Experimental Eye Research

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.

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.

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: 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.

Achromatopsia (ACHM) is a rare autosomal recessive retinal disorder characterized by absent cone photoreceptor function from early life, leading to severe visual impairment. Mutations in genes involved in the cone phototransduction cascade frequently result in elevated cyclic guanosine monophosphate (cGMP) levels and activation of stress pathways, including endoplasmic reticulum (ER) stress and the unfolded protein response. Targeting common downstream mechanisms rather than individual mutations may provide a broadly applicable therapeutic strategy. Here, we investigated whether pharmacological inhibition of valosin-containing protein (VCP), a key regulator of ER and protein homeostasis, can prevent cone degeneration in the spontaneous cone photoreceptor function loss 1 (cpfl1) mouse model of ACHM. Organotypic culture of retinal explants from cpfl1 mice were treated with the selective VCP inhibitor ML240. Cone survival, cell death, opsin expression and localization were assessed by TUNEL assay, immunohistochemistry, and quantitative image analysis. ML240 treatment significantly increased cone density and improved cone opsin expression and trafficking to the outer segments (OSs) in cpfl1 explants compared to controls. Importantly, rhodopsin trafficking in rod photoreceptors was unaffected, indicating that VCP inhibition did not impair normal rod phototransduction. These findings demonstrate that VCP inhibition by ML240 effectively preserves cone photoreceptors and improves cone-specific functional markers in the cpfl1 model. Targeting VCP may represent a mutation-independent therapeutic strategy for preventing cone death in ACHM.

Purpose: To develop an interpretable automated pipeline for dome-shaped macula (DSM) detection and measurement from optical coherence tomography (OCT) B-scans. Methods: We analyzed one eye from each of 300 children aged 4-18 years in the Shanghai Child and Adolescent Large-scale Eye Study High Myopia (SCALE-HM) cohort (50 DSM-positive; 100 test eyes). A U-Net pretrained on OCTA-500 and fine-tuned on the development set segmented the retinal pigment epithelium (RPE) boundary. A geometric module located the apex, fitted a tangent-line baseline, and computed perpendicular height in physical coordinates. An eye was DSM-positive if any gradable B-scan had height >= 50 um. Two masked graders evaluated the test set; a third adjudicated disagreements. Results: The area under the receiver operating characteristic curve was 0.927 (95% confidence interval [CI], 0.850-0.986), sensitivity was 82.1% (95% CI, 64.4%-92.1%), and specificity was 100% (95% CI, 94.9%-100%) against the adjudicated reference. In concordant-positive eyes, automated height agreed with manual measurements (intraclass correlation coefficient, 0.949; mean absolute error, 6.9 um; bias, -5.7 um, versus Doctor A). Mean RPE-boundary error on pediatric test scans was 1.54 um. Conclusions: The pipeline may be considered a reliable and reproducible tool for automated DSM detection and height measurement on radial OCT, with each value traceable to landmarks on the source B-scan. External validation across populations, devices, and acquisition protocols is needed. Translational Relevance: The pipeline provides an objective and reproducible method to detect DSM and quantify dome height, width, and orientation across OCT meridians. Because each measurement can be verified on the source B-scan, it enables standardized DSM assessment.

Purpose: To evaluate the associations of the numerical AI-CO center index and visually classified central dark-area extent with Humphrey 10-2 mean deviation (MD). Methods: This single-center retrospective study included 320 eyes of 169 patients with AI-CO imaging, broad-field 24-2F or 30-2 testing, and 10-2 testing. Both visual field examinations were performed within 365 days before or after AI-CO imaging. Two ophthalmologists independently classified central dark-area extent into five Levels (0-4). Analyses assessed Spearman correlations, areas under the receiver operating characteristic curve (AUCs), distributions across final central dark-area Levels, and interrater reliability. Patient-cluster bootstrap confidence intervals and a one-eye-per-patient sensitivity analysis addressed inclusion of both eyes. Results: AI-CO center correlated with 10-2 MD (rho=0.829; 95% CI, 0.773-0.873). AUCs for 10-2 MD below -2, -6, and -12 dB were 0.902, 0.939, and 0.961, respectively. The numbers of eyes in Levels 0-4 were 66, 57, 94, 53, and 50; median 10-2 MD values were -0.59, -1.48, -3.54, -6.24, and -18.15 dB, respectively. Quadratic-weighted kappa was 0.970 (95% CI, 0.958-0.981). Sensitivity analyses yielded broadly similar findings. Conclusions: The numerical AI-CO center index and visually classified central dark-area extent were associated with central visual field damage. Assessing the extent of dark regions in the central image alongside the numerical index may provide useful information for interpreting central visual field status from the AI-CO display.

