Experimental Eye Research

Cone photoreceptors are essential for daylight vision, and their degeneration has more profound visual consequences than rod loss in retinal degenerative diseases. Metabolic dysfunction is closely associated with cone degeneration, however the relatively small population of cones in mice and humans have limited our understanding of cone-specific metabolism. Here, we leveraged cone-dominant and cone-degeneration mouse models including Nrl-/-, Cnga3-/-, and high-dose Triiodothyronine (T3) treatment to investigate cone-specific metabolism and their metabolic impacts on the retinal pigment epithelium (RPE). Across models, cone-dominant retinas consistently showed lower pyruvate abundance alongside increases in glutathione, purines, pentose phosphate pathway intermediates, and one-carbon metabolites. Increases in several key amino acids were also associated with higher cone abundance, such as proline, arginine, alanine, valine, leucine, and hypotaurine. Strikingly, aminoadipate, an intermediate in lysine catabolism, was the most robustly changed metabolite in the retina, showing highly consistent increases across models. Relative cone increases were also associated with metabolic changes in the RPE/choroid. Like the retina, RPE/choroids showed consistent increases in aminoadipate, proline, and hypotaurine, as well as xanthosine and betaine, alongside decreases in uracil. Moreover, proteomic analysis of Nrl-/- mice showed decreases in many key metabolite transporters in the RPE/choroid, including carriers for glucose, lactate, aspartate, glutamate, serine, lysine, taurine, and proline. Collectively, these findings further our understanding of cone-specific metabolism and highlight potential cone-specific metabolic vulnerabilities in retinal degeneration.

Iris biomechanics plays a key role in primary angle-closure glaucoma (PACG). Soft tissue biomechanics is heavily influenced by tissue water content; therefore, accurately assessing water content is needed to describe iris biomechanics in a physiologically relevant manner. Yet, accurate experimental measurements of iris water content are rare in the literature. Therefore, our objective was to experimentally assess iris water content and potential sex differences in New Zealand White rabbit iris--a suitable model for studying iris biomechanics. We also examined the effect of exposure to phosphate-buffered saline (PBS) on iris water content, given its common use to maintain hydration during soft-tissue biomechanical testing. Our results showed slightly higher water content in males (89.2% {+/-} 2.3%) than in females (87.7% {+/-} 2.2%) (p = 0.022), while we found no significant differences in gross iris morphology between the sexes, indicating subtle compositional differences. Furthermore, PBS incubation had a minimal effect on water content only in female irides. Overall, our results indicated high iris water content in both sexes compared with anatomically adjacent ocular tissues and other soft connective tissues. These results improve our understanding of iris composition and physiology and can serve as a baseline for constructing biomechanical models to assess its role in PACG.

Pathogenic variants in the myocilin (MYOC) gene are the most common cause of Mendelian open-angle glaucoma. In 2022, the Clinical Genome Resource (ClinGen) Glaucoma Variant Curation Expert Panel (VCEP) published rule specifications for MYOC variant interpretation, including a pilot study of 81 variants. Here, we present the results of curating 271 MYOC variants reported in people with open-angle glaucoma using updated specification rules. Of all the variants, 11 were classified as benign (B), 45 as likely benign (LB), 166 as variants of uncertain significance (VUS), 35 as likely pathogenic (LP), and 14 as pathogenic (P). All LP/P variants were located within the conserved olfactomedin domain encoded by exon 3. The updated variant curation guidelines from the Glaucoma VCEP increased the number of clinically definitive classifications from 28% (74/265) to 39% (105/271), with 95% (41/43) of reclassified variants moving to greater clinical relevance. Functional evidence was lacking for 93% (154/166) of VUS. Additional functional evidence could further enhance classification by halving (85/166) the proportion of those classified as VUS. These findings highlight the role of rule calibration and rigorous functional evidence assessment toward improving variant classification with clinical utility for patients.

