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物动力蛋白的突变是神经元疾病的病因，包括遗传性运动神经元变性和沙克-玛丽-图斯病。更广泛地说，我们知道动力蛋白驱动功能的缺陷，如逆行轴突运输，涉及神经退行性疾病的致病机制，包括肌萎缩性侧索硬化症（ALS）、亨廷顿舞蹈症和阿尔茨海默氏症。然而，涉及的具体机制尚不清楚。动力蛋白是一种多效性细胞马达，在神经元中具有多种不同的作用。在这里，我们将关注这样一种假设，即动力蛋白驱动的降解细胞器（包括溶酶体和自噬体）的逆行运输缺陷是这些疾病特征的轴突变性的主要原因。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，主要集中在以下三个方面：(1)神经退行性变性过程中逆行轴突转运是如何改变的？我们假设JNK和Cdk5通路的病理改变导致轴突运输过程中相反微管运动的失调。我们将通过多种ALS模型的原代神经元水泡转运的定量活细胞成像来验证这一假设。然后，我们将利用单分子分辨率的体外重构方法，从机制上剖析激酶错误调节如何影响运动功能。这些研究将验证一个模型，即反向马达的协调中断是导致沿轴突运输改变的主要缺陷。(2)自噬体在神经元中发生和装载的途径是什么？我们假设神经元中的自噬遵循一种维持细胞稳态所需的刻板和空间调节途径。我们将使用定量活细胞成像技术研究初级感觉和运动神经元的自噬体生物发生和货物装载，重点关注动力蛋白和优神经蛋白的作用。然后我们将确定这条通路对细胞的反应，以解决这条通路在细胞应激反应中上调能力有限的假设。(3)动力蛋白驱动的自噬缺陷是如何导致轴突变性的？我们假设活跃的、动力蛋白驱动的自噬体运输与功能密切相关，并且运输缺陷将导致老化细胞器和聚集蛋白的缺陷降解。我们将使用实时成像、生化和细胞分析来确定沿轴突的自噬体运输缺陷如何导致神经退行性变，以及不同的动力蛋白突变如何以不同的方式扰乱细胞功能，导致不同的临床表现。细胞质动力蛋白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）和沙科-玛丽-图斯病（20型），但其机制仍有待确定。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.