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Function of LOXHD1 in mechanosensory hair cells

LOXHD1 在机械感觉毛细胞中的功能

基本信息

批准号：
9756363
负责人：
Nicolas Grillet
金额：
$ 42.7万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9756363
关键词：
Actin-Binding Protein Actins Address Adult Affect Alleles Antibodies Auditory Auditory Physiology Binding Biological Assay C-terminal Calcium Cell physiology Cells Cochlea Complementary DNA Complex Cytoskeleton Data Defect Detection Development Diffusion Down-Regulation Drug usage Electrophysiology (science)Elements Equilibrium Etiology Exons Family Fluorescence Recovery After Photobleaching Functional disorder Genes Genetic study Goals Hair Hair Cells Hearing Human Hybrids Image Impairment In Vitro Investigation Ions Knock-in Mouse Labyrinth Life Link Lipids Lipoxygenase Maintenance Measures Membrane Membrane Lipids Messenger RNA Missense Mutation Molecular Morphology Mouse Strains Mus Mutant Strains Mice Mutate Mutation Nonsense Mutation Pathway interactions Patients Phenotype Phosphatidic Acid Phosphatidylinositol 4,5-Diphosphate Physiology Property Proteins Publishing RNA Interference RNA Splicing Reporter Reporting Research Residual state Sensory Speed Structure Surface Synapses Testing Time Transgenic Organisms Translating Variant Viral Work age related alpha Toxin base calcium indicator deaf deafness experimental study gene function hearing impairment improved in vivo mechanotransduction mutant novel overexpression postnatal premature progressive hearing loss sound

项目摘要

SUMMARY Our long-term goal is to understand the molecular function of genes responsible for deafness. Here we study the gene Loxhd1 (for lipoxygenase homology domain 1) causing a non-syndromic form of hearing loss in mouse and humans (DFNB77). In humans, the impairment onset can be either progressive or congenital. Interestingly all DFNB77 patients carry at least one allele with a missense mutation. The function of LOXHD1 is unknown. LOXHD1 protein is a suite of evolutionary-conserved PLAT (Polycystin Lipoxygenase Alpha-Toxin) domains, known in other proteins to bind to lipids and proteins. In the inner ear, the LOXHD1 expression is restricted to hair cells with a high level of expression starting after the first postnatal week. LOXHD1 localizes at the membrane-cytoskeleton interface of stereocilia. A missense mutation in mouse Loxhd1 leads to hair cell dysfunction while not affecting the hair bundle structure. Based on published work and new preliminary data we propose that LOXHD1 is necessary for hair bundle mechanosensitivity after the first week of life, and forms an intra-stereociliary connection between the membrane and the actin-cytoskeleton. Immunolocalization studies suggest LOXHD1 is absent at the stereocilia tips where mechanotransduction occurs. We will test whether LOXHD1 regulates at long range the mechanotransduction activity by 1) controlling the maintenance/localization of core elements of the mechanotransduction machinery, and/or 2) perturbing the stereocilia membrane properties. We plan to address the central hypothesis of this proposal by pursuing the following specific aims: (SA1): Characterize the auditory phenotypes of nonsense (DFNB77-like) and missense mutations in Loxhd1-defective mice. The novel Loxhd1-Stop mutant we have generated carries a nonsense mutation in the same exon mutated in the missense mutant. We will compare the phenotypes of the two mutants on the Loxhd1 mRNA integrity, on the maturation of the hair cell and the auditory physiology. (SA2): Characterize the mechanotransduction (MET) defect in the Loxhd1-defective mice. After an electrophysiology analysis of the hair cell MET in the mutants we will investigate the underlying mechanisms: We will test if LOXHD1 is required for MET by controlling the targeting or maintenance of core-MET components. Alternatively, LOXHD1 could influence MET by affecting the stereociliary membrane properties. We will measure the lipid diffusion in the stereocilia membrane by Fluorescence Recovery After Photobleaching. (SA3): Identification of LOXHD1 molecular partners. We will further delineate the molecular pathways for the functions of LOXHD1. First, we will confirm and better localize LOXHD1 in stereocilia with a PLAT10 antibody and newly generated HA-tag knock-in mice. In parallel, we will define which lipid PLAT10 can bind to in vitro and the in vivo relevance of these interactions. Via a double-hybrid screen, we found a protein interactor of PLAT10 which is linked to the actin-cytoskeleton and expressed in the stereocilia bundle. We will test the extent to which this interactor accounts for LOXHD1 function.

总结