Increased tonic cannabinoid CB1R activity and brain region-specific desensitization of CB1R Gi/o signaling axis in mice with global genetic knockout of monoacylglycerol lipase

Abstract

In mammalian brain, monoacylglycerol lipase (MAGL) is the primary enzyme responsible for terminating signaling function of the endocannabinoid 2-arachidonoylglycerol (2-AG). Previous in vivo studies with mice indicate that both genetic and chronic pharmacological inactivation of MAGL result in 8-30-fold increase of 2-AG concentration in the brain, causing desensitization and downregulation of cannabinoid CB1 receptor (CB1R) activity, leading to functional and behavioral tolerance. However, direct evidence for reduced CB1R activity in the brain is lacking. In this study, we used functional autoradiography to assess basal and agonist-stimulated CB1R-dependent Gi/o protein activity in multiple brain regions of MAGL-KO mice in comparison to their wild-type (WT) littermates. In addition, the role of endogenous cannabinoids in basal CB1R signaling was assessed after comprehensive pharmacological blockade of 2-AG hydrolysis by determining the contents of endocannabinoids (eCBs) in WT and MAGL-KO brain tissues by LC/MS/MS technology. To show whether lack of MAGL cause compensatory alterations in the serine hydrolase activity, we compared serine hydrolase pattern of WT and MAGL-KO using activity-based protein profiling. Consistent with studies using chronic pharmacological MAGL inactivation in vivo, we observed a statistically significant decrease of CB1R-Gi/o signaling in most of the studied brain regions. In MAGL-KO brain sections, elevated 2-AG levels were mirrored to heightened basal CB1R-dependent Gi/o-activity, as well as, dampened agonist-evoked responses in several brain regions. The non-selective serine hydrolase inhibitor methylarachidonoylfluorophosphonate (MAFP) was able to significantly elevate 2-AG levels in brain sections of MAGL-KO mice, indicating that additional serine hydrolases possess 2-AG hydrolytic activity in MAGL-KO brain sections.

The cannabinoid system and pain

“…Full inhibition of MAGL via JZL184 produces many cannabinoid-like behaviours (Long et al, 2009a), suggesting that this approach may share some of the unwanted side-effects of cannabinoids. Furthermore, several studies have now revealed that sustained global elevation of 2-AG via genetic deletion of MAGL or persistent blockade of MAGL activity with enzyme inhibitors produces functional antagonism of the brain EC system, resulting in profound downregulation and desensitization of CB 1 receptors in nociception-associated regions, and a loss of analgesic phenotype (Chanda et al, 2010; Imperatore et al, 2015; Navia-Paldanius et al, 2015; Schlosburg et al, 2010). Chronic MAGL blockade may also result in physical dependence, since a CB 1 antagonist precipitated behavioural symptoms of withdrawal following repeated high dose JZL184 treatment (Schlosburg et al, 2009).…”

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections – Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils

“…Such studies should be of wide interest to increase knowledge on the myriad roles that SHs likely play in the CNS. Based on studies from this and other laboratories [14,[19][20][21][22][23][24][25][26][27], gel-ABPP using brain membranes typically detects 20-30 SH bands, depending on Fig. 6 High-resolution imaging of TAMRA-FP hotspots and TAMRA-FP hotspot clusters and their inhibitor sensitivity in glioma.…”

“…We cross-validated the tissue-ABPP findings in gelbased ABPP using homogenates of glioma and control brain. In addition, we used rat cerebellar membranes to facilitate comparison, as previous gel-based ABPP has substantially relied on SH profiling in rodent brain membrane proteomes [14,[19][20][21][22][23][24][25][26][27]. Building on such studies from this and other laboratories [14,[19][20][21][22][23][24][25][26][27], we could identify many of the SH bands based on inhibitor sensitivity and mobility pattern in SDS-PAGE.…”

“…Position of molecular weight markers (in kDa) is indicated on the gel. Based on previous studies from this and other laboratories [14,[19][20][21][22][23][24][25][26][27] many healthy brain-resident SHs were identified, as indicated at left. Note comparable activities of KIAA1363 and LYPLA1/2 doublets (black asterisk) in control brain and glioma.…”

Tissue-ABPP enables high-resolution confocal fluorescence imaging of serine hydrolase activity in cryosections – Application to glioma brain unveils activity hotspots originating from tumor-associated neutrophils

“…A straightforward starting point could be comparative tissue-ABPP of an individual SH, e.g. the principal endocannabinoid hydrolase MAGL using brain sections of wild-type and MAGL-deficient mice, as MAGL knockout does not lead to compensatory changes in the activity of other SHs, as evidenced by gel-based ABPP (Navia-Paldanius et al, 2015). Competitive tissue-ABPP would offer a complementary approach for sections where MAGL is chemically inactivated with highly selective and ultra-potent inhibitors, such as JJKK-048 and KML29 (Aaltonen et al, 2013;Bachovchin and Cravatt, 2012).…”