Mechanistic analysis of axonal transport defects in motor neuron degenerative dis

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种以运动神经元变性和死亡为特征的致命且无法治愈的疾病。散发性和遗传性肌萎缩侧索硬化症都遵循一种共同的致病途径，目前尚不清楚。可以解释ALS和其他类似神经退行性疾病中观察到的运动神经元特异性细胞死亡的一个假设是，运动神经元对轴突运输的缺陷特别敏感。沿轴突的主动运输是由微管马达蛋白驱动的，而多种运动蛋白驱动顺行运输，而细胞质动力蛋白及其激活剂dynactin是唯一驱动逆行运输的马达。动力蛋白或动力蛋白的显性突变足以引起运动神经元疾病，表明轴突运输在维持健康运动神经元方面的重要性。我们现在有数据直接表明在多种运动神经元疾病模型中存在显著的逆行运输缺陷，包括具有良好特征的家族性ALS小鼠模型；在这些模型中，动力蛋白定位和功能缺陷作为疾病发病机制的早期事件发生。我们将研究轴突运输缺陷与神经变性的联系机制，重点是逆行运输效率和被运输货物性质的变化。我们假设，这些变化共同作用，导致神经元中生存和死亡信号的平衡改变，导致神经元退化和细胞死亡。为了验证这一假设，我们将追求三个具体目标。在特定的目标1中，我们将观察动力蛋白介导的转运在疾病发生和发展过程中是如何改变的。我们将通过分析从小鼠神经退行性疾病模型中分离出的蛋白质和细胞器的运动性来探讨其具体机制。在特定的目标2中，我们将使用动态蛋白的蛋白质组筛选，比较野生型和变性神经元中由动力蛋白活跃转运的货物。我们假设，动力蛋白物质的变化，特别是信号分子，将导致生存和死亡信号之间的平衡发生变化。最后，在具体目标3中，我们将使用神经退行性疾病的细胞模型来研究轴突运输缺陷是如何导致神经退行性疾病的。我们将研究运输机械缺陷和正在运输的货物的变化，以及细胞器运输和蛋白质降解缺陷的相对贡献。本文提出的研究将使人们更清楚地了解轴突运输在运动神经元退行性疾病中的作用。随着越来越多的退行性和老年性疾病，包括亨廷顿病和阿尔茨海默病，观察到细胞内运输的中断，很可能这里提出的机制研究将提供更广泛适用于我们对神经元退化的理解的见解。与公共健康相关：蛋白质、囊泡和细胞器沿着神经元延长的轴突的主动运动称为轴突运输。这种转运对神经元的健康和功能至关重要，在肌萎缩侧索硬化症(ALS或Lou Gehrig病)等疾病中，这一过程中的缺陷会导致运动神经元退化，导致肌肉萎缩。在这里，我们建议研究轴突运输的缺陷，无论是由驱动轴突运输的马达蛋白突变直接引起的，还是由其他突变蛋白的表达间接引起的，导致退行性疾病的机制。

DESCRIPTION (provided by applicant): Abstract Amyotrophic lateral sclerosis (ALS) is a fatal and incurable disease characterized by the degeneration and death of motor neurons. Both sporadic and inherited forms of ALS follow a common pathogenic pathway, which is still not understood. One hypothesis that may explain the motor neuron-specific cell death observed in ALS and other similar neurodegenerative diseases is that motor neurons are uniquely sensitive to defects in axonal transport. Active transport along the axon is driven by microtubule motor proteins; while multiple kinesins drive anterograde transport, cytoplasmic dynein and its activator dynactin are the only motor driving retrograde transport. Dominant mutations in either dynein or dynactin are sufficient to cause motor neuron disease, demonstrating the importance of axonal transport in maintaining healthy motor neurons. We now have data directly demonstrating significant defects in retrograde transport in multiple models of motor neuron disease, including a well- characterized mouse model of familial ALS; defects in dynein localization and function occur as an early event in disease pathogenesis in these models. We will examine the mechanisms linking defects in axonal transport to neurodegeneration, focusing on alterations in both the efficiency of retrograde transport and the nature of the cargos being transported. We hypothesize that these changes act together to lead to alterations in the balance of survival and death signals in the neuron, resulting in neuronal degeneration and cell death. To test this hypothesis, we will pursue three specific aims. In Specific Aim 1, we will look at how dynein-mediated transport is altered during disease onset and progression. We will investigate the specific mechanisms involved by analyzing the motility of proteins and organelles isolated from mouse models of neurodegenerative disease. In Specific Aim 2, we will compare the cargos that are actively transported by dynein in wild type and degenerating neurons, using proteomic screens for dynein cargos. We hypothesize that changes in dynein cargos, especially signaling molecules, will result in alterations in the balance between survival and death signals. Finally, in Specific Aim 3, we will use cellular models of neurodegenerative disease to investigate how defects in axonal transport lead to neurodegeneration. We will examine the relative contributions of defects in the transport machinery and alterations in the cargo being transported, as well as defects in organelle trafficking and protein degradation. The studies proposed here will lead to a clearer understanding of the role of axonal transport in motor neuron degenerative disease. As disruption of intracellular trafficking has been observed in a growing number of degenerative and aging diseases, including Huntington's and Alzheimer's Disease, it is likely that the mechanistic studies proposed here will provide insights that are more broadly applicable to our understanding of neuronal degeneration. PUBLIC HEALTH RELEVANCE: The active movement of proteins, vesicles, and organelles along the extended axons of neurons is called axonal transport. This transport is essential for the health and function of the neuron, and defects in the process cause motor neuron degeneration leading to muscle atrophy in diseases such as Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease). Here, we propose to investigate the mechanisms by which defects in axonal transport, caused either directly by mutations in the motor proteins that drive this transport, or indirectly by the expression of other mutant proteins, lead to degenerative disease.