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Y. Tang, X. Liu, R.K. Zoltoski, L.A. Novak, R.A. Herrera, I. Richard, J.R. Kuszak, N.M. Kumar; Calpain 3 Disruption Delays Cataract Formation in the 1293 Cx46 KO Mouse Lens . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4107.
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To determine the role of γ–crystallin degradation & protease calpain 3 in cataract formation in the 129 α3Cx46 KO mice.
129 α3Cx46 KO and Calpain 3 KO mice were mated to generate homozygote double knockout (dKO) mice. Lens homogenates from 2, 5, 8 and 11–week mice (wild type, α3Cx46 KO & dKO) were analyzed by western blots using antibodies to αA, αB, and γ–crystallin, MIP and α8Cx50. Various ion levels, including calcium, potassium, & sodium, were measured by atomic absorption spectroscopy. Lenses from 7 to 11 weeks of mice were examined by visual observation, laser scan analysis & histological methods.
Visual observation of the lenses from the different KO mice indicated a significant delay in the formation of a cataract in the dKO mice, compared to the α3Cx46 KO mice. α3Cx46 KO mice develop a nuclear cataract soon after birth around 2 weeks. However, in the dKO mice, a cataract was not visually observed until 7–8 weeks and the appearance of the cataract type was different from a nuclear cataract in being more pulverulent and distributed throughout the lens. Western blot analysis of γ–crystallin indicated a lack of significant cleavage of γ–crystallin in the lenses from dKO. This is in contrast to the generation of a proteolytic fragment of γ–crystallin that occurs and is associated with the nuclear cataract in α3Cx46 KO mice. Laser scan analysis confirmed that the lenses of dKO mice were functionally unimpaired at least until 7–8 weeks. Ion level measurements of the lenses showed a temporal increase in levels of calcium ions in dKO mice that had a time course similar to that observed for the α3Cx46 KO mice. Preliminary histological analysis of lens sections indicated subtle changes in the dKO lens.
The lack of γ–crystallin cleavage in dKO mice indicated that proteolysis of γ–crystallin in the lenses of α3Cx46 KO mice is dependent on calpain 3 isoforms. The increase of calcium ion concentration in the lenses of dKO mice indicated that increased levels of this ion in the lenses of dKO mice is insufficient to cause the formation of a cataract, at least until the age of 7 weeks. The observation of a cataract at later stages in the dKO lenses may be due to activation of other proteases. These results are consistent with the hypothesis that the loss of α3Cx46 leads to increased levels of Ca ions and this increase activates Lp82/85, resulting in the formation of a nuclear cataract.
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