Mechanisms of Mammalian Genetic Hearing Loss

项目总结 这项工作旨在了解人类耳聋基因编码蛋白质的机制， TMPRSS3，导致毛细胞死亡和听力丧失。毛细胞被顶端紧密连接蛋白包围。 复合体，在覆盖毛细胞顶端的内淋巴和内淋巴之间形成屏障 外淋巴，覆盖毛细胞的基底外侧。内淋巴含有高钾 外淋巴中钾离子浓度低，电荷量大 电势。顶端紧密连接的破坏导致内淋巴K+的通透性和死亡 感觉毛细胞。编码紧密连接蛋白的多个基因的变异会导致人类耳聋和 导致毛细胞在耳蜗内电位快速升高的过程中迅速退化。这一独特的时空 毛细胞死亡的模式与编码丝氨酸蛋白酶的基因变体相似， TMPRSS3.我们的初步数据显示，TMPRSS3的缺失破坏了顶端紧密连接的形成。我们 假设TMPRSS3通过维持紧密连接屏障来防止毛细胞退化 毛细胞之间通过紧密连接相关蛋白底物的蛋白质分解。此应用程序的目标是 明确TMPRSS3基因缺失导致紧密连接功能紊乱的生物学机制。在……里面 Aim1，我们将在体内测试TMPRSS3介导的毛细胞死亡是否依赖于耳蜗内电位和 我们将确定TMPRSS3中紧密连接蛋白的位置和/或蛋白水解性切割是否发生改变- 毛细胞缺乏。利用免疫组织化学、生化和超微结构技术，我们将确定 TMPRSS3的缺失是如何改变紧密连接的。目的2我们将在体内使用AAV介导的基因传递 以确定TMPRSS3功能是否依赖于蛋白酶，以及听力损失变体TMPRSS3是否具有功能。 在目标3中，我们将在人类干细胞来源的内耳有机体中使用多组学方法来确定 TMPRSS3调控的转录和蛋白质组途径。通过实现这些目标，我们不仅将 加深我们对TMPRSS3分子机制和内部蛋白酶底物的理解 耳朵，但也获得了对紧密连接的动态调节的洞察。这有可能影响到 遗传性耳聋有多种形式。

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.