Experimental Eye Research

Purpose: To identify transcript-level variants in crystallin genes in paediatric patients with unilateral cataracts. Methods: Anterior capsulorhexis (n=12) from patients underwent surgical management of congenital unilateral cataracts was collected. Total RNA was isolated from lens epithelial cells, and complementary DNA (cDNA) was synthesized. Full-length RNA transcripts of 10 lens-specific crystallin genes were PCR-amplified and analysed via Sanger sequencing. Identified transcript variants were further validated using genomic DNA (gDNA) through Sanger sequencing. In addition, the full-length (~7,535 bp) CRYBA1 genomic region was sequenced using Oxford Nanopore Technology. Results: Aberrant low molecular weight (LMW) amplicons (~370 bp) of the CRYBA1 transcript were identified in three patients presented with unilateral cataract. Of 3 patients, 2 had persistent fetal vasculature (PFV) and 1 had pre-existing posterior capsular defect (PPCD). Sanger sequencing revealed a precise loss of exons 2 to 4 in the CRYBA1 RNA transcript. No coding, splice-site, or large deletion variants were detected in the genomic DNA of the patients or their parents. In silico analysis predicted two possible truncated proteins arising from these alternatively spliced transcripts: one comprising the first 11 amino acids of the N-terminal region with a loss of all Greek key motifs, and another comprising 90 amino acids encoded by exons 5 and 6, initiated from an alternative start codon in exon 5, and loss of Greek key motifs 1 & 2. Conclusion: The precise skipping of exons 2 to 4, consistent with canonical splicing signals (5-prime-GU...AG-3-prime), in the absence of genomic alterations, suggests the presence of alternatively spliced (AS) CRYBA1 transcripts in human lenses. This is the first report documenting AS-CRYBA1 transcripts in association with childhood cataracts with PFV and PPCD.

Early glaucoma detection and treatment are critical to prevent irreversible blindness. Glaucoma polygenic risk scores (PRS) offer an effective approach for stratifying disease risk and are increasingly available in clinical practice. However, the psychosocial impact of receiving glaucoma PRS results is currently unknown. As such, this study investigated short-term psychosocial outcomes of disclosing glaucoma PRS to individuals over 50 years from the general population. Individuals from the bottom 10%, middle 45 to 55%, and top 10% of PRS scores were invited to receive their results and complete surveys before and 2 weeks after receiving results to assess anxiety, test-related distress, decisional regret, recall and understanding. Of invited participants, 51.7% (136/263) enrolled with 133 completing both surveys. Two weeks after disclosure, PRS recall was high (78.2%), although PRS knowledge remained limited. Privacy concerns were moderate, not differing across PRS groups (X^2 = 4.17, p = .124). Small reductions in glaucoma-related anxiety (z = -2.93, p = .003), generalised anxiety (z = -3.75, p < .001) and stress (z = -2.49, p = .013) were observed following disclosure. While scores remained within normal ranges, higher glaucoma-related anxiety (t = -2.36, p = .020), higher negative emotions (X^2 = 20.80, p < .001), and lower positive experience (F = 5.70, p = .004) were seen for high-risk participants compared to lower-risk participants. Decisional regret was low and did not differ across PRS groups (X^2 = 0.28, p = .869). These findings support the psychosocial safety of glaucoma PRS testing while highlighting the need for improved education and longer-term follow-up to support clinical implementation.

