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. ? ? ?
