Michele K. McKinney

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.

Discovery and Characterization of a Highly Selective FAAH Inhibitor that Reduces Inflammatory Pain

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAHs serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as determined by activity-based protein profiling; raises brain anandamide levels for up to 24 hr; and produces significant cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

A Second Fatty Acid Amide Hydrolase with Variable Distribution among Placental Mammals

Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and α-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species.

Evidence for Distinct Roles in Catalysis for Residues of the Serine-Serine-Lysine Catalytic Triad of Fatty Acid Amide Hydrolase

Fatty acid amide hydrolase (FAAH) is a mammalian amidase signature enzyme that inactivates neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The recent determination of the three-dimensional structures of FAAH and two distantly related bacterial amidase signature enzymes indicates that these enzymes employ an unusual serine-serine-lysine triad for catalysis (Ser-241/Ser-217/Lys-142 in FAAH). Mutagenesis of each of the triad residues in FAAH has been shown to severely reduce amidase activity; however, how these residues contribute, both individually and in cooperation, to catalysis remains unclear. Here, through a combination of site-directed mutagenesis, enzyme kinetics, and chemical labeling experiments, we provide evidence that each FAAH triad residue plays a distinct role in catalysis. In particular, the mutation of Lys-142 to alanine indicates that this residue functions as both a base involved in the activation of the Ser-241 nucleophile and an acid that participates in the protonation of the substrate leaving group. This latter property appears to support the unusual ability of FAAH to hydrolyze amides and esters at equivalent rates. Interestingly, although structural evidence indicates that the impact of Lys-142 on catalysis probably occurs through the bridging Ser-217, the mutation of this latter residue to alanine impaired catalytic activity but left the amide/ester hydrolysis ratios of FAAH intact. Collectively, these findings suggest that FAAH possesses a specialized active site structure dedicated to a mechanism for competitive amide and ester hydrolysis where nucleophile attack and leaving group protonation occur in a coordinated manner dependent on Lys-142.

Fatty acid amide hydrolase shapes NKT cell responses by influencing the serum transport of lipid antigen in mice

The potent regulatory properties of NKT cells render this subset of lipid-specific T cells a promising target for immunotherapeutic interventions. The marine sponge glycolipid alpha-galactosylceramide (alphaGalCer) is the proto-typic NKT cell agonist, which elicits this function when bound to CD1d. However, our understanding of the in vivo properties of NKT cell agonists and the host factors that control their bioactivity remains very limited. In this report, we isolated the enzyme fatty acid amide hydrolase (FAAH) from mouse serum as an alphaGalCer-binding protein that modulates the induction of key effector functions of NKT cells in vivo. FAAH bound alphaGalCer in vivo and in vitro and was required for the efficient targeting of lipid antigens for CD1d presentation. Immunization of Faah-deficient mice with alphaGalCer resulted in a reduced systemic cytokine production, but enhanced expansion of splenic NKT cells. This distinct NKT response conferred a drastically increased adjuvant effect and strongly promoted protective CTL responses. Thus, our findings identify not only the presence of FAAH in normal mouse serum, but also its critical role in the tuning of immune responses to lipid antigens by orchestrating their transport and targeting for NKT cell activation. Our results suggest that the serum transport of lipid antigens directly shapes the quality of NKT cell responses, which could potentially be modulated in support of novel vaccination strategies.

Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase.

Background and purpose:  Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH.

Clickable, photoreactive inhibitors to probe the active site microenvironment of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the endocannabinoid anandamide (AEA) and several other bioactive lipid amides. The catalytic mechanism of FAAH has been largely elucidated, and structural models of the enzyme suggest that it may recruit its hydrophobic substrates directly from the lipid bilayer of the cell. Testing this hypothesis, however, requires new tools to explore FAAH-substrate interactions in native cell membranes. Here, we have addressed this problem by creating clickable, photoreactive inhibitors that probe the microenvironment surrounding the FAAH active site. We show that these probes can be used directly in cell membranes, where distinct crosslinked adducts are observed for inhibitors that are buried within versus exposed to the external environment of the FAAH active site.