Lipidomic mapping of the cellular anatomy of brain endocannabinoid metabolism

描述(申请人提供)：基于质谱学(MS)的代谢组学方法的最新发展正在帮助建立脂分子作为神经系统中信号转导的关键媒介。例如，内源性大麻素(ECB)2-花生四烯基甘油(2-AG)是连接内源性大麻素系统(ECS)和大脑中促炎二十烷类物质的关键代谢中枢。通过这种方式，2-AG信号通路调节疼痛、焦虑、神经炎症和神经变性，因此吸引了相当大的药物兴趣用于神经系统疾病的治疗。然而，脑部2-AG的全球增加也会导致一系列不良副作用。2-AG代谢酶的多样性和差异性分布可能为针对特定的脑2-AG商店提供一种手段，以充分利用基于2-AG的治疗药物的开发，并具有可接受的安全性。我们假设，抑制不同的2-AG生物合成和降解酶将调节特定脑细胞间隔中的ECS/二十烷类信号网络，以产生抗炎和神经保护作用，同时限制全球ECS激动剂或拮抗剂所观察到的有害后果。这项建议描述了利用新产生的工具选择性地破坏2-AG代谢酶的实验，以及基于MS的脂质组学的能力，以定义开发基于ECB的治疗的新的酶靶点。首先，我们将绘制小鼠大脑中2-AG降解途径的细胞解剖图。具体地说，我们将使用靶向和非靶向的脂肪组谱来检查主要的脑2-AG水解酶(MAGL、ABHD6和ABHD12)在神经元和胶质细胞中的区划，以及表征这些酶基因缺失后细胞类型特定的代谢变化。其次，我们将确定大脑中两种主要的2-AG生物合成酶--二酰基甘油脂肪酶(DAGL)a和b的抑制是否可以选择性地调节大脑ECS/二十聚糖网络。通过利用DAGLa或b基因敲除小鼠的优势，我们将绘制这两种酶在神经系统中的细胞分布图，并评估它们的失活如何在基础上和在神经炎症的实验范例中改变ECB/二十烷类化合物的池。最后，我们将对2-AG在脑内的解剖分布进行基于MS的代谢成像。我们将使用一种新的纳米结构-引发质谱学(NIMS)成像平台来表征2-AG代谢酶抑制后不同脑区的2-AG水平的分布。综上所述，该项目将推动应用尖端脂质组学技术的前沿，以确定ECS/二十烷类信号网络的细胞和解剖分区及其在脑细胞间通信中的作用，并有助于开发基于ECB的治疗神经系统疾病的新疗法。

DESCRIPTION (provided by applicant): Recent developments in mass spectrometry (MS)-based metabolomic approaches are helping to establish lipid molecules as critical mediators of signal transduction in the nervous system. The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG), for example, serves as a key metabolic hub connecting the endogenous cannabinoid system (ECS) and pro-inflammatory eicosanoid production in the brain. In this manner, 2-AG signaling pathways modulate pain, anxiety, neuroinflammation, and neurodegeneration and have thus attracted considerable pharmaceutical interest for the treatment of nervous system diseases. Global increases in brain 2-AG, however, can also cause a range of undesirable side-effects. The multiplicity and differential distribution of 2-AG metabolic enzymes may provide a means to target specific brain 2-AG stores to fully capitalize on the development of 2-AG-based therapeutics with acceptable safety profiles. We hypothesize that the inhibition of distinct 2-AG biosynthetic and degradative enzymes will modulate the ECS/eicosanoid signaling network in specific brain cellular compartments to produce anti-inflammatory and neuroprotective effects, while limiting detrimental consequences observed with global ECS agonism or antagonism. This proposal describes experiments that leverage newly generated tools to selectively disrupt 2-AG metabolic enzymes together with the power of MS-based lipidomics to define novel enzymatic targets for the development of eCB-based therapeutics. First, we will map the cellular anatomy of 2-AG degradation pathways in the mouse brain. Specifically, we will use targeted and untargeted lipidomic profiling to examine the compartmentalization of the major brain 2-AG hydrolases (MAGL, ABHD6 and ABHD12) in neurons vs. glia, as well as to characterize cell type-specific metabolic alterations following genetic deletion of these enzymes. Second we will determine whether the inhibition of diacylglycerol lipases (DAGL) a and b, the two main 2-AG biosynthetic enzymes in the brain, can selectively modulate the brain ECS/eicosanoid network. By taking advantage of DAGLa or b knockout mice, we will map the cellular distribution of these two enzymes in the nervous system and assess how their inactivation alters pools of eCBs/eicosanoids basally and in experimental paradigms of neuroinflammation. Finally, we will carry out MS-based metabolic imaging of the anatomical distribution of 2-AG in the brain. We will use a novel Nanostructure-initiator Mass Spectrometry (NIMS) imaging platform to characterize the distribution of 2-AG levels in different brain regions basally and after inhibitio of 2-AG metabolic enzymes. In summary, this project will push the frontiers of applying cutting- edge lipidomic technologies to characterize the cellular and anatomical compartmentalization of the ECS/eicosanoid signaling network and its role in brain intercellular communication, as well as aid the development of novel eCB-based therapeutics for the treatment of nervous system diseases.