ASSEMBLY AND AXONAL TRANSPORT OF NEUROFILAMENT PROTEINS

神经丝被认为在许多人类神经退行性疾病的病因学中发挥核心作用，最著名的是肌萎缩侧索硬化症。这些疾病的特点是在受影响的神经元的轴突中大量积累神经丝，导致轴突变性。这些疾病中神经丝的堆积被认为是由于缓慢的轴突运输机制的改变导致的，该机制将细胞骨架和胞浆蛋白从细胞体中的合成部位沿着轴突移动。然而，人们对这些机制知之甚少，也存在争议。主要问题涉及细胞骨架蛋白的组装位置和它们移动的形式。聚合物运输假说认为，细胞体和近端轴突是细胞骨架蛋白及其运动形式的主要组装部位。聚合物运输假说认为，细胞体和近端轴突是主要的组装部位，细胞骨架蛋白以移动聚合物的形式运输。相反，细胞骨架蛋白是以低聚物亚单位的形式运输的，这些亚单位沿着轴突和轴突尖端局部组装。为了验证这些假说，将在培养的神经元中研究神经丝蛋白的组装和轴突运输，这是有利的，因为它们可以直接观察和实验。拟议的实验将解决两个具体目标。具体目标1，免疫荧光和免疫电子显微镜将与定量数字图像分析相结合，以确定生物素化和内源性神经丝蛋白在神经元中的组装位置。对于特定目标2，包括包括轴突收缩在内的新策略将与对活细胞中荧光神经丝蛋白的直接观察相结合，以确定神经丝蛋白的运输形式。这项研究的长期目标是确定神经丝蛋白在轴突中移动的机制，以及在某些神经退行性疾病中导致神经丝积累的机制。通过测试关于神经丝组装和轴突运输的特定假设，这里提出的研究代表着朝着这一目标迈出的重要一步。

Neurofilaments are thought to play a central role in the etiology of a number of human neurodegenerative diseases, most notably amyotrophic lateral sclerosis. These disorders are characterized by massive accumulations of neurofilaments in the axons of affected neurons, leading to axonal degeneration. The accumulation of neurofilaments in these diseases is 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 cell body. However, these mechanisms are poorly understood and controversial. The principal issue concerns the site of assembly of cytoskeletal proteins and the form in which they move. The polymer transport hypothesis proposes that the cell body and proximal axons are principal sites of assembly of cytoskeletal proteins and the form in which they move. The polymer transport hypothesis proposes that the cell body and proximal axon are principal sites of assembly and that cytoskeletal proteins are transported in the form of moving polymers. In contrast, the cytoskeletal proteins are transported in the form of subunits of oligomers that assemble locally along the axon and the axon tip. To test these hypotheses, the assembly and axonal transport of neurofilament proteins will be investigated in cultured neurons, which are advantageous because of their accessibility to direct observation and experimentation. The proposed experiments will address two specific aims. For Specific Aim 1, immunofluorescence and immunoelectron microscopy will be combined with quantitative digital image analysis to determine the sites of assembly of biotinylated and endogenous neurofilament proteins in neurons. For Specific Aim 2, novel strategies that include constriction that includes constriction of axons will be combined with direct observation of fluorescent neurofilament proteins in living cells to determine the form in which neurofilament proteins are transported. The long-term goal of this research is to determine the mechanism by which neurofilament proteins move in axons and the mechanisms that lead to the accumulation of neurofilaments in certain neurodegenerative diseases. By testing specific hypotheses on the assembly and axonal transport of neurofilaments, the studies proposed here represent an important step toward this goal.