AXONAL TRANSPORT OF NEUROFILAMENTS

描述（由申请人提供）：神经丝的细胞质积累是许多人类神经退行性疾病的标志性病理特征，最明显的是肌萎缩性侧索硬化症。这些神经丝状堆积被认为是由缓慢轴突运输机制的变化引起的，轴突运输将细胞骨架和胞质蛋白从它们在神经细胞体中的合成部位沿轴突移动。我们最近在培养的神经细胞中观察到神经丝蛋白的轴突转运缓慢。蛋白质以丝状结构的形式移动，可能代表单一的神经丝聚合物。与人们普遍认为的慢轴突运输是一种缓慢的、同步的、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度移动，接近快速轴突运输的速度，而且这种运动也是罕见的、双向的和高度异步的。基于这些观察，我们提出了一种新的慢轴突运输模型，其中实际运动速度很快，但由于快速运动被长时间的停顿打断，整体速度很慢。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Aim 1中，我们将测试移动细丝代表单一神经丝聚合物的假设。我们期望这些实验也能揭示这些细丝运动的轨迹。在Aim 2中，我们将验证这样的假设，即运动和静止细丝在特定表位的磷酸化状态不同，它们与特定微管运动蛋白的关联也不同。在Aim 3中，我们将验证快速移动的细丝被递送到生长轴突尖端的假设，其数量足以支持轴突生长过程中轴突神经细丝阵列的细化。我们还将验证以下假设：生长锥是纤维运动方向频繁逆转的地方，而远端轴突中纤维运动的频率和/或方向受到轴突生长速度的调节。我们研究的长期目标是确定神经丝蛋白沿轴突运输的机制和调控，以及导致神经丝状神经病中神经丝积累的机制。

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.