, on key markers of RPE senescence and aging with potential relevance to AMD.
Methods: RPE flat mounts and retinal cryosections were analyzed from Pnpla2-/- and Pnpla2+/+ mice aged 3 months. Senescence-associated β-galactosidase (SA-β-gal) activity was assessed in flat mounts. DAPI was used to quantify RPE cells with single or multiple nuclei. Immunohistofluorescence was carried out to assess RPE tight junctions and expression of senescence and AMD markers using antibodies to zonula occludens 1 (ZO-1), phospho-histone (P-γ-H2AX), apolipoprotein E (ApoE), and high mobility group box 1 (HMGB1). Fundus imaging was acquired, and electroretinography (ERG) assessed visual function.
Results: Pnpla2-/- RPE exhibited increased SA-β-gal activity, multinucleation of the population, and the translocation of HMGB1 from nucleus to cytoplasm, indicative of cellular senescence. Tight junctions were disrupted. The number of P-γ-H2AX-positive RPE cells increased by 50%, suggesting increased DNA damage. ApoE levels were elevated in Bruch's membrane and subretinal regions. At 3 months of age, attenuation of ERG c-wave amplitude was observed in both Pnpla2-/- and Pnpla2+/- mice. By 7 months of age, Pnpla2+/- mice exhibited continued attenuation of ERG c-wave amplitude and developed white spots.
Conclusions: Pnpla2 deficiency accelerates cellular features of RPE aging and generates AMD-like features. These findings underscore the importance of PNPLA2 in mitigating AMD progression and highlight its significance in retinal health and degeneration.

DOI: https://doi.org/10.1167/iovs.66.12.11

Int J Ophthalmol. 2025 ;18(9): 1626-1639

Integration of multi-omics data reveals dysregulated RNA methylation in retinal pigment epithelium drives age-related macular degeneration.

Ya-Jun Gong, Zhi-Lin Zou, Kai-Rui Qiu, Qiang Wang, Xiao-Lai Zhou.

AIM: To investigate the role of RNA methylation in retinal pigment epithelial (RPE) cells in age-related macular degeneration (AMD).
METHODS: RNA methylation-related gene expression profiles of AMD patient and normal control retinal pigment epithelium were evaluated by single-cell transcriptome from 34 samples (11 from normal donors and 23 from AMD patients). The causal relationship between RNA methylation dysfunction and AMD was analyzed by summary-data-based Mendelian randomization (SMR) using AMD GWAS data and multi-omics quantitative trait loci (QTL), including expression QTLs (eQTLs), protein QTLs (pQTLs), splicing QTLs (sQTLs), and m6A-QTLs (mQTLs). Additionally, machine learning models were applied to validate the causal association between RNA methylation dysfunction and AMD using Bulk RNA sequencing data from 31 normal donors and 37 AMD patients.
RESULTS: The single-cell transcriptome data analysis revealed massive dysregulation of RNA methylation-related gene expression in the RPE of AMD patients. SMR revealed causal associations between key RNA methylation regulators (METTL3, NSUN6, and MRM1, etc.) and AMD onset. Machine learning models further validated these findings and demonstrated a high accuracy of AMD risk prediction by using the above-identified RNA methylation-related genes: METTL3, NSUN6, and MRM1. Furthermore, METTL3 and NSUN6 were found to have a protective effect, while MRM1 was associated with an increased risk of AMD.
CONCLUSION: The results reveal the implication of dysregulation of RNA methylation-related gene expression in the RPE of AMD patients and further demonstrated a causal association between RNA methylation-related genes (METTL3, NSUN6, and MRM1) and AMD. These findings highlight the importance of RNA methylation in the pathogenesis of AMD and offer potential biomarkers and therapeutic targets for AMD management.

Keywords: RNA methylation; age-related macular degeneration; retinal pigment epithelium

DOI: https://doi.org/10.18240/ijo.2025.09.03

Invest Ophthalmol Vis Sci. 2025 Sep 02. 66(12): 2

Nkx3.2 Inhibits Retinal Degeneration by Modulating Inflammation and Cell Death in RPE.

Minsun Park, Seung-Won Choi, Da-Un Jeong, Hae-Sol Shin, Jiyeon Han, Hye-Kyoung Oh, Jun-Ho Jang, Nari Ryu, Hyunjin Jeong, Young-Do Song, Kyoung Yul Seo, Sanghyuk Lee, Dae-Won Kim.

