Palmitoylethanolamide in CNS health and disease - Semantic Scholar

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Pharmacological Research 86 (2014) 32–41

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Review

Palmitoylethanolamide in CNS health and disease Giuseppina Mattace Raso, Roberto Russo, Antonio Calignano ∗ , Rosaria Meli Department of Pharmacy, University of Naples “Federico II”, Naples, Italy

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Article history: Received 12 March 2014 Received in revised form 7 May 2014 Accepted 8 May 2014 Available online 17 May 2014 Keywords: Acylethanolamide Peroxisome proliferator-activated receptor Neurodegenerative disease Inflammation Pain Behavior Food intake

a b s t r a c t The existence of acylethanolamides (AEs) in the mammalian brain has been known for decades. Among AEs, palmitoylethanolamide (PEA) is abundant in the central nervous system (CNS) and conspicuously produced by neurons and glial cells. Antihyperalgesic and neuroprotective properties of PEA have been mainly related to the reduction of neuronal firing and to control of inflammation. Growing evidence suggest that PEA may be neuroprotective during CNS neurodegenerative diseases. Advances in the understanding of the physiology and pharmacology of PEA have potentiated its interest as useful biological tool for disease management. Several rapid non-genomic and delayed genomic mechanisms of action have been identified for PEA as peroxisome proliferator-activated receptor (PPAR)-␣ dependent. First, an early molecular control, through Ca+2 -activated intermediate- and/or big-conductance K+ channels opening, drives to rapid neuronal hyperpolarization. This is reinforced by the increase of the inward Cl− currents due to the modulation of the gamma aminobutyric acid A receptor and by the desensitization of the transient receptor potential channel type V1. Moreover, the gene transcription-mediated mechanism sustains the long-term anti-inflammatory effects, by reducing pro-inflammatory enzyme expression and increasing neurosteroid synthesis. Overall, the integration of these different modes of action allows PEA to exert an immediate and prolonged efficacious control in neuron signaling either on inflammatory process or neuronal excitability, maintaining cellular homeostasis. In this review, we will discuss the effect of PEA on metabolism, behavior, inflammation and pain perception, related to the control of central functions and the emerging evidence demonstrating its therapeutic efficacy in several neurodegenerative diseases. © 2014 Published by Elsevier Ltd.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct and indirect mechanisms of action of PEA in CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological and therapeutic role of PEA in CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammation and adaptive immune response in the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PEA and acute and chronic pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PEA in CNS disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of PEA and PPAR␣ on metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AE, acylethanolamide; PEA, palmitoylethanolamide; CNS, central nervous system; PPAR, peroxisome proliferator-activated receptor; BKCa, calciumactivated intermediate- and/or big-conductance potassium channels, IKCa; GABA, gamma-aminobutyric acid; OEA, oleoylethanolamide; FAAH, fatty acid amide hydrolase; AEA, anandamide; CB, cannabinoid receptor; NF-␬B, nuclear factor-␬B; GPR, G protein-coupled receptor; TRPV1, transient receptor potential channel type V1; 2-AG, 2arachidonoylglycerol; DRG, dorsal root ganglia; StaR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 side-chain cleavage; AD, Alzheimer’s disease; NOS, nitric oxide synthase; COX-2, cyclooxygenase-2; OVX, ovariectomized; AMPK, AMP-activated protein kinase. ∗ Corresponding author at: via D. Montesano, 49-80131 Naples, Italy. Tel.: +39 081 678441; fax: +39 081 678403. E-mail address: [email protected] (A. Calignano). http://dx.doi.org/10.1016/j.phrs.2014.05.006 1043-6618/© 2014 Published by Elsevier Ltd.

G. Mattace Raso et al. / Pharmacological Research 86 (2014) 32–41

Introduction Palmitoylethanolamide (PEA) belongs to the class of fatty acid ethanolamides (or acylethanolamides, AEs), formed “on demand” from membrane phospholipids [1]. The existence of AEs in the mammalian brain has been known for decades, but only in the last years the putative biological functions of the most abundant AE species have been delineated [2]. The presence of AEs and their cognate precursors in various tissues and their pharmacological properties suggest that these molecules play a role as paracrine or autocrine regulators of peripheral functions, therefore AEs were initially called Autacoid Local Injury Antagonism Amides or ALIAmides [3]. Later on, their possible role as neuromodulators was proposed by several research groups after finding that AEs are produced in discrete areas of the brain under both physiological and pathological conditions [4]. In fact, all AEs accumulate during neuronal injury, and in particular PEA and oleoylethanolamide (OEA) have neuroprotective effects [5]. PEA is abundant in the central nervous system (CNS) and conspicuously produced by glial cells [2,6,7]. PEA has been studied extensively for its anti-inflammatory and neuroprotective effects, mainly in models of peripheral neuropathies [8,9]. In tissues PEA levels depend on enzymatic formation mainly from N-palmitoylethanolamine-phospholipids and on its degradation by fatty acid amide hydrolase (FAAH) [10] or N-acylethanolamine-hydrolyzing acid amidase (NAAA) in inflammatory status [11]. Considering that NAAA preferentially hydrolyzes PEA over other AEs, selective NAAA inhibitors that may increase local levels of endogenous PEA were expected to be anti-inflammatory and analgesic drugs [11]. The availability of specific inhibitors of FAAH has allowed a better understanding of the effects mediated by increased levels of AEs at the central level [12]. These inhibitors can increase levels of all AEs including the classic cannabinomimetic arachidonoylethanolamide or anandamide (AEA). Therefore, their concomitant increase can synergistically contribute to several pharmacological effects. In the past it was proposed the idea that PEA was a cannabinoid receptor (CB)2 agonist [13], conversely, Lo Verme et al. [14] showed that PEA had no effect in CB2 knockout mice. To date, it is widely recognized that the main PEA pharmacological effects are mediated by activation of peroxisome proliferatoractivated receptor (PPAR)-␣ [15]. Initially identified as a receptor for peroxisome-stimulating plasticizers in the liver, PPAR-␣ is an ubiquitous transcription factor, activated by various endogenous fatty acid derivatives, including PEA and its monounsaturated analog, oleoylethanolamide (OEA) [14]. The discovery of PPAR-␣ in distinct areas of the brain, has opened a new scenario to explore the possible activity of these AEs in the central nervous system [16]. PPARs are regulators of gene networks, which control pain and inflammation, by switching off the nuclear factor-␬B (NF-␬B) signaling cascade, a key element in the transcription of genes, leading to the synthesis of proinflammatory mediators [17]. These receptors are responsible for the delayed genomic effects of AEs. Beside PPAR-␣ PEA can activate several different receptors and inhibit some ion channels involved in rapid response to neuronal firing, e.g., vanilloid receptor and K+ channels (Kv4.3, Kv1.5) [18]. Recently, the discovery that PEA, through activation of PPAR␣, stimulates de novo neurosteroid synthesis [19], suggests that two separate but converging mechanisms could contribute to the central effect of PEA, an early molecular control through calcium-activated intermediate- and/or big-conductance potassium channels (IKCa and BKCa) opening, silencing neuronal firing [15], and thereafter a reinforcing effect mediated by gene transcription and hence neurosteroid synthesis [20,21]. It is noteworthy that neuronal hyperpolarization by K+ efflux is also reinforced by inward

