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.     

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.     

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.     

AlphaA-crystallin expression prevents gamma-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens

Cataracts, the loss of lens transparency, are the leading cause of human blindness. The zebrafish embryo, with its transparency and relatively large eyes, is an excellent model for studying ocular disease in vivo. We found that the zebrafish cloche mutant, both the cloche(m39) and cloche(S5) alleles, which have defects in hematopoiesis and blood vessel development, also have lens cataracts. Quantitative examination of the living zebrafish lens by confocal microscopy showed significant increases in lens reflectance. Histological analysis revealed retention of lens fiber cell nuclei owing to impeded terminal differentiation. Proteomics identified gamma-crystallin as a protein that was substantially diminished in cloche mutants. Crystallins are the major structural proteins in mouse, human and zebrafish lens. Defects in crystallins have previously been shown in mice and humans to contribute to cataracts. The loss of gamma-crystallin protein in cloche was not due to lowered mRNA levels but rather to gamma-crystallin protein insolubility. AlphaA-crystallin is a chaperone that protects proteins from misfolding and becoming insoluble. The cloche lens is deficient in both alphaA-crystallin mRNA and protein during development from 2-5 dpf. Overexpression of exogenous alphaA-crystallin rescued the cloche lens phenotype, including solubilization of gamma-crystallin, increased lens transparency and induction of lens fiber cell differentiation. Taken together, these results indicate that alphaA-crystallin expression is required for normal lens development and demonstrate that cataract formation can be prevented in vivo. In addition, these results show that proteomics is a valuable tool for detecting protein alterations in zebrafish.     

Determination of calcium, sodium, potassium and magnesium concentrations in human senile cataractous lenses

Cataractous lenses have been found to have a distribution of the intracellular ionic environment, the concentrations of potassium and magnesium decreasing and the concentrations of sodium and calcium increasing relative to the cytosol of most cells. This arises as a result of changes to lens membrane characteristics causing an increase in lens membrane permeability. These changes have been found to be initiated as a result of normal ageing of the human lens. In this study, total Ca2+, K+, Na+ and Mg2+ contents have been determined in human normal and cataractous lenses using atomic absorption and flame emission spectroscopy. The normal human lens Ca2+ is between 0.15 and 0.5 miromol g(-1) fresh lens weight; in senile cataracts the value increased up to 9.31 micromol g(-1) ( p < 0.0001). The normal levels of Na+, Mg2+ and K+ are 20, 5.5 and 60 micromol g(-1) respectively; these changed to 136.10, 3.60 and 9.33 micro mol g(-1), respectively in cataractous senile human lenses ( p < 0.002, p < 0.002 and p < 0.01). The remarkable differences in these elements may play some role in cataractogenesis.     

Glucosylation of human lens protein and cataractogenesis.

Examination of glucosylation of lens protein was conducted utilizing tritiated BH₄^?. The overall results indicate that approximately 0.20 moles of tritium were incorporated per mole of protein. Similar results were obtained with normal and senile cataractous lenses with varying degrees of opacity. Furthermore no difference in the ³H incorporation was observed between soluble and insoluble protein fractions derived from these lenses. Investigation of selected polypeptides isolated from the senile cataracts gave comparable results. Protein isolated from diabetic lenses had only slightly higher levels of tritium incorporation, giving an average value of 0.27 moles per mole of protein. Analyses of the tritiated products indicate that approximately 50% of the incorporation is probably due to reduction of other types of compounds. These results suggest that glucosylation does not appear to be a primary factor in cataract formation.     

