Investigative Opthalmology & Visual Science

Peroxisomes are ubiquitous organelles that compartmentalize metabolic reactions including lipid catabolism and cellular detoxification. Pathogenic variants in PEX1 and PEX6 disrupt essential peroxisome functions and cause profound neurodegenerative diseases called peroxisome biogenesis disorders (PBDs). Despite retinal degeneration and blindness occurring frequently in PBDs, precisely how impaired peroxisome activity disrupts retinal function remains to be fully explored. To address this, we differentiated PEX1-/-, PEX6-/-, and wildtype human induced pluripotent stem cells into retinal pigment epithelium (iRPE) to study the consequences of peroxisome dysfunction in this disease-relevant cell type. Despite exhibiting impaired peroxisome matrix protein import, PEX1-/- and PEX6-/-iRPE had comparable morphology, tight junctions, and expression of proteins characteristic of RPE compared to wildtype iRPE. Targeted lipid profiling revealed reduced docosahexaenoic acid, a polyunsaturated fatty acid (PUFA) essential for retinal function, and elevated lipid species exclusively metabolized by peroxisomes in PEX1-/- and PEX6-/- iRPE. Following a photoreceptor outer segment (POS) challenge, PEX1-/- and PEX6-/- iRPE demonstrated disrupted PUFA retroconversion and lipid droplet accumulation. Additionally, PEX1-/- and PEX6-/-iRPE had impaired rhodopsin degradation, lysosomal dysfunction, and reduced transepithelial electrical resistance. These findings suggest that dysregulated POS metabolism in the RPE is a potential mechanism driving retinal degeneration in patients with PBDs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/701576v2_ufig1.gif" ALT="Figure 1"> View larger version (99K): org.highwire.dtl.DTLVardef@bf2389org.highwire.dtl.DTLVardef@b62e1forg.highwire.dtl.DTLVardef@8e0b21org.highwire.dtl.DTLVardef@17cb332_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic summarizing the consequences of PEX1 and PEX6 knockout on iRPE biology, including the presence of import-incompetent peroxisomes, impaired {omega}3 and {omega}6 fatty acid retroconversion, lipid droplet accumulation, and defective photoreceptor outer segment phagocytosis.

Purpose: Exfoliation glaucoma (XFG) is the most common secondary glaucoma. Prior studies suggest a higher incidence in women and links to reproductive history, implying estrogen-related pathways. Metabolomic data also indicated inverse associations with steroid-related plasma metabolites, suggesting steroid involvement in XFG pathogenesis. Methods: We conducted a nested case-control study within the Nurses' Health Study (NHS) (1980-2018), NHSII (1989-2019), and Health Professionals Follow-up Study (1986-2018), with 217 XFG suspect (XFGS)/XFG cases and 217 matched controls (62 men and 372 women). We evaluated 18 endogenous steroids and five steroid classes using conditional logistic regression. Secondary analyses examined effect modifications by age and residential latitude, and heterogeneity by disease severity (XFGS vs. XFG). Metabolite set enrichment analysis (MSEA) was used for class-level associations. Multiple comparisons were addressed using the number of effective tests (NEF) for individual steroids and false discovery rate (FDR) for steroid classes. Results: No individual steroid or steroid class met NEF- or FDR-adjusted significance thresholds, overall or by sex. Nonetheless, across both sexes, MSEA demonstrated a non-significant inverse trend between androgen levels and XFG/XFGS risk (FDR=0.22), with 11-ketotestosterone showing a nominal inverse association (OR=0.54; 95%CI=0.31-0.93; P=0.03). Progestogens showed enrichment scores in the positive trend (FDR=0.31), with a borderline positive association between progesterone and XFG/XFGS (OR=2.21; 95%CI=1.00-4.87; P=0.05). Conclusions: Although we observed no statistically significant associations with steroids after correction for multiple testing, the suggestive patterns for androgens and progestogens support the possibility of steroid-related pathways in XFG etiology and support further evaluation in larger studies.