Glaucoma is an optic neuropathy characterized by progressive retinal degeneration. Population genetic analyses describe an association of variants in angiopoietin-like protein 7 (ANGPTL7) with lower intraocular pressure (IOP) and decreased glaucoma susceptibility. In addition to its reported role in the trabecular meshwork (TM), we demonstrate that ANGPTL7 is also expressed within disease-relevant cell types of the human posterior eye, specifically prelaminar optic nerve head astrocytes (ONHA). Contrasting with previously published findings, we observe no difference in IOP between Angptl7-/- mice and wild-type littermates. Despite absence of an IOP phenotype, loss of ANGPTL7 protects from IOPinduced retinal ganglion cell (RGC) damage in a rodent glaucoma neurodegeneration model. ANGPTL7 may signal through LAIR1-positive immune cells, potentially contributing to pro-inflammatory signaling and phenotypic modulation of resident immune cells, with evidence suggesting that oligomerization of ANGPTL7 is necessary for its activity. Comprehensively, our data highlight an unappreciated role of ANGPTL7 in modulating neuroinflammation in the posterior eye, contributing to glaucoma progression.

Abstract Purpose To apply a mathematical model integrating vascular permeability, lesion transmural pressure, choroidal excess pressure, and wall stability to incomplete fluid response and massive submacular hemorrhage in neovascular age-related macular degeneration. Methods Permeability was calculated from aqueous vascular endothelial growth factor (VEGF). Windkessel gave usual lesion pressure from choriocapillaris pressure and high inflow from an arterial-level puncture pressure used as a working value. Choroidal excess pressure was calculated from choroidal thickness and a pachy index as increased outflow resistance. Exudative change used a revised Starling equation scaled to early central-retinal-thickness reduction, with 40-m threshold. A relative rupture-hazard index was calculated from Laplace wall stress and wall stability. VEGF, pressures, and that reduction were from the literature. Pressure was isolated from permeability and choroidal excess. Results In the model, raising lesion pressure from choroidal-excess lesion pressure to high inflow increased untreated exudative change from 150 to 760 m. Residual under saturation was 64 m as a lower-end reference (Monte Carlo 69 - 286 m; median, 170) and 90 m with high-hazard choroidal excess; both above 40 m. Reducing choroidal thickness in the thickened choroidal-excess state added 13 - 14 m, smaller than that residual. Lowering lesion pressure from high inflow to usual lesion pressure (21 to 10 mmHg) reduced the index to 0.64 of the high-hazard reference, and to 0.34 with wall stabilization. Conclusion Lesion transmural pressure due to high inflow left residual exudation even after permeability suppression with anti-VEGF. Fluid resolution on optical coherence tomography may not parallel a reduction in the rupture-hazard index, depending on wall stability.