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chloride currents, sustained by the positive modulation of gammaaminobutyric acid (GABA)(A) receptors [20]. Therefore, PEA may influence several biological functions and pathological conditions, such as epilepsy and pain, at least in part, through GABA(A) receptors modulation. In fact, using experimental models in mice, it has been observed that pharmacological blockade of neurosteroid biosynthesis reduces PEA’s ability to alleviate pain-related behaviors elicited by chemical tissue damage or acute inflammation produced by carrageenan. In particular, in the spinal cord, PEA causes an increase in the levels of allopregnanolone, with which it shares a similar pharmacological profile [19]. The anti-inflammatory actions of PEA, leading to a reduction of peripheral and central sensitization, are mediated by neuronal and non-neuronal cells [22]. The latter comprise glia (in particular, astrocytes and microglia) as well as peripheral and central mast cells. In particular, mast cells in the CNS have been shown to play a pivotal role in inflammatory and neurodegenerative diseases [8]. Emerging evidence suggests that the cross-talk between mast cells and glia has an important role in neuroinflammation, exacerbating the acute inflammatory response, accelerating neurodegenerative disease progression and promoting pain perception [22]. In this context, PEA can function in maintaining cellular homeostasis, not only by inhibiting mast cell activation in the CNS and regulating microglial cell activity, but also by blocking peripheral mast cell activation and hence signaling pathways from the periphery to the brain [22]. PEA has been detected in the CNS and its concentration shows significant changes during pathological conditions, as shown in glutamate-treated neocortical neurons ex vivo [23], in cortex after CNS injury [9,24] and in microdialysis fluid from a patient with stroke [25]. Although the role of PEA in the CNS remains in part unexplored, in this review we will discuss the main evidence addressing the physiological and pharmacological properties of PEA in CNS. The complex and generous profile of PEA activity may thus explain its broad potential in treating different central disorders related to pain and inflammation.

Direct and indirect mechanisms of action of PEA in CNS Evidence indicates that PEA is an important anti-inflammatory, analgesic, and neuroprotective mediator acting at several molecular targets in both central and peripheral nervous systems [14,17]. Synthesis and degradation of PEA occur in various cell types, including those relevant for chronic pain and inflammation signaling, such as immune cells, neurons and microglia [22]. Initially, it was found that in some cases, PEA could potentiate the effect of AEA on CB or vanilloid receptor 1 [27–29]. This so-called “entourage effect” could be mediated by PEA competitive inhibition of AEA hydrolysis on FAAH [30] and/or direct allosteric effect of PEA on transient receptor potential channel type V1 (TRPV1), also known as the vanilloid receptor type 1 [31,32]. However, PEA is not a ‘classical’ endocannabinoid, according to the current pharmacological classification rules [33]. Most of the biological functions of AEA and 2-arachidonoylglycerol (2-AG), two major endocannabinoids, are mediated by the two G protein coupled CB1 and CB2, but there has been a steady stream of pharmacological evidence for the existence of other orphan receptors, to date considered additional cannabinoid receptors [34], such as G protein-coupled receptor (GPR)55 and GPR119 [35,36]. PEA is reported to have affinity for orphan receptors such as GPR55 [37], since PEA was shown to mediate GTP gamma S formation in cells transfected with the human cDNA for this receptor [35]. GPR55 activation has been suggested to account for some of the non-CB1, non-CB2 effects reported for certain cannabinoid ligands [38]. Moreover, it has been demonstrated that PEA could also bind GPR119 [36]. This receptor is expressed at

