Changes in lens proteins induced at the early stage of cataractogenesis in cac (Nakano) mice

Changes in lens proteins induced at the early stages of cataractogenesis in cac (Nakano) mice were investigated by two-dimensional gel electrophoresis (2D-PAGE). The 2D-PAGE profile of lens proteins in 21-day-old cac mice differed from that in 27-day-old normal mice, even though the appearance of 'pin-head' nuclear opacity (26-day-old) had not yet been observed in the lenses. Especially noticeable were great differences in the polypeptides associated with the alpha- and beta- crystallin subfractions, the appearances of which corresponded to an increase in a ratio of the amounts of Na+ to that of K+ in the lenses of defective mice. No dramatic decrease in the gamma-crystallin fraction was observed until the mature cataract stage.     

Age and cataract-related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens.

Heavy molecular weight (HMW) proteins were detected in normal and cataractous mouse lenses. The HMW aggregates increased with the age of the lens in normal mouse. Alpha and β-crystallins were detected by immunodiffusion in the HMW fractions from normal and Nakano mice. No γ-crystallin could be detected in these aggregates by immunodiffusion; however, a slight amount of this crystallin was detected using the radioimmunoassay. The polypeptide composition of the HMW proteins was different in the Nakano mouse from the normal. By SDS polyacrylamide gel electrophoresis, a polypeptide of 27 000 mol. wt. was evident in the Nakano HMW material that was not present in the normal HMW protein, but a 15 000 mol. wt. band was absent in the Nakano.Two other differences were seen with the Nakano lens. First, the water insoluble lens protein was extremely high. By 90 days, about two thirds of the protein was insoluble in these lenses. Secondly, the sharp drop in γ-crystallin at the time of complete opacification of the lens was in part a result of the leakage of this protein into the anterior chamber of these mice. By radioimmunoassay, the level of γ-crystallin in the Nakano aqueous humor at the time of the cataract was greater than 100 ng per microliter. These data demonstrate that crystallins are converted to the insoluble proteins and some diffuse out of the lens during cataract formation.     

Cataract formation by a semiquinone metabolite of acetaminophen in mice: possible involvement of Ca(2+)and calpain activation

Acetaminophen, an analgesic/antipyretic, is metabolized by hepatic cytochrome P450 to N -acetyl- p -benzoquinone imine (NAPQI), which is transported by blood circulation to the eye and induces anterior cortical cataract in mice. In this study we injected NAPQI into the anterior chamber of mouse eye and investigated time-dependent cellular responses in the lens. After a lag period of about 2 hr following NAPQI injection, lens opacification as determined by measurement of light scattering by the lens became evident and progressively increased thereafter. There was no difference in the profile of opacity development between a P450-inducer responsive mouse strain and a non-responsive strain. During the lag period, a marked increase in free intracellular Ca(2+)in the lens epithelium was observed at 1 hr by confocal fluorescence microscopy with a Ca(2+)probe. Concurrent with the free Ca(2+)increase, there was a 300% rise in the activity of the non-lysosomal neutral protease calpain in the lens at 1 hr after NAPQI injection. Evidence indicated degradation of vimentin in the lens in which calpain activity was enhanced. Co-injection of calpain inhibitors (N-Ac-Leu-Leu-norleucinol and N-Ac-Leu-Leu-methioninal) with NAPQI protected animals completely from cataract development, although a rise in free intracellular Ca(2+)in the lens epithelium was still observed. Lenses from the protected mice did not exhibit enhanced calpain activity. These results suggest the following sequence of events as a possible mechanism of NAPQI-induced cataract. NAPQI introduced in the anterior chamber of the eye enters the lens epithelial cells and disturbs Ca(2+)homeostasis with a resultant rise in free intracellular Ca(2+)which in turn activates calpain in the epithelium. The neutral protease then degrades cellular proteins (e.g. cytoskeletal proteins) and initiates anterior cortical cataract formation.     

Retarded and distinct progress of lens opacification in congenic hereditary cataract mice, Balb/c-nct/nct

The Nakano cataract gene, nct, was introduced into Balb/c mice by repeated backcrosses to elucidate the possible effects of background genes on its expression. The resulting congenic Balb/c-nct/nct mice were characterized by retarded and sporadic cataract formation with a tendency of further retardation in males and by the different disease process of cataract as compared with Nakano mice. The age of 50% cataract incidence was 60 days in females and 90 days in males compared with 22 days in Nakano mice, and lens opacification commenced in a diffuse, mild form at the cortex in congenic but in a pin-head, intense form at the core in Nakano mice. Sex hormones seemed to be involved in the difference in cateractogenesis between male and female mice. Microphthalmia was slighter in degree in Balb/c background mice. The results indicated that the nct-dependent cataractogenesis may be influenced by background genes and some non-hereditary factors. Balb/c-nct/nct mice will provide a new type of hereditary cataract model.     

