Moderate caloric restriction delays cataract formation in the Emory mouse

Eye lens senile cataract is a major cause of blindness, affecting the elderly in particular. The etiology of the disorder has been elusive, and attempts to delay the onset of senile cataracts have been unsuccessful. The need for more information is underscored by epidemiologists who estimate that the ability to delay cataract formation in humans by only 10 years would eliminate the need for 50% of the cataract extractions performed annually in the United States. The Emory mouse provides the best model for human senile cataracts. Feeding Emory mice a diet that was restricted in calories by approximately 21% delayed the onset of cataracts. This is the first study that demonstrates in vivo the delay of senile-type cataracts. In these animals, aging and cataracts are associated with diverse changes in the proportion of various proteins (particularly 21, 22, 31-34 kDa) and with transformation of proteins from a soluble to an insoluble state. In advanced cataracts, there is a loss of total protein. Within a cataract grade, there is no difference between restricted and nonrestricted animals in relative proportion of specific lens proteins or in amounts of total or soluble proteins. The transition from a clear to cataractous lens appears when the soluble-to-total protein ratio falls below about 0.58. The exclusive use of gamma-crystallin as an indicator of lens viability is questioned. To the extent that cataract formation is due to lens protein oxidation and/or an inability to proteolytically remove damaged protein, it would appear that caloric restriction results in enhanced protection against lens oxidative stress or in prolonged proteolytic function.     

Increased O-GlcNAc causes disrupted lens fiber cell differentiation and cataracts

Diminished proteolytic functionality in the lens may cause cataracts. We have reported that O-GlcNAc is an endogenous inhibitor of the proteasome. We hypothesize that in the lens there is a cause-and-effect relationship between proteasome inhibition by O-GlcNAc, and cataract formation. To demonstrate this, we established novel transgenic mouse models to over-express a dominant-negative form of O-GlcNAcase, GK-NCOAT, in the lens. Expression of GK-NCOAT suppresses removal of O-GlcNAc from proteins, resulting in increased levels of O-GlcNAc in the lenses of our transgenic mice, along with decreased proteasome function. We observed that transgenic mice developed markedly larger cataracts than controls and lens fiber cell denucleation was inhibited. Our study suggests that increased O-GlcNAc in the lens could lead to cataract formation and attenuation of lens fiber cell denucleation by inhibition of proteasome function. These findings may explain why cataract formation is a common complication of diabetes since O-GlcNAc is derived from glucose.     

TSR1突变导致先天性白内障及其在晶状体中的表达

先天性白内障(congenital cataract,CC)是一种罕见的晶状体发育异常疾病,主要表现为晶状体部分或完全浑浊。先天性白内障遗传异质性高,已鉴定的致病基因多达266个。本研究在一个中国先天性白内障家系中通过全基因组测序及Sanger测序验证,筛查到一个新的先天性白内障候选致病基因TSR1,与家系疾病表型共分离。通过minigene实验证实该变异影响TSR1基因mRNA剪接。Western blotting、免疫荧光和RT-PCR实验证实TSR1在人晶状体上皮细胞SRA01/04、年龄相关性白内障患者晶状体前囊膜组织、24周人胎眼晶状体和小鼠晶状体中表达。通过对iSyTE数据库的分析发现,Tsr1在小鼠的胚胎期和不同发育时期的晶状体中都有表达,且在晶状体特异性CBP:p300双敲除小鼠中Tsr1表达下调。提取在CBP:p300双敲除小鼠晶状体中与Tsr1具有相同表达模式的一组基因进行蛋白质-蛋白质相互作用网络(protein-protein interaction,PPI)分析,结果表明筛选出6个基因与Tsr1存在直接相互作用。GO功能分析表明Tsr1参与核糖体的组装,还可能在MAPK-Erk信号通路中发挥作用,为进一步明确Tsr1在晶状体中的功能提供了有价值的研究线索。     

Crystallins and hereditary cataracts: molecular mechanisms and potential for therapy

Hereditary childhood cataracts can arise from single-point mutations in genes encoding crystallins, the major protein components of the lens. The cataracts are most commonly inherited by an autosomal dominant mechanism. The nature of the changes in the lens resulting from these point mutations in crystallin genes has not been fully characterised. While aggregation and light scattering associated with expression of the mutant crystallin protein may be an end point, it is also necessary to determine the progression of changes induced at the level of development and differentiation. A key finding in recent work is that cell death or cytotoxicity is associated with mutations in alpha A-crystallin. The variable morphology or localisation of the cataract in different pedigrees, even with the identical crystallin gene mutation, has led to the idea that other environmental or genetic factors interact to give the final lens phenotype. The study of mechanisms of formation of hereditary cataracts may lead to a greater understanding of the mechanisms that lead to age-related cataracts, a very common cause of blindness in the ageing population.     

