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frontiers in REVIEW ARTICLE BEHAVIORAL NEUROSCIENCE published: 14 March 2012 doi: 10.3389/fnbeh.2012.00009 The endocannabinoid system: an overview Natalia Battista 12*, Monia Di Tommaso’, Monica Baris and Mauro Maccarrone 12 Department of Biomedical Sciences, University of Teramo, Teramo, Italy European Center for Brain Research (CERC)/Santa Lucia Foundation, Rome, Italy Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata" Rome, Italy Edited by: Upon the identification of anandamide (AEA) in the porcine brain, numerous studies Patrizia Campolongo, Universita contributed to the current state of knowledge regarding all elements that form the degli Studi di Roma La Sapienza, Italy endocannabinoid system (ECS)." How this complex system of receptors, ligands, and Reviewed by: enzymes is integrated in helping to regulate fundamental processes at level of central Tiziana Bisogno, National Research nervous and peripheral systems and how its regulation and dysregulation might counteract Council, Italy disturbances of such functions, is nowadays still under investigation. However, the Bruno M. Fonseca, Instituto de most recent advances on the physiological distribution and functional role of ECS Biologia Molecular e Celular da Universidade do Porto, Portugal allowed the progress of various research tools aimed at the therapeutic exploitation *Correspondence: of endocannabinoid (eCB) signaling, as well as the development of novel drugs with Natalia Battista, Department of pharmacological advantages. Here, we shall briefly overview the metabolic and signal Biomedical Sciences, University of transduction pathways of the main eCBs representatives, AEA, and 2-arachidonoylglycerol Teramo, Piazza A. Moro, 45, Teramo (2-AG), and we will discuss the therapeutic potential of new ECS-oriented drugs. 64100, Italy. -mail: Keywords: anandamide, 2-arachidonoylglycerol, endocannabinoids, metabolic pathways, signal transduction ENDOCANNABINOID SYSTEM: METABOLISM AND acid amide hydrolase-1 (FAAH-1) termed FAAH-1 like anan- TARGETS OF ENDOCANNABINOIDS damide transporter (FLAT) has been reported in neural cells Starting from 1992, when anandamide (AEA) was identified (Fu et al., 2011). After re-uptake, the biological activity of eCBs for the first time in the porcine brain (Devane et al., 1992), is ended by a FAAH, for AEA (Mckinney and Cravatt, 2005), numerous studies contributed to the current state of knowledge and/or by a specific monoacylglycerol lipase (MAGL), for 2- regarding all elements that form the "endocannabinoid system AG (Dinh et al., 2002). Additionally, other enzymes showing (ECS)" (Maccarrone et al., 2010). Endocannabinoids (eCBs) are "amidase signature," such as FAAH-2 (Wei et al., 2006) and the lipid mediators, isolated from brain and peripheral tissues that N-acylethanolamine-hydrolyzing acid amidase (NAAA) (Tsuboi include amides, esters, and ethers of long chain polyunsaturated et al., 2005), which belongs to the choloylglycine hydrolase family, fatty acids; they mimic the action of A’-tetrahydrocannabinol might bind with low affinity and hydrolyse AEA to release arachi- (THC) in different biological processes. Until now, the most donic acid and ethanolamine. Also cyclooxygenase-2 (COX-2), bioactive eCBs are anandamide (arachidonylethanolamide; AEA) different lipoxygenase (LOX) isozymes and cytochrome P450 are and 2-arachidonoylglycerol (2-AG), yet the eCBs family includes able to accept AEA and 2-AG as a substrate, leading to the biosyn- also virodhamine, noladin ether, and N-arachidonoyldopamine thesis of prostaglandin-ethanolamides (Kozak et al., 2002) and (NADA), besides homo-linolenylethanolamide (HEA), docosate- -glyceryl esters (Kozak et al., 2001), hydroxy-anandamides, and traenylethanolamide (DEA), and other cognate compounds such hydroxyleicosatetraenoyl-glycerols (van der Stelt et al., 2002), as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) respectively. For a comprehensive review on alternative pathways (Figure 1). of eCBs see and Rouzer and Marnett (Rouzer and Marnett, 2011). CBs are released "on demand" from membrane phos- eCBs act principally through cannabinoid receptors, that include pholipid precursors and, although AEA synthesis might be type-1 and type-2 (CB, and CB2) receptors; more recently, it has due to several metabolic routes (Muccioli, 2010), N-acylpho- been highlighted the ability of some CB and non-CB ligands to sphatidylethanolamine-specific phospholipase D (NAPE-PLD) is bind also to GPR55 (Glucksmann and Weich, 1999; Wise and currently considered the major enzyme responsible for AEA Brown, 2001; Drmota et al., 2004; Pertwee, 2007; Ryberg et al., production (Okamoto et al., 2009), whereas a specific phos- 2007; Lauckner et al., 2008), thus suggesting that the latter pro- pholipase C followed by the activity of the sn-1-diacylglycerol tein might act as a novel "type-3 (CB3)" cannabinoid receptor lipase (DAGL) is responsible for 2-AG synthesis (Ueda et al., (Moriconi et al., 2010). CB receptors are members of the large 2011). The cellular uptake from the extracellular to the intra- family of heptahelical G protein coupled receptors (GPCRs), acti- cellular space is ascribed to a purported "endocannabinoid vate Gi/o proteins (Pertwee et al., 2010). Anatomical studies have membrane transporter (EMT)" that is likely to take up both revealed that these receptors display a highly divergent pattern AEA and 2-AG. However, while there is wide experimental evi- of distribution throughout the organism: CBj mainly present in dence to support the concept that AEA transport across mem- the central nervous system (Herkenham et al., 1991) and, on the branes is protein-mediated, conclusive evidence of its molecular other hand, CB2 mainly distributed in peripheral and immune identity is still lacking. Very recently, a partly truncated fatty cells (Munro et al., 1993). This topographical dichotomy has been Frontiers in Behavioral Neuroscience March 2012 | Volume 6 | Article 9 | 1 

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Battista et al. The endocannabinoid system: an overview W-Arachidonoylethanolamine 2-Arachidonoylglycerol (Anandamide, AEA) (2-AG) -OH – NH N-Arachidonoyldopamine 2-Arachidonoylglycerylether O-Arachidonoylethanolamine (NADA) (Noladin ether) (Virodhamine) OH LOH N-Palmitoylethanolamine (PEA) N-Oleoylethanolamine (OEA) FIGURE 1 | Chemical structures of biologically active eCBs and of the eCB-like compounds. AEA 2-AG AA Glycerol AIRPV1 NAPE PLD EtNHa PAGL CBRS FAAHOH PPAR MAGL Inside Outside FIGURE 2 | Schematic representation of the main elements that hydrolyzed by a cytosolic monoacylglycerol lipase (MAGL) that releases constitute the endocannabinoid system. The synthesis of glycerol and AA. A purported endocannabinoid membrane transporter (EMT) W-arachidonoyl-ethanolamine (AEA) is due to the activity of a NAPE-specific clears AEA and 2-AG from the extracellular space, and takes them up into the phospholipase D (NAPE-PLD), whereas a fatty acid amide hydrolase (FAAH) is cell. Both AEA and 2-AG trigger several signal transduction pathways by responsible for its intracellular degradation to ethanolamine (EtNH2) and acting at their targets, CB1, CB2, GPR55, and nuclear PPARs. AEA, but not arachidonic acid (AA). 2-Arachidonoylglycerol (2-AG) is released from 2-AG, binds intracellularly also TRPV1, and thus it is also designated as a true membrane lipids through the activity of diacylglycerol lipase (DAGL), and it is endovanilloid. revised by a number of studies documenting the presence of CB] and De Petrocellis, 2010), is an alternative target for AEA, but not in several non-neuronal cells and tissues (Gong et al., 2006), and for 2-AG. More recently, also nuclear receptors like the peroxi- of CB2 in the brain stem (van Sickle et al., 2005) and in neuronal some proliferator-activated receptors (PPARs) have been added cells upon exogenous insults (Viscomi et al., 2009). In addition, to the list of eCBs targets, activated under physiological and the non-selective cationic channel type-1 vanilloid receptor (tran- pathological conditions (Pistis and Melis, 2010). A schematic rep- sient receptor potential vanilloid 1, TRPV1), usually activated by resentation of eCBs, their receptors, biosynthetic and catabolic capsaicin and by noxious stimuli-like heat and protons (Di Marzo enzymes, as well as putative transporter, is depicted in Figure 2. Frontiers in Behavioral Neuroscience March 2012 | Volume 6 | Article 9 | 2 

