Epidemiology and cerebrovascular events related to cervical and intracranial arteries dissection: the experience of the city of Pisa.

Spontaneous dissection of cervical arteries (sCAD) is a major cause of ischemic stroke in young patients, with an incidence varying from 1.7 to 3/100,000/year for extracranial internal carotid artery (ICAD) and 1 to 1.9/100,000/year for extracranial vertebral artery (VAD). Reliable epidemiological data on stroke incidence related to sCAD are scarce in Italy. This study aims to evaluate the incidence, clinical features, and outcome of cerebrovascular events related to sCAD and spontaneous intracranial arteries dissections (sIAD) in the city of Pisa (Italy). We retrospectively analyzed consecutive patients admitted between December 1997 and June 2015 with a diagnosis of stroke, TIA, or Bernard-Horner syndrome due to acute cervical or intracranial artery dissection. Considering that our hospital collects presumptively all patients hospitalized with sCAD coming from the referral geographical area, data may provide a good approximation to real incidence of sCAD in our population. Clinical and radiological features, acute treatment and outcome were collected. Seventy-seven cases were included (mean age 48.1±10.4 years, range 23-77,72.7% males), 66 residents in the district of Pisa. Crude incidence rate of cerebrovascular events due to intra or extracranial dissection was 1.88/100,000/year. The incidence of ICAD was 0.80/100,000/year and 0.43/100,000/year for VAD. Stroke occurred in 76.6% of patients. VAD was more prone to cause ischemic stroke and present with cervical pain or focal signs (p < 0.01) than ICAD group, which had older age at onset. sIAD were more frequent in the posterior circle (p = 0.01) and more associated with ischemic lesions. A good outcome (mRS 0-2) was observed in 79% of patients. This is the first epidemiological attempt to investigate impact of sCAD and sIAD in Italy.

Functional and structural analysis of four novel mutations of CYP21A2 gene in Italian patients with 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients.

Visualization of membrane localization and the functional state of CB2R pools using matched agonist and inverse agonist probe pairs.

The diversity of physiological roles of the endocannabinoid system has turned it into an attractive yet elusive therapeutic target. However, chemical probes with various functionalities could pave the way for a better understanding of the endocannabinoid system at the cellular level. Notably, inverse agonists of CB2R - a key receptor of the endocannabinoid system - lagged behind despite the evidence regarding the therapeutic potential of its antagonism. Herein, we report a matched fluorescent probe pair based on a common chemotype to address and visualize both the active and inactive states of CB2R, selectively. Alongside extensive cross-validation by flow cytometry, time-lapse confocal microscopy, and super-resolution microscopy, we successfully visualize the intracellular localization of CB2R pools in live cells. The synthetic simplicity, together with the high CB2R-selectivity and specificity of our probes, turns them into valuable tools in chemical biology and drug development that can benefit the clinical translatability of CB2R-based drugs.

Endocannabinoid signaling in Alzheimer’s disease: current knowledge and future directions.

The importance of the endocannabinoid system (ECS) in the modulation functions of the central nervous system has been extensively investigated during the last few years. In particular, accumulated evidence has implicated ECS in the pathophysiology of Alzheimer’s disease (AD), that is a progressive, degenerative, and irreversible disorder characterized by the accumulation in the brain of beta-amyloid fragments forming insoluble plaques, and of intracellular neurofibrillary tangles (NTFs) associated with synaptic and neuronal loss. In all the processes involved in the formation of both plaques and NFTs, the key-role played by the ECS has been documented. Here, we review current knowledge and future directions of ECS modulation both in animal models of AD and in human tissues, underlying the role of endocannabinoid signaling in the development of AD hallmarks. Overall, the available data suggest that next generation therapeutics might target distinct ECS elements, for instance CB2 receptor or fatty acid amide hydrolase, as a promising approach to halt or at least to slow down disease progression.

Long-term use of HU210 adversely affects spermatogenesis in rats by modulating the endocannabinoid system.

