Investigative Opthalmology & Visual Science

PurposeHealthy corneas resist colonization by virtually all microbes yet contact lens wear can predispose the cornea to sight-threatening infection with Pseudomonas aeruginosa. Here, we explored how lens wear changes corneal epithelium transcriptional responses to P. aeruginosa and its impact on bacterial gene expression. MethodsMale and female C57BL/6J mice were fitted with a contact lens on one eye for 24 h. After lens removal, corneas were immediately challenged for 4 h with P. aeruginosa. A separate group of naive mice were similarly challenged with bacteria. Bacteria-challenged eyes were compared to uninoculated naive controls as was lens wear alone. Total RNA-sequencing determined corneal epithelium and bacterial gene expression. ResultsPrior lens wear profoundly altered the corneal response to P. aeruginosa, including: upregulated pattern-recognition receptors (tlr3, nod1), downregulated lectin pathway of complement activation (masp1), amplified upregulation of tcf7, gpr55, ifi205, wfdc2 (immune defense) and further suppression of efemp1 (corneal stromal integrity). Without lens wear, P. aeruginosa upregulated mitochondrial and ubiquinone metabolism genes. Lens wear alone upregulated axl, grn, tcf7, gpr55 (immune defense) and downregulated Ca2+-dependent genes necab1, snx31 and npr3. P. aeruginosa exposure to prior lens wearing vs. naive corneas upregulated bacterial genes of virulence (popD), its regulation (rsmY, PA1226) and antimicrobial resistance (arnB, oprR). ConclusionPrior lens wear impacts corneal epithelium gene expression altering its responses to P. aeruginosa and how P. aeruginosa responds to it favoring virulence, survival and adaptation. Impacted genes and associated networks provide avenues for research to better understand infection pathogenesis.

The primate retina has evolved regional specialisations for specific visual functions. The macula is specialised towards high acuity vision and is an area that contains an increased density of cone photoreceptors and signal processing neurons. Different regions in the retina display unique susceptibility to pathology, with many retinal diseases primarily affecting the macula. To better understand the properties of different retinal areas we conducted an untargeted metabolomics analysis on full thickness punches from three different regions (macula, temporal peri-macula and periphery) of primate retina. Half of all metabolites identified showed differential abundance in at least one comparison between the three regions. The unique metabolic phenotype of different retinal regions is likely due to the differential distribution of different cell types in these regions reflecting the specific metabolic requirements of each cell type. Furthermore, mapping metabolomics results from macula-specific eye diseases onto the region-specific distributions of healthy primate retina revealed differential abundance defining systemic metabolic dysregulations that were region specific, highlighting how our results may help to better understand the pathobiology of retinal diseases with region specificity.

ObjectiveMitochondrial tricarboxylic acid (TCA) cycle is central to energy production and redox balance in the eye, which must sustain high metabolic activity to support vision. Retinal neurons, the retinal pigment epithelium (RPE), cornea, and lens each have distinct physiological roles and metabolic demands, yet the absolute concentrations of key TCA intermediates and their variation by tissue, sex, and time of day are not well-defined. MethodsTargeted gas chromatography-mass spectrometry was employed to quantify the absolute concentrations of TCA cycle metabolites in mouse ocular tissues collected at 10 AM and 2 PM to capture diurnal variations. Key metabolite ratios were subsequently calculated to provide insight into TCA cycle dynamics across eye tissues. ResultsThe retina showed the highest concentrations of TCA metabolites among all ocular tissues, particularly succinate, citrate, and malate, consistent with its high energy demands. The RPE/choroid demonstrated well-balanced intermediates with the highest -ketoglutarate (-KG)/Isocitrate ratio, reflecting its efficient mitochondrial oxidation and reductive carboxylation. Corneal metabolism was featured by dominant malate, especially in females, suggesting a metabolic adaptation for redox regulation and oxidative stress defense. The lens had uniformly low metabolite levels except for succinate, indicating minimal mitochondrial activity under physiologically low oxygen conditions. Notably, both the cornea and lens showed significant sex-dependent and diurnal variations in TCA cycle intermediates. ConclusionThis study demonstrates distinct tissue-specific mitochondrial metabolism in the eye, reflecting the unique functional and biochemical demands of each tissue. These metabolic signatures may underlie their susceptibility to mitochondrial dysfunction in various ocular diseases.

