Differential control of 2-AG’s activity at CB1R by ABHD6 and MAGL

摘要 大脑中含量最丰富的内源性大麻素(ECB)--2-花生四烯酸甘油(2-AG)被两种 酶：单甘油脂肪酶(MAGL)和α/β水解酶结构域-包含6个不同的 水解性和亚细胞定位(分别为突触前和突触后)。因此， 选择性抑制每种酶导致2-AG-CB1R信号的不同时空增强 大脑，以及潜在的协同治疗益处。 我们最近收集的结果表明，2-AG产量的刺激增加是通过使用 GRABeCB2.0，一种最近开发的2-AG传感器。值得注意的是，代谢性受体介导了2-AG的增加 是由ABHD6控制的，而2-AG的离子亲和性受体依赖性增加不受ABHD6控制。 这些结果提出了MAGL如何控制依赖刺激的2-AG生产的问题？ 证明受体依赖的2-AG的产生和CB1R信号活性的增加是 由ABHD6和MAGL控制的差异化将提供额外的机械区分级别 在这些欧洲央行水解酶之间。为了加深我们对ABHD6和ABHD6各自作用的理解 MAGL在控制大脑中的2-AG-CB1R信号转导方面，我们发起了一项努力，现在已经成功地验证了 GRABeCB2.0在培养神经细胞中的药理学特征及几种可增加2-AG的刺激物 制作。基于这一前提，我们提出了以下两个关于小鼠神经元的问题 原代培养和纹状体切片使用活细胞荧光显微镜，双光子显微镜和 电生理学： 目的1：哪些刺激能增加神经元中2-AG的产生和GRABeCB2.0信号？ 目的2：ABHD6和MAGL如何不同地控制2-AG-CB1R中刺激依赖性的增加 神经元中的信号？ 完成这项新的R21拨款提案中概述的工作将提供对 ABHD6和MAGL分别在脑内控制2-AG-CB1R信号转导中的作用。我们的长期目标是 为了增加我们对ABHD6和ABHD6和ABHD6在分子、细胞和系统水平上的差异的理解 MAGL控制大脑中的ECB信号，这是一项有助于开发新疗法的工作，减少了 经典大麻素激动剂可能产生的滥用和不良影响。

Summary The most abundant endocannabinoid (eCB) in brain, 2-arachidonoyl glycerol (2-AG), is inactivated by two enzymes: monoacylglycerol lipase (MAGL) and α/β hydrolase domain-contain 6 (ABHD6) that differ in their hydrolyzing activities and subcellular localization (presynaptic and postsynaptic, respectively). Accordingly, selective inhibition of each enzyme results in different spatiotemporal enhancement of 2-AG-CB1R signaling in the brain, and potentially synergistic therapeutic benefits. We recently gathered results showing that stimulated increase in 2-AG production is reliably measured using GRABeCB2.0, a recently developed 2-AG sensor. Remarkably, metabotropic receptor mediated increase in 2-AG is controlled by ABHD6, whereas ionotropic receptor-dependent increase in 2-AG is not controlled by ABHD6. These results raise the question of how MAGL controls stimulation-dependent 2-AG production? Demonstrating that receptor-dependent increases in 2-AG production and activity at CB1R signaling are differentially controlled by ABHD6 and MAGL would provide an additional level of mechanistic distinction between these eCB-hydrolyzing enzymes. To increase our understanding of the respective roles of ABHD6 and MAGL in controlling 2-AG-CB1R signaling in brain, we initiated an effort and have now successfully validated the GRABeCB2.0 pharmacological profile in neural cells in culture and identified several stimuli that increase 2-AG production. Based on this premise, we propose to address the following two questions in mouse neurons in primary culture and striatal slices using live-cell fluorescence microscopy, two-photon microscopy and electrophysiology: Aim 1: Which stimuli increase 2-AG production and GRABeCB2.0 signal in neurons? Aim 2: How do ABHD6 and MAGL differentially control stimuli-dependent increases in 2-AG-CB1R signaling in neurons? Completion of the work outlined in this new R21 grant proposal will provide a comprehensive understanding of the respective role of ABHD6 and MAGL in controlling 2-AG-CB1R signaling in the brain. Our long-term goal is to increase our understanding of the molecular, cellular, and system’s level differences by which ABHD6 and MAGL control eCB signaling in brain, a body of work that will help develop novel therapeutics with reduced potential for abuse and adverse effects produced by classic cannabinoid agonists.