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Battista et al. The endocannabinoid system: an overview eCBS AND THEIR SIGNAL TRANSDUCTION PATHWAYS The activation of GPR55, the purported "CB;" cannabi- The signal transduction pathways coupled to CB, TRPV1, and noid receptor, has been linked to (1) intracellular Ca2+ increase PPAR receptors are summarized in Table 1. Among the effects (Lauckner et al., 2008); (2) activation of the small GTPase pro- elicited by eCBs by binding to CB receptors, we should recall teins RhoA, Rac, and Cdc42 (Ryberg et al., 2007; Henstridge Ca2+ channels inhibition (including N-, P/Q-, and L-type chan- et al., 2009), and (3) ERK phosphorylation (Oka et al., 2007, nels), inhibition of adenylyl cyclase and subsequent decrease of 2009). Additionally, by triggering PPARs, eCBs exert a variety CAMP-dependent protein kinase, which leads to decreased phos- of long-term effects via genomic mechanisms and rapid non- phorylation of the K channels, regulation of ionic currents, acti- genomic actions, which are opposite to those evoked by activation vation of focal adhesion kinase, stimulation of mitogen-activated of "classical" surface cannabinoid receptors (Pistis and Melis, protein kinase (MAPK) cascades (Pertwee, 2006), and specifically 2010). As a consequence, PPARs activation affects several phys- ERK, p38 MAPK cascades (Derkinderen et al., 2001; Gertsch et al., iological and pathological processes, such as lipid metabolism, 2004), and the stimulation of additional intracellular pathways energy balance, and feeding behavior, neuroprotection, epilepsy, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway circadian rhythms, inflammation, addiction, and cognitive func- through CB2 (Molina-Holgado et al., 2002). tions (Pistis and Melis, 2010). However, AEA can also act as a Unlike CB2, CB1 receptors are associated to special mem- modulator of other signaling pathways and, in fact, it has been brane microdomains, called "lipid rafts" (LR) that modulate observed that muscarinic and glutamate receptors have allosteric CB1-dependent signaling pathways. The functional relationship sites for AEA binding (Lanzafame et al., 2004). In this context, between CB] and LR is affected by cholesterol content; in par- it should be underlined that there are several findings show- ticular, membrane cholesterol enrichment in both primary and ing that eCBs modulate the signaling of several neuropeptides immortalized cell lines reduces the binding to CB, and subse- and hormones (Manzanares et al., 1999; Beinfeld and Connolly, quent G-protein dependent signaling through adenylyl cyclase 2001; Ghozland et al., 2002). This highly complex network of and MAPK (Bari et al., 2005). Moreover, the disruption of LRs by interactions is reflected in the multifaceted modulatory effects cholesterol depletion modifies AEA-induced endocytosis of CB1, of eCBs on the regulation of brain and behavioral functions which apparently loses the capacity to be directed toward the (Lopez-Moreno et al., 2008). lysosomal compartment. Therefore, LRs, besides representing a favorable platform to regulate CBj signaling, might also represent PHYSIOLOGICAL ACTIONS OF ECS AND THERAPEUTIC a cellular device for its intracellular trafficking (Sarnataro et al., PERSPECTIVES 2005; Dainese et al., 2007). The general model to explain the neu- The presence of ECS in vertebrates, mammals, and humans romodulatory actions of AEA involves the release of eCBs from implies a role in several physiological processes, including a postsynaptic neuron upon