Purpose To evaluate the safety, tolerability, and preliminary bioactivity of a single suprachoroidal injection of AXT107 (Gersizangitide), a multimodal integrin-disrupting peptide that acts on both the VEGFA pathway and angiopoietin-Tie2 pathway, in eyes with neovascular age-related macular degeneration (nAMD). Design Phase 1/2a, open-label, dose-escalation clinical trial. Participants Fifteen subjects with active nAMD, including 11 treatment-experienced partial responders to intravitreal anti-vascular endothelial growth factor (VEGF) therapy and 4 treatment-naive subjects; 14 eyes met the protocol-specified baseline imaging criterion and were included in efficacy analyses. Methods Study eyes received a single suprachoroidal injection of AXT107 microparticles at doses of 0.125, 0.25, or 0.5 mg and were followed for 40 weeks. Protocol-specified rescue intravitreal anti-VEGF treatment was permitted from week 12 based on protocol-specified best-corrected visual acuity (BCVA) or central subfield thickness (CST) criteria; anti-VEGF treatment before Week 12 was investigator-initiated. Primary outcomes were ocular and systemic safety through week 40; secondary outcomes included change in CST, change in BCVA, and time to first protocol-specified rescue injection. Main Outcome Measures Incidence of ocular and systemic adverse events, intraocular pressure (IOP), CST, BCVA, and time to first protocol-specified rescue injection. Results Twenty subjects were screened and 15 enrolled across 4 U.S. sites; 3 eyes received 0.125 mg, 3 received 0.25 mg, and 9 received 0.5 mg of AXT107. No serious adverse events related to AXT107 occurred, and no cases of endophthalmitis, retinal detachment, suprachoroidal hemorrhage, or clinically significant intraocular inflammation were observed through week 40. No clinically meaningful intraocular pressure (IOP) elevations occurred at any dose, and no subject required chronic IOP-lowering therapy. Among the 14 eligible eyes included in efficacy analyses, 2 eyes (01-04 and 01-05) completed 40 weeks without protocol-specified anti-VEGF rescue, and 1 eye (02-04) first received protocol-specified rescue at week 36. At the last on-treatment visit, BCVA changes ranged from +15 to -31 ETDRS letters; 3 eyes gained[&ge;]5 letters, 3 gained[&ge;]10 letters, and 1 gained[&ge;]15 letters. Among the 14 eligible eyes, anatomical improvements in CST were observed in at least 1 eye in each dose cohort. The most durable responses were seen in treatment-experienced eyes receiving 0.5 mg (median durability of 36 weeks), whereas treatment-naive 0.5-mg eyes showed more variable courses and generally earlier protocol-specified rescue in this small cohort. Conclusions In this first-in-human study, a single suprachoroidal injection of AXT107 up to 0.5 mg was well tolerated through Week 40, with no drug-related serious adverse events, no clinically significant intraocular inflammation, and no sustained IOP elevations. After a single suprachoroidal AXT107 injection, 2 eyes completed 40 weeks without protocol-specified rescue anti-VEGF treatment, and 1 additional eye received its first protocol-specified rescue treatment at Week 36. The exploratory observation that durability appeared greatest in treatment-experienced eyes supports further controlled study of AXT107, particularly in treatment-experienced nAMD populations.

Purpose: Age is the leading factor for age-related macular degeneration (AMD), in which retinal pigmented epithelium (RPE) atrophy is a hallmark. The nuclear factor-erythroid 2-related factor 2 (Nrf2), a major anti-stress transcription factor, decreases in AMD, suggesting bioenergetic stress interacts with age during AMD pathogenesis. We profiled visual function and gene expression of the retina and RPE-choroid with Nrf2 deletion and aging. Methods: We tested 3mo and 14mo Nrf2 null (Nrf2 KO) and Nrf2 WT mice for optokinetic tracking (OKT) and dark-adapted electroretinography (ERG) responses. Eyes were harvested for retina outer nuclear layer (ONL) thickness analysis and retina and RPE-choroid cup RNA isolation for RNAseq profiling. Results: Visual acuity was reduced with aging with lower values in the Nrf2 KO vs Nrf2 WT comparison at 14mo. We observed aged-related reduction of a-, b-, and c-wave ERG amplitudes in both WT and KO mice. Age-related c-wave decline was exacerbated by Nrf2 loss. By RNAseq, two-way ANOVA of RPE-choroid samples revealed differentially expressed genes (DEGs) with age and genotype main effects, as well as DEGs with age and genotype interaction effects. Age DEGs were over-represented in pathways related to inflammation and phagocytosis. Genotype DEGs were associated with alterations in antioxidant and inflammatory processes. Retina RNAseq showed age and genotype DEGs. Conclusions: Ablation of Nrf2 exacerbates age-related declines suggesting that decrease in Nrf2 with aging has functional consequences for inflammation/resiliency. We provide retina and RPE-choroid bulk-RNAseq datasets from the same cohort of mice that can be interrogated to better understand degeneration of retina/RPE-choroid.