Crystalline lens induction of lipid peroxidation

The level of lipid peroxidation products (LPP) was determined in the aqueous humor from the anterior chamber of patients with cataract and donor eyes. The content of LPP in senile cataract aqueous humor was shown to be significantly increased. To determine the possible mechanism of LPP increase in aqueous humor, human lenses at different stages of cataract as well as transparent human and rabbit lenses were incubated for 3 hours in 3.0 ml medium containing liposomes (0.5 mg/ml) prepared from phospholipids from the egg yolk and 0.14 M NaCl + 0.01 M TRIS-HCl buffer, pH 7.4). Corrections were made for phospholipid autooxidation. The level of LPP accumulation in the medium was determined by MDA assay. The rate of LPP production increased significantly in transparent lenses and in early senile cataract, as compared to controls and advanced (mature) cataracts. EDTA (1 mM), superoxide dismutase (114 u/sample), catalase (900 u/sample), chelated iron (III): Fe3+-ADP addition to the incubation medium depressed the level of LPP accumulation. This suggests the participation of Fe2+, O2-., H2O2 in the mechanism of LPP production in the lens. The induction of lipid peroxidation in the lens can be significant for leukotriene and prostaglandin synthesis in the eye.     

A temperature-sensitive mutation of Crygs in the murine Opj cataract

In Opj, an inherited cataract in mice, opacity is associated with a mutation in Crygs, the gene for gammaS-crystallin, the first mutation to be associated with this gene. A single base change causes replacement of Phe-9, a key hydrophobic residue in the core of the N-terminal domain, by serine. Despite this highly non-conservative change, mutant protein folds normally at low temperature. However, it exhibits a marked, concentration-dependent decrease in solubility, associated with loss of secondary structure, at close to physiological temperatures. This is reminiscent of processes thought to occur in human senile cataracts in which normal proteins become altered and aggregate. The Opj cataract is progressive and more severe in Opj/Opj than in Opj/+. Lens histology shows that whereas fiber cell morphology in Opj/+ mice is essentially normal, in Opj/Opj, cortical fiber cell morphology and the loss of maturing fiber cell nuclei are both severely disrupted from early stages. This may indicate a loss of function of gammaS-crystallin which would be consistent with ideas that members of the betagamma-crystallin superfamily may have roles associated with maintenance of cytoarchitecture.     

A study of a hereditary cataract in the mouse

This report presents a study of cataracts seen in a random-bred strain of Swiss mice with Balb/c mice used as a control group. The embryonic development, and histological and slit lamp observations of the lenses in the two groups of animals are contrasted. The cataract is dominant in its inheritance (Tissot, '62). It appears either unilaterally or bilaterally as a dense white opacity in the lens substance. The earliest sign of abnormal formation occurs at 14 days of embryonic development. This is associated with a defect in the primary lens fibers formation. Progressive degeneration of these fibers occurs until they are reduced to a mass of cellular debris seen at the last day of gestation. The secondary fibers are also laid down in an abnormal manner. The normal lamellar arrangement of the secondary fibers is not seen in cataractous lenses. The abnormal lens fiber development leads to progressive vacuolization. The mature cataract seen in the adult is filled with many vacuoles, the largest ones occurring at the equatorial region. The nuclear region consists of a clumpy eosinophilic mass with scattered calcified areas. The rate of growth of the secondary fibers is different from that of the normal group. Most of the mature cataracts in the adult contain a vascularized epithelium. There are three possible areas of primary involvement which may lead to the development of the cataract. This are: (1) A defect in the development of the primary lens fibers; (2) A defect in the development of the secondary lens fibers; (3) An abnormal lens epithelium which may interfere with nutrition of the lens and thus initiate cataract formation.     

The Evaluation of the Oxidative Stress Parameters in Nondiabetic and Diabetic Senile Cataract Patients

The aim of the present study was to evaluate the copper (Cu), zinc (Zn), malondialdehyde (MDA), glutathione (GSH), and advanced oxidation protein products (AOPP) levels and superoxide dismutase (SOD) activities in diabetic senile cataract. Ten patients with diabetic senile cataract and ten patients with nondiabetic senile cataract (control group) were included in this study. AOPP, MDA, and GSH levels and SOD activity were measured by a spectrophotometric method. Serum, lens Cu, and Zn levels were measured by an atomic absorption spectrophotometric method. Both the lens and serum Zn and Cu levels between the two groups were not significantly different (p > 0.05). GSH, AOPP, and MDA levels and the SOD activities in the diabetic senile cataract group were significantly increased as compared to the control group (p < 0.05). Oxidative stress is one of the major factors which may lead to the early cataract formation. Oxidative events are of great importance in diabetic complications and, particularly in the lens, may have a role in the pathogenesis of cataract associated with diabetes mellitus as exhibited in this study.     

