Defining Antigenic Targets of Autoimmune Sensorineural Hearing Loss

描述（由申请人提供）：该项目的目标是确定称为CTL 2的膜转运蛋白在自身免疫性听力损失（阿勒）中的作用，我们最近发现该蛋白是内耳中抗体诱导听力损失的靶点。CTL 2功能似乎是毛细胞存活所必需的，因为抗体与该分子的细胞外结构域的结合导致毛细胞死亡、瘢痕形成和听力损失。我们推测，CTL 2也是导致自身免疫性感音神经性听力损失的人抗体的靶点，因为阿勒患者具有与内耳支持细胞结合的抗体，其分布模式与抗CTL 2单克隆抗体KHRI-3所定义的分布模式相同。这些抗体还结合由KHRI-3从内耳提取物中沉淀的68-72 kDa蛋白。此外，患者血清中支持细胞抗体的存在与免疫抑制性皮质类固醇治疗后的听力改善显著相关。CTL 2是一种具有10-11个跨膜结构域的膜糖蛋白，由高度保守的基因编码。在这个提议中，我们将测试的假设，CTL 2是阿勒的目标。Cochlin是内耳中另一种丰富的蛋白质，与CTL 2相关，也可能是自身抗体的靶点。我们将在哺乳动物细胞和昆虫细胞中表达CTL 2和cochlin蛋白，并开发ELISA和蛋白质印迹法用于确定阿勒患者中针对这些蛋白质的抗体的频率。我们将用表达CTL 2或cochlin的细胞免疫动物，以确定这是否会诱导抗体和免疫介导的听力损失。为了更好地理解CTL 2在内耳中的功能作用，我们将评估CTL 2亚型和cochlin在发育中的内耳中表达的时间和位点，并确定在内耳中是否存在亚型偏好。我们将创建由内耳启动子驱动的异常CTL 2基因的转基因小鼠，以确定CTL 2是否对内耳和听力的正常发育至关重要。这些研究将有助于我们了解CTL 2在内耳中的功能。我们还将深入了解抗体诱导阿勒的机制，并确定CTL 2和/或cochlin是否可用于检测听力损失患者的病原性抗体，以便更有效地管理此类患者的治疗，以避免进一步的听力损失。自身免疫性听力损失被怀疑是快速进行性听力损失的原因之一，通常会导致深度耳聋。诊断和管理是困难的，因为不存在有效的诊断测试。如果没有这样的测试，用免疫抑制药物治疗患者是危险的。如果CTL 2或cochlin的抗体是阿勒的原因，那么我们的表达系统可用于检测和监测这些抗体以进行诊断并指导治疗，这可以防止损伤并保护听力。

DESCRIPTION (provided by applicant): The goal of this project is to determine the role in autoimmune hearing loss (AHL) of a membrane transporter protein called CTL2 that we recently discovered as the target of antibody induced hearing loss in the inner ear. CTL2 function appears to be essential for hair cell survival since binding of antibody to the extracellular domain of this molecule leads to hair cell death, scar formation, and hearing loss. We postulate that CTL2 is also the target of human antibodies that cause autoimmune sensorineural hearing loss since patients with AHL have antibodies that bind to inner ear supporting cells with the same distribution pattern as that defined by KHRI-3, an anti-CTL2 monoclonal antibody. These antibodies also bind to the 68-72 kDa protein precipitated by KHRI-3 from inner ear extracts. Furthermore, the presence of supporting cell antibodies in patients' sera is significantly associated with hearing improvement after immunosuppressive corticosteroid treatment. CTL2 is a membrane glycoprotein with 10-11 membrane spanning domains that is encoded by a highly conserved gene. In this proposal we will test the hypothesis that CTL2 is the target of AHL. Cochlin, another abundant protein in the inner ear, associates with CTL2 and may also be a target of autoantibodies. We will express the CTL2 and cochlin proteins in mammalian cells and insect cells and develop ELISA and western blot assays for determining the frequency of antibodies to these proteins in patients with AHL. We will immunize animals with cells expressing CTL2 or cochlin to determine if this will induce antibodies and immune mediated hearing loss. To better understand the functional role of CTL2 in the inner ear, we will assess the timing and locus of expression of CTL2 isoforms and cochlin in the developing inner ear and determine if there is an isoform preference in the inner ear. We will create transgenic mice with aberrant CTL2 genes driven by inner ear promoters to determine if CTL2 is essential for the normal development of the inner ear and for hearing. These studies will help us to understand the function of CTL2 in the inner ear. We will also gain insight into the mechanism of antibody induced AHL and determine if CTL2 and/or cochlin can be used to detect pathogenic antibodies in patients with hearing loss so that the treatment of such patients can be managed more effectively to avoid further hearing loss. Autoimmune hearing loss is suspected to be one cause of rapidly progressive hearing loss that often leads to profound deafness. Diagnosis and management are difficult because an effective diagnostic test does not exist. Without such a test it is dangerous to treat patients with immunosuppressive drugs. If antibodies to CTL2 or cochlin are a cause of AHL, then our expression systems can be used to detect and monitor these antibodies for diagnosis and to guide treatment, which could prevent damage and preserve hearing.