Recent societal acceptance of cannabinoids as recreational and therapeutic drugs has posed a potential hazard to male reproductive health. Mammals have a highly sophisticated endogenous cannabinoid (ECS) system that regulates male (and female) reproduction and exo-cannabinoids may influence it adversely. Therefore it is imperative to determine their effects on male reproduction so that men can make informed choices as to their use. Here, an animal model was used to administer HU210, a synthetic analogue of Δ9-tetrahydrocannabinol (THC) and potent cannabinoid receptor (CB) agonist to determine its effects on reproductive organ weights, spermatogenesis, testicular histology and sperm motility. Its effects on the physiological endocannabinoid system were also investigated. Spermatogenesis was markedly impaired with reductions in total sperm count after 2 weeks of exposure. Spermatogenic efficiency was depleted, and Sertoli cell number decreased as exposure time increased with seminiferous tubules showing germ cell depletion developing into atrophy in some cases. Sperm motility was also adversely affected with marked reductions from 2 weeks on. HU210 also acted on the sperm's endocannabinoid system. Long-term use of exo-cannabinoids has adverse effects on both spermatogenesis and sperm function. These findings highlight the urgent need for studies evaluating the fertility potential of male recreational drug users. HU210, a selective agonist for CB1 and CB2 cannabinoid receptors impairs spermatogenesis and sperm motility and deregulates the endocannabinoid system.

Pin1 contribution to Alzheimer's disease: transcriptional and epigenetic mechanisms in patients with late-onset Alzheimer's disease.

BACKGROUND: Neurofibrillary tangles and senile plaques are hallmarks of Alzheimer's disease (AD) although the molecular basis of their coexistence remains elusive. The peptidyl-prolyl cis/trans isomerase Pin1 acts on both tau and amyloid precursor protein to regulate their functions by influencing tau phosphorylation and amyloid precursor protein processing. OBJECTIVE: In order to identify potential biomarkers for AD in easily accessible cells and to gain insight into the relationship between the brain and peripheral compartments in AD pathology, we investigated Pin1 expression and activity in the peripheral blood mononuclear cells of subjects with late-onset AD (LOAD) and age-matched controls (CT). METHODS: Gene and protein expression, promoter methylation, Ser(16) phosphorylation and activity of Pin1 were evaluated in 32 samples from subjects with LOAD and in 28 samples from CT. RESULTS: In LOAD subjects, there was a statistically significant reduction in Ser(16) phosphorylation (-30%; p = 0.041) and promoter methylation (-8%; p = 0.001), whereas Pin1 expression was significantly increased (+74%; p = 0.018). CONCLUSION: The modifications of Pin1 found in LOAD subjects support its involvement in the pathogenesis of the disease with an important role being played by epigenetic mechanisms.

Alterations of the endocannabinoid system in an animal model of migraine: evaluation in cerebral areas of rat.

Endocannabinoids are involved in the modulation of pain and hyperalgesia. In this study we investigated the role of the endocannabinoid system in the migraine model based on nitroglycerin-induced hyperalgesia in the rat. Male rats were injected with nitroglycerin (10 mg/kg, i.p.) or vehicle and sacrificed 4 h later. The medulla, the mesencephalon and the hypothalamus were dissected out and utilized for the evaluation of activity of fatty acid amide hydrolase (that degrades the endocannabinoid anandamide), monoacylglycerol lipase (that degrades the endocannabinoid 2-arachidonoylglycerol), and binding sites specific for cannabinoid (CB) receptors. The findings obtained show that nitroglycerin-induced hyperalgesia is associated with increased activity of both hydrolases and increased density of CB binding sites in the mesencephalon. In the hypothalamus we observed an increase in the activity of fatty acid amide hydrolase associated with an increase in density of CB binding sites, while in the medulla only the activity of fatty acid amide hydrolase was increased. Anandamide also proved effective in preventing nitroglycerin-induced activation (c-Fos) of neurons in the nucleus trigeminalis caudalis. These data strongly support the involvement of the endocannabinoid system in the modulation of nitroglycerin-induced hyperalgesia, and, possibly, in the pathophysiological mechanisms of migraine.

A new methodological approach for in vitro determination of the role of DNA methylation on transcription factor binding using AlphaScreen® analysis: Focus on CREB1 binding at hBDNF promoter IV.

