New Target of Oxidative Stress Regulation in Cochleae: Alternative Splicing of the p62/Sqstm1 Gene

Abstract

We investigated oxidative stress and antioxidant response in the p62/Sqstm1-Keap1-Nrf2 pathway in C57BL/6 mice cochleae during age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL), and the function of full-length and variant p62 in the regulation of Nrf2 activation. Groups of young (2 months), old (13–14 months), control, and acoustic trauma (AT) mice were examined cochlear damage and oxidative stress as follows: auditory brainstem response and hair cell counts; malondialdehyde (MDA) levels measured by assay kit and 7,8-dihydro-8-oxoguanine (8-oxoG) detected by immunohistochemistry. Full-length and variant p62 were examined for expression in cochleae, hippocampus (HIP), and auditory cortex (AC) using immunoblotting. Keap1-Nrf2 pathway activation was based on immunoblotting of nuclear Nrf2 and quantitative real-time PCR of Nrf2 target genes HO-1/NQO-1. The oxidative function of full-length and variant p62 was examined in HEI-OC-1 cells by flow cytometry. The results showed hearing loss, and cochlear hair cell loss was associated with MDA accumulation and 8-oxoG expression during ARHL and NIHL. Nrf2 showed no obvious changes in nuclear protein. Expression levels mRNA for HO-1 and NQO1 were lower in old mice and mildly greater in AT Mice. The expression of p62 splicing variant lacking the Keap1-interacting region was greater than full-length p62 in cochleae. However, the expression of p62 splicing variant was lesser than full-length p62 in HIP and AC. For HEI-OC-1 cells, overexpression of full-length p62 decreased ROS levels induced by H₂O₂. Oxidative stress is closely related to ARHL and NIHL. Changing the ratio of full-length to variant p62 protein expression may be a new target to reduce the level of oxidative stress in cochleae.