遗传性白内障动物模型的研究进展

白内障是严重影响公众健康的重大疾病。人群中, 大部分遗传性白内障是外显率较高的常染色体显性遗传, 但也有X连锁和常染色体隐性遗传存在。随着分子生物学技术的发展, 出现了许多白内障遗传动物模型, 这些模型有助于揭示白内障的发病机制, 为晶状体的发育和生理学研究提供新的见解, 还有助于进一步了解遗传、环境和营养等因素对晶状体的作用方式, 并为白内障遗传病的诊断和治疗提供依据。文章综述了遗传性白内障动物模型的相关基因、突变形式及其研究进展。     

Expression changes in mRNAs and mitochondrial damage in lens epithelial cells with selenite

An overdose of sodium selenite induces cataracts in young rats. The mid-stage events producing the cataract include calpain-induced hydrolysis and precipitation of lens proteins. Apoptosis in lens epithelial cells has been suggested as an initial event in selenite cataracts. Expression levels of two genes associated with apoptosis were altered in lens epithelial cells from selenite-injected rats. The purpose of the present experiment was to perform a more comprehensive search for changes in expression of mRNAs in lens epithelial cells in order to more fully delineate the early events in selenite-induced cataracts. Lens epithelial cells were harvested at 1 and 2 days after a single subcutaneous injection of sodium selenite (30 mumol/kg body weight) into 12-day-old rats. Gene expression was analyzed using a commercial DNA array (Rat Genome U34A GeneChip array, Affymetrix). Of approximately 8000 genes assayed by hybridization, 13 genes were decreased and 27 genes were increased in the rat lens epithelial cells after injection of selenite. Some of the up-regulated genes included apoptosis-related genes, and a majority of the down-regulated genes were mitochondrial genes. Previously observed changes in expression of EGR-1 mRNA were also confirmed. Changes in the expression patterns of mRNAs were also confirmed by RT-PCR. To determine the mechanism for damage of lens epithelial cells (alpha TN4 cell) by culture in selenite, leakage of cytochrome c from mitochondria was measured. Selenite caused significant leakage of cytochrome c into the cytosol of alpha TN4 cells. Our data suggested that the loss of integrity of lens epithelial cells by selenite might be caused by preferential down-regulation of mitochondrial RNAs, release of cytochrome c, and impaired mitochondrial function. Up-regulation of mRNAs involved in maintenance of DNA, regulation of metabolism, and induction of apoptosis may also play roles.     

Role of the non-enzymatic glycosylation of protein in the formation of human cataracts

We have measured the free epsilon amino groups in soluble and insoluble proteins of clear human lenses and diabetic and non-diabetic senile cataractous lenses. The free epsilon amino groups content of soluble and insoluble proteins was significantly lower in diabetic cataracts than in clear lenses and non diabetic senile cataracts. Our results seem to demonstrate that non-enzymatic glycosylation of lens protein could play a role in the pathogenesis of cataract in diabetes.     

Degradation of crystallins from a psoriatic patient undergoing PUVA therapy

Comparative physico-chemical and spectroscopic analyses were carried out in human lens proteins obtained from extracts of normal, senile and PUVA cataracts. Mass recovery analysis reveals a large protein concentration loss in the PUVA cataract relative to the normal lens and senile cataract. This protein loss parallels an increase in the degraded polypeptide chains. However, the tryptophan content (2.1 mol/mol of 20 kDa protein subunit) and the apparent fluorescence quantum yield (phi f = 0.056) of the tryptophan residues which are believed to be involved in the development of UV-induced cataracts are unchanged after age-related alterations and/or in vivo photochemistry associated with psoralen (8MOP) photosensitized reactions.     