PurposeOculocutaneous albinism type 1 (OCA1) is an inherited disorder caused by tyrosinase (TYR) gene mutations. Affected individuals experience visual impairment and severe photosensitivity from ocular hypomelanosis, with no current treatments. We evaluated the safety and efficacy of a TYR-encoding adeno-associated virus (AAV) vector in healthy rhesus macaques as a potential OCA1 treatment. MethodsA novel AAV2-based capsid (ATX002) was packaged with the human VMD2 promoter and TYR (hTYR) fused with mGreenLantern (mGL). Two adult rhesus macaques were injected with ATX002-hVMD2-hTYR-mGL subretinally (OD) and intravitreally (OS). Safety and efficacy were assessed via comprehensive ophthalmic examination, fundus photography, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinography at baseline and defined timepoints up to 12 weeks post-injection, followed by post-mortem immunohistochemistry (IHC). ResultsBoth subretinal doses induced localized hypermelanosis by 3 weeks post-injection, which persisted through the study endpoint and was accompanied by measurable thickening of the retinal pigment epithelium (RPE) on SD-OCT. Histological IHC confirmed successful RPE transduction via robust mGL fluorescence, corroborating in vivo findings by revealing localized RPE hyperplasia and transgene-expressing cells adjacent to regions of de novo hypermelanosis. Intravitreal delivery did not induce any changes to the RPE. Transient uveitis was observed but successfully managed with anti-inflammatory treatment. ConclusionsSubretinal AAV-TYR delivery is a safe and effective approach with the potential to induce RPE pigmentation. These findings support the use of AAV-TYR gene therapy for OCA1, demonstrating efficacy and a manageable safety profile in a large-animal model, and provide a critical bridge toward human clinical translation.

Purpose: Animal models of myopia typically induce monocular refractive shifts via form deprivation (FD) or lens-induced myopia (LIM), modeling susceptibility to myopia, but with potentially limited applicability to childhood myopia. Here we describe a novel, genetically diverse mouse model of naturally occurring refractive error (NORE) with three distinct refractive phenotypes: hyperopic, myopic, and intermediate. Methods: C57BL/6J mice were mated to 129S2/SvPasCrl mice to create F1 or F2 offspring. Refractive errors in male and female F1 (N=21) and F2 (N=101) mice were assessed on postnatal days (P) 28 and 42 using photorefractometry. In a subset of mice (N=30 - 40), corneal radius of curvature, axial ocular dimensions, retinal and visual function were assessed. Results: F2 mice were classified as NORE with either hyperopic (RE [&ge;] 0 diopters (D) at P28 and P42), myopic (RE<0D at P28 and P42) or intermediate (RE<0D at P28 and RE [&ge;] 0D at P42) refractions based on individual trajectories. All ocular parameters changed with age, with significantly slower growth in axial length and vitreous chamber depth in the intermediate versus myopic mice (p<0.05). Lens thickness was smaller in the myopic group at P28. Differences in refraction were not attributed to variances in retinal function or dopamine signaling. Conclusions: NORE mice represent a novel, genetically diverse wild-type mouse model that, unlike traditional models, does not require interventions such as FD or LIM to induce myopia. NORE mice provide a valuable tool for future investigations of genetic and environmental mechanisms and targeted therapeutic strategies for refractive errors.

Col8a2Q455K/Q455K (Q455K) mice exhibit features of early-onset Fuchs endothelial corneal dystrophy (FECD), including decreased endothelial cell density (ECD) and guttae formation. Within the context of these clinical features, this study longitudinally evaluates ferroptosis in Q455K and wild-type (WT) mice using in vivo imaging, PCR and immunohistochemistry. Fifty-six Q455K and 56 WT mice were evaluated from 3 to 24 months of age with in vivo confocal microscopy; ECD and guttae were measured. Ferroptosis marker expression was determined with PCR and immunohistochemistry (IHC). Data were analyzed using two-way ANOVA with Tukeys post hoc test, Chi-square test and a paired t-test. The ECD significantly decreased in both groups from 3 to 24 months of age, but more markedly in Q455K (2285-/+317 to 1012-/+58 cells/mmSquare) versus WT mice (2714-/+139 to 2057-/+149 cells/mmSquare, P<0.0001). Guttae were observed exclusively in Q455K mice beginning at 3 months of age and increased over time (P=0.0003). The Q455K mice demonstrate guttae at the vertices of corneal endothelial cells rather than their centers (74.3% vs. 25.7%P<0.001). Expression of ferroptosis-related genes (Tfrc, Slc40a1, Ftl1, Gpx4) were significantly increased in the Q455K versus WT mice (P<0.05). Furthermore, corresponding protein expression (transferrin receptor 1, ferroportin, ferritin and glutathione peroxidase 4) was significantly elevated adjacent to guttae in Q455K versus WT mice (P<0.05). These findings implicate guttae in the initiation of ferroptosis as it relates to the pathophysiology of FECD and provide an optimal window for testing novel FECD therapies using this murine model, particularly those that target ferroptosis.