ObjectiveTo determine the profile of inflammation-related proteins and complement system factors in serum of CRB1-associated inherited retinal dystrophies (CRB1-IRDs). DesignA case-control study. Subjects, Participants, and/or ControlsA cohort of 30 Dutch CRB1-IRD patients and 29 Dutch healthy controls (HC) (Cohort I), and a second cohort of 123 CRB1-IRD patients from 14 countries and 1292 controls (Cohort II) were used in this study. MethodsQuantitative 370-plex targeted proteomics in blood plasma and genotyping of the single nucleotide variant (SNV) rs7535263 in the CFH gene. Main Outcome MeasuresPlasma concentrations of inflammation-related proteins and the genotype of the SNV rs7535263. ResultsCRB1-IRD patients showed increased plasma levels of complement system and coagulation cascade proteins compared to healthy controls. Complement Factor I [CFI], Serpin Family D1 [SERPIND1], and Complement Factor H [CFH] were significantly elevated (q<0.05, adjusted for age and sex), which correlated (Pearsons correlation coefficient >0.6) with higher levels of plasma Complement Component 3 [C3] (q = 0.064). The most enriched pathway in patients was the "Complement cascade" (R-HSA-166658, Padj = P = 3.03 x 10-15). An analysis of the genotype of CFH variant rs7535263, which is in close physical proximity to the CRB1 gene and is associated with other retinal conditions by influencing plasma complement levels, revealed significantly skewed allele distribution specifically in Dutch patients (A allele of rs7535263, odds ratio (OR) [95%CI = 2.85 [1.35-6.02], P = 6.19 x 10-3), but not in a global case-control cohort (P = 0.12). However, CRB1 missense variants that are common in patients display strong linkage disequilibrium (LD) with rs7535263 in CFH in the UK Biobank (D = 0.97 for p.(Cys948Tyr); D = 1.0 for p.(Arg764Cys)), indicating that genetic linkage may influence plasma complement factor levels in CRB1-IRD patients. After accounting for the CFH genotype in the proteomic analyses, we also detected significantly elevated plasma levels of Complement Factor H Related 2 [CFHR2] in CRB1-IRD patients (q = 0.04). ConclusionsCRB1-IRDs are characterized by changes in plasma levels of complement factors and proteins of the innate immune system, which is influenced by common functional variants in the CFH-CFHR locus. This indicates that innate immunity is implicated in CRB1-IRDs.

PurposeTo compare the plasma metabolic profile of patients with a CRB1-associated inherited retinal degeneration (CRB1-IRD) with healthy controls (HCs). DesignA case-control study. MethodsPlasma concentration of 619 metabolites was measured with the MxP(R) Quant 500 Kit in 30 patients with a CRB1-IRD and 29 HCs. We fitted a linear regression model with adjustments for age and sex based on the concentration of metabolites in {micro}M ({micro}mol/L), or on the sums and ratios of metabolites, to determine differences between patients and controls. ResultsOver-representation of pathways among metabolites associated strongest to CRB1-IRDs (P < 0.05, n = 62) identified amino acid pathways (such as beta-alanine, histidine, and glycine/serine) and bile acid biosynthesis, driven by a decrease in deoxycholic acid derivatives produced by gut microbiota. Enrichment analysis of metabolic classes across the plasma metabolic profile further identified significant positive enrichment for lipid metabolites glycerophospholipids, cholesterol esters, and ceramides, and significant depletion for bile acid metabolites. Further investigation of the sums and ratios (i.e., metabolism indicators) ascertained a significant decrease in intestinal microbial-dependent secondary bile acid classes. ConclusionsLipid metabolic alterations and decreased microbiota-related secondary bile acid concentrations indicate significant alterations in gut metabolism in patients with a CRB1-IRD.