Regional distribution of free calcium in selenite cataract: relation to calpain II

The purpose of this experiment was to assess the roles of free, intracellular calcium and calcium-dependent neutral protease (calpain II, EC.34.22.17) in selenite nuclear cataract. Free calcium ion concentrations within lens nuclear fibers during selenite cataractogenesis increased to 3 microM on day 2 post-injection (clear lens) and to 108 microM at day 4 (nuclear cataract). Calpain II is known to be activated in vitro by calcium levels above 50 microM. Calpain II activity was present in the lens nucleus at time periods preceding formation of selenite cataract. These data suggested that after selenite injection, calpain II was activated by elevated free calcium in the nucleus, and that calpain II-induced proteolysis of nuclear proteins was an important mechanism in selenite cataract. Calpain II levels were also observed to decrease in the nucleus during selenite cataractogenesis, probably due to autolysis. This was supported by the finding that incubation of purified lens calpain II with 100 microM calcium caused partial inactivation of the protease.     

Naphthoquinone-Induced cataract in mice: possible involvement of Ca2+ release and calpain activation.

N-acetyl-p-benzoquinone imine (NAPQI), a semiquinone metabolite of acetaminophen, produces cataract in mice. Naphthalene is biotransformed to the cataractogenic metabolite 1,2-naphthoquinone (NQ). Intracameral injection of NAPQI elicits a rapid increase in free intracellular Ca2+ in the lens epithelium and calpain activation before lens opacification begins. In order to test whether the cellular response is a common feature of quinone-induced cataracts, we injected in this work 1,2-naphthoquinone (NA) in the anterior chamber of mouse eye and followed cellular responses in the lens prior to opacity development. A marked rise in free intracellular Ca2+ in the lens epithelium and concurrent activation of calpain were observed within 1 hr after NQ injection preceding lens opacity development. These results support the suggestion that Ca2+ release and calpain activation are involved in the mechanism of quinone-induced cataractogenesis.     

Morphological study on cataractogenesis of the Nakano mouse lens

· Background: Although some histopathological features on the Nakano mouse lens have been pointed out by a few investigators, there seem to have been no detailed studies on the sequential changes that occur. · Methods: We used the following two approaches: (1) Observation of the whole lens by dissection microscopy and (2) light and electron microscopic examination of the sectioned lens specimen. · Results: (1) The Nakano mouse lens showed sustained transparency up to 19 days after birth, fine opacity at the 20th day, and development of a mature cataract around the 30th day. In addition, although the Y-shaped posterior suture was normal at the 15th day, bending of the suture line appeared around the 19th day. (2) The cataractous lens revealed degeneration of the epithelial cells and adjacent anterior cortical fibers at the 10th day. Swelling of the anterior cortical fibers became prominent, and swelling of the posterior cortical fibers occurred by the 15th day. Upon separation of the suture around the 20th day, fine opacity occurred in the perinuclear zone, which extended to the anterior cortex and finally led to the formation of a mature cataract. · Conclusions: These results indicate that epithelial degeneration is a major feature of cataract in the Nakano mouse, and the subsequent lens fiber swelling and posterior sutural separation are the underlying causes of the development of opacity. Received: 2 April 1998 Revised version received: 15 June 1998 Accepted: 23 July 1998     

Cellular events preceding acetaminophen cataractogenesis studied by confocal fluorescence microscopy.

Acetaminophen (APAP) is biotransformed by hepatic cytochrome P450 (CYP) enzymes to the cataractogenic metabolite N-acetyl-p-benzoquinone imine (NAPQI). In the previous studies in which NAPQI was injected into the anterior chamber of mouse eye, we observed mitochondrial dysfunction and disturbances in Ca2+ homeostasis in the lens epithelium, and activation of the nonlysosomal neutral protease calpain. In this work we investigated whether intraperitoneal injection of APAP elicits similar cellular responses in the lens epithelium prior to the onset of lens opacity development. Following APAP injection, reactive oxygen species generation, intracellular free Ca2+ increase and calpain activation in the lens epithelium were determined in situ by fluorescence confocal microscopy. It was found that cellular events in the lens prior to the onset of opacification were essentially identical to those elicited by NAPQI. In addition, lens calpain activities were characterized based on their Ca2+ requirement and several calpain inhibitors were shown to prevent cataract development.     