Photoreceptor outer segments are sensory cilia whose maintenance depends on a balance between basal disc renewal and tip shedding, controlled by intraflagellar transport and axonemal microtubule organization. Microtubule plus-end proteins regulate microtubule dynamics and are strong candidates for roles in this process. In this study, mCherry-tagged EB1, EB3, and DCX were overexpressed in zebrafish (Danio rerio) cone photoreceptors under a cone-specific promoter. Eyes were examined at 5 and 10 dpf, and eyecup depth, diameter, and cone photoreceptor area were quantified relative to uninjected controls. At 5 dpf, all three constructs produced eyes indistinguishable from those of controls. By 10 dpf, all three constructs significantly increased eye cup depth and cone photoreceptor area. EB1 and DCX also significantly increased eye cup diameter. EB1 and, more severely, EB3 also caused retinal holes, mainly in the retinal pigment epithelium and at the outer nuclear/outer plexiform layer, along with misshapen cells near the inner plexiform layer. DXC did not cause retinal holes, but, like EB1 and EB3, produced enlarged, bulbous cone outer segments. The results show that overexpression of any of the three +TIPs results in a similar eye and photoreceptor overgrowth phenotype, while also producing construct-specific defects: EB1 and EB3 disrupt the broader retinal architecture, whereas DCX produces enlarged eyes. The shared outer segment hypertrophy suggests an imbalance between cargo delivery at the basal end and shedding of the distal tips. The organomegaly may reflect altered progenitor signaling in the ciliary marginal zone.

Retinopathy of prematurity (ROP) is caused by hyperoxic exposure of prematurely born infants. The mouse model of oxygen-induced retinopathy (OIR) recapitulates pathological features of both phase I and phase II ROP. We here looked at the retinal proteins that change in response to hyperoxia in phase I of the mouse model of OIR. Using tandem mass tag labeled proteomics, we found several differentially expressed proteins (DEPs) in phase I of OIR. Of all the DEPs, we investigated the role of previously unknown protein NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2). NDUFA4L2 protein and its paralog NDUFA4 are both mitochondrial complex I proteins; however, here we demonstrate that NDUFA4L2 changes in both phases of OIR, with no changes in its paralog NDUFA4, implying its unique function in pathophysiology of the disease. We demonstrate that NDUFA4L2 is an oxygen-sensitive protein and regulates retinal endothelial cell migration by rescuing isocitrate dehydrogenase flux impaired by hyperoxia in phase I of OIR.

Purpose: Photoreceptor Kv2.1/Kv8.2 voltage-gated potassium channels carry an outward potassium current, helping to set the resting membrane potential and to shape dim light responses. Because potassium flux in the outer retina influences extracellular osmolarity and fluid distribution, we hypothesized that Kv2.1/Kv8.2 channels also contribute to fluid homeostasis in this region of the retina. Methods: OCT imaging was performed in Kv8.2 heterozygous (Het) and knockout (KO) mice aged 4-7 weeks under dark- and light-adapted conditions. Light-dark differences in the distance between the external limiting membrane (ELM) and retinal pigment epithelium (RPE) ({Delta}ELM-RPE) were calculated to quantify light-evoked expansion of the subretinal space (SRS). As a secondary outcome, outer nuclear layer (ONL) thickness was also measured under both lighting conditions. Retinal gene expression was assessed by RNA-seq and droplet digital RT-PCR. Retinal protein expression was determined by western blotting and immunolabeling. Results: {Delta}ELM-RPE was significantly reduced in Kv8.2 KO mice compared with Het controls, indicating reduced SRS hydration. ONL thickness exhibited a small but significant light-dark change that was different between genotypes. Transcriptomic analyses revealed upregulation of osmosensitive genes, including osmolyte transporters and aquaporins. AQP1 protein expression in photoreceptors increased. Conclusions: These findings reveal a previously unrecognized role for Kv2.1/Kv8.2 channels in outer retinal fluid homeostasis and support a model in which photoreceptor potassium efflux contributes to osmotic water movement into the subretinal space.