Age-related macular degeneration (AMD) is among the leading causes of blindness worldwide. Early AMD is characterized by dysfunction in the choroid, including early dropout of endothelial cells and increased deposition of the complement cascade's membrane attack complex (MAC) in the choriocapillaris. In this study, we used a single-cell RNA sequencing-based approach with barcoded antibodies to measure abundance of the MAC and other surface proteins at single cell resolution on RPE/choroid samples from four aged human donor eyes. We included antibodies to detect the MAC, CD34, CD45, and complement regulators CD55 and CD59, in addition to control antibodies. Our analysis of these data revealed cell clusters with expected gene expression profiles and antibody-based detection of CD34 and CD45 congruent with transcriptome-based cell identity. We also detected surface complement regulators CD55 and CD59 across a wide variety of cell types. Across endothelial cells, surface CD55 and CD59 appeared more abundant on venous clusters, and abundance of each was correlated with expression of a third complement regulator, clusterin (CLU). The MAC was detected on a variety of cell types, but was most abundant on the surface of cells in the macrophage family, smooth muscle cells, and pericytes. We confirmed these findings by identifying MAC deposition on choriocapillaris pericytes using immunohistochemistry for MAC, endothelial, and pericyte markers. Ultimately, these data showcase a valuable new approach to analyze gene expression and surface complement in human donor eyes, and provide novel insight into patterns of MAC deposition and complement protection in the aging human choroid.

Purpose: Retinal ganglion cell (RGC) death following optic nerve injury is driven by transcriptional programs involved in apoptosis, neuroinflammation, and other regulated cell death pathways; however, the early transcriptomic changes initiating these processes remain poorly defined. We used rRNA-depleted total RNA sequencing to characterize early alterations in protein-coding genes, long noncoding RNAs (lncRNAs), and alternative splicing events in mouse retinas following optic nerve crush (ONC) and silicone oil-induced ocular hypertension under-detected (SOHU), prior to substantial RGC loss. Methods: Whole retinas were collected 6 hours after ONC and 5 days after SOHU induction. Differentially expressed protein-coding genes and lncRNA-classified transcripts were identified using total RNA sequencing, followed by functional enrichment, lncRNA-associated pathway analysis, alternative splicing analysis, and fluorescent in situ hybridization (FISH) validation. Results: In SOHU retinas, hundreds of protein-coding genes and lncRNA-classified transcripts were differentially expressed, with enrichment of immune activation, extracellular matrix remodeling, complement, phagocytic signaling, and suppression of mitochondrial energy metabolism pathways. Similarly, in ONC retinas, many protein-coding genes and lncRNA-classified transcripts were differentially expressed, with a prominent chemokine, cytokine-dominant inflammatory response. FISH validation supported injury-associated upregulation of Prss56 and Lif. Both injury models exhibited substantial transcript-level alterations, with widespread alternative splicing changes detected. Conclusions: Early optic nerve injury induces rapid transcriptional changes involving protein-coding genes, lncRNAs, and transcript isoform regulation before substantial RGC loss. These findings highlight early alterations in both coding and noncoding RNA landscapes following retinal injury and provide a valuable resource of candidate genes and pathways for future validation in RGC degeneration and neuroprotection.

Most recessive inherited retinal dystrophies are, in principle, amenable to gene augmentation, yet only one such therapy has received regulatory approval. We sought to identify tractable autosomal recessive retinitis pigmentosa (RP) genes that could be treated using existing non-proprietary adeno-associated virus (AAV) components. More than 100 RP-associated genes were prioritized according to cellular expression, coding-sequence size, and the availability of cell type-specific promoters, yielding 19 candidate genes expressed predominantly in rods and/or retinal pigment epithelium (RPE). In rhesus monkey eyes, the human RHO and BEST1 promoters drove rod- and RPE-specific expression, respectively, whereas the GFAP and RLBP1 promoters were limited by absent Muller glial expression or dose-associated RPE toxicity. We selected PDE6B as a proof-of-concept gene and evaluated AAV8-RHO-PDE6B after neonatal subretinal delivery. The vector effectively preserved outer nuclear layer structure, electroretinography responses, and visually guided behaviors in rd1 and rd10 mice for at least 6 months. Dose-ranging studies identified retinal abnormalities at the higher doses, whereas the lower doses were comparatively well tolerated in eyes of mice and rhesus monkeys. Together, these findings define a preclinical framework for developing gene-augmentation vectors for a subset of early-stage autosomal recessive RP patients.