Cysteine protease inhibitor E64 reduces the rate of formation of selenite cataract in the whole animal

The purpose of this experiment was to test the effectiveness of E64 in prevention of selenite nuclear cataract in the whole animal. E64 is an inhibitor of cysteine proteases such as calpain (EC.3.4.22.17). In the whole animal, daily intraperitoneal injection of E64 was mildly effective in slowing the rate of formation of selenite nuclear cataract, although prevention was not permanent. Frequency of the nuclear cataract in selenite group at 5 days post selenite injection was significantly decreased from 40% to 17% in the selenite + E64 group, and the density of cataract in the Se + E64 group was reduced. However, crystallins and calpain were still degraded in the selenite + E64 group. E64 was more effective against selenite cataract when present continuously during lens culture, where it slowed the rate of formation of nuclear opacity. Amelioration of cataract occurred both in vitro and in vivo even though lens calcium concentrations were elevated. The results supported the idea that application of calpain inhibitor is beneficial in prevention of rodent selenite cataracts.     

Biosynthesis of proteoglycans by lens epithelial cells of cataractous mouse (Nakano strain)

The capsules (with epithelial cells attached) of lenses from normal and cataractous mice (Nakano strain) were biosynthetically labeled in vitro with radioactive precursors. The labeled macromolecules were chromatographed on a Sepharose CL-4B column and analyzed by specific enzyme digestion. The incorporation of [3H]-proline and [3H]-glucosamine into macromolecules was comparable in the cataract and normal capsules, while that of [35S]-sulfate was reduced by 60% in the cataract capsules, indicating that the proteoglycan synthesis was specifically decreased in the cataract lens. Glycosaminoglycan analyses showed an increased synthesis of hyaluronic acid and decreased synthesis of heparan sulfate in the cataract capsules. It is possible that the alterations in the synthetic level and glycosaminoglycan components of proteoglycan affect the permeabilities of macromolecules to lens capsule and lead to cataract in Nakano mouse lens.     

Hydration and elevated calcium alone do not produce xylose nuclear cataract: role of proteolysis by calpain.

The purpose of this experiment was to determine the contribution of calpain proteolytic enzyme (EC 3.4.22.17) in the formation of nuclear cataract during lens culture in xylose. Increased lens calcium was found to be required for formation of xylose nuclear cataract in our culture system. Inhibition of calpain by the cysteine protease inhibitor E64 was effective in slowing the formation of nuclear cataract, even though lens calcium and hydration were markedly elevated. These results showed that hydration and elevated calcium alone do not produce xylose nuclear cataract, and they indicated that calpain proteolysis may be necessary for xylose nuclear cataract in the rat lens.     

Biochemical evidence for conversion to milder form of hereditary mouse cataract by different genetic background

Congenic hereditary cataract mice, BALB/c-nct/nct, were established by introducing the nct gene from Nakano into BALB/c mice. These mice developed a milder cortical form of cataract which developed sporadically and later in life than in Nakano mice. Combined use of BALB/c and BALB/c-nct/nct mice enables biochemical comparison of normal clear lenses, congenic clear lenses which are destined to be opacified some time later, and opacified lenses in the same genetic and aging statuses. We compared the age-related changes in water content and water-soluble and -insoluble fractions among these three types of lenses. Congenic clear lenses and opaque lenses were more similar to BALB/c normal clear lenses and Nakano opaque ones, respectively, in these parameters. These results suggest, in addition to formation of aggregated crystallins and their accumulation in water-insoluble fractions, that decreased protein synthesis, increased protein degradation and augmented leakage of crystallin might have a significant role in the nct-induced lens opacification.     

Swelling of the lens fibers.

Lens cells of congenital mouse cataract (Nakano and Fraser strains) and galactose-fed rats were studied by scanning electron microscopy. Similarly lens cells of normal young mice and rats were examined as controls. Normal lenses of young rodents consist of lens fibers in all maturation stages which have been demonstrated in humans and in monkeys. Lens cells in the congenitally cataractous lenses are irregular in size and shape in the earlier stage of the cataract formation. Swelling of the lens cell occurs corresponding to the occurrence of early optical opacity. Swelling of the cell occurs segmentally in congential cataractous lenses; in the apical ends (Fraser) and in the posterior ends (Nakano). Similar swelling of the lens cell is observed in the main cell body in the superficial lens cortex of galactose-fed rats. However, numerous intercellular cysts are formed by the accumulation of fluid which may have been pumped out of the cells before the cells became degenerative. These numerous shrunken fibers are present among swollen cells in both congenital and galactose-induced cataractous lenses.     

