Human Cataract Research Articles

Generation of Lens Progenitor Cells and Lentoid Bodies from Pluripotent Stem Cells: Novel Tools for Human Lens Development and Ocular Disease Etiology.

In vitro differentiation of human pluripotent stem cells (hPSCs) into specialized tissues and organs represents a powerful approach to gain insight into those cellular and molecular mechanisms regulating human development. Although normal embryonic eye development is a complex process, generation of ocular organoids and specific ocular tissues from pluripotent stem cells has provided invaluable insights into the formation of lineage-committed progenitor cell populations, signal transduction pathways, and self-organization principles. This review provides a comprehensive summary of recent advances in generation of adenohypophyseal, olfactory, and lens placodes, lens progenitor cells and three-dimensional (3D) primitive lenses, "lentoid bodies", and "micro-lenses". These cells are produced alone or "community-grown" with other ocular tissues. Lentoid bodies/micro-lenses generated from human patients carrying mutations in crystallin genes demonstrate proof-of-principle that these cells are suitable for mechanistic studies of cataractogenesis. Taken together, current and emerging advanced in vitro differentiation methods pave the road to understand molecular mechanisms of cataract formation caused by the entire spectrum of mutations in DNA-binding regulatory genes, such as PAX6, SOX2, FOXE3, MAF, PITX3, and HSF4, individual crystallins, and other genes such as BFSP1, BFSP2, EPHA2, GJA3, GJA8, LIM2, MIP, and TDRD7 represented in human cataract patients.

Expression of oxysterols in human lenses: Implications of the sterol pathway in age-related cataracts

Lanosterol, an oxysterol molecule, has been proposed to help maintain lens transparency by inhibiting the formation of protein aggregates. This sterol is produced by the enzyme lanosterol synthase and is part of a metabolic pathway that forms cholesterol as a final step. Abnormalities in lanosterol synthase are responsible for congenital cataracts. The αA-crystallin protein, which acts as a molecular chaperone to lanosterol synthase, has been reported to have anti-protein aggregation, anti-inflammatory and anti-apoptotic properties. In this work, we evaluated the correlation of lanosterol synthase and αA-crystallin in human cataractous lenses with the grade of opacity, as well as the expression of lanosterol synthase, farnesyl DPP, geranyl synthase and squalene epoxidase genes. Lanosterol synthase and αA-crystallin were overexpressed in cataractous lenses as well as farnesyl-DP synthase, squalene epoxidase, lanosterol synthase and geranyl synthase genes in cataratous lenses in comparison with normal lenses. Our data confirm that lanosterol synthase and the sterol pathway are upregulated in cataractous lenses. This argues for a functional role of the oxysterol pathway and its products as an important mediator in the pathogenesis of human cataracts.

Deficiency of Jamc Leads to Congenital Nuclear Cataract and Activates the Unfolded Protein Response in Mouse Lenses.

PurposeThe malfunction of junctional adhesion molecule C (JAM-C) has been reported to induce congenital cataract in humans and mice; however, specific characters and the mechanism of this cataract are still unclear. This study aimed to characterize abnormal lens development in Jamc knockout mice and clarify the underlying mechanism.Methods Jamc knockout mice backcrossed onto the C57BL/6 genetic background were used for this research. Slit-lamp and darkfield images showed the cataract phenotype of Jamc−/− mice. Hematoxylin and eosin staining was performed to visualize the morphological and histological features. RNA sequencing was applied to detect differentially expressed genes. Quantitative RT-PCR, western blot, and immunofluorescence were used to determine the level of unfolded protein response (UPR)-related genes. TUNEL staining was utilized to label cell death.Results Jamc knockout mice exhibited nuclear cataract with abnormal lens morphology and defective degradation of nuclei and organelles in lens fiber cells. Compared with wild-type control mice, the expression level of BiP, CHOP, TRIB3, and CHAC1, genes involved in endoplasmic reticulum stress and the UPR, were highly upregulated in Jamc−/− lenses, suggesting that abnormal lens development was accompanied by UPR activation. Moreover, increased cell death was also found in Jamc−/− lenses.ConclusionsCongenital nuclear cataract caused by Jamc deficiency is accompanied by defective degradation of nuclei and organelles in lens fiber cells, lens structure disorder, and UPR activation, suggesting that JAM-C is required for maintaining normal lens development and that UPR activation is involved in cataract formation in Jamc-deficient lenses.