BackgroundExcessive oxidative stress and related chronic, sub-clinical inflammation is linked causally to the development and progression of degenerative diseases of the retina including diabetic retinopathy, age-related macular degeneration and glaucoma, leading causes of blindness worldwide. The above responses may be related directly to dysregulated retinal immunity and are potentiated by the combined actions of native retinal cells (e.g., retinal pigment epithelial (RPE) and microglial cells) and immune cells infiltrating from the periphery. Maintaining tight regulation of these cells such that effective control of pathogens is accomplished yet uncontrolled inflammation and consequent tissue damage is prevented is extremely important. However, the molecular mechanisms that control this delicate balance are poorly understood. We hypothesize that the hydroxycarboxylic acid receptor 2 (HCAR2/GPR109A) may play an important role. HCAR2/GPR109A has been shown to regulate immune cell responses that potentiate anti-inflammatory signaling upon its activation in various tissues as evidenced principally by suppressed pro-inflammatory cytokine secretion in various experimental model systems. We have demonstrated HCAR2/GPR109A expression in RPE, microglia and endothelial cells and, our in vitro studies support that the receptor elicits anti-inflammatory signaling in these cell types. However, the functional relevance of HCAR2/GPR109A expression and its activation in the retina of the living animal has not been demonstrated definitively. This is the purpose of the present study. MethodsRetinal function was evaluated temporally in wildtype (Hcar2/Gpr109a +/+, WT) and knockout (Hcar2/Gpr109 -/-, KO) mice via electroretinography (ERG). Fundoscopic imaging, spectral domain-optical coherence tomography (OCT), fluorescein angiography and post-mortem histological analyses were additionally performed to evaluate retinal health. Gene microarray, RT-qPCR studies, ingenuity analyses and proteome pathway mapping were performed to evaluate potential key differences in the molecular signatures of WT and KO mouse retinas. Leukostasis and flow cytometric assays were performed to demonstrate the in vivo impact of HCAR2/GPR109A expression and its therapeutic activation on pro-inflammatory immune cell trafficking in retina. ResultsLongitudinal studies revealed progressive anomalies in retinal morphology and function in HCAR2/GPR109A knockout mice that impacted the entire retina. Gene expression and protein interactome analyses revealed differences in gene and protein expression consistent with the increased immune reactivity and infiltration of bone-marrow derived immune cells detected in KO mouse retinas. Studies conducted in an acute model of retinal (endotoxin-induced) inflammation revealed that targeting the receptor via intraperitoneal administration of agonist, beta-hydroxybutyrate, limits immune cell activation, infiltration and related inflammation in WT retinas. ConclusionsThe present studies demonstrate a central role for HCAR2/GPR109A in regulating the complex interplay between resident retinal cells and peripheral immune cells and, the potential therapeutic utility that targeting the receptor holds with respect to preventing and treating inflammatory retinal diseases. HighlightsOxidative stress and inflammation are major causative factors in degenerative retinal diseases stemming from numerous causes (e.g., aging, diabetes, sickle cell). Thus, identifying new targets and developing strategies to counter these factors to prevent and treat retinal degeneration is important. The present in vivo study demonstrates convincingly the principal role of the hydroxycarboxylic acid receptor 2 (HCAR2/GPR109A) as a major regulator of retinal immune responses under normal conditions and therefore, as a target with extremely high potential for therapeutic modulation of these responses in retinal disease.

LC3b (Map1lc3b) plays an essential role in canonical autophagy and is one of several components of the autophagy machinery that mediates non-canonical autophagic functions. Phagosomes are often associated with lipidated LC3b, to pro-mote phagosome maturation in a process called LC3-associated phagocytosis (LAP). Specialized phagocytes such as mammary epithelial cells, retinal pigment epithelial (RPE) cells, and sertoli cells utilize LAP for optimal degradation of phagocytosed material, including debris. In the visual system, LAP is critical to maintain retinal function, lipid homeostasis and neuroprotection. In a mouse model of retinal lipid steatosis - mice lacking LC3b (LC3b-/-), we observed increased lipid deposition, metabolic dysregulation and enhanced inflammation. Herein we present a non-biased approach to determine if loss of LAP mediated processes modulate the expression of various genes related to metabolic homeostasis, lipid handling, and inflammation. A comparison of the RPE transcriptome of WT and LC3b-/- mice revealed 1533 DEGs, with ~73% upregulated and 27% down-regulated. Enriched gene ontology (GO) terms included inflammatory response (upregulated DEGs), fatty acid metabolism and vascular transport (downregulated DEGs). Gene set enrichment analysis (GSEA) identified 34 pathways; 28 were upregulated (dominated by inflammation/related pathways) and 6 were downregulated (dominated by metabolic pathways). Analysis of additional gene families identified significant differences for genes in the solute carrier family, RPE signature genes, and genes with potential role in age-related macular degeneration. These data indicate that loss of LC3b induces robust changes in the RPE transcriptome contributing to lipid dysregulation and metabolic imbalance, RPE atrophy, inflammation, and disease pathophysiology.

