Function of LOXHD1 in mechanosensory hair cells

LOXHD1 在机械感觉毛细胞中的功能

• 批准号: 10238112
• 负责人: Nicolas Grillet
• 金额: $ 41.24万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 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/10238112
• 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

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.

概括 我们的长期目标是了解负责耳聋的基因的分子功能。我们在这里学习 基因loxhd1（用于脂氧合酶同源域1）导致非混合性听力损失形式 老鼠和人类（DFNB77）。在人类中，损害的发作可以是进步的或先天性的。 有趣的是，所有DFNB77患者至少携带一个具有错义突变的等位基因。 loxhd1的功能是 未知。 LoxHD1蛋白是一套进化保存的平台（Polycystin lipoxygoxyase alpha-Toxin） 在其他蛋白质中已知的结构域与脂质和蛋白质结合。在内耳中，loxhd1的表达为 仅限于毛细胞以高度表达的毛细胞从第一个星期后开始。 LOXHD1本地化 在立体膜的膜细胞骨架界面上。鼠标LOXHD1中的错义突变导致毛细胞 功能障碍，同时不影响发束结构。根据已发表的工作和新的初步数据 我们建议LOXHD1在生命的第一周后对于头发束机械效应是必需的，并形成了 膜与肌动蛋白 - 骨骨架之间的肌内偏置连接。免疫定位 研究表明，在发生机械转移的立体尖端，没有LoxHD1。我们将测试 loxHD1是否在远距离调节机械转导活动1）控制 机械传输机械的核心元素的维护/定位和/或2）扰动 立体膜膜特性。我们计划通过追求该提案的中心假设 以下特定目的：（SA1）：表征胡说八道的听觉表型（类似于DFNB77）和 LOXHD1缺陷小鼠的错义突变。我们生成的新型LoxHD1-Stop突变体载有A 在错义突变体中突变的同一外显子中的无意义突变。我们将比较 LOXHD1 mRNA完整性上的两个突变体，关于毛细胞和听觉生理学的成熟。 （SA2）：表征LOXHD1缺陷小鼠中的机械转移（MET）缺陷。之后 对突变体中毛细胞的电生理分析我们将研究基本机制： 我们将通过控制Core-Met的靶向或维护来测试是否需要LOXHD1进行MET 成分。或者，LOXHD1可以通过影响立体膜特性来影响MED。 我们将通过荧光恢复后测量立体膜膜中的脂质扩散 光漂白。 （SA3）：LOXHD1分子伙伴的识别。我们将进一步描述 LOXHD1功能的分子途径。首先，我们将在 立体胶质具有柏拉图10抗体和新产生的HA-TAG敲入小鼠。并行，我们将定义哪个 脂质平台可以与体外结合以及这些相互作用的体内相关性。通过双杂交屏幕，我们 找到了与肌动蛋白 - 细胞骨架相关的蛋白质相互作用者，并在立体中表达 捆。我们将测试该交互者在多大程度上解释LOXHD1函数的程度。