Usher proteins in the inner ear structure and function

描述(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性仍然未知，这阻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(USH)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。USH是一种影响人类听力和视觉的神经感觉障碍。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手段。这一特殊应用的目的是确定一种新发现的USH蛋白CIB2在立体纤毛束形成、钙稳态和MET中的作用，以及CIB2功能丧失导致听力障碍的机制。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定位于小鼠内耳内外毛细胞的纤毛纤毛的上部。基于强大的初步数据，这一建议的中心假设是，CIB2是一种钙缓冲蛋白，在内耳毛细胞的机械感觉立体纤毛中维持钙离子稳态所必需的。因此，CIB2功能的丧失将影响立体纤毛束的结构和机械敏感性，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，这一知识将在分子水平上提高我们对听觉MET和钙稳态过程的理解，这最终将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使命中与发展基础知识相关的部分相关，这可能有助于减轻人类残疾的负担。这一假说将通过追求三个特定的目标来验证：(1)阐明Cib2F91S小鼠听力损失的机制，(2)阐明CIB2在立体纤毛束的发育和功能中的作用，以及(3)阐明CIB2与内耳毛细胞立体纤毛中存在的已知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、MET电流、适应、立体纤毛中钙离子浓度、细胞骨架和超微结构变化的影响。我们的研究将使用当代的遗传学、分子、细胞、组织化学和生理学技术。这种方法是创新的，因为CIB2是一种新的USH蛋白，因此，它在听觉和视觉过程中的作用从未被研究过。这项拟议的研究意义重大，首先，因为它有望从机制上理解CIB2如何参与内耳毛细胞立体纤毛的结构发展和功能。其次，这项拟议的研究可能会揭示立体纤毛中钙缓冲的机制和生理意义，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.