Purpose: To investigate the association between mitochondrial DNA (mtDNA) copy number (CN) and the rate of visual field progression in patients with primary open-angle glaucoma. Design: Retrospective cohort study Participants: 200 patients with primary open-angle glaucoma (POAG) with a mean of 11 years of longitudinal clinical data and DNA extracted from peripheral blood. Methods: DNA extraction was performed on peripheral blood leukocytes collected from 200 POAG patients who had an average follow-up of 11 years. Mitochondrial CN was quantified by multiplex quantitative TaqMan PCR using custom primers and probes against cytochrome b and beta-2 microglobulin as measures of mitochondrial and nuclear genes, respectively. Univariable and multivariable linear regression models were used to evaluate the association between mtDNA CN and rates of visual field progression, quantified by the slope of standard automated perimetry (SAP) mean deviation (MD) over time. Main Outcome Measures: Association of mtDNA CN with MD slope. Results: Mean mtDNA CN was 107.6 copies per cell (interquartile range, 70.5 to 126.7). Higher ln(mtDNA CN) was associated with slower rates of MD loss in both univariable (beta = 0.29, p < 0.001) and multivariable analyses (beta = 0.25, p = 0.003). Higher peak IOP was independently associated with faster visual field progression in both analyses. A significant interaction was observed between mtDNA CN and peak IOP (beta = -0.054, p < 0.001), with the association between lower mtDNA CN and faster SAP progression being more pronounced at lower levels of peak IOP. Conclusion: Lower mtDNA CN was associated with faster SAP progression, with the association being most pronounced at lower levels of peak IOP. These findings support mtDNA CN as a potential systemic biomarker of mitochondrial genomic reserve and susceptibility to visual field loss.

Purpose: To characterize the biomechanical response of the optic nerve head (ONH) in myopic versus non-myopic individuals with normal-tension glaucoma (NTG), and to evaluate the association between ONH strains and the rate of visual field (VF) progression. Design: Retrospective clinical cohort study. Participants: A total of 118 NTG eyes from 77 patients with [&ge;] 2 years of follow-up and 5 VF tests were enrolled, including 66 eyes with myopia and 52 eyes without. Methods: Each participant underwent three-dimensional (3D) optical coherence tomography (OCT) imaging of the ONH under three mechanical loading conditions: 20{degrees} adduction, 30{degrees} abduction and acute intraocular pressure (IOP) elevation by applying a 0.80 N force to the lower eyelid. Effective strains across the global ONH complex and within specific segmented tissues (pre-lamina, lamina cribrosa, choroid, and sclera) were quantified using a validated 3D strain-mapping algorithm. Results: Myopic NTG eyes exhibited significantly higher ONH strains compared to non-myopic eyes under acute IOP elevation (10.1{+/-}2.7% vs. 9.3{+/-}1.8%, P = 0.044) and abduction (9.2{+/-}2.3% vs. 8.4{+/-}2.1%, P = 0.048). In the non-myopic group, ONH strains varied significantly across conditions (P < 0.001), with adduction (9.9{+/-}2.0%) and acute IOP elevation (9.3{+/-}1.8%) eliciting greater strains than abduction (8.4{+/-}2.1%). These condition-dependent variations were less pronounced in the myopic group (10.1{+/-}2.7% vs. 9.8{+/-}2.7% vs. 9.2{+/-}2.3%, P = 0.07). Multivariable linear mixed-effects analysis revealed that in myopic NTG, higher ONH strains induced by adduction were significantly associated with faster MD decline (estimate: -0.139 dB/year per 1% increase, P = 0.03, adjusted for age, spherical equivalent and baseline perimetric mean defect). Conversely, larger strains during acute IOP elevation were associated with slower progression (estimate: 0.184 dB/year per 1% increase, P = 0.004). Tissue-specific analysis confirmed that adduction-induced strains in the choroid and pre-lamina tissue as well as IOP-induced strains across all ONH tissues were significantly correlated with the rate of perimetric progression in myopic eyes (all P < 0.05). Conclusions: In this cohort, greater adduction-induced ONH strain was associated with faster perimetric deterioration in myopic NTG. These findings support further investigation of gaze-related optic nerve traction as a potential non-IOP-related biomechanical factor in myopic NTG. Keywords: Normal tension glaucoma, Myopia, Optic nerve head, Ocular biomechanics, Visual field Progression, adduction.

