Human Cataract Research Articles

The Synergistic Effects of Polyol Pathway-Induced Oxidative and Osmotic Stress in the Aetiology of Diabetic Cataracts.

Cataracts are the world's leading cause of blindness, and diabetes is the second leading risk factor for cataracts after old age. Despite this, no preventative treatment exists for cataracts. The altered metabolism of excess glucose during hyperglycaemia is known to be the underlying cause of diabetic cataractogenesis, resulting in localised disruptions to fibre cell morphology and cell swelling in the outer cortex of the lens. In rat models of diabetic cataracts, this damage has been shown to result from osmotic stress and oxidative stress due to the accumulation of intracellular sorbitol, the depletion of NADPH which is used to regenerate glutathione, and the generation of fructose metabolites via the polyol pathway. However, differences in lens physiology and the metabolism of glucose in the lenses of different species have prevented the translation of successful treatments in animal models into effective treatments in humans. Here, we review the stresses that arise from hyperglycaemic glucose metabolism and link these to the regionally distinct metabolic and physiological adaptations in the lens that are vulnerable to these stressors, highlighting the evidence that chronic oxidative stress together with osmotic stress underlies the aetiology of human diabetic cortical cataracts. With this information, we also highlight fundamental gaps in the knowledge that could help to inform new avenues of research if effective anti-diabetic cataract therapies are to be developed in the future.

Longitudinal study of microphthalmia in connexin 50 knockout mice using spectral-domain optical coherence tomography.

Connexin 50 (Cx50) mediated signaling is essential for controlling the lens growth and size. Cx50 mutations cause microphthalmia, smaller lenses, and cataracts in humans and animals. These ocular defects have never been investigated in live Cx50 mutant mice by using non-invasive imaging techniques. Here, we report a longitudinal study of the ocular defects in Cx50 knockout (Cx50KO) mice from the ages of 3 weeks to 12 months by using spectral-domain optical coherence tomography (SD-OCT). The anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL) were measured along the visual axis and adjusted with corresponding refractive indices. The SD-OCT image data confirm age-related reductions of LT and AL in live Cx50KO mice compared to age-matched wild-type (WT) controls, and the reduction values are comparable to the in vitro measurements of Cx50KO eyeballs and lenses reported previously. Moreover, reductions of ACD were observed in Cx50KO mice at all ages studied while VCD changes are statistically insignificant in comparison to the WT controls. Therefore, Cx50KO's microphthalmia with small lens is selectively associated with delayed ACD development but not the vitreous formation. This work supports the notion that lens size and/or growth is important for anterior chamber development.

Loss of αBa-crystallin, but not αA-crystallin, increases age-related cataract in the zebrafish lens

The vertebrate eye lens is an unusual organ in that most of its cells lack nuclei and the ability to replace aging protein. The small heat shock protein α-crystallins evolved to become key components of this lens, possibly because of their ability to prevent aggregation of aging protein that would otherwise lead to lens opacity. Most vertebrates express two α-crystallins, αA- and αB-crystallin, and mutations in each are linked to human cataract. In a mouse knockout model only the loss of αA-crystallin led to early-stage lens cataract. We have used the zebrafish as a model system to investigate the role of α-crystallins during lens development. Interestingly, while zebrafish express one lens-specific αA-crystallin gene (cryaa), they express two αB-crystallin genes, with one evolving lens specificity (cryaba) and the other retaining the broad expression of its mammalian ortholog (cryabb). In this study we used individual mutant zebrafish lines for all three α-crystallin genes to determine the impact of their loss on age-related cataract. Surprisingly, unlike mouse knockout models, we found that the loss of the αBa-crystallin gene cryaba led to an increase in lens opacity compared to cryaa null fish at 24 months of age. Loss of αA-crystallin did not increase the prevalence of cataract. We also used single cell RNA-Seq and RT-qPCR data to show a shift in the lens expression of zebrafish α-crystallins between 5 and 10 days post fertilization (dpf), with 5 and 6 dpf lenses expressing cryaa almost exclusively, and expression of cryaba and cryabb becoming more prominent after 10 dpf. These data show that cryaa is the primary α-crystallin during early lens development, while the protective role for cryaba becomes more important during lens aging. This study is the first to quantify cataract prevalence in wild-type aging zebrafish, showing that lens opacities develop in approximately 25% of fish by 18 months of age. None of the three α-crystallin mutants showed a compensatory increase in the expression of the remaining two crystallins, or in the abundant βB1-crystallin. Overall, these findings indicate an ontogenetic shift in the functional importance of individual α-crystallins during zebrafish lens development. Our finding that the lens-specific zebrafish αBa-crystallin plays the leading role in preventing age-related cataract adds a new twist to our understanding of vertebrate lens evolution.