Connexin Mediated Cataract Prevention in Mice

Cataracts, named for any opacity in the ocular lens, remain the leading cause of vision loss in the world. Non-surgical methods for cataract prevention are still elusive. We have genetically tested whether enhanced lens gap junction communication, provided by increased α3 connexin (Cx46) proteins expressed from α8(Kiα3) knock-in alleles in Gja8^tm1(Gja3)Tww mice, could prevent nuclear cataracts caused by the γB-crystallin S11R mutation in Crygb^S11R/S11R mice. Remarkably, homozygous knock-in α8(Kiα3/Kiα3) mice fully prevented nuclear cataracts, while single knock-in α8(Kiα3/−) allele mice showed variable suppression of nuclear opacities in Crygb^S11R/S11R mutant mice. Cataract prevention was correlated with the suppression of many pathological processes, including crystallin degradation and fiber cell degeneration, as well as preservation of normal calcium levels and stable actin filaments in the lens. This work demonstrates that enhanced intercellular gap junction communication can effectively prevent or delay nuclear cataract formation and suggests that small metabolites transported through gap junction channels protect the stability of crystallin proteins and the cytoskeletal structures in the lens core. Thus, the use of an array of small molecules to promote lens homeostasis may become a feasible non-surgical approach for nuclear cataract prevention in the future.     

Reversible `fingerprint' cataract secondary to diabetes mellitus

Transient diabetic cataracts are generally located in the anterior and posterior subcapsular regions of the lens. The following case report describes a reversible diabetic cataract with a `fingerprint' like shape in the anterior adult nucleus. The etiology is unclear but may relate to protein modification and/or osmotic changes in the lens.     

晶状体生化的研究 平阳霉素诱发大鼠白内障的研究

 本文报道间日经皮下注射平阳霉素(Pingyangmycin)(25μg/10g体重)于出生后四日之乳鼠可诱发白内障,其诱发率为84.6％。患白内障时晶状体中可溶性蛋白质含量明显降低。经Sephadex G-200柱层析可见βH,γ晶体蛋白降低,α晶体蛋白则相对升高。经10％聚丙烯酰胺凝胶电泳及等电聚焦电泳可见γ晶体蛋白改变明显,这些结果和我们用半乳糖及亚硒酸钠诱发白内障所得结果一致。     

Defining a link between gap junction communication, proteolysis, and cataract formation

Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.     

NMR study of the state of water in the human lens during cataract development

Water proton spin-spin relaxation times (T2) and the content of bound, "non-freezable" at -9 degrees C water in both normal human lenses and human lenses of different stages of cataract progression (cataracta incipiens, nondum matura, mature hypermatura) were measured by NMR spin echoes method. By the stage of cataracta nondum matura, increase of bound water content and simultaneous, almost half decrease of the relaxation time (T2), were observed. However, on the following stages of cataract evaluation (almost mature, mature cataracts) a gradual decrease of bound water content is noted, but only for the mature cataract stage the water content significantly differs from that of the normal one. On the stage of hypermature cataract the presence of two unexchanged with each other fractions of water is found. The obtained data are explained by lens protein reconstructions during the cataract progression.     

The enzymatic activities of GTP cyclohydrolase, sepiapterin reductase, dihydropteridine reductase and dihydrofolate reductase; and tetrahydrobiopterin content in mammalian ocular tissues and in human senile cataracts

The enzymatic activities of GTP cyclohydrolase, sepiapterin reductase, dihydropterin reductase and dihydrofolate reductase were determined in the ocular tissues of rat, rabbit, calf and human. The enzymatic activities of the pteridine biosynthesis and the content of tetrahydropteridine (BH4) were higher in retina and ciliary body-iris as compared with lens tissue in all mammalian species tested. The activities of the pteridine synthesizing enzymes and BH4 content were decreased in human senile cataracts as compared with age-matched clear human lenses. The loss of BH4 may result in lenticular proteins more susceptible to oxidation and contribute to high molecular weight protein formation in cataracts.     