The peripapillary retinal nerve fibre layer (pRNFL) thickness and Bruch's membrane opening-minimum rim width (BMO-MRW) are three-dimensional retinal biomarkers for glaucoma. We aimed to demonstrate that AI-derived thickness from two-dimensional fundus images can act as proxies, enabling the discovery of novel neurodegenerative loci. AI-derived pRNFL and BMO-MRW strongly correlated with OCT-derived thickness (r: 0.69 and 0.79, respectively). After validation, these phenotypes were predicted in two cohorts lacking disc-centred OCT: UK Biobank and the Canadian Longitudinal Study on Aging. The predicted phenotypes showed strong genetic correlations with directly measured phenotypes from a previous study for both phenotypes (0.70 and 0.96, respectively). This data increased statistical power, identifying 29 loci for pRNFL thickness and 122 loci for BMO-MRW, including 14 loci that were independent of VCDR. We observed shared and sector-specific thickness loci overlapping glaucoma loci and revealed loci independent of known risk factors. Together, these results emphasise that multidimensional inferences can be drawn from 2D imaging, enabling downstream genetic analyses.

Activated leukocyte cell adhesion molecule A (Alcama) plays a role in axonal guidance, cell differentiation, and retinal lamination in a developing retina and was identified as a marker for activated Muller glial cells in adult zebrafish. However, its spatiotemporal localization and its involvement in retina regeneration remains unclear. Here we induced focal photoreceptor damage in zebrafish using laser photocoagulation and examined the expression and localization of Alcama at different time points post lesion. Immunohistochemistry in wild type fish and Tg(kdrl-EGFP) fish showed Alcama localized to the blood retina barrier with increased expression in Muller glial end feet and radial processes in a regenerating retina. To confirm its role in retina regeneration, alcama expression was transiently knocked down using morpholinos in adult fish. Scanning laser ophthalmoscopy, Zpr1 immunostaining and EdU staining showed delayed retina regeneration in alcama knockdown fish, indicating a possible role for Alcama in zebrafish retina regeneration.

Fuchs endothelial corneal dystrophy (FECD) is a disease of the corneal endothelium (CE) characterized by the loss of corneal endothelial cells (CECs) and guttae formation, ultimately resulting in corneal edema and vision loss. FECD primarily affects the central CE while sparing the peripheral CE, however the underlying mechanism contributing to the spatial differences remain unknown. Oxidative stress has been increasingly recognized as a key contributor to the pathogenesis of FECD, with CECs being particularly susceptible to damage from reactive oxygen species (ROS), high metabolic activity and ultraviolet induced DNA damage. The non-proliferative nature of CECs, along with the accumulation of oxidative damage can ultimately lead to CEC loss, a key feature of FECD. In this study, we induced oxidative stress with hydrogen peroxide (H2O2) on ex-vivo corneal specimens and observe increased cell death in the central region compared to the peripheral CE. To investigate these underlying differences, we performed bulk RNA sequencing (RNA-seq) on the central and peripheral regions of CE from FECD and normal cadaveric donors. Pathway analysis identified an enrichment of genes involved in collagen and extracellular matrix between the central and peripheral regions of CE in both normal and FECD, as well as between normal and FECD CE. Intriguingly, we identified the long non-coding RNA (lncRNA), NEAT1 as a top differentially expressed gene, with reduced expression in the central CE compared to the peripheral CE and lower expression in FECD compared with normal CE. Using corneal endothelial cell lines and ex-vivo specimens from FECD patients and normal cadavers, we found decreased NEAT1 expression levels in FECD and increased susceptibility to H2O2-induced oxidative stress. We observed that NEAT1 knockdown in normal and FECD cells exacerbated H2O2-mediated oxidative stress, and that NEAT1 overexpression protected FECD cells. We report in this study, a novel insight in the spatial differences in gene expression in the CE and identify reduced expression of NEAT1 in the central CE as a potential contributor to oxidative stress-related cell death in FECD. These findings provide novel insight into FECD pathogenesis and why FECD pathology preferentially affects the central CE. Antioxidants targeting NEAT1 signaling could be developed into novel therapeutics aimed at preventing FECD pathogenesis.