Postponement of the opacification of lentoid bodies derived from human induced pluripotent stem cells after lanosterol treatment-the first use of the lens aging model in vitro in cataract drug screening.

Purpose: Our previous study observed that human induced pluripotent stem cell (HiPSC)-derived lentoid bodies (LBs) became cloudy with extended culture time, partially mimicking the progress of human age-related cataracts (ARCs) in a dish. In the present study, lanosterol, a potential anticataract drug, was used to further verify the value of this model in drug screening for cataract treatment. Methods: Mature LBs on day 25, which were differentiated from HiPSCs using the “fried egg” method, were continually cultured and treated with either dimethyl sulfoxide (control) or lanosterol. The LBs’ shape and opacity alterations were examined using light microscopy and mean gray value evaluation. The soluble and insoluble proteins were examined through SDS-PAGE gel electrophoresis combined with Coomassie blue staining. The protein aggregations were examined with immunofluorescence. Results: The mature LBs became cloudy with an extended culture time, and the opacification of the LBs was partially prevented by lanosterol treatment. There was less increase in insoluble proteins in the lanosterol-treated LBs than in the control group. There were also fewer cells containing aggregated protein (αA‐crystallin and αB‐crystallin) puncta in the lanosterol-treated LBs than in the control LBs. Conclusion: It was found that the opacification of LBs could be delayed by lanosterol treatment, which could be achieved by reducing protein aggregation, suggesting a promising HiPSC-derived drug-screening model for Age-related cataract.

A Study of Diabetic and Non-diabetic Human Cataract Lens by Scanning Electron Microscopy

Published on:August 2022Journal of Young Pharmacists, 2022; 14(3):302-304Original Article | doi:10.5530/jyp.2022.14.59 Authors:Swathy Govindaswamy1,*, Dhivya Sundaram21Department of Pharmacy Practice, KMCH College of Pharmacy, Coimbatore, Tamil Nadu, INDIA.2Department of Biomedical Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, INDIA. Abstract:Background: This research aimed to ascertain the differences in morphological manifestation in the human diabetic cataractous lens compared to a non-diabetic cataractous lens by Scanning Electron Microscopy (SEM). Materials and Methods: Lens fibers were prefixed in glutaraldehyde and subsequently post-fixed in a Hexamethyldisilazane and examined by SEM. The SEM images gave a comparative morphological impression of the ongoing structural alterations during the degeneration process of human non-diabetic cataracts and diabetic cataract lenses. Results: Diabetic cataractous lens fibers were identified by more chaotic longitudinal fiber splitting, porosity, and granulation of the lens fiber membrane and opening (distortion) of the lens fiber interdigitation system than their non-cataractous diabetic counterparts. Diabetic cataract lenses showed an increased rate of nucleus compaction compared to non-diabetic cataract lenses. Conclusion: The absence of Finger-like and flap projections and the disappearance of the ball and socket system is prominent in diabetic cataract lenses, suggesting the hyperglycaemic effect in the degeneration of lens fibers and further contributes to the early onset of cataracts in diabetes compared to non-diabetic.Keywords: Cataract, Diabetic complications, Morphology, Scanning electron microscopy.

Oxidation of active cysteines mediates protein aggregation of S10R, the cataract-associated mutant of mouse GammaB-crystallin.

The Ser10 to Arg mutation in mouse γB-crystallin (MGB) has been associated with protein aggregation, dense nuclear opacity, and the degeneration of fiber cells in the lens core. Overexpression of the gap junction protein, connexin 46 (Cx46), was found to suppress the nuclear opacity and restore normal cell-cell contact. However, the molecular basis for the protein aggregation and related downstream effects were not evident from these studies. Here, we provide a comparison of the structures and solution properties of wild type MGB and the S10R mutant in vitro and show that, even though the mutation does not directly involve cysteine residues, some cysteines in the mutant protein are activated, leading to the enhanced formation of intermolecular disulfide-crosslinked protein aggregates relative to the wild-type. This occurs even as the protein structure is essentially unaltered. Thus, the primary event is enhanced protein aggregation due to the disulfide crosslinking of the mutant protein. We suggest that these aggregates eventually get deposited on fiber cell membranes. Since the gap junction protein, Cx46 is involved in the transport of reduced glutathione, we posit that these deposits interfere in Cx46-mediated glutathione transport and facilitate the oxidative stress-mediated downstream changes. Overexpression of Cx46 suppresses such oxidative aggregation. These studies provide a plausible explanation for the protein aggregation and other changes that accompany this mutation. If indeed cysteine oxidation is the primary event for protein aggregation also in vivo, then the S10R mutant mouse, which is currently available, could serve as a viable animal model for human age-onset cataract.

