(858) Mapping the molecular landscape of noise-induced cochlear synaptopathy using RNA sequencing

Introduction: To define the molecular responses underlying cochlear excitotoxicity and synaptopathy.

Methods: A 3D in vitro cochlear explant model using postnatal day 2–3 mouse cochleae was employed to interrogate molecular changes following excitotoxic injury that mimics noise trauma and neurotrophin treatment. Explants were exposed to kainic acid, kainic acid followed by neurotrophin-3 (NT3), or control media. Synapse quantification was performed by immunohistochemistry using antibodies against Myo7a, CtBP2, and GluR2, with synapses defined by co-localization of CtBP2 and GluR2. Additional explants from each condition were homogenized for bulk RNA sequencing, followed by bioinformatic analysis in Python.

Results: Synapse quantification demonstrated significant synapse loss following kainic acid–induced excitotoxicity (2 synapses/IHC) compared to controls (19 synapses/IHC, p < 0.05). In contrast, explants treated with NT3 after excitotoxic exposure showed substantial synaptic recovery (18 synapses/IHC), which was not significantly different from controls (p > 0.05). Bulk RNA-seq revealed both shared and condition-specific transcriptional changes across NT3, kainic acid, and control conditions, with the NT3 vs control comparison capturing the largest and most overlapping gene set (124 of 202 total genes). Gene set enrichment analysis demonstrated that the kainic acid condition was dominated by GO terms related to regulation of the regulated secretory pathway, neurotransmitter secretion, and calcium-ion–regulated exocytosis, consistent with activation of calcium-permeable glutamate receptors. In contrast, NT3 treatment was enriched for GO terms associated with regulation of RNA metabolic processes, RNA-mediated gene silencing, and signal transduction in response to DNA damage, suggesting activation of transcriptional and epigenetic regulatory programs.

Conclusions: These findings indicate that excitotoxic injury triggers calcium-handling and secretory responses, whereas NT3 treatment promotes RNA- and gene regulatory pathways consistent with a broader injury response.