TRPM3-mediated inhibition of active Na-K transport by the ocular lens epithelium

Purpose: The TRPM3 channel is selectively expressed in the lens epithelium, a monolayer at the anterior lens surface, but undetectable in fiber cells that make up the lens bulk. A point mutation in TRPM3 gene (substitution of isoleucine by methionine at codon 65; I65M) causes early onset autosomal dominant cataract in humans and in mice. From studies on transgenic mice, it is apparent the I65M TRPM3 mutation disrupts lens Na-K homeostasis. However, the normal role of lens TRPM3 in the lens is currently unknown. Here, we explore the lens response to TRPM3 activation. Methods: Studies were carried out using intact mouse lenses, cultured mouse lens epithelial cells and a human lens epithelium cell line (HLEB3). Active Na-K transport was studied by using rubidium (Rb) as a tracer for potassium entry. Rb was detected by atomic absorption spectrophotometry. Intracellular calcium was measured in nonconfluent single cells by Fura-2 ratiometric imaging. Results: Rb uptake was measured over a period of 10 min in intact mouse lenses exposed to a TRPM3 agonist CIM0216 (10 μM). CIM0216 reduced the rate of Rb uptake by ~20% (Ctrl 6.4±0.4 vs CIM 4.5±0.4 mmoles/kg lens dry weight/10 min, n=7, p<.01). The concentration dependence of the response to CIM0216 (1 to 50 μM) was examined in primary cultured mouse lens epithelium and Rb uptake was reduced by CIM0216 concentrations > 10 μM. The TRPM3 agonist reduced Rb uptake to a similar extent (~20%) in mouse lens epithelium, HLEB3 cells and HLEB3 cells that had been stably transfected with the I65M mutant TRPM3. However, the mutant cells responded to CIM0216 at a 10-fold lower concentration than WT mouse and HLEB3 cells. The Rb uptake response to CIM0216 was significantly inhibited by a TRPM3 antagonist, primidone (50 μM), and was abolished when cells were bathed in calcium-free solution. To study calcium entry, Fura2-loaded mouse lens epithelial cells were exposed to the TRPM3 agonist pregnenolone (100 μM). Pregnenolone caused an immediate increase in cytoplasmic calcium that was inhibited by primidone (Peak calcium concentration Ctrl 201±37 vs primidone 137±8 nM, n=5, p<.001). In cultures lens epithelium, agents that increase cellular cAMP, including IBMX, forskolin and 8p-CPT-cAMP, all caused a marked reduction of Rb uptake. Conclusions: TRPM3 activation causes a significant reduction in the rate of active Na-K transport and the response is associated with calcium entry. Because most lens Rb uptake is ouabain-sensitive, the response points to Na,K-ATPase inhibition. Studies are under way to test whether the Na-K transport response to TRPM3 activation involves cAMP signaling. This work was supported by NIH grants R01EY029171 and R01EY009532. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aβ in the Lens of the Eye: A Noninvasively Detectable Early Biomarker of Alzheimer’s Disease β‐Amyloidopathy in Humans and Transgenic Mouse Model

