The endocannabinoid system in neurodegeneration

Endocannabinoids are bioactive lipids, that comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the best studied endocannabinoids, and act as agonists of cannabinoid receptors. Thus, AEA and 2-AG mimic several pharmacological effects of the exogenous cannabinoid delta9-tetrahydrocannabinol, the psychoactive principle of hashish and marijuana. It is known that the activity of endocannabinoids at their receptors is limited by cellular uptake through specific membrane transporters, followed by intracellular degradation by a fatty acid amide hydrolase (for AEA and partly 2-AG) or by a monoacylglycerol lipase (for 2-AG). Together with AEA, 2-AG and congeners, the proteins that bind, transport and metabolize these lipids form the "endocannabinoid system". This new system will be briefly presented in this review, in order to put in a better perspective the role of the endocannabinoid pathway in neurodegenerative disorders, like Parkinson's disease, Huntington's disease, and multiple sclerosis. In addition, the potential exploitation of antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, as next-generation therapeutics will be discussed.     

Critical enzymes involved in endocannabinoid metabolism

Investigations of the pathways involved in the metabolism of endocannabinoids have grown exponentially in recent years following the discovery of cannabinoid receptors (CB) and their endogenous ligands, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The in vivo biosynthesis of AEA has been shown to occur through several pathways mediated by N-acylphosphatidylethanolamide-phospholipase D (NAPE-PLD), a secretory PLA(2) and PLC. 2-AG, a second endocannabinoid is generated through the action of selective enzymes such as phosphatidic acid phsophohydrolase, diacylglycerol lipase (DAGL), phosphoinositide-specific PLC (PI-PLC) and lyso-PLC. A putative membrane transporter or facilitated diffusion is involved in the cellular uptake or release of endocannabinoids. AEA is metabolized by fatty acid amidohydrolase (FAAH) and 2-AG is metabolized by both FAAH and monoacylglycerol lipase (MAGL). The author presents an integrative overview of current research on the enzymes involved in the metabolism of endocannabinoids and discusses possible therapeutic interventions for various diseases, including addiction.     

Cannabinoid drugs and enhancement of endocannabinoid responses: strategies for a wide array of disease states

The endogenous cannabinoid system has revealed potential avenues to treat many disease states. Medicinal indications of cannabinoid drugs including compounds that result in enhanced endocannabinoid responses (EER) have expanded markedly in recent years. The wide range of indications covers chemotherapy complications, tumor growth, addiction, pain, multiple sclerosis, glaucoma, inflammation, eating disorders, age-related neurodegenerative disorders, as well as epileptic seizures, traumatic brain injury, cerebral ischemia, and other excitotoxic insults. Indeed, a great effort has led to the discovery of agents that selectively activate the cannabinoid system or that enhance the endogenous pathways of cannabinergic signaling. The endocannabinoid system is comprised of three primary components: (i) cannabinoid receptors, (ii) endocannabinoid transport system, and (iii) hydrolysis enzymes that break down the endogenous ligands. Two known endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), are lipid molecules that are greatly elevated in response to a variety of pathological events. This increase in endocannabinoid levels is suggested to be part of an on-demand compensatory response. Furthermore, activation of signaling pathways mediated by the endogenous cannabinoid system promotes repair and cell survival. Similar cell maintenance effects are elicited by EER through inhibitors of the endocannabinoid deactivation processes (i.e., internalization and hydrolysis). The therapeutic potential of the endocannabinoid system has yet to be fully determined, and the number of medical maladies that may be treated will likely continue to grow. This review will underline studies that demonstrate medicinal applications for agents that influence the endocannabinoid system.     

Endocannabinoids control spasticity in a multiple sclerosis model.

Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated with nerve damage, increased levels of the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and of the AEA congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these compounds were found in normal and non-spastic CREAE mice. While exogenously administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of endocannabinoid re-uptake and hydrolysis-probably through the enhancement of endogenous levels of AEA, and, possibly, 2-arachidonoyl glycerol-significantly ameliorated spasticity to an extent comparable with that observed previously with potent cannabinoid receptor agonists. These studies provide definitive evidence for the tonic control of spasticity by the endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which exhibit little psychotropic activity.     

