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.

项目摘要 这项工作旨在了解人类耳聋基因编码的蛋白质， TMPRSS 3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，其在覆盖毛细胞顶侧的内淋巴和 外淋巴，覆盖毛细胞的基底外侧。内淋巴含有高钾 外淋巴液中钾离子浓度低，电荷量高， 电势顶端紧密连接的破坏导致内淋巴K+的渗透性和细胞的死亡。 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， 导致耳蜗内电位快速上升期间毛细胞快速变性。这个独特的时间 毛细胞死亡的模式模拟了在编码丝氨酸蛋白酶的基因中的变体中所看到的， TMPRSS 3.我们的初步数据显示TMPRSS 3的缺失破坏了顶端紧密连接的形成。我们 假设TMPRSS 3通过维持紧密连接屏障来防止毛细胞变性 通过紧密连接相关蛋白质底物的蛋白质水解在毛细胞之间进行。这个应用程序的目标是 确定TMPRSS 3缺失如何导致紧密连接功能破坏的生物学机制。在 目的1，我们将测试TMPRSS 3介导的毛细胞死亡是否依赖于体内耳蜗内电位， 我们将确定在TMPRSS 3中紧密连接蛋白的位置和/或蛋白水解切割是否改变， 缺乏毛细胞利用免疫组织化学、生物化学和超微结构技术， TMPRSS 3的缺失如何物理改变紧密连接。目的2：利用腺相关病毒介导的体内基因传递技术 以确定TMPRSS 3功能是否是蛋白酶依赖性的以及听力损失变体TMPRSS 3是否是功能性的。 在目标3中，我们将在人类干细胞来源的内耳类器官中使用多组学方法来确定 TMPRSS 3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 进一步了解TMPRSS 3的分子机制和蛋白酶底物， 耳朵，而且还获得深入了解紧密连接的动态调节。这有可能影响到 遗传性耳聋的多种形式。