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Shylaja Hegde, Om P Srivastava; Molecular Mechanism of Development of Congenital Nuclear Cataract in βA3 Conditional Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2016;57(12):729.
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© ARVO (1962-2015); The Authors (2016-present)
To understand the molecular mechanism of development of congenital nuclear cataract in lens-specific βA3-crystallin conditional knockout (cKO) mice.
Previously, we have generated a βA3 complete KO mouse model using EUCOM gene trap cassette in exon 4 of mouse CRYAB1 gene. In the present study, the homozygous βA3 KO were crossed with Flip mice, followed by crossing with Mlr-10 cre mice. Cataract development was confirmed by Micron-IV Slit-lamp microscopy. Proteins from 1-month-old lenses were analyzed by mass spectrometric and western blot methods. Immunohistochemistry and immunofluorescence were used to determine the cellular defects.
Results showed that similar to βA3 complete KO mice, lens-specific cKO mice of βA3-crystallin also developed congenital nuclear cataract. In cKO lenses, the concentration of water insoluble proteins was increased compared to age-matched wild type (WT) lenses. On separation of lens proteins by low speed (800 g) and high speed (5000 g) centrifugation, the sedimented fractions showed an increased accumulation of protein fragments (<20 kDa) in cKO lenses compared to WT lenses. Further, MS/MS analysis of these fragments identified a number of differentially fragmented proteins compared to WT lenses; and the most interesting was the increased accumulation of fragments of Calpain 3, Histone 4 and Phakinin. This was further confirmed by western blot analysis. Lens epithelial cells (LEC) from cKO lenses showed similar cellular defects as observed in complete βA3 KO lenses, which included impaired lysosomal clearance, increased calcium levels and calpain activation
Most of the defects observed in lenses of complete βA3 KO were also found in lenses of cKO of βA3-crystallin. Using cKO lenses, we further confirmed the clapain-3 activation. Overall, this study will provide an insight on events that leads to the development of nuclear cataract in βA3 cKO lenses, i.e. whether the increased calcium level in the absence of βA3-crystallin leads to two independent parallel events (lysosomal defects and calpain-3 activation) or a sequential event (increased calcium activates the calpain that in turn causes lysosomal membrane permeabilization and defective lysosomal waste clearance).
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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