Muscarinic Receptor Type 3 Regulation of Oligodendrocyte Progenitor Differentiation

3 型毒蕈碱受体对少突胶质祖细胞分化的调节

基本信息

批准号：
10316180
负责人：
Fraser James Sim
金额：
$ 34.89万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10316180
关键词：
Acetylcholine Address Adult Atrophic Auditory Brainstem Responses Axon CD4 Positive T Lymphocytes Calcium Cell Differentiation process Childhood Cholinergic Fibers Confocal Microscopy Data Demyelinating Diseases Demyelinations Dependence Development Electron Microscopy Engraftment Flow Cytometry Glutamates Goals Human Image Impairment In Vitro Kinetics Knock-out Knowledge Lead Lesion LoxP-flanked allele Lysophosphatidylcholines Mediating Molecular Multiple Sclerosis Mus Muscarinic Acetylcholine Receptor Muscarinic Agonists Muscarinics Myelin Nerve Degeneration Neurons Neurotransmitters Oligodendroglia Outcome Pathologic Permeability Pre-Clinical Model Production Receptor Signaling Regulation Reporter Rodent Role STIM1 gene Shivering Signal Transduction Source Spinal Cord Spinal Cord Lesions T-Lymphocyte Testing Therapeutic Transplantation antagonist cell type cellular development cholinergic neuron clinical translation experimental study human model improved leukodystrophy live cell microscopy macrophage mouse model myelination new therapeutic target novel oligodendrocyte progenitor optogenetics pre-clinical prevent receptor recruit regenerative therapy remyelination repaired single-cell RNA sequencing stem cells therapeutic target translational potential voltage

项目摘要

In multiple sclerosis, impaired oligodendrocyte differentiation limits remyelination and leads to axonal atrophy and neurodegeneration. Identified in several compound screens, non-selective muscarinic receptor (MR) antagonists improve myelin repair in rodents. Understanding the mechanisms by which these antagonists act and the role of muscarinic acetylcholine (ACh) signaling in OPCs is of paramount importance to the clinical translation of this approach. Importantly, our preliminary studies provide direct evidence for a role of M3R function in OPC differentiation. Using a novel combination of lentiviral M3R KD and human OPC transplantation, we found that ex vivo M3R KD increases oligodendrocyte production following engraftment in shiverer/rag2 mice, a preclinical model of childhood hypomyelination. Furthermore, we show that conditional M3R knockout increased OPC differentiation following spinal cord demyelination. While these data establish a clear functional role of M3R in OPCs, that cannot be compensated by other MR subtypes, there are gaps in knowledge regarding the potential of M3R targeting to improve functional and structural myelin repair, the role of MR-induced store-operated calcium-entry (SOCE), and the cellular sources of ACh following demyelination. The three aims in this proposal address related aspects of MR signaling to better define and characterize therapeutic targets to improve myelin repair. Aim 1: Determine the importance of M3R signaling in human OPCs following transplantation and in resident OPCs during remyelination in adult CNS. We will test the hypothesis that blocking M3R signaling in OPCs will augment and improve pre-clinical outcomes of transplant-mediated repair and enhance spontaneous remyelination. Aim 2: Determine the mechanisms by which M3R regulates differentiation of hOPCs. We will test the hypothesis that MR signaling is mediated by oscillatory SOCE. We will use optogenetic SOCE (OptoSTIM1) and conditional deletion of Stim1 to determine the functional role of SOCE in OPCs as an effector of M3R signaling and during remyelination. Aim 3: Determine the functional sources of ACh during remyelination. We will test the hypothesis that ACh synthesized by infiltrating T cells and neighboring cholinergic fibers impair OPC differentiation and remyelination. We will use ChAT-GFP reporter and ChAT floxed mice to characterize and then ablate ChAT expression from demyelinating spinal cord lesions. In summary, this project will establish the therapeutic utility of specifically targeting M3R to improve myelin repair, the mechanisms by which M3R acts to block OPC differentiation, and the functional sources of ACh during remyelination. In addition to the now-established role of activity-dependent differentiation and myelination, these studies will begin to characterize the novel concept that some neurotransmitters such as ACh act to prevent untimely OPC differentiation. Lastly, by establishing that M1/3R acts primarily via SOCE, we will provide proof-of-concept that SOCE could itself be a common mechanism by which OPC differentiation is pathologically delayed.

在多发性硬化症中，少突胶质细胞分化限制的受损降压限制并导致轴突萎缩和神经变性。在几个化合物筛选中鉴定出非选择性毒蕈碱受体（MR）拮抗剂改善啮齿动物的髓磷脂修复。了解这些拮抗剂的作用机制毒蕈碱乙酰胆碱（ACH）在OPC中的作用对于临床至关重要这种方法的翻译。重要的是，我们的初步研究为M3R的作用提供了直接的证据 OPC分化的功能。使用慢病毒M3R KD和人类OPC的新型组合移植，我们发现在植入后，离体M3R KD增加了少突胶质细胞的产生 Shiverer/rag2小鼠，一种儿童降低旋转的临床前模型。此外，我们表明了条件 M3R敲除增加脊髓脱髓鞘后的OPC分化。这些数据建立了一个 M3R在OPC中的明确功能作用，无法通过其他MR亚型补偿，其中存在差距关于M3R靶向提高功能和结构髓磷脂修复的潜力的知识， MR诱导的储藏式钙进入（SOCE）和脱髓鞘后ACH的细胞来源。这该提案中的三个目的涉及MR信号的相关方面，以更好地定义和表征治疗改善髓磷脂修复的靶标。目标1：确定M3R信号在人类OPC中的重要性成人中枢神经系统恢复后，移植和常驻OPC中的居民OPC。我们将测试阻止的假设 OPC中的M3R信号传导将增加并改善移植介导的修复和临床前结果增强自发性再髓。目标2：确定M3R调节分化的机制 HOPC。我们将检验以下假设：MR信号传导是由振荡SOCE介导的。我们将使用光遗传学 SOCE（optostim1）和STIM1的条件缺失，以确定SOCE在OPC中的功能作用 M3R信号的效应因子和在再生期间。目标3：确定ACH的功能源再髓。我们将测试通过浸润T细胞和相邻胆碱能合成的ACH的假设纤维会损害OPC的分化和再生。我们将使用CHAT-GFP记者，然后聊天小鼠表征并从脱髓鞘的脊髓病变中灌溉聊天表达。总之，这个项目将建立专门针对M3R以改善髓磷脂修复的治疗效用，该机制通过 M3R的作用可阻止OPC分化，以及在再生过程中ACH的功能来源。此外为了依靠活动依赖性分化和髓鞘的作用，这些研究将开始表征了一些新颖的概念，即某些神经递质（例如ACH）的作用以防止不合时宜的OPC 分化。最后，通过确定M1/3R主要通过SOCE起作用，我们将提供概念证明 SOCE本身可能是一种常见的机制，通过病理学延迟OPC分化。