AbstractBackgroundAmyloid‐ß (Aß) accumulation in brain is an early hallmark of Alzheimer’s disease (AD). We discovered Aß deposition, ß‐amyloidopathy, and colocalizing supranuclear cataracts (SNC) in lenses from people with AD (Goldstein LE et al., Lancet, 2003) and Down syndrome (DS; Moncaster JA et al., PLoS One, 2010), but not other neurodegenerative diseases or normal aging. These findings spurred development of an investigational drug‐device lens Aß eye scanner (Sapphire II, Cognoptix) that combines an Aß‐binding fluorescent ligand (Aftobetin) and specialized laser scanning ophthalmoscope for noninvasive measurement of lens Aß (FDA Breakthrough Device, 2021). Early clinical results indicate that lens Aß is an ideal biomarker for early AD detection. Here, we investigated Aß accumulation and amyloidopathy in lens and brain.MethodTg2576 transgenic (Tg+) mice express Swedish mutant human amyloid precursor protein (hAPP‐Swe), human Aß (hAß), ß‐amyloidopathy, and cognitive deficits (Hsiao et al., Science, 1996). Non‐transgenic (Tg–) littermates were used as controls. Lenses were examined by ex vivo lens stereomicroscopy. Brains and lenses were analyzed by Aß immunohistochemistry, amyloid histopathology, anti‐Aß immunogold electron microscopy, tryptic digest mass spectrometry, anti‐hAß ELISA/immunoblotting. Quasi‐elastic light scattering hAß‐lens protein aggregation analysis.ResultTg2576 Tg+ mice, but not Tg– controls, age‐dependently express hAPP, accumulate hAß, and develop hAß molecular pathology in the lens and exhibit age‐dependent Aß supranuclear opacification that recapitulates lens pathology and SNC phenotype expression in human AD and DS. We detected hAß in conditioned medium from Tg+ but not Tg– lens explant cultures. hAß potently promotes mouse lens protein aggregation in vitro.ConclusionThese results support mechanistic (genotype‐phenotype) linkage between age‐dependent Aß pathology and AD‐related phenotypes in lens and brain. Collectively, our findings identify Aß pathology as the shared molecular etiology of age‐dependent supranuclear cataracts associated with two human diseases (AD, DS) and homologous murine cataracts in the Tg2576 transgenic mouse model of AD. These results represent the first evidence of AD‐related Aß pathology outside the brain and support lens Aß as an optically‐accessible biomarker for early detection and longitudinal monitoring of AD.

Aβ in the Lens of the Eye: A Noninvasively Detectable Early Biomarker of Alzheimer’s Disease β‐Amyloidopathy in Humans and Transgenic Mouse Model

AbstractBackgroundAmyloid‐β (Aβ) accumulation in brain is an early hallmark of Alzheimer’s disease (AD). We discovered Aβ deposition, β‐amyloidopathy, and colocalizing supranuclear cataracts (SNC) in lenses from people with AD (Goldstein LE et al., Lancet, 2003) and Down syndrome (DS; Moncaster JA et al., PLoS One, 2010), but not other neurodegenerative diseases or normal aging. These findings spurred development of an investigational drug‐device lens Aβ eye scanner (Sapphire II, Cognoptix) that combines an Aβ‐binding fluorescent ligand (Aftobetin) and specialized laser scanning ophthalmoscope for noninvasive measurement of lens Aβ (FDA Breakthrough Device, 2021). Early clinical results indicate that lens Aβ is an ideal biomarker for early AD detection. Here, we investigated Aβ accumulation and amyloidopathy in lens and brain.MethodTg2576 transgenic (Tg+) mice express Swedish mutant human amyloid precursor protein (hAPP‐Swe), human Aβ (hAβ), β‐amyloidopathy, and cognitive deficits (Hsiao et al., Science, 1996). Non‐transgenic (Tg–) littermates were used as controls. Lenses were examined by ex vivo lens stereomicroscopy. Brains and lenses were analyzed by Aβ immunohistochemistry, amyloid histopathology, anti‐Aβ immunogold electron microscopy, tryptic digest mass spectrometry, anti‐hAβ ELISA/immunoblotting. Quasi‐elastic light scattering hAβ‐lens protein aggregation analysis.ResultTg2576 Tg+ mice, but not Tg– controls, age‐dependently express hAPP, accumulate hAβ, and develop hAβ molecular pathology in the lens and exhibit age‐dependent Aβ supranuclear opacification that recapitulates lens pathology and SNC phenotype expression in human AD and DS. We detected hAβ in conditioned medium from Tg+ but not Tg– lens explant cultures. hAβ potently promotes mouse lens protein aggregation in vitro.ConclusionThese results support mechanistic (genotype‐phenotype) linkage between age‐dependent Aβ pathology and AD‐related phenotypes in lens and brain. Collectively, our findings identify Aβ pathology as the shared molecular etiology of age‐dependent supranuclear cataracts associated with two human diseases (AD, DS) and homologous murine cataracts in the Tg2576 transgenic mouse model of AD. These results represent the first evidence of AD‐related Aβ pathology outside the brain and support lens Aβ as an optically‐accessible biomarker for early detection and longitudinal monitoring of AD.

