Endogenous ligands for cannabinoid receptors ("endocannabinoids") include the lipid transmitters anandamide and 2-arachidonoylglycerol (2-AG). Endocannabinoids modulate a diverse set of physiological processes and are tightly regulated by enzymatic biosynthesis and degradation. Termination of anandamide signaling by fatty acid amide hydrolase (FAAH) is well characterized, but less is known about the inactivation of 2-AG, which can be hydrolyzed by multiple enzymes in vitro, including FAAH and monoacylglycerol lipase (MAGL). Here, we have taken a functional proteomic approach to comprehensively map 2-AG hydrolases in the mouse brain. Our data reveal that approximately 85% of brain 2-AG hydrolase activity can be ascribed to MAGL, and that the remaining 15% is mostly catalyzed by two uncharacterized enzymes, ABHD6 and ABHD12. Interestingly, MAGL, ABHD6, and ABHD12 display distinct subcellular distributions, suggesting that they may control different pools of 2-AG in the nervous system.

Phospholipase A2 (PLA2) enzymes are considered the primary source of arachidonic acid for cyclooxygenase (COX)-mediated biosynthesis of prostaglandins. Here, we show that a distinct pathway exists in brain, where monoacylglycerol lipase (MAGL) hydrolyzes the endocannabinoid 2-arachidonoylglycerol to generate a major arachidonate precursor pool for neuroinflammatory prostaglandins. MAGL-disrupted animals show neuroprotection in a parkinsonian mouse model. These animals are spared the hemorrhaging caused by COX inhibitors in the gut, where prostaglandins are instead regulated by cytosolic-PLA2. These findings identify MAGL as a distinct metabolic node that couples endocannabinoid to prostaglandin signaling networks in the nervous system and suggest that inhibition of this enzyme may be a new and potentially safer way to suppress the proinflammatory cascades that underlie neurodegenerative disorders.

Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor desensitization in the nervous system. Here we show that a similar form of functional antagonism is produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of MAGL. Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity, and desensitization of brain CB1 receptors. These data contrasted with blockade of fatty acid amide hydrolase (FAAH), an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and associated with agonism and functional antagonism of the brain cannabinoid system, respectively.

The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB 1 cannabinoid receptors on neurons and CB 2 cannabinoid Correspondence should be addressed to N.S. (nstella@uw.edu). 11 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Neuroscience website. Competing Financial Interests:The authors declare no competing financial interests.Reprints and permissions information is available online at http://www.nature.com/reprintsandpermissions/. NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2011 February 1. Published in final edited form as:Nat Neurosci. 2010 August ; 13(8): 951-957. doi:10.1038/nn.2601. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript receptors on microglia. Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase α-β-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB 2 -mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activitydependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB 1 -dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.In the nervous system, the endocannabinoids (eCBs) arachidonoylethanolamide (anandamide) and 2-AG are produced and inactivated by neurons and glia 1,2 . The production of eCBs increases in response to specific stimuli, including membrane receptor activation, ion channel opening and calcium influx 2 . eCBs are inactivated by cellular uptake followed by intracellular enzymatic hydrolysis 3,4 . The balance between this production and inactivation dictates the levels of extracellular eCB accumulation and the ensuing activation of CB 1 receptors expressed by neurons (regulating neurotransmitter release) and CB 2 receptors expressed by microglia (regulating their motility and ability to produce immunomodulators) [4][5][6][7] . Thus, the enzymatic steps that control the production and inactivation of eCBs constitute promising molecular targets for indirectly modulating CB 1 and CB 2 receptor activity, and thereby controlling neurotransmission and neuroinflammation.Of all the steps that control the accumulation of eCBs, the hydrolytic enzymes that inactivate anandamide and 2-AG represent the most promising pharmacological and genetic targets for fine-tuning the local accumulation of these lipid transmitters. Inhibition of fatty acid amide hydrolase (FAAH) increases...

Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110þ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (>80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activitybased assay in a library-versus-library format to screen 70þ SHs against 140þ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.enzymology | mass spectrometry | profiling | proteomics A major challenge facing biological researchers in the 21st century is the functional characterization of the large number of unannotated gene products identified by genome sequencing efforts (1). Many proteins partly or completely uncharacterized with respect to their biochemical activities belong to expansive, sequence-related families (2). Although such membership can inform on the general mechanistic class to which a protein belongs (e.g., enzyme, receptor, or channel), it is insufficient to predict specific biochemical and physiological functions, which require knowledge of substrates, ligands, and interacting biomolecules. On the contrary, membership within a large protein family can even present a barrier to achieving these goals by frustrating the implementation of standard genetic and pharmacological methods to probe protein function. For example, targeted gene disruption of one member of a protein superfamily may result in cellular compensation from other family members.Problems are also encountered when attempting to develop specific inhibitors and/or ligands for uncharacterized members of large protein families, where at least two major experimental issues must be addressed. First, there is an intrinsic difficulty facing ligand discovery for uncharacterized proteins, which often lack the functional information required to develop high-quality assays for compound screening. Creative solutions to this problem have emerged for specific protein classes, such as G-protein coupled receptors (GPCRs) (3) and kinases (4, 5), where generic assays have been develop...

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Advances in human genetics are leading to the discovery of new disease-causing mutations at a remarkable rate. Many such mutations, however, occur in genes that encode for proteins of unknown function, which limits our molecular understanding of, and ability to devise treatments for, human disease. Here, we use untargeted metabolomics combined with a genetic mouse model to determine that the poorly characterized serine hydrolase α/β-hydrolase domain-containing (ABHD)12, mutations in which cause the human neurodegenerative disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, and cataract), is a principal lysophosphatidylserine (LPS) lipase in the mammalian brain. ABHD12 −/− mice display massive increases in a rare set of very long chain LPS lipids that have been previously reported as Toll-like receptor 2 activators. We confirm that recombinant ABHD12 protein exhibits robust LPS lipase activity, which is also substantially reduced in ABHD12 −/− brain tissue. Notably, elevations in brain LPS lipids in ABHD12 −/− mice occur early in life (2-6 mo) and are followed by age-dependent increases in microglial activation and auditory and motor defects that resemble the behavioral phenotypes of human PHARC patients. Taken together, our data provide a molecular model for PHARC, where disruption of ABHD12 causes deregulated LPS metabolism and the accumulation of proinflammatory lipids that promote microglial and neurobehavioral abnormalities.lipidomics | neuroinflammation G enome mapping and sequencing have facilitated determination of the genetic basis for over 3,500 inherited diseases in humans (1), with additional pathogenic mutations still being discovered. For many Mendelian disorders, however, the molecular and cellular mechanisms that link genotype to disease phenotype remain poorly understood. This problem is perhaps most apparent for diseases where the causative mutations occur in genes that encode for proteins with unannotated or poorly characterized functions. Here, we focus on one such disease -the neurodegenerative disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, and cataract).PHARC [Mendelian Inheritance in Man (MIM) no. 612674; Online Mendelian Inheritance in Man database, http://omim.org] is a rare, autosomal recessive disorder that causes polymodal sensory and motor defects associated with demyelination of sensomotor neurons, retinal dystrophy, and cerebellar atrophy (2). PHARC symptoms are slowly progressive and begin in the childhood or teenage years; heterozygous carriers are unaffected (3). In 2010, PHARC was reported to be caused by homozygous mutations in ABHD12, which codes for the poorly characterized serine hydrolase enzyme α/β-hydrolase domain-containing (ABHD)12 (3). To date, five distinct ABHD12 mutations have been identified in patients with PHARC, all of which are expected to lead to complete loss of ABHD12 expression (3, 4). PHARC, therefore, likely represents a human ABHD12 null model.We demonstrated previously that ABHD12 is a membranebound e...

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