Human Cataract Formation Research Articles

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.

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.

A simplified method for producing human lens epithelial cells and light-focusing micro-lenses from pluripotent stem cells

Here we describe a modified method for harvesting tens-of-millions of human lens epithelial-like cells from differentiated pluripotent stem cell cultures. To assess the utility of this method, we analysed the lens cell population via: light microscopy; single cell RNA-sequencing and gene ontology analyses; formation of light-focusing micro-lenses; mass spectrometry; and electron microscopy. Both individually and collectively, the data indicate this simplified harvesting method provides a large-scale source of stem cell-derived lens cells and micro-lenses for investigating human lens and cataract formation.

Myricetin Prevents Cataract Formation by Inhibiting the Apoptotic Cell Death Mediated Cataractogenesis.

BackgroundThe current research work aimed to explore the protective role of myricetin against cataractogenesis in humans, in terms of its anti-apoptotic potential.Material/MethodsHuman eye lens epithelial cells were exposed to oxidative stress by treating with hydrogen peroxide (H2O2). The levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) were determined using standard detection kits. DAPI (4′,6-diamidino-2-phenylindole), AO/EB (acridine orange/ethidium bromide) and Annexin V/propidium iodide (PI) staining assays were used for the assessment of cell apoptosis. Western blotting was used to examine the protein concentrations.ResultsThe exposure of human epithelial eye lens cells to H2O2 led to significant accumulation of reactive oxygen species molecules. Treatment of the H2O2-stressed epithelial cells with myricetin caused significant (P<0.05) increased levels of SOD, CAT, and GSH. Western blot analysis also showed a significant (P<0.05) increase in the expression of SOD, CAT, and GSH levels in human epithelial eye lens cells. Additionally, myricetin administration to H2O2-treated epithelial eye lens cells caused a significant decline in cell apoptosis ratio. The induction of apoptosis was associated with upregulation of Bax and downregulation of Bcl-2.ConclusionsThe results of this study showed the potential of myricetin in protecting the apoptosis driven cataract formation in humans.

The hypoattenuating ocular lens on CT is not always due to cataract formation.

Hypoattenuating ocular lenses on CT have been described with cataract formation in humans, however published studies are currently lacking regarding this finding in veterinary patients. The purpose of this retrospective and prospective study was to describe the varying CT appearances of the ocular lens in vivo, and investigate the causes for CT density variations in a population of cats and dogs. A total of 102 canine and feline patients with CT of the head acquired at the authors' hospital between May 2011 and March 2019 were included. A bilateral hypoattenuating halo surrounding an isoattenuating to mildly hypoattenuating core was described in the ocular lens center of every cat in which a Philips brand proprietary image construction filter was used. A similar but more varied hypoattenuating region was noted in the lenses of 45.8% of dogs where the same filter was applied, as well as 43.8% of dogs with a second, similar filter. Ophthalmic examination of three live cats and one dog with hypoattenuating lenses demonstrated normal lens translucency, excluding the presence of cataract. The effect of different proprietary filters on lens appearance was also described in three fresh cadavers with normal lenses identified on ophthalmic, macroscopic, and microscopic examination. Etiology of the hypoattenuating areas within the ocular lens was not conclusively determined. Recognition that such a variant may be seen in the absence of cataract is important, in order to prevent misdiagnosis.

Structural and functional characterization of a missense mutant of human γS-crystallin associated with dominant infantile cataracts