PurposeTo better understand the etiologic pathways in glaucoma, we aimed to identify pre-diagnostic plasma metabolites associated with glaucoma risk. MethodsIn a case-control study from the Nurses Health Study (NHS), NHSII and Health Professionals Follow-Up Study (HPFS), 599 incident primary open-angle glaucoma (POAG) cases (mean time between blood draw and diagnosis was 10.3 years) were 1:1 matched to 599 controls. Plasma metabolites were measured with LC-MS/MS at the Broad Institute (Cambridge, MA, USA); 367 metabolites from 17 metabolite classes passed quality control analyses. For comparison, in a cross-sectional study in the UK Biobank, 168 NMR metabolites (Nightingale, Finland; version 2020) were measured in serum samples from 2,238 prevalent glaucoma cases and 44,723 controls. Metabolites were probit-score transformed for normality; multiple logistic regression was used to identify metabolites associated with POAG in NHS/NHSII/HPFS and glaucoma in UK Biobank. In NHS/NHSII/HPFS, we also used Metabolite Set Enrichment Analysis to identify metabolite classes associated with POAG. All analyses adjusted for established glaucoma risk factors. False discovery rate (FDR) and number of effective tests (NEF) were used to adjust for multiple comparisons. ResultsNine metabolite classes were associated (FDR<0.05) with POAG in NHS/NHSII/HPFS: triglycerides, diglycerides, two lysophospholipids classes [lysophosphatidylcholines and lysophosphatidylethanolamines], and two phospholipid class [phosphatidylethanolamines and phosphatidylcholines] were positively associated, while cholesteryl esters, carnitines, and organic acids and derivatives were inversely associated with POAG risk; further adjustment for covariates minimally altered the results. These associations were particularly stronger for POAG with paracentral visual field loss. In the UK Biobank, notably, triglycerides and phospholipids (from which lysophospholipids are derived through hydrolysis), were confirmed to be associated (p<0.05) with higher glaucoma risk. Also, in the UK Biobank, the metabolites of tyrosine, glucose, and glutamine were positively associated (NEF<0.2) while 3-hydroxybutyrate, acetate, citrate, pyruvate, and lactate (the latter 4 being anionic organic acids) were inversely associated with glaucoma (NEF<0.05). ConclusionsHigher levels of glycerides (diglycerides and triglycerides) and phospholipids were adversely associated with glaucoma in both the NHS/NHSII/HPFS and the UK Biobank, suggesting that they play an important role in glaucoma pathogenesis. PRECISHigher glyceride and phospholipid levels in pre-diagnostic plasma was associated with glaucoma risk in three cohorts and were associated with prevalent glaucoma in the UK Biobank. Altered lipid metabolism may be etiologically important in glaucoma.

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

Macular corneal dystrophy (MCD) is classified as corneal stromal dystrophy. In this study, we retrospectively reviewed the surgical outcomes of 118 MCD patients receiving surgical treatment in the past 30 years and found patients receiving penetrating keratoplasty had the lowest recurrence rate 13.75%, compared with 40.91% patients receiving deep anterior lamellar keratoplasty and 25% receiving phototherapeutic keratectomy. Transcriptomic analysis in human corneal single-cell sequencing atlas found the MCD pathogenic gene CHST6 was abundant in corneal endothelium rather than other cell types. CHST6 protein showed a similar expression pattern to its mRNA. The mouse homologous gene Chst5 was 120-fold higher in corneal endothelium than in the epithelial and stromal layers. Mice with specifically Chst5 knockdown in the endothelial layer by microinjection of the adeno-associated virus serotype 9 - shRNA plasmids into the anterior chamber, rather than Chst5 knockdown into the stroma, showed MCD-like phenotypes. Corneal opacification and abnormally larger collagen fibrils were observed in the endothelial Chst5 knockdown mice. The same corneal characteristics were observed after overexpressing human CHST6 mutant R50H in the mouse endothelium. These observations indicating the pathogenesis of MCD is more related to the corneal endothelium rather than the stroma. Significance StatementOur study gave evidence that corneal endothelium contributing more to the macular corneal dystrophy (MCD) development, rather than other cell types in the cornea. We proposed penetrating keratoplasty might serve as a more proper surgical treatment for MCD according to the recurrence rate analysis. We also provided a novel method to construct MCD mouse model.