The retinal pigment epithelium (RPE) consists of polarized epithelial cells, serving as a support system for photoreceptor maintenance, where the polarity is contributed by the distribution of syntaxins (STX) within the cell. STX3, a known regulator of apical trafficking in epithelial cells, was previously understood to be absent in human RPE cells, with its functions thought to be compensated by STX1A. However, our results on SNARE mRNA expression profile in RPE detected the presence of 2 splice variants of STX3. Further investigation in donor retina, primary hRPE, and ARPE-19 cells revealed detectable levels of STX3 mRNA and protein. STX3 knockdown in ARPE19 resulted in a significant reduction of tight junction (TJ) proteins, compromising TJ assembly, highlighting the critical role of STX3 in maintaining RPE integrity. In addition, immunoprecipitation followed by LC-MS/MS analysis revealed that STX3 and STX1A have a distinct novel protein interactome in RPE. This study identified unique and shared interactants for STX3 and STX1A, suggesting a broader role for RPE beyond its traditional photoreceptor support function. This further emphasises the biological significance of STX1A and STX3 in maintaining retinal homeostasis, which could facilitate the development of novel therapeutic strategies for retinal disorders. SignificanceThis study identified the presence of STX3 in the human RPE cells, which was previously reported to be absent. Further, we demonstrated that STX3 knockdown in ARPE19 cells disrupted TJ assembly, highlighting its potential role in preserving RPE cell polarity and structural integrity, challenging the notion that STX3 functions were thought to be compensated by STX1A. Moreover, immunoprecipitation followed by LC-MS/MS analysis in RPE identified the protein interaction networks of both STX1A and STX3. Interestingly, unique and shared interactants, including proteins associated with neuronal plasticity, indicated unidentified functions of STX3 and STX1A in RPE. This suggests that they might perform both overlapping and distinct functions for maintaining RPE cell integrity and thus retinal homeostasis. Overall, our preliminary findings challenge the established view that STX3 is non-existent in RPE cells and initiate new directions for exploring the multifaceted and potentially non-redundant functions of STX3 in RPE.

PurposeMuller cell (MC) morphology and markers were investigated using histology and immunohistochemistry in an eye with clinically documented multifocal geographic atrophy (GA) and correlated with clinical images. MethodsThe donor was followed clinically for five years and last examined six years before death. The superior posterior pole retina was dissected and immunolabeled with antibodies against glial fibrillary acidic protein (GFAP; activated MCs and astrocytes) and glutamine synthetase (GS, MC) and Ulex Europaeus Agglutinin-1 lectin (blood vessels) before embedding for JB-4 cross section analysis. The inferior macula was cryopreserved. Cryosections were immunolabeled with MC homeostatic and activation markers. Transmission electron microscopy (TEM) of the fellow eye was used to study ultrastructure changes. ResultsGross examination demonstrated mottled retinal pigment epithelium (RPE) over presumably calcified drusen. In the submacular retina, MC processes surrounding both drusen and outer retinal pigmented lesions created a large subretinal membrane. Cryosection analysis demonstrated persistence of aquaporin 4 and GS in MCs with both proteins prominently expressed in the subretinal membrane. Increased MC S100B and GFAP expression were also observed in the atrophic area as well as the OJZ. Cryosection labeling and TEM confirmed the MC encasing calcified drusen and RPE debris as well as invading basal laminar deposits. ConclusionsThis multifocal GA case demonstrates how MC activation and structural changes surrounding individual drusen could coalesce, contributing to photoreceptor loss. MCs penetrating basal laminar deposits and encasing calcified drusen suggests that they are attempting to clear these and/or protect the retina from harmful contents.

