Molecular Organization and Function of Paranodal Axo-Glial Junctions

描述（由申请人提供）：有髓鞘轴突的标志是它们组织成分子上不同的结构域，这是快速传播作用的先决条件 潜力结旁结构域通过涉及轴突接触蛋白相关蛋白（Caspr）、接触蛋白（Cont）和神经胶质神经成束蛋白NF 155的相互作用建立轴-神经胶质分隔连接（AGSJ）。节点域由神经元NF 186、电压门控钠（Nav）通道和锚蛋白G（AnkG）（一种细胞骨架衔接蛋白）组织。我们发现，Caspr和NF 155的丢失导致AGSJS的丢失，araparanodal蛋白的错误定位，结旁轴突细胞骨架的解体和有髓轴突的变性，但结的组织仍然相对不受影响。最近，我们发现带4.1B，一个paranodal/acutaparanodal细胞骨架蛋白，是必不可少的稳定AGSJS和acutaparanodal组织。我们在这里表明，Whirlin，另一种细胞骨架蛋白，是所需的旁结压实和细胞骨架的稳定性。在进一步的研究中，我们证明了节点NF 186的缺失废除了节点处的Nav通道和AnkG的聚集，允许侧翼的结旁AGSJ侵入节点空间。最重要的是，我们在这里表明，在体内损失的AnkG不会取消节点的形成，但可能会影响节点的稳定性。虽然关于轴突结构域的组成和组织已经取得了重大进展，但仍然存在关于这些结构域的跨膜组分如何与局部细胞骨架相互作用以启动结构域组织并确保轴突结构的长期稳定性和维持的基本问题。基于我们已发表的和初步的研究，我们的中心假设是，跨膜组件和局部轴突细胞骨架的轴突域组织，其稳定性和功能是至关重要的。我们将使用遗传，分子和细胞生物学方法，以确定特定的作用paranodes，节点及其相关的细胞骨架蛋白在域的稳定和维护，和恢复的关键轴突结构域的效力，通过重新表达NF 155和NF 186在逐渐变弱的成年小鼠突变体。我们将在以下具体目标中实现我们的目标：（1）在成年期细胞骨架支架蛋白和NF 155的丢失对结旁AGSJs的维持和功能有什么后果？(2)在成年期，结节细胞骨架蛋白和NF 186的丢失对有髓轴突中的结节的稳定性和功能有什么影响？以及（3）成年有髓轴突是否具有长时间的无序结构域结构，能够重新组织轴突结构域以恢复神经传导？总的来说，我们的研究将提供直接影响轴突结构域形成和维持的机制，以及这些结构如何重组和神经功能恢复的见解。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（MS））如何导致轴突结构紊乱的理解，并指导治疗干预措施的发展。

DESCRIPTION (provided by applicant): 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.