UPR Activation and the Down–Regulation of α-Crystallin in Human High Myopia-Related Cataract Lens Epithelium

Purpose

To investigate the expression of αA- and αB-crystallin and the unfolded protein response in the lens epithelium of patients with high myopia-related cataracts.

Methods and Materials

The central portion of the human anterior lens capsule together with the adhering epithelial cells, approximately 5 mm in diameter, were harvested and processed within two hours after cataract surgery from high myopia-related (spherical equivalent ≥-10.00 diopters) and age-related cataract patients or from high myopia but non-cataractous patients (tissue were collected from ocular trauma patients with high myopia and lens trauma). Anterior lens samples from fresh cadaver normal human eyes were used as normal control (collected within 6 hours from death). Real-time PCR was performed to detect the mRNA levels of α-crystallins as well as unfolded protein response (UPR)-related GRP78, spliced-XBP1, ATF4 and ATF6. Western blot analysis was used to determine the protein level of α-crystallin, GRP78, p-IRE1α, p-eIF2α and ATF6.

Results

In the lens epithelium of the high myopia-related cataract group and the age related cataract group, the mRNA and soluble protein expression of αA- and αB-crystallin were both decreased; additionally, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased relative to the normal control.

Conclusion

These results suggest the significant loss of soluble α-crystallin and the activation of the UPR in the lens epithelium of patients with high myopia-related cataract, which may be associated with the cataractogenesis of high myopia-related cataract.     

Effect of high-glucose levels on protein oxidation in cultured lens cells,and in crystalline and albumin solution and its inhibition by vitamin B6 and N-acetylcysteine: its possible relevance to cataract formation in diabetes

Diabetic patients have elevated levels of glucose in their blood and other body fluids. This project studied the effect of high-glucose concentrations (HG) on the protein oxidation in cultured lens cells and in crystalline protein solution. In addition, we also examined the effect of HG on the oxidation and turbidity (aggregation) of albumin protein solution. This study also examined whether vitamin B₆ [pyridoxine (P), pyridoxamine (PM)] or n-acetylcysteine (NAC) is capable of preventing protein oxidation similar to that seen in cataracts. For cell culture studies, rabbit lens cells were cultured in control or HG medium at 37°C for 2 d. For studies with protein solution, a buffered solution of serum albumin or crystalline protein was incubated with normal glucose (5 mM) or HG (50–100 mM) in a water bath at 37°C for 4 d. All treatments were carried out with and without the addition of P, PM, or NAC. We found significantly higher levels of carbonyl protein (an index of protein oxidation) in HG-treated compared with normal glucose-treated lens cells and in crystalline protein solution. P, PM, and NAC significantly decreased the protein oxidation in lens cells and crystalline protein solution. We also found significantly higher levels of protein oxidation and turbidity (an index of protein aggregation) and its inhibition by P, PM, and NAC in HG-treated compared with normal glucose-treated albumin solution. This suggests that HG can cause the oxidation and modification of proteins in the lens, and that vitamin B₆ and NAC supplementation may be helpful in slowing the oxidation of lens proteins. This study explains the cause of early cataract development and the potential benefit of supplementation with vitamin B₆ and NAC in the prevention of the development of cataract among the diabetic population.     

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.     

先天性与后天性大鼠白内障晶体中游离氨基酸含量变化的研究

用药物诱发大鼠先天性白内障动物模型,检测了晶体中１８种游离氨基酸（ＦＡＡ）含量,并与大鼠硒性和半乳糖性白内障晶体中ＦＦＡ含量进行了比较,发现：仔一代和仔二代先天性白内障晶体中各ＦＡＡ含量接近;先天性白内障晶体内,酪氨酸和羟脯氨酸含量明显高于正常对照组（Ｐ＜０．０５）;谷氨酸,甘氨酸等９种ＦＡＡ明显低于硒性白内障（Ｐ＜０．０５）;而酪氨酸高于半乳糖性白内障组,半胱氨酸等５种ＦＡＡ含量均低于半乳糖性白内障组。同时还发现：硒性和半乳糖性白内障晶体内一些ＦＡＡ变化与以往报道不同,这些现象提示先天性与后天性白内障病变过程可能不同。