PurposeHyperosmolar stress (HOS) is a major contributor to corneal epithelial cell damage in dry eye disease. We have previously shown that HOS damages mitochondria and impairs cell metabolism in corneal epithelial cells. Small extracellular vesicles (sEVs) are cell-derived lipid envelopes that are present in all body fluids, including tears. Prior studies suggest that sEV release and composition may be linked with changes in cell metabolism. In this study, we tested the effects of HOS on sEV release and composition, and found that sEV cargo may reflect early, underlying changes in dry eye disease. MethodsTelomerase-immortalized human corneal epithelial (hTCEpi) cells were treated with 450 mOsm NaCl for five days to induce chronic HOS. sEVs were isolated using differential centrifugation followed by iodixanol density gradient flotation. Particle number was determined using Nanoparticle Tracking Analysis (NTA). Mass spectrometry was used to assess the sEV proteome, and selected proteins were validated by immunoblot. Proteome pathways were analyzed using KEGG and CORUM. ResultsPathway analysis revealed an increase in metabolic proteins and proteasome components in sEV cargo released from hTCEpi cells exposed to HOS. These proteins were increased more than fourfold in HOS-sEVs. Examination of proteins involved in the endosomal pathway and NTA further confirmed an increase in HOS-sEV release. ConclusionOur findings suggest a potential mechanism whereby corneal epithelial cells exposed to HOS retain proteins involved in maintaining tissue integrity, while simultaneously releasing unneeded proteins involved in cell metabolism. The presence of metabolic proteins in sEVs may serve as early indicators of dry eye disease.

Primary open-angle glaucoma (POAG) disproportionately affects individuals of African ancestry, yet rare coding variation in this population remains understudied. To address this gap, we performed a multi-cohort exome-wide meta-analysis across POAAGG, PMBB, All of Us, and UK Biobank, including 4,815 POAG cases and 22,922 controls of genetically inferred African ancestry. Although no gene reached exome-wide significance, we identified several suggestive gene-level associations driven by rare variants (minor allele frequency [≤]0.1% or singletons),including signals in SRF, BLTP3A, METTL2A, and KRT10. Among these, SRF demonstrated the strongest association and was driven by rare missense variants with moderate effect sizes. Given its role in cytoskeletal organization and actin dynamics; processes central to trabecular meshwork function and intraocular pressure regulation SRF represents a biologically plausible candidate gene. Notably, these genes have not been previously highlighted in predominantly European ancestry POAG association studies, suggesting potential ancestry-specific rare variant contributions. Overall, our findings highlight the critical importance of investigating rare coding variation in POAG, in disproportionately affected populations to deepen understanding of POAG etiology and genetic risk.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in ageing populations, with oxidative stress recognised as a key pathogenic driver. The dietary antioxidant and cytoprotectant, L-ergothioneine (ET), is avidly accumulated in many tissues, especially the eye. However its relationship to AMD has not been investigated. Here, we examined ETs distribution in ocular tissue and assessed circulating and intraocular ET levels in patients with neovascular AMD. Compared with ocularly-normal age-matched individuals, AMD patients exhibited significantly lower serum ET; elevated levels of ET metabolites, hercynine and ETSO, which may be generated by oxidative stress; and elevated levels of serum allantoin, a product of oxidative damage to urate in humans. Levels of ET in aqueous humour in AMD patients were marginally lower than cataractous patients who are already known to have significantly lower ET levels than healthy eyes. High ET levels were seen in human ocular tissues concentrating in regions vulnerable to oxidative injury, including the lens, retina, retinal pigment epithelium, and choroid, supporting a physiological protective role of ET in the eye. These findings identify the strong association between low ET levels and AMD, warranting further studies to determine whether ET supplementation can modify AMD risk or progression.

Age-related macular degeneration (AMD) is a common, complex disease affecting older individuals that can lead to severe vision loss. It is characterized by early anatomical changes in the retina, retinal pigment epithelium (RPE), and choroid, especially in the central (macular) region. AMD can progress to severe atrophy and/or pathologic angiogenesis that leads to visual decline. Over 30 genetic loci have been identified as contributing to AMD risk; however, the mechanisms by which genetic variants affect pathology has not been thoroughly explored. In this report we examined single-nucleus gene expression in the retina, RPE and choroid of 88 individuals categorized by AMD stage, as well as 37 previously published samples. Genotyping was performed on 1.8 million SNPs, with additional SNPs imputed, on each donor to identify expression quantitative trait loci (eQTLs). We found that two AMD-risk loci (PILRB and ARMS2/HTRA1) affected the expression of PILRB and HTRA1, respectively. The risk allele of PILRB was associated with increased PILRB RNA in cones, fibroblasts, choroidal macrophages, and RPE, whereas the HTRA1 risk locus was associated with decreased HTRA1 RNA in the RPE. We also identified an age-related decrease in complement inhibitors in the choriocapillaris, a tissue susceptible to complement mediated damage in AMD.

