The underlying molecular mechanisms of age-related hearing loss (ARHL) in humans and many strains of mice have not been fully characterized. This common age-related disorder is assumed to be closely associated with oxidative stress. Here, we demonstrate that mTORC1 signaling is highly and specifically activated in the cochlear neurosensory epithelium (NSE) in aging mice, and rapamycin injection prevents ARHL. To further examine the specific role of mTORC1 signaling in ARHL, we generated murine models with NSE-specific deletions of Raptor or Tsc1, regulators of mTORC1 signaling. Raptor-cKO mice developed hearing loss considerably more slowly than WT littermates. Conversely, Tsc1 loss led to the early-onset death of cochlear hair cells and consequently accelerated hearing loss. Tsc1-cKO cochleae showed features of oxidative stress and impaired antioxidant defenses. Treatment with rapamycin and the antioxidant N-acetylcysteine rescued Tsc1-cKO hair cells from injury in vivo. In addition, we identified the peroxisome as the initial signaling organelle involved in the regulation of mTORC1 signaling in cochlear hair cells. In summary, our findings identify overactive mTORC1 signaling as one of the critical causes of ARHL and suggest that reduction of mTORC1 activity in cochlear hair cells may be a potential strategy to prevent ARHL.

Glutathione synthetase (GSS) catalyzes the final step in the synthesis of glutathione (GSH), a well-established antioxidant. Research on the specific roles of the Gss gene during spermatogenesis remains limited due to the intricate structure of testis. In this study, we identified pachytene spermatocytes as the primary site of GSS expression and generated a mouse model with postnatal deletion of Gss using Stra8-Cre (S8) to investigate the role of GSS in germ cells. The impact of Gss knockout on reducing male fertility is age-dependent and caused by ferroptosis in the testis. The 2-month-old S8/Gss^−/− male mice exhibited normal fertility, due to a compensatory increase in GPX4, which prevented the accumulation of ROS. With aging, there was a decline in GPX4 and an increase in ALOX15 levels observed in 8-month-old S8/Gss^−/− mice, resulting in the accumulation of ROS, lipid peroxidation, and ultimately testicular ferroptosis. We found that testicular ferroptosis did not affect spermatogonia, but caused meiosis disruption and acrosome heterotopia. Then the resulting aberrant sperm showed lower concentration and abnormal morphology, leading to reduced fertility. Furthermore, these injuries could be functionally rescued by inhibiting ferroptosis through intraperitoneal injection of GSH or Fer-1. In summary, Gss in germ cells play a crucial role in the resistance to oxidative stress injury in aged mice. Our findings deepen the understanding of ferroptosis during spermatogenesis and suggest that inhibiting ferroptosis may be a potential strategy for the treatment of male infertility.

Significance: The mechanistic target of rapamycin (mTOR) plays a central role in growth, metabolism, and aging. It is assembled into two multiprotein complexes, namely, mTORC1 and mTORC2. We previously demonstrated the efficacy of sirolimus in ARHL in mice by decreasing mTORC1. However, the aspect of mTORC2 regulation in the cochlea is poorly characterized. Herein, based on pharmacological and genetic interventions, we found that a high dose of sirolimus resulted in severe hearing loss by reducing the mTORC2/AKT signaling pathway in the cochlea. Furthermore, selective activation of mTORC2 could protect against hearing loss induced by acoustic trauma and cisplatin-induced ototoxicity. Hence, the therapeutic activation of mTORC2 in conjunction with decreasing mTORC1 might represent a promising and effective strategy in preventing hearing loss.

Mitochondrial dynamics is essential for maintaining the physiological function of the mitochondrial network, and the disorders of it leads to a variety of diseases. Our previous study identified mitochondrial dynamics controlled anti-tumor immune responses and anxiety symptoms. However, how mitochondrial dynamics affects auditory function in the inner ear remains unclear. Here, we show that deficiency of FAM73a or FAM73b, two mitochondrial outer membrane proteins that mediate mitochondrial fusion, lead to outer hair cells damage and progressive hearing loss in FVB/N mice. Abnormal mitochondrial fusion causes elevated oxidative stress and apoptosis of hair cells in early stage. Thereafter, the activation of macrophages and CD4⁺ T cell is found in the mutant mice with the increased expression of the inflammatory cytokines IL-12 and IFN-γ compared to control mice. Strikingly, dramatically decreased number of macrophages by LCCA treatment alleviates the hearing loss of mutant mice. Collectively, our finding highlights that FAM73a or FAM73b deficiency affects hair cells survival by disturbing mitochondrial function, and the subsequent immune response in the cochleae worsens the damage of hair cells.