Contribution of COMT2 to auditory processing in mammalian sensory hair cells

项目摘要/摘要 听力损失是人类最普遍的感觉障碍，并导致严重的质量损失。 受苦受难的人的生活。哺乳动物的耳蜗通过转换声音来启动对声音的感知。 以极高的灵敏度、速度和精度将电波转换成电信号。机械转导在体内的作用 感觉毛细胞导致电化学信号，从而诱导神经递质从 特殊的带状突触，启动听觉处理的神经信号。分子 毛细胞中听觉处理机制的组成还不是很清楚，而 具体的分子相互作用如何导致声音处理的生理事件尚不清楚。 已有100多种非综合征形式的遗传性耳聋被描述，但其中许多基因 与这些突变相关的基因是未知的或没有很好地描述。儿茶酚-O-的突变 甲基转移酶2(COMT2)可导致人类非综合征性隐性耳聋(DFNB63)。我们的实验室 由ENU突变产生的一种小鼠突变体(称为‘Add’)，在该突变体中，comt2的点突变， 老鼠与人类基因同源，会导致深度耳聋。Comt2基因在头发中的表达 内耳中的细胞，但COMT2对小鼠和 人类是完全未知的。本提案的目的是研究COMT2在 外周听觉系统和COMT2在听觉加工中的作用。假设是 COMT2通过调节突触功能参与毛细胞功能和听觉处理 耳蜗管。AIM 1将利用原位杂交、免疫组织化学和免疫金电子 用显微镜精确表征comt2的细胞特异性和时间表达模式 小鼠内耳和听性脑干的mRNA及COMT2蛋白的亚细胞定位 在感觉毛细胞中。AIM 2将使用CRISPR/Cas9技术来生成和验证构成 Comt2基因缺失小鼠和Comt2基因缺失小鼠，以便于研究Comt2功能的丧失 外周听觉功能和听觉处理。AIM 3将分析comt2突变体以确定 Comt2在感觉毛细胞中促进听觉处理的机制。通过 询问听觉系统中的Comt2表达模式和功能，这项工作将 有助于更好地理解听觉加工的机制，生理作用 COMT2，以及人类DFNB63的病因。

Project Summary/Abstract Hearing loss is the most prevalent sensory deficit in humans and causes a significant toll on the quality of life of those afflicted. The mammalian cochlea initiates the perception of sound by converting sound waves into electrical signals with exquisite sensitivity, speed and precision. Mechanotransduction in sensory hair cells leads to electrochemical signals that induce neurotransmitter release from specialized ribbon synapses, initiating the neural signal for auditory processing. The molecular composition of the auditory processing machinery in hair cells is not well understood, and the nature of how specific molecular interactions lead to physiological events for the processing of sound is unclear. Over 100 nonsyndromic forms of genetic hearing loss have been described but many of the genes linked with these mutations are unknown or not well characterized. Mutations in Catechol-O- methyltransferase 2 (COMT2) cause nonsyndromic recessive deafness (DFNB63) in humans. Our lab generated by ENU-mutagenesis a mouse mutant (termed `Add') in which a point mutation in Comt2, the mouse homolog of the human gene, leads to profound deafness. Comt2 mRNA is expressed by hair cells in the inner ear but the manner in which COMT2 contributes to auditory processing in mice and humans is completely unknown. The objective of this proposal is to investigate the role of COMT2 in the peripheral auditory system and the contribution of COMT2 to auditory processing. The hypothesis is that COMT2 contributes to hair cell function and auditory processing by regulating synapse function in the cochlea. Aim 1 will utilize in situ hybridization, immunohistochemistry and immunogold electron microscopy to precisely characterize the cell-specific and temporal expression patterns of Comt2 mRNA in the mouse inner ear and auditory brainstem and the subcellular localization of COMT2 protein in sensory hair cells. Aim 2 will use CRISPR/Cas9 technologies to generate and validate constitutive Comt2 null mice and floxed Comt2 mice to facilitate studies that will examine loss of Comt2 function on peripheral auditory function and auditory processing. Aim 3 will analyze Comt2 mutants to determine the mechanism by which Comt2 contributes to auditory processing in sensory hair cells. By interrogating Comt2 expression patterns and functions within the auditory system, this work will contribute to a better understanding of mechanisms of auditory processing, physiological roles of COMT2, and etiologies of DFNB63 in humans.