PurposeElevated intraocular pressure (IOP) due to increased outflow resistance through the trabecular meshwork (TM) is a major risk factor for primary open-angle glaucoma. Outflow through the TM is segmental, consisting of high flow (HF) and low flow (LF) regions. Here, we investigate how ocular hypertension impacts segmental outflow using a dexamethasone (DEX) mouse model and compare TM stiffness between HF and LF regions. MethodsNanoparticles containing DEX or vehicle were injected twice weekly in 2-4-month-old C57BL/6J mice (n=14), and IOP was measured weekly. At week 4, mouse eyes were perfused in vivo with fluorescent nanospheres to assess flow patterns and the circumferential percentage of high, intermediate, and low flow regions in each eye. Sagittal sections were collected from HF and LF regions, and atomic force microscopy (AFM) was used to measure tissue stiffness. Immunofluorescent labeling was used to compare fibronectin and -SMA protein levels. ResultsDEX treatment significantly elevated IOP by an average of 33.3% and altered tracer distribution but not the percentage of HF and LF regions around the circumference. No significant differences in TM stiffness were detected between DEX-treated and control mice, or between HF and LF regions. Increased fibronectin in LF regions of DEX-treated eyes suggested subtle TM structural changes that were not detected by AFM. ConclusionsDexamethasone alters segmental flow distribution and may impact cell contractility rather than ECM stiffness to cause IOP elevation in young mice. These findings better characterize the nature of segmental outflow and TM mechanics in this model of steroid-induced glaucoma.

PurposeSubretinal drusenoid deposits (SDDs) are a distinct entity in age-related macular degeneration (AMD) and associated with photoreceptor impairment during progression. Their early impact on photoreceptors remains incompletely understood. This study examined photoreceptor reflectivity during the phase when SDDs were not clinically detectable on optical coherence tomography (OCT) using adaptive optics scanning laser ophthalmoscopy (AOSLO). DesignLongitudinal observational study. ParticipantsPatients with intermediate AMD. MethodsSix eyes of four patients with intermediate AMD and predominantly SDDs underwent multimodal imaging 3-4 times over 3.5 years. Individual SDDs were graded using an OCT-based 3-stage system at each time point. Cross-sectional retinal structure and photoreceptor reflectivity at the location where the new SDDs developed during follow-up were evaluated using OCT and AOSLO. Main Outcome MeasuresPhotoreceptor reflectivity change prior to and during SDD development. ResultsForty-eight retinal locations where new dot-type SDDs developed during follow-up were identified. AOSLO revealed reduced photoreceptor reflectivity in these regions before OCT demonstrated clinically evident deposits (stage [≥] 1) between the ellipsoid zone and the retinal pigment epithelium at the corresponding sites. The mean time to development of stage 1, stage 2, and stage 3 SDDs was 11.78 {+/-} 5.01, 17.40 {+/-} 6.08, and 18.72 {+/-} 4.08 months, respectively. ConclusionsHigh-resolution adaptive optics confocal imaging enables detection of photoreceptor optical property alterations at a stage when SDDs are not yet evident on OCT. This finding underscores the exceptional sensitivity of photoreceptors to minimal structural or functional perturbations during SDD formation and defines an early window for potential intervention.

