Assembly and Maintenance of the Axonal Cytoskeleton in Cultured Neurons

近年来，一些新的神经元中间丝物种的存在已被证实。Shea博士已经证明，“66kDa神经元中间丝”，也被称为α -连接蛋白，是由NB2a/d1细胞组成性表达的，并在分化过程中在神经鞘基部和轴突丘内显示出独特的分布。谢伊博士将通过两种已经用于轴突细胞骨架动力学研究的方法来调节处于不同分化阶段的细胞中的这种蛋白质：1)抑制这种神经元中间丝物种与其他细胞骨架成分的易位、组装和相互作用，2)通过反义寡核苷酸处理细胞来抑制这种蛋白质的合成。每隔一小时，通过相衬显微镜检查细胞的神经突生长、神经突缩回、稳定性的改变，以及66kDa神经元中间丝/ α -间连蛋白和其他轴突细胞骨架抗原在细胞内定位的免疫细胞化学。这些分析将有助于确定神经突生长、持续延伸或稳定是否需要NF-66。比较H-200下调66kDa神经元中间丝/ α -间连蛋白后获得的结果，将提供有关这种新型神经元中间丝物种在轴突生长和成熟中所起作用的信息。

In recent years, the presence of several novel neuronal intermediate filament species has been documented. Dr. Shea has shown that the "66kDa neuronal intermediate filament," also known as alpha-internexin, is constitutively expressed by NB2a/d1 cells, and displays a unique distribution within the neuritic base and axon hillock during differentiation. Dr. Shea will modulate this protein in cells at various stages of differentiation by two methods already in use for the study of axonal cytoskeletal dynamics: 1) inhibiting the translocation, assembly, and interaction of this neuronal intermediate filament species with other cytoskeletal components, and 2) inhibiting the synthesis of this protein by treating cells with antisense oligonucleotides. Cells will be examined at hourly intervals by phase-contrast microscopy for alterations in neurite outgrowth, neurite retraction, compromise of stability, and immunocytochemistry for intracellular localization of the 66kDa neuronal intermediate filament/alpha-internexin as well as other axonal cytoskeletal antigens. These analyses will help determine whether or not NF-66 is required for neurite outgrowth, continued elongation, or stabilization. Comparison of results obtained following down-regulation of the 66kDa neuronal intermediate filament/alpha-internexin with H-200 will provide information regarding the role played by this novel neuronal intermediate filament species in axonal outgrowth and maturation.