REPS2 downregulation facilitates FGF-induced adhesion and migration in human lens epithelial cells through FAK/Cdc42 signaling and contributes to posterior capsule opacification

Posterior capsular opacification (PCO) can cause postoperative visual loss after cataract surgery. Residual human lens epithelial cell (HLEC) proliferation, migration, epithelial-mesenchymal transition (EMT) and synthesis of extracellular matrix (ECM) are the entitative reasons for PCO. Low expression of Ral-binding protein 1-associated Eps domain-containing 2 (REPS2) and high levels of basic fibroblast growth factor (b-FGF) were observed in the lens and postoperative aqueous humor of cataract patients. REPS2 was identified as a negative regulator in growth factor signaling; however, its function in HLECs is unknown. This was first investigated in the present study by evaluating REPS2 expression in anterior lens capsules from cataract patients, a mouse cataract model, and HLE-b3 cells. The biological function of REPS2 in HLE-B3 cells was assessed by REPS2 silencing and Cell Counting Kit 8, wound healing, Transwell migration, F-actin staining, G-protein pulldown and western blot assays. In the present study, REPS2 was significantly downregulated in human and mouse cataract capsules and H2O2-treated HLE-B3 cells. REPS2 knockdown increased fibronectin, type I collagen, and α-smooth muscle actin expression levels and stimulated HLECs proliferation and migration; these effects were enhanced by FGF treatment and accompanied with focal adhesion kinase (FAK) phosphorylation, cell division cycle 42 (Cdc42) activation, focal adhesion protein upregulation, and F-actin cytoskeleton reorganization. However, treatment with the FAK inhibitor PF573228 abolished these effects. Thus, REPS2 downregulation in cataract HLECs induces their proliferation and facilitates FGF-induced ECM synthesis, EMT, cell adhesion and migration by activating FAK/Cdc42 signaling, which may underlie PCO pathogenesis.

The E3 Ligase RNF157 Inhibits Lens Epithelial Cell Apoptosis by Negatively Regulating p53 in Age-Related Cataracts.

PurposeAge-related cataract (ARC) is a major cause of vision impairment worldwide. The E3 ubiquitin ligase RING finger protein 157 (RNF157) is involved in regulating cell survival and downregulated in human cataractous lens samples. However, the function of RNF157 in cataracts remains unclear. This study aimed to determine the role of RNF157 in ARC.MethodsReal-time polymerase chain reaction (PCR) and Western blotting were used to analyze the expression of RNF157 in clinical lens capsules, rat cataract models, and oxidative stress cell models. Western blot analysis and flow cytometry were used to evaluate cell apoptosis. Co-IP assay, protein stability assay, and ubiquitination assay were used to detect the interaction between RNF157 and its substrate p53.ResultsThe expression of RNF157 was downregulated in human cataract samples, UVB-induced rat cataract model, and H2O2-treated human lens epithelial cells (LECs). Ectopic expression of RNF157 protected LECs from H2O2-induced apoptosis. In contrast, knockdown of RNF157 enhanced oxidative stress-induced apoptotic cell death. Moreover, silence of RNF157 in the rat ex vivo lens model exacerbated lens opacity. Mechanistically, RNF157 causes ubiquitination and degradation of the tumor antigen p53. Overexpression of p53 eliminated the antiapoptotic effects of RNF157, whereas p53 knockdown rescued RNF157 silencing-induced cell death.ConclusionsOur findings revealed that reduced RNF157 expression promoted LEC apoptosis by upregulating p53 in cataracts, suggesting that the regulation of RNF157 expression may serve as a potential therapeutic strategy for cataracts.

