Role of axoglial CAMs in the organization of myelinated axons

描述(申请人提供)：髓鞘覆盖被Ranvier结节分开的节段的轴突。在下层轴突中，Na+通道聚集在Ranvier结节处，与邻近旁结区(JXP)的K+通道之间有一个特殊的轴突旁结节连接(PNJ)。这种组织对于神经冲动的跳跃运动是必不可少的，它的干扰导致了在脱髓鞘人类疾病中常见的病理生理变化。在周围神经系统(PNS)中，髓鞘雪旺细胞通过未知的机制调节轴突膜的这种精确组织。我们以前已经发现了几种细胞黏附分子，包括gliomedin、CASPR、Caspr2和Necl4，它们在沿着髓鞘单位纵轴的特定位置介导轴突接触。这些CaM在有髓轴突的组织中起着重要的作用：胶质样蛋白聚集在节点上的Na+通道，CASPR参与PNJ和JXP分别限制Na+和K+通道分布的膜屏障的产生，Caspr2作为维持JXP处K+通道的支架，Necl蛋白组织节点间膜。这项研究的目的是研究这些轴突凸轮在沿着有髓轴突产生功能域过程中潜在的分子机制。首先，将使用从GLDN-/-小鼠分离的髓鞘雪旺细胞/神经元培养进行互补实验，以确定Gliomedin的通道聚集活性是如何受到调节的。其次，将采用转基因挽救方法来检验这样的假设，即PNJ通过形成依赖于轴突细胞骨架的膜屏障来限制Na+通道在结节间隙的分布。第三，将采取类似的体内方法来研究Caspr2和Tag-1之间的相互作用模式，并揭示这些分子在JXP招募和保持KV1通道的潜在机制。最后，我们将通过分析缺失Necl4的突变小鼠以及缺失与Necl4共存的髓鞘雪旺细胞中的细胞骨架连接蛋白4.1G的小鼠周围神经的形态和分子组织来研究Necl蛋白在有髓轴突组织中的作用。总之，拟议的实验将提供有关轴突和髓鞘形成细胞协调分化的重要信息，这使得有髓纤维能够最大限度地提高其传导速度。因此，更好地了解这些机制可能会导致新的途径来恢复脱髓鞘轴突的功能，并可能被证明对设计脱髓鞘疾病的治疗策略很有用。

DESCRIPTION (provided by applicant): The myelin sheath covers the axon in segments that are separated by the nodes of Ranvier. In the underlying axons Na+ channels are clustered at the nodes of Ranvier, separated by a specialized axoglial paranodal junction (PNJ) from K+ channels that are found at the nearby juxtaparanodal region (JXP). This organization is essential for the saltatory movement of the nerve impulses, and its disturbance results in pathophysiological changes often seen in demyelinating human disorders. In the peripheral nervous system (PNS), myelinating Schwann cells regulate this precise organization of the axonal membrane through unknown mechanisms. We have previously identified several cell adhesion molecules (CAMs), including gliomedin, Caspr, Caspr2, and Necl4, which mediate axoglial contact at specific sites along the longitudinal axis of the myelin unit. These CAMs play important roles in the organization of myelinated axons: gliomedin cluster Na+ channels at nodes, Caspr is involved in the generation of a membrane barrier at the PNJ that restricts the distribution of Na+ and K+ channels to the nodes and JXP, respectively, Caspr2 serves as a scaffold that maintains K+ channels at the JXP, and Necl proteins organize the internodal membrane. The objective of the proposed research is to study the molecular mechanisms underlying the function of these axoglial CAMs in the generation of functional domains along myelinated axons. First, complementation experiments will be carried out using myelinating Schwann cells/neuron cultures isolated from gldn-/- mice to determine how the channels clustering activity of gliomedin is being regulated. Secondly, a transgenic rescue approach will be taken to examine the hypothesis that the PNJ restricts the distribution of Na+ channels to the nodal gap by forming a membrane barrier, which depends on the presence of the axonal cytoskeleton. Third, a similar in vivo approach will be taken in order to examine the mode of interaction between Caspr2 and TAG-1 and to reveal the underlying mechanisms by which these molecules recruit and retain Kv1 channels at the JXP. Finally, we will study the role of Necl proteins in the organization of myelinated axons by analyzing the morphology and the molecular organization of peripheral nerves of mutant mice lacking Necl4, as well as mice lacking the cytoskeletal linker protein 4.1G, which colocalizes and associates with Necl4 in myelinating Schwann cells. Altogether, the proposed experiments will provide important information about the coordinated differentiation of axons and myelin-forming cells, which allow myelinated fibers to maximize their conduction velocity. Better understanding these mechanisms may thus lead to new routes to the restoration of function in demyelinated axons and may prove useful in the design of therapeutical strategies for demyelinating disorders.