Purpose: To identify the expression of Nkx3.2 in retinal pigment epithelium (RPE) and evaluate its physiological role in association with retinal degeneration.
Methods: Nkx3.2 expression in RPE was examined by biochemical and histological analyses. Various in vitro and in vivo assays were employed to reveal the molecular mechanisms by which Nkx3.2 regulates inflammatory responses and cell survival in RPE. In addition, by investigating multiple animal models, the biological significance of Nkx3.2 in retinal degeneration was assessed.
Results: Nkx3.2 expression was verified in human cadaveric and mouse eye tissues and shown to be regulated by aging and oxidative stress. Mouse model analyses demonstrated retina protection activity of Nkx3.2 against aging, oxidative stress, vascular endothelial growth factor (VEGF) hyperactivation, and laser-induced damage. In vitro studies showed that Nkx3.2 downregulates pro-inflammatory cytokines and chemokines, but it upregulates anti-inflammatory factors. In addition, Nkx3.2 induced proteasomal degradation of receptor-interacting protein kinase 3 (RIP3), which, in turn, inhibited necroptosis. Consistent with these results, transcriptome analysis of mouse retina tissues indicated that Nkx3.2 can modulate gene expression profiles related to inflammatory responses, cell death, and visual function under oxidative stress.
Conclusions: Nkx3.2 can suppress inflammatory responses and necroptic cell death in RPE. By employing these mechanisms, Nkx3.2 may play a significant role in inhibiting retinal degeneration caused by aging and oxidative stress.

DOI: https://doi.org/10.1167/iovs.66.12.2

Life Sci. 2025 Aug 29. pii: S0024-3205(25)00568-5. [Epub ahead of print]380 123933

Dysregulation of the TNF-α-OTUD3-PPARγ signaling axis exacerbates retinal oxidative stress and inflammation in diabetic retinopathy.

Jialin Zhang, Zhen Wang, Junjun Liu, Wenchang Xu, Lu Lu, Na Zhou, Yongfeng Song.

AIMS: Diabetic retinopathy (DR) is one of the major complications of diabetes. In addition to hyperglycemia, various mechanisms contribute to the development of microvascular damage to the retina, which have not been fully elucidated. The aim of this study was to investigate Ovarian tumor domain-containing protein 3 (OTUD3)'s protection against DR by targeting peroxisome proliferator-activated receptor γ (PPARγ)-mediated dysfunction and identifying therapeutic strategies.
MATERIALS AND METHODS: We conducted clinical analysis of 208 type 2 diabetes mellitus (T2DM) patients with OTUD3 genotyping, combined with diabetic homozygous mutated (Otud3-/-) mouse models and retinal pigment epithelium (RPE) cell lines (OTUD3 knockdown/mutation).
KEY FINDINGS: We found increased hyperreflective foci (HRF) associated with an increased immune activation in diabetic Otud3-/- mice compared to Otud3 wild-type (Otud3+/+) mice. OTUD3 knockdown or mutated cells showed increased cell dysfunction and oxidative stress markers under inflammatory conditions. Further upstream transcription factors predict analysis suggest PPARγ as the potential target of OTUD3. Finally, we found PPARγ agonist could rescue the phenotype in RPE cells characterized by increased ROS levels, enhanced migration, and elevated apoptosis resulting from OTUD3 loss of function through knockdown or mutation.
SIGNIFICANCE: Our study offers novel insights into how deubiquitylase OTUD3 maintains the normal function of the retina by deubiquitylating PPARγ and provides a novel therapeutic target for this vision-threatening diabetic complications.

Keywords: Diabetic retinopathy; OTUD3; Oxidative stress; PPARγ; TNF-α

DOI: https://doi.org/10.1016/j.lfs.2025.123933

Biology (Basel). 2025 Aug 07. pii: 1014. [Epub ahead of print]14(8):

Bidirectional Hypoxic Extracellular Vesicle Signaling Between Müller Glia and Retinal Pigment Epithelium Regulates Retinal Metabolism and Barrier Function.

Alaa M Mansour, Mohamed S Gad, Samar Habib, Khaled Elmasry.

