Molecular Organization and Function of Paranodal Axo-glial Junctions

结旁轴突-神经胶质连接的分子组成和功能 有髓轴突被组织成分子上和功能上不同的区域，其定义由 允许快速跳跃动作电位传播的特定蛋白质复合体。旁结域 包含由轴突接触素相关蛋白1建立的轴突-胶质连接(AGJ) (Cntnap1或Caspr1)，以及Contactin(Cntn)和髓鞘/神经胶质束蛋白155(NF155)。节点域 (Ranvier的结节)由神经元神经束蛋白186(NF186)、电压门控钠(NAV)通道、 和两个细胞骨架蛋白Ankyrin G(AnkG)和βIV Spectrin(βIVSpec)。在髓鞘相关的 疾病时，这一区域结构受损，导致神经传导和肌肉减少或丧失。 软弱。我们的工作证明了上述蛋白质在组织、成熟和 轴突域的维护。我们在βIVSpec中使用了一种新的遗传策略来进行节点域重组 突变体，使结节功能得以恢复并防止运动功能障碍。重要的是，最近 进一步鉴定与严重AGJ和髓鞘缺陷相关的人CNTNAP1突变 强调AGJ蛋白在神经结构和功能中的重要性。我们已经产生了新的鼠标 人CNTNAP1三种特异性突变模型(Cys323Arg、Arg388Pro和Arg764Cys)这些单曲 Cntnap1的氨基酸变化影响其稳定性、转运和与Cntn的相互作用，并导致中断 结旁区域，并导致严重的运动障碍。虽然已经取得了重大进展 关于轴突域的组织；仍然存在与以下方面有关的根本首要问题 AGJ与神经肌肉健康：人类突变对AGJ的形成和生理有什么影响 轴突的特性？轴突域的进行性破坏如何影响轴突及其肌肉 令人兴奋？神经活性下降如何改变神经肌肉接头(NMJ)并导致肌肉 萎缩？在这个修订的应用程序中，我们将使用人类CNTNAP1突变和轴突的小鼠模型 领域拆分和重组模型，以剖析组织基础的机制， 轴突域的维护和修复。我们的具体目标是解决知识方面的三个关键差距：(1) 小鼠Cntnap1人类突变对其结构/功能、AGJ形成和轴突有何影响 结构域组织；以及这些Cntnap1突变是否导致功能表型的丧失和/或获得？(2)如何 NF186/AnkG突变体中轴突结构域的时间轴与神经功能减退有关吗 传导导致进行性运动障碍？以及(3)进行节点域重组和恢复 Biv Spectrin突变体的神经传导是否能预防/逆转肌肉萎缩？总体而言，我们的研究将 极大地提高了我们对人类AGJ和髓鞘相关病理如何影响轴突和 神经肌肉健康；并可能为功能恢复的时间表和潜力提供关键的见解 为未来对这些破坏性神经病理进行治疗干预的途径。

Molecular Organization and Function of Paranodal Axo-glial Junctions Myelinated axons are organized into molecularly and functionally distinct domains defined by the presence of specific protein complexes that allow rapid saltatory action potential propagation. The paranodal domain contains the axo-glial junctions (AGJs) that are established by axonal Contactin-associated protein 1 (Cntnap1 or Caspr1), and Contactin (Cntn) and myelin/glial Neurofascin 155 (NF155). The nodal domains (nodes of Ranvier) are organized by neuronal Neurofascin 186 (NF186), voltage-gated sodium (Nav) channels, and two cytoskeletal scaffolding proteins Ankyrin G (AnkG) and βIV Spectrin (βIVSpec). In myelin-related diseases, this domain structure is compromised, leading to a decrease or loss of nerve conduction and muscle weakness. Our work demonstrated specific functions of the above proteins in the organization, maturation and maintenance of axonal domains. We used a novel genetic strategy for nodal domain reorganization in βIVSpec mutants, which allowed restoration of nodal function and prevented motor dysfunction. Importantly, recent identification of human CNTNAP1 mutations that are associated with severe AGJ and myelin defects further highlight the importance of AGJ proteins in nerve structure and function. We have generated new mouse models of three specific human CNTNAP1 mutations (Cys323Arg, Arg388Pro and Arg764Cys). These single amino acid changes in Cntnap1 affect its stability, transport and interactions with Cntn, and lead to disruption of the paranodal domains, and cause severe motor disability. While significant advances have been made regarding the organization of axonal domains; there still remain fundamental overarching questions related to AGJs and neuromuscular health: What impact do human mutations have on AGJ formation and physiological properties of axons? How does progressive disruption of axonal domains affect axons and the muscles they innervate? How does declining nerve activity change neuromuscular junctions (NMJs) and lead to muscle atrophy? In this revised application, we will use mouse models of human CNTNAP1 mutations and axonal domain disorganization and reorganization models to dissect the mechanisms that underlie the organization, maintenance and restoration of axonal domains. Our Specific Aims address three key gaps in knowledge: (1) What impact do human mutations in mouse Cntnap1 have on its structure/function, AGJ formation and axonal domain organization; and do these Cntnap1 mutations cause loss and/or gain of function phenotypes? (2) How does the timeline of axonal domain disorganization in NF186/AnkG mutants correlate with decline in nerve conduction leading to progressive motor disability? And (3) Does nodal domain reorganization and restoration of nerve conduction in bIV Spectrin mutants prevent/reverse muscle atrophy? Collectively, our studies will significantly advance our understanding of how human AGJ- and myelin-related pathologies impact axonal and neuromuscular health; and may offer critical insights into the timelines of functional restoration and potential avenues for future therapeutic interventions for these devastating neuropathologies.