Aqueous Lumican Correlates with Central Retinal Thickness in Patients with Idiopathic Epiretinal Membrane: A Proteome Study.

Idiopathic epiretinal membrane (iERM) is a pathological fibrocellular change in the vitreoretinal junction over the macular area; however, possible pathogenic mechanisms remain unclear. Changes in the differential protein composition of the aqueous humor (AH) may represent potential molecular changes associated with iERM. To gain new insights into the molecular mechanisms of iERM pathology, a sensitive label-free proteomics analysis was performed to compare AH protein expressions in patients with cataracts with or without iERM. This study employed nanoflow ultra-high-performance liquid chromatography-tandem mass spectrometry to investigate protein compositions of the AH obtained from individual human cataract eyes from 10 patients with iERM and 10 age-matched controls without iERM. Eight proteins were differentially expressed between the iERM and control samples, among which six proteins were upregulated and two were downregulated. A gene ontology (GO) analysis revealed that iERM was closely associated with several biological processes, such as immunity interactions, cell proliferation, and extracellular matrix remodeling. Additionally, multiple proteins, including lumican, cyclin-dependent kinase 13, and collagen alpha-3(VI) chain, were correlated with the central retinal thickness, indicating a multifactorial response in the pathogenic process of iERM. Changes in the AH level of lumican between iERM and control samples were also confirmed by an enzyme-linked immunosorbent assay. In conclusion, several pathological pathways involved in iERM were identified in the AH by a proteomic analysis, including immune reactions, cell proliferation, and remodeling of the extracellular matrix. Lumican is a potential aqueous biomarker for predicting iERM development and monitoring its progression. More clinical parameters also need to be identified to complete the analysis, and those could provide additional targets for treating and preventing iERM.

Roles of Eph-Ephrin Signaling in the Eye Lens Cataractogenesis, Biomechanics, and Homeostasis.

The eye lens is responsible for fine focusing of light onto the retina, and its function relies on tissue transparency and biomechanical properties. Recent studies have demonstrated the importance of Eph-ephrin signaling for the maintenance of life-long lens homeostasis. The binding of Eph receptor tyrosine kinases to ephrin ligands leads to a bidirectional signaling pathway that controls many cellular processes. In particular, dysfunction of the receptor EphA2 or the ligand ephrin-A5 lead to a variety of congenital and age-related cataracts, defined as any opacity in the lens, in human patients. In addition, a wealth of animal studies reveal the unique and overlapping functions of EphA2 and ephrin-A5 in lens cell shape, cell organization and patterning, and overall tissue optical and biomechanical properties. Significant differences in lens phenotypes of mouse models with disrupted EphA2 or ephrin-A5 signaling indicate that genetic modifiers likely affect cataract phenotypes and progression, suggesting a possible reason for the variability of human cataracts due to Eph-ephrin dysfunction. This review summarizes the roles of EphA2 and ephrin-A5 in the lens and suggests future avenues of study.

