Preventing hair cell loss by regulating prestin's function

描述(申请人提供)：哺乳动物，包括人类，由于其高灵敏度和尖锐的频率选择性，需要外毛细胞(OHC)的扩增。内耳细胞也是内耳中最脆弱的细胞，与大多数感音神经性听力损失有关。虽然通过干细胞替代或基因疗法再生OHC在未来提供了一个令人兴奋的选择，但防止毛细胞丢失仍然是处理毛细胞丢失的最直接和最现实的方法，包括再生的毛细胞。因此，我们的提案将重点放在毛细胞的维护上，特别是外毛细胞。OHC的死亡与已知的破坏细胞的活性氧物种(ROS)的产生密切相关。为什么与Corti器官中的其他细胞相比，毛细胞更容易受到ROS的影响，这一点尚不完全清楚。OHC的主要功能是通过它们的躯体电动来放大机械信号。这种独有的特性是由一种名为prestin的独特马达蛋白执行的，这种蛋白只在毛细胞中表达。过去，研究OHC的扩增机制和防止OHC的丢失被认为是两个独立的研究领域。出乎意料的是，我们发现prestin(499-prestin突变体)的非功能点突变会导致OHC死亡。此外，prestin不仅与参与ROS生成的蛋白质有关，而且它本身可能具有氧化还原酶活性，这可能是OHC防御系统的一部分，以减少ROS。这些数据表明，Prestin的功能与毛细胞的脆弱性之间存在密切联系。这项建议的目的是了解这一联系，并随后寻找更好的战略来防止中心的流失。目的本研究的目的是在体外系统地研究Prestin的氧化还原酶活性，以揭示为什么外毛细胞是Corti器官中最脆弱的细胞。这些知识将使我们能够制定更好的战略来防止OHC损失。在AIM II中，我们将比较wt-prestin和突变的499-prestin表达细胞的细胞特性。随后开发的监测prestin功能和细胞脆弱性之间联系的系统和方法(S)将被用于测试不同的抗OHC损失剂对prestin功能的影响。从AIM II获得的信息将有助于我们在细胞水平上了解prestin的功能与细胞死亡之间的关系，并为选择更好的方法保护OHC提供指导。AIM III将专注于通过小分子化合物文库筛选程序寻找新化合物。我们的目的是寻找新的化合物，这些化合物可能对Prestin的氧化还原酶能力具有特定和有效的影响，因此可用于防止OHC损失。将使用各种细胞、生化和分子生物学方法。我们预防毛细胞丢失的策略是基于对prestin功能的了解，这是毛囊细胞所独有的。这种创新的方法不仅将提供关于耳蜗放大的基本机制的关键信息，而且还将导致对与OHC死亡相关的过程的更深层次的理解。后者可能导致直接治疗或预防OHC损害。这项拟议的工作是翻译研究的第一步，它将耳蜗生理学的基本机制与听力障碍的治疗联系起来。 与公共健康相关：外毛细胞构成耳蜗放大器，对提供高灵敏度和频率选择性的听力至关重要。它们也是内耳中最脆弱的细胞，参与了影响数百万人听力的大多数感觉神经性听力损失。Prestin是外毛细胞的马达蛋白，是耳蜗放大所必需的。这项建议的目的是研究Prestin对耳朵中氧化应激的保护作用。这些研究收集的数据不仅将在分子水平上扩大Prestin作为基于OHC的耳蜗放大器的知识，还将加深对外毛细胞丢失相关机制的理解，并可能为预防OHC损伤提供新的方法。

DESCRIPTION (provided by applicant): Mammals including humans need amplification by outer hair cells (OHCs) for their high sensitivity and sharp frequency selectivity. OHCs are also the most vulnerable cells in the inner ear and are involved in a majority of sensorineural hearing loss. Although OHC regeneration through either stem cell replacement or gene therapy provides an exciting option in the future, preventing hair cell loss is still the most direct and realistic approach to deal with hair cell loss, and including regenerated ones. Therefore, our proposal will focus on the maintenance of hair cells, particularly outer hair cells. OHC death is strongly connected with the generation of reactive oxygen species (ROS) that are known to destroy cells. Why OHCs, as compared to other cells in the organ of Corti, are more vulnerable to ROS is not fully understood. The main function of OHCs is to amplify mechanical signals through their somatic electromotility. This exclusive property is executed by a unique motor protein called prestin, which is expressed only in OHCs. In the past, studying OHC amplification mechanisms and preventing OHC loss were considered as two separate research fields. Unexpectedly, we discovered that non-functional point mutations in prestin (499-prestin mutant) leads to OHC death. In addition, prestin is not only associated with proteins involved in ROS generation, but prestin itself may have redox enzymatic activity, which could be a part of the OHC defense system to minimize ROS. These data indicate a close connection between prestin's function and the vulnerability of OHCs. The goal of this proposal is to understand this link, and subsequently to search for better strategies to prevent OHCs loss. Aim I of this proposal is designed to systematically characterize prestin's redox enzymatic activity in an in vitro system in order to reveal why the OHCs are the most vulnerable cells in the organ of Corti. This knowledge will allow us to develop a better strategy to prevent OHC loss. In Aim II, we will compare cell properties between wt-prestin and mutant 499-prestin expressing cells. Subsequent development of a system and method(s) to monitor the connection between prestin's function and cell vulnerability will then be used to test the effects of different anti-OHC loss reagents on prestin's functions. The information obtained from AIM II will help us to understand the relationship between prestin's function and cell death at the cellular level and provide guidance for selecting better approaches to protect OHCs. Aim III will focus on a search for new compounds through small molecule compound library screening procedures. Our purpose is to find novel compounds that may have specific and potent effects on prestin's redox enzymatic capacity, and are therefore usable for preventing OHC loss. Various cellular, biochemical and molecular biological methods will be used. Our strategies to prevent hair cell loss are based on knowledge derived from understanding prestin's function, which is unique to OHCs. This innovative approach will not only provide crucial information about basic mechanisms of cochlear amplification but it will also lead to a deeper understanding of processes associated with OHC death. The latter may lead to direct treatments or prevention of OHC damage. The proposed work is a first-step in translational research, which bridges basic mechanisms of cochlear physiology and treatments for hearing impairment. PUBLIC HEALTH RELEVANCE: Outer hair cells constitute the cochlear amplifier, essential for providing high sensitivity and frequency selectivity of hearing. They are also the most vulnerable cells in the inner ear and are involved in a majority of sensorineural hearing loss that affects the hearing of millions of people. Prestin, the motor protein of outer hair cells, is required for cochlear amplification. The goal of this proposal is to investigate prestin's protection role against oxidative stress in the ear. Data collected from these studies will not only expand knowledge of prestin as the OHC-based cochlear amplifier at the molecular level, but also produce a deeper understanding of mechanisms associated with outer hair cells loss, and may offer new methods for the prevention of OHC damage.