PINK1 Protects Auditory Hair Cells and Spiral Ganglion Neurons from Cisplatin-induced Ototoxicity via Inducing Autophagy and Inhibiting JNK Signaling Pathway

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Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) gene encodes a serine/threonine kinase, which acts as a molecular sensor of mitochondrial health necessary for mitochondrial quality control. The present study was designed to examine whether PINK1 expressed in C57BL/6 murine cochlea and HEI-OC1 cells and, if so, to investigate the possible mechanisms underlying the action of PINK1 in cisplatin-induced death of sensory hair cells (HCs) and spiral ganglion neurons (SGNs) in vitro. The expression pattern of PINK1, formation of parkin particles, and autophagy were determined by immunofluorescent staining. The expressions of PINK1, LC3B, cleaved-caspase 3 and p-JNK were measured by Western blotting. The levels of reactive oxygen species (ROS) were evaluated by DCFH-DA and Mito-Sox Red staining. The mitochondrial membrane potential was detected by Tetramethylrhodamine methyl ester perchlorate (TMRM) and Rhodamine 123. Cell viability and apoptosis were examined by CCK8 assay, TUNEL staining and Annexin V Apoptosis Detection Kit, respectively. We found that PINK1 was widely expressed in the cytoplasm in HCs, SGNs, stria vascularis of C57BL/6 cochlea and HEI-OC1 cells and, notably, the expression level in cochlear HCs and SGNs of postnatal day 4 (P4) mice was higher than that in adult mice. Moreover, treatment with 30μM cisplatin elicited the formation of ROS, which, in turn, led to PINK1 activation, parkin recruitment, autophagy formation and JNK pathway relevant to apoptosis in HEI-OC1 cells, HCs, and SGNs. Meanwhile, co-treatment with ROS scavenger N-acetyl-L-cysteine (NAC) or H2O2 consumer catalase-polyethylene glycol (PEG-catalase) inhibited parkin recruitment, alleviated autophagy formation, and mitigated JNK pathway related apoptosis. In addition, PINK1 silencing resulted in a lower level of autophagy, but, a higher mortality in HEI-OC1 cells treated with cisplatin. Taken together, data from this work reveal that PINK1 possesses the protective effect via induction of autophagy and resistance of apoptosis under cisplatin stimulus in sensory HCs and SGNs, implying that PINK1 might serve as an important regulator of cisplatin-elicited ototoxicity.

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