PurposeAnti-vascular endothelial growth factor (anti-VEGF) therapy is the standard of care for neovascular age-related macular degeneration (AMD), yet many patients exhibit persistent retinal degeneration, fibrosis, and incomplete therapeutic response. The molecular pathways underlying this incomplete response remain poorly understood. We sought to identify VEGF-independent signaling pathways active in the vitreous of anti-VEGF-treated AMD patients. MethodsWe performed multiplex antibody-based proteomic profiling of 1,000 human proteins in vitreous samples from patients with neovascular AMD receiving anti-VEGF therapy (n=8) and comparative controls (n=6). Differential protein expression was assessed using one-way ANOVA, followed by gene ontology and pathway enrichment analyses. Drug-target relationships were evaluated to identify potential opportunities for therapeutic repositioning. ResultsWe identified 107 differentially expressed proteins (p<0.05), including key regulators of immune signaling, angiogenesis, and metabolism. Notably, multiple components of cytotoxic lymphocyte pathways were dysregulated, including IL-21R, SIGLEC-7, CTLA4, and IL-2-associated signaling. Enrichment analyses revealed significant activation of pathways related to T-cell activation, interleukin signaling, and leukocyte-mediated cytotoxicity. These immune signatures persisted despite suppression of VEGF signaling. Several clinically available immunomodulatory agents--including abatacept, sirolimus, and dupilumab--targeted pathways identified in this dataset. ConclusionsAnti-VEGF-treated neovascular AMD exhibits persistent cytotoxic immune signaling in the vitreous, suggesting that VEGF-independent immune mechanisms may contribute to ongoing retinal damage and incomplete therapeutic response. These findings provide a rationale for combination therapeutic strategies targeting both angiogenic and immune pathways in AMD.

ImportanceGenome-wide association studies have identified hundreds of common single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) associated with primary open-angle glaucoma (POAG) risk, though these variants have modest effect sizes and individually may have minor contributions to disease development. As whole-genome sequencing data is becoming more readily available, structural variants and other complex genomic features can be interrogated for contribution to disease risk. ObjectiveTest the association of structural variants in known glaucoma loci with disease risk. DesignCross-sectional study. SettingA multicenter cohort of individuals from the United States who contributed genomic and electronic health record data to the All of Us Research Program. ParticipantsPOAG case/control cohorts were generated in the All of Us Researcher Workbench using age (>40 for cases, >65 for controls) and ICD 9/10 diagnosis codes. Main Outcomes and MeasuresLogistic regression analyses adjusted for age, sex, and the top 10 principal components of ancestry were used to test association of structural variants within 500 kilobases of 309 known open-angle glaucoma risk loci. The significance threshold after Bonferroni correction was set at p<1.6x10-4. Results516 POAG cases and 18,716 controls of European ancestry from the All of Us v8 data release were included in the analysis. Mean age was 77.0 years among cases and 74.7 years among controls. Females comprised 45.7% of cases and 56.5% of controls. An 8,732 base pair deletion upstream of PITX2 (chr4:110680827-110689558) was associated with 7.3-fold higher odds of POAG (95% confidence interval: 2.9-18.5, p= 2.4x10-5, variant carrier frequency= 1.6% in cases and 0.25% in controls). Functional annotation identified multiple enhancers overlapping the deletion, suggesting that this structural variant likely impacts gene regulation and expression. Conclusion and RelevanceWhole genome sequencing data captures rare structural variants with large effect sizes that are missed by conventional SNP and indel genotyping approaches, enabling improved POAG risk stratification. These data also expand the phenotypic spectrum of structural variation in the PITX2 locus from childhood glaucoma to adult-onset disease, where age at diagnosis and clinical severity may be influenced by the extent of disrupted regulatory elements.