Unbalanced redox status network as an early pathological event in congenital cataracts

The lens proteome undergoes dramatic composition changes during development and maturation. A defective developmental process leads to congenital cataracts that account for about 30% of cases of childhood blindness. Gene mutations are associated with approximately 50% of early-onset forms of lens opacity, with the remainder being of unknown etiology. To gain a better understanding of cataractogenesis, we utilized a transgenic mouse model expressing a mutant ubiquitin protein in the lens (K6W-Ub) that recapitulates most of the early pathological changes seen in human congenital cataracts. We performed mass spectrometry-based tandem-mass-tag quantitative proteomics in E15, P1, and P30 control or K6W-Ub lenses. Our analysis identified targets that are required for early normal differentiation steps and altered in cataractous lenses, particularly metabolic pathways involving glutathione and amino acids. Computational molecular phenotyping revealed that glutathione and taurine were spatially altered in the K6W-Ub cataractous lens. High-performance liquid chromatography revealed that both taurine and the ratio of reduced glutathione to oxidized glutathione, two indicators of redox status, were differentially compromised in lens biology. In sum, our research documents that dynamic proteome changes in a mouse model of congenital cataracts impact redox biology in lens. Our findings shed light on the molecular mechanisms associated with congenital cataracts and point out that unbalanced redox status due to reduced levels of taurine and glutathione, metabolites already linked to age-related cataract, could be a major underlying mechanism behind lens opacities that appear early in life.

Cellular Localization of FOXO3 Determines Its Role in Cataractogenesis

The transcription factor FOXO3 is a core regulator of cellular homeostasis, stress response, and longevity. The cellular localization of FOXO3 is closely related to its function. Here, we explored the role of FOXO3 in cataract formation. FOXO3 showed nuclear translocation in lens epithelial cells arranged in a single layer on lens capsule tissues from both human cataract and N-methyl-N-nitrosourea (MNU)-induced rat cataract, also in MNU-injured human lens epithelial cell lines (HLECs). FOXO3 knockdown inhibited the MNU-induced increase in expression of genes related to cell cycle arrest (GADD45α and CCNG2) and apoptosis (Bak and TP53). H2 is highly effective in reducing oxidative impairments in nuclear DNA and mitochondria. When we applied H2 to MNU-injured HLECs, FOXO3 underwent cleavage by MAPK1 and translocated into mitochondria, thereby increasing the transcription of oxidative phosphorylation-related genes (MTCO1, MTCO2, MTND1, and MTND6) in HLECs. Furthermore, H2 mediated the translocation of FOXO3 from the nucleus to the mitochondria within the lens epithelial cells of cataract capsule tissues of rats exposed to N-methyl-N-nitrosourea (MNU). This intervention ameliorated MNU-induced cataracts in the rat model. In conclusion, we demonstrated the correlation between the localization of FOXO3 and its function in cataract formation. We also determined that H2 protects HLECs from injury by leading FOXO3 mitochondrial translocation via MAPK1 activation. Mitochondrial FOXO3 can increase mtDNA transcription, stabilize mitochondrial function in HLECs.

HSP90β prevents aging-related cataract formation through regulation of the charged multivesicular body protein (CHMP4B) and p53

Cataract is a leading ocular disease causing global blindness. The mechanism of cataractogenesis has not been well defined. Here, we demonstrate that the heat shock protein 90β (HSP90β) plays a fundamental role in suppressing cataractogenesis. HSP90β is the most dominant HSP in normal lens, and its constitutive high level of expression is largely derived from regulation by Sp1 family transcription factors. More importantly, HSP90β is significantly down-regulated in human cataract patients and in aging mouse lenses, whereas HSP90β silencing in zebrafish causes cataractogenesis, which can only be rescued by itself but not other HSP90 genes. Mechanistically, HSP90β can directly interact with CHMP4B, a newly-found client protein involved in control of cytokinesis. HSP90β silencing causes upregulation of CHMP4B and another client protein, the tumor suppressor p53. CHMP4B upregulation or overexpression induces excessive division of lens epithelial cells without proper differentiation. As a result, these cells were triggered to undergo apoptosis due to activation of the p53/Bak-Bim pathway, leading to cataractogenesis and microphthalmia. Silence of both HSP90β and CHMP4B restored normal phenotype of zebrafish eye. Together, our results reveal that HSP90β is a critical inhibitor of cataractogenesis through negative regulation of CHMP4B and the p53-Bak/Bim pathway.