High prevalence of cataracts in birds with pheomelanin-based colouration

The crystalline lens of the eyes of vertebrates focuses light on the retina. Therefore, maintaining the lens clear is necessary for proper visual function. However, oxidative damage to proteins of the lens leads to opacification and lens dysfunction, termed cataract. Antioxidants thus have a role in avoiding the development of cataracts through their reduction of oxidative stress, and glutathione (GSH), a key intracellular antioxidant, belongs to the primary antioxidant defence mechanism of the lens. Other physiological mechanisms that require GSH may compete with the antioxidant mechanism of the eye. Pheomelanin is a main type of melanin, the most common pigment in vertebrates, and its synthesis consumes GSH. Here, we use data on 81 bird species to test the hypothesis that species producing large amounts of pheomelanin should have diminished capacity to use GSH to protect their eyes and, as a consequence, higher prevalence of cataracts. As predicted, the proportion of pheomelanic plumage was positively associated with the proportion of individuals with cataracts across species, suggesting that production of pheomelanin may have profound fitness consequences, as birds with cataracts have limited ability to perform vital activities. This constitutes the first comparative study of cataracts in wild animals.     

Altered chaperone-like activity of alpha-crystallins promotes cataractogenesis

Despite the enormous number of studies demonstrating changes in the chaperone-like activity of α-crystallins in vitro, little is known about how these changes influence life-long lens transparency in vivo. Using the γB-crystallin I4F mutant protein as a target for αA-crystallins, we examined how cataract phenotypes are modulated by interactions between α-crystallins with altered chaperone-like activities and γB-I4F proteins in vivo. Double heterozygous α-crystallin knock-out αA(+/-) αB(+/-) mice with a decreased amount of α-crystallins were used to simulate reduced total α-crystallin chaperone-like activity in vivo. We found that triple heterozygous αA(+/-) αB(+/-) γB(I4F/+) mice developed more severe whole cataracts than heterozygous γB(I4F/+) mice. Thus, total chaperone-like activity of α-crystallins is important for maintaining lens transparency. We further tested whether mutant αA-crystallin Y118D proteins with increased chaperone-like activity influenced the whole cataract caused by the γB-I4F mutation. Unexpectedly, compound αA(Y118D/+) γB(I4F/+) mutant lenses displayed severe nuclear cataracts, whereas the lens cortex remained unaffected. Thus, the synergistic effect of αA-Y118D and γB-I4F mutant proteins is detrimental to the transparency only in the lens core. α-Crystallins with different chaperone-like activities are likely required in the lens cortex and nucleus for maintaining transparency.     

A juvenile-onset, progressive cataract locus on chromosome 3q21-q22 is associated with a missense mutation in the beaded filament structural protein-2

Juvenile-onset cataracts are distinguished from congenital cataracts by the initial clarity of the lens at birth and the gradual development of lens opacity in the second and third decades of life. Genomewide linkage analysis in a multigenerational pedigree, segregating for autosomal dominant juvenile-onset cataracts, identified a locus in chromosome region 3q21.2-q22.3. Because of the proximity of the gene coding for lens beaded filament structural protein-2 (BFSP2) to this locus, we screened for mutations in the coding sequence of BFSP2. We observed a unique C-->T transition, one that was not observed in 200 normal chromosomes. We predicted that this led to a nonconservative R287W substitution in exon 4 that cosegregated with cataracts. This mutation alters an evolutionarily conserved arginine residue in the central rod domain of the intermediate filament. On consideration of the proposed function of BFSP2 in the lens cytoskeleton, it is likely that this alteration is the cause of cataracts in the members of the family we studied. This is the first example of a mutation in a noncrystallin structural gene that leads to a juvenile-onset, progressive cataract.     

Diverse roles of Eph/ephrin signaling in the mouse lens

Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout (-/-) mice often display anterior polar cataracts while EphA2(-/-) lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5(-/-) lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5(-/-) lenses. Moreover, the disruption of membrane-associated β-catenin and E-cadherin junctions is observed in ephrin-A5(-/-) lens central epithelial cells. In contrast, EphA2(-/-) lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates β-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells.     