Alzheimer's disease amyloid-β pathology in the lens of the eye

Neuropathological hallmarks of Alzheimer's disease (AD) include pathogenic accumulation of amyloid-β (Aβ) peptides and age-dependent formation of amyloid plaques in the brain. AD-associated Aβ neuropathology begins decades before onset of cognitive symptoms and slowly progresses over the course of the disease. We previously reported discovery of Aβ deposition, β-amyloidopathy, and co-localizing supranuclear cataracts (SNC) in lenses from people with AD, but not other neurodegenerative disorders or normal aging. We confirmed AD-associated Aβ molecular pathology in the lens by immunohistopathology, amyloid histochemistry, immunoblot analysis, epitope mapping, immunogold electron microscopy, quantitative immunoassays, and tryptic digest mass spectrometry peptide sequencing. Ultrastructural analysis revealed that AD-associated Aβ deposits in AD lenses localize as electron-dense microaggregates in the cytoplasm of supranuclear (deep cortex) fiber cells. These Aβ microaggregates also contain αB-crystallin and scatter light, thus linking Aβ pathology and SNC phenotype expression in the lenses of people with AD. Subsequent research identified Aβ lens pathology as the molecular origin of the distinctive cataracts associated with Down syndrome (DS, trisomy 21), a chromosomal disorder invariantly associated with early-onset Aβ accumulation and Aβ amyloidopathy in the brain. Investigation of 1249 participants in the Framingham Eye Study found that AD-associated quantitative traits in brain and lens are co-heritable. Moreover, AD-associated lens traits preceded MRI brain traits and cognitive deficits by a decade or more and predicted future AD. A genome-wide association study of bivariate outcomes in the same subjects identified a new AD risk factor locus in the CTNND2 gene encoding δ-catenin, a protein that modulates Aβ production in brain and lens. Here we report identification of AD-related human Aβ (hAβ) lens pathology and age-dependent SNC phenotype expression in the Tg2576 transgenic mouse model of AD. Tg2576 mice express Swedish mutant human amyloid precursor protein (APP-Swe), accumulate hAβ peptides and amyloid pathology in the brain, and exhibit cognitive deficits that slowly progress with increasing age. We found that Tg2576 trangenic (Tg+) mice, but not non-transgenic (Tg–) control mice, also express human APP, accumulate hAβ peptides, and develop hAβ molecular and ultrastructural pathologies in the lens. Tg2576 Tg+ mice exhibit age-dependent Aβ supranuclear lens opacification that recapitulates lens pathology and SNC phenotype expression in human AD. In addition, we detected hAβ in conditioned medium from lens explant cultures prepared from Tg+ mice, but not Tg– control mice, a finding consistent with constitutive hAβ generation in the lens. In vitro studies showed that hAβ promoted mouse lens protein aggregation detected by quasi-elastic light scattering (QLS) spectroscopy. These results support mechanistic (genotype-phenotype) linkage between Aβ pathology and AD-related phenotypes in lens and brain. Collectively, our findings identify Aβ pathology as the shared molecular etiology of two age-dependent AD-related 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 point to lens Aβ as an optically-accessible AD biomarker for early detection and longitudinal monitoring of this devastating neurodegenerative disease.

Physics-aware learning and domain-specific loss design in ophthalmology.

The human cataract, a developing opacification of the human eye lens, currently constitutes the world's most frequent cause for blindness. As a result, cataract surgery has become the most frequently performed ophthalmic surgery in the world. By removing the human lens and replacing it with an artificial intraocular lens (IOL), the optical system of the eye is restored. In order to receive a good refractive result, the IOL specifications, especially the refractive power, have to be determined precisely prior to surgery. In the last years, there has been a body of work to perform this prediction by using biometric information extracted from OCT imaging data, recently also by machine learning (ML) methods. Approaches so far consider only biometric information or physical modelling, but provide no effective combination, while often also neglecting IOL geometry. Additionally, ML on small data sets without sufficient domain coverage can be challenging. To solve these issues, we propose OpticNet, a novel optical refraction network based on an unsupervised, domain-specific loss function that explicitly incorporates physical information into the network. By providing a precise and differentiable light propagation eye model, physical gradients following the eye optics are backpropagated into the network. We further propose a new transfer learning procedure, which allows the unsupervised pre-training on the optical model and fine-tuning of the network on small amounts of surgical patient data. We show that our method outperforms the current state of the art on five OCT-image based data sets, provides better domain coverage within its predictions, and achieves better physical consistency.

RNA-binding proteins and post-transcriptional regulation in lens biology and cataract: Mediating spatiotemporal expression of key factors that control the cell cycle, transcription, cytoskeleton and transparency

