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

基本信息

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
项目类别：
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

项目摘要

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.

概括大脑中最丰富的内源性大麻素 (eCB) 2-花生四烯酰甘油 (2-AG) 可被两种物质灭活酶：单酰基甘油脂肪酶 (MAGL) 和 α/β 水解酶结构域包含 6 (ABHD6)，其不同之处在于水解活性和亚细胞定位（分别为突触前和突触后）。选择性抑制每种酶会导致 2-AG-CB1R 信号传导在不同时空上增强大脑，以及潜在的协同治疗益处。我们最近收集的结果表明，2-AG 产量的刺激增加可以通过以下方法可靠地测量： GRABeCB2.0，最近开发的 2-AG 传感器值得注意的是，代谢型受体介导 2-AG 的增加。受 ABHD6 控制，而 2-AG 的离子型受体依赖性增加不受 ABHD6 控制。这些结果提出了一个问题：MAGL 如何控制刺激依赖性 2-AG 的产生？证明 2-AG 产量和 CB1R 信号传导活性的受体依赖性增加是由 ABHD6 和 MAGL 进行差动控制将提供额外级别的机械区别这些 eCB 水解酶之间的关系，以增加我们对 ABHD6 和 ABHD6 各自作用的了解。 MAGL 在控制大脑中的 2-AG-CB1R 信号传导方面，我们发起了一项努力，现已成功验证 GRABeCB2.0 在培养的神经细胞中的药理学特征，并确定了几种增加 2-AG 的刺激基于这个前提，我们建议解决小鼠神经元中的以下两个问题。使用活细胞荧光显微镜、双光子显微镜和电生理学：目标 1：哪些刺激会增加神经元中 2-AG 的产生和 GRABeCB2.0 信号？目标 2：ABHD6 和 MAGL 如何差异控制 2-AG-CB1R 的刺激依赖性增加神经元中的信号传导？完成这项新的 R21 拨款提案中概述的工作将提供对 ABHD6 和 MAGL 在控制大脑中 2-AG-CB1R 信号传导方面各自的作用是我们的长期目标。增加我们对 ABHD6 和 ABHD6 的分子、细胞和系统水平差异的理解 MAGL 控制大脑中的 eCB 信号传导，这项工作将有助于开发具有减少功能的治疗小说经典大麻素激动剂潜在的滥用和不良反应。