Characterization of lens defects in transgenic mice expressing a human R116C A–crystallin mutant

Abstract

Abstract: : Purpose: An R116C missense mutation in human alpha–A crystallin is associated with autosomal dominant cataract in humans. We previously showed that ectopic expression of human R116C alpha–A crystallin leads to lens defects in transgenic mice. In the present study, we characterized the morphological and biochemical features in three independent founder lines in order to further explore possible mechanisms leading to lens defects associated with this mutation. Methods: Lens phenotypes were recorded from three independent transgenic lines expressing human R116C alpha–A crystallin under control of the 409 bp mouse alpha–A crystallin gene promoter. A line expressing a relatively high level of wild–type human alpha–A crystallin was also produced as a transgene control. Lens phenotypes were determined and scored at ages up to six months. Differences in the occurrence and severity of abnormalities were tested using the Kruskal–Wallis rank sum test. Results: Seven different morphological abnormalities were found to be associated with expression of the mutant R116C alpha–A crystallin. In contrast, expression of wild–type human alpha–A crystallin was not associated with these abnormalities. Formation of highly stained membrane structures, as well as numerous vacuoles and vesicles, was observed in fiber cells of the inner cortex of R116C transgenic mice. Immunohistochemical staining indicated that the ectopic wild–type human alpha–A crystallin is evenly distributed in mouse lens, whereas the mutant R116C crystallin is unevenly distributed and produces a granulated pattern. Western blotting studies showed that the insoluble fraction from lens homogenates of R116C transgenic mice contained increased levels of the mutant protein, consistent with the hypothesis that the mutation is associated with an increase in membrane binding capacity compared to wild type. Conclusions: Expression of the human R116C alpha–A crystallin transgene leads to a range of defects in the lens. Increased formation of vacuoles and vesicles in the fiber cells is associated with the expression of mutant alpha–A crystallin. Enhanced membrane binding by mutant alpha–A crystallin may contribute to formation of fiber cell abnormalities.

Keywords: crystallins • cataract • transgenics/knock–outs