Development of the ocular lens – a transparent tissue capable of sustaining frequent shape changes for optimal focusing power – pushes the boundaries of what cells can achieve using the molecular toolkit encoded by their genomes. The mammalian lens contains broadly two types of cells, the anteriorly located monolayer of epithelial cells which, at the equatorial region of the lens, initiate differentiation into fiber cells that contribute to the bulk of the tissue. This differentiation program involves massive upregulation of select fiber cell-expressed RNAs and their subsequent translation into high amounts of proteins, such as crystallins. But intriguingly, fiber cells achieve this while also simultaneously undergoing significant morphological changes such as elongation – involving about 1000-fold length-wise increase – and migration, which requires modulation of cytoskeletal and cell adhesion factors. Adding further to the challenges, these molecular and cellular events have to be coordinated as fiber cells progress toward loss of their nuclei and organelles, which irreversibly compromises their potential for harnessing genetically hardwired information. A long-standing question is how processes downstream of signaling and transcription, which may also participate in feedback regulation, contribute toward orchestrating these cellular differentiation events in the lens. It is now becoming clear from findings over the past decade that post-transcriptional gene expression regulatory mechanisms are critical in controlling cellular proteomes and coordinating key processes in lens development and fiber cell differentiation. Indeed, RNA-binding proteins (RBPs) such as Caprin2, Celf1, Rbm24 and Tdrd7 have now been described in mediating post-transcriptional control over key factors (e.g. Actn2, Cdkn1a (p21Cip1), Cdkn1b (p27Kip1), various crystallins, Dnase2b, Hspb1, Pax6, Prox1, Sox2) that are variously involved in cell cycle, transcription, cytoskeleton maintenance and differentiation in the lens. Furthermore, deficiencies of these RBPs have been shown to result in various eye and lens defects and/or cataract. Because fiber cell differentiation in the lens occurs throughout life, the underlying regulatory mechanisms operational in development are expected to also be recruited for the maintenance of transparency in aged lenses. Indeed, in support of this, TDRD7 and CAPRIN2 loci have been linked to age-related cataract in humans. Here, I will review the role of key RBPs in the lens and their importance in understanding the pathology of lens defects. I will discuss advances in RBP-based gene expression control, in general, and the important challenges that need to be addressed in the lens to define the mechanisms that determine the epithelial and fiber cell proteome. Finally, I will also discuss in detail several key future directions including the application of bioinformatics approaches such as iSyTE to study RBP-based post-transcriptional gene expression control in the aging lens and in the context of age-related cataract.

Defect of LSS Disrupts Lens Development in Cataractogenesis.

Congenital cataract is one of the leading causes of blindness in children worldwide. About one-third of congenital cataracts are caused by genetic defects. LSS, which encodes lanosterol synthase, is a causal gene for congenital cataracts. LSS is critical in preventing abnormal protein aggregation of various cataract-causing mutant crystallins; however, its roles in lens development remain largely unknown. In our study, we generated a mouse model harboring Lss G589S mutation, which is homologous to cataract-causing G588S mutation in human LSS. LssG589S/G589S mice exhibited neonatal lethality at postal day 0 (P0), whereas these mice showed severe opacity in eye lens. Also, we found that cataract was formed at E17.5 after we examined the opacity of embryonic lens from E13.5 to E18.5. Moreover, disrupted lens differentiation occurred at E14.5 prior to formation of the opacity of eye lens, shown as delayed differentiation of lens secondary fiber and disordered lens fiber organization. In addition, RNA-seq analysis indicated that cholesterol synthesis signaling pathways were significantly downregulated. Overall, our findings provide clear evidence that a mouse model harboring a homozygous Lss G589S mutation can recapitulate human congenital cataract. Our study points out that LSS functions as a critical determinant of lens development, which will contribute to better understanding LSS defects in cataractogenesis and developing therapies for cataracts.

Pathogenesis of Age-Related Cataract: A Systematic Review of Proteomic Studies

Aims: The aim of this systematic review is to identify all the available data on human lens proteomics with a critical role in age-related cataract formation in order to elucidate the physiopathology of the aging lens. Methods:: We searched on Medline and Cochrane databases. The search generated 328 manuscripts. We included nine original proteomic studies that investigated human cataractous lenses. Results: Deamidation was the major age-related post-translational modification. There was a significant increase in the amount of αA-crystallin D-isoAsp58 present at all ages, while an increase in the extent of Trp oxidation was apparent in cataract lenses when compared to aged normal lense. During aging, enzymes with oxidized cysteine at critical sites included GAPDH, glutathione synthase, aldehyde dehydrogenase, sorbitol dehydrogenase, and PARK7. Conclusion: D-isoAsp in αA crystallin could be associated with the development of age-related cataract in humans by contributing to the denaturation of a crystallin and decreasing its ability to act as a chaperone. Oxidation of Trp may be associated with nuclear cataract formation in humans, while the role of oxidant stress in age-related cataract formation is dominant.