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particularly high levels in the gut and also found in immune cells. Initially considered a “third” endocannabinoid receptor, despite sharing little homology with CBs, it has been linked to inflammatory processes as well [39]. Previously, Overton and colleagues [36] have shown that OEA could be an agonist for GPR119, while PEA and AEA displayed very weak affinity. This receptor is particularly known for its involvement in glucagon-like peptide-1 secretion from enteroendocrine cells in the gut and hence glucose homeostasis [34]. One study has also reported minor expression of GPR119 in the CNS [40], but at present, no clear function for this receptor in the brain has been reported. Lo Verme and colleagues [14,15] have characterized PEA as a ligand and agonist of PPAR-␣, that is also the target of other AEs, such as OEA and stearoylethanolamide, with EC50 values dependent on the type of assay and animal species. The anorexic effect of OEA, known to be mediated by activation of PPAR-␣ in the intestine [41], and its weight losing effect is abolished in PPAR-␣ knockout mice [14]. On the other hand, PEA shows anti-inflammatory and analgesic effects mediated by activation of PPAR-␣ [14,15], reducing NF-␬B activation and pro-inflammatory enzyme synthesis [17]. The rapid effects of PEA may not be mediated through PPAR␣ transcription activity but, instead, through PPAR-␣-dependent non-genomic activation of protein kinases [15,42]. Our results suggested that ligand-activated PPAR-␣ could suppress pain responses by altering the gating properties of IKCa and BKCa channels in dorsal root ganglia (DRG) neurons, in agreement either with the presence of PPAR-␣ in this first relay station of pain or with the suppression of nocifensive behavior even when PEA was locally administered in the paw [15]. Recently, it has been shown that AEs inhibit Kv1.5 and Kv4.3 channels by interacting with an extracellular binding site in fibroblast cell and CHO culture model, respectively [43,44]. Interestingly, in the brain, Kv1.5 channel is involved in activation of microglial and dendritic cells [45,46] and Kv4.3 channels, found in hippocampal interneurons and in pyramidal and GABAergic cortical neurons, are involved in rhythmic activity, controlling synaptic plasticity [47,48]. Beside the mechanisms above mentioned, the analgesic activity of PEA may also be ascribed to a more direct action on mast cells, via an autacoid local injury antagonism mechanism [3], combining a dual activity both on neurons of nociceptive pathway and on non neuronal cells, such as mast cells in the periphery and glia in the spinal cord. Recently, the demonstration that PEA-induced allopregnanolone synthesis in astrocytes in PPAR-␣-dependent manner, opened a new field to better understand PEA effects in CNS [21]. Indeed, PEA effects were blunted by a selective PPAR-␣ antagonist, or by siRNA-mediated knock down of PPAR-␣ [21]. Previously, we have also showed that PEA modulates pentobarbital-evoked hypnosis by activating PPAR-␣ and inducing de novo neurosteroid synthesis. In particular, an increase in allopregnanolone levels was found in the brainstem of PEA-treated mice, leading to a reinforcement of the hypnotic effect of pentobarbital, due to a positive modulation of GABA(A) receptor by the hormone [19]. Consistently, the antinociceptive activity of PEA was partially reduced in two models of acute and persistent pain, the formalin test and carrageenan-induced paw edema, when 5␣-reductase and cytochrome P450 side-chain cleavage (P450scc) were inhibited. In both models, PEA administration in challenged mice specifically restored the expression of two proteins involved in neurosteroidogenesis, the steroidogenic acute regulatory protein (StAR) and P450scc in the ipsilateral horns of spinal cord, where an increase in allopregnanolone was found [20]. Among neuroactive steroids, allopregnanolone seems to share many of the effects showed by PEA, including analgesic, anticonvulsant, and antiallodynic activities [14,15,49–51]. This hormone has been recognized to be a positive activator of the GABA(A) receptor, increasing the inward

Cl− currents that lead to neuronal hyperpolarization, also achieved by PEA through the opening of the IKCa and BKCa channels. In summary, the main direct and indirect mechanisms of action of PEA are illustrated in Fig. 1.

Physiological and therapeutic role of PEA in CNS Inflammation and adaptive immune response in the CNS Inflammation often accompanies tissue injury and the pathogenesis of many chronic disease, including those of autoimmune nature [22,52]. Local functional interplay between nervous and immune systems can finely regulate the severity and duration of the inflammatory response. Dysregulation of neuroimmune interactions can prompt the propagation and exacerbation of inflammatory reactions. Pro-inflammatory cytokines play a pivotal role in this crosstalk, as they regulate host responses to infection, inflammation and reactions to stress or trauma. Astrocytes, and even more microglia, constitute an important source of inflammatory mediators in several pathological conditions ranging from chronic pain [53,54] and epilepsy [55] to neurodegenerative disorders, such as Alzheimer’s disease (AD) [56–59], Parkinson’s disease [60,61] and amyotrophic lateral sclerosis [62] and they may also contribute to schizophrenia, depression and other psychiatric disorders [63,64]. Indeed, microglia is an interesting player in the modulation of neurogenesis both in healthy and injured brain. Microglial cells are activated by antigens or changes in the brain homeostasis, transforming innate immunity into an adaptive immune response, recruiting systemic immune cells, scavenging dead cells and secreting factors involved in neuron survival [65]. However, microglia-mediated neuroinflammation is able to compromise healthy brain in senescence [66]; altered and prolonged activation of glia can induce autoimmune response, contributing to brain injury and neuronal cell death [67], providing a link between neuroinflammation and neuropathic pain [22]. Both microglia and astrocytes also respond to pro-inflammatory signals derived from peripheral immune cells, such as mast cells [22]. Like macrophages, these cells reside in the brain of many species, and move into the healthy brain through the blood brain barrier, but cross mainly the barrier in inflammatory and pathological conditions. In the brain mast cells, not only produce a wide spectrum of inflammatory mediators, but they are also capable of phagocytosis and antigen presentation, and can modulate the adaptive immune response in astrocyte and microglia. The physiological role and pharmacological properties of PEA in the CNS are less clear than those of AEA and 2-AG. The levels of PEA measured in whole mouse or rat brain (100–550 pmol/g) [9,68–70] were found to be increased after pathophysiological stimuli. In particular, beside neurons [71], astrocytes are also able to produce PEA in a ratio of 2:1 relative to AEA [6]. Moreover, PEA is produced and hydrolysed by microglia [7], inhibits mast cell activation [13,72] and its levels are increased in glutamate-treated neocortical neurons ex vivo [23] and in cortex after CNS injury [9,24,25], as well as in muscle dialysate from women with chronic neck–shoulder pain [73]. Furthermore, PEA reduces mast cell activation by local autacoids mechanism and decreases neuron loss caused by mast cell activation, and not by induction of nitric oxide synthase (NOS) in astrocytes [74]. Moreover, PEA is able to reduce oxidative stress in challenged C6 glioma cells and this effect is in part mediated by neurosteroid synthesis [21], suggesting a role for PEA in peroxide removal from the brain counteracting the effects of reactive oxygen species damage that may be involved in several neuropathological disorders [75]. Recently, PEA was found to control mast cell

