Critical role of TPRN rings in the stereocilia for hearing

Inner ear hair cells detect sound vibration through the deflection of mechanosensory stereocilia. Cytoplasmic protein TPRN has been shown to localize at the taper region of the stereocilia, and mutations in TPRN cause hereditary hearing loss through an unknown mechanism. Here, using biochemistry and dual stimulated emission depletion microscopy (STED) imaging, we showed that the TPRN, together with its binding proteins CLIC5 and PTPRQ, formed concentric rings in the taper region of stereocilia. The disruption of TPRN rings, triggered by the competitively inhibiting the interaction of TPRN and CLIC5 or the exogenous TPRN overexpression, led to stereocilia degeneration and severe hearing loss. Most importantly, restoration of the TPRN rings was able to rescue the damaged auditory function of Tprn knockout mice by exogenously expressing TPRN at an appropriate level in HCs via promoter recombinant adeno-associated virus (AAV). In summary, our results reveal highly structured TPRN rings near the taper region of stereocilia that are crucial for stereocilia function and hearing. Besides, TPRN rings restoration in stereocilia by AAV-Tprn effectively repaired the damaged hearing, which laid down the foundation for clinical application of AAV-mediated gene therapy in patients with TPRN mutation.