A major proportion of retinal disease-causing genes are related to the primary cilium, a microtubule-based signalling organelle essential for multiple developmental pathways. Previous work has shown that the primary cilium plays a crucial role in the development of the retinal pigment epithelium (RPE) affecting homeostasis and function, in particular phagocytosis. We used a cell biology approach to analyse the influence of ciliary genes on RPE phagocytosis and dissect the underlying molecular mechanisms. We found that loss of ciliary trafficking via depletion of Ift20 and Ift88 in RPE-J cells resulted in impaired phagocytosis, specifically by reducing photoreceptor outer segments binding, changes in apical membrane morphology and altered mitochondrial metabolism, whereas loss of Bbs6 showed no functionality phenotype. In addition, proteomics revealed mis-regulated pathways and targets, through which new phagocytosis-related proteins were identified. Our data highlight the role of primary cilia proteins in RPE function and metabolism, essential for visual health.

Cyclase-associated actin cytoskeleton regulatory protein 2 (CAP2) is a conserved actin-binding protein that promotes actin filament (F-actin) turnover by disassembling ADF/cofilin-decorated filaments, supporting F-actin remodeling in differentiating cells and tissues. In the ocular lens, the actin cytoskeleton is critical for maintaining tissue biomechanical properties during fiber cell maturation. To assess CAP2s role in the lens, we examined lens-specific CAP2 knockout (CAP2cKO) mice at cellular and tissue levels. CAP2cKO lenses were normal in size, shape, and transparency but exhibited increased stiffness under compression and enhanced recovery after load removal. While total actin levels and F-actin were unchanged, immunofluorescence revealed higher levels of Tropomodulin 1 (Tmod1, F-actin pointed-end capping), Tropomyosin3.5 (Tpm3.5, F-actin stabilizing), and T-plastin (F-actin bundling) in F-actin-rich membrane protrusions of mature fibers, while -actinin-1 (F-actin cross-linking) was reduced. These findings suggest that CAP2 loss disrupts F-actin remodeling, promoting filament stabilization through Tpm3.5 binding, Tmod1 capping and T-plastin bundling. Consequently, F-actin networks become stiffer and more resilient, altering lens biomechanics. This study provides the first evidence that CAP2 regulates cell biomechanical properties in a non-muscle tissue through modulation of F-actin-associated proteins. Summary statementCAP2 loss alters the actin cytoskeleton through increased F-actin stabilization by Tmod1 and Tpm3.5, and filament bundling by T-plastin, leading to stiffer lenses.

Keratoconus (KC) is a common eye disease where the cornea undergoes degenerative thinning and steepening. The absence of biomarkers for early diagnosis prior to the onset of overt corneal phenotypes and the lack of curative treatments rooted in a fundamental understanding of KC biology remain significant challenges. To address these issues, we investigated the role of unresolved oxidative stress in KC pathogenesis. Malondialdehyde (MDA) a lipid peroxidation byproduct that accumulates during oxidative stress was significantly elevated in the tears of KC patients compared to unaffected controls and positively correlated with maximal keratometry (Kmax), a measure of KC severity. Similarly, the secreted antioxidant glutathione peroxidase 3 (GPX3), was significantly increased in patient tears, and strongly correlated with Kmax. In a cell culture model of oxidative stress, KC corneal stromal cells displayed increased apoptosis and suboptimal activation of NRF2, a transcription factor master regulator of antioxidant genes. Conversely, inhibition of NRF2 in donor stromal cells elicited KC-like cellular phenotype, whereas sulforaphane, an NRF2 booster restored antioxidant gene expression and the deposition of cornea-typical collagens. Our study identified cellular antioxidant signaling dysregulations in keratoconus where sulforaphane treatment may be restorative. Consistent increases in patient tear MDA and GPX3 present these as promising biomarkers for KC diagnosis and severity predictions.