Infantile cataracts constitute one of the most important causes of childhood blindness worldwide. Human γS-crystallin is the most abundant protein in the eye lens cortex. A missense mutant of human γS-crystallin, Y67N (abbreviated hereafter as γS-Y67N) is recently reported to be associated with dominant infantile cataracts. To understand the structural basis for γS-Y67N to cause lens opacification, we constructed, expressed and purified γS-Y67N and its wild-type (abbreviated hereafter as γS-WT) and studied the structural and functional differences between them in solution using circular dichroism (CD), differential scanning calorimetry (DSC), fluorescence spectroscopy and extrinsic spectral probes. Extensive equilibrium characterization indicate that replacement of the highly conserved Tyr at 67th position by Asn distorts the conserved Tyr corner at the second Greek key motif in the N-terminal domain (NTD) and leads to substantial loss of structural stability. Our intrinsic fluorescence quenching results reveal differential in-vitro refolding kinetics identifying partially folded kinetic intermediates for both proteins. Extrinsic fluorescence studies further reveal loosening up of the compact structure of γS-crystallin upon mutation associated with enhanced aggregation. As Ca2+ homeostasis is a crucial regulator of lens transparency, we further investigated the Ca2+-binding properties of γS-WT and γS-Y67N by isothermal titration calorimetry (ITC) to identify lens Ca2+ distribution in health and in disease. Overall, our results highlight the vital role of conserved Tyr corners in stabilizing Greek key motifs and provide useful structural and functional insights into the mechanism of cataract formation in humans.

Correction to: A zebrafish model of foxe3 deficiency demonstrates lens and eye defects with dysregulation of key genes involved in cataract formation in humans.

The authors noticed that Fig.5A and B aspect ratios appeared sub-optimal in the online published version. This has now been changed.

A zebrafish model of foxe3 deficiency demonstrates lens and eye defects with dysregulation of key genes involved in cataract formation in humans.

The Forkhead box E3 (FOXE3) gene encodes a transcription factor with a forkhead/winged helix domain that is critical for development of the lens and anterior segment of the eye. Monoallelic and biallelic deleterious sequence variants in FOXE3 cause aphakia, cataracts, sclerocornea and microphthalmia in humans. We used clustered regularly interspaced short palindromic repeats/Cas9 injections to target the foxe3 transcript in zebrafish in order to create an experimental model of loss of function for this gene. Larvae that were homozygous for an indel variant, c.296_300delTGCAG, predicting p.(Val99Alafs*2), demonstrated severe eye defects, including small or absent lenses and microphthalmia. The lenses of the homozygous foxe3 indel mutants showed more intense staining with zl-1 antibody compared to control lenses, consistent with increased lens fiber cell differentiation. Whole genome transcriptome analysis (RNA-Seq) on RNA isolated from wildtype larvae and larvae with eye defects that were putative homozygotes for the foxe3 indel variant found significant dysregulation of genes expressed in the lens and eye whose orthologues are associated with cataracts in human patients, including cryba2a, cryba1l1, mipa and hsf4. Comparative analysis of this RNA-seq data with iSyTE data identified several lens-enriched genes to be down-regulated in foxe3 indel mutants. We also noted upregulation of lgsn and crygmxl2 and downregulation of fmodb and cx43.4, genes that are expressed in the zebrafish lens, but that are not yet associated with an eye phenotype in humans. These findings demonstrate that this new zebrafish foxe3 mutant model is highly relevant to the study of the gene regulatory networks conserved in vertebrate lens and eye development.

Telomere Attrition in Human Lens Epithelial Cells Associated with Oxidative Stress Provide a New Therapeutic Target for the Treatment, Dissolving and Prevention of Cataract with N-Acetylcarnosine Lubricant Eye Drops. Kinetic, Pharmacological and Activity-Dependent Separation of Therapeutic Targeting: Transcorneal Penetration and Delivery of L-Carnosine in the Aqueous Humor and Hormone-Like Hypothalamic Antiaging Effects of the Instilled Ophthalmic Drug Through a Safe

