A Ring-like Protein Complex In The Taper Region Is Critical For Stereocilia Maintenance And Hearing Function

Deafness is one of the most common sensory defects in clinic.About half of sensorineural deafness is caused by genetic mutations.The genetic mutation causes damage to the cochlear epithelium,leading to the death of hair cells.At present,there is still no effective treatment for sensorineural deafness caused by hair cell injury.Inner ear hair cells(HCs)are sound receptors in auditory epithelium,and they could sense and transmit sound signals through the stereocilia on their surface.The stereocilia mainly contain parallel F-actin filaments that are cross-linked by F-actin related proteins.The diameter of the stereocilia narrows rapidly at the junction with the HCs’ body and this region is called the taper region,which provides rigid support for the standing of the stereocilia.TPRN(TAPERIN)is named for its location in the taper region.TPRN is encoded by the Tprn gene,and mutations in Tprn cause hereditary hearing loss in mice and humans,but the underlying molecular mechanisms have not been elucidated.In this study,we found that TPRN formed a ring structure in the taper region of the root of the stereocilia which was stable under the interaction of CLIC5,and the complex formed by the TPRN and CLIC5 was arranged in a concentric ring structure by using thestimulated emission depletion microscopy(STED).Further,competitive inhibition of their interaction or disruption of the TPRN ring structure in Tprn-knockout mice led to stereocilia damage and severe hearing loss.In addition,we also found that the expression level of Tprn is crucial for the maintenance of ring structure and hearing,and too little or too much expression of Tprn can lead to the loss of ring structure and serious stereocilia damage.Tprn at an appropriate level in Tprn-knockout mice via Anc80L65,an adeno-associated virus targeting hair cells,carrying modified type I hammerhead ribozyme or CMV173 promoter,was able to restore Tprn’s ring structure and restore hearing function to some extent,and maintain hearing recovery for at least 2 months,suggesting that hearing loss caused by the ring loss of TPRN can be restored after the ring structure restoration.In summary,our results reveal that a highly cyclic structural complex near the conical region of the static cilia is essential for the function of the static cilia,and its disruption causes hearing impairment;Meanwhile,moderate overexpression of exogenous Tprn mediated by HHR or CMV173 promoters restored hearing function in Tprn-knockout mice,suggesting that gene therapy mediated by HHR and CMV173 promoters may prevent human hearing loss caused by Tprn dysfunction.In summary,our results reveal a highly structured compartment near the taper region of stereocilia that is crucial for stereocilia function and that is disrupted in cases of hearing loss.Meanwhile,appropriate overexpression of exogenous Tprn mediated by HHR or CMV173 promoter can restore hearing function in Tprn-knockout mice,which suggests that HHR/CMV173-mediated gene therapy has the potential to prevent hearing loss in humans with Tprn disfunction.