BACKGROUND: DNA methylation plays a relevant role in the regulation of gene transcription, but currently the exact quantification of transcription factors binding to methylated DNA is not being determined. The binding of the transcription factor cAMP response element-binding protein-1 to its cognate CpG containing motif is known to be impaired upon methylation. It thus represents a paradigmatic system to experimentally verify the validity of a new in vitro method to measure the role of methylation on DNA/transcription factors binding. METHOD: An AlphaScreen® assay was developed to quantitatively measure the contribution of DNA CpG methylation on the interaction with transcription factors. The method was validated measuring the variation in affinity of cAMP response element-binding protein-1 and its recognition motif in human Brain-derived neurotrophic factor gene exon IV promoter as a function of CpG methylation. RESULTS: For the first time, a quantitative direct correlation between DNA methylation and transcription factors binding is reported showing a dramatic reduction in binding affinity between fully methylated and non-methylated DNA. COMPARISON WITH EXISTING METHODS: This methodology allows to directly measure DNA/transcription factors binding ability as a function of DNA methylation levels thus improving not quantitative methods available today. Moreover, it allows to work with purified proteins and oligonucleotides without need of chromatin. CONCLUSIONS: The present methodology is suggested as a new analytical tool for the quantitative determination of the effect of CpG methylation on the interaction of gene promoters with transcription factors regulating gene expression, a key epigenetic mechanism implicated in many physiological and pathological conditions.

Role in anxiety behavior of the endocannabinoid system in the prefrontal cortex.

In the present study we explored with a multidisciplinary approach, the role of anandamide (AEA) in the modulation of anxiety behavior at the level of the prefrontal cortex (PFC). Low doses of the metabolically stable AEA analog, methanandamide, microinjected into the PFC, produced an anxiolytic-like response in rats, whereas higher doses induced anxiety-like behaviors. Pretreatment with the selective antagonist of CB1 or TRPV1 receptors (AM251 and capsazepine, respectively) suggested that the anxiolytic effect evoked by AEA might be due to the interaction with the CB1 cannabinoid receptor, whereas vanilloid receptors seem to be involved in AEA anxiogenic action. When AEA contents in the PFC were increased by microinjecting the selective inhibitor of fatty acid amide hydrolase (FAAH), URB597, we observed an anxiolytic response only at low doses of the compound and no effect or even an anxiogenic profile at higher doses. In line with this, a marked decrease of AEA levels in the PFC, achieved by lentivirus-mediated local overexpression of FAAH, produced an anxiogenic response. These findings support an anxiolytic role for physiological increases in AEA in the PFC, whereas more marked increases or decreases of this endocannabinoid might lead to an anxiogenic response due to TRPV1 stimulation or the lack of CB1 activation, respectively.

5-Lipoxygenase and anandamide hydrolase (FAAH) mediate the antitumor activity of cannabidiol, a non-psychoactive cannabinoid.

It has been recently reported that cannabidiol (CBD), a non-psychoactive cannabinoid, is able to kill glioma cells, both in vivo and in vitro, independently of cannabinoid receptor stimulation. However, the underlying biochemical mechanisms were not clarified. In the present study, we performed biochemical analysis of the effect of CBD both in vivo, by using glioma tumor tissues excised from nude mice, and in vitro, by using U87 glioma cells. In vivo exposure of tumor tissues to CBD significantly decreased the activity and content of 5-lipoxygenase (LOX, by approximately 40%), and of its end product leukotriene B4 ( approximately 25%). In contrast cyclooxygenase (COX)-2 activity and content, and the amount of its end product prostaglandin E2, were not affected by CBD. In addition, in vivo treatment with CBD markedly stimulated ( approximately 175%) the activity of fatty acid amide hydrolase (FAAH), the main anandamide-degrading enzyme, while decreasing anandamide content ( approximately 30%) and binding to CB1 cannabinoid receptors ( approximately 25%). In vitro pre-treatment of U87 glioma cells with MK-886, a specific 5-LOX inhibitor, significantly enhanced the antimitotic effect of CBD, whereas the pre-treatment with indomethacin (pan-COX inhibitor) or celecoxib (COX-2 inhibitor), did not alter CBD effect. The study of the endocannabinoid system revealed that CBD was able to induce a concentration-dependent increase of FAAH activity in U87 cells. Moreover, a significantly reduced growth rate was observed in FAAH-over-expressing U87 cells, compared to wild-type controls. In conclusion, the present investigation indicates that CBD exerts its antitumoral effects through modulation of the LOX pathway and of the endocannabinoid system, suggesting a possible interaction of these routes in the control of tumor growth.

Degradation of endocannabinoids in chronic migraine and medication overuse headache.

