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_version / Biochemistry / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/161554 |
| Date | January 2012 |
| Creators | Tam, Chung-nga., 談頌雅. |
| Contributors | Sham, MH, Song, Y |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B4786994X |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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