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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。耳石，CaCO 3和蛋白质的微晶，是个体感知线性加速度和重力以进行空间定位和平衡控制所必需的。这一系统的功能障碍是一个严重的临床问题，尤其是在老年人中，但对耳石生物合成的调节机制知之甚少。耳石似乎处于动态平衡状态，极易受到衰老、感染、疾病和基因突变的不利影响，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异性蛋白组分的相互作用决定了它们的位点特异性形成;（2）耳石蛋白稳态对正常耳石功能至关重要;（3）OC 90是耳石的主要蛋白组分，通过螯合Ca而对耳石的形成至关重要。OC 90突变小鼠将表现出内耳的功能缺陷，前庭是本建议的重点。具体来说，在这个建议中，我们将（1）确定其他鼠耳石蛋白及其细胞来源，以帮助确定可能有助于耳石形成的因素。我们将通过在PS10蛋白芯片上共沉淀来鉴定OC 90、耳凝胶蛋白和耳石、其包埋膜或感觉上皮中的其他蛋白之间的任何相互作用，以检验假设#1。(2)我们将通过H4芯片上的蛋白质谱分析来研究是否破坏耳锥蛋白稳态导致了衰老（C57 BI/6 J）和退化性耳锥（头倾斜小鼠）中的耳锥异常和功能障碍，并研究胚胎和新生幼崽中耳锥蛋白沉积的变化，这些蛋白沉积破坏了钙稳态（倾斜，PMCA-/-小鼠）。(3)然后，我们将通过检查OC 90表达和Ca转运的空间协调来检验假设#3，并通过制造两个突变小鼠系，一个为空，另一个仅携带一个功能性（PLA 2L）结构域，来研究OC 90在耳石形成和重力感测以及耳蜗功能中的作用。结合未来复杂的行为，生理和基因/蛋白质分析研究，我们将展示OC 90在身体平衡中的作用，特定位点形成的分子机制，耳石的调节和适当的维护。因此，我们将有助于了解内耳发育，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.