PurposeTo systematically evaluate ocular biometric and systemic laboratory factors associated with cataract in highly myopic eyes and to characterize potential nonlinear associations using an interpretable machine learning approach, thereby providing deeper mechanistic insights into the pathogenesis of highly myopic cataract. DesignA cross-sectional study encompassed 770 eyes of 594 patients with high myopia from Eye & ENT Hospital of Fudan University. SubjectsThe non-cataract control group included 458 eyes while the cataract group contained 312 eyes. MethodsDemographic traits, ocular biometric and systemic laboratory factors were gathered while features with over 30% of missing data were excluded. Composite indices were obtained through calculation. Multiple machine learning models were compared to investigate the association between features and highly myopic cataract, and the random forest (RF) model was chosen and fine-tuned. Feature selection was carried out by means of Shapley additive explanations (SHAP) and non-linear relationships were probed using SHAP dependence diagrams and confirmed with partial dependence plots. Main Outcome Measures(1) The Area Under the Curve (AUC) and other metrics of multiple machine learning models; (2) Top feature importance of the final simplified RF model; (3) Overall trends between features and highly myopic cataract; (4) Potential inflection points of top continuous features. ResultsA simplified fine-tuned RF model with 17 features reached stable discriminative performance, with a mean AUC of 0.762 (95%CI: [0.731, 0.794]) among 10 independent testing sets. Age and axial length (AL) turned out to be the most influential features which had non-linear relationships highly myopic cataract, with an inflection point seen around 65.75 (95%CI: [63.72, 67.79]) years for age and 30.55 (95% CI: [29.22, 31.88]) mm for axial length respectively, while the ratio of anterior chamber depth to axial length (ACD/AL) was associated with highly-myopic cataract in a U-shape. Ocular biometric factors were more strongly related to highly myopic cataract than systemic laboratory factors. ConclusionsOcular biometric factors, especially age, AL, and composite indices like ACD/AL, have strong and non-linear connections with highly myopic cataract. These results emphasize the significance of ocular structural arrangement in cataract within highly myopic eyes and indicate that interpretable data-driven methods could offer clinically relevant understandings regarding its phenotypic description.

PurposeTo investigate the effects of morning and evening narrowband blue light exposure on axial length, and to examine the short-term effect of morning blue light combined with myopic defocus on axial length. MethodsFor objective 1, 18 individuals underwent 60 minutes of narrowband blue light exposure (460nm) in the morning (9:00-11:00AM) and evening (5:00-7:00PM) of the same day. The axial length values were normalized to the average of the morning and evening axial length values. For objective 2, 27 young adults were exposed to 60 minutes of narrowband blue light and broadband white light while wearing a +3.00 D lens over the right eye. Axial length was measured using Lenstar LS900. ResultsA significant reduction in axial length was observed after exposure to morning blue light compared to evening blue light (-10.0{+/-}3.96{micro}m vs.-0.67{+/-}3.30{micro}m; p=0.02), whereas no such effect was observed with broadband white light exposure (0.0{+/-}3.53 {micro}m vs. -2.50{+/-}4.23{micro}m, p=0.70). While the broadband white light exposure did not alter the normal diurnal variation in axial length (+2.35{+/-}1.82{micro}m vs.-6.25{+/-}2.21{micro}m, p=0.04), blue light diminished such a pattern (-4.12{+/-}1.72{micro}m vs. - 2.00{+/-}2.00{micro}m, p=0.48). The myopic defocus did not influence axial length under either narrowband blue or broadband white light conditions. ConclusionThe short-term narrowband blue light exposure led to a significant decrease in axial length in the morning than evening exposure, with a likely influence on the diurnal rhythm of axial length. Morning blue light exposure with lens-induced myopic defocus did not provide additional short-term modulation of axial length.

Induced pluripotent stem cell-derived retinal pigment epithelium (iPSC RPE) has become a widely used model for studying the mechanisms of age-related macular degeneration (AMD). However, the nutrient composition of currently used RPE culture media is highly variable, posing a major challenge to reproducibility in RPE metabolism and phenotype. We systematically investigate how six distinct nutrient microenvironments shape RPE phenotype, function and metabolism in both iPSC RPE and fetal RPE (fRPE). These included MEM, DMEM-HG/F12 basal media, physiological human plasma-like medium (HPLM) supplemented with FBS or B27, and X-VIVO 10. Although canonical RPE markers were expressed across all conditions, B27 supplementation and X-VIVO 10 increased RPE cell size, hexagonality, and transepithelial resistance. Culture in HPLM+FBS induced accumulation of lipid droplets and sub-RPE deposits, whereas X-VIVO 10 resulted in the formation of large intracellular vacuoles. B27 supplementation enhanced basal respiration, while X-VIVO 10 increased glycolytic capacity. Amino acid consumption was broadly conserved across media types, including complete depletion of proline in all conditions by 48 hours; however, lipid and nucleotide metabolism varied substantially between conditions. Notably, B27 supplementation in specific media types reversed the net direction of several metabolites, with creatine, serine and taurine shifting from consumption to production, while riboflavin and guanine shifted from production to consumption. These findings establish the nutrient environment as a key determinant of RPE phenotype, function and metabolism. Our work provides a valuable resource for media selection and interpretation of cellular and metabolic phenotypes relevant to RPE disease modeling.

