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

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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.