Chronic migraine (CM) is frequently associated with medication overuse headache (MOH). The endocannabinoid system plays a role in modulating pain including headache and is involved in the common neurobiological mechanism underlying drug addiction and reward system. Anandamide (AEA) and 2-arachidonoylglycerol are the most biologically active endocannabinoids, which bind to both central and peripheral cannabinoid receptors. The level of AEA in the extracellular space is controlled by cellular uptake via a specific AEA membrane transporter (AMT), followed by intracellular degradation by the enzyme AEA hydrolase (fatty acid amide hydrolase, FAAH). AMT and FAAH have also been characterized in human platelets. We assayed the activity of AMT and of FAAH in platelets isolated from four groups of subjects: MOH, CM without MOH, episodic migraine and controls. AMT and FAAH were significantly reduced in CM and MOH, compared to either controls or episodic migraine group. This latter finding was observed in both males and females with CM and MOH. Changes observed in the biochemical mechanisms degrading endogenous cannabinoids may reflect an adaptative behaviour induced by chronic headache and/or drug overuse.

Cortical expression of brain derived neurotrophic factor and type-1 cannabinoid receptor after striatal excitotoxic lesions.

An involvement of one particular neurotrophin, namely, the brain-derived neurotrophic factor (BDNF), has been demonstrated in the pathophysiology Huntington's disease. Type-1 cannabinoid (CB1) receptor has been postulated to upregulate BDNF gene transcription. To better understand the relationship between CB1 and BDNF levels in a situation where the striatum is degenerating, we studied, by dual label immunofluorescence, the distribution of CB1 and BDNF in cortical neurons projecting to the striatum in our rat quinolinic acid model of striatal excitotoxicity. We completed our study with quantitative analyses of BDNF protein levels and CB1 binding activity in the cortex. We show that, 2 weeks post lesion, cortical neurons contain more BDNF compared with controls and to earlier time points. Such BDNF up-regulation coincides with a higher binding activity and an increased protein expression of CB1. We suggest that after excitotoxic lesions, CB1 might, at least transiently, upregulate BDNF in the attempt to rescue striatal neurons from degeneration.

CB2 receptors in reproduction.

Cannabinoids have been always identified as harmful drugs because of their negative effects on male and female reproduction. The discovery of the 'endocannabinoid system (ECS)', composed of bioactive lipids (endocannabinoids), their receptors and their metabolic enzymes, and the generation of mouse models missing cannabinoid receptors or other elements of the ECS, has enabled a wealth of information on the significance of endocannabinoid signalling in multiple reproductive events: Sertoli cell survival, spermatogenesis, placentation, fertilization, preimplantation embryo development, implantation and postimplantation embryonic growth. These studies have also opened new perspectives in clinical applications, pointing to the ECS as a new target for correcting infertility and for improving reproductive health in humans. This review will focus on the involvement of type-2 cannabinoid (CB2) receptors in reproductive biology, covering both the male and female sides. It will also discuss the potential relevance of the immunological activity of CB2 at the maternal/foetal interface, as well as the distinctiveness of CB2 versus type-1 cannabinoid (CB1) receptors that might be exploited for a receptor subtype-specific regulation of fertility. In this context, the different signalling pathways triggered by CB1 and CB2 (especially those controlling the intracellular tone of nitric oxide), the different activation of CB1 and CB2 by endogenous agonists (like anandamide and 2-arachidonoylglycerol) and the different localization of CB1 and CB2 within membrane subdomains, termed 'lipid rafts', will be discussed. It is hoped that CB2-dependent endocannabinoid signalling might become a useful target for correcting infertility, in both men and women.

Good news for CB1 receptors: endogenous agonists are in the right place.

Endocannabinoids are endogenous ligands of brain-type (CB1) and spleen-type (CB2) cannabinoid receptors. N-Arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are prototype members of the fatty acid amides and the monoacylglycerols, two groups of endocannabinoids. Unlike CB1, CB2 receptors do not reside within 'caveolae', specialized membrane microdomains that are well-known modulators of the activity of a number of G protein-coupled receptors. In this issue of the British Journal of Pharmacology, Rimmerman and coworkers demonstrate that 2-AG is entirely localized in the caveolae of dorsal root ganglion cells, where also part of AEA (approximately 30%) can be detected. However, most of AEA (approximately 70%) was detected in non-caveolae fractions, that is where CB2 receptors are localized. The different interaction of AEA and 2-AG with membrane microdomains might have significant implications for endocannabinoid-dependent autocrine and/or retrograde-paracrine signalling pathways. It also raises an important question about the structural determinants responsible for a different localization of two apparently similar endocannabinoids within lipid bilayers.