Purpose: To assess the feasibility of quantitative fundus autofluorescence (FAF) measurement in early age-related macular degeneration (AMD) using the freely available ImageJ software, to characterize signal intensity across FAF patterns, and to evaluate interobserver reproducibility in pattern classification. Methods: Single-center, non-blinded, retrospective, consecutive-case analytical study. FAF images acquired with Spectralis OCT+HRA (Heidelberg Engineering) from patients with early dry AMD seen at a tertiary referral center between January 2010 and September 2016 were analyzed. A standardized 300x300-pixel region of interest (ROI) centered on the fovea was evaluated in ImageJ v2.0.0-rc54/1.51h (Fiji distribution). Mean, minimum, and maximum autofluorescence (AF) pixel intensity were recorded. Each image was independently classified according to the Bindewald classification system by two graders; a third senior grader adjudicated discordances. Cohen's kappa (k) was used to assess interobserver agreement. Results: Of 423 patients with available FAF studies, 107 had dry AMD; 45 met quality and diagnostic criteria for early AMD and were included in the quantitative analysis. Mean age was 73.47 +/- 8.1 years; 62.2% were female. Mean FAF intensity was 120.26 (range 74.76-160.79); mean minimum was 32.07 (range 3-63) and mean maximum was 205.80 (range 125-255). Seven of eight Bindewald patterns were identified; the stippled pattern was absent. The most frequent pattern was minimal changes (31.1%), followed by increased focal (24.4%) and patchy (15.6%). Reticular pattern showed the highest mean AF (143.8), while lace pattern showed the lowest (88.4). Interobserver agreement for Bindewald pattern classification was almost perfect (k = 0.969; 95% CI, 0.908-1.000; p < 0.001). Agreement for lesion extent was moderate (k = 0.531) and for foveal involvement was substantial (k = 0.622). Conclusions: Quantitative FAF evaluation of early AMD using ImageJ is feasible and reproducible. ImageJ represents a cost-free alternative for multimodal retinal image analysis, with potential for automated screening applications in resource-limited settings. Keywords: age-related macular degeneration; fundus autofluorescence; ImageJ; quantitative autofluorescence; image analysis; Bindewald classification; interobserver agreement

The morphogenesis of the optic cup provides a robust system for studying how two apposed epithelial monolayers with distinct properties fold and stretch in a coordinated manner to form the primordial eye. While much research has been conducted on the temporal dynamics of retinal neuroepithelium invagination, the spatial organization and stretching of the retinal pigment epithelium has received less attention. The fish species Astyanax mexicanus offers a unique model to examine the mechanisms of optic tissue morphogenesis through a comparative lens, as it exhibits natural variation in eye development between its river-dwelling and cave-adapted morphs. Using quantitative 3D imaging of optic cups from both morphs, we found that RPE morphogenesis involves transient, graded, and anisotropic cell stretching that patterns the epithelium during optic cup shaping. Analyses of RPE nuclear spacing and cell morphology showed that tissue stretching gradually increases along the proximo-distal axis, suggesting maximal tension in the elongated distal RPE cells aligned along the optic cup meridians. Furthermore, nuclear volumes and apical surface areas of RPE cells scaled spatially along the same axis, independently of endoreplication. In the cavefish natural mutant, RPE expansion was delayed by over six hours and proximal stretching exhibited altered isotropy, indicative of disrupted temporal coordination and suggesting modified mechanical constraints. These results demonstrate that RPE morphogenesis is a highly heterogeneous process from a spatiotemporal perspective, offering new insights into the study of the biomechanical principles of eye development in vertebrates. Summary statementThis study reveals the emergence of cell morphology gradients within the retinal pigment epithelium during morphogenesis of the eye in two distinct populations of the same species of fish.

