Usher proteins in the inner ear structure and function

描述（由申请人提供）：内耳毛细胞立体纤毛的许多基本成分的分子特征仍然未知，这妨碍了我们对声音机械传导（MET）的精确机制的理解。为此，对Usher综合征（一种影响人类听力和视力的神经感觉障碍）的基因/蛋白质决定因素的研究在阐明内耳转导机制的分子组成方面取得了丰硕成果。我们研究的长期目标是充分了解USH蛋白如何参与内耳的生物过程，作为开发预防和治疗这种双重神经感觉障碍的新策略的一种手段。这项特殊应用的目的是确定新发现的USH蛋白CIB2在立纤毛束形成、Ca2+稳态和MET中的作用，以及CIB2功能丧失引起的听力损伤的机制。CIB2蛋白由CIB2编码，CIB2是58个人类家族中Usher综合征1型和非综合征性耳聋的致病基因。CIB2定位于小鼠内耳内毛细胞和外毛细胞的立体纤毛上部。基于强有力的初步数据，本提案的中心假设是CIB2是维持内耳毛细胞机械感觉立体纤毛中Ca2+稳态所需的Ca2+缓冲蛋白。因此，CIB2功能的丧失将影响立体纤毛束的结构和力学敏感性，从而导致听力损失。这项研究的基本原理是，一旦知道CIB2在内耳中的功能，这一知识将提高我们在分子水平上对听觉MET和Ca2+稳态过程的理解，这将最终有助于开发预防或逆转听力损失的治疗药物。因此，拟议的研究与NIH的使命有关，即发展基础知识，这可能有助于减轻人类残疾的负担。这一假设将通过三个具体目标来验证：(1)阐明Cib2F91S小鼠听力损失的机制，(2)阐明CIB2在立纤毛束发育和功能中的作用，以及(3)阐明CIB2与内耳毛细胞立纤毛中已知的USH蛋白之间的潜在相互作用。我们的实验方法是确定CIB2缺失对内耳形态学、MET电流、适应性、立纤毛中Ca2+浓度、细胞骨架和超结构改变的影响。我们的研究将采用当代遗传、分子、细胞、组织化学和生理技术。该方法具有创新性，因为CIB2是一种新型USH蛋白，因此其在听觉和视觉过程中的作用从未被研究过。这项研究具有重要意义，首先，因为它有望为CIB2如何参与内耳毛细胞立体纤毛的结构发育和功能提供机制理解。其次，该研究可能揭示Ca2+缓冲在立体纤毛中的机制和生理意义，这在听觉MET中至关重要。

DESCRIPTION (provided by applicant): The molecular identities of many essential components of the hair cell stereocilia in the inner ear are still unknown, precluding our understanding of the precise mechanisms of the mechanotransduction (MET) of sound. To this end, studies of the gene/protein determinants of Usher Syndrome (USH), a neurosensory disorder affecting both hearing and vision in humans, have been fruitful in elucidating the molecular components of transduction machinery in the inner ear. The long-term goal of our research is to understand fully how USH proteins are involved in the biological processes of the inner ear as a means of developing new strategies to prevent and treat this dual neurosensory disorder. The objective of this particular application is to determine the role of a newly identifid USH protein, CIB2, in stereocilia bundle formation, Ca2+ homeostasis and MET, as well as the mechanisms underlying hearing impairment caused by the loss of CIB2 function. The CIB2 protein is encoded by CIB2, the causative gene for Usher syndrome type 1 and non-syndromic deafness in 58 human families. CIB2 is localized at the upper part of stereocilia of both inner and outer hair cells in the mouse inner ear. Based on strong preliminary data, the central hypothesis of this proposal is that CIB2 is a Ca2+-buffering protein required for the maintenance of Ca2+ homeostasis in the mechanosensory stereocilia of the inner ear hair cells. Loss of CIB2 function, then, will affect both the structure and mechanosensitivity of stereocilia bundles, resulting in hearing loss. The rationale for the proposed research is that once the function of CIB2 in the inner ear is known, this knowledge will improve our understanding of auditory MET and Ca2+ homeostasis processes at the molecular level, which will ultimately aid in developing therapeutic agents for preventing or reversing hearing loss. Thus, the proposed research is relevant to that part of NIH's mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human disability. This hypothesis will be tested by pursuing three specific aims: (1) elucidate the mechanism of hearing loss in Cib2F91S mice, (2) elucidate the role of CIB2 in the development and function of stereocilia bundles, and (3) elucidate potential interactions between CIB2 and known USH proteins present in the inner ear hair cell stereocilia. Our experimental approach is to define the consequences of loss of CIB2 on inner ear morphology, the MET current, adaptation, Ca2+ concentration in the stereocilia, cytoskeletal and ultra-structural alterations. Our studies will employ contemporary genetic, molecular, cellular, histochemical and physiological techniques. The approach is innovative, because CIB2 is a novel USH protein, therefore, its role in the hearing and vision processes has never been investigated. The proposed research is significant, first, because it is expected to provide mechanistic understanding of how CIB2 is involved in the structural development and function of inner ear hair cell stereocilia. Secondly, the proposed study will likely uncover the mechanisms and physiological significance of Ca2+ buffering in stereocilia, which is critically important in auditory MET.