Endocannabinoids (eCBs) function as retrograde signaling molecules at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-␣ (DAGL␣) and -␤ (DAGL␤) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their respective contribution to this and to eCB signaling has not been tested. In the present study, we show ϳ80% reductions in 2-AG levels in the brain and spinal cord in DAGL␣ Ϫ/Ϫ mice and a 50% reduction in the brain in DAGL␤ Ϫ/Ϫ mice. In contrast, DAGL␤ plays a more important role than DAGL␣ in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGL␤ Ϫ/Ϫ mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABAmediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGL␣ Ϫ/Ϫ animals and relatively unaffected in DAGL␤ Ϫ/Ϫ animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGL␣ Ϫ/Ϫ and/or DAGL␤ Ϫ/Ϫ mice. These findings provide the first evidence that DAGL␣ is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.

The diacylglycerol lipases (DAGLs) hydrolyse diacylglycerol to generate 2-arachidonoylglycerol (2-AG), the most abundant ligand for the CB 1 and CB 2 cannabinoid receptors in the body. DAGL-dependent endocannabinoid signalling regulates axonal growth and guidance during development, and is required for the generation and migration of new neurons in the adult brain. At developed synapses, 2-AG released from postsynaptic terminals acts back on presynaptic CB 1 receptors to inhibit the secretion of both excitatory and inhibitory neurotransmitters, with this DAGL-dependent synaptic plasticity operating throughout the nervous system. Importantly, the DAGLs have functions that do not involve cannabinoid receptors. For example, 2-AG is the precursor of arachidonic acid in a pathway that maintains the level of this essential lipid in the brain and other organs. This pathway also drives the cyclooxygenase-dependent generation of inflammatory prostaglandins in the brain, which has recently been implicated in the degeneration of dopaminergic neurons in Parkinson's disease. Remarkably, we still know very little about the mechanisms that regulate DAGL activity-however, key insights can be gleaned by homology modelling against other a/b hydrolases and from a detailed examination of published proteomic studies and other databases. These identify a regulatory loop with a highly conserved signature motif, as well as phosphorylation and palmitoylation as post-translational mechanisms likely to regulate function.

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The diacylglycerol lipases (DAGLalpha and DAGLbeta) synthesize 2-arachidonoylglycerol (2-AG), a full agonist at cannabinoid receptors. Dynamic regulation of DAGL expression underpins its role in axonal growth and guidance during development, retrograde synaptic signalling at mature synapses, and maintenance of adult neurogenesis. We show here that DAGLalpha expression is dramatically down-regulated when neural stem (NS) cells are differentiated toward a gamma-aminobutyric acidergic neuronal phenotype. To understand how DAGLalpha expression might be controlled, we sought to identify the core promoter region and regulatory elements within it. The core promoter was identified and shown to contain both an enhancer and a suppressor region. Deletion analysis identified two elements, including a GC-box, that specifically promote expression in NS cells. Bioinformatic analysis identified three candidate transcription factors that might regulate DAGLalpha expression in NS cells by binding to the GC box; these were specificity protein 1 (Sp1), early growth response element 1 (EGR1), and zinc finger DNA-binding protein 89 (ZBP-89). However, Sp1 was the only factor that could bind to the GC-box. A specific mutation within the GC-box that inhibited Sp1 binding reduced DAGLalpha promoter activity in NS cells. Likewise, a dominant negative Sp1 was shown to bind to the GC-box and to suppress DAGLalpha promoter activity specifically in NS cells. Finally, like DAGLalpha, Sp1 was down-regulated during neuronal differentiation. A full characterization of the DAGLalpha promoter will help to elucidate the upstream pathways that regulate DAGLalpha expression in NS cells and their progeny.