Abstract Purpose: To expand the clinical and mechanistic understanding of the +57C>T seed-region mutation in miR-184 causing EDICT (endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning) syndrome. Design: Cross-sectional analysis and laboratory confirmation Participants: 18 members of a four-generation family with known +57C>T miR-184 status Methods: We used optical biometry, corneal topography, and medical history to characterize the clinical phenotype. Carrier effects on ocular biometric measurements were estimated using polygenic linear mixed models incorporating a pedigree-derived kinship matrix, adjusted for age and sex. Patient-derived and control induced pluripotent stem cells (iPSCs) were generated and differentiated into corneal endothelial cells (CECs). Main Outcome Measures: Axial length, keratometry (in diopters), white-to-white corneal diameter, topography mapping, central and peripheral corneal thickness, and history of retinal detachment or corneal transplant were compared between mutation carriers and noncarriers, adjusting for age and sex. Cellular analysis was conducted with immunostaining (ZO-1, ATP1A1), morphometric quantification, qRT-PCR of endothelial markers, and transendothelial electrical resistance (TEER). Results: 10 of 18 family members were heterozygous for +57C>T, with retinal detachment occurring in 5/10 affected individuals compared to 0/8 unaffected individuals (p=0.04). Affected eyes had 2.2 mm shorter axial length (p=0.02), 9.3 D steeper mean keratometry (p=0.004), 1.6 mm smaller horizontal corneal diameter (p=0.0001), and 139-micrometer thinner central corneas (p=0.003). Mutant iPSC-derived CECs were associated with irregular borders, increased cell and nucleus area, widened intercellular gaps, disrupted ATP1A1 membrane localization, and reduced barrier function on TEER (all p<0.05). Gene expression analysis showed downregulation of COL4A1, COL4A3, and AQP1 with upregulation of COL8A1. Conclusions: The miR-184 +57C>T mutation produces a broad ocular phenotype that includes smaller, thinner corneas and microphthalmia. Mechanistically, it disrupts CEC junctional integrity, extracellular matrix and pump-related genes, supporting a role for miR-184 in coordinated anterior-posterior eye morphogenesis.

Glaucoma is a group of eye diseases that affects 4 million people in the US and is one of the leading causes of vision loss due to damage to the eyes optic nerve (ON) which is composed of axons from retinal ganglion cells (RGCs) that transmit visual information to the brain. Injury to the ON often triggers RGC death and subsequent loss of visual function. Despite its increasing prevalence worldwide, effective therapies for glaucoma remain elusive. Notably, the thirteen-lined ground squirrel (TLGS) exhibits intrinsic neuroprotection during hibernation; however, reproducing this protective state pharmacologically has proven challenging. To elucidate the metabolic mechanisms underlying this resilience, we conducted untargeted metabolomic analyses on TLGS retinas at 6 hours, 3 days, and 7 days following ON crush. Retinas from awake and hibernating animals were compared to identify temporal and state-dependent metabolic signatures. Distinct metabolomic profiles were observed in hibernating animals relative to their awake counterparts. Pathway analyses revealed coordinated regulation of amino acid, lipid, and purine metabolism that likely contributes to hibernation-induced resilience. Furthermore, our findings indicate that hibernating TLGS retinas increase exosome biogenesis, prompting in vitro validation using TLGS-derived exosomes, which demonstrated robust neuroprotective and anti-inflammatory effects. Proteomic and transcriptomic characterization of exosomal cargo identified conserved miRNAs, mRNAs, and proteins implicated in redox balance, cytoskeletal stabilization, and stress-response regulation. Collectively, these data support the hypothesis that metabolic reprogramming and exosome-mediated intercellular signaling underlie hibernation-associated neuroprotection. Modulating these pathways may provide a blueprint for novel therapeutic strategies to mitigate neurodegeneration and promote recovery following optic nerve injury. Graphical AbstractIllustration depicting state-dependent metabolic responses to optic nerve crush (ONC) injury in Thirteen-lined Ground Squirrels (TLGS). In Awake animals, injury triggers enhanced ATP production through the TCA cycle, leading to excessive reactive oxygen species (ROS) generation and subsequent retinal ganglion cell (RGC) death. In contrast, Hibernating animals shift toward lipid metabolism and utilize ATP for the biosynthesis of ceramides and sphingolipids, promoting membrane integrity and exosomal signaling. Additionally, a range of metabolites associated with hibernation-linked neuroprotection are elevated, contributing to enhanced RGC survival. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/733742v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@1ef3a7eorg.highwire.dtl.DTLVardef@e9293dorg.highwire.dtl.DTLVardef@192729forg.highwire.dtl.DTLVardef@1a32cb5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Purpose: To investigate corneal subbasal nerve plexus alterations using in vivo corneal confocal microscopy (IVCM) in patients with Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) and Spastic Paraplegia Type 7 (SPG7). Methods: This cross-sectional pilot study included eight ARSACS patients, five SPG7 patients, and twenty age- and sex-matched healthy controls. All participants underwent neurological and ophthalmological examination followed by central corneal imaging using IVCM. Quantitative corneal nerve parameters were analyzed with automated software, and correlations with clinical severity scales were assessed. Results: The mean age was 34.2 +/- 3.4 years in controls, 34.5 +/- 0.7 years in the ARSACS group, and 38.2 +/- 3.5 years in the SPG7 group. Corneal nerve branch density (CNBD) and corneal nerve total branch density (CTBD) were significantly lower in ARSACS and SPG7 patients compared with healthy controls. CNFD, CNFL, CNFA, CNFW, and CNFrD were lower in ARSACS and SPG7 patients compared with healthy controls; however, these differences did not reach statistical significance. No statistically significant differences in IVCM parameters were detected between ARSACS and SPG7 patients. Spearman correlation analysis did not show significant correlations between corneal nerve parameters and FARS, SARA, ADL scores, or disease duration. Conclusion: IVCM revealed reduced corneal nerve branching parameters in patients with ARSACS and SPG7. These findings indicate involvement of the corneal subbasal nerve plexus and support the potential role of corneal confocal microscopy as a non-invasive ocular imaging modality for evaluating peripheral neural alterations in hereditary spastic ataxias.