Susceptibility of ovine lens crystallins to proteolytic cleavage during formation of hereditary cataract

PURPOSE: To produce two-dimensional electrophoresis (2-DE) maps for ovine crystallins and examine changes in ovine crystallins during cataract formation. METHODS: Soluble and insoluble fractions were isolated from normal, whole lenses of 26-week-old sheep, the proteins separated by 2-DE, and the spots digested with trypsin and subjected to tandem mass spectral analysis. Spot identifications were made by using mass spectrometry data from each spot digestion and data from 2-DE maps of proteins from soluble and insoluble cortices of 10-month-old ovine lens. Ovine alphaA-, alphaB-, and betaB3-crystallin cDNAs were sequenced, whereas other ovine crystallins were identified by using bovine sequences. Proteins were then isolated from whole lenses of 26-week-old lambs with mature hereditary cataracts, and the changes in the crystallins were determined by 2-DE. The masses of truncated crystallins were determined after elution from 2-DE gels. RESULTS: The ovine lens contained the normal complement of crystallins and, similar to other mammalian lenses, underwent partial proteolysis of betaB1-, betaA3-, and betaB3-crystallin during maturation. Cataract development was associated with enhanced truncation of alpha- and beta-crystallins. C-terminal truncations of alphaA- and alphaB-crystallin and N-terminal truncation of betaB2-crystallin were observed as well as a loss of gamma-crystallin. CONCLUSIONS: These data provide the first 2-DE gel maps for ovine lens crystallins and indicated that ovine lens crystallins are truncated during lens maturation. The differences in proteolysis appearing in normal and cataractous lenses suggested that calpain isoforms may be differentially activated during lens maturation and cataract. The ovine hereditary cataract is a useful nonrodent model to study the role of calpain proteolysis in cataract formation.     

Immunohistochemical study of calpain-mediated α-crystallin proteolysis in the UPL rat hereditary cataract

The UPL (Upjohn Pharmaceutical Limited) rat is a dominant hereditary cataract model that develops early-onset cataracts (E-type) in rats homozygous for the trait, and late-onset cataracts (L-type) in heterozygous rats. Using antibodies specific to the calpain-proteolyzed forms of α-crystallin, we determined their immunohistochemical localization of the L- and E-rat lenses. Immunoreactivity indicating the proteolyzed forms was detected and found restricted to degenerated lens fibers of the mature stage of the L-rat cataract. Lenses from E-rats, which have abnormally elongated lens fibers during the fetal period, had proteolyzed α-crystallin forms at 1 week of age. The results of this present study indicate that calpain-mediated proteolysis of α-crystallin occurred in the UPL rat lenses during cataract formation and that calpain may be an important factor in the development of complete lens opacification.     

Lp82 is the dominant form of calpain in young mouse lens

The purpose of this study was to characterize Lp82 calpain in normal mouse. Lp82 is a lens-specific, calcium-activated isozyme from the calpain super family of cysteine proteases (EC 34.22.17). RT-PCR and molecular cloning were performed on total RNA from 12 day-old mice. Lp82 and m-calpain protein levels and proteolytic activities in lenses were measured by casein zymography, immunoblotting, and ELISA after partial purification by DEAE-HPLC.The 2334-bp cDNA encoding for mouse Lp82 contained a single large open reading frame encoding a protein of 709 amino acid residues with a calculated molecular weight of 82.2 kDa and a predicted pI of 5.8. The amino acid sequence of mouse lens Lp82 was 99% homologous to rat lens Lp82. As in rat, mouse lens Lp82 showed a unique N -terminus and deletion of the IS1 and IS2 regions. In contrast to rat, Lp82 was the dominant calpain in young mouse lens. Lp82 was lens-specific, and the lens nucleus contained the highest specific activity of Lp82 and very little m-calpain. Endogenous Lp82 in lens soluble proteins was activated by addition of calcium and caused limited proteolysis of crystallins even in the presence of large amounts of recombinant domain I from the natural calpain inhibitor calpastatin. Loss of Lp82 protein accompanied aging of mouse lens. Lp82 may be responsible for a major portion of crystallin proteolysis occurring during normal lens development and maturation, or during cataract formation in young mice.     