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Fig. 1. Direct and indirect mechanisms of action of PEA. The indirect mechanism would involve PEA potentiation of AEA effects through (a) a competitive inhibition of AEA metabolism by FAAH, leading to an increase in AEA levels and its binding to CB1 (b); an allosteric activity on TRPV1, increasing AEA affinity to this receptor, and inducing later TRPV1 desensitization. (c) Through a PPAR-␣ dependent non-genomic mechanism, PEA increases the gating properties of IKCa and BKCa channels, resulting in a fast reduction of neuronal firing. Moreover, PPAR-␣ activation, through a genomic mechanism, increases the expression of StaR and P450scc, involved in cholesterol transfer into the mitochondria and its metabolism in pregnanolone, respectively. The resulting increase in allopregnanolone levels leads to a positive allosteric activation of GABA(A) receptors, an increase in Cl− currents and a reinforcing effect on the reduction of neuronal firing. PEA anti-inflammatory effect appears to be related to a cytoplasmatic complex, that reduces NF-␬B transcription activity, dampening the transcription of pro-inflammatory gene. PEA, palmitoylethanolamine; AEA, anandamide; FAAH, fatty acid amide hydrolase; PA palmitic acid, EA, ethanolamine; CB1, cannabinoid receptor 1; TRPV1, transient receptor potential channel type V1; PPAR-␣, peroxisome proliferatoractivated receptor-␣; I/BKCa, calcium-activated intermediate- or big-conductance potassium channels; StaR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 side-chain cleavage; GAGA-A, gamma-aminobutyric acid receptor A; NF-␬B, nuclear factor-␬B.

degranulation and reduce nerve fiber formation in carrageenininduced granuloma, modulating mast cell activation in close proximity to nerves fibers, resulting in the reduction of mechanical allodynia [76]. The control exerted by PEA on mast cell activation is also reinforced by evidence showing that PEA significantly reduced the production and the release of several mediators by mast cells, such as TNF␣ and neurotrophic factors, like nerve growth factor, in an in vivo model of neuropathic pain [77]. A central mechanism for PPAR-␣ agonists, including PEA and GW7647, has been shown in controlling peripheral inflammation. In fact, we showed anti-inflammatory and anti-edemigenic effects of central administration of these compounds, in mice subjected to carrageenan-induced paw edema. The increased PPAR-␣ levels and the reduced expression of cyclooxygenase (COX)-2 and inducible NOS (iNOS) were evidenced in the spinal cord, which represents the main relay station of the neural firing between the inflamed area and the CNS. Indeed we found that PEA suppressed NF-␬B signaling in DRG nuclei, which may explain the mechanisms by which PEA down-regulates COX-2 and iNOS expression in sciatic nerves. We assume that these enzymes are an outcome measure of NF-␬B nuclear activity in DRG, because protein levels in nerve fibers are strictly dependent on DNA transcription in DRG cell bodies [17,78]. PEA is also able to attenuate the degree of peripheral inflammation in another animal model of peripheral nerve injury, the chronic constriction injury, which is associated to a profound local inflammatory response that involves T cells and macrophages [79]. After nervous system trauma, PEA reduces

edema and macrophage infiltration [80], evaluated as CD86 positive cells [81], responsible to produce high levels of oxidative metabolites (e.g., nitric oxide and superoxide) and proinflammatory cytokines [82]. Therefore, the antihyperalgesic and neuroprotective properties of PEA are related to its anti-inflammatory effect and its ability to prevent macrophage infiltration in the nerve. Overall these observations suggest a key role of PEA in maintaining cellular homeostasis when faced with external stressors or pathological stimuli provoking inflammation response and tissue damage. PEA and acute and chronic pain Although pain perception is thought to be controlled mainly by neurotransmitter systems that operate within the CNS, antinociceptive mechanisms also occur in peripheral tissues. In addition to its known anti-inflammatory activity, PEA elicited analgesia in acute and inflammatory pain [17,83–85]. In fact, it has been reported that exogenous administration of PEA exerted antinociceptive effects in various models of inflammatory and neuropathic pain in the mouse, such as carrageenan-induced hyperalgesia [20,78], and chronic constriction injury [81]. Evidence indicates that, in animal models of neuropathic pain, hyperalgesia and allodynia were characterized by an increase in classical endocannabinoids (AEA and 2-AG) in nuclei involved in descending nociceptive pathways, as well as other brainstem regions more involved in the emotional components of chronic pain, while PEA levels were significantly decreased [86]. These

