Mechanisms of Mammalian Genetic Hearing Loss

哺乳动物遗传性听力损失的机制

• 批准号: 10660134
• 负责人: Rick F Nelson
• 金额: $ 61.3万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660134
• 关键词: 3-Dimensional; Adaptor Signaling Protein; Address; Adult; Affect; Apical; Audiology; Biochemical; Biological; Cell Death; Cell Survival; Cell physiology; Cells; Cessation of life; Charge; Child; Cochlea; Cochlear Implants; Cut protein; Data; Development; Endolymph; Epitopes; Frequencies; Gene Delivery; Gene Mutation; Gene Proteins; Genes; Genetic Complementation Test; Genotype; Goals; Hair Cells; Hearing; Hour; Human; In Vitro; Knowledge; Labyrinth; Link; Location; Mammalian Genetics; Mediating; Membrane; Molecular; Mutant Strains Mice; Mutation; Organ of Corti; Organoids; Pathway interactions; Patients; Pattern; Peptide Hydrolases; Perilymph; Permeability; Phenotype; Physiological; Play; Potassium; Preventive treatment; Process; Protein Truncation; Proteins; Proteolysis; Proteomics; Regulation; Reporting; Role; Sensory; Sensory Hair; Serine Protease; Side; Techniques; Testing; Tight Junctions; United States; Variant; Work; curative treatments; deafness; design; electrical potential; genetic deafness; genetic variant; hearing impairment; hereditary hearing loss; human stem cells; in vivo; induced pluripotent stem cell; insight; link protein; mouse model; multiple omics; mutant; mutant mouse model; novel; overexpression; postnatal; preservation; prevent; protein complex; single-cell RNA sequencing; targeted treatment; transcriptomics; treatment strategy

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.

项目概要 这项工作旨在了解人类耳聋基因编码的蛋白质的机制， TMPRSS3，导致毛细胞死亡和听力损失。毛细胞被顶端紧密连接蛋白包围 复合物，在内淋巴之间形成屏障，覆盖毛细胞的顶端侧 外淋巴，覆盖毛细胞的基底外侧。内淋巴含有大量钾 浓度和高电荷，而外淋巴液钾浓度低和低 电势。心尖紧密连接的破坏导致内淋巴 K+ 的渗透性和死亡 感觉毛细胞。编码紧密连接蛋白的多个基因的变异导致人类耳聋和 导致耳蜗内电位快速上升期间毛细胞快速退化。这种独特的时空 毛细胞死亡的模式与编码丝氨酸蛋白酶的基因变异相似， TMPRSS3。我们的初步数据表明，TMPRSS3 的缺失会破坏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变性 毛细胞之间通过紧密连接相关蛋白底物的蛋白水解作用。该应用程序的目标是 定义 TMPRSS3 缺失如何导致紧密连接功能破坏的生物学机制。在 Aim1，我们将测试 TMPRSS3 介导的毛细胞死亡是否依赖于体内耳蜗电位，以及 我们将确定 TMPRSS3 中紧密连接蛋白的位置和/或蛋白水解切割是否发生改变 毛细胞缺陷。使用免疫组织化学、生化和超微结构技术，我们将确定 TMPRSS3 的缺失如何在物理上改变紧密连接。目标 2 我们将使用 AAV 介导的体内基因传递 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衍生的内耳类器官中使用多组学方法来确定 TMPRSS3 调控的转录组和蛋白质组途径。通过实现这些目标，我们不仅 促进我们对 TMPRSS3 内部分子机制和蛋白酶底物的理解 耳，还可以深入了解紧密连接的动态调节。这有可能影响 遗传性耳聋有多种形式。