ObjectiveTo assess how whole genome sequencing and varying phenotype definitions influence genetic discovery for primary open-angle glaucoma (POAG) in a diverse population. DesignAncestry-stratified genome-wide association studies (GWASs) and cross-ancestry meta-analyses of POAG cases and controls using two phenotype definitions. ParticipantsCases (age>40) and controls (age>65) were identified in the National Institutes of Health All of Us Research Program v8 data release and sub-divided into genetically inferred ancestral groups. Using the relaxed phenotype (ICD codes only), case/control counts were: European (1,846/84,654), African (1,042/15,966), and Latino/Admixed American (305/10,167). Using the stringent phenotype (ICD codes and evidence of glaucoma treatment in the electronic health record), case/control counts were: European (1,528/79,276), African (862/14,076), and Latino/Admixed American (250/9,668). Cross-ancestry meta-analyses included 3,193 cases/110,787 controls for the relaxed phenotype and 2,640 cases/103,020 controls for the stringent phenotype. MethodsGWASs were conducted within European, African, and Latino/Admixed American ancestry groups individually using firth logistic regression with age, sex, and the top 10 genotype principal components included as covariates. The ancestry-stratified GWASs were then meta-analyzed using a fixed-effects, inverse variance-weighted approach. Main Outcome MeasuresIdentification of genome-wide significant loci (P < 5x10-8) for POAG using different phenotype definitions and ancestry groups. ResultsKnown POAG risk loci (e.g., TMCO1, CDKN2B-AS1, and GMDS) reached genome-wide significance in both the European GWASs and cross-ancestry meta-analyses (odds ratio (OR) range: 1.19-1.38). A novel risk locus near CYP2A7 (rs76935404[T], OR = 1.35) was identified in the African ancestry GWAS using the stringent phenotype definition. Effect sizes for known POAG risk loci from prior large-scale meta-analyses strongly correlated with effect sizes in this study (Pearson r = 0.75-0.84, P < 1 x 10- for all). The strength and consistency of these correlations support the robustness of the findings. ConclusionsThis study demonstrates the value of whole genome sequencing, diverse ancestry inclusion, and phenotypic refinement in uncovering novel POAG genetic risk loci. The findings underscore the need to prioritize both genetic diversity and refined case/control definitions to advance understanding of this complex ocular disease. PrecisThis study identifies a novel primary open-angle glaucoma risk locus in individuals of African ancestry using whole genome sequencing and varying phenotype definitions in the diverse All of Us Research Program dataset.

In recent years, researchers have paid increasing attention on potential associations between respiratory and ocular diseases. To examine whether asthma is independently associated with macular degeneration (MD) and whether asthma can serve as a predictor of MD using data from the English Longitudinal Study of Ageing (ELSA). Data from the 2020-2021 wave of ELSA were analyzed. Statistical tests were performed on participants baseline characteristics. Multivariable logistic regression, stratified analysis, ROC curve analysis, smoothing curves and sensitivity analysis were conducted to assess the association, stability, predictive performance, dose-response relationship and robustness. A total of 6,703 participants were included. After adjusting for covariates, age and asthma were significantly associated with MD (p < 0.05), while diet and sex were not. Asthma was consistently linked to increased MD risk across three regression models (OR > 1, p < 0.05), with the association persisting in stratified analyses. ROC analysis showed moderate predictive performance (AUC = 0.757), and a positive dose-response relationship was observed. Sensitivity analyses confirmed the robustness of the association. Asthma may independently increase MD risk, providing novel insights into their relationship and implications for clinical risk stratification and preventive strategies.

