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 (WT) mice exhibited permanent threshold shifts, persistent ABR wave I amplitude reduction, and significant loss of both ribbon synapses and outer hair cells (OHC) in the basal turn. Unexpectedly, Vglut3-KO mice also exhibited significant synapse loss despite their inability to release glutamate, revealing glutamate-independent mechanisms of synaptopathy. Proteomic analysis of noise-exposed cochleae identified 62 differentially expressed proteins enriched in synaptic and extracellular matrix pathways, including upregulation of Rps6ka1, Cplx2, and Cdk5, and downregulation of Ppa2. Comparative profiling of Vglut3-KO mice further revealed 233 differentially expressed proteins, with widespread downregulation of synaptic, mitochondrial, and ECM pathways. 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, with VGLUT3 expression restored across all cochlear turns as confirmed by immunofluorescence. 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 OHC loss limiting gene therapy outcomes, underscoring the need for comprehensive otoprotective strategies in hereditary deafness patients awaiting treatment.