Visual impairment broadly impacts the ability of affected people to maintain their function and to remain independent during their daily occupations as they grow older. Visual impairment affects survival of older patients, quality of life, can affect a person's self-ranking of health, may be associated with social and functional decline, use of community support services, depression, falls, nursing home placement, and increased mortality. It has been hypothesized that senile cataract may serve as a marker for generalised tissue aging, since structural changes occurring in the proteins of the lens during cataract formation are similar to those which occur elsewhere as part of the aging process. The published analysis revealed a strong age-dependent relationship between undergoing cataract surgery and subsequent mortality. Nuclear opacity, particularly severe nuclear opacity, and mixed opacities with nuclear were significant predictors of mortality independent of body mass index, comorbid conditions, smoking, age, race, and sex. The lens opacity status is considered as an independent predictor of 2-year mortality, an association that could not be explained by potential confounders. Telomeres have become important biomarkers for aging as well as for oxidative stress-related disease. The lens epithelium is especially vulnerable to oxidative stress. Oxidative damage to the cuboidal epithelial cells on the anterior surface of the lens mediated by reactive oxygen species and phospholipid hydroperoxides can precede and contribute to human lens cataract formation. The erosion and shortening of telomeres in human lens epithelial cells in the lack of telomerase activity has been recognized as a primary cause of premature lens senescence phenotype that trigger human cataractogenesis. In this study we aimed to be focused on research defining the mechanisms that underlie linkages among telomere attrition in human lens epithelial cells associated with oxidative stress, biology of the lens response to oxidative damages, aging and health, cataract versus neuroendocrine regulation and disease. The cumulative results demonstrate that carnosine, released ophthalmically from the patented 1% Nacetylcarnosine prodrug lubricant eye drops, at physiological concentration might remarkably reduce the rate of telomere shortening in the lens cells subjected to oxidative stress in the lack of efficient antioxidant lens protection. Carnosine promotes the protection of normal cells from acquiring phenotypic characteristics of cellular senescence. The data of visual functions (visual acuity, glare sensitivity) in older adult subjects and older subjects with cataract treated with 1% N-acetylcarnosine lubricant eye drops showed significant improvement as compared, by contrast with the control group which showed generally no improvement in visual functions, with no difference from baseline in visual acuity and glare sensitivity readings. N-acetylcarnosine derived from the lubricant eye drops may be transported into the hypothalamic tuberomammillary nucleus (TMN) histamine neurons and gradually hydrolyzed. The resulting L-histidine may subsequently be converted into histamine, which could be responsible for the effects of carnosine on neurotransmission and hormone-like antiaging and anti-cataract physiological function. The research utilizing the N-acetylcarnosine lubricant eye drops powerful therapeutic platform provides the findings related to the intraocular uptake exposure sources as well as a timing dosage and duration systemic absorption of said preparation from the conjunctional sac reaching the hypothalamus with activities transfer into the hypothalamic-neuroendocrine pathways affecting across the hypothalamus metabolic pathway the telomere biology and cataract disease occurrence, reversal and prevention and the average expected lifespan of an individual. Such findings can be translated into clinical practice and may provide a basis for personalized cataract disease and aging prevention and treatment approaches.

Site specific oxidation of amino acid residues in rat lens γ-crystallin induced by low-dose γ-irradiation

Although cataracts are a well-known age-related disease, the mechanism of their formation is not well understood. It is currently thought that eye lens proteins become abnormally aggregated, initially causing clumping that scatters the light and interferes with focusing on the retina, and ultimately resulting in a cataract. The abnormal aggregation of lens proteins is considered to be triggered by various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, that occur during the aging process. Such modifications, which are also generated by free radical and reactive oxygen species derived from γ-irradiation, decrease crystallin solubility and lens transparency, and ultimately lead to the development of a cataract. In this study, we irradiated young rat lenses with low-dose γ-rays and extracted the water-soluble and insoluble protein fractions. The water-soluble and water-insoluble lens proteins were digested with trypsin, and the resulting peptides were analyzed by LC-MS. Specific oxidation sites of methionine, cysteine and tryptophan in rat water-soluble and -insoluble γE and γF-crystallin were determined by one-shot analysis. The oxidation sites in rat γE and γF-crystallin resemble those previously identified in γC and γD-crystallin from human age-related cataracts. Our study on modifications of crystallins induced by ionizing irradiation may provide useful information relevant to human senile cataract formation.

