Modulating ATOH1 and POU4F3 Pathways to Enhance Hair Cell Regeneration and Inhibit Ferroptosis in Cochlear Support Cells

Hearing loss affects millions worldwide, yet effective regenerative treatments remain limited. This study systematically investigated how the ATOH1/POU4F3 axis drives supporting cell reprogramming and hair cell regeneration while suppressing autophagy-dependent Ferroptosis in cochlear hair cells. We combined single-cell RNA Sequencing of mouse cochlear tissue across developmental stages with bulk transcriptomics, protein-protein interaction analysis, and pathway enrichment to identify key regulators. Using lentiviral overexpression and silencing, ChIP-qPCR, dual-luciferase reporter assays, Western blotting, immunofluorescence, and EdU labeling, we demonstrated that ATOH1 directly activates POU4F3, upregulates MYO7A expression, enhances supporting cell proliferation, and promotes mitotic entry. In an oxidative stress model, activation of this pathway reduced Autophagy flux, lipid peroxidation, and Fe²⁺ levels, improving cell survival. In a cisplatin-induced hearing loss mouse model, modulating the ATOH1/POU4F3 axis significantly improved ABR and DPOAE thresholds and restored hair cell structure and arrangement, confirmed by SEM and immunostaining. Together, these findings provide new mechanistic insights into how transcriptional regulation links regeneration and cell death resistance, highlighting a promising therapeutic target for hearing restoration.

Pou domain; atonal homolog 1; autophagy‐dependent ferroptosis; class 4; hair cell regeneration; hearing loss; reprogramming; transcription factor 3.