Hyperbaric oxygen as a model of lens aging in the bovine lens: The effects on lens biochemistry, physiology and optics

Age related nuclear (ARN) cataracts in humans take years to form and so experimental models have been developed to mimic the process in animals as a means of better understanding the etiology of nuclear cataracts in humans. A major limitation with these animal models is that many of the biochemical and physiological changes are not typical of that seen in human ARN cataract. In this review, we highlight the work of Frank Giblin and colleagues who established an in vivo animal model that replicates many of the changes observed in human ARN cataract. This model involves exposing aged guinea pigs to hyperbaric oxygen (HBO), which by causing the depletion of the antioxidant glutathione (GSH) specifically in the lens nucleus, produces oxidative changes to nuclear proteins, nuclear light scattering and a myopic shift in lens power that mimics the change that often precedes cataract development in humans. However, this model involves multiple HBO treatments per week, with sometimes up to a total of 100 treatments, spanning up to eight months, which is both costly and time consuming. To address these issues, Giblin developed an in vitro model that used rabbit lenses exposed to HBO for several hours which was subsequently shown to replicate many of the changes observed in human ARN cataract. These experiments suggest that HBO treatment of in vitro animal lenses may serve as a more economical and efficient model to study the development of cataract. Inspired by these experiments, we investigated whether exposure of young bovine lenses to HBO for 15 h could also serve as a suitable acute model of ARN cataract. We found that while this model is able to exhibit some of the biochemical and physiological changes associated with ARN cataract, the decrease in lens power we observed was more characteristic of the hyperopic shift in refraction associated with ageing. Future work will investigate whether HBO treatment to age the bovine lens in combination with an oxidative stressor such as UV light will induce refractive changes more closely associated with human ARN cataract. This will be important as developing an animal model that replicates the changes to lens biochemistry, physiology and optics observed in human ARN cataracts is urgently required to facilitate the identification and testing of anti-cataract therapies that are effective in humans.

Modeling congenital cataract in vitro using patient-specific induced pluripotent stem cells

Congenital cataracts are the leading cause of childhood blindness. To date, surgical removal of cataracts is the only established treatment, but surgery is associated with multiple complications, which often lead to visual impairment. Therefore, mechanistic studies and drug-candidate screening have been intrigued by the aims of developing novel therapeutic strategies. However, these studies have been hampered by a lack of an appropriate human-disease model of congenital cataracts. Herein, we report the establishment of a human congenital cataract in vitro model through differentiation of patient-specific induced pluripotent stem cells (iPSCs) into regenerated lenses. The regenerated lenses derived from patient-specific iPSCs with known causative mutations of congenital cataracts (CRYBB2 [p. P24T] and CRYGD [p. Q155X]) showed obvious opacification that closely resembled that seen in patients’ cataracts in terms of opacification severity and disease course accordingly, as compared with lentoid bodies (LBs) derived from healthy individuals. Increased protein aggregation and decreased protein solubility corresponding to the patients’ cataract severity were observed in the patient-specific LBs and were attenuated by lanosterol treatment. Taken together, the in vitro model described herein, which recapitulates patient-specific clinical manifestations of congenital cataracts and protein aggregation in patient-specific LBs, provides a robust system for research on the pathological mechanisms of cataracts and screening of drug candidates for cataract treatment.

Mutation of the EPHA2 Tyrosine-Kinase Domain Dysregulates Cell Pattern Formation and Cytoskeletal Gene Expression in the Lens.

Genetic variations in ephrin type-A receptor 2 (EPHA2) have been associated with inherited and age-related forms of cataract in humans. Here, we have characterized the eye lens phenotype and transcript profile of germline Epha2 knock-in mutant mice homozygous for either a missense variant associated with age-related cataract in humans (Epha2-Q722) or a novel insertion-deletion mutation (Epha2-indel722) that were both located within the tyrosine-kinase domain of EPHA2. Confocal imaging of ex vivo lenses from Epha2-indel722 mice on a fluorescent reporter background revealed misalignment of epithelial-to-fiber cell meridional-rows at the lens equator and severe disturbance of Y-suture formation at the lens poles, whereas Epha2-Q722 lenses displayed mild disturbance of posterior sutures. Immunofluorescent labeling showed that EPHA2 was localized to radial columns of hexagonal fiber cell membranes in Epha2-Q722 lenses, whereas Epha2-indel722 lenses displayed disorganized radial cell columns and cytoplasmic retention of EPHA2. Immunoprecipitation/blotting studies indicated that EPHA2 formed strong complexes with Src kinase and was mostly serine phosphorylated in the lens. RNA sequencing analysis revealed differential expression of several cytoskeleton-associated genes in Epha2-mutant and Epha2-null lenses including shared downregulation of Lgsn and Clic5. Collectively, our data suggest that mutations within the tyrosine-kinase domain of EPHA2 result in lens cell patterning defects and dysregulated expression of several cytoskeleton-associated proteins.

