Mechanistic analysis of axonal transport defects in neurodegenerative disease

神经退行性疾病轴突运输缺陷的机制分析

• 批准号: 9617503
• 负责人: Erika L Holzbaur
• 金额: $ 45.83万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9617503
• 关键词: Address; Affect; Afferent Neurons; Aging; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Autophagocytosis; Autophagosome; Axon; Axonal Transport; Back; Biochemical; Biogenesis; Cell physiology; Cellular Assay; Cellular Stress; Charcot-Marie-Tooth Disease; Clinical; Data; Defect; Degenerative Disorder; Degradation Pathway; Disease; Dynein ATPase; Goals; Heritability; Homeostasis; Human; Huntington Disease; Image; In Vitro; Intervention; Intracellular Transport; Kinesin; Lead; Length; Link; Lysosomes; MAPK8 gene; Mediating; Microtubules; Modeling; Molecular Motors; Motor; Motor Neurons; Movement; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organelles; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Phosphotransferases; Proteins; Regulation; Regulatory Pathway; Resolution; Role; Spinal Muscular Atrophy; Testing; Therapeutic; Vesicle; Vesicle Transport Pathway; axonal degeneration; dynactin; experimental study; gene therapy; genetic analysis; insight; live cell imaging; meter; motor neuron degeneration; mouse model; neuron loss; protein aggregate; reconstitution; response; retrograde transport; single molecule; stressor

Project Summary Mutations in cytoplasmic dynein or its activator dynactin are causative for neuronal diseases including heritable forms of motor neuron degeneration and Charcot­Marie­Tooth disease. More broadly, we know that defects in dynein­driven functions such as retrograde axonal transport are involved in the pathogenic mechanisms of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntington’s, and Alzheimer’s. However, the specific mechanisms involved remain unclear. Dynein is a pleiotropic cellular motor with multiple distinct roles in the neuron. Here we will focus on the hypothesis that defects in the dynein­driven retrograde transport of degradative organelles including lysosomes and autophagosomes are major contributors to the axonal degeneration that characterize these diseases. The goal of this proposal is to understand the specific mechanisms linking defects in dynein function to neurodegeneration, focusing on the following three aims: (1) How is retrograde axonal transport altered during neurodegeneration? We hypothesize that pathological alterations in the JNK and Cdk5 pathways lead to the dysregulation of opposing microtubule motors during axonal transport. We will test this hypothesis using quantitative live cell imaging of vesicular transport in primary neurons from multiple models of ALS. Then, we will mechanistically dissect how kinase mis­regulation affects motor function using in vitro reconstitution approaches with single molecule resolution. These studies will test the model that a disruption in the coordination of oppositely­oriented motors is the primary defect leading to altered transport along the axon. (2) What are the pathways for autophagosome biogenesis and cargo­loading in the neuron? We hypothesize that autophagy in the neuron follows a stereotypical and spatially regulated pathway that is required to maintain cellular homeostasis. We will examine autophagosome biogenesis and cargo­loading in primary sensory and motor neurons using quantitative live cell imaging, focusing on the roles of dynein and optineurin. Then we will determine how this pathway responds to cellular stressors, to address the hypothesis that this pathway has a limited ability to up­regulate in response to cellular stress. (3) How do defects in dynein­driven autophagy lead to degeneration of the axon? We hypothesize that the active, dynein­driven transport of autophagosomes is tightly linked to function, and that defects in transport will lead to defective degradation of aging organelles and aggregated proteins. We will use live imaging and biochemical and cellular assays to determine how defects in autophagosome transport along the axon contribute to neurodegeneration and how distinct dynein mutations differentially perturb cellular functions, leading to disparate clinical manifestations. Mutations in cytoplasmic dynein are sufficient to cause human neurodegenerative diseases including spinal muscular atrophy (SMA­LED) and Charcot­Marie­Tooth disease (Type 2O), but the mechanisms involved remain to be determined. Progress on these aims should offer new opportunities for therapeutic approaches or clinical intervention.

项目摘要 细胞质动力蛋白或其激活剂动力蛋白的突变是神经元疾病的病因，包括遗传性疾病。 运动神经元变性和夏科氏玛丽牙病。更广泛地说，我们知道， 动力蛋白驱动的功能，如逆行轴突运输，参与了 神经退行性疾病，包括肌萎缩侧索硬化症（ALS）、亨廷顿舞蹈症和阿尔茨海默氏症。 然而，所涉及的具体机制仍不清楚。动力蛋白是一种多效性细胞马达， 在神经元中扮演不同的角色。在这里，我们将集中在动力蛋白的缺陷驱动逆行的假设， 包括溶酶体和自噬体在内的降解细胞器的运输是导致细胞内蛋白质降解的主要原因。 轴突变性是这些疾病的特征。本提案的目的是了解具体的 动力蛋白功能缺陷与神经退行性变之间的联系机制，重点关注以下三个目标：（1） 神经退行性变时轴突逆行运输是如何改变的？我们假设， JNK和Cdk5通路的改变导致微管马达的失调， 轴突运输我们将使用定量活细胞成像的囊泡运输测试这一假设， 来自多种ALS模型的原代神经元。然后，我们将从机制上剖析激酶是如何错误调节的， 使用具有单分子分辨率的体外重建方法影响运动功能。这些研究 我将测试这个模型，即相反方向的马达协调的中断是主要缺陷 导致沿轴突的运输沿着改变。(2)自噬体生物合成的途径是什么， 神经元中的货物装载？我们假设神经元中的自噬遵循一种刻板的， 空间调节的途径，需要维持细胞内稳态。我们将检查自噬体 使用定量活细胞成像在初级感觉和运动神经元中的生物发生和货物运输负载， 着重于动力蛋白和视神经磷酸酶的作用。然后，我们将确定这一途径如何响应细胞 应激源，以解决这一假设，即该途径具有有限的能力，以上调对细胞应激的反应。 应力(3)动力蛋白驱动的自噬缺陷是如何导致轴突变性的？我们 假设自噬体的主动、动力蛋白驱动的转运与功能紧密相关， 运输缺陷将导致老化细胞器和聚集蛋白质的降解缺陷。我们将使用 活体成像和生物化学及细胞分析，以确定自噬体转运中的缺陷如何沿着 轴突导致神经变性以及不同动力蛋白突变如何不同地干扰细胞 功能，导致不同的临床表现。细胞质动力蛋白的突变足以导致 人类神经退行性疾病，包括脊髓性肌萎缩症（SMA）和夏科氏玛丽牙萎缩症 疾病（2O型），但涉及的机制仍有待确定。这些目标的进展应 为治疗方法或临床干预提供了新的机会。