Brain regional distribution of endocannabinoids: implications for their biosynthesis and biological function

The amounts, in nine different rat brain regions, of the two endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoylglycerol (2-AG), and of the putative AEA precursor N-arachidonoyl-phosphatidylethanolamine (NArPE), were determined by isotope-dilution gas chromatography-mass spectrometry and compared to the number of cannabinoid binding sites in each region. The distribution of NArPE, reported here for the first time, exhibited a good correlation with that of AEA, the former metabolite being 3-13 times more abundant than the endocannabinoid in all regions. The highest amounts of both metabolites (up to 358.5 and 87 pmol/g wet weight tissue, respectively) were found in the brainstem and striatum, and the lowest in the diencephalon, cortex, and cerebellum. These data support the hypothesis that, in the brain, AEA is a metabolic product of NArPE and may reach levels compatible with its proposed neuromodulatory function. The brain distribution of 2-AG, also described in this study for the first time, was found to correlate with that of AEA with levels ranging from 2.0 to 14.0 nmol/g (in the diencephalon and brainstem, respectively). The distribution of the endocannabinoids did not match exactly with that of cannabinoid binding sites, suggesting either that these compounds are not necessarily produced near their molecular targets, or that they play functional roles additional to the activation of cannabinoid receptors. Regional differences in the ligand/receptor ratios may also lead to predict corresponding differences in the efficiency of receptor activation, as shown by previous studies.     

The endocannabinoid 2-arachidonoylglycerol (2-AG) and metabolizing enzymes during rat fetoplacental development: A role in uterine remodelling

The main endocannabinoids (EC) identified in mammalian tissues are N-arachidonoylethanolamide (AEA, anandamide), and 2-arachidonoylglycerol (2-AG). AEA levels are critical in pregnancy, especially during implantation, decidualization, and placental development. As 2-AG functions in pregnancy are still largely undefined, we hypothesized that it may also have a role during fetoplacental development. We showed that 2-AG is not only present in the rat mesometrial decidua and plasma during fetoplacental development, but that both 2-AG synthesizing (diacylglycerol lipase) and degradation (monoacylglycerol lipase) enzymes are expressed by decidual cells. While lower concentrations of 2-AG induced apoptosis of rat primary decidual cells, via the CB1 receptor, higher concentrations induced a dramatic effect on cell morphology, cell viability and lactate dehydrogenase release, triggered through a mechanism independent of CB1. This study provides evidences that 2-AG fluctuation in maternal tissues throughout normal pregnancy is primarily regulated by its metabolizing enzymes. Together, these data supports the hypothesis that a deregulation of the endocannabinoid system through aberrant cannabinoid signalling may impact normal uterine remodelling process and consequently normal pregnancy.     

Biosynthesis and degradation of the endocannabinoid 2-arachidonoylglycerol

2-Arachidonoylglycerol (2-AG) is a monoacylglycerol (MAG) molecule containing an esterified arachidonic acid chain at sn-2 position of the glycerol backbone. Together with structurally similar N-arachidonoylethanolamine (anandamide), 2-AG has been extensively studied as an endogenous ligand of cannabinoid receptors (an endocannabinoid) in brain and other mammalian tissues. Accumulating evidence demonstrates that the endocannabinoid system, including the central-type cannabinoid receptor CB1 and 2-AG, is responsible for synaptic retrograde signaling in the central nervous system. As 2-AG is rapidly formed from membrane phospholipids on cellular stimuli and degraded to arachidonic acid and glycerol, the enzymes catalyzing its biosynthesis and degradation are believed to play crucial roles in the regulation of its tissue levels. The major biosynthetic pathway appears to consist of sequential hydrolyses of inositol phospholipids via diacylglycerol (DAG) by β-type phospholipase C and DAG lipase, while MAG lipase is a principal enzyme in the degradation. In this short review, we will briefly outline rapid advances in enzymological research on the biosynthetic and degradative pathways of 2-AG.     

