Abstract

To investigate the expression changes of ATP6V1B2 downregulation in Dominant Deafness-Onychodystrophy syndrome (DDOD syndrome), its impact on auditory function, and the underlying molecular mechanisms, thereby clarifying the role of ATP6V1B2 in maintaining inner ear auditory function. RNA interference was used to downregulate ATP6V1B2 expression in HEI-OC1 cells. A DDOD syndrome mouse model was established via cochlear microinjection of ATP6V1B2-specific morpholino, while control mice injected with a scramble morpholino. The expression and localization of ATP6V1B2 in the mouse inner ear were assessed by quantitative real-time polymerase chain reaction (RT-qPCR), immunohistochemistry, and Western blot. Auditory brainstem response (ABR) was measured to evaluate auditory function at different ages (4–36 weeks). Lysosomal H⁺-ATPase (vacuolar-type ATPase [V-ATPase]) and cathepsin-D (Cath-D) activities were detected using enzyme activity assays. Western blot and immunofluorescence were used to analyze lysosomal-associated proteins (LAMP1, LAMP2, TPC1, TPC2) and autophagy-and-apoptosis-related proteins (LC3, Caspase-3, Bcl-2, Bax). Apoptosis, reactive oxygen species (ROS) levels, and mitochondrial membrane potential were quantified by flow cytometry and staining with 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), respectively. In DDOD model mice, ATP6V1B2 mRNA and protein levels were significantly reduced in the inner ear, with decreased localization in key auditory regions such as cochlear hair cells and spiral ganglion neurons (SGNs), which was accompanied by SGN loss. ABR thresholds were markedly elevated after 28 weeks, indicating progressive auditory dysfunction. Mechanistically, cochlear V-ATPase and Cath-D activities were significantly reduced, while lysosomal membrane protei