An optimized spectrophotometric assay reveals increased activity of enzymes involved in 2‐arachidonoyl glycerol turnover in the cerebral cortex of a rat model of Alzheimer’s disease

The endocannabinoid system is implicated in a plethora of neuropsychiatric disorders. However, it is technically challenging to assess the turnover of 2‐arachidonoyl glycerol (2‐AG), the principal endocannabinoid molecule in the brain. Two recent studies showed that diacylglycerol lipase α (DAGLα), an enzyme chiefly responsible for the cerebral production of 2‐AG, also accepts the surrogate chromogenic substrate 4‐nitrophenyl butyrate (4‐NPB). Here, we aimed to optimize this spectrophotometric assay for ex vivo brain tissue, in particular, rat cerebrocortical homogenates, to measure the activity of the major enzymes responsible for the production and degradation of 2‐AG. The initial velocity of 4‐NPB hydrolysis was dependent on protein, substrate, and Ca2+ concentrations, and was sensitive to the non‐selective serine hydrolase inhibitor, methoxy arachidonyl fluorophosphonate, the DAGLα inhibitors, OMDM188, tetrahydrolipstatin, and RHC80267, as well as the monoacylglycerol lipase (MAGL) inhibitor, JZL184, respectively. Next, we tested the usefulness of this assay in ex vivo brain tissue of rat models of human health conditions known to affect cerebrocortical 2‐AG production, i.e. pathological stress and sporadic Alzheimer's disease (AD). In rats submitted to chronic restraint stress, cortical CB1R density was significantly decreased, as assessed with radioligand binding. Nevertheless, 4‐NPB hydrolysis remained at control levels. However, in rats 4 weeks after intracerebroventricular injection with streptozotocin – an established model of sporadic AD –, both CB1R levels and 4‐NPB hydrolysis and its DAGL‐ and MAGL‐dependent fractions were significantly increased. Altogether, we optimized a simple complementary ex vivo technique for the quantification of DAGL and MAGL activity in brain samples. 2‐AG is the principal endocannabinoid molecule. 2‐AG signaling is altered in many brain diseases, thus, targeting the key 2‐AG turnover enzymes, DAGLα and MAGL is an emerging therapeutic strategy. We optimized a technique for ex vivo brain homogenates, using the surrogate chromogenic DAGLα and MAGL substrate, 4‐NPB. We observed increased DAGL and MAGL activity in the cerebral cortex of a rat model of sporadic AD. This assay also can facilitate the discovery of novel DAGLα and MAGL inhibitors.