AXONAL TRANSPORT OF NEUROFILAMENTS

描述（由申请人提供）：神经丝的细胞质积聚是许多人类神经退行性疾病的标志性病理特征，最显著的是肌萎缩性侧索硬化症。这些神经丝积聚被认为是由缓慢轴突运输机制的变化引起的，该机制将细胞骨架和胞质蛋白质从它们在神经细胞体中的合成位点沿沿着移动。我们最近在培养的神经细胞中观察到神经丝蛋白的缓慢轴突运输。这些蛋白质以丝状结构的形式移动，可能代表单个神经丝聚合物。与广泛认为的慢轴突运输是一种缓慢、同步和完全顺行的运动的观点相反，我们发现细丝实际上以非常快的速率移动，接近快轴突运输的速率，并且运动也是罕见的、双向的和高度异步的。基于这些观察结果，我们提出了一个新的模型，慢轴突运输的实际速度是快的，但整体速度是缓慢的，因为快速的运动被长时间的停顿中断。在这个应用中，我们建议使用活细胞荧光成像策略来测试这个假设的特定方面。在目标1中，我们将检验移动的神经丝代表单个神经丝聚合物的假设。我们希望这些实验也能揭示出细丝沿着移动的轨迹。在目标2中，我们将测试的假设，即移动和静止的细丝在特定的表位磷酸化状态不同，他们在与特定的微管马达蛋白的关联不同。在目标3中，我们将测试的假设，即快速移动的丝被交付到生长轴突的尖端，在足够的数量，以支持轴突生长过程中的轴突神经丝阵列的阐述。我们还将测试的假设，即生长锥是一个网站的灯丝运动的方向经常逆转，并在远端轴突的频率和/或方向性的灯丝运动调节响应轴突生长的速度。我们的长期研究目标是确定神经丝蛋白沿沿着轴突转运的机制和调节以及导致神经丝神经病中神经丝积聚的机制。

DESCRIPTION (provided by applicant): Cytoplasmic accumulations of neurofilaments are a hallmark pathological feature of a number of human neurodegenerative diseases, most notably amyotrophic lateral sclerosis. These neurofilamentous accumulations are thought to be caused by changes in the mechanisms of slow axonal transport, which move cytoskeletal and cytosolic proteins along axons from their site of synthesis in the nerve cell body. We have recently observed the slow axonal transport of neurofilament protein in cultured nerve cells. The proteins move in the form of filamentous structures that may represent single neurofilament polymers. Contrary to the widely held view that slow axonal transport is a slow, synchronous and exclusively anterograde movement, we found that the filaments actually move at very fast rates, approaching the rate of fast axonal transport, and that the movements are also infrequent, bi-directional and highly asynchronous. Based on these observations, we have proposed a new model for slow axonal transport in which the actual rate of movement is fast, but the overall rate is slow because the rapid movements are interrupted by prolonged pauses. In this application, we propose to use live-cell fluorescence imaging strategies to test specific aspects of this hypothesis. In Aim 1 we will test the hypothesis that the moving filaments represent single neurofilament polymers. We expect that these experiments will also reveal the tracks along which the filaments move. In Aim 2 we will test the hypothesis that moving and stationary filaments differ in their phosphorylation state at specific epitopes and that they differ in their association with specific microtubule motor proteins. In Aim 3 we will test the hypothesis that rapidly moving filaments are delivered to the tip of growing axons in sufficient quantity to support the elaboration of the axonal neurofilament array during axon growth. We will also test the hypothesis that the growth cone is a site of frequent reversals in the direction of filament movement and that the frequency and/or directionality of filament movements in the distal axon is regulated in response to the rate of axon growth. The long-term goal of our research is to determine the mechanism and regulation of neurofilament protein transport along axons and the mechanisms that lead to the accumulation of neurofilaments in neurofilamentous neuropathies.