Molecular mechanism of the initial process of neuronal aging.-perturbation in transport and polymerization-depolymerization dynamics of axonal cytoskeleton.

对轴突神经丝聚合-解聚和转运的调控机制进行了研究，获得了以下新发现：1)。腹腔注射β， β′-亚氨基二丙腈（IDPN）后24小时内，大鼠坐骨运动轴突NF-H亚基（表观分子量20万）磷酸化瞬间降低。这早于IDPN引起的神经系统症状的首次出现（注射后约3天），也早于IDPN治疗大鼠运动轴突内神经丝运输的紊乱（注射后约1-2天）。当在磷酸酶抑制剂存在的情况下制备时，与不含这些抑制剂的情况相比，含有1% Triton-100的缓冲液处理后的NF-H的溶解度增加到总溶解度的20%左右。相比之下，无论是否存在磷酸酶抑制剂，NF-L亚基（表观分子量68,000）都保持不溶性。当NF-H的n端结构域被体外c- amp依赖性蛋白激酶磷酸化时，它与NF-L亚基一起溶解，表明整个神经丝解聚。对轴突神经丝进行标记并分析其转运情况时，NF-H的溶解度在标记的神经丝蛋白转运峰处最小，在转运的前端和尾部最大，表明神经丝的转运形式与NF-H的溶解度密切相关。

Studies on the regulatory mechanism of polymerization-depolymerization and transport of axonal neurofilament was carried out and the following new findings were obtained.1).Within 24 hours after intra-peritoneal injection of beta, beta'-iminodipropionitrile (IDPN), phosphorylation of NF-H subunit (apparent molecular weight 200,000) was transiently decreased in the rat sciatic motor axons. This is earlier than the first appearance of neurological symptoms caused by IDPN (about 3 days after injection), and than the disturbance of neurofilament transport within the motor axons of IDPN-treated rat (about 1-2 days after injection).2).When preparation was performed in the presence of phosphatase inhibitors, the solubility of NF-H after treatment with the buffer containing 1% Triton-100 was increased to about 20% of the total, compared to that in the absence of those inhibitors. In contrast NF-L subunit (apparent molecular weight 68,000) remained insoluble irrespective of the presence or absence of phosphatase inhibitors.When N-terminal domain of NF-H was phosphorylated by c-AMP-dependent protein kinase in vitro, it was solubilized together with NF-L subunit, suggesting depolymerization of whole neurofilament.When axonal neurofilaments were labeled and their transport was analyzed, solubility of NF-H was minimum at the peak of transport of labeled neurofilament protein, and maximum at the leading edge and the tail of the transport, suggesting the close association between transported form of neurofilament and solubility of NF-H.