Mechanistic analysis of axonal transport defects in motor neuron degenerative dis

摘要 肌萎缩侧索硬化症(ALS)是一种以退行性变为特征的致命的不治之症。 以及运动神经元的死亡。散发性和遗传性肌萎缩侧索硬化症都遵循一种常见的致病因素 途径，这仍然是不清楚的。一种可能解释运动神经元特异性细胞的假说 在ALS和其他类似的神经退行性疾病中观察到的死亡是运动神经元 对轴突运输中的缺陷特别敏感。沿着轴突的主动运输是由 微管马达蛋白；当多个动蛋白驱动顺行转运时，细胞质动力蛋白和 它的激活剂dynactin是唯一驱动逆行运输的马达。两个基因中的任何一个都存在显性突变 动力蛋白或动力蛋白足以引起运动神经元疾病，证明了 轴突运输对维持健康运动神经元的作用。我们现在有数据直接证明 运动神经元病的多种模型在逆行运输方面存在显著缺陷，包括- 家族性肌萎缩侧索硬化症的特征小鼠模型；动力蛋白定位和功能缺陷发生在 这些模型中疾病发病机制的早期事件。我们将研究连接缺陷的机制 在轴突运输向神经退行性变方面，重点研究了两者逆行效率的改变 运输和所运输货物的性质。我们假设这些变化起作用 共同导致神经元中生存和死亡信号平衡的改变，导致 神经元变性和细胞死亡。为了验证这一假设，我们将追求三个具体目标。在……里面 具体目标1，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如何改变 进步。我们将通过分析蛋白质的运动性来研究其具体的机制。 以及从神经退行性疾病小鼠模型中分离出的细胞器。在具体目标2中，我们将 比较野生型和变性神经元中由动力蛋白主动转运的货物， 使用蛋白质组筛查动力蛋白货物。我们假设动力系统中货物的变化，尤其是 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， 在特定的目标3中，我们将使用神经退行性疾病的细胞模型来研究缺陷如何 在轴突运输中导致神经退行性变。我们将检查缺陷在 运输机械和运输货物的变化，以及细胞器的缺陷 贩运和蛋白质降解。这里提出的研究将导致对 轴突运输在运动神经元退行性疾病中的作用。因为细胞内的干扰 在越来越多的退行性和老年性疾病中观察到贩运，包括 亨廷顿氏症和阿尔茨海默氏症，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变性的理解的见解。蛋白质、囊泡和细胞器沿着神经元延伸的轴突的主动运动称为 轴突运输。这种运输对于神经元的健康和功能以及过程中的缺陷是必不可少的。 在肌萎缩侧索硬化症等疾病中导致运动神经元退化导致肌肉萎缩 (ALS或Lou Gehrig病)。在这里，我们建议研究轴突缺陷的机制 运输，要么直接由驱动这种运输的马达蛋白突变引起，要么间接由 其他突变蛋白的表达，会导致退行性疾病。

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. 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.