COBRE: BOYS TOWN NAT RES HOSP: P4: FORMATION & REGULATION OF OTOCONIA

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。耳石是碳酸钙和蛋白质的结晶，个体需要感知直线加速度和重力来进行空间定向和平衡控制。这一系统的功能障碍是一个严重的临床问题，尤其是在老年人，但对调节耳锥生物合成的机制知之甚少。耳石处于动态平衡状态，对衰老、感染、疾病和基因突变的不利影响非常敏感，但这些问题的分子机制在很大程度上是未知的。我们的总体假设是：(1)耳石及其包埋膜的多种特定蛋白质组分的相互作用决定了其位置特异性的形成；(2)耳石蛋白的稳态对正常的耳石功能是必不可少的；(3)OC90是耳石的主要蛋白质组分，它通过隔离钙而形成耳石。OC90突变小鼠将表现出内耳功能缺陷，前庭是这项提议的重点。具体地说，在这项建议中，我们将(1)确定其他小鼠耳锥蛋白及其细胞来源，以帮助确定可能有助于耳锥形成的因素。我们将通过PS10蛋白芯片上的共沉淀来确定OC90、耳明胶和耳锥、其嵌入膜或感觉上皮中其他蛋白质之间的任何相互作用，以检验假设#1。(2)我们将通过H4芯片上的蛋白质图谱来检测耳胶蛋白平衡失调是否导致衰老(C57BI/6J)和退行性耳锥(头部倾斜小鼠)的异常和功能障碍，并检测扰乱钙稳态的胚胎和新生幼鼠耳锥蛋白沉积的变化。(3)然后，我们将通过检测OC90表达和钙转运的空间协调来检验假设#3，并通过建立两个突变的小鼠系，一个是空的，另一个只携带一个功能域(PLA2L)，以研究OC90在耳锥形成和重力感知以及耳蜗功能中的作用。结合未来复杂的行为学、生理学和基因/蛋白质谱研究，我们将展示OC90在身体平衡中的作用，以及位置特异性形成、调节和适当维持耳锥的分子机制。因此，我们将有助于了解内耳的发育、功能、生理和病理。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Otoconia, crystallites of CaCO3 and proteins, are required for an individual to sense linear acceleration and gravity for spatial orientation and balance control. Dysfunction of this system is a critical clinical problem, particulady in the elderly, yet little is known about the mechanisms that regulate otoconial biosynthesis. Otoconia appear to be in a dynamic state of equilibrium highly susceptible to adverse effects of aging, infections, diseases and genetic mutations, but the molecular mechanisms of these problems are largely unknown. Our overall hypotheses are that (1) the interplay of multiple specific protein components of otoconia and their embedding membrane determines their site-specific formation; (2) Otoconial protein homeostasis is essential to normal otoconia function; and (3) OC90, a major protein component of otoconia, is essential for the formation of otoconia by sequestering Ca. OC90 mutant mice will manifest a functional deficit of the inner ear, with the vestibule as the focus of this proposal. Specifically in this proposal, we will (1) identify other murine otoconial proteins and their cellular origin, to help identify factors that may contribute to otoconia formation. We will identify any interactions among OC90, otogelin, and other proteins in otoconia, its embedding membrane or the sensory epithelium by co-precipitation on PS10 protein chips to test hypothesis #1. (2) We will examine whether disrupted otoconin homeostasis caused the abnormal and malfunctioning otoconia in aging (C57BI/6J) and degenerating otoconia (head-tilt mice) by protein profiling on H4 chips, and examine changes in the deposition of otoconial proteins in embryos and newborn pups that have disrupted calcium homeostasis (tilt, PMCA-/- mice). (3) Then we will test hypothesis #3 by examining the spatial coordination of OC90 expression and Ca transportation, and by making two mutant mouse lines, one null and the other carting only one functional (PLA2L) domain, to study the role of OC90 in otoconia formation and gravity sensing, as well as in cochlea function. Combined with future sophisticated behavioral, physiological and gene/protein profiling studies, we will demonstrate the role of OC90 in bodily balancing, the molecular mechanisms of site-specific formation, regulation and proper maintenance of otoconia. Thus we will contribute to understanding inner ear development, function, physiology and pathology.