Molecular Organization and Function of Paranodal Axo-Glial Junctions

有髓轴突的特点是它们组织成分子上不同的区域，这是 动作电位的快速传播。旁结节区域建立轴突-神经胶质间隔连接 (AGSJs)通过轴突联系蛋白相关蛋白(CASPR)、联系蛋白(Cont)和 神经胶质束蛋白NF155。结节结构域由神经元NF186、电压门控钠(NAV)组成 通道，以及Ankyrin G(AnkG)，一种细胞骨架适配器蛋白。我们发现CASPR和NF155的损失 结果导致AGSJs丢失，旁结节蛋白定位错误，旁结节组织紊乱 轴突细胞骨架和有髓轴突变性，但结节组织相对保留 不受影响。最近，我们证明了Band4.1B，一种结旁/旁结节细胞骨架蛋白，是必不可少的 为了AGSJ和旁联结组织的稳定性。我们在这里展示了Whirlin，另一种细胞骨架 蛋白质，是结旁致密和细胞骨架稳定所必需的。在进一步的研究中，我们证明了 节点NF186的丢失消除了节点上NAV通道和AnkG的聚集，从而允许侧翼 旁结节AGSJ侵入结节空间。最重要的是，我们在这里展示了体内AnkG的缺失 不会取消节点形成，但可能会影响节点的稳定性。在取得重大进展的同时， 关于轴突域的组成和组织，仍然存在一些基本问题 关于这些结构域上的跨膜成分如何与局部细胞骨架相互作用 领域组织，并确保轴突体系结构的长期稳定和维护。基于我们的 根据已发表的和初步的研究，我们的中心假设是跨膜成分和 局部轴突细胞骨架对轴突域的组织、稳定性和功能至关重要。我们 将使用遗传、分子和细胞生物学方法来确定偏阳极、节点和 其相关细胞骨架蛋白在结构域稳定和维持中的作用以及修复的效果 NF155和NF186在逐渐虚弱的成年小鼠突变体中的重新表达对关键轴突结构域的影响。我们 将在以下具体目标中实现我们的目标：(1)细胞骨架丢失的后果是什么 支架蛋白和成年期间NF155的丢失对结旁AGSJ的维持和功能有何影响？ (2)成年后结节细胞骨架蛋白的丢失和NF186的丢失对 有髓轴突中结节的稳定性和功能？(3)为成体有髓轴突，周期延长 无序的区域结构能够重新组织轴突区域以恢复神经传导？ 总而言之，我们的研究将提供与轴突域直接相关的机制 以及如何重组这些结构和恢复神经功能。在 未来，这些研究将促进我们对脱髓鞘疾病(如多发性硬化症)的理解 (Ms)导致轴突域紊乱，并指导治疗干预措施的发展。

The hallmark of myelinated axons is their organization into molecularly distinct domains, a pre-requisite for the rapid propagation of action potentials. The paranodal domains establish the axo-glial septate junctions (AGSJs) through interactions involving axonal Contactin-associated protein (Caspr), Contactin (Cont), and glial Neurofascin NF155. The nodal domain is organized by neuronal NF186, voltage gated sodium (Nav) channels, and Ankyrin G (AnkG), a cytoskeletal adaptor protein. We showed that loss of Caspr and NF155 results in loss of AGSJs, mislocalization of the juxtaparanodal proteins, disorganization of the paranodal axonal cytoskeleton, and degeneration of myelinated axons, but nodal organization remains relatively unaffected. Recently, we showed that Band 4.1B, a paranodal/ juxtaparanodal cytoskeletal protein, is essential for the stability of AGSJs and juxtaparanodal organization. We show here that Whirlin, another cytoskeletal protein, is required for paranodal compaction and cytoskeletal stability. In further studies, we demonstrated that loss of nodal NF186 abolished clustering of Nav channels and AnkG at the nodes, allowing the flanking paranodal AGSJs to invade the nodal space. Most importantly, we show here that in vivo loss of AnkG does not abolish node formation, but may affect nodal stability. While significant advancements have been made regarding the composition and organization of axonal domains, there still remain fundamental questions regarding how the transmembrane components at these domains interact with local cytoskeleton to initiate domain organization, and to ensure long-term stability and maintenance of axonal architecture. Based on our published and preliminary studies, our central hypothesis is that the transmembrane components and local axonal cytoskeleton are critical for axonal domain organization, their stability and function. We will use genetic, molecular and cell biological methods to determine the specific role of paranodes, nodes and their associated cytoskeletal proteins in domain stabilization and maintenance, and the efficacy of restoration of key axonal domains by re-expression of NF155 and NF186 in progressively weak adult mouse mutants. We will accomplish our goals in the following specific aims: (1) What are the consequences of loss of cytoskeletal scaffolding proteins and loss of NF155 during adult life on the maintenance and function of paranodal AGSJs? (2) What are the consequences of loss of nodal cytoskeletal proteins and loss of NF186 during adult life on the stability and function of nodes in myelinated axons? and (3) Are adult myelinated axons with extended periods of disorganized domain structure able to re-organize axonal domains to restore nerve conduction? Collectively, our studies will provide insights that bear directly on the mechanisms by which axonal domains are formed and maintained, and how these structures can be reorganized and nerve function restored. In the future, these studies will advance our understanding of how demyelinating diseases, such as multiple sclerosis (MS) lead to axonal domain disorganization and guide the development of therapeutic interventions.