Beyond Excitotoxicity: Glutamate-Independent Mechanisms Underlie Noise-Induced Synaptopathy and Compromise Gene Therapy for Hereditary Deafness

Noise-induced hearing loss (NIHL) is classically attributed to glutamate excitotoxicity, yet the precise requirement for synaptic glutamate release remains untested using genetic approaches. Here, we investigated noise-induced cochlear damage using Vglut3 knockout ( Vglut3 -KO) mice, which lack glutamatergic transmission from inner hair cells. Following 116 dB SPL noise exposure, wild-type mice exhibited permanent threshold shifts, persistent ABR wave I amplitude reduction, and significant ribbon synapse loss at the 32 kHz region. Unexpectedly, Vglut3 -KO mice also exhibited significant synapse loss despite their inability to release glutamate, revealing glutamate-independent mechanisms of synaptopathy. Proteomic analysis identified 62 differentially expressed proteins in noise-exposed cochleae, with significant enrichment in synaptic structures and extracellular matrix components, including up-regulation of Rps6ka1 , Cplx2 , and Cdk5 , and downregulation of Ppa2 . Importantly, noise exposure severely compromised AAV8- Vglut3 gene therapy efficacy in Vglut3 -KO mice: while unexposed mutants achieved complete functional recovery, noise-exposed animals failed to attain normal hearing thresholds. These findings demonstrate that high-intensity noise induces synaptopathy through both excitotoxic and non-excitotoxic mechanisms involving synaptic and ECM disruption, and that acoustic trauma creates persistent pathological changes limiting gene therapy outcomes, underscoring the need for comprehensive otoprotective strategies in hereditary deafness patients awaiting treatment.