Monia Di Tommaso

The endocannabinoid system: an overview

Upon the identification of anandamide (AEA) in the porcine brain, numerous studies contributed to the current state of knowledge regarding all elements that form the “endocannabinoid system (ECS).”How this complex system of receptors, ligands, and enzymes is integrated in helping to regulate fundamental processes at level of central nervous and peripheral systems and how its regulation and dysregulation might counteract disturbances of such functions, is nowadays still under investigation. However, the most recent advances on the physiological distribution and functional role of ECS allowed the progress of various research tools aimed at the therapeutic exploitation of endocannabinoid (eCB) signaling, as well as the development of novel drugs with pharmacological advantages. Here, we shall briefly overview the metabolic and signal transduction pathways of the main eCBs representatives, AEA, and 2-arachidonoylglycerol (2-AG), and we will discuss the therapeutic potential of new ECS-oriented drugs.

The role of endocannabinoids in gonadal function and fertility along the evolutionary axis.

Endocannabinoids are natural lipids able to bind to cannabinoid and vanilloid receptors. Their biological actions at the central and peripheral level are under the tight control of the proteins responsible for their synthesis, transport and degradation. In the last few years, several reports have pointed out these lipid mediators as critical signals, together with sex hormones and cytokines, in various aspects of animal and human reproduction. The identification of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in reproductive cells and tissues of invertebrates, vertebrates and mammals highlights the key role played by these endogenous compounds along the evolutionary axis. Here, we review the main actions of endocannabinoids on female and male reproductive events, and discuss the interplay between them, steroid hormones and cytokines in regulating fertility. In addition, we discuss the involvement of endocannabinoid signalling in ensuring a correct chromatin remodeling, and hence a good DNA quality, in sperm cells.

The Novel Reversible Fatty Acid Amide Hydrolase Inhibitor ST4070 Increases Endocannabinoid Brain Levels and Counteracts Neuropathic Pain in Different Animal Models

The effect of the enol carbamate 1-biphenyl-4-ylethenyl piperidine-1-carboxylate (ST4070), a novel reversible inhibitor of fatty acid amide hydrolase (FAAH), was investigated for acute pain sensitivity and neuropathic pain in rats and mice. Brain enzymatic activity of FAAH and the endogenous levels of its substrates, anandamide (AEA; N-arachidonoylethanolamine), 2-arachidonoylglycerol (2-AG), and N-palmitoylethanolamine (PEA), were measured in control and ST4070-treated mice. ST4070 (10, 30, and 100 mg/kg) was orally administered to assess mechanical nociceptive thresholds and allodynia by using the Randall-Selitto and von Frey tests, respectively. Neuropathy was induced in rats by either the chemotherapeutic agent vincristine or streptozotocin-induced diabetes, whereas the chronic constriction injury (CCI) model was chosen to evaluate neuropathy in mice. ST4070 produced a significant increase of nociceptive threshold in rats and counteracted the decrease of nociceptive threshold in the three distinct models of neuropathic pain. In diabetic mice, ST4070 inhibited FAAH activity and increased the brain levels of AEA and PEA, without affecting that of 2-AG. The administration of ST4070 generated long-lasting pain relief compared with pregabalin and the FAAH inhibitors 1-oxo-1[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL135) and cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-ylester (URB597) in CCI neuropathic mice. The antiallodynic effects of ST4070 were prevented by pretreatment with cannabinoid type 1 and cannabinoid type 2 receptor antagonists and by the selective peroxisome proliferator-activated receptor α antagonist [(2S)-2-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl]amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester (GW6471). The administration of ST4070 generated long-lasting neuropathic pain relief compared with pregabalin and the FAAH inhibitors OL135 and URB597. Taken together, the reversible FAAH inhibitor ST4070 seems to be a promising novel therapeutic agent for the management of neuropathic pain.

Differences in the Endocannabinoid System of Sperm from Fertile and Infertile Men