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results indicate not only a role for endocannabinoid elevation in lessening pain perception, but also an involvement of PEA, whose decrease may modulate pain threshold. On the other hand at the peripheral level, Jhaveri et al. [87] found significantly decreased levels of both AEA and PEA in the hindpaw at the peak of carrageenan-induced hyperalgesia, possibly related to either increased metabolism of AEA and PEA or their decreased synthesis. Importantly, the observed elevation of AEA or PEA, via inhibition of FAAH or COX-2, was associated with anti-nociceptive effects, which were blocked by GW6471, a PPAR-␣ antagonist, implicating one more time this receptor in pain transmission [87]. To date, the majority of studies on the role of PPAR-␣ in pain processing have focused on peripheral or spinal sites of action of endogenous ligands of the receptor, while relatively few studies have investigated the role of supraspinal PPAR-␣ signaling in pain, showing its role in reducing pain responding [17,78,85]. A significant anti-inflammatory and anti-hyperalgesic action of endogenous and synthetic PPAR-␣ agonists has been demonstrated through central administration [17,78]. In mice receiving icv PEA, the reduced levels of PPAR-␣ in DRG following carrageenan treatment were restored to basal levels, suggesting that supraspinal administration of PEA modulates PPAR-␣ expression in DRG through a descending pathway [17]. These observations agree with our previous report, in which we found that supraspinal injection of PEA reduced COX-2 and iNOS expression at spinal level after peripheral inflammatory challenge [78]. Thus, the reduction of PPAR-␣ expression at several sites in the central (spinal) or peripheral nervous system (DRG) are proposed to be involved in peripheral damage and related hyperalgesia signaling and maintenance. More recently, Okine et al. [88] have studied the modulating effect of pain perception by PPAR-␣ in medial prefrontal cortex. This area, in fact, is involved in supraspinal affective and cognitive modulation of pain [89–91] and in addition it shows high expression of PPAR-␣ [16]. In this study, the authors found a reduction in PEA and OEA levels in formalin-induced nociception, and that a PPAR-␣ antagonist is able to lessen pain responses. The authors hypothesize in the medial prefrontal cortex a permissive role for PPAR-␣ never described before, for pain perception [88]. A conceivable interpretation, according with their data, is the blunting of presynaptic inhibition of PPAR-␣, through its antagonist, causing an increase in serotoninergic and noradrenergic tones, that are systems known to be controlled by prefrontal cortex in modulating gate threshold at spinal level. All PPAR subtypes (alpha, beta/delta and gamma) have been found in the CNS, where they exhibit specific patterns of localization [16], including DRG sensory neurons and glial cells. As such, PEA may advocate several strategies to achieve analgesic effects including ion channels activity [15,92], and activation of nuclear receptors [15], or involvement of other targets [77]. In fact, PEA has been suggested to increase the activity and/or reduce the degradation of endogenous agonists of CB receptors, the so-called ‘entourage’ effect’ [30], reinforcing their actions at the level of other targets, such as the TRPV1 channels [93]. In TRPV1transfected cells, PEA potentiates AEA effects on [Ca2+ ]i responses, an effect attributed to its ability to increase TRPV1 affinity for AEA [27], or to interfere with AEA synthesis [14], rather than to the inhibition of its hydrolysis [29,94]. Molecular experiments aimed at investigating the mechanism underpinning PEA analgesic activity, revealed that this AE at low concentrations significantly reduces TRPV1 currents induced by capsaicin in F11 cells through a PPAR-␣-mediated mechanism [95]. Thus, it is conceivable that the mechanism by which PEA could modulate ion channels current involves both big and intermediate potassium channels, and TRPV1 desensitization. Accordingly, changes in the endogenous levels of PEA have been shown to alter the acute [Ca2+ ]i responses evoked by depolarizing conditions in

sensory neurons [92], underlying also in this model a rapid PPAR␣-mediated non-genomic mechanism. Another evidence for a role of PEA in the CNS is the ability of this compound to modulate neurosteroidogenesis in the spinal cord in pain conditions [20]. The involvement of de novo neurosteroid synthesis in the modulation of pain behavior by PEA was investigated in two models of acute and persistent pain, the formalin test and carrageenan-induced paw edema. The antinociceptive activity of PEA was blunted in PPAR-␣ deficient mice, and partially reduced when the animals were treated with aminoglutethimide or finasteride, implying that de novo neurosteroid synthesis is involved in the antinociceptive effect of PEA. Accordingly, in the spinal cord, allopregnanolone levels were increased by PEA treatment both in formalin- and carrageenan- challenged mice. PEA administration restored the expression of two proteins involved in neurosteroidogenesis, StaR and P450scc, in the ipsilateral horns of spinal cord, without affecting expression of these proteins in the contralateral side [20]. These data provide new information about the pleiotropic mechanisms by which PEA influences pain behavior. Very recent studies support the existence of complex interrelationships between PPARs and oxytocin (OXT), which would play an important role in the regulation of pain, food intake and lipid metabolism [96,97]. OXT is a neuropeptide that is produced in the supraoptic and paraventricular nuclei of the hypothalamus and released into circulation by way of magnocellular neurons extending down to the posterior pituitary. OXT is released from paraventricular neurons projects throughout the CNS to the amygdala, the striatum, the raphe nuclei, and the superficial and deep lamina of the dorsal horn, sites, these, at which pain signals are modulated in a dynamic manner. Therefore, another PPARmediated mechanism of PEA could also include the production and/or release of OXT. Our preliminary unpublished data show a reversion by atosiban, an inhibitor of OXT receptor, of PEA antinociceptive effect in formalin test in mice. This is consistent with recent data by Romano et al. [96], showing that OEA produces an increase of OXT immunoreactivity in the neurohypophysis and in the paraventricular nucleus. Although this hypothetical mechanism requires further study to be demonstrated, overall, these findings propose a novel AEs role in controlling OXT neurosecretion. Accordingly, our recent results show that central administration (icv) of OXT produces a significant antihyperalgesic effect due to endocannabinoid activation [98]. Several papers have shown that OXT peptide levels in humans and in animals have a relationship with pain and analgesia [99]. Yang [100] demonstrated that in cerebral spinal fluid and plasma the concentration of OXT was lowered in patients with acute and low back pain and that OXT administration relieves low back pain. PEA in CNS disorders The observation that PEA may be protective in a delayed postglutamate paradigm of excitotoxic death [8] opened the way to several in vivo and in vitro studies on the neuroprotective properties of PEA. The accumulation of this substance in brain tissue following injury suggested a neuroprotective function, which seemed to be related to its general cellular actions [5]. Moreno et al. [16] provided an immunohistochemical analysis of PPARs and RXRs in the CNS in the adult rat brain in areas involved in complex processes, such as aging, neurodegeneration, learning and memory, where a role for PEA in neuroprotection was observed [26]. The growth of knowledge about the molecular mechanisms underlying AD has highlighted the role of neuroinflammation in the pathophysiology of this disorder. AD is classically characterized by the deposit of misfolded proteins: the extracellular accumulation of beta amyloid peptide (A␤), and the formation of intracellular neurofibrillary

