Investigating the role of cannabinoid receptors in oligodendrocyte development

项目摘要 髓鞘是一种保护鞘，它包裹着神经元轴突，促进快速跳跃式传导， 维持轴突的完整性并提供代谢支持它是由专门的神经胶质细胞制造的， 中枢神经系统（CNS）中的少突胶质细胞（OL）。髓磷脂的重要性在 神经系统疾病，如多发性硬化症（MS），其中髓鞘的破坏可导致不适当的 神经元的功能，并可能最终导致轴突变性沿着其他症状。而 因此，髓鞘的重要性是显而易见的，目前没有治疗选择来预防或帮助髓鞘再生， 部分原因是髓鞘形成的分子机制尚未完全了解。但 内源性大麻素系统（ECS）作为治疗多种疾病的潜在靶点， 神经系统疾病，包括MS。然而，ECS之间的关系还没有明确的认识， 和OL发育或髓鞘形成。旨在了解驱动OL的分子机制 我们的实验室对培养的大鼠原代OLs进行了蛋白质组学分析， 通过遗传方法增加髓鞘形成。在一系列蛋白质中， 具有增加的髓鞘的OL是两种ECS酶，FAAH 1和DAGL-1。这一增长发生在OL期间 前体细胞（OPC）分化，表明ECS可能在OL发展的这个阶段发挥作用。这 模型得到了我在激活ECS的斑马鱼中的初步体内研究的支持。对于这项建议，我的目标是 明确CB1和CB2在OL发育和髓鞘形成中的作用， 受体操作，使用CRISPR-Cas9介导的基因组编辑进行仔细的基因组分析， 多维受体激活和实时成像。在目标1中，我将使用一系列药理学和 遗传学方法来剖析CB1和CB2在OL发育和髓鞘形成的各个阶段中的作用。 在目标2中，我将通过钙成像研究CB1激活的下游信号作用，使用 新的光笼化配体，其在遗传上靶向于OL谱系细胞。

Project Summary Myelin is the protective sheath that wraps around neuronal axons to facilitate fast, saltatory conduction, maintain axon integrity, and provide metabolic support. It is made by specialized glial cells called oligodendrocytes (OL) in the central nervous system (CNS). The importance of myelin is highlighted in neurological diseases such as multiple sclerosis (MS), where disruption of myelin can result in improper neuronal function and may ultimately lead to axonal degeneration along with other symptoms. While the importance of myelin is thus evident, there are currently no treatment options to prevent or aid in remyelination, in part because the molecular mechanisms of myelin formation are not fully understood. However, the endocannabinoid system (ECS) has gained increasing interest as a potential target to treat several neurological diseases, including MS. Yet there is no clear understanding in the relationship between the ECS and OL development or myelination. Aiming to understand of the molecular mechanisms that drive OL development and myelination, our lab conducted a proteomic analysis of cultured rat primary OLs in which myelination was increased by genetic methods. Among the list of proteins that were significantly upregulated in OLs with increased myelin were two ECS enzymes, FAAH1 and DAGL-. This increase occurred during OL precursor (OPC) differentiation, suggesting the ECS may play a role at this stage of OL development. This model is supported by my preliminary in vivo studies in zebrafish activating the ECS. For this proposal, I aim to define the role of CB1 and CB2 in OL development and myelination through continued pharmacological receptor manipulation, careful genomic analysis using CRISPR-Cas9 mediated genome editing, and precise multidimensional receptor activation and live imaging. In Aim 1, I will use a range of pharmacological and genetic approaches to dissect the roles of CB1 and CB2 in various stages of OL development and myelination. In Aim 2, I will investigate the downstream signaling effects of CB1 activation through calcium imaging, using a novel photocaged ligand that is genetically targeted to OL lineage cells.