Endocannabinoid structure-activity relationships for interaction at the cannabinoid receptors

Anandamide (N -arachidonoylethanolamine) was the first ligand to be identified as an endogenous ligand of the G-protein coupled cannabinoid CB1 receptor. Subsequently, two other fatty acid ethanolamides, N -homo- gamma -linolenylethanolamine and N -7,10,13,16-docosatetraenylethanolamine were identified as endogenous cannabinoid ligands. A fatty acid ester, 2-arachidonoylglycerol (2-AG), and a fatty acid ether, 2-arachidonyl glyceryl ether also have been isolated and shown to be endogenous cannabinoid ligands. Recent studies have postulated the existence of carrier-mediated anandamide transport that is essential for termination of the biological effects of anandamide. A membrane bound amidohydrolase (fatty acid amide hydrolase, FAAH), located intracellularly, hydrolyzes and inactivates anandamide and other endogenous cannabinoids such as 2-AG. 2-AG has also been proposed to be an endogenous CB2 ligand. Structure-activity relationships (SARs) for endocannabinoid interaction with the CB receptors are currently emerging in the literature. This review considers cannabinoid receptor SAR developed to date for the endocannabinoids with emphasis upon the conformational implications for endocannabinoid recognition at the cannabinoid receptors.     

Enhancement of endocannabinoid signaling by fatty acid amide hydrolase inhibition: A neuroprotective therapeutic modality

AimsThis review posits that fatty acid amide hydrolase (FAAH) inhibition has therapeutic potential against neuropathological states including traumatic brain injury; Alzheimer's, Huntington's, and Parkinson's diseases; and stroke.Main methodsThis proposition is supported by data from numerous in vitro and in vivo experiments establishing metabolic and pharmacological contexts for the neuroprotective role of the endogenous cannabinoid (“endocannabinoid”) system and selective FAAH inhibitors.Key findingsThe systems biology of endocannabinoid signaling involves two main cannabinoid receptors, the principal endocannabinoid lipid mediators N-arachidonoylethanolamine (“anandamide”) (AEA) and 2-arachidonoyl glycerol (2-AG), related metabolites, and the proteins involved in endocannabinoid biosynthesis, biotransformation, and transit. The endocannabinoid system is capable of activating distinct signaling pathways on-demand in response to pathogenic events or stimuli, thereby enhancing cell survival and promoting tissue repair. Accumulating data suggest that endocannabinoid system modulation at discrete targets is a promising pharmacotherapeutic strategy for treating various medical conditions. In particular, neuronal injury activates cannabinoid signaling in the central nervous system as an intrinsic neuroprotective response. Indirect potentiation of this salutary response through pharmacological inhibition of FAAH, an endocannabinoid-deactivating enzyme, and consequent activation of signaling pathways downstream from cannabinoid receptors have been shown to promote neuronal maintenance and function.SignificanceThis therapeutic modality has the potential to offer site- and event-specific neuroprotection under conditions where endocannabinoids are being produced as part of a physiological protective mechanism. In contrast, direct application of cannabinoid receptor agonists to the central nervous system may activate CB receptors indiscriminately and invite unwanted psychotrophic effects.     

Characterization of biotin-anandamide, a novel tool for the visualization of anandamide accumulation

Anandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist of both cannabinoid and vanilloid receptors. During the last two decades, its metabolic pathways and biological activity have been investigated extensively and relatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that has the same lipophilicity of the parent compound. In addition, by means of biochemical assays and fluorescence microscopy, we show that b-AEA is accumulated inside the cells in a way superimposable on that of AEA. Conversely, b-AEA does not interact or interfere with the other components of the endocannabinoid system, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEA hydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEA could be a very useful probe for visualizing the accumulation and intracellular distribution of this endocannabinoid.