High levels of anandamide, an endogenous cannabinoid, block the growth of sheep preimplantation embryos by inducing apoptosis and reversible arrest of cell proliferation.

BACKGROUND: The process of implantation is mediated by various molecules, one of which is anandamide (AEA), a lipid signalling ligand belonging to the family of endocannabinoids. AEA exerts its effects on implantation by binding to the Type 1 Cannabinoid Receptor (CB1-R), expressed in both blastocysts and uterus. We wanted to know whether the endocannabinoid signalling system was present also in the sheep reproductive tract and which kind of effect(s) AEA had on the development of sheep blastocysts in vitro. METHODS: We analysed the expression and activity of the endocannabinoid system in sheep reproductive tracts and blastocysts. Hatched sheep blastocysts were then exposed to AEA and its effect(s) were determined by TUNEL assay and by measuring the rate of necrosis and 5-bromo-deoxyuridine incorporation. RESULTS: We show that the AEA signalling system is present in sheep and that high concentrations of AEA induce apoptosis and inhibit cell proliferation via a CB1-R-dependent mechanism. Indeed, AEA effects were blocked when sheep blastocysts were cultured in the presence of the CB1-R antagonist SR161417A. Moreover, AEA inhibition of cell proliferation was reversible, as arrested embryos resumed a normal growth rate upon AEA removal from the medium. CONCLUSIONS: Our results suggest that disturbed regulation of AEA signalling via CB1-R may be associated with pregnancy failure. AEA could lower the quality of blastocysts by inducing apoptosis and inhibiting cell proliferation, thus making them incompetent for implantation.

Oxidative Stress in Cerebral Small Vessel Disease Dizziness Patients, Basally and After Polyphenol Compound Supplementation.

BACKGROUND: Leukoaraiosis (LA) is a common radiological finding in elderly, frequently associated with several clinical disorders, including unexplained dizziness. The pathogenesis of LA is multifactorial, with a dysfunction of cerebral microcirculation resulting in chronic hypoperfusion and tissue loss, with oxidative stress involved in this cascade. OBJECTIVE: The aim of this study was to analyse some oxidative stress biomarkers in a cohort of LA patients. METHOD: Fifty-five consecutive patients (33 males, median age 75 years) with LA were recruited. In a subgroup of 33 patients with LA and unexplained dizziness, we have then performed an open study to evaluate if 60-day supplementation with a polyphenol compound may modify these biomarkers and influence quality of life, analysed with the Dizziness Handicap Inventory (DHI) scale. RESULTS: At baseline, blood oxidative stress parameters values were outside normal ranges and compared to matched healthy controls. After the two months supplementation, we observed a significant decrement of advanced oxidation protein products values and a significant improvement of DHI. CONCLUSION: Oxidative stress biomarkers may be useful to detect redox imbalance in LA and to provide non-invasive tools to monitor disease status and response to therapy.

Endothelium and Oxidative Stress: The Pandora's Box of Cerebral (and Non-Only) Small Vessel Disease?

Common cerebral small vessel disease (cSVD) abnormalities are a common neuroradiological finding, especially in the elderly. They are associated with a wide clinical spectrum that leads to an increasing disability, impaired global function outcome and a reduced quality of life. A strong association is demonstrated with age and hypertension and other common vascular risk factors, including diabetes mellitus, dyslipoproteinemia, smoking, low vitamin B12 level, and hyperomocysteinemia. Although these epidemiological associations suggest a systemic involvement, etiopathogenetic mechanisms remain unclear. This review focuses on the potential role of endothelial dysfunction and oxidative stress in the pathogenic cascade leading to cSVD. We stressed on the central role of those pathways, and suggest the importance of quantifying the cerebral (and non-only) "endotheliopathic and oxidative load" and its clinical presentation that could lead to a better determination of vascular risk degree. In addition, understanding underlying pathogenic mechanisms could allow us to slow down the progression of vascular damage and, therefore, prevent the disability due to reiterated microvascular damage.

