Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss

The Food and Drug Administration–approved drug sirolimus, which inhibits mechanistic target of rapamycin (mTOR), is the leading candidate for targeting aging in rodents and humans. We previously demonstrated that sirolimus could treat ARHL in mice. In this study, we further demonstrate that sirolimus protects mice against cocaine-induced hearing loss. However, using efficacy and safety tests, we discovered that mice developed substantial hearing loss when administered high doses of sirolimus. Using pharmacological and genetic interventions in murine models, we demonstrate that the inactivation of mTORC2 is the major driver underlying hearing loss. Mechanistically, mTORC2 exerts its effects primarily through phosphorylating in the AKT/PKB signaling pathway, and ablation of P53 activity greatly attenuated the severity of the hearing phenotype in mTORC2-deficient mice. We also found that the selective activation of mTORC2 could protect mice from acoustic trauma and cisplatin-induced ototoxicity. Thus, in this study, we discover a function of mTORC2 and suggest that its therapeutic activation could represent a potentially effective and promising strategy to prevent sensorineural hearing loss. More importantly, we elucidate the side effects of sirolimus and provide an evaluation criterion for the rational use of this drug in a clinical setting.

With increasing progress and a rapid improvement in the living standards of individuals in society, considerable importance is being given to hearing problems. Therefore, it is of great significance to study the occurrence and understand the mechanisms underlying the development of hearing disorders to effectively prevent, detect, and treat deafness. Among the hearing-impaired population, sensorineural deafness accounts for the majority of cases of hearing loss. Studies have shown that almost 50% of hereditary sensorineural deafness is caused by mutations in hair cell (HC) enriched genes, although HCs constitute only approximately one-tenth of the cells in the sensory epithelium of the cochlea (1). These factors responsible for injury, including genetic defects, can lead to irreversible damage and the loss of cochlear HCs, which is the main cause of sensorineural hearing loss (2). Therefore, understanding the mechanism of the development and survival of auditory HCs and being able to restore the function of cochlear sensory epithelium is an ideal approach in auditory reconstruction and also the primary focus in the field of otology.Mechanistic target of rapamycin (mTOR), now defined as the mechanistic target of sirolimus, is a highly conserved serine/threonine protein kinase, which controls the growth of cells and organisms induced by growth factors and nutrients. The mTOR is assembled into two multiprotein complexes, namely, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which can be distinguished based on their related proteins and their sensitivity to sirolimus (3). Evidence indicates that mTORC1 is mainly responsible for cell growth and proliferation in response to growth factors, nutrients, or stress, and that the two main downstream targets of mTORC1, namely, p70S6 kinase (S6K) and elongation factor 4E binding protein (4E-BP1), are key regulators of cap-dependent protein translation (4, 5). Although the mechanisms that regulate mTORC1 are well understood, the aspect of regulation of mTORC2 is relatively poorly characterized. The mTORC2 signaling is insensitive to nutrients; however, it responds to growth factors, such as insulin, through poorly defined mechanism(s) that require(s) PI3K (6). Experiments in yeast and cultured mammalian cells have indicated that mTORC2 plays a role in the regulation of the actin cytoskeleton (7, 8). The mTORC2 controls several members of the AGC kinase subfamily, including Akt, protein kinase C-α (PKC-α), and serum and glucocorticoid-induced protein kinase 1 (SGK1) (9–11). Although mTORC2 is relatively insensitive to acute sirolimus treatment, recent studies have shown that prolonged exposure to sirolimus can inhibit the mTORC2 complex assembly (12–14).As one of the central regulators of cellular activities, the mTOR signaling pathway has been attracting increasing attention in recent years (4, 15). Several studies now focus on the function of mTOR in metabolic tissues, largely because these tissues are particularly sensitive to the three inputs (nutrients, insulin, and energy status) that control mTOR (16, 17). Additionally, abnormalities in the mTOR signaling pathway can lead to conditions such as epilepsy (18, 19), diabetes mellitus (20, 21), tuberous sclerosis syndrome (22, 23), and tumors (24, 25). Nevertheless, existing knowledge regarding the functioning and regulation of the mTOR pathway in the maintenance of the auditory system and homeostasis is limited. To date, the available evidence suggests that auditory disorders result from changes in the mTOR signaling pathway (26–28). Recently, our study group provided evidence that inhibition of mTORC1 resulting from an intraperitoneal (i.p.) injection of 1 mg/kg sirolimus or from a genetic disorder leading to a decrease in mTORC1 activity via deficiency of Raptor attenuated age-related hearing loss (ARHL) in C57BL/6J mice, whereas overactivation of the mTORC1 signaling pathway in the neurosensory cells (NSE) caused premature HC death and progressive hearing loss (29). However, as a Food and Drug Administration (FDA)-approved drug, although an i.p. injection of sirolimus in wild-type (WT) C57BL/6J mice can prevent ARHL, some caveats must be considered when translating this finding to a clinical setting. Accordingly, the efficacy and safety of sirolimus should be comprehensively understood prior to its use in treating ARHL. While studying the effects of different doses of the drug, we serendipitously found that high doses of injected sirolimus could result in a significant loss of hearing in mice. Coincidentally, a recent study reports that the inhibition of mTOR by sirolimus results in dose-dependent damage of auditory HCs cultured ex vivo (30). Collectively, these findings suggest the complex functions of the mTOR signaling pathway in the auditory sensory epithelium, especially in auditory HCs. It is currently unknown whether mTOR acts through mTORC1, through mTORC2, or through the synergistic action of these two complexes in the development and survival of HCs. Neither the genetic nor the pharmacological manipulation of mTOR kinase is adequate to distinguish the role of these two complexes in cochlear HCs. In addition, the correlation between mTORC1 and mTORC2 in cochlear HCs and the downstream molecular regulatory signals remains to be elucidated.