The ultimate cause of blindness in glaucoma is the death of retinal ganglion cells, and understanding the mechanism behind retinal ganglion cell loss during glaucoma could lead to the development of novel treatments for glaucoma. Endothelin-1 has been shown to mediate retinal ganglion cell death during glaucoma through impairment of mitochondrial function. Retinal ganglion cells are highly metabolically active, and susceptible to oxidative damage and decreased respiratory capacity. Mitophagy is the process whereby damaged mitochondria are degraded to prevent further propagation of oxidative damage. The current study evaluates the effect of endothelin-1 on mitophagy in retinal ganglion cells. Electron microscopy revealed endothelin-1 administration lead to a decrease in healthy mitochondria in the optic nerve. The MitoQC mouse was used to evalute mitophagy in response to endothelin-1, along with immunohistochemical analysis of mitophagy proteins. Mitophagy follows different trends in the optic nerve and retinal ganglion cell bodies following endothelin-1 administration, mitophagy was increased in the optic nerve but decreased in the retina following endothelin administration. With elevation of intraocular pressure, mitophagy was increased in the retina but decreased in the optic nerve. In retinal ganglion cells, parkin expression and activation was unchanged 24 hours after endothelin-1 administration, but was decreased 72 hours following endothelin-1 administration. Taken together, these results suggest that endothelin-1 impacts mitophagy through parkin-independent mechanisms in retinal ganglion cell bodies, and the ganglion cell bodies and optic nerve appear to have different responses to endothelin-1.

The multiligand endocytic receptor, megalin (LRP2), is expressed in the retinal pigment epithelium (RPE) and patients lacking the receptor develop high myopia. Despite its established role in retinal development, the contribution of megalin to retinal homeostasis in the normally developed/mature eye remains poorly understood. Here, we investigated megalin function using an inducible knockout mouse (KO) model and human iPSC-derived RPE with megalin knockdown (KD) to distinguish post-developmental homeostatic functions from developmental effects. In vivo, megalin ablation caused progressive retinal degeneration and visual impairment, with morphological abnormalities in the RPE but no changes in myopia-associated ocular phenotypes including axial length and intraocular pressure. Proteomic profiling of megalin-KO RPE revealed reduction of autophagy-related proteins. In line with this, megalin deficiency was associated with accumulation of pro-cathepsin D, and perturbed rhodopsin turnover. This was supported in vitro, where trafficking of photoreceptor outer segment (POS) containing phagosomes to lysosomes was reduced, suggesting disturbed phagosome maturation. Megalin KD did not measurably impair initial uptake of POS discs, but delayed rhodopsin degradation, indicating defective post-ingestion processing. Together, these findings establish megalin as a key regulator of retinal homeostasis in the mature eye by controlling phagosome-lysosome fusion in the RPE and suggest that megalin dysfunction contributes to slowly progressive retinal degeneration. This positions megalin as a potential therapeutic target in lysosomal degenerative diseases in the retina.

