Clotrimazole-Mediated Autophagy to Protect Against Cisplatin-Induced Ototoxicity via the AMPK/mTOR/TFEB Pathway in Mice

Background: Cisplatin is an effective chemotherapeutic agent, but its clinical use is limited by dose-dependent ototoxicity that leads to irreversible sensorineural hearing loss. Accumulating evidence implicates impaired autophagy–lysosomal homeostasis in cisplatin-induced ototoxicity. Transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, represents a promising therapeutic target. Clotrimazole, an FDA-approved antifungal drug with emerging cytoprotective properties, has not been investigated for its potential to mitigate cisplatin-induced ototoxicity. Methods: We evaluated the protective effects of clotrimazole using House Ear Institute-Organ of Corti 1 cells, cochlear explants, and an adult C57BL/6J mouse model of transtympanic cisplatin ototoxicity. Apoptosis, reactive oxygen species (ROS), and autophagy flux were assessed using biochemical assays and imaging. RNA-sequencing was performed to identify transcriptional pathways regulated by clotrimazole. TFEB dependence was verified using small interfering RNA knockdown and pharmacological inhibition of AMP-activated protein kinase (AMPK). Cochlear function was assessed using auditory brainstem responses (ABRs), and hair-cell and synapse survival were quantified by immunofluorescence. Results: Clotrimazole significantly reduced cisplatin-induced apoptosis, ROS generation, and calcium overload. Transcriptomic profiling and functional assays revealed robust activation of autophagy. Clotrimazole promoted AMPK activation, suppressed mTORC1 signaling, and enhanced TFEB nuclear translocation. TFEB or AMPK inhibition abrogated these protective effects. In vivo , intratympanic clotrimazole preserved hair cell survival, maintained ribbon synapses, and significantly reduced ABR threshold shifts. Conclusions: Clotrimazole protects against cisplatin-induced ototoxicity by activating the AMPK–mTOR–TFEB axis and restoring autophagy lysosomal homeostasis. These findings support TFEB-targeted autophagy activation as a promising therapeutic strategy for preventing cisplatin-induced hearing loss. Antioxid. Redox Signal. 44, 928–950.