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization

Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide–mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways.

Selenite cataracts: Activation of endoplasmic reticulum stress and loss of Nrf2/Keap1-dependent stress protection

Cataract-induced by sodium selenite in suckling rats is one of the suitable animal models to study the basic mechanism of human cataract formation. The aim of this present investigation is to study the endoplasmic reticulum (ER) stress-mediated activation of unfolded protein response (UPR), overproduction of reactive oxygen species (ROS), and suppression of Nrf2/Keap1-dependent antioxidant protection through endoplasmic reticulum-associated degradation (ERAD) pathway and Keap1 promoter DNA demethylation in human lens epithelial cells (HLECs) treated with sodium selenite. Lenses enucleated from sodium selenite injected rats generated overproduction of ROS in lens epithelial cells and newly formed lens fiber cells resulting in massive lens epithelial cells death after 1–5days. All these lenses developed nuclear cataracts after 4–5days. Sodium selenite treated HLECs induced ER stress and activated the UPR leading to release of Ca2+ from ER, ROS overproduction and finally HLECs death. Sodium selenite also activated the mRNA expressions of passive DNA demethylation pathway enzymes such as Dnmt1, Dnmt3a, and Dnmt3b, and active DNA demethylation pathway enzyme, Tet1 leading to DNA demethylation in the Keap1 promoter of HLECs. This demethylated Keap1 promoter results in overexpression of Keap1 mRNA and protein. Overexpression Keap1 protein suppresses the Nrf2 protein through ERAD leading to suppression of Nrf2/Keap1 dependent antioxidant protection in the HLECs treated with sodium selenite. As an outcome, the cellular redox status is altered towards lens oxidation and results in cataract formation.

UVA Light-excited Kynurenines Oxidize Ascorbate and Modify Lens Proteins through the Formation of Advanced Glycation End Products: IMPLICATIONS FOR HUMAN LENS AGING AND CATARACT FORMATION

Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320-400 nm, 100 milliwatts/cm(2), 45 min to 2 h), young human lenses (20-36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.

In vivo substrates of the lens molecular chaperones αA-crystallin and αB-crystallin.

αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin.

A Novel p.G112E Mutation in BFSP2 Associated with Autosomal Dominant Pulverulent Cataract with Sutural Opacities

Purpose: To identify the genetic defect in a Chinese family with bilateral pulverulent sutural cataract.Materials and methods: A three-generation family with congenital cataract was recruited in the study. The study protocol followed the principles of the Declaration of Helsinki. Detailed family history and clinical data were recorded. Genomic DNA was extracted from peripheral blood leukocytes. Candidate gene sequencing was performed to identify the disease-causing mutation. The effects of amino acid changes on the structure and function of proteins were predicted by bioinformatics analysis.Results: All affected individuals presented pulverulent opacities in the embryonal nucleus and sutures. Direct candidate gene sequencing revealed a heterozygous c. 335 G>A variation in the beaded filament structural protein 2(BFSP2) gene, which resulted in the replacement of a highly conserved glycine by glutamic at codon 112 (p. G112E). Haplotype analysis indicated that the affected members shared a common haplotype with markers near BFSP2. This mutation co-segregated with all affected individuals and was not observed in unaffected members or in 120 ethnically matched controls. Bioinformatic analyses confirmed that the mutation altered the hydrophobic and secondary structure of the protein around the substitution site.Conclusions: We report a novel mutation (p.G112E) in the BFSP2 gene, underscoring the physiological importance of the beaded filament protein and supporting its role in human cataract formation.