A cardinal feature of nonexudative age-related macular degeneration (AMD) is the accumulation of drusen between the retinal pigment epithelium (RPE) and Bruch's membrane, yet the mechanisms that initiate and promote drusen formation remain to be fully understood. Hydroxyapatite (HAP), a calcium-phosphate mineral identified within human drusen, has been proposed to contribute to drusen nucleation and is associated with increased likelihood of RPE atrophy. However, mechanistic studies providing insight into RPE-associated mineralization have been limited by a lack of easy-to-use and manipulatable culture models. Here, we established a rapid, inducible ARPE-19-based model of HAP mineralization. We found that ARPE-19 cells cultured in minimum essential media (MEM) supplemented with nicotinamide (NIC), sodium phosphate, and ascorbic acid (NaPAA) reproducibly formed HAP positive deposits, whereas mineralization was not observed while using standard DMEM/F12 medium, DMEM/F12 with NaPAA, MEM-NIC medium, or MEM-NIC medium with alternative osteogenic supplements (such as beta-glycerophosphate, ascorbic acid, and dexamethasone). Scanning electron microscopy with energy dispersive X ray spectroscopy demonstrated that the formed deposits were enriched in calcium (Ca) and phosphorus (P) with Ca/P ratios comparable to drusen-associated HAP deposits from AMD donor tissue. ARPE-19-based HAP deposits were also closely associated with neutral lipid-rich material, staining positive for BODIPY around their core structure. Bulk RNA sequencing of mineralized ARPE-19 cells revealed extracellular matrix, angiogenic, inflammatory, and metabolic transcriptional changes in the absence of strong enrichment of canonical osteogenic pathways. Co-treatment of mineralizing cells with either pyrophosphate, EDTA, or sodium citrate significantly reduced mineral deposition, but these approaches were not able to reverse it. Together, these findings establish a tractable RPE mineralization model that recapitulates key features of AMD-associated HAP and provides a platform for investigating mechanisms of drusen-associated mineral formation.

Background and Objective: High-speed and high-resolution retinal imaging using adaptive optics line confocal ophthalmoscopy (AOLCO) resolves individual red blood cells (erythrocytes) flowing through the smallest retinal capillaries, but quantifying their velocity first requires removing linear and nonlinear distortions caused by eye motion that may result in a displacement of tens to hundreds of pixels between frames. We present a computational pipeline that stabilizes AOLCO capillary video and measures erythrocyte velocity in true physical units, delivered as an interactive 3D Slicer extension and a batch command-line tool sharing one analysis core. Methods: Our video analysis pipeline has four stages: preprocessing, registration, postprocessing, and velocity measurement. During preprocessing, illumination is corrected, and closed-eye frames are identified. During registration, each frame is aligned to a reference frame using affine and, when needed, B-Spline registration, initialized by our robust and fast (GPU-based) translation-registration algorithm to accommodate large eye movements (e.g., microsaccades). During postprocessing, adjacent registered frames are differenced to suppress stationary structures, and a 2D projection of the frame-difference video reveals the capillary network. During velocity measurement, an operator manually identifies capillary segments on the 2D projection image. Spatiotemporal images are then generated along these segments from the frame-difference video, and blood-flow velocity curves are obtained from these images using the Radon transform. Results: We validate the velocimetry of the pipeline against synthetic ground-truth flow data, and the experiments show that the velocimetry recovers the speed within an error of 4% The GPU-based translation registration algorithm aligns the large eye motions (~90 um per frame on average) and runs about 3.6 x (100 Hz) to 38 x (400 Hz) faster than the baseline method. Across the full cohort (2344 videos, 57 subjects, 30 Hz to 400 Hz), our registration approach improves structural similarity (SSIM) of the video frames on every acquisition from 0.73 to 0.86. The recovered blood flow velocities are physiologically plausible, of the same order as published AO measurements. Conclusions: Our computational pipeline enables AOLCO velocimetry on the capillary-level.

