Background Age-related hearing loss (ARHL) is the most prevalent sensory deficit in older adults, yet its molecular progression remains poorly resolved. Few cochlear single-cell studies have integrated cell–cell communication, gene regulatory network (GRN) reconstruction, and cross-cohort bulk validation into one temporal framework. Methods We reanalyzed publicly available single-nucleus RNA sequencing data from C57BL/6 J mouse cochleae at 3, 12, and 24 months and cross-validated against two independent bulk microarray cohorts. Ligand–receptor interactions were inferred and validated at the ligand-expression level. GRNs were reconstructed and perturbed in silico; and feature selection extracted cross-scale inflammatory features. Results ARHL did not follow a linear decline. A cochlea-wide type I interferon response peaked at 12 months and resolved by 24. Apolipoprotein E followed a three-phase trajectory, baseline, compensatory upregulation, collapse, across spiral ganglion neurons, inner hair cells (IHCs), macrophages, and fibrocytes. SLIT–ROBO axon-guidance signaling shut down in these cells, alongside neurotrophic and Notch withdrawal. In silico perturbation identified T-box transcription factor 2 (Tbx2) as a protective GRN node: knockout drove 81.2% of IHCs toward aging and overexpression 74.6% toward youth. A 13-gene inflammatory panel showed midlife activation and end-stage exhaustion in macrophages, mirroring the interferon peak. Conclusion These findings support a two-stage ARHL model, midlife immune activation followed by end-stage synaptic, lipid, and axon-guidance collapse, positioning the midlife window and Tbx2-dependent IHC maintenance as candidate intervention targets, pending in vivo validation.