PurposeIn age-related macular degeneration (AMD) and Sorsbys fundus dystrophy (SFD), lipid-rich deposits known as drusen accumulate under the retinal pigment epithelium (RPE). Drusen may contribute to photoreceptor and RPE degeneration in AMD and SFD. We hypothesize that stimulating {beta}-oxidation in RPE will reduce drusen accumulation. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate {beta}-oxidation and diminish lipid accumulation in fatty liver disease. In this report we test the hypothesis that an ACC inhibitor, Firsocostat, limits the accumulation of lipid deposits in cultured RPE cells. MethodsWe probed metabolism and cellular function in mouse RPE-choroid, human fetal- derived RPE cells, and induced pluripotent stem cell-derived RPE cells. We used 13C6-glucose and 13C16-palmitate to determine the effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. 13C labeling of metabolites in these pathways were analyzed using gas chromatography-linked mass spectrometry. We quantified ApoE and VEGF release using enzyme-linked immunosorbent assays. Immunostaining of sectioned RPE was used to visualize ApoE deposits. RPE function was assessed by measuring the trans-epithelial electrical resistance (TEER). ResultsACC inhibition with Firsocostat increases fatty acid oxidation and remodels lipid composition, glycolytic metabolism, lipoprotein release, and enhances TEER. When human serum is used to induce sub-RPE lipoprotein accumulation, fewer lipoproteins accumulate with Firsocostat. In a culture model of Sorsbys fundus dystrophy, Firsocostat also stimulates fatty acid oxidation, improves morphology, and increases TEER. ConclusionsFirsocostat remodels intracellular metabolism and improves RPE resilience to serum-induced lipid deposition. This effect of ACC inhibition suggests that it could be an effective strategy for diminishing drusen accumulation in the eyes of patients with AMD.

PurposeThe human 10q26 locus is a major genetic risk factor for age-related macular degeneration (AMD). Fine mapping by linkage and large-scale genome-wide association studies (GWAS) has narrowed this region to a 30-kb interval encompassing the ARMS2 and HTRA1 genes. However, the causative gene(s), risk variants, and underlying pathogenic mechanisms remain unresolved. MethodsLong non-coding RNA (lncRNA) candidates within the ARMS2-HTRA1 region were identified using human postmortem retinal RNA-seq data and public databases (NCBI, Ensembl). Candidate transcripts were validated by RT-PCR and Sanger sequencing. Published single-cell RNA-seq datasets were analysed to define cell type-specific expression, and RNA levels were compared between AMD and non-AMD donor retinas. Additionally, expression changes were assessed in human iPSC-derived retinal pigment epithelium (RPE) cells exposed to cigarette smoke extract (CSE) and paraquat (PQT). ResultsWe identified and validated a lncRNA, HTRA1-AS1, and its transcript variants (ENST00000647969.1) within the ARMS2 locus. HTRA1-AS1 overlaps ARMS2 and is transcribed in the antisense orientation. It is predominantly expressed in rod photoreceptors, Muller glia and Choroid/RPE, and its retinal expression was significantly reduced in AMD compared with controls (43 AMD donors vs. 44 controls, p = 0.007). By contrast, HTRA1 mRNA showed no significant difference (p = 0.121). Furthermore, ENST00000647969.1, HTRA1-AS1 and ARMS2 expression increased dramatically, up to 101-fold, 8-fold and 75-fold, respectively, in induced pluripotent stem cells (iPSC)-derived RPE cells following cigarette smoke extract (CSE)-induced oxidative stress but showed no significant change after paraquat treatment. ConclusionThese findings suggest that HTRA1-AS1, a dysregulated lncRNA within the ARMS2 locus, may act as a non-coding element contributing to transcriptional mis-regulation underlying AMD pathogenesis.

