Benjamin F. Cravatt

Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase

The medicinal properties of marijuana have been recognized for centuries, but clinical and societal acceptance of this drug of abuse as a potential therapeutic agent remains fiercely debated. An attractive alternative to marijuana-based therapeutics would be to target the molecular pathways that mediate the effects of this drug. To date, these neural signaling pathways have been shown to comprise a cannabinoid receptor (CB1) that binds the active constituent of marijuana, tetrahydrocannabinol (THC), and a postulated endogenous CB1 ligand anandamide. Although anandamide binds and activates the CB1 receptor in vitro, this compound induces only weak and transient cannabinoid behavioral effects in vivo, possibly a result of its rapid catabolism. Here we show that mice lacking the enzyme fatty acid amide hydrolase (FAAH−/−) are severely impaired in their ability to degrade anandamide and when treated with this compound, exhibit an array of intense CB1-dependent behavioral responses, including hypomotility, analgesia, catalepsy, and hypothermia. FAAH−/−-mice possess 15-fold augmented endogenous brain levels of anandamide and display reduced pain sensation that is reversed by the CB1 antagonist SR141716A. Collectively, these results indicate that FAAH is a key regulator of anandamide signaling in vivo, setting an endogenous cannabinoid tone that modulates pain perception. FAAH may therefore represent an attractive pharmaceutical target for the treatment of pain and neuropsychiatric disorders.

Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines

The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.

Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects

2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for anandamide, is mediated by fatty acid amide hydrolase (FAAH) and, for 2-AG, is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors produce a select subset of the behavioral effects observed with CB1 agonists, intimating a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metabolism. Here, we report a potent and selective inhibitor of MAGL, JZL184, that, upon administration to mice, raises brain 2-AG by 8-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia, and hypomotility. These data indicate that 2-AG endogenously modulates several behavioral processes classically associated with the pharmacology of cannabinoids and point to overlapping and unique functions for 2-AG and anandamide in vivo.

Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry

Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chemical proteomic strategy to characterize enzyme function directly in native biological systems on a global scale. Here, we review the basic technology of ABPP, the enzyme classes addressable by this method, and the biological discoveries attributable to its application.

Quantitative reactivity profiling predicts functional cysteines in proteomes

Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.

Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis

Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity-phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals. PAPERFLICK:

Identification of protein pheromones that promote aggressive behaviour

Mice use pheromones, compounds emitted and detected by members of the same species, as cues to regulate social behaviours such as pup suckling, aggression and mating. Neurons that detect pheromones are thought to reside in at least two separate organs within the nasal cavity: the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). Each pheromone ligand is thought to activate a dedicated subset of these sensory neurons. However, the nature of the pheromone cues and the identity of the responding neurons that regulate specific social behaviours are largely unknown. Here we show, by direct activation of sensory neurons and analysis of behaviour, that at least two chemically distinct ligands are sufficient to promote male–male aggression and stimulate VNO neurons. We have purified and analysed one of these classes of ligand and found its specific aggression-promoting activity to be dependent on the presence of the protein component of the major urinary protein (MUP) complex, which is known to comprise specialized lipocalin proteins bound to small organic molecules. Using calcium imaging of dissociated vomeronasal neurons (VNs), we have determined that the MUP protein activates a sensory neuron subfamily characterized by the expression of the G-protein Gαo subunit (also known as Gnao) and Vmn2r putative pheromone receptors (V2Rs). Genomic analysis indicates species-specific co-expansions of MUPs and V2Rs, as would be expected among pheromone-signalling components. Finally, we show that the aggressive behaviour induced by the MUPs occurs exclusively through VNO neuronal circuits. Our results substantiate the idea of MUP proteins as pheromone ligands that mediate male–male aggression through the accessory olfactory neural pathway.

Endocannabinoid Hydrolysis Generates Brain Prostaglandins That Promote Neuroinflammation

A new tissue-specific pathway for the synthesis of proinflammatory prostaglandins is described. 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.

Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system

Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. 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 Mgll (encoding MAGL). Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, 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.

