NMDA受体在少突胶质前体细胞分化和髓鞘化/再髓鞘化中的作用

Myelin is critical for the normal function of the central nervous system (CNS). Oligodendrocyte precursor cell (OPC) is the cell source of this structure. Demyelination exists in a wide variety of CNS diseases shuch as MS/PVL that are currently no cure. During development, OPCs originate from restricted zone and migrate for a long distance to populate throughout the white matter, where they differentiate into mature oligodendrocytes (OLs) and myelinate axons in the CNS. Remyelination in adult after demyelination is, to a large extend, a recapitulation of this process. Thus, a thorough understanding of the molecular mechanisms that control OPC migration, differentiation, and myelination would be of great significance. Under the support of Young Scholar Project of NSFC, we have used multiple experimental systems including Boyden transwell, matrix-gel cell mass, SVZ tissue explants, and brain slice cultures to demonstrate that NMDA stimulation promotes the migration of OPCs. We also revealed that this effect was mediated by calcium dependent NMDA receptor (NMDAR)-Tiam1 interaction and the consequent Rac1-ERK1/2 activation signal pathway. This study implicates that NMDAR might play a critical role in direction of OPCs migration during development. And the applicant has published three articles as first author in Glia, JBC, and Mol Endocrinol. In spite of the above findings, however, the question whether NMDAR also involved in regulation of OPC differentiation and myelination/remyelination still remains to be elucidated. This is a most attractive issue as differentiation of OPCs into mature OLs is a prerequisite for both developmental myelination and adult remyelination and impairment of this process has been suggested to be a major reason for remyelination failure. We then set out to test it and our preliminary data showed that NMDARs were expressed in OPCs of all developmental stages, and NMDA specifically promoted the differentiation of OPC from O1+ immature to MBP+ mature OLs. It was also found that NMDAR activation increased the number of myelin segments in OPC-DRG co-cultures. In this proposal, we aim to confirm the role and reveal the underlying mechanism of NMDAR in OPC differentiation and myelination by using of both pharmacological tools and lentivirus-mediated gene knock down. Particularly, we will further test the in vivo function of NMDAR during myelination and remyelination in both OL linage cell specific NR1 knock out and knock in transgenic mice. Lysolecithin and cuprizone induced demyalintion in brain slice cultures and intact animals respectively will be introduced. And various techniques including immunostaining, confocal imaging, immunobloting, LFB staining and electromicroscopy will be used to examine the OPC differentiation and the extent of myelination/remyelination. This study may highlight the role of NMDAR in OPC differentiation and myelination/remyelination, and implicate a novel therapeutic target for demyelinating disease.

髓鞘结构是维持中枢神经系统正常功能重要保证。发育过程中少突胶质前体细胞(OPC)自其局限的发生区域迁移遍布至全脑白质，并分化为成熟的少突胶质细胞(OL)包裹轴突形成髓鞘。成体脱髓鞘后再髓鞘化很大程度上是这一发育事件的重演，充分揭示OPC迁移、分化和髓鞘化过程的分子调控机制对于深入认识脱髓鞘疾病的发病机理，寻找有效的治疗策略具有重要意义。在青年基金的资助下，申请者首次发现 NMDA受体(NR)通过Tiam1分子介导的信号转导对OPC迁移起到促进作用，以第一作者在Glia, JBC, Mol Endocrinol杂志发表论文。然而，NR是否在OPC的分化和髓鞘化/再髓鞘化过程中也起到关键作用尚待阐明。本项目将以OPC纯化培养、OPC-DRG共培养、脑片培养、铜腙脱髓鞘等为实验模型，通过基因敲减、特别是OL特异性转基因小鼠等方法，利用激光共聚焦、LBF染色，电镜等检测手段，深入阐明这一科学问题。

髓鞘结构是维持中枢神经系统（CNS）正常功能重要保证，脱髓鞘性神经疾病广泛存在且目前尚无有效治疗。发育过程中少突胶质前体细胞(OPC)自其局限的发生区域迁移遍布至全脑白质，并分化为成熟的少突胶质细胞(OL)包裹轴突形成髓鞘。成体脱髓鞘后再髓鞘化很大程度上是这一发育事件的重演，充分揭示OPC迁移、分化和髓鞘化过程的分子调控机制对于深入认识脱髓鞘疾病的发病机理，寻找有效的治疗策略具有重要意义。NMDA受体是一种及其重要的离子型谷氨酸受体，其在中枢神经神经系统中的重要性众所周知，一般认为它主要在神经元上发挥作用，而其在胶质细胞上功能的却鲜有所知。近年来的研究发现在成熟少突胶质细胞上表达功能性的NMDA受体并且可能介导了缺血条件下的髓鞘损伤，然而人们对它的正常生理功能却知之甚少。项目负责人在前一个青年基金的资助下，发现 NMDA受体可以通过Tiam1分子介导的信号转导对OPC迁移起到促进作用。而在本青年-面上连续项目的资助下，我们则进一步展开深入研究，探索了该受体是否在OPC的分化和髓鞘化/再髓鞘化过程中也起到关键作用。本项目通过利用包括OPC纯化培养、OPC-DRG共培养、脑片培养、铜腙脱髓鞘等在内的多种实验模型，采取慢病毒介导的基因沉默等实验技术，以激光共聚焦、LBF染色，电镜等等为检测观察手段，获得了以下的研究发现： 1.少突胶质前体细胞有功能性NMDA受体的表达；2.NMDA刺激能显著促进OPC的分化，包括增加其细胞突起的分支复杂程度和髓鞘相关蛋白的表达,该作用通过NMDA受体实现；3.NMDA受体介导的促OPC分化作用主要发生在细胞成熟的后期阶段，即由O1+的非成熟OL阶段向MBP+阳性的成熟OL阶段的转换； 4.NMDA受体是通过激活mTOR信号通路从而实现其促OPC分化作用的；5.NMDA受体激活能促进DRG-OPC共培养体系中的体外髓鞘化；6.阻断NMDA受体活性能抑制铜腙诱导脱髓鞘后的体内自发再髓鞘化。我们的以上研究结果揭示了NMDA受体参与OPC分化和髓鞘形成与再生的调节，大大拓展了人们对NMDA受体在神经系统中除神经元以外的细胞中作用的认识。通过本项目的实施，我们完成了既定的研究目标，现已在GLIA（IF=5.99）杂志上发表了三篇标注本项目基金资助的原始论著，项目负责人进一步获得面上项目一项，获得上海市科技进步二等奖一项，并入选了上海市青年科技启明。