Lipidomic mapping of the cellular anatomy of brain endocannabinoid metabolism

描述（由申请人提供）：基于质谱（MS）的代谢组学方法的最新发展有助于将脂质分子确定为神经系统中信号转导的关键介质。例如，内源性大麻素（eCB）2-花生四烯酸甘油（2-AG）是连接内源性大麻素系统（ECS）和大脑中促炎性二十烷酸产生的关键代谢中心。以这种方式，2-AG信号传导途径调节疼痛、焦虑、神经炎症和神经变性，并因此吸引了相当大的用于治疗神经系统疾病的药学兴趣。然而，大脑2-AG的整体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以提供靶向特定脑2-AG储存的手段，以充分利用具有可接受的安全性特征的基于2-AG的治疗剂的开发。我们假设，不同的2-AG生物合成和降解酶的抑制将调节特定脑细胞区室中的ECS/类花生酸信号传导网络，以产生抗炎和神经保护作用，同时限制全局ECS激动或拮抗作用所观察到的有害后果。该提案描述了利用新生成的工具选择性地破坏2-AG代谢酶的实验，以及基于MS的脂质组学的力量，以定义用于开发基于eCB的疗法的新酶靶点。首先，我们将绘制小鼠大脑中2-AG降解途径的细胞解剖图。具体而言，我们将使用靶向和非靶向脂质组学分析来检查神经元与神经胶质中主要脑2-AG水解酶（MAGL，ABHD 6和ABHD 12）的区室化，以及表征这些酶基因缺失后的细胞类型特异性代谢改变。其次，我们将确定是否抑制二酰基甘油脂肪酶（DAGL）a和B，两个主要的2-AG生物合成酶在大脑中，可以选择性地调节大脑ECS/类花生酸网络。通过利用DAGLa或B基因敲除小鼠，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改变eCB/类花生酸的库基础和神经炎症的实验范例。最后，我们将进行基于MS的代谢成像的解剖分布的2-AG在大脑中。我们将使用一种新的纳米结构引发剂质谱（NIMS）成像平台来表征基础和抑制2-AG代谢酶后不同脑区2-AG水平的分布。总之，该项目将推动应用尖端脂质组学技术的前沿，以表征ECS/类花生酸信号网络的细胞和解剖学区室化及其在脑细胞间通讯中的作用，并帮助开发用于治疗神经系统疾病的新型基于eCB的疗法。

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.