Purpose: Oxidative stress is thought to contribute to the development of age-related cataracts (ARCs), but the mechanisms by which oxidative damage leads to the opacification of the lens remain unclear. Previous studies suggest that oxidative stress can disrupt lens proteostasis. Therefore, it was hypothesized that ARCs arise from proteomic changes driven by an age-associated decline in oxidative stress defenses that interact with the lens proteostatic state. To test this hypothesis, proteomic analyses of lenses exposed to oxidative stress were performed to examine oxidative and proteostatic stress responses in vivo. Methods: Cataract formation was induced by injecting hydrogen peroxide into the aqueous humor of adult zebrafish. nrf2fh318/fh318 zebrafish were used to model the reduced oxidative stress protection observed in aged human lenses, while cryaba-/- zebrafish were used to model impaired lens proteostasis. Resulting opacities in WT, cryaba-/-, nrf2fh318/fh318, and cryaba-/-; nrf2fh318/fh318 lenses were quantified and proteomic changes in the cortex were analyzed using data independent acquisition Parallel Accumulation Serial Fragmentation mass spectrometry. Results: Hydrogen peroxide treatment induced the formation of cortical cataracts. Proteomic results showed that, dependent on genotype and day, oxidative stress activates the unfolded and mitochondrial unfolded protein responses. Additional changes were also observed in energy metabolism, Ca2+ homeostasis, protein degradation, and cytoskeletal and extracellular matrix remodeling pathways. Conclusions: Treated zebrafish lenses successfully model ARC and mass spectrometry proteomics identified the unfolded and mitochondrial unfolded protein responses as potential therapeutic targets for ARC.

PurposeFuchs endothelial corneal dystrophy (FECD) is a progressive disease, causing premature death of corneal endothelial cells (CECs). Iron-dependent lipid peroxidation and ferroptosis mediate cell death in FECD. We aimed to determine whether FECD progression is mediated by derangements in ferritinophagy - a form of autophagy that degrades ferritin to release labile ferrous iron - and whether ultraviolet A (UVA) exposure drives FECD progression by activating ferritinophagy. MethodsEndothelium-Descemet membrane (EDM) tissues were collected from patients with end-stage FECD undergoing endothelial keratoplasty and from healthy age-matched donor corneas. Separately, immortalized FECD and healthy control CEC lines were cultured. Cellular levels of NCOA4 production and LC3 activation, both markers of ferritinophagy, were quantified using western blotting and PCR. UVA-exposed immortalized cells were plated on coverslips, stained for immunohistochemistry (IHC), and analyzed using confocal microscopy. Corneal endothelial peels were stained and analyzed using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). ResultsSurgically explanted FECD CECs showed significantly increased levels of NCOA4 compared to healthy controls. LC3 activation was increased in FECD immortalized CECs; UV exposure further increased LC3 activation. Additionally, UVA exposure showed trends of increased expression of NCOA4 in immortalized FECD and healthy CECs. On IHC of FECD surgical explant tissue, ferritin was decreased markedly, NCOA4 localized in a dramatic punctate pattern, and both ferritin and LC3 localized within cell nuclei. Spectrometry images showed higher iron levels correlating with areas of higher FECD disease burden. ConclusionsOur results demonstrate ferritinophagy in FECD indicated by the increase of NCOA4 and LC3 ferritinophagy markers in FECD patient and cell culture models. Our finding that UVA activates ferritinophagy implicates this mechanism in UVA-mediated FECD progression. Altogether, aberrant iron dysregulation associated with FECD and ferroptosis may be mediated by ferritinophagy, providing a biomarker to assess disease severity as well as a potential target for future medical therapeutics.

