Functional Analyses of Tricellular Tight Junctions in Hearing and Deafness

描述（由申请人提供）：遗传性听力损失在每个人群中都很普遍，对听力受损者的生活构成了重大挑战。86个不同的基因位点（DFNB）与大家庭中隐性遗传性听力损失分离有关，其中38个基因位点的突变基因已被确定。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理解是不完整的。基因功能的体内研究是填补这一知识空白的重要一步，小鼠是了解对人类听力重要的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，是如何参与器官发生和耳功能的。更具体地说，我们希望了解由紧密连接蛋白Tricellulin突变引起的听力障碍的机制和病理生理，TRIC是DFNB49位点上非综合征性耳聋的致病基因。内耳中的三胞蛋白形成独特的、结构复杂的、细长的三细胞紧密连接（tTJs），这被认为是控制三个细胞之间这些接触点的渗透性。我们的中心假设是，三胞蛋白对内耳正常功能所需的上皮屏障的形成至关重要。在这里，我们提出了两种突变小鼠模型TricR497X和Tricflox- ex3的产生的初步数据。trick p.R497X突变纯合的小鼠在P30岁时出现严重的听力丧失，这些小鼠的毛细胞在大约P12时开始退化。我们计划通过两个具体目标来验证我们的假设：(1)阐明TricR497X小鼠耳聋的潜在原因；(2)确定Tricellulin在内耳TJ复合物的发育、维持和组装中的功能。我们的实验方法是确定三胞蛋白缺失对内耳形态、离子屏障、耳蜗电位、细胞骨架和超微结构改变的影响。我们的研究将采用最先进的遗传、分子、组织学和生理学技术。提出的工作的基本原理是，它将提供一个更好的理解细胞和分子功能的三胞蛋白。在这个项目完成后，我们将了解a)耳聋由Tricellulin功能丧失引起的机制及其病理生理，b) Tricellulin在TJ生物发生和屏障形成中的更广泛的作用。对听觉系统相关基因的更全面的了解将为治疗这种神经感觉缺陷的潜在治疗干预措施的发展提供基础。

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.