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tangles. Scuderi et al. [101] demonstrated that PEA exhibits antiinflammatory properties able to counteract A␤-induced astrogliosis. In particular, PEA reduced the A␤-induced activation of p38 and JNK, and transcription factors, NF-␬B and AP-1. This evidence helps to identify the molecular apparatus by which PEA contributes to down-regulate both astroglial reaction and pro-inflammatory signal overproduction. Subsequently, a first in vivo study by D’Agostino et al. [102] showed the ability of PEA to rescue experimentally induced memory deficit, evidencing the obligatory role of PPAR-␣ for the neuroprotective effect of PEA against amyloid toxicity. The finding that GW7647, a synthetic PPAR-␣ agonist, mimics the procognitive effect of PEA add further support to the notion that this nuclear receptor is the primary molecular target of PEA, confirming the physiopathological role of PEA/PPAR-␣ signaling in the CNS [17,78]. Peroxisomes have a crucial role for reactive oxygen species formation and lipid metabolism, and their importance in brain physiopathology is well established. The authors hypothesize that PEA acting at PPAR-␣ could increase the number of peroxisomes and/or the activity of the peroxisomal matrix protein catalase, counteracting the redox perturbation following the amyloid excess. Further in vitro study evaluated the neuroprotective effect of PEA on astrocyte activation and neuronal loss in models of A␤ neurotoxicity in mixed neuroglial cultures and organotypic hippocampal slices via PPAR-␣ [103]. The anti-inflammatory properties of PEA show that the reduction of reactive gliosis subsequently induces a marked rebound of neuroprotective effect on neurons. More recently, these protective effects of PEA in rat neuronal cultures and organotypic hippocampal slices challenged with A␤ were investigated. PEA reduced A␤-induced astrocyte activation showing a protective effect on neurons, reverted by a selective PPAR-␣ antagonist [104]. In experimental models of neurodegenerative diseases, such as Huntington’s disease in R6/2 mice, Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEVIDD) model or experimental autoimmune encephalomyelitis (EAE), reminiscent of chronic relapsing and chronic progressive forms of multiple sclerosis (MS) in mice [105,106], it has been observed an increase in PEA levels in CNS [107], which could be interpreted as an adaptive effort to preserve functions and integrity during the development of neurodegenerative damage. Moreover, PEA administration resulted in a reduction of motor disability in the mice subjected to a chronic model of MS, accompanied by an anti-inflammatory effect [107]. Therefore, it has been suggested that drugs inhibiting PEA degradation might be used to treat neurodegenerative diseases [105,108,109]. The fact that PPAR-␣ agonists, mainly by counteracting inflammatory and immune processes, show beneficial effects in mice with EAE [110,111] is in favor of the idea that PEA might induce its beneficial effect through the activation of this receptor [112]. The possible role of PEA in Parkinson’s disease has also been investigated [113]. Peripheral administration of PEA reduced 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced glial activation, and restored tyrosine hydroxylase and dopamine transporter expression in the substantia nigra, two effects that are blunted in PPAR-␣−/− mice. Consistently, a similar effect was shown for iNOS expression, which is reduced by PEA in the substantia nigra. Furthermore, chronic administration of PEA reversed MPTP-associated motor deficits, as revealed by the analysis of forepaw step width and percentage of faults [113]. As previously reported, PEA has been also demonstrated to be effective following central administration, such as intracerebroventricular, intra-periaqueductal gray, intra-rostral ventromedial medulla, or spinal cord laminal application in rodents [78,85,114], showing responsiveness of several CNS areas to PEA. A neuroprotective effect of PEA has been also substantiated in mice

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with transient middle cerebral artery occlusion [115], and related to PPAR-␣ activation [116]. A modulation of AEA and PEA mobilization has been observed in response to spinal cord injury in rats. Lesion-induced increases of AEA and PEA levels occur in the early stage with an upregulation of N-arachidonoylphospatidylethanolamine phospholipase D and a downregulation of the degradative enzyme FAAH, while in delayed stage 2-AG increases [117]. In a model of spinal cord compression trauma in mice, systemic administration of PEA significantly reduced the severity of spinal cord trauma [118,119]. Recent articles have shown that peripheral administration of OEA [120] or acylethanolamide-enhancing FAAH inhibitor [121] improves memory consolidation in rats in a PPAR-␣-dependent manner. It is conceivable that PPAR-␣ activation in the peripheral system may reach the brain via the vagus nerve [120]. Starting from these observations, D’Agostino et al. [102] demonstrated that a daily treatment with PEA reduces learning and memory deficits caused by central injection of the pre-aggregated A␤25–35. PPAR-␣ role in PEA-induced effect was shown by PEA failure in PPAR-␣−/− mice in restoring learning and memory processes and reinforced by similar data obtained with other PPAR-␣ agonists in wt mice. Endogenous cannabinoids play a protective role also in CNS disorders associated with neuronal hyperexcitability and the endocannabinoid system is known to influence seizure activity in the hippocampus [122]. PEA has been shown to exert anticonvulsive effects in tonic convulsions [123,124]. Among neurosteroids, allopregnanolone shares many effects showed by PEA, including an anticonvulsant activity [49,51]. Neurosteroids are synthesized in the brain and have been demonstrated to modulate various cerebral functions. Allopregnanolone is a positive allosteric modulator of the GABA(A) receptor complex acting on a specific steroid recognition site. Neurosteroids antagonize generalized tonic–clonic seizures in various animal models of epilepsy [125]. Citraro et al. [126] have evaluated the effects of such compounds in a genetic animal model of absence epilepsy, the WAG/Rij rat. These animals were chronically implanted with five frontoparietal cortical electrodes for electrocorticogram recordings and bilateral guide cannulae into specific brain areas of the cortico-thalamic circuit in order to evaluate the effects of allopregnanolone on the number and duration of epileptic spike-wave discharges. Allopregnanolone was able to reduce the number and duration of spike-wave discharges when microinjected into the peri-oral region of the primary somatosensory cortex. Since it has been shown that PEA, through PPAR-␣ increases allopregnanolone levels in CNS and astrocytes [19,21], the effectiveness of PEA was tested in these animals [127]. The authors demonstrate that PEA has anti-absence properties and that such properties depend on PPAR-␣ and a possible indirect activation of CB1 by AEA was suggested. Other data support a role for endocannabinoid system in convulsive diseases, in fact AM374, a FAAH inhibitor, which increased endogenous levels of AEA and PEA in the brain, showed high anticonvulsant and neuroprotective effects in rats related to the increase in level of saturated and unsaturated AEs [128]. However, a further reasonable explanation can be suggested. The findings related to PEA-induction of neurosteroid synthesis in the CNS, and the similar pharmacological profile of allopregnanolone and PEA in several CNS disorders, are strongly suggestive of their synergistic effect on ion channel control. In fact, PEA per se acts through the opening of the IKCa and BKCa, and indirectly, through the synthesis of allopregnanolone, can increase GABA-induced inward chloride currents, leading overall to neuronal hyperpolarization. Finally, all those effects exerted by PEA could cooperate with AEA-induced TPRV1 densensitization in controlling absence, as evidenced by Citraro et al. [127] in WAG/Rij rat. Endogenous cannabinoids, AEA and 2-AG, and CB1 receptors are involved in the neuronal mechanisms underlying the