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

Insufficient oxygenation in the lamina cribrosa (LC) may contribute to axonal damage and glaucomatous vision loss. To understand the range of susceptibilities to glaucoma, we aimed to identify key factors influencing LC oxygenation and examine if these factors vary with anatomical differences between eyes. We reconstructed 3D, eye-specific LC vessel networks from histological sections of four healthy monkey eyes. For each network, we generated 125 models varying vessel radius, oxygen consumption rate, and arteriole perfusion pressure. Using hemodynamic and oxygen supply modeling, we predicted blood flow distribution and tissue oxygenation in the LC. ANOVA assessed the significance of each parameter. Our results showed that vessel radius had the greatest influence on LC oxygenation, followed by anatomical variations. Arteriole perfusion pressure and oxygen consumption rate were the third and fourth most influential factors, respectively. The LC regions are well perfused under baseline conditions. These findings highlight the importance of vessel radius and anatomical variation in LC oxygenation, providing insights into LC physiology and pathology. Pathologies affecting vessel radius may increase the risk of LC hypoxia, and anatomical variations could influence susceptibility. Conversely, increased oxygen consumption rates had minimal effects, suggesting that higher metabolic demands, such as those needed to maintain intracellular transport despite elevated intraocular pressure, have limited impact on LC oxygenation.

PURPOSETo spatially map aquaporin-5 (AQP5) expression in bovine lens, molecularly characterize cytoplasmic AQP5-containing vesicles in the outer cortex, and elucidate AQP5 membrane trafficking mechanisms. METHODSImmunofluorescence was performed on bovine lens cryosections using AQP5, TOMM20, COX IV, calnexin, LC3B, LIMP-2, and connexin-50 antibodies and the fluorescent lipid membrane dye CM-DiI. AQP5 plasma membrane insertion was defined via line expression profile analysis. Transmission electron microscopy (TEM) was performed on bovine lens tissue sections to define cytoplasmic organelle identity, morphology, and subcellular localization in cortical fiber cells. Bovine lenses were treated with 10 nM bafilomycin A1 or 0.1% dimethyl sulfoxide vehicle control in ex vivo culture to determine changes in AQP5 plasma membrane expression. RESULTSImmunofluorescence analysis revealed cytoplasmic AQP5 expression in bovine lens epithelial cells and differentiating fiber cells. In the bovine lens cortex, complete AQP5 plasma membrane insertion occurs at r/a 0.951 + 0.005. AQP5-containing cytoplasmic vesicles are spheroidal, tubular in morphology, express TOMM20, and contain LC3B and LIMP-2 as fiber cells mature. TEM analysis revealed spheroidal, tubular autophagosomes, autolysosomes, and lysosomes with degrading mitochondria. AQP5-containing cytoplasmic vesicles and autolysosomes dock and fuse with the plasma membrane. Bafiloymcin A1 treatment reduced AQP5 plasma membrane expression by 27%. CONCLUSIONSAQP5 localizes to spheroidal, tubular cytoplasmic vesicles in the differentiating bovine lens fiber cells. During fiber cell differentiation, these vesicles incorporate LC3B and fuse with LIMP-2-positive lysosomes. AQP5 trafficking to the plasma membrane occurs through lysosome secretion as a novel mechanism of AQP5 trafficking.

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

Retinal pigment epithelium (RPE) at the back of the eye is a monolayer of cells with an extensive network of gap junctions that contributes to retinal health in a multitude of ways. One of those roles is the phagocytosis of photoreceptor outer segments. This renewal is under circadian regulation and peaks after light onset. Connexin 43 (Cx43) is the most predominantly expressed gap junction protein in RPE. In this study, we examine how gap junctions and specifically, Cx43 phosphorylation, contribute to phagocytosis in both human embryonic stem cell derived RPE and mouse RPE monolayers. We show that both Rac1 and CDK5 have differences in protein localization at different points in phagocytosis, and that by using their effectors, the capability of RPE for phagocytosis changes. CDK5 has not yet been reported in RPE tissue, and here we show that it likely regulates Cx43 localization and resulting electrical coupling. We find that gap junctions in RPE are temporally highly dynamic during phagocytosis and that regulation of gap junctions via phosphorylation is likely critical for maintaining eye health.