Mechanisms of Mammalian Genetic Hearing Loss

PROJECT SUMMARY This work is designed to understand the mechanism of how the protein encoded by the human deafness gene, TMPRSS3, leads to hair cell death and hearing loss. Hair cells are surrounded by apical tight junction protein complexes, which form a barrier between the endolymph which covers the apical side of the hair cell and perilymph, which covers the basolateral side of the hair cell. The endolymph contains a high potassium concentration and high electrical charge, while the perilymph has low potassium concentration and low electrical potential. Disruption of the apical tight junctions leads to permeability of endolymph K+ and death of sensory hair cells. Variants in the multiple genes encoding tight junction proteins cause human deafness and result in rapid hair cell degeneration during the rapid rise in endocochlear potential. This unique temporal pattern of hair cell death mimics what is seen with variants in the gene encoding the serine protease, TMPRSS3. Our preliminary data shows that loss of TMPRSS3 disrupts apical tight junction formation. We hypothesize that TMPRSS3 functions to prevent hair cell degeneration by maintaining the tight junction barrier between hair cells through proteolysis of tight junction related protein substrates. The goal of this application is to define the biological mechanism of how loss of TMPRSS3 leads to disruption of tight junction function. In Aim1, we will test if TMPRSS3-mediated hair cell death is dependent on the endocochlear potential in vivo and we will determine if the location and/or proteolytic cleavage of tight junction proteins are altered in TMPRSS3- deficient hair cells. Using immunohistochemical, biochemical and ultrastructure techniques, we will determine how loss of TMPRSS3 physically alters tight junctions. Aim 2 we will use AAV-mediated gene delivery in vivo to determine if TMPRSS3 function is protease dependent and if hearing loss variants TMPRSS3 are functional. In Aim 3, we will use multiomic approaches in human stem cell-derived inner ear organoids to determine transcriptomic and proteomic pathways regulated by TMPRSS3. By accomplishing these aims we will not only advance our understanding of the molecular mechanism and protease substrates of TMPRSS3 in the inner ear, but also gain insights into the dynamic regulation of tight junctions. This has the potential to impact multiple forms for genetic deafness.