Pathogenesis of congenital cataract in a gamma-crystallin mutant mousemodel

Congenital cataract is a leading cause of visual disability among children worldwide. It has a heterogeneous genetic basis; the cellular and molecular mechanisms for cataractogenesis remain elusive. A spontaneously occurred autosomal dominant mouse mutant named Secc, which displays small eye, cataract and closed eyelid, has been obtained in our laboratory. By gene mapping and DNA sequencing, we identified a single nucleotide deletion at position 273 of the Cryga gene, leading to a frame-shift from the 3rd Greek Key motif of the A-crystallin (Cryga). The aim of this study is to investigate the pathogenic mechanisms underlying the development of cataract in the Secc mutant, as a disease model for understanding human congenital cataract. Initial phenotype analysis showed that cataract was initiated in E14.5 CrygaSecc mutant embryos, the nuclei of the primary lens fibres were scattered and failed to align in the equatorial region. By E16.5, the secondary lens fibre cells were abnormally arranged with poor lens suture formation. Apoptotic cells were found in the centre of the lens as shown by TUNEL assay, cytoskeleton and cell adhesion in the lens centre were disturbed as shown in immunohistochemistry analysis. Previously by western blotting it was found that mutant -crystallins were enriched in the insoluble fraction. I hypothesized that mutant A-crystallins might be misfolded and protein aggregates were then formed. In this study, aggregation was observed in semi-thin sections stained with toluidine blue. By co-staining using custom-made anti-Secc antibody, CrygaSecc protein was found to be ubiquitinated and was wrapped around by vimentin. Clearly, in the Secc mutant lens, aggresomes were formed for the disposal of the misfolded proteins and to maintain cell survival. However, ultimately cell death would occur in the mutant lens and contributed to cataract formation. It is known that misfolded proteins would trigger unfolded protein response (UPR) and heat shock protein (HSP) responses to facilitate folding and to prevent misfolded proteins from intoxicating the cell. In order to determine which stress response pathway was triggered, gene expression analysis by qRT-PCR was performed. The expression of genes involved in the UPR pathways including BiP, CHOP and spliced variant of XBP-1 were all up-regulated significantly in E14.5 and 16.5 mutant lenses. In addition, among different ER stress related genes, cytosolic chaperones and autophagy related genes, Hsp70 and BiP were upregulated, while Hsp40 and Hsp90aa were downregulated in the homozygotes. The results suggested that both UPR and HSP response pathways were triggered during cataractogenesis in the Secc mutant. In conclusion, mutant A-crystallin appeared to trigger UPR, HSPs and cell death in the fibre cells, while autophagy was not triggered. In the lens fibre cells, the ubiquitin-proteasomal pathway was utilized for the removal of misfolded CrygaSecc proteins. However, the stress perpetuated as the lens grew and produced more mutant proteins. The mutant cells lost their normal cell adhesion, failed to maintain the proper lens architecture, leading to cataract formation. Similar cellular mechanisms could be implicated in human congenital cataract or age-related cataract development.published_or_final_versionBiochemistryDoctoralDoctor of Philosoph