Function of LOXHD1 in mechanosensory hair cells

LOXHD1 在机械感觉毛细胞中的功能

• 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
• 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

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导致耳聋的基因的分子功能。我们在这里学习 基因 Loxhd1（脂氧合酶同源域 1）导致非综合征形式的听力损失 小鼠和人类（DFNB77）。在人类中，损伤可能是进行性的，也可能是先天性的。 有趣的是，所有 DFNB77 患者均携带至少一个具有错义突变的等位基因。 LOXHD1的功能是 未知。 LOXHD1 蛋白是一套进化保守的 PLAT（多囊蛋白脂氧合酶α-毒素） 结构域，已知在其他蛋白质中与脂质和蛋白质结合。在内耳中，LOXHD1 表达为 仅限于出生后第一周后开始高水平表达的毛细胞。 LOXHD1 本地化 在静纤毛的膜-细胞骨架界面。小鼠 Loxhd1 的错义突变导致毛细胞形成 功能障碍，同时不影响发束结构。基于已发表的工作和新的初步数据 我们认为，LOXHD1 对于出生第一周后的发束机械敏感性是必需的，并形成 膜和肌动蛋白细胞骨架之间的立体纤毛内连接。免疫定位 研究表明，发生机械转导的静纤毛尖端不存在 LOXHD1。我们将测试 LOXHD1 是否通过 1) 控制远距离调节力转导活性 机械传导机械核心元件的维护/本地化，和/或 2) 扰动 静纤毛膜特性。我们计划通过追求以下目标来解决该提案的中心假设： 以下具体目标：（SA1）：表征无意义的听觉表型（DFNB77 样）和 Loxhd1 缺陷小鼠的错义突变。我们生成的新型 Loxhd1-Stop 突变体携带 无义突变在同一外显子中突变为错义突变体。我们将比较表型 两个突变体对 Loxhd1 mRNA 完整性、毛细胞成熟和听觉生理机能的影响。 (SA2)：表征 Loxhd1 缺陷小鼠的机械转导 (MET) 缺陷。经过一个 对突变体中毛细胞 MET 进行电生理学分析，我们将研究其潜在机制： 我们将通过控制核心 MET 的靶向或维持来测试 MET 是否需要 LOXHD1 成分。另外，LOXHD1 可以通过影响立体纤毛膜特性来影响 MET。 我们将通过荧光恢复测量静纤毛膜中的脂质扩散 光漂白。 (SA3)：LOXHD1 分子伴侣的鉴定。我们将进一步划定 LOXHD1 功能的分子途径。首先，我们将确认并更好地本地化LOXHD1 带有 PLAT10 抗体的静纤毛和新生成的 HA 标签敲入小鼠。同时，我们将定义哪些 脂质PLAT10可以结合体外和体内这些相互作用的相关性。通过双混合屏幕，我们 发现了 PLAT10 的蛋白质相互作用蛋白，该蛋白与肌动蛋白细胞骨架相连并在静纤毛中表达 捆。我们将测试该交互器对 LOXHD1 功能的影响程度。