NEUROFILAMENT PROTEINS--ASSEMBLY AND AXONAL TRANSPORT

已知神经丝在许多疾病的病因学中起核心作用， 人类神经退行性疾病，最显著的是运动神经元疾病， 巨大轴突神经病 这些疾病的特点是大规模的 在受影响的神经元的轴突中神经丝的积累，形成 巨大的气球样囊肿并导致轴突变性。 的 这些疾病中神经丝的积累被认为是由 细胞骨架运动的慢轴突运输机制的变化 沿着轴突。 慢轴突运输的机制知之甚少， 争议 主要问题是关于集会地点的问题。 细胞骨架蛋白及其运动形式。 的情况下 神经丝蛋白，大量证据表明，细胞体 是组装的主要场所，这些蛋白质被运输到 沿着轴突组装成聚合物。 另外，它也被 提出，细胞骨架蛋白是沿着轴突在非- 聚合形式，并且组装的主要位点在轴突尖端。 为了验证这些假设，神经丝的组装和运输 将使用免疫荧光技术在培养的神经元中研究蛋白质 显微镜和显微注射技术，结合定量 数字图像分析 为了确定神经丝蛋白的组装位点，牛低- 将分子量神经丝蛋白（NF-L）显微注射到 培养的大鼠神经元和注射的蛋白质的组装将被 使用物种特异性单克隆抗体可视化。 此外该 新合成的高分子量神经丝的组装位点 蛋白（NF-H）将在培养的神经元中鉴定， 它在轴突生长过程中的延迟表达。 为了观察轴突 神经丝转运，神经元特异性 将抗体显微注射到培养的神经元中， 结合的抗体将通过免疫荧光显微术进行分析。 到 可视化神经丝蛋白的运输，无论形式如何， 当它们移动时，转运蛋白的积累将被 在实验应用轴突收缩研究。 这些研究将产生关于组装的重要新信息， 轴突运输的神经丝，并将奠定基础，为未来 培养神经元中慢轴突运输机制的研究， 神经丝在轴突中积聚的机制。 一个 对这些机制的理解将为研究这些疾病的病因提供新的线索。 神经纤维性神经病

Neurofilaments are known to play a central role in the etiology of a number of human neurodegenerative disorders, most notably motor neuron disease and giant axonal neuropathy. These disorders are characterized by massive accumulations of neurofilaments in the axons of affected neurons, forming giant balloon-like swellings and 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 proteins along axons. The mechanism of slow axonal transport is poorly understood and controversial. The principal issue concerns the site of assembly of cytoskeletal proteins and the form in which they move. In the case of neurofilament proteins, considerable evidence suggests that the cell body is a principal site of assembly and that these proteins are transported along the axon as assembled polymers. Alternatively, it has also been proposed that cytoskeletal proteins are transported along axons in a non- polymeric form and that the principal site of assembly is at the axon tip. To test these hypotheses, the assembly and transport of neurofilament proteins will be investigated in cultured neurons using immunofluorescence microscopy and microinjection techniques, in conjunction with quantitative digital image analysis. To identify the sites of assembly of neurofilament proteins, bovine low- molecular weight neurofilament protein (NF-L) will be microinjected into cultured rat neurons and the assembly of the injected protein will be visualized using species-specific monoclonal antibodies. In addition, the sites of assembly of newly synthesized high-molecular weight neurofilament protein (NF-H) will be identified in cultured neurons by taking advantage of its delayed expression during axon outgrowth. To visualize the axonal transport of neurofilaments, Fab fragments of neurofilament-specific antibodies will be microinjected into cultured neurons and the movement of the bound antibodies will be analyzed by immunofluorescence microscopy. To visualize the transport of neurofilament proteins regardless of the form in which they move, the accumulation of transported proteins will be investigated at an experimentally applied axonal constriction. These studies will yield important new information on the assembly and axonal transport of neurofilaments and will lay the groundwork for future studies on the mechanisms of slow axonal transport in cultured neurons, and on the mechanisms that cause neurofilaments to accumulate in axons. An understanding of these mechanisms will shed new light on the etiology of neurofilamentous neuropathies.