ASSEMBLY AND AXONAL TRANSPORT OF NEUROFILAMENT PROTEINS

神经丝蛋白的组装和轴突运输

• 批准号: 6312304
• 负责人: Anthony Brown
• 金额: $ 3万
• 依托单位: OHIO UNIVERSITY ATHENS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6312304
• 关键词: axon; biotin; embryo /fetus tissue /cell culture; fluoresceins; immunoelectron microscopy; immunofluorescence technique; laboratory rat; microinjections; neurofilament; neurofilament proteins; neuronal transport; polymers; protein biosynthesis; protein transport

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.

神经丝被认为在许多人类神经退行性疾病的病因学中起核心作用，最显著的是肌萎缩性侧索硬化症。这些病症的特征在于受影响神经元的轴突中神经丝的大量积聚，导致轴突变性。这些疾病中神经丝的积累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沿着轴突从它们在细胞体中的合成位点移动。然而，这些机制是知之甚少和有争议的。主要的问题涉及到细胞骨架蛋白的组装位点和它们的运动形式。聚合物运输假说提出，细胞体和近端轴突是细胞骨架蛋白组装的主要场所，也是它们移动的形式。聚合物运输假说提出，细胞体和近端轴突是组装的主要场所，并且细胞骨架蛋白以移动聚合物的形式运输。相比之下，细胞骨架蛋白以低聚物亚基的形式运输，这些低聚物亚基沿着轴突和轴突尖端局部组装。为了验证这些假设，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。拟议的实验将针对两个具体目标。对于特定目标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，以确定神经元中生物素化和内源性神经丝蛋白的组装位点。对于特定目标2，包括轴突收缩在内的收缩的新策略将与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导致某些神经退行性疾病中神经丝积累的机制。通过测试特定的假设的装配和轴突运输的神经丝，这里提出的研究代表了朝着这一目标迈出了重要的一步。