Selenite-Induced Cataract as a Model for Cataract Diseases: Limitations, Considerations, and Alternative Approaches

Cataract diseases, characterized by the opacification of the lens, pose a significant global health burden. Animal models are invaluable for understanding cataractogenesis and testing potential treatments [1]. The selenite-induced cataract model has been widely used due to its rapid onset and predictable progression. However, this model has limitations that must be considered. This article reviews the limitations of the selenite-induced cataract model and explores alternative approaches that better represent the complexity and natural course of human cataract diseases [2].

Distinct gene expression profiles underlie morphological and etiological differences in pediatric cataracts.

Pediatric cataract is a major cause of preventable childhood blindness worldwide. Although genetic mutations or infections have been described in patients, the mechanistic basis of human cataract development remains poorly understood. Therefore, gene expression of structural, developmental, profibrotic, and transcription factors in phenotypically and etiologically distinct forms of pediatric cataracts were evaluated. This cross-sectional study included 89 pediatric cataract subjects subtyped into 1) prenatal infectious (cytomegalovirus, rubella, and combined cytomegalovirus with rubella infection), 2) prenatal non-infectious, 3) posterior capsular anomalies, 4) postnatal, 5) traumatic, and 6) secondary, and compared to clear, non-cataractous material of eyes with the subluxated lenses. Expression of lens structure-related genes (Aqp-0, HspA4/Hsp70, CrygC), transcription factors (Tdrd7, FoxE3, Maf, Pitx 3) and profibrotic genes (Tgfβ, Bmp7, αSmA, vimentin) in surgically extracted cataract lens material were studied and correlated clinically. In cataract material, the lens-related gene expression profiles were uniquely associated with phenotype/etiology of different cataracts. Postnatal cataracts showed a significantly altered FoxE3 expression. Low levels of Tdrd7 expression correlated with posterior subcapsular opacity, whereas CrygC correlated significantly with anterior capsular ruptures. The expression of Aqp0 and Maf was elevated in infectious cataracts, particularly in CMV infections, compared to other cataract subtypes. Tgfβ showed significantly low expression in various cataract subtypes, whereas vimentin had elevated gene expression in infectious and prenatal cataracts. A significant association between lens gene expression patterns in phenotypically and etiologically distinct subtypes of pediatric cataracts suggests regulatory mechanisms in cataractogenesis. The data reveal that cataract formation and presentation is a consequence of altered expression of a complex network of genes.

FOXM1 promotes TGF-β2-induced injury of human lens epithelial cells by up regulating VEGFA expression.

To explore whether Fork head box protein M1 (FOXM1) is involved in TGF-β2-induced injury of human lens epithelial cells and its related mechanism. Human lens epithelium samples from cataract patients and healthy controls were collected. A cellular epithelial injury model was established by treating HLE-B3 cells with TGF-β2. QPCR, immunoblot assays were performed to detect the levels of FOXM1 in human cataract samples and the lens epithelial injury cell model. FOXM1 siRNA and pcDNA3.1-FOXM1 plasmids were transfected into the cells to knockdown and overexpress FOXM1, respectively. MTT and wound closure and transwell assays were performed to analyze cell proliferation and migration in HLE-B3 cells. Immunoblot assays were also conducted to detect the effects of FOXM1 on EMT, VEGFA and MAPK/ERK signaling. We found high expression of FOXM1 in lens tissues of cataract patients. Silencing of FOXM1 in TGF-β2-induced HLE-B3 cells suppressed cell proliferation, migration, and the EMT process. Mechanistically, we found that downregulation of FOXM1 inhibited the VEGFA/MAPK signaling pathway in TGF-β2-induced HLE-B3 cells. FOXM1 promoted TGF-β2-induced injury of human lens epithelial cells (hLECs) by promoting VEGFA expression. FOXM1 could be a potential drug target for the treatment of ocular diseases.