Retinal neurons rely on continuous oxygen supply to maintain function, yet some species, like goldfish, withstand prolonged hypoxia due to adaptations that remain unclear. Horizontal cells (HCs) are inhibitory interneurons of the retina that express ionotropic glutamate receptors (iGluRs) and secrete gama-aminobutyric acid (GABA), allowing them to shape visual signalling by feeding back to photoreceptors. We investigated whether iGluRs in dissociated goldfish HCs are suppressed during hypoxia, and whether this process is mediated by oxygen-sensitive activation of mitochondrial ATP-sensitive K+ (mKATP) channels. Through perforated patch-clamp electrophysiology, we determined that 20 min of hypoxia reduced peak inward current density associated with glutamate application by ~25% (P = 0.0059). When mKATP channels were selectively blocked with 100 microM glibenclamide, hypoxic suppression of iGluRs was abolished. Inhibiting mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter with ruthenium red, or blocking Ca2+ release through ryanodine receptors in the endoplasmic reticulum, also abolished the hypoxia-induced suppression. Similarly, inhibition of calmodulin with calmidazolium, or of protein phosphatase 1 and 2A (PP1/PP2A) activity with calyculin A, prevented the hypoxia-induced suppression of iGluRs. Taken together, these results indicate that mKATP activation is required to mediate an intracellular Ca2+ signal that downregulates iGluR activity during hypoxia. Through the reduction of iGluR activity, HCs may lower their metabolic demand, allowing them to maintain cell function during hypoxia. Additionally, GABA release from HCs, as measured by mass spectrometry, was not affected by hypoxia, and we propose that downregulation of iGluR may account for hypoxic suppression of the retina in hypoxia-tolerant organisms.

Non-coding variants altering mRNA splicing are increasingly recognized as important cause of Mendelian disorders. Deep intronic variants (DIV) that activate cryptic exons (CEs) cause ~20% inherited retinal diseases (IRD) cases, yet it remains challenging to predict intronic variant pathogenicity accurately, thus we used a high throughput splicing assay (HTSA) to measure the effects of rare deep intronic variants in recessive IRDs with one confirmed pathogenic variant. 640 very rare deep intronic variants were chosen in trans-position with known mutation from 76 patients. We tested them with HTSA, which consists of a split-GFP minigene, separated by an SMN1 gene intron into which the 270bp sequences flanking the DIVs were cloned. The plasmid library was transfected into 293HEK cells, and 48 hours later total RNA was extracted from the transfected cells. RNA transcripts produced by HTSA minigene were amplified by RT-PCR and sequenced. The intron sequences spliced between the two GFP exons were identified and quantified. 98 variants activated CE >100 times more in altered oligo compared to reference oligo and were classified as pathogenic in this experimental setting, validated in longer context, 26 variants were classified as VUS. Interestingly, only 6 from 90 variants (6,6%) were predicted to be pathogenic by in silico algorithms (Splice AI) which leaves room for significant improvement of prediction algorithms. The results of this experiment confirmed diagnosis for 50 of 78 patients (64%). So, 19,4% of deep intronic variants were shown to cause CE activation. This can lead to conclusion that significant portion of undiagnosed patients with recessive IRDs carry pathogenic intronic mutation which causes the disease.

