Node of Ranvier Assembly: Role of Axo-Glial Interactions

描述(申请人提供)：令人震惊的钠通道集中 是轴突和髓鞘形成之间复杂的相互作用的结果 神经胶质细胞，即三叉神经节的许旺细胞和中枢神经系统的少突胶质细胞。这个 人们对这些相互作用的本质以及它们的机制知之甚少。 哪些钠通道被定位并组装成一个更大的复合体 结节上的其他蛋白质。相关蛋白包括186kD的亚型 神经束素，它可能与覆盖的神经胶质突起上的受体结合，以及 连接细胞黏附分子和钠通道的锚蛋白G。该节点是 侧边有旁结点，可用于初始结点 形成，但可能调节成熟和通道密度在节点。我们 建议通过以下方式解决此模型的重要特征 定位于结节的蛋白质，覆盖的神经胶质过程的作用 侧翼结节及其神经束素在引导结节组装中的潜在作用 该节点。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标记活的神经元共培养细胞来运输到这个位置 FAB片段在经历髓鞘形成时，分析结节是否在 Wlds/Ola小鼠轴突横断及轴突转运的研究 NA通道和其他蛋白质到结节；ii)研究 ERM雪旺细胞在三叉神经节形成中的阻断作用 通过显性负面策略或使用Rho激酶抑制剂；三) 描述侧翼旁结节突起和交界处在 CASPR缺陷小鼠缺乏结节的发育和成熟 关注结节宽度、通道密度和钠通道的旁结节 亚型和iv)决定细胞黏附的L1家族的作用 分子，特别是神经束素，通过产生小鼠 一种有条件的、神经元特异性的神经筋膜蛋白基因敲除，并确定 它们表现出异常的起始节段、节点或旁阳极组织，如果 通过将这些小鼠与现有的NrCAM品系杂交，影响最小 用于分析双基因敲除的基因敲除小鼠。这些研究应该 对负责装配的机制提供重要的新见解 指导其组装的神经胶质信号的性质，并可能 明确伴随脱髓鞘障碍的功能缺陷和 中断正常的跳跃传导。

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.