USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS

USHERIN：结构和功能分析

• 批准号: 7524463
• 负责人: Dominic E. Cosgrove
• 金额: $ 31.95万
• 依托单位: FATHER FLANAGAN'S BOYS' HOME
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7524463
• 关键词: Acceleration; Actins; Animal Housing; Animal Model; Animals; Apical; Arrestin; Arrestins; Binding Proteins; Biochemical; Biological Assay; Blindness; Bos taurus; Brefeldin A; Cattle; Cell physiology; Cells; Clinical; Co-Immunoprecipitations; Cochlea; Complex; Condition; Confocal Microscopy; Data; Defect; Development; Disease; Ear; Environment; Epithelial Cells; Exposure to; Eye; Foundations; Genes; Genetic; Genetic Research; Hair Cells; Hearing; Hearing Impaired Persons; Heterogeneity; Hour; Human; Immunoblotting; Laboratories; Lead; Length; Light; Location; MYO7A gene; Maintenance; Mediating; Membrane; Methods; Modeling; Mus; Nature; Neural Retina; Neuroepithelial Cells; Nose; Numbers; Organ of Corti; Pathology; Patients; Phenotype; Photoreceptors; Predisposition; Process; Progress Reports; Protein Isoforms; Protein translocation; Proteins; Public Health; Rate; Receptor Cell; Relative (related person); Retina; Retinal; Retinal Degeneration; Role; Sensory; Shapes; Staging; Stereocilium; Structure; Sucrose; System; Testing; Therapeutic; Time; Tracheal Epithelium; Transducin; Transport Process; Transport Vesicles; Usher Syndrome; Vesicle; Vision; Wild Type Mouse; Work; base; cell type; deafness; exposed human population; in vivo; inhibitor/antagonist; intracellular protein transport; mouse model; neuroepithelium; novel; photoreceptor degeneration; protein function; protein localization location; research study; trafficking; transport inhibitor; visual cycle

DESCRIPTION: Usher syndrome is the leading genetic cause of deaf/blindness in the world. Progress in genetic research has revealed a high degree of heterogeneity for the disorder, with nine specific genes identified as causal for the three clinical sub-types. The genes encode an array of products and preliminary functional analysis points to a number of specific interactions that suggest the existence of a functional usher protein "interactome". The existence of the "interactome" in vivo has never been proven, and the critical subcellular compartments of usher protein function are still controversial. Mouse models for the various usher genes all display a developmental defect in stereocilia associated with disorganized structure in the cuticular plate, abnormal numbers of actin paracrystals, and variable lengths and shapes, all associated with deafness. None of the usher mouse models develop retinal degeneration, which has hampered progress in understanding RP associated with Usher syndrome. In the preliminary results of this proposal we demonstrate that the usher proteins are present in the vesicular sub-cellular fraction of tracheal epithelial cells and do indeed form a complex in vivo that sediments on sucrose gradients at around 50 Svedbergs. For three different usher mouse models (all that have been tested thus far) we show a defect in arrestin and transducin protein translocation in photoreceptors following exposure to light in dark adapted animals. We also demonstrate light-induced photo- receptor cell degeneration in these same animals, suggesting that the usher mice do indeed possess a robust retinal phenotype that is not revealed in the environment of animal housing facilities. These findings form the foundation for the central hypothesis of this proposal that usher proteins function to mediate translocation of proteins in ciliated neuroepithelial cells of the retina and the cochlea. Defects in this process lead to stereo- ciliary defects and photoreceptor cell degeneration. We will test this hypothesis in three specific aims. In the first aim we perform biochemical analysis of the vesicular complex in ciliated tracheal epithelium and retinal extracts, and perform subcellular localization experiments in photoreceptors and hair cells. In the second aim we will examine mouse models for all of the known subtypes of Usher syndrome for defects in protein trans- location and light induced photoreceptor cell degeneration and test the hypothesis directly using vesicle transport inhibitors in organotypic cultures of neural retina. In the third aim we will examine the role of vesicular transport in stereociliary development and maintenance by blocking vesicle transport in cultures of mouse organ of Corti and by immunohistological analysis of usher proteins in usher mouse models. Successful completion of these aims will define a cellular process that explains the defective development and function of ciliated neuroepithelium in the eye and ear that define Usher syndrome pathology. This work will help align the field and promote an acceleration of work aimed at understanding the specific disease mechanism, which is an essential first step towards the development of effective therapeutic strategies. PUBLIC HEALTH RELEVANCE Usher syndrome is the leading cause of deaf/blindness, and results in the destruction of sensory cells in the eye and the retina. Nine different genes have been identified, and the encoded proteins interact with each other, but the functional connection with hearing and vision is still unknown. This proposal tests the hypothesis that the functional connection is a process called protein translocation, which may pinpoint the defective process that causes deafness and blindness in Usher patients.