Analysis of amyloid-like secondary structure in the Cryab-R120G knock-in mouse model of hereditary cataracts by two-dimensional infrared spectroscopy.

αB-crystallin is a small heat shock protein that forms a heterooligomeric complex with αA-crystallin in the ocular lens. It is also widely distributed in tissues throughout the body and has been linked with neurodegenerative diseases such as Alzheimer’s, where it is associated with amyloid fibrils. Crystallins can form amorphous aggregates in cataracts as well as more structured amyloid-like fibrils. The arginine 120 to glycine (R120G) mutation in αB-crystallin (Cryab-R120G) results in high molecular weight crystallin protein aggregates and loss of the chaperone activity of the protein in vitro, and it is associated with human hereditary cataracts and myopathy. Characterizing the amorphous (unstructured) versus the highly ordered (amyloid fibril) nature of crystallin aggregates is important in understanding their role in disease and important to developing pharmacological treatments for cataracts. We investigated protein secondary structure in wild-type (WT) and Cryab-R120G knock-in mutant mouse lenses using two-dimensional infrared (2DIR) spectroscopy, which has been used to detect amyloid-like fibrils in human lenses and measure UV radiation-induced changes in porcine lenses. Our goal was to compare the aggregated proteins in this mouse lens model to human lenses and evaluate the protein structural relevance of the Cryab-R120G knock-in mouse model to general age-related cataract disease. In the 2DIR spectra, amide I diagonal peak frequencies were red-shifted to smaller wavenumbers in mutant mouse lenses as compared to WT mouse lenses, consistent with an increase in ordered secondary structure. The cross peak frequency and intensity indicated the presence of amyloid in the mutant mouse lenses. While the diagonal and cross peak changes in location and intensity from the 2DIR spectra indicated significant structural differences between the wild type and mutant mouse lenses, these differences were smaller than those found in human lenses; thus, the Cryab-R120G knock-in mouse lenses contain less amyloid-like secondary structure than human lenses. The results of the 2DIR spectroscopy study confirm the presence of amyloid-like secondary structure in Cryab-R120G knock-in mice with cataracts and support the use of this model to study age-related cataract.

Heat treatment of soluble proteins isolated from human cataract lens leads to the formation of non-fibrillar amyloid-like protein aggregates

The loss of crystallins solubility with aging and the formation of amyloid-like aggregates is considered the hallmark characteristic of cataract pathology. The present study was carried out to assess the effect of temperature on the soluble lens protein and the formation of protein aggregates with typical amyloid characteristics. The soluble fraction of lens proteins was subjected for heat treatment in the range of 40–60 °C, and the nature of protein aggregates was assessed by using Congo red (CR), thioflavin T (ThT), and 8-anilinonaphthalene-1-sulfonic acid (ANS) binding assays, circular dichroism (CD), Fourier-transform infrared (FT-IR) spectroscopy, and transmission electron microscopy (TEM). The heat-treated protein samples displayed a substantial bathochromic shift (≈15 nm) in the CR's absorption maximum (λmax) and increased ThT and ANS binding. The heat treatment of lens soluble proteins results in the formation of nontoxic, β-sheet rich, non-fibrillar, protein aggregates similar to the structures evident in the insoluble fraction of proteins isolated from the cataractous lens. The data obtained from the present study suggest that the exposure of soluble lens proteins to elevated temperature leads to the formation of non-fibrillar aggregates, establishing the role of amyloid in the heat-induced augmentation of cataracts pathology.