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Abstract #82326 Published in IGR 20-4
Glaucoma-Associated Mutations in the Optineurin Gene Have Limited Impact on Parkin-Dependent Mitophagy
Chernyshova K; Inoue K; Yamashita SI; Fukuchi T; Kanki TInvestigative Ophthalmology and Visual Science 2019; 60: 3625-3635
PURPOSE: Glaucoma results in progressive degeneration of the optic nerve and irreversible vision loss. Several mutations in the gene encoding optineurin (OPTN), the receptor for Parkin-dependent mitochondrial autophagy (mitophagy), are associated with glaucoma and amyotrophic lateral sclerosis (ALS). ALS mutations in the ubiquitin-binding domain of OPTN impair Parkin-dependent mitophagy. However, the effects of glaucoma mutations in this region remain unknown. We examined the impact of glaucoma-associated OPTN mutations on Parkin-dependent mitophagy. METHODS: The mitochondria-localized, pH-sensitive fluorescent protein mito-Keima was used to monitor mitophagy. HeLa cells expressing Parkin were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or oligomycin/antimycin A (O/A) to induce Parkin-dependent mitophagy. Two complementary mitophagy receptors, OPTN and NDP52, were deleted in HeLa cells expressing mito-Keima and Parkin (DKO_HeLa). The mutant OPTN genes were re-introduced into DKO_HeLa cells using retroviruses or through transfection. Mitophagy activity and OPTN localization were evaluated via microscopic analyses. OPTN binding to ubiquitin was examined using an immunoprecipitation assay. RESULTS: Parkin-dependent mitophagy was inhibited in DKO_HeLa cells. INTRODUCTION: of two glaucoma mutations in the ubiquitin-interacting region of OPTN restored mitophagy in CCCP-treated DKO_HeLa cells, whereas the two ALS mutations failed to replicate this effect. Under treatment with CCCP, the two glaucoma-mutant OPTN proteins normally translocated to mitochondria and bound to ubiquitinated proteins. Furthermore, five additional glaucoma-mutant OPTN proteins restored CCCP-induced mitophagy. Moreover, treatment with O/A exhibited similar results. CONCLUSIONS: Glaucoma-mutant OPTN proteins retain their normal properties as mitophagy receptors, suggesting that mutations in the OPTN gene cause glaucoma through a mechanism independent of mitophagy defects.
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Classification:
3.4.2 Gene studies (Part of: 3 Laboratory methods > 3.4 Molecular genetics)
