Cytoskeletons and cell death-associated proteins expressed in neuronal cells in the neurofilament gene-manipulated mice

神经丝蛋白由三种蛋白质组成，分别是NF-L、NF-M和NP-H，其中，NF-M和NP-H具有其他中间丝蛋白所没有的独特的延伸和磷酸化的羧基末端结构域。与美国加州大学(UCSD)和纽约大学的研究人员合作，我们培育了不表达这些亚单位蛋白的基因敲除小鼠，以及仅缺少NF-M或/和NF-H的羧基末端尾巴的敲除小鼠，以了解每个NF亚单位蛋白和扩展的NF-M或/和NF-H的羧基末端末端的缺失如何与神经元中的NF组织、轴突结构、神经纤维的轴突运输、其他细胞骨架和膜结合细胞器的组织以及其他NF亚单位蛋白的表达有关。当NF-L被删除时，神经元内没有组装成神经网络。这样的轴突要细得多，但提供了更多的微管和线粒体。与NF-H相比，NF-M的缺失对轴突口径和组织结构的影响更为显著，提示NF-M对神经纤维的功能更为重要。由于核因子-M的羧基末端对核因子的组织和功能更重要，我们只删除了核因子-M或/和核因子-H的这个尾部结构域，不出所料，发现核因子-M的尾部对核因子的组织更加必要。然而，当同时缺失NF-M和NF-H尾巴时，轴浆组织发生了更剧烈的变化，如NF排列不规则，微管和线粒体密度增加。这些结果表明，构成神经纤维之间的交叉桥的NF-M和NP-H尾巴对神经纤维的组织和轴突功能是必需的，也与通过改变线粒体功能来调节神经细胞死亡有关。

Neurofilaments (NFs) are composed of three proteins, NF-L,NF-M and NP-H, and NF-M and NP-H are provided with uniquely extended and phosphorylated carboxyl-terminal tail domains that do not appear in other intermediate filament proteins. In collaboration with American researchers at California (UCSD) and New York Universities we generated knockout mice that do not express each of these subunit proteins as well as knockin mice that lack only the carboxyl-terminal tail of NF-M or/and NF-H, to know how the deletions of each NF subunit protein and of the extended carboxy-terminal tails of NF-M or/and NF-H are related with NF organization in the neuron, axonal structure, axonal transport of NFs, organizations of other cytoskeletons and membrane-bound organelles, and expression of other NF subunit proteins. When NF-L was deleted, NFs were not assembled in neurons. Such axons were much thinner but provided with many more microtubules and mitochondria. Compared with NF-H, the deletion of NF-M had more effective influence on axonal calibers and NF organization, suggesting that NF-M is more essential for the NF function. Since the carboxyl-terminal tail of NF-M appears to be more important for NF organization and function, we deleted only this tail domain of NF-M or/and NF-H, and found as expected that NF-M tail was more necessary for the NF organization. However, more drastic changes in axoplasmic organizations, such as irregularity of NF alignment, Increase in density of microtubules as well as mitochondria, were obtained when both NF-M and NF-H tails were simultaneously deleted. These results suggest that both NF-M and NP-H tails, which form crossbridges between NFs, are necessary for NF organization and axonal functions, and are also associated with regulation of neuronal cell death through the change in function of mitochondria.