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Shylaja Hegde, Rebecca Vance, Om P Srivastava; Determination of Functional Role of βA3/A1-Crystalline in Lens Development using βA3/A1 Knockout Mouse Models. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2601.
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© ARVO (1962-2015); The Authors (2016-present)
The aims of the study were to: (i) generate a complete CRYβA3/A1 Knockout (KO), and a lens- specific conditional CRYβA3/A1 (cKO) mouse models, and (ii) characterize the associated molecular and cellular defects in the lenses of these KO mice.
Complete CRYβA3/A1 mouse was generated by floxing the exon 4 of the CRYβA3/A1 gene, which disrupts the splicing of exon 4. For generation of cKO, βA3/A1 KO flip-positive mice were crossed with lens specific cre Tg (Cryaa-cre) 10 Mlr mice (cre expression in epithelial and fiber cells at embryonic day 9-13 and P0-21) and Tg (Cryaa-cre) 39 Mlr mice (cre expression only in epithelial cells from embryonic day 9-19.5 and P0-2). The molecular and cellular defects in lenses of complete KO mice were analyzed by western blot-, mass spectrometric- , immuno-histochemical- and transmission electron microscopic methods. Lens epithelial cell (LEC) culture derived from the wild type, heterozygous (HET) and homozygous (HOM) KO mice were analyzed by immunofluorescence methods.
Compared to WT and HET KO mice, the lenses of HOM KO mice showed 30% reduction in lens size, and developed nuclear cataract with distorted suture lines within a month. Further analyses of lenses by Western blot-, immunohistochemical- and immunofluorescent methods showed increased accumulation of LC3-II and p62 protein in HOM KO mice relative to WT and heterozygous mice, suggesting an impaired autophagy in HOM KO mice. In addition, the expression and arrangement of cytoskeletal proteins such as spectrin, beta-actin and tubulin were altered in HOM KO mice.
The CRYβA3/A1 KO mice develop a congenial nuclear cataract, which most likely due to impairment of autophagic cargo clearance. Our further studies focused on lens specific cKO are expected to unravel the precise function of βA3/A1-crystallin during lens development.
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