Purpose: The neural retina and retinal pigment epithelium/choroid (RPE/Cho) are metabolically coupled. Metabolic dysfunction of the RPE/Cho contributes to photoreceptor death in age-related macular degeneration (AMD). This study aimed to determine how aging, a major risk factor for AMD, alters the proteome and metabolic pathways in the retina and RPE/Cho. Methods: We performed quantitative proteomics on neural retina and RPE/Cho from young (8-week) and old (73-week) C57BL/6J mice. The differentially expressed proteins between old and young, as well as between the retina and RPE/Cho, were visualized using volcano plots and heatmaps, and functional analyses were assessed by Gene Ontology enrichment tools. Results: Aging altered the expression of 85 retinal proteins and 272 RPE/Cho, with 16 shared between tissues. In the aged retina, proteins associated with NADP(H) metabolism, neurodegeneration, and immunity were mostly upregulated, while proteins in lipid metabolism, transcription, and epigenetic regulation decreased. In the aged RPE/Cho, all 21 significantly altered lysosomal enzymes and transport proteins increased, together with complement components, and photoreceptor outer segment proteins. These changes accompanied increases in proteins associated with lipid metabolism and protection against lipid peroxidation, while several glutathione metabolism enzymes decreased. Seven shared proteins involved in lipid and NADP(H) metabolism, changed in opposite directions between retina and RPE/Cho. Comparison of the retinal and RPE/Cho proteomes identified 3073 differentially abundant proteins, of which 2452 maintained tissue-specific differences across both age groups, while approximately 17% showed age-dependent shifts in tissue enrichment. These shifts involved proteins associated with metabolism, extracellular matrix, visual function, lysosomal pathways, and innate immunity. Within the metabolic proteome, several glycolytic and fatty acid oxidation proteins showed greater relative enrichment in RPE/Cho with age, whereas glucose transporters and mitochondrial complexes shifted toward the retina. Conclusions: Overall, these findings show that aging differentially remodels the retina and RPE/Cho proteome, accompanied by altered metabolic specialization between the two tissues.

Fuchs Endothelial Corneal Dystrophy (FECD) is characterized by progressive corneal endothelial cell loss and the formation of corneal guttae. Currently, there is a global shortage of donor corneas and new strategies are needed to reduce the need for corneal transplantation. While adeno-associated viruses (AAVs) have the capacity to deliver anti-apoptotic genes to human corneal endothelial cells (CECs), this has not been fully explored as a therapeutic strategy for FECD. In this study, we evaluated the transduction efficiency of self-complementary (sc-) and single-stranded (ss-) AAV2 serotypes in human CECs and ex vivo tissues and assessed whether AAV-mediated expression of Bcl-xL could attenuate apoptosis in FECD. Seventeen scAAV2 serotypes were screened for transduction efficiency in normal human CECs via green fluorescent protein (GFP) expression. The top 4 AAV2 serotypes were further evaluated in FECD cell lines, healthy cadaveric donor specimens, and FECD patient specimens. FECD cell lines were transduced with anti-apoptotic ssAAV2/5-Bcl-xL (AAV2/5-CAG-eGFP-P2A-BCLXL) and treated with etoposide to induce apoptosis. We found that scAAV2/5 demonstrated high transduction efficiency across all normal and FECD cell lines and tissues. We observed that ssAAV2/5-mediated expression of Bcl-xL provided significant protection against etoposide-induced apoptosis in FECD CECs (71.68%{+/-}0.69 vs 23.96%{+/-}8.88%, p=0.018). Our findings show that AAVs have the potential for therapeutic gene delivery to the human corneal endothelium, and that targeting the Bcl-xL mediated apoptotic pathway can be further explored as a therapeutic for FECD.

Purpose. To determine which proposed keratoconus (KC) mechanisms carry its genetic susceptibility and how it relates to corneal structure, and which describe its corneal tissue. Methods. Six mechanism modules and two attribution sets (transforming growth factor-{beta} [TGF-{beta}] and canonical type-2 atopy) were tested for enrichment in three independent European KC genome-wide association studies (GWAS) and in GWAS of central corneal thickness (CCT) and corneal resistance factor (CRF), with colocalization at matrix loci. Tissue expression was pooled across nine arms from eight studies with control-source meta-regression; activator protein-1 (AP-1)/immediate-early regulatory activity was scored in 107 donors, spatial expression in 21, and serum responses in human and rabbit stromal cells. Results. Extracellular matrix (ECM) was the only module enriched in all three KC sources (P [&le;] 5.46 x 10-) and for CCT, CRF, and thickness-independent CRF (P [&le;] 2.07 x 10-). COL5A1 shared causal variants with CCT in all three sources (posterior probability 0.985-0.991), and FNDC3B and COL1A1 with CRF or its thickness-independent component (0.961-0.986). Pooled, KC corneas had lower general and corneal-local inflammatory and stress/immediate-early expression (log2 fold-change -0.97, -0.98, -0.82; P [&le;] 0.049). In whole-cornea and stromal tissue, ECM expression reversed between postmortem-donor and pathological controls (log2 fold-change +0.36, -2.34), whereas inflammatory and TGF-{beta} programs were lower against both. AP-1/immediate-early regulatory activity was lower in four donor cohorts (Hedges' g -1.50) and stress/immediate-early expression in two spatial cohorts; serum raised immediate-early readouts in human and rabbit cells. Conclusions. KC combines matrix-mechanical genetic susceptibility with a low-inflammatory, low-immediate-early corneal state whose matrix expression direction depends on the control source. Because this susceptibility is genetically shared with corneal thickness and resistance, interventions on the inherited disease are judged by corneal structure and mechanics.