Although MAP1LC3B/LC3B (LC3) is best known as a cytoplasmic marker of autophagosome biogenesis, a substantial pool of LC3 resides in the nucleus and shuttles dynamically between nuclear and cytoplasmic compartments, yet the composition and regulation of this nuclear interactome remain poorly defined. Using mass spectrometry-based proteomic profiling of nuclear GFP-LC3 immunoprecipitates from primary human trabecular meshwork (TM) cells, a mechanosensitive ocular cell type, we identified a reproducible nuclear LC3 interactome enriched for proteins containing LC3-interacting region (LIR) and expanded LIR (xLIR) motifs and reported nuclear localization. Among these, clathrin heavy chain (CLTC) emerged as a previously unrecognized nuclear LC3 partner that localizes to the nucleus and colocalizes with nuclear LC3 puncta. CLTC depletion reduced basal LC3-II levels, consistent with a role in autophagosome biogenesis, and markedly impaired nuclear LC3 accumulation induced by both nuclear export blockade with leptomycin B and cyclic mechanical stretch, without altering total CLTC abundance, indicating that CLTC actively promotes LC3 nuclear translocation rather than serving as a passive scaffold. Strikingly, mechanical stress-induced nuclear LC3 trafficking, but not its basal component, was selectively impaired in TM cells derived from glaucoma patients, despite comparable CLTC levels, pointing to a defect in coupling this trafficking pathway to mechanotransduction rather than in the core transport machinery itself. Together, these findings establish nuclear LC3 trafficking as an actively regulated, CLTC-dependent process linked to cytoskeletal and vesicular machinery, and implicate its dysregulation in mechanically stressed glaucomatous cells, providing a framework for understanding how autophagy intersects with nuclear homeostasis, mechanotransduction, and glaucoma pathogenesis.

Age-related macular degeneration (AMD) is the leading cause of irreversible central vision loss in older adults. The highest genetic risk for AMD maps to Chromosome 10q26. However, due to strong linkage disequilibrium between two genes, ARMS2 and HTRA1 located in this region, it has been difficult to determine whether one or both genes contribute to the AMD-disease risk. Functional studies of ARMS2 have been hampered due to the lack of animal models, as orthologs are only found in the higher primates and the gene is absent in rodents. To model ARMS2 biology in vivo, we used CRISPR-Cas9 to insert the complete human ARMS2 gene carrying the A69S risk variant (rs10490924), into a region of homology between humans and mice, on mouse Chromosome 7, 3.5 kb upstream of Htra1, preserving its native promoter, spacing, and orientation. We also generated a mouse model bearing its wild-type counterpart ARMS2A69 (ARMS2WT). RNA analysis revealed tissue- and cell-type-specific pattern of ARMS2 expression enriched in neural tissues, testis, and eye, and within the eye, in retinal horizontal cells and the retinal pigment epithelium (RPE). ARMS2 expression was consistent with human expression data and unaltered by the A69S mutation. By 12 months, ARMS2A69S mice develop AMD-relevant phenotypes including increased fundus spots, A69S-specific thinning of the outer nuclear layer, and reduced scotopic ERG amplitudes. Transcriptomic profiling of postnatal day 14 retina and RPE-enriched fractions revealed reduced mitochondrial oxidative-phosphorylation genes, corroborated by reduced retinal mtDNA copy number and lower maximal respiration, alongside preliminary results that suggest sphingolipid remodeling in the RPE. This humanized model links the ARMS2 A69S variant to mitochondrial dysfunction and provides an in vivo platform for defining AMD mechanisms and testing therapies.

