NEUROFILAMENT PROTEINS--ASSEMBLY AND AXONAL TRANSPORT

已知神经丝在许多疾病的病因中起核心作用。 人类神经退行性疾病，最明显的运动神经元病和 巨大轴索神经病。这些疾病的特点是大量 神经细丝在受影响神经元的轴突中堆积，形成 巨大的气球样肿胀并导致轴突变性。这个 在这些疾病中神经丝的堆积被认为是由 移动细胞骨架的慢轴突运输机制的变化 沿着轴突的蛋白质。 对轴突缓慢运输的机制了解甚少， 有争议的。主要问题涉及到集会的地点 细胞骨架蛋白及其运动形式。在.的情况下 神经丝蛋白，相当多的证据表明细胞体 是组装的主要部位，这些蛋白质被运输到 沿着轴突组装成聚合物。或者，它也一直是 提出细胞骨架蛋白是沿着轴突在非 聚合体形式，组装的主要部位在轴突顶端。 为了验证这些假说，神经丝的组装和运输 将使用免疫荧光技术研究培养神经元中的蛋白质。 显微镜和微注射技术，结合定量 数字图像分析。 为了确定神经丝蛋白的组装位置，牛低密度脂蛋白 分子量神经丝蛋白(NF-L)将被显微注射到 培养的大鼠神经元和注射的蛋白质的组装将 使用物种特异性的单抗进行可视化。此外， 新合成的高分子量神经丝的组装部位 蛋白质(核因子-H)将在培养的神经元中被鉴定 其在轴突生长过程中的延迟表达。可视化轴突的步骤 神经细丝的运输，神经细丝特异的Fab片段 抗体将被显微注射到培养的神经元中，并 结合的抗体将通过免疫荧光显微镜进行分析。至 可视化神经丝蛋白的运输，无论其形式是什么。 它们移动，运输的蛋白质的积累将是 在实验中应用轴突收缩进行了研究。 这些研究将产生关于组装和 神经细丝的轴突运输，将为以后的研究奠定基础 培养神经元轴突运输缓慢机制的研究 关于导致神经丝在轴突中堆积的机制。一个 对这些机制的理解将为进一步了解该病的病因提供新的线索。 神经丝状神经病。

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.