stimulation, then the back diffusion appetite, cancer, cardiovascular diseases, fertility, immune func- to presynaptic terminals, where AEA activates CB1 receptors, thus tions, memory, neuroprotection, and pain modulation (Ligresti modulating neuronal membrane permeability to Caffe K ions et al., 2009; Maccarrone et al., 2010) (Figure 3). and the activity of adenylyl cyclase. The final outcome is a mod- In the last 10 years, it has become clear that a dysregula- ified action of neurotransmitters (Di Marzo and De Petrocellis, tion of ECS is connected to pathological conditions, and thus 2010). its modulation through inhibition of metabolic pathways and/or agonism or antagonism of its receptors has an enormous potential for research and intervention in multiple areas of human health. Table 1 | Signal transduction pathways triggered by eCBs at different target receptors. Receptor Effect Neuroprotection CB1 and CB2 | Adenylyl cyclase Memory 1 Focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK Immune functions Cancer ERK, p38 through CB1, and P13K/Akt through CB2 1 K+ channels I Ca2+ channels GPR55 Intracellular [Ca2+] Appetite RhoA, Rac, and Cdc42 Cardiovascular disease 1 ERK phosphorylation TRPV1 1 Intracellular [Ca2+ ] Fertility 1 Caspases Cytochrome c release 1 Mitochondrial uncoupling 1 Pro-apoptotic kinases PPARs 1 ROS 1 Tyrosine kinases FIGURE 3 | The involvement of ECS in some pathophysiological conditions. Adiponectin and lipoprotein lipase Frontiers in Behavioral Neuroscience March 2012 | Volume 6 | Article 9 | 3 

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Battista et al. The endocannabinoid system: an overview Therefore, based on the therapeutic potential of THC, known and O-1966, two selective CB2 agonists, administrated 1 h before since centuries as medicine for its palliative effects in several transient middle cerebral artery occlusion, significantly decreased pathologies, plant-derived cannabinoids, synthetic cannabinoids, the mobilization of white blood cells and their adherence to vas- and eCBs have been tested as novel therapeutics in a wide range cular endothelial cells, reduced the infarct size, and improved of clinical trials. motor function after transient focal ischemia (Zhang et al., 2007, The neuroprotective effect of eCBs might be mediated by 2009). either CB1- or CB2-dependent mechanisms. Research studies According to these observations, pain management is prefer- using cb, knock-out mice showed an increased mortality ably handled using CB2 agonists, such as HU-308 and AM-1241, rate and an increased infarct area in cerebral ischemia mod- which display significant relief in inflammatory and neuropathic els (Parmentier-Batteur et al., 2002). It has been reported that pain models, without exhibiting central nervous system side the administration of the CB1 synthetic agonist WIN 55.212-2 effects (Hanus et al., 1999; Yao et al., 2006). In this context, new attenuated the neurological damage and reduced infarct size in selective CB2 receptor modulators, designed by Glaxo Smith Kline artery occlusion induced in rats (Nagayama et al., 1999), and as derivatives of pyrimidinecarboxamide, have been tested as additionally it reduced the glial damage after hypoxic-ischemic good clinical candidates to treat inflammatory, acute, and chronic brain injury in preterm lambs (Alonso-Alconada et al., 2010). The pain (Giblin et al., 2007, 2009). presence of CB2-positive cells in the brain during injury and in In the past, several reports documented that the selective inflammatory neurodegenerative disorders might provide a novel pharmacologic antagonism of the CB, receptor improves lipid strategy for cannabinoid-mediated intervention against stroke- abnormalities associated with obesity, as well as neurodegenera induced neurodegeneration, without the unwanted psychoactive tive diseases and nicotine or alcohol dependence (Centonze et al., effects of CB1 receptor stimulation (Cunha et al., 2011). 