Regulation of DAG lipase activity : implications for 'on-demand' endocannabinoid signalling

The diacylglycerol lipases (DAGL α and β) are key enzymes in the biosynthesis of 2-AG, the major endocannabinoid (eCB) in the brain. 2-AG acts on CB1 and/or CB2 receptors and DAGL-dependent eCB signalling regulates a large number of responses including axonal growth during development, as well as neurogenesis and retrograde synaptic plasticity in the adult. The enzymes also play a major role in driving pathogenic inflammatory responses via a DAGL/MAGL pathway that generates arachidonic acid as a precursor to prostaglandin synthesis. DAGL antagonists are being developed as novel therapeutics based on their ability to regulate eCB-mediated signalling and/or inflammatory responses, but the mechanisms underlying the regulation of these enzymes is poorly understood. The DAGLs appear to display ‘on-demand’ synthesis, generating increasing amounts of 2-AG in response to cellular messengers. Using a bioinformatics approach, we have postulated that phosphorylation is key mechanism for regulation of DAGL function. We overexpressed each enzyme in U2OS cells that harbour the Tango assay system. We showed the transgenic DAGLs to be expressed at the membrane, and DAGLα to be active using surrogate substrates. We measured an eCB-dependent CB1 response in the Tango assay, with evidence for kinase activation-dependent eCB signalling, but only a portion of this response appeared to be DAGL-dependent. As a result, we are pursuing a genetic strategy to systematically ‘switch off’ endogenous eCB production. We first targeted the DAGLs using the CRISPR/Cas9 system, using both wild-type and nickase Cas9. Our next strategy will be to knockout other eCB-producing enzymes in these cells to tease out which enzymes are resulting in the eCB-CB1 activation in the Tango assay. This will also provide us with a ‘parent’ cell line to support future mutagenesis studies to understand which (if any) phospho-sites are important for DAGL regulation.