Ubiquitination of Ku70 by Parkin promotes apoptosis of lens epithelial cells.

Oxidative damage-triggered apoptosis in lens epithelial cells is considered as a main risk factor in the pathogenesis of age-related cataracts. Ku70 is a key factor in the DNA repair process of double-strand breaks. In the present study, we aimed to investigate the role of Ku70 and its related E3 ubiquitin ligase in lens epithelial cell apoptosis. The levels of Ku70 in the anterior lens capsules of human cataracts and Emory mice were lower compared to controls. H2 O2 treatment resulted in decreased expression of Ku70 through accelerating Ku70 ubiquitination. Parkin, an E3 ubiquitin ligase, could interact with Ku70 and promote the ubiquitination and degradation of this protein. In addition, ubiquitinated Ku70 was regulated by ubiquitin-proteasome, autophagy-lysosome and mitophagy pathways. Ectopic expression of Ku70 protected SRA01/04 cells from H2 O2 -induced apoptosis, whereas silencing Ku70 exhibited the opposite trend. Co-transfected with Parkin non-ubiquitinatable Ku70 mutant could maintain its anti-apoptosis ability, whereas wild-type Ku70 failed. Moreover, Ku70 might facilitate mitochondrial fusion by increasing the expression of Mitofusin 1/2. The present study revealed that Parkin-mediated Ku70 ubiquitination facilitated H2 O2 -induced lens epithelial cell apoptosis through alleviating mitochondrial fusion, which could provide potential targets for age-related cataract treatment.

High-Throughput Transcriptomics of Celf1 Conditional Knockout Lens Identifies Downstream Networks Linked to Cataract Pathology.

Defects in the development of the ocular lens can cause congenital cataracts. To understand the various etiologies of congenital cataracts, it is important to characterize the genes linked to this developmental defect and to define their downstream pathways that are relevant to lens biology and pathology. Deficiency or alteration of several RNA-binding proteins, including the conserved RBP Celf1 (CUGBP Elav-like family member 1), has been described to cause lens defects and early onset cataracts in animal models and/or humans. Celf1 is involved in various aspects of post-transcriptional gene expression control, including regulation of mRNA stability/decay, alternative splicing and translation. Celf1 germline knockout mice and lens conditional knockout (Celf1cKO) mice develop fully penetrant cataracts in early postnatal stages. To define the genome-level changes in RNA transcripts that result from Celf1 deficiency, we performed high-throughput RNA-sequencing of Celf1cKO mouse lenses at postnatal day (P) 0. Celf1cKO lenses exhibit 987 differentially expressed genes (DEGs) at cut-offs of >1.0 log2 counts per million (CPM), ≥±0.58 log2 fold-change and <0.05 false discovery rate (FDR). Of these, 327 RNAs were reduced while 660 were elevated in Celf1cKO lenses. The DEGs were subjected to various downstream analyses including iSyTE lens enriched-expression, presence in Cat-map, and gene ontology (GO) and representation of regulatory pathways. Further, a comparative analysis was done with previously generated microarray datasets on Celf1cKO lenses P0 and P6. Together, these analyses validated and prioritized several key genes mis-expressed in Celf1cKO lenses that are relevant to lens biology, including known cataract-linked genes (e.g., Cryab, Cryba2, Cryba4, Crybb1, Crybb2, Cryga, Crygb, Crygc, Crygd, Cryge, Crygf, Dnase2b, Bfsp1, Gja3, Pxdn, Sparc, Tdrd7, etc.) as well as novel candidates (e.g., Ell2 and Prdm16). Together, these data have defined the alterations in lens transcriptome caused by Celf1 deficiency, in turn uncovering downstream genes and pathways (e.g., structural constituents of eye lenses, lens fiber cell differentiation, etc.) associated with lens development and early-onset cataracts.

Whole-exome sequencing prioritizes candidate genes for hereditary cataract in the Emory mouse mutant.