Background: Mitochondrial dysfunction is an emerging metabolic hallmark of age-related diseases, yet tools to directly profile mitochondrial pathways and test metabolic interventions in the living human eye remain limited. Multi-omics ocular liquid biopsy enables real-time proteomic and metabolomic profiling of the intraocular microenvironment, complementing systemic biomarkers and imaging surrogates. Here, we used this approach to define mitochondrial and tricarboxylic acid (TCA) cycle dysregulation in geographic atrophy (GA) and to assess whether oral -ketoglutarate (-KG) supplementation can modulate mitochondrial metabolites within the eye. Methods: Mitochondrial and TCA cycle-related proteins were profiled in aqueous humor (AH) samples from patients with GA using DNA-aptamer-based proteomics. In a phase 0 study, a second cohort undergoing sequential cataract surgery provided paired AH samples collected at first-eye surgery and at second-eye surgery after interim -KG supplementation. These samples underwent targeted metabolomic profiling using hydrophilic interaction liquid chromatography coupled with mass spectrometry. Results: In GA, 64 mitochondrial proteins were differentially expressed, including coordinated TCA-cycle deficiencies marked by reduced expression of enzymes regulating TCA entry and flux, including PDHB and DLST. In the phase 0 cohort, oral -KG supplementation significantly increased intraocular -KG levels and the -KG-to-succinate ratio (P < 0.05), with coordinated shifts across TCA intermediates consistent with enhanced TCA cycle flux. Conclusions: AH proteomics demonstrated mitochondrial pathway depletion in GA, consistent with reduced oxidative bioenergetic capacity. AH metabolomics provided first-in-human in vivo evidence that systemic -KG supplementation can modify intraocular metabolites and may enhance intraocular energy metabolism. These findings support ocular liquid biopsy as a precision-health framework for per-patient biomarker-guided metabolic trials in GA.

PurposeTo investigate whether the geometric shape of the globe, sagittal length (SL) and Posterior Radius of curvature (rPC), differ between patients with rhegmatogenous retinal detachment (RD) and macular hole (MH), and to determine if these parameters offer better diagnostic differentiation than axial length (AL) alone. MethodsThis retrospective study included 20 MH and 20 RD patients. Groups were axial-length matched. Ocular biometry was performed with ultrasound to measure AL and SL. The PR was calculated. Due to the non-normal distribution of the data, the Mann-Whitney U test was used for continuous variables and Fishers Exact Test for gender distribution. ResultsThere was no significant difference between the MH and RD groups in terms of age (median: 33.8 vs 32.6 years; p=1.00), gender (p=0.341), or AL (27.65 mm vs 28.58 mm; p=0.39). However, RD eyes showed significantly higher SL (median: 25.70 mm vs 22.90 mm; p=0.001) and a significantly flatter rPC (median: 28.25 mm vs 24.30 mm; p < 0.001). ConclusionRD eyes exhibit a distinct vertical equatorial expansion and posterior flattening that is not present in MH eyes, despite similar axial lengths. These geometric differences suggest that SL and PR are structural risk factors in vitreoretinal disease.

Rhodopsin (RHO) P23H is one of the most common mutations causing autosomal dominant retinitis pigmentosa (adRP), yet the relationship between retinal electrophysiology, structure and visually guided behaviour in rodent models remains unclear. We characterised changes in heterozygous P23H (Sakami line) mice and P23H line 3 (P23H-3) rats using full-field electroretinography (ERG), optomotor response (OMR) assays and, in rats, optical coherence tomography (OCT). ERG assessed rod- and cone-mediated responses relative to wild-type controls, whereas OMR under scotopic and photopic conditions quantified contrast sensitivity and visual acuity. In P23H mice, scotopic ERG responses were significantly reduced from postnatal day 16 and declined further from 4 months. Scotopic OMR contrast sensitivity remained largely preserved until 2 months, and photopic acuity was comparable to wild-type up to 6 months. In 13-week-old P23H-3 rats, ERG amplitudes were significantly reduced, and OCT revealed retinal thinning. OMR showed a decline in contrast sensitivity at 7 and 15 weeks, whereas photopic acuity was maintained. Thus, in both models, electrophysiological and structural abnormalities precede detectable OMR deficits, with implications for the selection of outcome measures in preclinical studies. Summary StatementThis study compares electrical and behavioural measures of vision in rodent models of inherited blindness, revealing that retinal dysfunction appears well before measurable vision loss.