Cataract induction by products of lipid peroxidation

Liposome suspension prepared from the unsaturated phospholipids exposed to lipid peroxidation (LPO) induced posterior subcapsular cataracts after injection into the posterior vitreous of rabbit eyes. In the background of this model lies a type of lens opacity formed during retinal degeneration when toxic peroxide substances diffuse anteriorly through the vitreous body resulting in vitreous opacities and complicated cataracts. Saturated liposomes (prepared from beta-oleoyl-gamma-palmitoyl) L-alpha-lecithin) did not induce lens opacities, which is the evidence that a lipid peroxidation mechanism may be responsible for the posterior cataracts. Along with cataract formation accumulation of LPO fluorescent products in vitreous, aqueous humor and lens was observed. It was followed by a decreased level of reduced glutathione in the lens. The obtained results strongly support the hypothesis of LPO initial role in cataracts.     

A Missense Mutation in CRYBB2 Leads to Progressive Congenital Membranous Cataract by Impacting the Solubility and Function of βB2-Crystallin

Congenital cataract is a major cause of visual impairment and childhood blindness. The solubility and stability of crystallin proteins play critical roles in maintaining the optical transparency of the lens during the life span. Previous studies have shown that approximately 8.3%∼25% of congenital cataracts are inherited, and mutations in crystallins are the most common. In this study, we attempted to identify the genetic defect in a four-generation family affected with congenital cataracts. The congenital cataract phenotype of this four-generation family was identified as membranous cataract by slit-lamp photography. Mutation screening of the candidate genes detected a heterozygous c.465G→C change in the exon6 of the βB2-crystallin gene (CRYBB2) in all family members affected with cataracts, resulting in the substitution of a highly conserved Tryptophan to Cystine (p.W151C). The mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis and found that the transition resulted in the absence of a BslI restriction site in the affected members of the pedigree. The outcome of PolyPhen-2 and SIFT analysis predicted that this W151C mutation would probably damage to the structure and function of βB2-crystallin. Wild type (wt) and W151C mutant βB2-crystallin were expressed in human lens epithelial cells (HLECs), and the fluorescence results showed that Wt-βB2-crystallin was evenly distributed throughout the cells, whereas approximately 34.7% of cells transfected with the W151C mutant βB2-crystallin formed intracellular aggregates. Taken together, these data suggest that the missense mutation in CRYBB2 gene leads to progressive congenital membranous cataract by impacting the solubility and function of βB2-crystallin.     

Protective effects of carnosine on dehydroascorbate-induced structural alteration and opacity of lens crystallins: important implications of carnosine pleiotropic functions to combat cataractogenesis

The high level of dehydroascorbic acid (DHA) in the lenticular tissue is an important risk factor for the development of age-related cataracts. In this study, the effects of DHA on structure and function of lens crystallins were studied in the presence of carnosine using gel mobility shift assay, different spectroscopic techniques, and lens culture analysis. The DHA-induced unfolding and aggregation of lens proteins were largely prevented by this endogenous dipeptide. The ability of carnosine to preserve native protein structure upon exposure to DHA suggests the essential role of this dipeptide in prevention of the senile cataract development. Although the DHA-modified α-crystallin was characterized by altered chaperone activity, functionality of this protein was significantly restored in the presence of carnosine. The increased proteolytic instability of DHA-modified lens proteins was also attenuated in the presence of carnosine. Furthermore, the assessment of lens culture suggested that DHA can induce significant lens opacity which can be prevented by carnosine. These observations can be explained by the pleiotropic functions of this endogenous and pharmaceutical compound, notably by its anti-glycation and anti-aggregation properties. In summary, our study suggests that carnosine may have therapeutic potential in preventing senile cataracts linked with the increased lenticular DHA generation, particularly under pathological conditions associated with the oxidative stress.     

Role of nonenzymatic glycosylation in experimental cataract formation

The relevance of nonenzymatic glycosylation of lens proteins to cataract formation was studied in rats on a normal and high galactose diet, treated with and without sorbinil, an aldose reductase inhibitor. All galactosemic rats not receiving sorbinil had cataracts; none receiving sorbinil had cataracts. Lens homogenate was treated with a 200 fold molar excess of [³H]-borohydride and the extent of glycosylation was estimated from radioactivity incorporation and quantitation of hexitol-lysine adduct after extensive dialysis. We found no differences in the radioactivity uptake nor the amounts of hexitol-lysine in the lenses of galactosemic rats treated with and without sorbinil. Thus, nonenzymatic glycosylation was not responsible for the sugar-induced cataracts.