The retina is highly sensitive to oxygen and blood supply, and hypoxia plays a key role in retinal diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Müller glial cells, which are essential for retinal homeostasis, respond to injury and hypoxia with reactive gliosis, characterized by the upregulation of the glial fibrillary acidic protein (GFAP) and vimentin, cellular hypertrophy, and extracellular matrix changes, which can impair retinal function and repair. The retinal pigment epithelium (RPE) supports photoreceptors, forms part of the blood-retinal barrier, and protects against oxidative stress; its dysfunction contributes to retinal degenerative diseases such as AMD, retinitis pigmentosa (RP), and Stargardt disease (SD). Extracellular vesicles (EVs) play a crucial role in intercellular communication, protein homeostasis, and immune modulation, and have emerged as promising diagnostic and therapeutic tools. Understanding the role of extracellular vesicles' (EVs') signaling machinery of glial cells and the retinal pigment epithelium (RPE) is critical for developing effective treatments for retinal degeneration. In this study, we investigated the bidirectional EV-mediated crosstalk between RPE and Müller cells under hypoxic conditions and its impact on cellular metabolism and retinal cell integrity. Our findings demonstrate that RPE-derived extracellular vesicles (RPE EVs) induce time-dependent metabolic reprogramming in Müller cells. Short-term exposure (24 h) promotes pathways supporting neurotransmitter cycling, calcium and mineral absorption, and glutamate metabolism, while prolonged exposure (72 h) shifts Müller cell metabolism toward enhanced mitochondrial function and ATP production. Conversely, Müller cell-derived EVs under hypoxia influenced RPE metabolic pathways, enhancing fatty acid metabolism, intracellular vesicular trafficking, and the biosynthesis of mitochondrial co-factors such as ubiquinone. Proteomic analysis revealed significant modulation of key regulatory proteins. In Müller cells, hypoxic RPE-EV exposure led to reduced expression of Dyskerin Pseudouridine Synthase 1 (DKc1), Eukaryotic Translation Termination Factor 1 (ETF1), and Protein Ser/Thr phosphatases (PPP2R1B), suggesting alterations in RNA processing, translational fidelity, and signaling. RPE cells exposed to hypoxic Müller cell EVs exhibited elevated Ribosome-binding protein 1 (RRBP1), RAC1/2, and Guanine Nucleotide-Binding Protein G(i) Subunit Alpha-1 (GNAI1), supporting enhanced endoplasmic reticulum (ER) function and cytoskeletal remodeling. Functional assays also revealed the compromised barrier integrity of the outer blood-retinal barrier (oBRB) under hypoxic co-culture conditions. These results underscore the adaptive but time-sensitive nature of retinal cell communication via EVs in response to hypoxia. Targeting this crosstalk may offer novel therapeutic strategies to preserve retinal structure and function in ischemic retinopathies.

Keywords: ECIS; GFAP; Müller glia; blood–retinal barrier; exosomes; extracellular vesicles; hypoxia; proteomics; retinal pigment epithelium; retinopathy

DOI: https://doi.org/10.3390/biology14081014

Acta Ophthalmol. 2025 Sep 02.

A model for de novo pigmentation of amelanotic retinal pigment epithelial cells.

Santosh Gupta, Lyubomyr Lytvynchuk, Taras Ardan, Hana Studenovska, Georgina Faura, Lars Eide, Ljubo Znaor, Slaven Erceg, Knut Stieger, Jan Motlik, Goran Petrovski.

PURPOSE: Retinal Pigment Epithelial (RPE) cells perform critical functions in the visual cycle. Their melanin pigmentation, which is organized into specialized compartments - melanosomes, is highly critical for proper vision. A chemical method to induce pigmentation in a non-pigmented model of ARPE-19 cells was applied using L-DOPA as a repurposed drug from the current treatment of Parkinson's disease.
METHODS: L-DOPA was optimized for its toxic effect on ARPE-19 cells along with pigmentation development. Gene expression and immunocytochemistry confirmed upregulation of melanogenesis-related genes and proteins. Melanosomes were characterized by TEM.
RESULTS: We found 1000 μM L-DOPA to induce pigmentation of ARPE-19 cells by Day 3, and achieve full pigmentation by Day 5. By Day 5, L-DOPA at 1000 μM induced mitochondrial and nuclear DNA damage. However, the gene expression of RPE-specific markers (tyrosinase, TYRP1, CRALBP, PEDF) was significantly different in L-DOPA-treated ARPE-19 cells compared to non-treated ones. Positive expression for Tyrosinase enzyme was confirmed by ICC on both Day 3 and Day 5 of L-DOPA treatment. Transmission electron microscopy showed the de novo melanosome formation with ultrastructural features of various stages of maturity (Stage I to IV), apical-basal polarity and melanosome localization on the apical side of the L-DOPA-treated ARPE-19 cells.
CONCLUSION: Our study showed that L-DOPA treatment could induce de novo melanosome formation in amelanotic RPEs. We propose a newer approach of developing an ex vivo model for de novo pigmentation of RPE cells with cell-specific modification and culture condition optimization.

Keywords: L‐DOPA; RPE; in vitro model; melanosomes; pigmentation

DOI: https://doi.org/10.1111/aos.17572