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rewarding effects of most drugs of abuse, including nicotine [129,130]. To clarify the role of dopamine neurons in the mediation of the anti-addicting properties of CB1, some authors studied either CB1 antagonists or, conversely, brain endocannabinoid levels enhancement induced by FAAH inhibition. They showed that the enhancement of brain AEs levels inhibited the responses of ventral tegmental area dopamine neurons to nicotine, rather than blocking CB1 receptors [42]. More importantly, the noncannabinoid AEs, OEA and PEA, play a novel and unsuspected role, as PPAR-␣ agonists, in the negative regulation of dopamine neurons responses to nicotine. Importantly, PPAR-␣-mediated “anti-addictive” properties seem to be limited to nicotine [131]. A plausible explanation for this effect is most likely because the ultimate target downstream PPAR-␣ activation in ventral tegmental area dopamine cells is the phosphorylation of nicotinic receptors containing ␤2 subunits (␤2*-nAChRs) [132]. Thereby, nuclear PPAR-␣ tonically regulates ␤2*-nAChRs, controlling dopamine neuron firing activity. These findings strongly support PPAR-␣ as new therapeutic targets for disorders associated with unbalanced dopamine–acetylcholine systems and its agonists and modulators, including PEA, as effective drugs. Role of PEA and PPAR˛ on metabolism PPAR-␣ is considered a target of drugs useful for the treatment of hypertriglyceridemia. Indeed, PPAR-␣ agonists have been shown to regulate metabolic activities, leading to fatty acid catabolism [133] and PPAR-␣ is expressed at substantial levels in tissues with a high rate of fatty acid oxidation, such as liver, heart and skeletal muscle, where it regulates fatty acid homeostasis [134–137]. All isoforms of PPAR were shown to reveal specific patterns of localization in different areas of the brain and spinal cord at neuronal and glial cell level [16,138,139]. Interestingly, PPAR-␤/␦ is widely distributed, while ␣ and ␥ isotypes show a more constrained pattern of expression [16]. Moreover, the injection of a PPAR-␣ agonist into the hypothalamus was shown to induce expression of PPAR-␣ target genes and reduce food intake [140]. The majority of the tissues expressing PPAR-␣ plays a role in whole-body metabolic homeostasis and is incline to inflammation when metabolism is impaired, a condition which may or not promote pathological conditions, such as obesity, non alcoholic steatohepatitis, type 2 diabetes or cardiovascular diseases. The anti-inflammatory effect of several PPAR-␣ agonists suggested the potentiality to target this receptor in metabolic disorders often associated to the activation of inflammatory pathways. Therefore, it is conceivable that PPAR-␣ may have a key role in all those diseases where inflammation is a major determinant of complications including overweight and obesity, underlining the relationship between nutrition, metabolic organs, and vascular tissues [141–143]. In fact, a finely regulated interaction between inflammation and metabolic system exists [144]. Chronic low grade of inflammation, associated by increased systemic levels of inflammatory cytokines/adipokines, could lead to detrimental effects modulated by PPAR-␣ [145] not only on metabolic organs. Rodriguez de Fonseca et al. [146] evaluated the effect of AEs on feeding behavior, demonstrating a significant anorexic effect of OEA and, although to a lesser degree, a reduction of food intake by PEA. Later, OEA effects on feeding behavior and weight loss were ascribed to the activation of PPAR-␣ [41]. Conversely, the mechanism underlying PEA effect on feeding behavior was not elucidated. PEA, as OEA, binds PPAR-␣, but is unable to exert on food intake and lipolysis, effects similar to those of OEA, as shown by Rodriguez de Fonseca [146], possibly because of its lower potency. However, PEA activity at PPAR-␣ is efficacious enough not only to induce profound

