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

ABHD6 和 MAGL 对 CB1R 上 2-AG 活性的差异控制

• 批准号: 10664172
• 负责人: Nephi Stella
• 金额: $ 19.44万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10664172
• 关键词: 2-arachidonylglycerol; ABHD6 gene; Address; Adverse effects; Affect; Agonist; Applications Grants; BDKRB2 gene; Biological Models; Biosensor; Bradykinin; Brain; CNR1 gene; Cannabinoids; Cell Line; Cells; Chronic; Corpus striatum structure; Development; Drug Addiction; Electrophysiology (science); Endocannabinoids; Enzymes; Fluorescence Microscopy; Goals; Hydrolase; MAGL inhibitor; Maps; Measures; Mediating; Molecular; Monoacylglycerol Lipases; Mus; N-Methylaspartate; Neurons; Presynaptic Terminals; Production; Property; Receptor Signaling; Research; Role; Signal Transduction; Slice; Stimulus; Synaptic Transmission; System; Testing; Therapeutic; Time; Transfection; Veratridine; Work; abuse liability; cell type; endocannabinoid signaling; lipoprotein lipase; nervous system disorder; novel; novel therapeutics; pharmacologic; postsynaptic; presynaptic; receptor; sensor; side effect; small molecule; spatiotemporal; tool; two photon microscopy

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.

总结 脑中最丰富的内源性大麻素（eCB）2-花生四烯酸甘油（2-AG）可被两种 酶：单酰基甘油脂肪酶（MAGL）和α/β水解酶结构域6（ABHD 6），它们在其功能上不同， 水解活性和亚细胞定位（分别为突触前和突触后）。因此，委员会认为， 每种酶的选择性抑制导致2-AG-CB 1 R信号传导在不同的时空增强， 大脑，以及潜在的协同治疗益处。 我们最近收集的结果表明，刺激增加2-AG生产是可靠的测量，使用 GRABeCB2.0是最近开发的2-AG传感器。值得注意的是，代谢型受体介导的2-AG增加 由ABHD 6控制，而2-AG的离子型受体依赖性增加不受ABHD 6控制。 这些结果提出了一个问题，如何MAGL控制刺激依赖的2-AG生产？ 证明CB 1 R信号传导的2-AG产生和活性的受体依赖性增加， ABHD 6和MAGL的差异控制将提供额外水平的机械区别 在这些eCB水解酶之间。为了加深我们对ABHD各自作用的理解6， MAGL在控制大脑中的2-AG-CB 1 R信号传导方面，我们发起了一项努力，现在已经成功地验证了 GRABeCB 2.0在培养的神经细胞中的药理学特征，并鉴定了几种增加2-AG的刺激物 生产基于这一前提，我们提出在小鼠神经元中解决以下两个问题， 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：哪些刺激增加神经元中2-AG的产生和GRABeCB 2.0信号？ 目的2：ABHD 6和MAGL如何不同地控制2-AG-CB 1 R的刺激依赖性增加 神经元中的信号 完成这项新的R21赠款提案中概述的工作将全面了解 ABHD 6和MAGL在控制脑中2-AG-CB 1 R信号传导中的各自作用。我们的长期目标是 增加我们对ABHD 6和ABHD 7在分子、细胞和系统水平差异的理解， MAGL控制大脑中的eCB信号传导，这是一项有助于开发新疗法的工作， 滥用的可能性和由经典大麻素激动剂产生的副作用。