USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS

USHERIN:结构和功能分析

基本信息

  • 批准号:
    7857910
  • 负责人:
  • 金额:
    $ 30.66万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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:亚瑟综合症是世界上导致耳聋/失明的主要遗传原因。遗传学研究的进展揭示了这种疾病的高度异质性,有9个特定基因被确定为三种临床亚型的病因。这些基因编码一系列的产物,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导蛋白“相互作用组”。体内“相互作用组”的存在从未被证实,而且usher蛋白功能的关键亚细胞区室仍然存在争议。各种usher基因的小鼠模型都显示出静纤毛的发育缺陷,这些缺陷与表皮板结构紊乱、肌动蛋白旁晶体数量异常以及长度和形状可变有关,所有这些都与耳聋有关。Usher小鼠模型均未出现视网膜变性,这阻碍了对Usher综合征相关RP的了解进展。在这个建议的初步结果中,我们证明了引导蛋白存在于气管上皮细胞的囊泡亚细胞组分中,并且确实在体内形成了一种复合物,该复合物在约50 Svedbergs的蔗糖梯度上沉积。对于三种不同的usher小鼠模型(到目前为止已经测试过),我们发现在黑暗适应动物中暴露于光后,光感受器中的arrestin和transducin蛋白易位存在缺陷。我们还证明了在这些相同的动物中光诱导的光感受器细胞变性,这表明usher小鼠确实具有在动物饲养设施的环境中未显示的稳健的视网膜表型。这些发现形成了该提议的中心假设的基础,即引导蛋白的功能是介导视网膜和耳蜗的纤毛神经上皮细胞中的蛋白质易位。这一过程中的缺陷导致立体纤毛缺陷和感光细胞变性。我们将在三个具体目标中检验这一假设。在第一个目标中,我们进行纤毛气管上皮和视网膜提取物中的泡状复合物的生化分析,并进行光感受器和毛细胞中的亚细胞定位实验。在第二个目标中,我们将检查Usher综合征所有已知亚型的小鼠模型的蛋白质移位缺陷和光诱导的感光细胞变性,并在神经视网膜的器官型培养物中直接使用囊泡转运抑制剂测试假设。在第三个目标中,我们将研究囊泡运输在静纤毛的发展和维护中的作用,通过阻断小鼠Corti器官培养液中的囊泡运输,并通过免疫组织学分析usher小鼠模型中的usher蛋白。这些目标的成功完成将定义一个细胞过程,解释定义Usher综合征病理学的眼和耳中纤毛神经上皮的发育和功能缺陷。这项工作将有助于调整该领域,并促进加速旨在了解特定疾病机制的工作,这是制定有效治疗策略的重要第一步。Usher综合征是耳聋/失明的主要原因,并导致眼睛和视网膜中感觉细胞的破坏。目前已鉴定出9种不同的基因,编码的蛋白质相互作用,但与听觉和视觉的功能联系仍然未知。这项提议验证了一个假设,即功能性连接是一个称为蛋白质易位的过程,这可能会查明导致Usher患者耳聋和失明的缺陷过程。

项目成果

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Dominic E. Cosgrove其他文献

Dominic E. Cosgrove的其他文献

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{{ truncateString('Dominic E. Cosgrove', 18)}}的其他基金

RESCUE OF ALPORT SYNDROME OTOPATHOLOGY
阿尔波特综合征的拯救 耳病理学
  • 批准号:
    9214327
  • 财政年份:
    2016
  • 资助金额:
    $ 30.66万
  • 项目类别:
SIGNALING MECHANISMS IN REGULATION OF TUMOR ANGIOGENESIS
肿瘤血管生成调节的信号机制
  • 批准号:
    8279120
  • 财政年份:
    2010
  • 资助金额:
    $ 30.66万
  • 项目类别:
USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS
USHERIN:结构和功能分析
  • 批准号:
    6794110
  • 财政年份:
    2002
  • 资助金额:
    $ 30.66万
  • 项目类别:
USHERIN--FUNCTION, EXPRESSION, AND ROLE IN PATHOGENESIS
USHERIN——功能、表达和发病机制中的作用
  • 批准号:
    6589749
  • 财政年份:
    2002
  • 资助金额:
    $ 30.66万
  • 项目类别:
USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS
USHERIN:结构和功能分析
  • 批准号:
    7524463
  • 财政年份:
    2002
  • 资助金额:
    $ 30.66万
  • 项目类别:
USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS
USHERIN:结构和功能分析
  • 批准号:
    7640521
  • 财政年份:
    2002
  • 资助金额:
    $ 30.66万
  • 项目类别:
USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS
USHERIN:结构和功能分析
  • 批准号:
    6926069
  • 财政年份:
    2002
  • 资助金额:
    $ 30.66万
  • 项目类别:
USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS
USHERIN:结构和功能分析
  • 批准号:
    6543888
  • 财政年份:
    2002
  • 资助金额:
    $ 30.66万
  • 项目类别:
USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS
USHERIN:结构和功能分析
  • 批准号:
    8077368
  • 财政年份:
    2002
  • 资助金额:
    $ 30.66万
  • 项目类别:
USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS
USHERIN:结构和功能分析
  • 批准号:
    8277962
  • 财政年份:
    2002
  • 资助金额:
    $ 30.66万
  • 项目类别:

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