2-arachidonoylglycerol levels are increased in leukocytospermia and correlate with seminal macrophages.

Evidence has been produced that macrophages can actively generate endocannabinoids (eCBs) in response to inflammatory stimuli. As eCBs are involved in the control of several physiological processes, including reproduction, here, we explored whether seminal levels of the eCBs, N-arachidonoylethanolamine (AEA), and 2-arachidonoylglycerol (2-AG), were higher in the presence of leukocytospermia, and were correlated with semen concentration of macrophages. The content of AEA and 2-AG was measured by high-performance liquid chromatography/mass spectrometry in seminal plasma of ejaculates from 18 leukocytospermic patients (>1 × 106 leukocytes/mL) and 21 normozoospermic controls. In the same ejaculates, round cells were phenotyped by flow-cytometry as leukocytes (CD45+), macrophages (CD14+), and activated macrophages (CD14+, HLA-DR+). The levels of 2-AG, but not of AEA, were significantly higher in ejaculates from leukocytospermic patients than in controls and exhibited a significant correlation with semen concentration of macrophages and activated macrophages. Significant associations of 2-AG with macrophages and activated macrophages persisted after adjustment for semen volume and sperm concentration. In conclusion, here we provide evidence that seminal plasma levels of 2-AG are higher in the presence of leukocytospermia, as a marker of macrophages activation. Further studies are warranted to elucidate possible clinical implications.

Human adipose tissue binds and metabolizes the endocannabinoids anandamide and 2-arachidonoylglycerol.

Endocannabinoids are a group of biologically active endogenous lipids that have recently emerged as important mediators in energy balance control. The two best studied endocannabinoids, anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the endogenous ligands of the central and peripheral cannabinoid receptors. Furthermore, AEA binds to the transient receptor potential vanilloid type-1 (TRPV1), a capsaicin-sensitive, non-selective cation channel. The synthesis of these endocannabinoids is catalyzed by the N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and the sn-1-selective diacylglycerol lipase (DAGL), whereas their degradation is accomplished by the fatty acid amide hydrolase (FAAH) and the monoglyceride lipase (MGL), respectively. We investigated the presence of a functional endocannabinoid system in human adipose tissue from seven healthy subjects. Subcutaneous abdominal adipose tissue underwent biochemical and molecular biology analyses, aimed at testing the expression of this system and its functional activity. AEA and 2-AG levels were detected and quantified by HPLC. Real time PCR analyzed the expression of the endocannabinoid system and immunofluorescence assays showed the distribution of its components in the adipose tissue. Furthermore, binding assay for the cannabinoid and vanilloid receptors and activity assay for each metabolic enzyme of the endocannabinoid system gave clear evidence of a fully operating system. The data presented herein show for the first time that the human adipose tissue is able to bind AEA and 2-AG and that it is endowed with the biochemical machinery to metabolize endocannabinoids.

New insights into endocannabinoid degradation and its therapeutic potential.

Endocannabinoids are amides, esters and ethers of long chain polyunsaturated fatty acids, which act as new lipidic mediators. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of (-)-Delta9-tetrahydrocannabinol (THC), the active principle of Cannabis sativa preparations like hashish and marijuana. The activity of AEA and 2-AG at their receptors is limited by cellular uptake through an anandamide membrane transporter (AMT), followed by intracellular degradation. A fatty acid amide hydrolase (FAAH) is the main AEA hydrolase, whereas a monoacylglycerol lipase (MAGL) is critical in degrading 2-AG. Here, we will review growing evidence that demonstrates that these hydrolases are pivotal regulators of the endogenous levels of AEA and 2-AG in vivo, overall suggesting that specific inhibitors of AMT, FAAH or MAGL may serve as attractive therapeutic targets for the treatment of human disorders. Recently, the N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), which synthesizes AEA from N-arachidonoylphosphatidylethanolamine (NArPE), and the diacylglycerol lipase (DAGL), which generates 2-AG from diacylglycerol (DAG) substrates, have been characterized. The role of these synthetic routes in maintaining the endocannabinoid tone in vivo will be discussed. Finally, the effects of inhibitors of endocannabinoid degradation in animal models of human disease will be reviewed, with an emphasis on their ongoing applications in anxiety, cancer and neurodegenerative disorders.