Pathogenesis of congenital cataract in a gamma-crystallin mutant mousemodel

Abstract

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.