Microglial impact on remyelination

项目摘要 增强髓鞘再生是在多发性硬化症患者脱髓鞘后恢复脑功能的关键策略。 硬化症（MS）患者;然而，尽管共同努力，刺激MS脑髓鞘再生的能力， 仍然难以捉摸虽然促进少突胶质细胞前体分化的信号通路有 已经确定，正在调查的实验环境不复制限制机制， MS特异性炎性CNS损伤后髓鞘再生。目前的建议是建立在我们的新模型上的 使用从MS患者产生的致病性重组抗体（rAbs）的脱髓鞘/髓鞘再生。 髓鞘特异性MS rAbs在体内和离体指导补体介导的脱髓鞘，所有这些都 与小胶质细胞活化相关的自发修复。然而，脱髓鞘的外植体， 持续暴露于髓鞘特异性MS rAb不能激活小胶质细胞，并且少突胶质细胞的成熟被抑制。 压抑类似地，在rAb介导的脱髓鞘阻断后， 少突胶质细胞成熟阻止活跃的髓鞘再生。使用单细胞RNASeq（scRNASeq）对 从髓鞘再生外植体分离的小胶质细胞，我们确定了转录不同的小胶质细胞亚群， 与成功或失败的髓鞘再生有关。因此，我们假设小胶质细胞信号是至关重要的， 在从早期髓鞘形成到活跃髓鞘形成的过渡过程中， 少突胶质细胞和髓鞘特异性MS自身抗体调节这些信号以阻止髓鞘再生。测试 我们的假设，我们提出了三个互补的具体目标。在目标1中，我们将评估小胶质细胞和 在MS rAb介导的脱髓鞘的体内模型中的少突胶质细胞反应，并比较这些反应 与毒素介导的脱髓鞘中所见的相似。丘脑内或胼胝体注射髓鞘特异性MS rAb + HC将与药理学小胶质细胞耗竭和慢性 施用MS rAb以验证小胶质细胞应答对完整神经系统中髓鞘再生的影响。 系统在溶血卵磷脂诱导的脱髓鞘后将进行类似的研究， 小胶质细胞活化和髓鞘再生的不同时间过程。在目标2中，我们将研究 脱髓鞘、小胶质细胞反应和少突胶质细胞再生的原位活体成像 皮质脱髓鞘这种对髓鞘丢失、小胶质细胞活化和髓鞘再生的实时分析将是 与铜腙介导的脱髓鞘相比。最后，在目标3中，我们将研究 使用髓鞘脱髓鞘的离体小脑切片，小胶质细胞影响髓鞘再生的机制- 特异性rAb加人补体（HC）。我们将重点研究几个小胶质细胞基因的作用， 通过scRNASeq鉴定的预期促进或损害髓鞘再生的基因。外观正常的白色物质 将评估具有不同程度脱髓鞘和髓鞘再生的MS病变组织，以确定 功能重要的小胶质细胞亚群的丰度和定位。这些研究的结果将 提供了新的机制控制髓鞘再生炎症损伤后的见解。此外该 所获得的知识可以确定新的治疗方法， 修复.

PROJECT SUMMARY Enhancing remyelination is a critical strategy for restoring brain function after demyelination in multiple sclerosis (MS) patients; however, despite concerted efforts, the ability to stimulate remyelination in MS brain has remained elusive. While signaling pathways that promote oligodendrocyte precursor differentiation have been identified, the experimental milieux under investigation do not replicate the mechanisms limiting remyelination following MS-specific inflammatory CNS injuries. The current proposal builds on our new models of demyelination/remyelination using pathogenic recombinant antibodies (rAbs) generated from MS patients. Myelin-specific MS rAbs direct complement-mediated demyelination in vivo and ex vivo, all of which spontaneously repair in association with microglial activation. However, demyelinated explants that are continuously exposed to myelin-specific MS rAb fail to activate microglia, and oligodendrocyte maturation is inhibited. Similarly, targeted depletion of microglia following rAb-mediated demyelination blocks oligodendrocyte maturation preventing active remyelination. Using single cell RNASeq (scRNASeq) on microglia isolated from remyelinating explants, we identified transcriptionally distinct microglial subsets that are associated with successful or failed remyelination. Hence, we hypothesize that microglial signals are critical for oligodendrocyte responses during the transition from early myelinating to actively myelinating oligodendrocyte, and myelin-specific MS autoantibody modulates these signals to arrest remyelination. To test our hypothesis, we propose three complementary specific aims. In Aim 1, we will evaluate microglial and oligodendrocyte responses in in vivo models of MS rAb-mediated demyelination and compare those responses to those seen in toxin-mediated demyelination. Intrathalamic or corpus callosum injection of myelin-specific MS rAb plus HC will be performed in conjunction with pharmacologic microglial depletion and chronic administration of MS rAb to validate the impact of microglial responses on remyelination in the intact nervous system. Comparable studies will be done following lysolecithin-induced demyelination, which has a very different time course of microglial activation and remyelination. In Aim 2, we will study the dynamics of demyelination, microglial responses and oligodendrocyte regeneration in situ using intravital imaging following cortical demyelination. This real-time analysis of myelin loss, microglial activation and remyelination will be compared to that seen following cuprizone-mediated demyelination. Finally, in Aim 3, we will investigate the mechanisms by which microglia impact remyelination using ex vivo cerebellar slices demyelinated with myelin- specific rAb plus human complement (HC). We will focus on investigating the role of several microglial genes identified by scRNASeq that are expected to promote or impair remyelination. Normal appearing white matter and MS lesion tissue with varying degrees of demyelination and remyelination will be evaluated to determine the abundance and localization of functionally-important microglial subsets. The results of these studies will provide insights into novel mechanisms controlling remyelination after inflammatory injury. In addition, the knowledge gained may identify novel therapeutic approaches that will result in clinically-meaningful myelin repair.