离子通道在带髓鞘轴突中的转运机制

Action potentials (APs) propagating along axons play a central role in cell-to-cell communication in the nervous system. AP firing minimally requires sequential activation of two types of voltage-gate ion channels, Na+ (Nav) and K+ (Kv) channels. Although these channels have been extensively studied, how they are properly transported into axons-a prerequisite for AP firing-remains poorly understood. Crucial for efficient initiation and saltatory conduction of APs along myelinated axons, Nav channels are clustered at the axon initial segment and nodes of Ranvier through ankyrin-G (AnkG). Genome-wide association studies identified AnkG as a risk gene for bipolar disorder. In contrast, Kv1 channels are clustered in juxtaparanodal (JXP) regions flanking the nodes to prevent repetitive firing. A JXP cell adhesion protein, Caspr2, is involved in clustering Kv1 channels and implicated in autism spectrum disorder. Due to the lack of a suitable myelin coculture system, dynamic transport of channels and their associated proteins has never been visualized along myelinated axons. Although we previously showed that Kv1.2 axonal transport requires KIF3A/kinesin-2, how AnkG and Nav channels are transported into axons remains a mystery. In our recent studies, we found that AnkG directly binds to KIF5, raising the intriguing possibility that AnkG links Nav channels to KIF5 motors. Importantly, we established a novel myelin coculture of hippocampal neurons, allowing us to directly visualize channel transport along myelinated central axons. Based on our preliminary data, we hypothesize that crucial for proper AP firing, Nav channels are transported by KIF5 via AnkG to the AIS and nodes of Ranvier, whereas Kv1 channels are transported by KIF3 to JXP regions. Nodal and JXP complexes partially pre-assemble during transport. Using a multidisciplinary approach, including protein biochemical assays, hippocampal myelin coculture, imaging, patch-clamp recording, and conditional knockout mice, we will (1) determine interactions of AnkG, KIF5, and Nav channels, (2) elucidate dynamics of transport of Nav and Kv channels along myelinated axons, and (3) determine in vivo specificity of KIF-mediated transport of these axonal ion channels. This project will not only contribute to a better understanding of axonal transport of ion channels, but also provide novel mechanistic insights into pre-assembly of nodal and JXP complexes at the level of intracellular transport.

钠(Nav)和钾(Kv)离子通道的顺序激活和不同定位是神经冲动的启动和跳跃传导的产生机制。 Nav通道和锚蛋白-G(AnkG)聚集在有髓鞘轴突起始段和郎飞氏结， 而Kv1通道聚集在节两侧(JXP)。 这些通道的突变可导制人的神经疾病。 AnkG为躁郁症风险基因， 而JXP粘附蛋白CASPR2的突变与自闭症相关。 此项目将验证一个新的假说：Nav和其他节点蛋白由分子马达KIF5通过AnkG转运，而Kv1和JXP蛋白则由KIF3转运。　其中，节点和JXP蛋白在不同的囊泡中预组装。 将采用蛋白生化分析，神经髓鞘共培养，影像，电生理和小鼠模型，(1)检验AnkG, KIF5和其他节点蛋白的结合; (2)观察Nav和Kv1通道及相关蛋白在有髓轴突中的动态转运; (3)确定两种分子马达转运这些蛋白的体内特异性。　结果将有助于发展对一些神经和精神疾病的早期诊断和治疗。

本项目旨在探索钾离子通道（kv）和钠离子通道（nav）在带髓鞘轴突中的转运机制，按照计划执行，已经发表了2篇SCI论文。快速发放动作电位神经元能激发高达1000赫兹的动作电位（APs），在声音定位、运动协调和认知等重要脑功能中发挥关键作用。本项目研究中，我们报告了kv3电压门控钾离子通道（kv）和钠离子通道（nav）通道的协同作用是诱导和维持神经元快速发放动作电位所必需的。电压钳分析显示，kv3/nav电流比与神经元快速发放动作电位之间存在较强的相关性。单独表达kv3通道可以将30%-60%的动作电位慢发放（ss）神经元转化为快速发放动作电位神经元。相反，nav1.2或nav1.6与kv3.1或kv3.3共同表达，而不是单独表达或与kv1.2共同表达，以100%效率将神经元的慢发放转化为快发放。而且，基于RNA测序的全基因组分析显示，kv3/nav比率和kv3表达水平与动作电位的最大频率密切相关。因此，神经元动作电位的快速发放是通过适当平衡kv3和nav通道的活动来建立的，并且可以通过通道生物物理特征和定位模式进一步进行精细调节。本项目的主要结果近期发表在cell出版社的第一本开放性期刊《iScience》上（参考结题论文1）。在上述研究的基础上，为了进一步探索调节钾离子通道是否能够影响髓鞘损伤后的修复效率，我们采用培养少突胶质细胞前体细胞OPC筛选了多种分子，发现compound 3能够在体外增强OPC的增生和分化效率。进一步采用髓鞘损伤小鼠模型，证实compound 3能够在髓鞘损伤动物模型上增强损伤髓鞘的修复效率，并提高了髓鞘修复的质量（使新生髓鞘的厚度增加）。这一结果正在投稿中。在实验过程中，我们意外地发现，激活基底前脑胆碱能神经元对严重的全身性炎症-脓毒症具有显著的抗炎效应。该论文已经在Critical Care Medicine (Impact Factor 7.4) 上发表（参考结题论文2）。