Single‐Nucleus Transcriptomics Uncovers Xaf1‐Driven PANoptosis as a Therapeutic Target in Aminoglycoside‐Induced Hearing Loss

Aminoglycoside antibiotics are essential in managing many life-threatening diseases. However, their derivatives, such as neomycin, are associated with severe side effects such as persistent sensorineural hearing loss. Therefore, it is essential to elucidate the molecular and biochemical mechanisms of aminoglycoside-induced ototoxicity and identify targets for alleviating ototoxic injury. Here, we provide a detailed cochlear cell atlas of neomycin-induced acute and chronic ototoxicity-related changes through single-nucleus RNA sequencing profiling. Utilising this cochlear cell atlas, we used the Augur and scDist algorithms to evaluate cell-type-specific susceptibility to neomycin injury. We observed aberrant expression of X-linked inhibitor of apoptosis (Xiap)–associated factor 1 (Xaf1) in neomycin-exposed cochleae using the cochlear cell atlas, and we identified a novel role for Xaf1 in facilitating PANoptosis through overexpression and knockdown assays in vitro. Finally, we assessed the protective role of Xaf1 against neomycin-induced ototoxicity by Xaf1 knockdown in cochlear hair cells using adeno-associated virus-based gene delivery. Mechanistically, Xaf1 orchestrates PANoptosis activation through direct interaction with and transcriptional regulation of ZBP1, establishing its hierarchical position upstream in the signalling cascade. This study presents detailed cochlear cellular maps of neomycin-induced ototoxicity and serves as a valuable resource for identifying transcriptome-wide disease-driving perturbations at the single-cell level. More importantly, we identified Xaf1 as a critical target for modulating the PANoptosis pathway, offering a promising treatment strategy for aminoglycoside-induced ototoxicity.