The Emory cataract (Em) mouse mutant has long been proposed as an animal model for age-related or senile cataract in humans-a leading cause of visual impairment. However, the genetic defect(s) underlying the autosomal dominant Em phenotype remains elusive. Here, we confirmed development of the cataract phenotype in commercially available Em/J mice [but not ancestral Carworth Farms White (CFW) mice] at 6-8 months of age and undertook whole-exome sequencing of candidate genes for Em. Analysis of coding and splice-site variants did not identify any disease-causing/associated mutations in over 450 genes known to underlie inherited and age-related forms of cataract and other lens disorders in humans and mice, including genes for lens crystallins, membrane/cytoskeleton proteins, DNA/RNA-binding proteins, and those associated with syndromic/systemic forms of cataract. However, we identified three cataract/lens-associated genes each with one novel homozygous variant including predicted missense substitutions in Prx (p.R167C) and Adamts10 (p.P761L) and a disruptive in-frame deletion variant (predicted missense) in Abhd12 (p.L30_A32delinsS) that were absent in CFW and over 35 other mouse strains. In silico analysis predicted that the missense substitutions in Prx and Adamts10 were borderline neutral/damaging and neutral, respectively, at the protein function level, whereas, that in Abhd12 was functionally damaging. Both the human counterparts of Adamts10 and Abhd12 are clinically associated with syndromic forms of cataract known as Weil-Marchesani syndrome 1 and polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract syndrome, respectively. Overall, while we cannot exclude Prx and Adamts10, our data suggest that Abhd12 is a promising candidate gene for cataract in the Em/J mouse.

Age-related cataract: GSTP1 ubiquitination and degradation by Parkin inhibits its anti-apoptosis in lens epithelial cells

PurposeOxidative stress-induced apoptosis of lens epithelial cells (LECs) contributes to the pathogenesis of age-related cataract (ARC). The purpose of this research is to underlie the potential mechanism of E3 ligase Parkin and its oxidative stress-associated substrate in cataractogenesis. MethodsThe central anterior capsules were obtained from patients with ARC, Emory mice, and corresponding controls. SRA01/04 cells were exposed to H2O2 combined with cycloheximide (a translational inhibitor), MG-132 (a proteasome inhibitor), chloroquine (an autophagy inhibitor), Mdivi-1 (a mitochondrial division inhibitor), respectively. Co-immunoprecipitation was employed to detect protein-protein interactions and ubiquitin-tagged protein products. Levels of proteins and mRNA were evaluated by western blotting and quantitative RT-PCR assays. ResultsGlutathione-S-transferase P1 (GSTP1) was identified as a novel Parkin substrate. Compared with corresponding controls, GSTP1 was significantly decreased in the anterior lens capsules obtained from human cataracts and Emory mice. Similarly, GSTP1 was declined in H2O2-stimulated SRA01/04 cells. Ectopic expression of GSTP1 mitigated H2O2-induced apoptosis, whereas silencing GSTP1 aggregated apoptosis. In addition, H2O2 stimulation and Parkin overexpression could promote the degradation of GSTP1 through the ubiquitin-proteasome system, autophagy-lysosome pathway, and mitophagy. After co-transfection with Parkin, the non-ubiquitinatable GSTP1 mutant maintained its anti-apoptotic function, while wildtype GSTP1 failed. Mechanistically, GSTP1 might promote mitochondrial fusion through upregulating Mitofusins 1/2 (MFN1/2). ConclusionOxidative stress induces LECs apoptosis via Parkin-regulated degradation of GSTP1, which may provide potential targets for ARC therapy.

Damage resistance of gamma crystallins: Weathering heat, UV, and ionizing radiation.

The γ-crystallins are structural and refractive proteins of the vertebrate lens. They belong to the βγ-crystallin superfamily, which is characterized by an exceptionally stable double-Greek key fold. In the context of the lens, these proteins must remain stable and soluble for the lifetime of the organism. Recent work in my group focuses on understanding how γ-crystallins maintain functionality in the face of damage caused by aging and oxidation. I will present results on the impact of heat, UV light, and ionizing radiation on human γS-crystallin and cataract-associated variants. We find that the γ-crystallins are not particularly resistant to damage; on the contrary, they appear to be selected for the ability to remain soluble even when the protein surface is significantly modified and transient unfolding intermediates are populated. These have been investigated using a variety of biophysical techniques, including circular dichroism, mass spectrometry, fluorescence, and NMR spectroscopy. In particular, mass spectrometry measurements on irradiated samples show evidence of both deamidation and oxidative damage, while high-resolution NMR data allow us to locate protein regions and specific residues that are involved in conformational changes, altered dynamics, and protein-protein contacts. We will discuss proposed aggregation mechanisms mediated by different types of protein modification, as well as the relevance of these findings to human cataract disease.