硫醇转移酶基因敲除小鼠模型的建立及其白内障形成机制

目的:通过建立硫醇转移酶(TTase)基因敲除小鼠模型,观察其晶状体形态和生化方面随年龄的改变,探讨TTase在晶状体氧化还原系统中的重要作用及参与年龄相关性白内障形成的机制。方法:建立TTase基因敲除小鼠模型并进行基因型鉴定。裂隙灯观察TTase基因敲除型和野生型小鼠白内障随年龄的发生情况。检测小鼠晶状体中谷胱甘肽(GSH)含量。Western blot检测晶状体中氧化产物蛋白质二硫化物(PSSG)的变化。免疫共沉淀法鉴定形成PSSG的蛋白质。观察纯化的重组人晶状体TTase(RHLT)对PSSG的脱硫醇作用。结果:TTase基因敲除型小鼠和野生型小鼠白内障的发生均随年龄而增加,且主要表现为核性白内障。在TTase基因敲除型小鼠中,白内障最早从4月龄开始发生,9月龄时最为显著;野生型小鼠白内障最早从9月龄开始发生,12月龄时最为显著。两种基因型小鼠晶状体中GSH含量均随年龄增加而下降,9月龄TTase基因敲除型小鼠晶状体中GSH下降更为显著,且PSSG的表达明显高于野生型,主要表现为高分子聚合物。免疫共沉淀反应证实形成PSSG的蛋白质包含肌动蛋白(actin)和三磷酸甘油醛脱氢酶(GAPDH),这种积聚的PSSG可被GSH还原,且与纯化的RHLT反应更有效。结论:TTase基因敲除可以加速小鼠年龄相关性白内障的发生,这与晶状体中PSSG的积聚相关,且形成的PSSG可被TTase脱硫醇,证实TTase在防止年龄相关性白内障的发生中发挥重要作用。     

Studies on primary cultures of adult lens cells from normal and hereditary cataractous mice.

Lens epithelial cells from normal and congenital cataractous mice strains were cultured under similar conditions. Both normal and cataractous cells actively propagated and reached confluency on the eleventh day. These cells, thereafter, underwent morphological changes characterized by cell elongation, aggregation and formation of lentoid bodies at about 15 days.Electron microscopy revealed these lentoid bodies to consist of immature lens cells. These structures derived from cataractous cells had numerous vacuoles in the cytoplasm much more so than in the normal lens cells. In addition, some lentoid bodies closely resembled mature fibers of the intact lens. It was also demonstrated that these lentoid bodies showed positive immunofluorescence when reacted with fluorescent antiserum to γ-crystallin.There were certain differences observed between the cultured cells derived from normal lens and Nakano cataract. The disappearance of organelles and denucleation process were delayed in the lentoid bodies found in cultured Nakano cells when compared to normal cell culture. In addition a second type of lentoid body, although present as a minor population, was observed in the Nakano cell culture. Other subtle differences were observed during the course of culturing normal and cataractous lens cells.     

Dietary caloric restriction may delay the development of cataract by attenuating the oxidative stress in the lenses of Brown Norway rats

Dietary caloric restriction (CR) is the only experimental intervention that can reliably retard the development of cataract in a normal animal model. Here we have studied the possible mechanisms by which CR retards the age-related degeneration of the lens of Brown Norway rats. We have found that CR slowed protein insolubilization and blunted declines of the total soluble thiols, protein thiols, reduced glutathione and ascorbic acid levels in the lenses of old BN rats. From the lens protein point of view, the development of cataract in rat lenses has 3 stages: (1) the precipitation of gamma-crystallin, (2) the insolubilization of beta-crystallin, and (3) the final precipitation of alpha-crystallin which was saturated with other denatured lens proteins. A similar sequence is also observed when the lens proteins are subjected to oxidative stress in vitro. These data are the first to suggest that CR may retard the age-related degeneration of the lens by attenuating the oxidative stress in the lens. Since oxidative stress is likely a main cause of human cataract, CR intervention may be relevant to humans as well.     

Glutathione reductase from human cataract lenses can be revived by reducing agents and by a molecular chaperone, alpha-crystallin

PURPOSE: The aim of this study was to investigate how glutathione reductase (GR) loses its activity during cataract formation and whether it is possible to revive it back to the normal levels. METHOD: In this study, endogenous as well as synthetic reducing systems (GSH, TTase, DTT, captopril) and alpha-crystallin at different concentrations were incubated with the soluble fraction of human cataract lens protein. The activity of glutathione reductase with or without the reducing agents and alpha-crystallin was tested, and the difference in activity gained was calculated. RESULTS: Five agents (GSH, DTT, TTase, captopril, alpha-low crystallin) were able to revive the activity of GR from human cataract lenses to different extents. CONCLUSION: This study shows that human lens GR activity was revived by different reducing agents as well as by a molecular chaperone (alpha-crystallin).