描述：Usher综合征是世界上导致耳聋/失明的主要遗传原因。遗传学研究的进展表明，这种疾病具有高度的异质性，有9个特定基因被确定为三个临床亚型的原因。这些基因编码了一系列的产物，初步的功能分析指出了一些特定的相互作用，这表明存在一个功能性的引座者蛋白“相互作用体”。活体内是否存在相互作用体一直没有得到证实，而关键的亚细胞亚细胞区段对迎接蛋白功能的影响仍存在争议。各种引导者基因的小鼠模型都显示出立毛发育缺陷，与角质板的无序结构有关，肌动蛋白副晶体的异常数量，以及不同的长度和形状，所有这些都与耳聋有关。没有一个引座者小鼠模型发生视网膜变性，这阻碍了理解与Usher综合征相关的RP的进展。在这项研究的初步结果中，我们证明了引座者蛋白存在于气管上皮细胞的囊泡亚细胞部分中，并且确实在体内形成了一种复合体，在大约50 Svedbergs的蔗糖梯度上沉积。对于三个不同的引座者小鼠模型(到目前为止已经测试过的所有模型)，我们发现在黑暗适应动物中暴露在光下的光感受器中arrestin和转导蛋白的蛋白转位存在缺陷。我们还在这些动物中证明了光诱导的光感受器细胞退化，这表明引座鼠确实具有在动物收容设施的环境中没有显示的健壮的视网膜表型。这些发现构成了这一建议的中心假设的基础，即引入蛋白质的功能来调节视网膜和耳蜗具纤毛的神经上皮细胞中蛋白质的移位。这一过程中的缺陷会导致立体纤毛缺陷和感光细胞退化。我们将在三个具体目标上检验这一假设。在第一个目标中，我们对纤毛气管上皮和视网膜提取物中的囊泡复合体进行生化分析，并在光感受器和毛细胞中进行亚细胞定位实验。在第二个目标中，我们将检验所有已知亚型Usher综合征的小鼠模型，以寻找蛋白质转位缺陷和光诱导的光感受器细胞退化，并在神经视网膜的器官培养中直接使用囊泡运输抑制剂来检验这一假说。在第三个目标中，我们将通过阻断小鼠Corti器官培养中的小泡运输和通过免疫组织学方法分析Usher小鼠模型中的Usher蛋白，来研究小泡运输在立体纤毛发育和维持中的作用。这些目标的成功完成将定义一个细胞过程，解释眼睛和耳朵中纤毛神经上皮的发育和功能缺陷，这定义了亚瑟综合征的病理。这项工作将有助于调整这一领域，并促进加快旨在了解具体疾病机制的工作，这是制定有效治疗战略的重要第一步。与公共卫生相关亚瑟综合征是导致失聪/失明的主要原因，并导致眼睛和视网膜中的感觉细胞被破坏。目前已鉴定出9个不同的基因，编码的蛋白质相互作用，但与听觉和视觉的功能联系仍不清楚。这项提议验证了这样一种假设，即这种功能连接是一种被称为蛋白质易位的过程，这可能会精确定位导致Usher患者耳聋和失明的缺陷过程。