Node of Ranvier Assembly: Role of Axo-Glial Interactions

描述（由申请人提供）：Na+通道的显著浓度 轴突和髓鞘之间复杂的相互作用 神经胶质细胞，即PNS中的施万细胞和CNS中的少突胶质细胞。的 这些相互作用的性质知之甚少， Na+通道的靶向和组装成一个更大的复合物， 其他蛋白质在节点。相关的蛋白质包括186 kD的 神经成束蛋白，其可以结合覆盖的神经胶质过程上的受体，和 锚蛋白G，其将细胞粘附分子连接到Na+通道。该节点 两侧有结旁连接处，这是初始结所必需的 形成，但可以调节节点处的成熟和通道密度。我们 建议通过描述 将蛋白质靶向至淋巴结，神经胶质过程的作用， 侧翼节点和神经成束蛋白在指导组装的潜在作用， 节点。具体地说，我们将i）确定靶向于 节点从现有池重新分布和/或新合成， 通过用抗NrCAM标记神经元的活的共培养物运输到该位点 Fab片段，因为他们经历髓鞘形成，分析节点是否形成后， 横切Wlds/奥拉小鼠的轴突，并表征 Na+通道和其他蛋白质的节点; ii）调查的作用， ERM+雪旺细胞通过阻断其形成在PNS结形成中的过程 通过显性阴性策略或使用Rho激酶抑制剂; iii） 描述了侧翼结旁过程和连接在 在缺乏Caspr的小鼠中， 结旁连接，关注节点宽度、通道密度和Na+通道 亚型和iv）确定细胞粘附的L1家族的作用 分子，特别是神经成束蛋白，通过产生具有 有条件的，神经元特异性敲除神经成束蛋白， 它们表现出异常的起始节段、结或旁阳极组织，如果 通过将这些小鼠与现有的NrCAM系杂交， 敲除小鼠用于分析双敲除。这些研究应 为负责大会的机制提供重要的新见解 神经胶质信号的性质，指导其组装， 阐明伴随脱髓鞘疾病的功能缺陷， 中断正常跳跃传导。

DESCRIPTION (provided by applicant): The striking concentration of Na+ channels at the node results from complex interactions between axons and myelinating glial cells, i.e. Schwann cells in the PNS and oligodendrocytes in the CNS. The nature of these interactions are poorly understood as are the mechanisms by which Na+ channels are targeted to and assemble into a larger complex with other proteins at the node. Associated proteins include the 186 kD isoform of neurofascin, which may bind to receptors on overlying glial processes, and ankyrin G which links cell adhesion molecules to Na+ channels. The node is flanked by paranodal junctions, which are dispensible for initial node formation, but may regulate the maturation and channel density at the node. We propose to address important features of this model by characterizing the targeting of proteins to the node, the role of glial processes that overlie and flank the node and the potential role of neurofascin in directing assembly of the node. Specifically, we will i) determine whether proteins targeted to the node redistribute from existing pools and/or are newly synthesized and transported to this site by labeling live cocultures of neurons with anti-NrCAM Fab fragments as they undergo myelination, analyzing whether nodes form after transecting axons of the Wlds/Ola mouse, and characterizing axonal transport of Na+ channels and other proteins to the node; ii) investigate the role of the ERM+ Schwann cell processes in PNS node formation by blocking their formation by dominant negative strategies or the use of Rho kinase inhibitors; iii) characterize the role of the flanking paranodal processes and junctions in the development and maturation of the node in Caspr deficient mice which lack paranodal junctions focusing on node width, channel density and Na+ channel subtypes and iv) determine the role of the L1 family of cell adhesion molecules, in particular neurofascin, in node formation by generating mice with a conditional, neuron-specific knockout of neurofascin and determine whether they exhibit aberrant initial segment, node or paranode organization and, if minimally affected, by crossing these mice to an existing line of NrCAM knockout mice for analysis of the double knockouts. These studies should provide important new insights into the mechanisms responsible for the assembly of the node, the nature of the glial signals that direct its assembly and may clarify the functional deficits that accompany demyelinating disorders and interrupt normal saltatory conduction.