anti-inflammatory and antinociceptive effects [14], but also to exert metabolic actions. Our recent study demonstrates anorexic and fatlosing effects of a chronic treatment with PEA in ovariectomized (OVX) obese rats [147]. These effects are consistent with the recovery of the impairment of leptin receptor signaling at hypothalamic level, probably restoring leptin sensitivity. The PEA effect on central leptin signaling was associated to a reduction of leptin synthesis by adipose tissue and its serum level in OVX rats. All the alterations induced by estrogen loss, are hallmark of leptin resistance, a condition where the increase in circulating leptin level is accompanied by the unresponsiveness of the hypothalamic functional leptin receptor Ob-Rb, and hence a reduction of the anorectic effect of the hormone. The prevention of leptin resistance by PEA treatment has been shown by an increase in Ob-Rb expression in the hypothalamus, a reduction of the inhibitors of leptin signaling, increase pSTAT3, a downstream factor of leptin signaling transduction. The direct effect of PEA has been also shown in non obesogenic condition, in sham-operated animals, that treated with PEA, revealed an increase in hypothalamic leptin signaling. This data were also confirmed in SH-SY5Y neuroblastoma cell line, an in vitro model to study leptin signaling [148,149]. PEA reduction of body weight together with the reversion in leptin resistance has been related to the suppression of food intake by hypothalamic modulation of AMP-activated protein kinase (AMPK) activity and neuropeptide synthesis [147]. In fact, the restoration of leptin sensitivity at hypothalamic level is consistent with the increase in propriomelanocortin and the reduction of Agouti related protein transcripts. Beyond the central hypothalamic effect of PEA, our data support a peripheral effect of this ethanolamide on white adipose tissue. PEA, inducing an increase in AMPK activation in adipose tissue, inhibits pro-inflammatory cytokine synthesis driving polarization of macrophages to M2 phenotype. Accordingly with pAMPK increase in adipose tissue, PEA restores carnitine palmitoyl transferase 1 transcription, confirming its role in the modulation of catabolic pathways. Indeed, we have hypothesized that separate mechanisms contribute to the fat loosing effect of PEA, a peripheral effect on adipose tissue through the normalization of leptin synthesis and the increase of lipid catabolic pathway, and in addition at central level the normalization of leptin sensitivity and the modulation of propriomelanocortin and Agouti related protein, consistently with a reduction of food consumption. The central effect of PEA is reinforced by the evaluation of AEs in the cerebrospinal fluid and several areas of the CNS. Indeed, diurnal variations of AEs have been demonstrated: while in the cerebrospinal fluid there is an increase in the concentrations of AEA, OEA, and PEA during the light-on period, in the parenchymal brain tissues (i.e. pons, hippocampus, and hypothalamus) AEs accumulated during the light-off period [150]. The authors suggest that this timely distribution support the idea that the increase in tissue AEs concentration occurs when the animal is awake. Therefore, AEs would target all brain regions to regulate feeding and sleep behaviors. The evaluation of PEA, as well as OEA, levels in several tissues of obese Zucker fa/fa rats, deficient of leptin receptors, revealed that these animals showed a significant increase in both AEs in the brainstem, compared to wt rats, together with an increase in PPAR-␣ immune reactivity in neurons of the brainstem nuclei, and the nuclei of the solitary tract [151]. These findings strengthened the hypothesis of a direct action of AEs through PPAR-␣ activation in these brainstem regions. The alterations of PEA and OEA levels in Zucker rats, revealed in other peripheral tissues (i.e. duodenum, liver, pancreas, and adipose tissue), surely indicate a delicate interaction between central and peripheral signals in the control of food intake and body weight, even if these data cannot be clearly interpreted due to the lack of functional data.

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Conclusions The well-known peripheral anti-inflammatory properties of PEA have been widely recognized also in CNS. A clear role for PEA in central neuroinflammation has been delineated and the involvement of PPAR-␣ has also been determined. Indeed, all inflammatory-based disorders in CNS could benefit from this endogenous molecule, which in last years has been demonstrated to be safe and active through several molecular mechanisms, converging to lessen inflammatory signaling, that is one of the common starting point of progressive neurodegenerative diseases. Here, we reviewed findings highlighting multiple and converging mechanisms that account for the pleiotropic effects of PEA, validating its possible therapeutic efficacy. It is interesting to note that PPAR-␣ the main common molecular target for PEA, is the converging focus through which this endogenous molecule could exert its functions in metabolism, behavior, inflammation and pain perception. Therefore, PEA may be considered a multifunctional compound for an innovative approach against complex diseases, such as neurodegeneration, in which the ‘one-disease one-target’ and ‘onetarget one-compound’ paradigms may be advocated as a possible reason for the lack of really effective therapeutic tools. Conflict of interest The authors have no financial and commercial conflicts of interest. References [1] Cadas H, Gaillet S, Beltramo M, Venance L, Piomelli D. Biosynthesis of an endogenous cannabinoid precursor in neurons and its control by calcium and cAMP. J Neurosci 1996;16:3934–42. [2] Hansen HS, Moesgaard B, Hansen HH, Petersen G. N-Acylethanolamines and precursor phospholipids: relation to cell injury. Chem Phys Lipids 2000;108:135–50. [3] Aloe L, Leon A, Levi-Montalcini R. A proposed autacoids mechanism controlling mastocyte behavior. Agents Actions 1993;39:C145–7. [4] Balvers MG, Verhoeckx KC, Meijerink J, Wortelboer HM, Witkamp RF. Measurement of palmitoylethanolamide and other N-acylethanolamines during physiological and pathological conditions. CNS Neurol Disord Drug Targets 2013;12:23–33. [5] Hansen HS, Moesgaard B, Petersen G, Hansen HH. Putative neuroprotective actions of N-acyl-ethanolamines. Pharmacol Ther 2002;95:119–26. [6] Walter L, Franklin A, Witting A, Moller T, Stella N. Astrocytes in culture produce anandamide and other acylethanolamides. J Biol Chem 2002;277:20869–76. [7] Muccioli GG, Stella N. Microglia produce and hydrolyze palmitoylethanolamide. Neuropharmacology 2008;54:16–22. [8] Skaper SD, Buriani A, Dal Toso R, Petrelli L, Romanello S, Facci L, et al. The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons. Proc Natl Acad Sci USA 1996;93:3984–9. [9] Franklin A, Parmentier-Batteur S, Walter L, Greenberg DA, Stella N. Palmitoylethanolamide increases after focal cerebral ischemia and potentiates microglial cell motility. J Neurosci 2003;23:7767–75. [10] Cravatt BF, Giang DK, Mayfield SP, Boger DL, Lerner RA, Gilula NB. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature 1996;384:83–7. [11] Ueda N, Tsuboi K, Uyama T. Metabolism of endocannabinoids and related N-acylethanolamines: canonical and alternative pathways. FEBS J 2013;280:1874–94. [12] Kinsey SG, O’Neal ST, Long JZ, Cravatt BF, Lichtman AH. Inhibition of endocannabinoid catabolic enzymes elicits anxiolytic-like effects in the marble burying assay. Pharmacol Biochem Behav 2011;98:21–7. [13] Facci L, Dal Toso R, Romanello S, Buriani A, Skaper SD, Leon A. Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc Natl Acad Sci USA 1995;92:3376–80. [14] Lo Verme J, Fu J, Astarita G, La Rana G, Russo R, Calignano A, et al. The nuclear receptor peroxisome proliferator-activated receptor alpha mediates the antiinflammatory actions of palmitoylethanolamide. Mol Pharmacol 2005;67, 159. [15] Lo Verme J, Russo R, La Rana G, Fu J, Farthing J, Mattace-Raso G, et al. Rapid broad-spectrum analgesia through activation of peroxisome proliferatoractivated receptor-alpha. J Pharmacol Exp Ther 2006;319:1051–61.

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