ABSTRACT: Hearing loss (HL) is a global disability caused by irreversible damage to cochlear hair cells, essential for sound transduction. In mammals, the inability to regenerate these cells results in permanent auditory deficits. Early postnatal mice exhibit a limited regenerative potential from supporting cells, offering insights into possible therapeutic strategies. Noise-induced hearing loss (NIHL) is a widely used model for studying cochlear damage due to its shared molecular mechanisms with other forms of auditory dysfunction, including age-related hearing loss (presbycusis). Comprehensive molecular characterization of the cochlea is essential to understanding its pathophysiology and exploring regenerative approaches. Proteomics, enabled by mass spectrometry, is a powerful tool for deciphering protein networks involved in hearing loss and regeneration. This study aims to characterize and compare the proteomic profiles of cochleae from normal-hearing adult mice, NIHL adult mice, and neonatal mice, to identify key proteins and pathways involved in auditory function, hearing loss, and potential regenerative processes. Mouse cochleae were harvested from three groups: normal-hearing adult mice (P28, NH), NIHL adult mice (P28, NIHL), and neonatal mice (P2-7, NN). Hearing function in adult mice was assessed using Brainstem Auditory Evoked Potentials (ABR) only in the adult mice group. Since the auditory process in mice begins between P12-P14, the test was not performed on neonates. Deafness was induced by overexposure to noise. For this, the animals were subjected to white noise at 130 dBSPL for a total of 24 hours in a closed field and sound isolation booth with an audio signal generator. In the normal hearing adult group, hearing was confirmed by the presence of ABR waves between 90-30 dB. In the NIHL group, audiological evaluation was performed before noise exposure to ensure good hearing and up to 24 hours after exposure to confirm HL, evidenced by the absence of waves up to 90 dB. Proteomic analysis using Nano LC-MS/MS was conducted, and proteins were identified and quantified using MaxQuant software. Global proteomes and differentially abundant proteins (DAPs) were analyzed using Perseus.The analysis revealed 1572 proteins in NH, 1605 in NIHL, and 2113 in the NN profiles, totaling 2413 unique proteins, with 81 DAPs between NH vs NIHL and 715 between NH vs NN. The results were validated through robust bioinformatics analyses, including gene and phenotype ontology, biological pathways, and protein-protein interaction networks. Special emphasis was placed on genes previously associated with human deafness, loci linked to hearing loss without identified genes, and druggability assessments to pinpoint potential therapeutic targets. Our findings were further validated through the integration of public omics datasets, allowing for cellular distribution analysis of DAP-encoding genes across major cochlear cell types. This integrative approach not only highlights key molecular pathways such as oxidative stress and sensory epithelium plasticity but also identifies promising targets for regenerative therapies. The dataset and insights derived from this study contribute significantly to understanding auditory pathophysiology and advancing therapeutic strategies for hearing restoration.
Ana Carla Batissoco