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C. Pang, L.-Y. Jia, N. Fan, D. Lam, G. Yam; Chemical Chaperones Correct Mutant Phenotype of Myocilin - A Potential Treatment for Glaucoma. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4072.
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To investigate the applicability of chaperone-assisted therapy for a repertoire of 13 myocilin mutants causing primary open-angle glaucoma.
Human trabecular meshwork cells were transfected to express recombinant FLAG/myc-tagged wildtype or glaucoma-causing mutant myocilin (R82C, C245Y, Q368X, P370L, T377M, D380A, R422C, R422H, C433R, Y437H, I477N, I477S and N480K). The cells were treated with small-molecule chemical chaperones, 4-phenylbutyric acid (4-PBA, 1 mM), trimethylamine N-oxide (TMAO, 100 mM) or deuterium oxide (D2O) in medium for 2 days. Extracellular myocilin in culture media was analyzed by immunoprecipitation for myc, followed by Western blotting for FLAG. Cells were lyzed with 0.5% Triton X-100 and separated into detergent soluble and insoluble fractions for further Western blotting for FLAG and housekeeping genes. Apoptosis was quantified by counting cells showing fragmented nuclei after immunofluorescence of FLAG and nuclear DAPI staining.
Chemical chaperones, 4-PBA, TMAO and D2O, reduced insoluble mutants and restored myocilin secretion in a mutation-dependent manner. Except Q368X and C433R, all variants showed improved secretion to variable extents. Reduced apoptosis was observed. The osmolytes TMAO and D2O were better than 4-PBA in improving myocilin solubility and secretion.
We showed that chemical chaperones alleviated mutant phenotypes of myocilin and were feasible to treat myocilin-caused primary open-angle glaucoma. Since these molecules are cell permeable, our results warrant a test of topical administration.
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