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Induction of mitophagy in the HEI-OC1 auditory cell line and activation of the Atg12/LC3 pathway in the organ of Corti
18 April 2018 (online)
Mitochondrial dysfunction has been reported in several neurodegenerative disorders. Since the possible involvement of mitochondria in acquired hearing loss remains uncertain, we focused on the study of mitophagy, a selective intracellular autophagic mechanism by which malfunctioning mitochondria are removed. We searched for molecular indicators of mitophagy within House Ear Institute-Organ of Corti-1 (HEI-OC1) cells as well as in the organ of Corti (OC) exposing HEI-OC1 cells and OC to a well-described mitophagy-inducing agent: the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and compared it with gentamicin. We used COXIV as mitochondrial marker as well as proteins associated with the autophagosome formation process: LC3, Atg5 and Atg12. The induction of mitophagy in HEI-OC1 cells was detected by objectivizing the translocation of fluorescence-tagged LC3 to mitochondria using confocal microscopy after 6-hour incubation with CCCP or gentamicin. CCCP treatment showed to induce the mitochondrial translocation of LC3. Gentamicin, on the other hand, generated no impact on LC3. Under the same conditions, protein levels of COXIV, Atg5/12 and LC3 were evaluated by an immunoblot analysis after 24-hour incubation. We demonstrated mitophagy after CCCP exposure in HEI-OC1 cells by showing a downregulation of COXIV in the immunoblot. We also demonstrated changes on Atg12 and LC3 proteins levels in both the OC and HEI-OC1 cells after CCCP exposure but not after gentamicin. Our data strongly suggest that gentamicin had no impact in the activation of mitophagy-neither in the HEI-OC1 cell line nor in the OC. Therefore, we speculate that mitophagic-independent mechanisms may underly aminoglycoside ototoxicity.