Pathogenic Mechanisms of Reduced Transport Initiation in Perry Syndrome

佩里综合征转运起始减少的致病机制

• 批准号: 9107239
• 负责人: Jeffrey John Nirschl
• 金额: $ 4.36万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9107239
• 关键词: Active Biological Transport; Address; Affect; Affinity Chromatography; Automobile Driving; Autophagocytosis; Autophagosome; Axon; Axonal Transport; Binding; Binding Proteins; Binding Sites; Biochemical; Brain; Brain-Derived Neurotrophic Factor; CLIP-170 gene; Cells; Cellular biology; Clinical; Complex; Cyclic AMP-Dependent Protein Kinases; Defect; Disease; Distal; Dynein ATPase; Endosomes; FOS gene; Genes; Genetic; Glycine; Health; Human; Immunoblotting; Immunofluorescence Immunologic; In Vitro; Invertebrates; Knowledge; Label; Lead; Link; Measures; Mediator of activation protein; Microfluidics; Mitochondria; Modeling; Molecular Conformation; Monitor; Motor; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Parkinsonian Disorders; Pathogenesis; Pathway interactions; Phosphorylation; Plus End of the Microtubule; Point Mutation; Pregnenolone; Process; Protein Complex Subunit; Protein Dephosphorylation; Proteins; Quantum Dots; Recombinants; Recruitment Activity; Regulation; Reporter; Resolution; Signal Transduction; Syndrome; Testing; Total Internal Reflection Fluorescent; Vesicle; Work; cell fixing; dynactin; insight; live cell imaging; mutant; neuronal cell body; neurosteroids; neurotrophic factor; novel strategies; novel therapeutics; prevent; protein complex; reconstitution; retrograde transport; single molecule; small molecule; trafficking

 DESCRIPTION (provided by applicant): Many neurodegenerative diseases, including Parkinson's disease (PD), show deficits in axonal transport. However, it is not clear how these defects contribute to pathogenesis. The primary motor driving long-distance retrograde axonal transport is cytoplasmic dynein, along with its required activator dynactin (dynein activator). Dynactin is a multi-protein complex that directly binds the dynein motors as well as the microtubule plus-end binding proteins EB1 and EB3 (EBs) and CLIP-170. The p150Glued subunit of dynactin directly interacts with EBs and CLIP-170 via the Cytoskeletal Associated Protein-Glycine Rich domain (CAP-Gly). Human mutations in the CAP-Gly domain of p150Glued reduce retrograde transport initiation from the distal axon and cause a rare, genetic form of Parkinsonism known as Perry syndrome. The precise mechanism and regulation of transport initiation however, are not known. In addition, there are no established mechanisms linking reduced retrograde transport initiation to neurodegeneration. I will address these questions in the following specific aims: In Aim 1, I will determine how Perry mutations affect the binding of p150Glued to CLIP-170 using cellular and biochemical approaches. Then, I will reconstitute the retrograde transport initiation complex in vitro and visualize transport initiatio of purified brain-derived vesicles using TIRF microscopy with single-molecule resolution. I predict that Perry syndrome mutations abrogate the ability of p150Glued to bind CLIP-170, thus preventing the CLIP-170 dependent recruitment of dynactin for transport initiation. In Aim 2, I will test how Perry mutations affect two major functions of retrograde transport, which are to facilitate autophagic clearance of proteins from the distal axon and to transport neurotrophic signals to the cell body. I hypothesize that Perry syndrome mutations will reduce autophagosome transport and/ or neurotrophic factor signaling. Disruption of either process individually, or both together, may explain the pathogenesis of Perry syndrome. I will test this hypothesis using live-cell imaging of transfected cortical neurons in microfluidic chambers. Finally, I will use the mechanistic insight gained from our biochemical studies to test whether small molecules can increase retrograde transport initiation and rescue the defects seen in neurons expressing Perry mutant p150Glued. Understanding the mechanism and regulation of transport initiation is crucial to define the pathogenic mechanisms in Perry syndrome. In addition, the new insights from this work may suggest novel approaches to modulate axonal transport with small molecules. Finally, since Perry syndrome shares many clinical and pathological hallmarks of Parkinson's, the knowledge gained from these studies may be relevant for sporadic Parkinson's disease.

 描述（由申请人提供）：许多神经退行性疾病，包括帕金森病（PD），显示出轴突运输缺陷，但是，尚不清楚这些缺陷如何促进发病机制，驱动长距离逆行轴突运输的主要动力是细胞质动力蛋白。及其所需的激活剂 dynactin（动力蛋白激活剂）是一种多蛋白复合物，可直接结合动力蛋白马达。以及微管正端结合蛋白 EB1 和 EB3 (EB) 以及 CLIP-170 dynactin 的 p150Glued 亚基通过细胞骨架相关蛋白富含甘氨酸结构域 (CAP-Gly) 直接与 EB 和 CLIP-170 相互作用。 p150Glued 的 CAP-Gly 结构域减少了远端轴突的逆行运输起始，并导致罕见的、然而，帕金森病（称为佩里综合征）的确切机制和调节尚不清楚。此外，还没有将逆行转运启动减少与神经变性联系起来的既定机制。在目标 1 中，我将确定 Perry 突变如何影响 然后，我将使用细胞和生化方法将 p150Glued 与 CLIP-170 结合，并在体外重建逆行转运起始复合物，并使用 TIRF 显微镜以单分子分辨率可视化纯化的脑源性囊泡的转运起始。消除 p150Glued 结合 CLIP-170 的能力，从而防止 CLIP-170 依赖的 dynactin 募集在目标 2 中，我将测试 Perry 突变如何影响逆行运输的两个主要功能，即促进远端轴突中蛋白质的自噬清除以及将神经营养信号传输到细胞体。单独或同时减少自噬体转运和/或神经营养因子信号传导可以解释佩里综合征的发病机制，我将使用微流体中转染的皮质神经元的活细胞成像来检验这一假设。最后，我将利用我们从生化研究中获得的机制见解来测试小分子是否可以增加逆行转运启动并挽救表达 Perry 突变体 p150Glued 的神经元中观察到的缺陷，了解转运启动的机制和调节对于定义转运启动至关重要。此外，这项工作的新见解可能会提出用小分子调节轴突运输的新方法。最后，由于佩里综合征具有帕金森病的许多临床和病理特征，因此从这些研究中获得的知识可能会有所帮助。与散发性帕金森病有关。