Purpose: Age-related Macular Degeneration (AMD), a degenerative disease of aging, leads to central vision loss and has a strong genetic risk. Genetic heritability, used to quantify genetic influence on a trait, has mainly focused on twin study designs but these are vulnerable to bias. Studying relatives beyond twins is necessary to bring clarity to the genetic burden of AMD and help focus the search for additional genetic risk loci. Methods: Through both single nucleotide polymorphism (SNP) and pedigree-based heritability methods, the heritability of AMD was analyzed using relationship informed analyses of families from an Amish population (n = 525). AMD status was determined using the Beckman grading scale (285 controls and 240 cases). An estimate of genetic relatedness preceded SNP heritability estimation, whereas the pedigree heritability model utilized genealogical reports. Primary models were adjusted for age, sex, and population structure. A comparison of SNP- and pedigree-based models followed heritability estimation. Sensitivity models adjusting for all possible combinations of three known strong AMD genetic risk variants were constructed. Results: SNP heritability is 55% +/- 13% (p= 9.87e-06) and the pedigree heritability is 49% +/- 18% (p= 3.06e-04). The sensitivity analyses revealed that the estimates were robust to changes in the inclusion of AMD variants as covariates. Conclusions: These heritability estimates support existing twin and SNP-based AMD heritability estimates and corroborate the substantial involvement of genetics in AMD. Adjusting for known AMD variants revealed that additional genetic contribution exists, supporting a large polygenic effect in AMD.

Traumatic optic neuropathy (TON) occurs due to direct or indirect injury to the optic nerve and is a significant cause of visual disability. So far, there is no effective treatment. The lack of understanding of the cellular mechanisms by which trauma induces inflammation and damage in retinal neurons is a critical knowledge gap in developing effective therapies. We have studied the role of the arginase 1 (A1) enzyme in this pathology. We have found previously that treatment with a long-acting form of human recombinant A1, pegylated A1 (PEG-A1) after optic nerve crush limits activation of retinal microglia and macrophages (M{Phi}) and reduces inflammation, thereby decreasing injury and protecting visual function. Here we report on studies designed to demonstrate the therapeutic efficacy of PEG-A1 in mouse models of direct and indirect TON and to elucidate the underlying mechanisms. We used ONC to model direct TON and sonication-induced trauma to the supraorbital rim to model indirect TON (SI-TON). At different times after injury, mice were treated with PEG-A1 which was delivered systemically by i.p. injection or locally by intravitreal injection. In order to assess the role of A1-induced activation of the ornithine/polyamine pathway in the protective effects of PEG-A1, some mice were treated with the ornithine decarboxylase (ODC) inhibitor, difluoromethylornithine (DFMO) immediately after the PEG-A1 treatment. Retinal function was determined by OptoMotry and electroretinography. Retinal injury and microglia/M{Phi} activation were assessed by immunofluorescence imaging. Expression of inflammatory cytokines was determined by Western blotting and quantitative RT PCR. Liquid chromatography mass spectrometry was used to analyze changes in arginase/ODC pathway metabolites. Results showed that PEG-A1 treatment improved neuronal survival and visual function whether delivered systemically or intravitreally. This neuroprotection was associated with decreased microglia/M{Phi} activation, decreased inflammatory cytokine expression, and increased formation of L-ornithine and putrescine. Furthermore, DFMO treatment blocked these effects, indicating that PEG-A1 limits retinal injury and preserves vision after ocular injury by activating ODC. ODC processes the arginase product L-ornithine to form polyamines which are known to promote reparative functions. Thus, PEG-A1 therapy offers a new strategy to limit trauma-induced vision loss and promote repair after TON.

