The aim of this study is to elucidate the detailed mechanism of endoplasmic reticulum (ER) stress-induced auditory cell death based on the function of the initiator caspases and molecular complex of necroptosis. Here, we demonstrated that ER stress initiates not only caspase-9-dependent intrinsic apoptosis along with caspase-3, but also receptor-interacting serine/threonine kinase (RIPK)1-dependent necroptosis in auditory cells. We observed the ultrastructural characteristics of both apoptosis and necroptosis in tunicamycin-treated cells under transmission electron microscopy (TEM). We demonstrated that ER stress-induced necroptosis was dependent on the induction of RIPK1, negatively regulated by caspase-8 in auditory cells. Our data suggested that ER stress-induced intrinsic apoptosis depends on the induction of caspase-9 along with caspase-3 in auditory cells. The results of this study reveal that necroptosis could exist for the alternative backup cell death route of apoptosis in auditory cells under ER stress. Interestingly, our data results in a surge in the recognition that therapies aimed at the inner ear protection effect by caspase inhibitors like zVAD-fmk might arrest apoptosis but can also have the unanticipated effect of promoting necroptosis. Thus, RIPK1-dependent necroptosis would be a new therapeutic target for the treatment of sensorineural hearing loss due to ER stress.

Age-related hearing loss (ARHL) is one of the most prevalent types of sensory decline in a superaging society. Although various studies have focused on the effect of oxidative stress on the inner ear as an inducer of ARHL, there are no effective preventive approaches for ARHL. Recent studies have suggested that oxidative stress-induced DNA damage responses (oxidative DDRs) drive cochlear cell senescence and contribute to accelerated ARHL, and autophagy could function as a defense mechanism against cellular senescence in auditory cells. However, the underlying mechanism remains unclear. Sodium arsenite (NaAsO₂) is a unique oxidative stress inducer associated with reactive oxygen species (ROS) that causes high-tone hearing loss similar to ARHL. Transcription factor EB (TFEB) functions as a master regulator of the autophagy‒lysosome pathway (ALP), which is a potential target during aging and the pathogenesis of various age-related diseases. Here, we focused on the function of TFEB and the impact of intracellular ROS as a potential target for ARHL treatment in a NaAsO₂-induced auditory premature senescence model. Our results suggested that short exposure to NaAsO₂ leads to DNA damage, lysosomal damage and mitochondrial damage in auditory cells, triggering temporary signals for TFEB transport into the nucleus and, as a result, causing insufficient autophagic flux and declines in lysosomal function and biogenesis and mitochondrial quality. Then, intracellular ROS derived from damaged mitochondria play a role as a second messenger to induce premature senescence in auditory cells. These findings suggest that TFEB activation via transport into the nucleus contributes to anti-senescence activity in auditory cells and represents a new therapeutic target for ARHL. We have revealed the potential function of TFEB as a master regulator of the induction of oxidative stress-induced premature senescence and the senescence-associated secretion phenotype (SASP) in auditory cells, which regulates ALP and controls mitochondrial quality through ROS production.

The etiology of hearing impairment following cochlear damage can be caused by many factors, including congenital or acquired onset, ototoxic drugs, noise exposure, and aging. Regardless of the many different etiologies, a common pathologic change is auditory cell death. It may be difficult to explain hearing impairment only from the aspect of cell death including apoptosis, necrosis, or necroptosis because the level of hearing loss varies widely. Therefore, we focused on autophagy as an intracellular phenomenon functionally competing with cell death. Autophagy is a dynamic lysosomal degradation and recycling system in the eukaryotic cell, mandatory for controlling the balance between cell survival and cell death induced by cellular stress, and maintaining homeostasis of postmitotic cells, including hair cells (HCs) and spiral ganglion neurons (SGNs) in the inner ear. Autophagy is considered a candidate for the auditory cell fate decision factor, whereas autophagy deficiency could be one of major causes of hearing impairment. In this paper, we review the molecular mechanisms and biologic functions of autophagy in the auditory system and discuss the latest research concerning autophagy-related genes and sensorineural hearing loss to gain insight into the role of autophagic mechanisms in inner-ear disorders.

Background: Mitochondrial substrate switching plays an important role in aging. The substrate metabolic rate is closely related to mitochondrial activity, as mitochondria are the primary site for substrate oxidation and ATP production. Different substrates (glucose, amino acids, and fatty acids) enter the mitochondria through distinct pathways and are metabolized at different rates, depending on the energy demand and cellular conditions. However, it remains unclear how the mitochondrial metabolic rate of these substrates affects auditory cellular function. This study aimed to characterize the substrate-dependent mitochondrial respiratory responses of cochlear cells under varying energy supply conditions and metabolic stress, focusing on glucose, amino acids, and fatty acids as representative energy sources. Methods: The oxygen consumption rate (OCR) was measured after substrate addition using an Agilent Seahorse XF24 Flux Analyzer In-House Ear Institute-Organ of Corti 1 (HEI-OC1) cells, and the maximum OCR (MOCR) was determined as part of the mitochondrial stress test. Statistical analyses were performed using analysis of variance (ANOVA). Results: The OCR increased significantly after glutamine (L-Gln) or palmitate addition. The MOCR after L-Gln addition was significantly higher than that after glutamic acid, glycine, and phenylalanine addition. The MOCR after pyruvate addition was significantly higher than that after glucose addition. However, there was no significant increase in the MOCR after fatty acid addition. Conclusions: Glucose is essential for basal metabolism but cannot rapidly meet sudden energy demands. Pyruvate and L-Gln serve as effective substrates for short-term, high-intensity energy demands. Fatty acids increase OCR through mitochondrial uncoupling effects, though their role may be limited in inner ear cells. These findings provide a foundation for exploring metabolic interventions to support cochlear function and hearing health.

Age-related hearing loss (ARHL) is one of the most prevalent types of sensory decline in a superaging society. Although various studies have focused on the effect of oxidative stress on the inner ear as an inducer of ARHL, there are no effective preventive approaches for ARHL. Recent studies have suggested that oxidative stress-induced DNA damage responses (oxidative DDRs) drive cochlear cell senescence and contribute to accelerated ARHL, and autophagy could function as a defense mechanism against cellular senescence in auditory cells. However, the underlying mechanism remains unclear. Sodium arsenite (NaAsO₂) is a unique oxidative stress inducer associated with reactive oxygen species (ROS) that causes high-tone hearing loss similar to ARHL. Transcription factor EB (TFEB) functions as a master regulator of the autophagy‒lysosome pathway (ALP), which is a potential target during aging and the pathogenesis of various age-related diseases. Here, we focused on the function of TFEB and the impact of intracellular ROS as a potential target for ARHL treatment in a NaAsO₂-induced auditory premature senescence model. Our results suggested that short exposure to NaAsO₂ leads to DNA damage, lysosomal damage and mitochondrial damage in auditory cells, triggering temporary signals for TFEB transport into the nucleus and, as a result, causing insufficient autophagic flux and declines in lysosomal function and biogenesis and mitochondrial quality. Then, intracellular ROS derived from damaged mitochondria play a role as a second messenger to induce premature senescence in auditory cells. These findings suggest that TFEB activation via transport into the nucleus contributes to anti-senescence activity in auditory cells and represents a new therapeutic target for ARHL. We have revealed the potential function of TFEB as a master regulator of the induction of oxidative stress-induced premature senescence and the senescence-associated secretion phenotype (SASP) in auditory cells, which regulates ALP and controls mitochondrial quality through ROS production.