Presence and distribution of l-kynurenine aminotransferases immunoreactivity in human cataractous lenses

To investigate the presence and distribution of l-kynurenine aminotransferases immunoreactivity in human and animal lenses during cataract formation. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, KAT II and KAT III on sections of 26 anterior capsules from patients undergoing surgical treatment of anterior subcapsular cataract (ASC) and 22 cataractous lenses from human eyes enucleated because of choroidal malignant melanoma. Additionally, the eyes of 11-month-old DBA/2J mice (6 eyes) were investigated (with KAT I and II). Ten clear human lenses and four BL6 mice lenses were used as controls. Spatial immunoreactivity patterns of enzymes were compared with Periodic Acid - Schiff (PAS)-stained sections. Immunohistochemical analysis revealed presence of KAT I, KAT II and KAT III in extracellular structures of all studied types of cataract in human eyes showing specific pattern of the stain. In cortical cataract, immunoreactivity was observed on cortical lens fibres. In nuclear cataract, KAT II revealed stronger and diffused staining than KAT I. Additionally, both KAT showed more pronounced staining at the edge of small clefts. In normal human lenses, KAT I, II and III, immunoreactivity was not observed. Presence of KAT I and KAT II in the intercellular substance of DBA/2J mice cataract was observed. In BL6 mice lenses without cataract, only weak KAT I and KAT II staining was observed. Presence of l-kynurenine aminotransferases in extracellular matrix (ECM) during human cataract formation suggests that products of l-kynurenine pathway might be involved in mechanisms of cataractogenesis.

Accelerated aging of Asp 58 in αA crystallin and human cataract formation

Racemisation of amino acids is one of the most abundant modifications in long-lived proteins. In this study racemisation of Asp 58 in the small heat shock protein, αA crystallin, was investigated. In normal human lenses, levels of l-isoAsp, d-isoAsp and d-Asp increased with age, such that by age 70 they accounted for approximately half of the total Asp at this site. Levels of d-isoAsp were significantly higher in all cataract lenses than age-matched normal lenses. The introduction of d-isoAsp in αA crystallin could therefore be associated with the development of cataract. Its more rapid formation in cataract lenses may represent an example of accelerated protein aging leading to a human age-related disease.

A Class I UV-blocking (senofilcon A) soft contact lens prevents UVA-induced yellow fluorescence and NADH loss in the rabbit lens nucleus in vivo

It is known that fluorescence, much of it caused by UVA light excitation, increases in the aging human lens, resulting in loss of sharp vision. This study used an in vivo animal model to investigate UVA-excited fluorescence in the rabbit lens, which contains a high level of the UVA chromophore NADH, existing both free and bound to λ-crystallin. Also, the ability of a Class I (senofilcon A) soft contact lens to protect against UVA-induced effects on the rabbit lens was tested. Rabbit eyes were irradiated with UVA light in vivo (100 mW/cm2 on the cornea) for 1 h using monochromatic 365 nm light. Irradiation was conducted in the presence of either a senofilcon A contact lens, a minimally UV-absorbing lotrafilcon A contact lens, or no contact lens at all. Eyes irradiated without a contact lens showed blue 365 nm-excited fluorescence initially, but this changed to intense yellow fluorescence after 1 h. Isolated, previously irradiated lenses exhibited yellow fluorescence originating from the lens nucleus when viewed under 365 nm light, but showed normal blue fluorescence arising from the cortex. Previously irradiated lenses also exhibited a faint yellow color when observed under visible light. The senofilcon A contact lens protected completely against the UVA-induced effects on fluorescence and lens yellowing, whereas the lotrafilcon A lens showed no protection. The UVA-exposure also produced a 53% loss of total NADH (free plus bound) in the lens nucleus, with only a 13% drop in the anterior cortex. NADH loss in the nucleus was completely prevented with use of a senofilcon A contact lens, but no significant protection was observed with a lotrafilcon A lens. Overall, the senofilcon A lens provided an average of 67% protection against UVA-induced loss of four pyridine nucleotides in four different regions of the lens. HPLC analysis with fluorescence detection indicated a nearly six-fold increase in 365 nm-excited yellow fluorescence arising from lens nuclear λ-crystallin after the in vivo UVA exposure. It is concluded that UVA-induced loss of free NADH (which fluoresces blue) may have allowed the natural yellow fluorescence of λ-crystallin and other proteins in the lens nucleus to become visible. Increased fluorescence exhibited by UVA-exposed λ-crystallin may have been the result of a UVA-induced change in the conformation of the protein occurring during the initial UVA-exposure in vivo. The results demonstrate the greater susceptibility of the lens nucleus to UVA-induced stress, and may relate to the formation of human nuclear cataract. The senofilcon A contact lens was shown to be beneficial in protecting the rabbit lens against effects of UVA light, including changes in fluorescence, increased yellowing and loss of pyridine nucleotides.