Subretinal photovoltaic implants provide central vision to patients blinded by atrophic age-related macular degeneration, with acuity limited by their 100-{micro}m pixels. Higher resolution requires smaller pixels incorporating three-dimensional electrodes, which can be fabricated by gold electroplating. However, the retinal response to exposed gold remains poorly characterized. Here, we evaluated gold biocompatibility on subretinal implants in Royal College of Surgeons rats and compared it with platinum- and titanium-coated surfaces. Although in-vivo optical coherence tomography revealed no overt structural disruption, gold implants induced cellular-scale anomalies, including abnormal morphology of rod bipolar cells, microglial accumulation near the implant, and increased cell death within days after implantation. These effects occurred across flat, pillar, and honeycomb geometries, indicating a material-rather than geometry-dependent response. By contrast, platinum- and titanium-coated implants showed substantially lower loss and morphological disruption of rod bipolar cells, together with markedly reduced microglial activation. These findings indicate that exposed gold surfaces can induce acute retinal inflammation and neuronal loss, whereas conformal platinum or titanium coatings substantially improve biocompatibility. Such coatings enable the development of three-dimensional subretinal prostheses with smaller pixels for improved visual resolution.

Pathogenic mutations in the ABCA4 gene cause several inherited retinal diseases, particularly Stargardt disease (STGD1). However, many missense variants remain classified as variants of uncertain significance (VUS) due to inconclusive evidence regarding their pathogenic impact. The missense VUS span across all the domains of ABCA4, with the majority found in the larger extracellular domains (ECDs). The largest uncharacterized region of ABCA4 is located in ECD1, where limited structural information and inconsistent computational predictions hinder clinical interpretation of missense VUS in this region. Here, we integrated in silico analysis with in vitro functional assays to evaluate the pathogenicity of VUS in this region and improve their diagnostic classification. Missense VUS in the ECD1 uncharacterized region were curated from ClinVar. Six multiallelic sites were identified in the uncharacterized region and 13 missense VUS on these multiallelic sites were characterized using the integrated analysis. In the in silico platform, the pathogenicity of the VUS were predicted using multiple algorithms, and the structural effects of the variants were analyzed compared to the wild type. Recombinant variants were expressed in virus-like particles (VLPs), and protein expression, membrane localization, and ATPase activity were quantified relative to wild type to identify potential disease-causing variants. From the integrated analysis, variants with pronounced structural destabilization, impaired membrane trafficking, and reduced or absent N-retinylidene-phosphatidylethanolamine (NRPE) substrate stimulated ATPase activities were identified as potentially deleterious. Notably, VUS at p.H193P and p.I214N showed loss of function, with p.I214N reflecting selectively impaired membrane targeting and p.H193P reflecting combined expression and trafficking defects. Additionally, NRPE-stimulated ATPase activities were impaired in VUS, p.V195L, p.V195I, p.D197H, p.I214F and p.N269S. Overall structural destabilization interfered with the NRPE-stimulated ATPase activities of p.N269S, while the lack of NRPE-stimulated ATPase activities of p.D197H, p.V195L, p.V195I and p.I214F are thought to be due to impaired NRPE interactions with ABCA4. All the VUS at p.R140, p.H193Y, p.D197N and p.N269H showed both the basal and NRPE-stimulated ATPase activities but less than that of the wild type, displaying a mild functional deficit. Together, these findings demonstrated that certain VUS within the unresolved ECD1 region disrupt ABCA4 stability and function, supporting their contribution to disease pathogenesis. This integrative approach highlights key residues likely to be pathogenic and advances the interpretation of VUS in inherited retinal disorders.

