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Abstract #24011 Published in IGR 11-3
Correction of the disease phenotype of myocilin-causing glaucoma by a natural osmolyte
Jia LY; Gong B; Pang CP; Huang Y; Lam DS; Wang N; Yam GHInvestigative Ophthalmology and Visual Science 2009; 50: 3743-3749
See also comment(s) by Terete Borras •
PURPOSE: To characterize a novel Asp384Asn (D384N) mutant myocilin (MYOC) that causes juvenile-onset open-angle glaucoma (JOAG) and investigate the correction of mutant phenotype by a natural osmolyte, trimethylamine N-oxide (TMAO). METHODS: A Chinese JOAG family was recruited and genomic DNA was extracted from peripheral blood obtained from 44 family members. Coding regions of the MYOC were sequenced. Two hundred individuals (>60 years old) without ocular hypertension or glaucoma were the control subjects. Full-length human wild-type MYOC cDNA was cloned in p3xFLAG-myc-CMV-25 and missense mutation was introduced by site-directed mutagenesis. Transfected human trabecular meshwork cells were treated with small-molecule chemical chaperones. Secreted MYOC was analyzed by combined immunoprecipitation-Western blot analysis. Intracellular myocilin was fractionated into Triton X-100-soluble and insoluble fractions, and analyzed by Western blot analysis. Intracellular aggregate and apoptosis were assayed by immunofluorescence. The effect of TMAO on subcellular myocilin distribution was analyzed by density gradient fractionation, followed by Western blot analysis. RESULTS: A novel c.1150G>A change of MYOC was identified. Screening of optineurin, WDR36, and CYP1B1 showed an absence of disease-causing polymorphisms. Mutated D384N myocilin had reduced solubility and was aggregation-prone and nonsecreted. Treatment of transfected cells with TMAO improved the solubility of the D384N mutant, which was corrected for secretion in a dose-response manner. TMAO reduced the distribution of the D384N mutant in the endoplasmic reticulum (ER), alleviated ER stress, and rescued cells from apoptosis. CONCLUSIONS: The results indicate that TMAO, with chaperoning activity, facilitated the folding and secretion of mutant MYOC. This therapeutic approach assisted by a chemical chaperone can be developed for treating glaucoma.
Dr. L.Y. Jia, Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, P.R. China
Classification:
9.1.2 Juvenile glaucoma (Part of: 9 Clinical forms of glaucomas > 9.1 Developmental glaucomas)
3.6 Cellular biology (Part of: 3 Laboratory methods)
11.14 Investigational drugs; pharmacological experiments (Part of: 11 Medical treatment)