Autophagy Requirements for Eye Lens Differentiation and Transparency.

Recent evidence points to autophagy as an essential cellular requirement for achieving the mature structure, homeostasis, and transparency of the lens. Collective evidence from multiple laboratories using chick, mouse, primate, and human model systems provides evidence that classic autophagy structures, ranging from double-membrane autophagosomes to single-membrane autolysosomes, are found throughout the lens in both undifferentiated lens epithelial cells and maturing lens fiber cells. Recently, key autophagy signaling pathways have been identified to initiate critical steps in the lens differentiation program, including the elimination of organelles to form the core lens organelle-free zone. Other recent studies using ex vivo lens culture demonstrate that the low oxygen environment of the lens drives HIF1a-induced autophagy via upregulation of essential mitophagy components to direct the specific elimination of the mitochondria, endoplasmic reticulum, and Golgi apparatus during lens fiber cell differentiation. Pioneering studies on the structural requirements for the elimination of nuclei during lens differentiation reveal the presence of an entirely novel structure associated with degrading lens nuclei termed the nuclear excisosome. Considerable evidence also indicates that autophagy is a requirement for lens homeostasis, differentiation, and transparency, since the mutation of key autophagy proteins results in human cataract formation.

Cataracts Across the Tree of Life: A Roadmap for Prevention and Biomedical Innovation

Spontaneous cataracts have been identified in the lenses of animals across a phylogenetically wide range of species. This can be a source of insights and innovation for human health professionals, but many persons may lack awareness of it. By providing a phylogenetic survey and analysis of species with cataract vulnerability, this paper demonstrates how a broad comparative perspective can provide critical information about environmental hazards to human visual health and can spark potential innovations in the prevention and treatment of cataracts in humans. Perspectives. Review and synthesis of selected literature with interpretation and perspective. We found 273 recorded cases of spontaneously occurring cataracts in 113 species of birds, 83 species of mammals, 30 species of actinopterygii fish, 10 species of amphibians, 6 species of reptiles, and 1 species of cephalopod. A phylogenetically wide range of species, including many living in and around human environments, are vulnerable to cataracts. These animals may serve as sentinels for human visual health. Variation in cataract vulnerability across species may also facilitate the identification of resistance-conferring physiologies, leading to accelerated innovation in the prevention and treatment of cataracts in humans.

A tapt1 knock-out zebrafish line with aberrant lens development and impaired vision models human early-onset cataract.

Bi-allelic mutations in the gene coding for human trans-membrane anterior-posterior transformation protein 1 (TAPT1) result in a broad phenotypic spectrum, ranging from syndromic disease with severe skeletal and congenital abnormalities to isolated early-onset cataract. We present here the first patient with a frameshift mutation in the TAPT1 gene, resulting in both bilateral early-onset cataract and skeletal abnormalities, in addition to several dysmorphic features, in this way further expanding the phenotypic spectrum associated with TAPT1 mutations. A tapt1a/tapt1b double knock-out (KO) zebrafish model generated by CRISPR/Cas9 gene editing revealed an early larval phenotype with eye malformations, loss of vision, increased photokinetics and hyperpigmentation, without visible skeletal involvement. Ultrastructural analysis of the eyes showed a smaller condensed lens, loss of integrity of the lens capsule with formation of a secondary lens and hyperplasia of the cells in the ganglion and inner plexiform layers of the retina. Transcriptomic analysis pointed to an impaired lens development with aberrant expression of many of the crystallin and other lens-specific genes. Furthermore, the phototransduction and visual perception pathways were found to be significantly disturbed. Differences in light perception are likely the cause of the increased dark photokinetics and generalized hyperpigmentation observed in this zebrafish model. In conclusion, this study validates TAPT1 as a new gene for early-onset cataract and sheds light on its ultrastructural and molecular characteristics.