Mechanistic analysis of axonal transport defects in neurodegenerative disease

描述（由申请人提供）：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，包括运动神经元变性和腓骨肌萎缩症的遗传形式。更广泛地说，我们知道动力蛋白驱动功能（例如逆行轴突运输）的缺陷与神经退行性疾病（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）的发病机制有关。然而，所涉及的具体机制仍不清楚。动力蛋白是一种在神经元中具有多种不同作用的多效性细胞运动蛋白。在这里，我们将集中在动力蛋白驱动的降解细胞器，包括溶酶体和自噬体的逆行运输的缺陷是轴突变性，这些疾病的特征的主要贡献者的假设。本研究的目的是了解动力蛋白功能缺陷与神经退行性变之间的具体机制，重点关注以下三个方面：（1）神经退行性变过程中逆行轴突运输是如何改变的？我们推测JNK和Cdk 5通路的病理改变导致轴突运输过程中微管马达的失调。我们将测试这一假设，使用定量活细胞成像的囊泡运输在初级神经元从多个模型的ALS。然后，我们将机械地剖析激酶的错误调节如何影响运动功能，使用体外重建方法与单分子分辨率。这些研究将测试的模型，在协调的相反取向的电机中断是主要的缺陷，导致改变运输沿着轴突。(2)神经元中自噬体生物合成和货物装载的途径是什么？我们假设神经元中的自噬遵循维持细胞内稳态所需的刻板和空间调节的途径。我们将使用定量活细胞成像来研究初级感觉和运动神经元中的自噬体生物发生和货物装载，重点是动力蛋白和视神经磷酸酶的作用。然后，我们将确定该途径如何响应cellula，以解决该途径在响应细胞应激时上调的能力有限的假设。(3)动力蛋白驱动的自噬缺陷是如何导致轴突退化的？我们假设自噬体的主动的、动力蛋白驱动的运输与功能紧密相关，并且运输缺陷将导致老化细胞器和聚集蛋白的降解缺陷。我们将使用实时成像和生物化学和细胞分析来确定自噬体运输缺陷如何沿着沿着轴突促进神经变性，以及不同的动力蛋白突变如何差异地扰乱细胞功能，导致不同的临床表现。细胞质动力蛋白的突变足以引起人类神经退行性疾病，包括脊髓性肌萎缩症（SMA-LED）和腓骨肌萎缩症（2 O型），但涉及的机制仍有待确定。这些目标的进展应该为治疗方法或临床干预提供新的机会。

DESCRIPTION (provided by applicant): Mutations in cytoplasmic dynein or its activator dynactin are causative for neuronal diseases including heritable forms of motor neuron degeneration and Charcot-Marie-Tooth disease. More broadly, we know that defects in dynein-driven 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 tha defects in the dynein-driven 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 mis-regulation 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 oppositely-oriented motors is the primary defect leading to altered transport along the axon. (2) What are the pathways for autophagosome biogenesis and cargo-loading 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 cargo-loading 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 cellula, to address the hypothesis that this pathway has a limited ability to up-regulate in response to cellular stress. (3) How do defects in dynein-driven autophagy lead to degeneration of the axon? We hypothesize that the active, dynein-driven 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 (SMA-LED) and Charcot-Marie-Tooth 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.