Objective: To investigate the performance of anterior segment (AS) OCT quantitative imaging of anterior chamber inflammation in uveitis patients with diverse demographics. Design: Prospective cross-sectional study. Participants: 144 adult patients managed at a tertiary care service in the UK Methods: Repeated swept-source ASOCT imaging was performed pre- and post-pupil dilation (i.e. 4 scan sets). Inflammation was quantified using a validated human in the loop automated image analysis pipeline, Minuscule Cell Detection (MCD), which identified and counted putative inflammatory cells on AS-OCT. Main Outcome Measures: Test-retest variability of ASOCT and diagnostic accuracy of various ASOCT derived measurands (minimum, maximum, median counts per cross sectional image, and total counts across volume image sets per eye or MINCC, MAXCC, MEDCC and TOTCC) versus Standardization of Uveitis Nomenclature (SUN) grading system as assessed by a uveitis specialist. Results: A total of 281 eyes were included in the analysis. Median age was 48 years (IQR 36 to 64). Strong test-retest measurand reliability was demonstrated, with a 95% tolerance interval ratio 0.3 to 3.0. The best diagnostic performances for SUN activity were observed with the MINCC threshold of 3 particles (negative predictive value for clinical activity of 89.8%, 95% CI 83.0 to 94.1). Associations between ASOCT measurands and patient age (adjusted coefficient 7.5 additional particles, 95% CI 0.5 to 14.6, p<0.04 for age over 60 years versus under 44), and pigment load (52.8, 11.8 to 92.9, p<0.01 in eyes with AC pigment versus without) were noted. Conclusions: ASOCT assessment of anterior chamber inflammation in uveitis meets current recommendations for quantitative imaging biomarkers, demonstrating strong repeatability, linearity with clinical assessment scores and stability with pupil dilation and patient characteristics of ethnicity and lens status. The absence of variability in diagnostic indices across derived measurands suggests similar performance across different acquisition protocols. Further longitudinal cross-platform studies are needed to determine limitations of use.

Cell replacement represents a potential treatment modality for retinal disorders characterized by photoreceptor loss. However, photoreceptor replacement approaches have not been clinically established. To take this forward, the main goal of this study was to systematically compare human photoreceptors of different ages and identify those that enable functional integration into the degenerative retina. Donor cells were isolated from iPSC-derived retinal organoids generated by a GMP-compliant protocol at differentiation days 120, 150, or 200 and transplanted subretinally into cone photoreceptor function loss 1 (Cpfl1) recipients, an inherited mouse model of cone degeneration. While younger photoreceptors showed slightly improved transplantation outcomes, donor photoreceptors of all culture stages displayed long-term survival, cone identity, structural integration into the host retina, and tight interactions with host Mueller glia, including formation of a continuous outer limiting membrane. Transplanted photoreceptors showed signs of advanced maturation, including correct polarization with generation of apical inner- and outer segments, while basal synapses were formed with host bipolar cells. Electrophysiological assessment of host retinal ganglion cells revealed light-evoked responses in transplant-containing regions, providing evidence for functional incorporation of human photoreceptors into the mouse neuro-retinal circuitry. Thus, GMP-compliant human iPSC-derived photoreceptors are stable over a wide range of differentiation stages and constitute a robust cell source for retinal transplantation and functional repair. The findings provide important prerequisites for the development of standardized procedures towards clinical translation of photoreceptor replacement in the retina.

Reactive nitrogen species (RNS), particularly peroxynitrite generated from the reaction of superoxide and nitric oxide, are implicated in thermally-induced oxidative stress but remain difficult to resolve in live coral cells. We optimized fluorescent dye strategies to directly quantify superoxide, nitric oxide, and peroxynitrite production in thermally stressed Pocillopora acuta cell suspensions. Thermal stress was associated with an increase in intracellular peroxynitrite concentration, but not in its precursors, nitric oxide and superoxide, highlighting challenges with the application of fluorescent probes and their controls to live coral cells. Compounds developed for mammalian systems often translate poorly to non-model systems such as corals: strong endogenous fluorescence and multiple membrane barriers within the coral symbiocyte, for instance, limited the function of the nitric oxide probe, DAF-2DA. Despite these limitations, the detection of peroxynitrite in live, thermally stressed P. acuta cells represents a step forward in understanding the mechanism of coral bleaching. We also outline strategies for improving the performance of commercial dyes in non-model systems, including media optimization with EDTA treatment to preserve both cell viability and probe performance.