The cornea is the outermost layer of the eye, most vulnerable to injury including from chemical exposure, either environmental, industrial or chemical weapons. For example, exposure of the eye to sulfur mustard (SM) can cause mustard gas keratopathy, resulting in devastating consequences for vision. Available treatments for corneal mustard keratopathy are either ineffective, or if used for a prolonged time, have deleterious side effects. Here, we aimed to harness the anti-inflammatory and anti-angiogenic peptide angiotensin-(1-7), known as Ang-(1-7), that is derived from the renin-angiotensin system (RAS). We designed and developed a protein-like polymer (PLP) displaying a brush of norleucine modified Ang (1-7) peptides (NLE Ang (1-7) PLP), which is a more potent derivative of Ang-(1-7), for administration as an eye drop. In a mouse model of nitrogen mustard (NM)-induced corneal injury, topical treatment and intraperitoneal (i.p.) injection of NLE Ang (1-7) PLP persistently minimized: (i) corneal haze; (ii) edema; (iii), expression of pro-inflammatory and pro-angiogenic genes; and (iv) neovascularization and conjunctivalization as evidenced after a prolonged treatment and recovery period. Collectively, our observations have demonstrated the efficacy of this novel polymer proteomimetic based eye drop treatment in reducing chemical agent-induced corneal injury as well as indicating its potential application in other ocular surface diseases associated with inflammation and angiogenesis.

Protein aggregates are a diverse array of intra- and extracellular structures containing misfolded proteins with limited macromolecular movement. Protein aggregation is induced by aging and cellular stress and is a hallmark of neurodegenerative diseases including Huntington's, Alzheimer's, and Parkinson's. Fluorescent tools to visualize global protein aggregation in whole organisms are, therefore, important for screening genes and compounds that may reduce or alter protein aggregation in connection with improved health outcomes. Fluorescent probes that report low fluidity structures in cells, such as molecular rotor dyes, are gaining popularity and overcome certain limitations of targeted markers such as tagged proteins or antibodies. However, permeability has been a key barrier preventing widespread use of such probes in whole organisms, including in C. elegans. To achieve visualization of non-specific protein aggregates in whole C. elegans nematodes without the need for strain engineering, we have developed a simple soaking protocol. Our protocol overcomes the permeability limitations of the commercially available molecular rotor dye, Proteostat, enabling widespread labeling of protein aggregates in live and post-fixed worms. Here, we present our novel approach and demonstrate quantification of protein aggregates in multiple tissues to detect differences in protein aggregation due to both genotype and treatment effects. We also outline anticipated future applications for this protocol, including its adaptation for other dyes, as well as limitations and special considerations for its use.

Background: In 2026, the US Food and Drug Administration (FDA) identified uveitis as a potential signal of serious risk for the B-cell maturation antigen (BCMA) x CD3 bispecific antibodies teclistamab and elranatamab. Uveitis in this setting may reflect opportunistic infection or immune-mediated inflammation, which require opposite management. Methods: We analyzed FDA Adverse Event Reporting System (FAERS) reports from July 2020 to June 2026 (8,690,601 reports after deduplication) using a case-non-case design, calculating reporting odds ratios (RORs) and the information component for primary-suspect reports. Comparators were belantamab mafodotin, talquetamab and CD3 bispecific antibodies used outside myeloma. Every primary-suspect uveitis report (n = 76) was classified as infectious, probable immune-mediated (hereafter immune-mediated) or indeterminate by a physician blinded to a rule-based algorithm. Results: Uveitis was reported in 18 of 2,995 teclistamab and 29 of 1,691 elranatamab reports (ROR 4.10 [95% CI 2.58-6.52] and 11.85 [8.21-17.12]). Of these 47 cases, 20 were infectious (18 cytomegalovirus chorioretinitis; median onset 92 days), and infection-specified uveitis was disproportionately reported for both agents and for CD3 bispecific antibodies used outside myeloma. Thirteen were immune-mediated, arose early (median onset 4 days) and were co-reported with cytokine release syndrome in six; immune-mediated uveitis showed a signal only for elranatamab (8 cases; ROR 3.59, 1.79-7.18), not for teclistamab or any comparator. Six of the eight elranatamab cases originated from Japan. Agreement between physician and algorithm was 98.7%. Conclusions: The FDA uveitis signal appears to comprise a late infectious uveitis shared by most T-cell-redirecting bispecific antibodies examined and an early immune-mediated uveitis that showed a signal only with elranatamab. These hypothesis-generating findings support prompt ophthalmological assessment of new visual symptoms and exclusion of cytomegalovirus retinitis before corticosteroids are started.