Purpose: To understand the functional impact of macular atrophy (MA) and fibrosis in eyes with treated neovascular age-related macular degeneration (nAMD) based on deep visual sensitivity losses. Design: Cross-sectional study. Participants: One-hundred and nine eyes from 97 individuals with nAMD that had been treated for >=12 months without significant retinal fluid. Methods: All participants underwent defect-mapping microperimetry (DMP) testing - a strategy optimized to quantify the spatial extent of deep visual sensitivity losses through single 10 decibel stimuli presentations - where 208 locations were sampled in the central 8 degrees radius region. Participants also underwent color fundus photography, fundus autofluorescence and OCT imaging, which were co-registered and manually annotated for MA and fibrosis to determine their two-dimensional pointwise extent at individual test locations, and global extent in the corresponding region where DMP was performed. Main Outcome Measures: Pointwise association between the presence of MA and fibrosis with missing a stimulus on DMP and global association between the extent of MA and fibrosis with the proportion of locations missed (PLM) on DMP. Results: At a pointwise level, the presence of MA and fibrosis were independently associated with an increased likelihood of missing a stimulus on DMP (odds ratio = 42.5 and 2.5 respectively; P < 0.001 for both). However, at a global level, only an increasing extent of MA was associated with a significantly higher PLM on DMP (P < 0.001), but not fibrosis (P <= 0.227). MA extent explained a substantial proportion of the variance in the PLM (R2 = 0.97) and there was a very strong correlation between the two parameters ({rho} = 0.93). Conclusions: This study confirmed the expected functional impact of MA and fibrosis on DMP testing at a pointwise level. However, it showed at the global level that only the extent of MA, and not fibrosis, was independently associated with functional loss on DMP, and that there was a very strong structure-function correlation based on MA. These findings thus suggest that evaluating MA alone may be sufficient for capturing structural changes strongly associated with the global extent of deep visual sensitivity losses in those with treated nAMD.

Purpose: To test whether juvenile open-angle glaucoma (JOAG) and primary congenital glaucoma (PCG) are polygenically continuous with primary open-angle glaucoma (POAG), using a multi-trait POAG polygenic risk score (PRS). Design: Retrospective cohort study. Participants: 3,102 open-angle glaucoma index cases, of whom 2,836 had a recorded age at diagnosis (52 PCG, 226 JOAG, and 2,558 POAG) and 999 European-ancestry controls from a glaucoma disease registry and a population-based cohort. Two replication cohorts comprised 70 JOAG and 31 PCG probands with 230 controls (Mass Eye and Ear, MEE), and 9 PCG and 120 POAG cases (Genetics of Glaucoma Study, GOGS). Methods: One index case per family was analysed. PRS were calculated from weighted SNPs and adjusted for ancestry. Cases were stratified by age at diagnosis (PCG <=3 years, JOAG >=4 and <40 years, POAG >=40 years) and presence of pathogenic/likely pathogenic variants ("Mendelian"). Groups were compared by Kruskal-Wallis test, with logistic regression and area under the curve for per-SD effects. Main Outcome Measures: PRS centile values compared across glaucoma subtypes and Mendelian and non-Mendelian subgroups. Results: Mean PRS was highest in cases diagnosed in the fourth decade of life. Among subgroups, it was highest in non-Mendelian JOAG (84.1%), exceeding controls (53.3%), Mendelian JOAG (68.9%), and non-Mendelian POAG (79.3%) (all P <= 0.003). Non-Mendelian JOAG was also the best discriminated from controls (OR 3.66 per SD increase, 95% CI 3.02-4.49; AUC 0.814, 95% CI 0.782-0.845). Neither PCG subgroup differed from controls. Among Mendelian cases, only MYOC p.Gln368Ter showed significantly elevated PRS (OR 2.49 per SD, 95% CI 1.89-3.34). In the MEE replication cohort, JOAG was associated with higher PRS (OR 2.12 per SD, 95% CI 1.50-3.07) while PCG was not (OR 0.90, P = 0.73). In GOGS, mean PRS was higher in POAG than PCG (84.8 vs 59.7 centile; P = 0.026). Conclusions: A POAG PRS predicts JOAG risk with discrimination comparable to or greater than POAG itself, supporting a shared polygenic architecture across juvenile and adult open-angle glaucoma. PCG showed no PRS elevation regardless of Mendelian status, indicating a distinct architecture for which rare-variant sequencing remains appropriate.