Enzymatic Pathways That Regulate Endocannabinoid Signaling in the Nervous System

Chemical signals, or neurotransmitters, represent the fundamental mode for intercellular communication in the nervous system. (1) The classical model for neurotransmitter action involves the uptake and storage of these small molecules into synaptic vesicles, release of vesicular contents into the synaptic cleft in response to depolarization of the presynaptic terminal by an action potential, binding of released neurotransmitters to cognate protein receptors on the postsynaptic (and presynaptic) terminal, and, finally, termination of signaling by protein-mediated uptake and degradation of neurotransmitters from the synaptic cleft. This model applies to a large number of well-studied neurotransmitters, including glutamate, γ-amino butyric acid (GABA), acetylcholine, and the monoamines, all of which represent aqueous solution-soluble molecules. More recently, lipids have emerged as an important class of chemical messengers in the nervous system that operate by a distinct mechanism. The hydrophobic nature of lipids precludes their stable uptake and storage into synaptic vesicles. Instead, lipid messengers appear to be biosynthesized and released by neurons at the moment of their intended action, which is often referred to as “on-demand” production. Similarly, the capacity of lipids to freely cross cell membranes places the burden of signal termination largely on the action of degradative enzymes. Lipid signaling systems are thus embedded within an elaborate collection of metabolic pathways, the composition and regulation of which ultimately establish the magnitude and duration of transmitter action. Here, we will review these general concepts as they relate to a specific class of lipid transmitters, the endogenous cannabinoids (endocannabinoids), and highlight how delineation of their cognate metabolic enzymes has been translated into the development of chemical and genetic tools to test the role that the endocannabinoid system plays in nervous system signaling and behavior. Endocannabinoids are defined as endogenous small molecules that activate the cannabinoid receptors CB1 and CB2, which are G-protein-coupled receptors that also recognize Δ9-tetrahydrocannabinol (THC), the psychoactive component of marijuana. (2, 3) The CB1 receptor is the major cannabinoid receptor in the nervous system and is responsible for mediating most of the neurobehavioral effects of THC. (4, 5) The CB2 receptor is predominantly expressed in immune cells, (6) where it appears to play a role in mediating the immunosuppressive effects of cannabinoids. Two principal endocannabinoids have been identified in mammals, N-arachidonoyl ethanolamine (anandamide) (7) and 2-arachidonoylglycerol (2-AG) (8, 9) (Figure 1). Each endocannabinoid also belongs to a much larger class of lipids, termed N-acyl ethanolamines (NAEs) and monoacylglycerols (MAGs), respectively, where individual members differ in the length and degree of unsaturation of their acyl chains (Figure 1). Several NAEs and MAGs have been ascribed potential biological activities in vivo; (10) however, most of these lipids do not serve as ligands for cannabinoid receptors, a property that appears to be restricted to polyunsaturated derivatives such as anandamide and 2-AG. Figure 1 Two principle endocannabinoids, N-arachidonoyl ethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), which are members of theN-acyl ethanolamine (NAE) and monoacylglycerol (MAG) classes of lipids, respectively. In the nervous system, endocannabinoids are hypothesized to act as retrograde messengers, being released by postsynaptic neurons and traversing the synaptic cleft to stimulate CB1 receptors on presynaptic termini (11, 12) (Figure 2). This model is supported by a large number of in vitro electrophysiological studies, (12) as well as by the restricted localization of the CB1 receptor to presynaptic structures in many regions of the nervous system. (13, 14) Once activated by endocannabinoids, CB1 receptors couple principally through the G i/G o class of G proteins to regulate calcium and potassium channels and reduce the probability of neurotransmitter release. (3) This suppression of neurotransmitter release can result in the inhibition or, paradoxically, disinhibition of neuronal circuits, depending on whether the CB1 receptor is expressed on glutamatergic or GABergic neurons. Figure 2 General model for endocannabinoid-based retrograde signaling. Upon release of neurotransmitter (e.g., glutamate), postsynaptic receptors (e.g., α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl- d-aspartic acid (NMDA)) and ... Despite sharing a common receptor and considerable structural similarity, anandamide and 2-AG can be distinguished by multiple factors. First, these endocannabinoids activate cannabinoid receptors to a differing degree in vitro, with anandamide displaying lower intrinsic efficacy than 2-AG, which results in the former lipid acting as a partial agonist (15) (it should be specified that the relevance of this distinction for signaling in vivo is unclear, especially when one considers that THC also acts as only a partial agonist for cannabinoid receptors in vitro (16)). Second, the endogenous quantities of anandamide and 2-AG differ dramatically, with the latter lipid being found at more than 100-fold higher concentrations in the nervous system. (16) Of course, these values are based on bulk tissue measurements of endocannabinoids, which almost certainly reflect a combination of metabolic and signaling pools of these lipids. Indeed, recent microdialysis studies have revealed that the extracellular concentrations of anandamide and 2-AG are nearly equivalent (within 2 5-fold), (17, 18) suggesting that, at least for the latter endocannabinoid, a large fraction of bulk tissue concentration may correspond to intracellular metabolic pools. Finally, and of greatest relevance for the subject of this review, anandamide and 2-AG are regulated by distinct biosynthetic and degradative pathways. Over the past decade, several excellent reviews have appeared that discuss endocannabinoid metabolism and signaling. (10, 19-23) Here, we will focus on the most recent advances in our understanding of the composition and regulation of endocannabinoid metabolic pathways, especially as pertains to the nervous system. A pervasive theme throughout this review will be the importance of developing selective genetic and pharmacological tools to specifically perturb individual enzymatic pathways to test their contribution to endocannabinoid metabolism, nervous system function, and, ultimately, mammalian physiology and behavior.