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（由适用提供）：许多神经退行性疾病，包括帕金森氏病（PD），显示了轴突运输中的定义。但是，尚不清楚这些缺陷如何促进发病机理。主要的运动驱动长距离逆行轴突转运是细胞质动力蛋白，以及其所需的激活剂Dynactin（Dynein激活剂）。 DynActin是一种多蛋白质复合物，可直接结合动力蛋白电机以及微管加末端结合蛋白EB1和EB3（EB）和夹170。 DynActin的P150GLOUD亚基通过细胞骨架相关蛋白 - 甘氨酸富含域（盖胶囊）直接与EBS和CLIP-170相互作用。 P150GLUED帽盖结构域中的人类突变减少了远端轴突的逆行运输计划，并引起一种罕见的遗传形式的帕金森氏症，称为佩里综合征。但是，尚不清楚运输计划的精确机制和调节。此外，没有建立的机制将减少的逆行运输计划与神经退行性关系联系起来。我将在以下具体目的中解决这些问题：在AIM 1中，我将确定佩里突变如何影响 使用细胞和生化方法结合P150GLE与夹170的结合。然后，我将在体外重新建立逆行运输倡议复合物，并使用具有单分子分辨率的TIRF显微镜可视化纯化的脑源性蔬菜的运输计划。我预测，佩里综合征突变消除了P150GLED结合夹170的能力，从而阻止了Dynactin依赖于夹的clip-170依赖于运输计划的能力。 AIM 2，我将测试佩里突变如何影响逆行转运的两个主要功能，这是为了促进远端轴突蛋白的自噬清除，并将神经营养信号转运到细胞体。我假设佩里综合征突变将减少自噬体的转运和/或神经营养因子信号传导。分别或两者兼而有之的过程都可以解释佩里综合征的发病机理。我将使用微流体室中翻译的皮质神经元的活细胞成像检验该假设。最后，我将利用从我们的生化研究中获得的机械见解来测试小分子是否可以增加逆行运输计划并挽救表达佩里突变体P150GLOUD的神经元中看到的缺陷。了解运输计划的机制和调节对于定义佩里综合征的致病机制至关重要。此外，这项工作的新见解可能暗示了用小分子调节轴突运输的新方法。最后，由于佩里综合症具有帕金森氏症的许多临床和病理标志，因此从这些研究中获得的知识可能与零星帕金森氏病有关。