Electron tomography of fiber cell cytoplasm and dense cores of multilamellar bodies from human age-related nuclear cataracts

Human nuclear cataract formation is a multi-factorial disease with contributions to light scattering from many cellular sources that change their scattering properties over decades. The aging process produces aggregation of cytoplasmic crystallin proteins, which alters the protein packing and texture of the cytoplasm. Previous studies of the cytoplasmic texture quantified increases in density fluctuations in protein packing and theoretically predicted the corresponding scattering. Multilamellar bodies (MLBs) are large particles with a core of crystallin cytoplasm that have been suggested to be major sources of scattering in human nuclei. The core has been shown to condense over time such that the refractive index increases compared to the adjacent aged and textured cytoplasm. Electron tomography is used here to visualize the 3D arrangement of protein aggregates in aged and cataractous lens nuclear cytoplasm compared to the dense protein packing in the cores of MLBs. Thin sections, 70 nm thick, were prepared from epoxy-embedded human transparent donor lenses and nuclear cataracts. Tilt series were collected on an FEI T20 transmission electron microscope (TEM) operated at 200 kV using 15 nm gold particles as fiducial markers. Images were aligned and corrected with FEI software and reconstructed with IMOD and other software packages to produce animated tilt series and stereo anaglyphs. The 3D views of protein density showed the relatively uniform packing of proteins in aged transparent lens nuclear cytoplasm and less dense packing of aged cataractous cytoplasm where many low-density regions can be appreciated in the absence of the TEM projection artifacts. In contrast the cores of the MLBs showed a dense packing of protein with minimal density fluctuations. These observations support the conclusion that, during the nuclear cataract formation, alterations in protein packing are extensive and can result in pronounced density fluctuations. Aging causes the MLB cores to become increasingly different in their protein packing from the adjacent cytoplasm. These results support the hypothesis that the MLBs increase their scattering with age and nuclear cataract formation.

A novel mutation impairing the tertiary structure and stability of γC-crystallin (CRYGC) leads to cataract formation in humans and zebrafish lens

Congenital cataract is one of the leading causes of human blindness. In this study, we identified a novel, heterozygous c.385G<T mutation in CRYGC that resulted in the substitution of a highly conserved glycine by cysteine at codon 129 (p.Gly129Cys) in a three-generation Chinese family with autosomal dominant congenital nuclear cataract by sequencing candidate genes. Using zebrafish as a model, we demonstrated that γC-crystallin p.Gly129Cys mutant caused the vacuole and the incomplete denucleation of lens, recapitulating the cataract phenotype in human beings. Molecular modeling and spectroscopic studies indicated that the mutation impaired the tertiary structure of the protein by modifying the H-bonding network in the C-terminal domain. The mutation led to a dramatic decrease in the thermal stability of γC-crystallin, and a significant increase in the propensity of aggregation when subject to storage at high concentrations, heat, and UV- irradiation stresses. Taken together, these results indicate that a novel γC-crystallin p.Gly129Cys mutation impaired the tertiary structure of the protein and caused cataract formation, which provides a new insight into how the mutation may affect the γC-crystallin structure, stability, and function. Our study also highlighted zebrafish as a valuable model tool for studying congenital inherited cataract.