Molecular anatomical analysis of neurofilament

本研究获得了四个有关神经丝组织的主要新结果。1.从牛脊髓中分离纯化的不同亚基组成的神经营养因子重组后，用快速冷冻深蚀刻云母电子显微镜观察，发现最大的神经营养因子亚基-H是核心丝之间形成交叉桥的最重要的成分。2.核因子-H是磷酸化程度最高的亚基，在轴突中磷酸化程度较高，而在树突或核周中磷酸化程度较低。此外，轴突网络在结构组织上与树突或核周型网络有很大的不同。根据这些事实和结果1，我们用不同的电子显微镜技术在体外检测了NF-H的磷酸化状态如何影响NF的结构，发现去磷酸化显著改变了交叉桥的结构。因此，我们提供了核因子-H羧基末端尾部结构域磷酸化依赖的结构变化的证据。3.为了阐明为什么核因子-H在轴突中被大量磷酸化，而在其他隔室中没有，我们用免疫金标记法检测了大鼠中枢神经元。我们在树突中发现，当神经营养因子单独出现时，其神经营养因子-H没有被磷酸化，而当神经营养因子捆绑在一起时，神经营养因子-H被磷酸化。结果表明，核因子-H的磷酸化是形成交叉桥的必要条件，而去磷酸化是核因子与其他蛋白质相互作用的必要条件。4.以上结果表明，根据核因子-H的磷酸化程度，交叉桥在结构上是可变的。为了在体内证实这一观点，我们比较了铝中毒后堆积在核周或树突中的神经营养因子和轴突神经营养因子。我们的检查表明，与轴突相比，堆积型神经纤维组的核心丝排列规则较少，交叉桥较少，核因子-H磷酸化程度较低。综上所述，我们的数据表明核因子-H的磷酸化对于De的形成是必要的

Four main new results concerning neurofilament (NF)organizations were obtained in the present study. 1. When NFs reassembeld in various combinations of NF subunits after purified from bovine spinal cord were examined by quick-freeze deep-etch mica electron microscopy, NF-H, the largest NF subunit, was found to be the most essential component for forming cross-bridges between core filaments. 2. NF-H, the most phosphorylated subunit, is heavily phosphorylated in the axon but poorly phosphorylated in the dendrite or perikaryon. Further, axonal NFs are considerably different in structural organization from the dendritic or perikaryal NFs. From these facts and the result 1, we examined with various electron microscopic techniques how NF-H phosphorylation state influences the NF structure in vitro, and found that crossbridge structures were significantly altered by dephosphorylation. Thus, we provide the evidence of phosphorylation-de-pendent structural change in the NF-H carboxy-terminal tail domain. 3. To clarify why NF-H is heavily phosphorylated in the axon but not in the other compartments, we examined rat central neurons with immunogold labeling. We found in dendrites that when NFs appeared singly their NF-H was not phosphorylated while when NFs were bundled NF-H became phosphorylated. The result suggests that phosphorylation of NF-H is necessary for forming crossbridges and unphosphorylation is necessary for NF interaction with other proteins. 4. From all above results, crossbridges are considered to be structurally changeable according to the NF-H phosphorylation degree. To confirm this idea in vivo, we compared NFs accumulated in perikaryon or dendrite following aluminum intoxication with axonal NFs. Our examination showed that core filaments have less regular alignment, fewer crossbridges and lower degree of NF-H phosphorylation in accumulated NFs than in axonal NFs. In conclusion, our data indicate that NF-H phosphorylation is necessary for the formation of de