0-3853 2007; Di Marzo, 2008). Following the good outcome obtained in Table 2 | Chemical structures and therapeutic potential of some ECS-targeted molecules. Chemical structure Compound ECS target Diseases References PF-04457845 FAAH Pain, Osteoarthritis Ahn et al., 2011 URB 597 Anxiety, Cannabis dependence, Bortolato et al., 2007 Hyperalgesia SR141716A CB1 Eating disorder Christopoulou and Kiortsis, 2011 WIN 55.212-2 Ischemic stroke, Brain injury Nagayama et al., 1999; Alonso-Alconada et al., 2010 HU-308 CB2 Neuropathic pain Hanus et al., 1999 GSK554418A Acute/chronic pain Giblin et al., 2009 GW842166X Inflammatory pain Giblin et al., 2007 Frontiers in Behavioral Neuroscience March 2012 | Volume 6 | Article 9 | 4 



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Battista et al. The endocannabinoid system: an overview various clinical trials, the best known CB, blocker SR141617A, (Ahn et al., 2011). It is noteworthy that FAAH inhibitors, because also called rimonabant (and commercially known as Acompliaof their own pharmacological properties, are attractive reme- was released on the worldwide market as anti-obesity drug, but dial also for cannabis dependence; in fact, they do not appear only few months later it was withdrawn because of increased rates to evoke tolerance following long-term administration, and they of depression, anxiety, and suicide among patients who received it do not display significant abuse liability (Clapper et al., 2009). (Christopoulou and Kiortsis, 2011). In addition, further concerns Table 2 reports some agonists, antagonists, and/or inhibitors of were raised considering the possible side effects of this weight ECS designed for the treatment of several pathological conditions. loss pill on the reproductive functions and human infertility (Bari et al., 2011). CONCLUSION Alternative strategies to treat pain syndromes, such as neu- Almost 20 years after the identification of AEA, all members of ropathic pain, fibromyalgia, but also spontaneous abortion, ECS are nowadays considered intriguing targets for the devel- headache, psychiatric disorders, and neurodegenerative diseases, opment of selective and specific compounds able to modulate are based on the enhacement of the eCB tone, through the inhi- human pathophysiology. A deeper and more detailed under- bition of eCBs-hydrolyzing enzymes (Lichtman and Chapman, standing of proteins involved in eCBs metabolism and signal- 2001). The most promising FAAH inhibitor seems to be URB597 transduction pathways could help to design compounds that (also named KDS-4103), which has biochemical and behavioral might prolong the activity of eCBs in a time- and site-dependent effects during both sub-acute and chronic treatments. In rodents, way, excluding undesired psychotropic effects, and to develop once-daily dosing of URB597 for five weeks elicits antidepress transgenic mice, where different ECS elements can be knocked sant effects in chronically stressed animals, without altering CB1 down or knocked in, allowing innovative therapeutic strategies in receptor mRNA levels (Bortolato et al., 2007). Pfizer and Vernalis a vast panorama of pathologies. pharmaceutical companies focused on FAAH as main target to design and develop new molecules (PF-04457845 and V158866, ACKNOWLEDGMENTS respectively), that are being tested in clinical studies as potential This work was partly supported by Fondazione TERCAS therapies for a range of pain disorders, including osteoarthritis (2009-2012 project to Mauro Maccarrone). REFERENCES Beinfeld, M. C., and Connolly, K. F. (2011). Endocannabinoid sys- Dinh, T. P., Freund, T. F., and Piomelli, Ahn, K., Smith, S. E., Liimatta, M. (2001). Activation of CBI cannabi- tem in cardiovascular disorders – D. (2002). A role for monoglyceride B., Beidler, D., Sadagopan, N., noid receptors in rat hippocam- new pharmacotherapeutic opportu lipase in 2-arachidonoylglycerol Dudley, D. T., Young, T., Wren, P., pal slices inhibits potassium-evoked nities. J. Pharm. Bioallied. Sci. 3, inactivation. Chem. Phys. 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