Functional Analyses of Tricellular Tight Junctions in Hearing and Deafness

描述(申请人提供)：遗传性听力损失在每个人群中都很普遍，并对听力障碍者的生活构成重大挑战。目前已发现86个不同的遗传基因座(DFNB)与隐性遗传性耳聋分离有关，其中38个基因座的突变基因已被鉴定。然而，对这些基因的功能以及它们如何解释听力损失表型的了解是不完整的。对基因功能的活体研究是填补这一知识空白的重要一步，而小鼠是了解人类听力重要基因功能的理想哺乳动物模型。我们的长期目标是了解紧密连接，包括紧密连接(TJ)蛋白，是如何参与耳朵的器官发生和功能的。更具体地说，我们希望了解DFNB49基因座上导致非综合征性耳聋的致病基因trc编码的紧密连接蛋白tricellin突变导致听力障碍的机制和病理生理学。内耳中的三细胞蛋白形成独特的、结构复杂的细长的三细胞紧密连接(TTJ)，被认为控制着三个细胞之间这些接触点的通透性。我们的中心假设是，三纤维素对于内耳正常运作所需的上皮屏障的形成至关重要。在这里，我们提供了两个突变小鼠模型TricR497X和Tricflx-EX3的生成的初步数据。TRIC基因p.R497X突变纯合的小鼠在P30岁时听力严重丧失，这些小鼠中的毛细胞在大约P12岁时开始退化。我们计划通过追求两个具体目标来验证我们的假设：(1)阐明TricR497X小鼠耳聋的根本原因；(2)确定毛细胞蛋白在内耳TJ复合体的发育、维持和组装中的功能。我们的实验方法是确定三纤维素丢失对内耳形态、离子屏障、耳蜗内电位、细胞骨架和超微结构变化的影响。我们的研究将使用最先进的遗传学、分子、组织学和生理学技术。这项拟议工作的基本原理是，它将提供对三纤维素的细胞和分子功能的更好的理解。在这个项目完成时，我们将了解a)毛细胞蛋白功能丧失导致耳聋的机制和由此产生的病理生理学，以及b)毛细胞蛋白在TJ生物发生和屏障形成中更一般的作用。对听觉系统中涉及的基因的更完整的了解将为开发潜在的治疗措施来治疗这种神经感觉缺陷提供基础。

DESCRIPTION (provided by applicant): Hereditary hearing loss is prevalent in every population and poses significant challenges to the lives of the hearing impaired. Eighty-six distinct genetic loci (DFNB) have now been linked to recessively inherited hearing loss segregating in large families, and the mutated genes for 38 of these loci have been identified. However, understanding of the function of the genes and how they account for a hearing loss phenotype is incomplete. In vivo studies of gene function are an important step in filling this knowledge gap, and the mouse serves as an ideal mammalian model for understanding the functions of genes that are important for hearing in humans. Our long-term goal is to understand how tight junctions, including tight junction (TJ) proteins, are involved in organogenesis and function of the ear. More specifically, we wish to understand the mechanisms and pathophysiology of hearing impairment caused by mutations in the tight junction protein Tricellulin, encoded by TRIC, the causative gene for nonsyndromic deafness at the DFNB49 locus. Tricellulin in the inner ear forms unique, structurally complex and elongated tricellular tight junctions (tTJs), which are presumed to control permeability at these contact points between three cells. Our central hypothesis is that Tricellulin is critical for the formation of the epithelial barriers required for the proper functioning of the inner ear. Here we present preliminary data on the generation of two mutant mouse models TricR497X and Tricflox- ex3. Mice homozygous for the p.R497X mutation of Tric have profound hearing loss by age P30 and the hair cells in these mice start to degenerate at approximately P12. We plan to test our hypotheses by pursuing two specific aims: (1) elucidate the underlying cause of deafness in TricR497X mice and (2) determine the functions of Tricellulin in the development, maintenance and assembly of the TJ complex in the inner ear. Our experimental approach is to define the consequences of loss of Tricellulin on the inner ear morphology, ionic barrier, endocochlear potential, cytoskeletal and ultrastructural alterations. Our studies will employ state-of-the art genetic, molecular, histological and physiological techniques. The rationale for the proposed work is that it will provide an enhanced understanding of the cellular and molecular function of Tricellulin. At the completion of this project, we will have acquired knowledge about a) the mechanism of deafness caused by loss of Tricellulin function and the resulting pathophysiology and b) the more general role of Tricellulin in TJ biogenesis and barrier formation. A more complete knowledge of the genes involved in the auditory system will provide a foundation for development of potential therapeutic interventions to treat this neurosensory deficit.