Function of LOXHD1 in mechanosensory hair cells

LOXHD1 在机械感觉毛细胞中的功能

• 批准号: 10468742
• 负责人: Nicolas Grillet
• 金额: $ 41.28万
• 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/10468742
• 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.

摘要 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(DFNB77)。在人类中，损害的发病可以是进行性的，也可以是先天性的。 有趣的是，所有DFNB77患者至少携带一个带有错义突变的等位基因。LOXHD1的功能是 未知。LOXHD1蛋白是一组进化保守的PLAT(Polycystin Lipoxygenase Alpha-toxx) 结构域，在其他蛋白质中已知可以与脂类和蛋白质结合。在内耳中，LOXHD1的表达是 仅限于出生后第一周开始高水平表达的毛细胞。LOXHD1本地化 在立体纤毛的膜-细胞骨架界面。小鼠Loxhd1错义突变导致毛细胞 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，在生命的第一周，LOXHD1是毛束机械敏感性所必需的，并形成 膜和肌动蛋白细胞骨架之间的立体纤毛连接。免疫定位 研究表明，在发生机械转导的立体纤毛尖端，LOXHD1缺失。我们将测试 LOXHD1是否通过1)控制 机械转换机械核心部件的维护/国产化，和/或2)扰乱 立体纤毛膜的性能。我们计划通过追求这一建议的核心假设来解决 以下具体目标：(SA1)：描述胡言乱语的听觉表型(类似DFNB77)和 Loxhd1缺陷小鼠的错义突变。我们产生的新的Loxhd1-Stop突变体携带一个 同一外显子的无义突变在错义突变体中发生突变。我们将比较这些基因的表型。 两个突变体对Loxhd1mRNA的完整性，对毛细胞的成熟和听觉生理的影响。 (SA2)：描述Loxhd1缺陷小鼠的机械转导(MET)缺陷。在一次 突变型毛细胞的电生理学分析我们将探讨其潜在的机制： 我们将通过控制CORE-MET的目标或维护来测试MET是否需要LOXHD1 组件。另外，LOXHD1还可以通过影响立体纤毛膜的性质来影响MET。 我们将用荧光回收法测量脂质在立体纤毛膜中的扩散。 光漂白。(SA3)：确定LOXHD1分子伙伴。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抗体的立体纤毛和新产生的HA标签敲入小鼠。同时，我们将定义哪些 脂类PLAT10在体外可以结合到体内，这些相互作用与体内的相关性。通过双混合屏幕，我们 发现了PLAT10的一个蛋白质相互作用元件，它与肌动蛋白-细胞骨架相连，并在立体纤毛中表达 捆绑。我们将测试这个交互角色在多大程度上影响了LOXHD1函数。

项目成果

Loxhd1 Mutations Cause Mechanotransduction Defects in Cochlear Hair Cells.

• DOI: 10.1523/jneurosci.0975-20.2021
• 发表时间: 2021-04-14
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Trouillet A;Miller KK;George SS;Wang P;Ali NE;Ricci A;Grillet N
• 通讯作者: Grillet N

A rare genomic duplication in 2p14 underlies autosomal dominant hearing loss DFNA58.

• DOI: 10.1093/hmg/ddaa075
• 发表时间: 2020-04
• 期刊: Human molecular genetics
• 影响因子: 3.5
• 作者: Karina Lezirovitz;G. A. Vieira-Silva;A. C. Batissoco;D. Levy;J. Kitajima;Alix Trouillet;Ellen Ouyang-