Male infertility is a major cause of problems for many couples in conceiving a child. Recently, lifestyle pastimes such as alcohol, tobacco and marijuana have been shown to have further negative effects on male reproduction. The endocannabinoid system (ECS), mainly through the action of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) at cannabinoid (CB1, CB2) and vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with a clear impact on male reproductive potential. Here, sperm from fertile and infertile men were used to investigate content (through LC-ESI-MS), mRNA (through quantitative RT-PCR), protein (through Western Blotting and ELISA) expression, and functionality (through activity and binding assays) of the main metabolic enzymes of AEA and 2-AG (NAPE-PLD and FAAH, for AEA; DAGL and MAGL for 2-AG), as well as of their binding receptors CB1, CB2 and TRPV1. Our findings show a marked reduction of AEA and 2-AG content in infertile seminal plasma, paralleled by increased degradation: biosynthesis ratios of both substances in sperm from infertile versus fertile men. In addition, TRPV1 binding was detected in fertile sperm but was undetectable in infertile sperm, whereas that of CB1 and CB2 receptors was not statistically different in the two groups. In conclusion, this study identified unprecedented alterations of the ECS in infertile sperm, that might impact on capacitation and acrosome reaction, and hence fertilization outcomes. These alterations might also point to new biomarkers to determine male reproductive defects, and identify distinct ECS elements as novel targets for therapeutic exploitation of ECS-oriented drugs to treat male fertility problems.

Regulation of male fertility by the endocannabinoid system

Mammalian conception is a complex process regulated by both sexual behavior and reproductive performance. Alcohol, marijuana and tobacco are among the main factors which affect negatively fertility in women and men. Several studies have demonstrated that marijuana impairs the male copulatory activity, and that smokers of this illegal drug show reduced fertility due, for instance, to decrease in sperm concentration, defective sperm function or alteration of sperm morphology. The discovery of endocannabinoids and all components responsible for their metabolism has allowed to collect valuable information on the effects of these endogenous lipids, able to mimic the actions of delta-9-tetrahydrocannabinol (THC), in reproductive functions. The purpose of this review is to describe the actions of cannabinoids and endocannabinoids on the control of procreation and hormonal release during the fertilization process in males.

Type-1 (CB1) Cannabinoid Receptor Promotes Neuronal Differentiation and Maturation of Neural Stem Cells

Neural stem cells (NSCs) are self-renewing cells that can differentiate into multiple neural lineages and repopulate regions of the brain after injury. We have investigated the role of endocannabinoids (eCBs), endogenous cues that modulate neuronal functions including neurogenesis, and their receptors CB1 and CB2 in mouse NSCs. Real-time PCR and Western blot analyses indicated that CB1 is present at higher levels than CB2 in NSCs. The eCB anandamide (AEA) or the CB1-specific agonist ACEA enhanced NSC differentiation into neurons, but not astrocytes and oligodendrocytes, whereas the CB2-specific agonist JWH133 was ineffective. Conversely, the effect of AEA was inhibited by CB1, but not CB2, antagonist, corroborating the specificity of the response. CB1 activation also enhanced maturation of neurons, as indicated by morphometric analysis of neurites. CB1 stimulation caused long-term inhibition of the ERK1/2 pathway. Consistently, pharmacological inhibition of the ERK1/2 pathway recapitulated the effects exerted by CB1 activation on neuronal differentiation and maturation. Lastly, gene array profiling showed that CB1 activation augmented the expression of genes involved in neuronal differentiation while decreasing that of stemness genes. These results highlight the role of CB1 in the regulation of NSC fate and suggest that its activation may represent a pro-neuronal differentiation signal.

Endocannabinoids Stimulate Human Melanogenesis via Type-1 Cannabinoid Receptor

Background: Endocannabinoids like anandamide (AEA) play a key role in skin biology. Results: At high concentrations, AEA induces apoptosis of primary human melanocytes through TRPV1 receptors, whereas at low concentrations, it stimulates melanogenesis through CB1 receptors. Conclusion: AEA regulates cell death or melanin synthesis through different pathways. Significance: The double effect of AEA on human melanocytes might have implications beyond skin biology. We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB1, CB2, and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μm) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB1-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 μm). This CB1-dependent activity was fully abolished by the selective CB1 antagonist SR141716 or by RNA interference of the receptor. CB1 signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB1 activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor.

Distinct modulation of the endocannabinoid system upon kainic acid-induced in vivo seizures and in vitro epileptiform bursting.

There is clear evidence on the neuroprotective role of the endocannabinoid (eCB) signaling cascade in various models of epilepsy. In particular, increased levels of eCBs protect against kainic acid (KA)-induced seizures. However, the molecular mechanisms underlying this effect and its age-dependence are still unknown. To clarify this issue, we investigated which step of the biosynthetic and catabolic pathways of the eCBs may be responsible for the eCB-mediated neuroprotection in the hippocampus of P14 and P56-70 KA-treated rats. We found that both anandamide and N-palmitoylethanolamine, together with their biosynthetic enzyme significantly increased in the hippocampus of younger KA-treated rats, while decreasing in adults. In contrast, the levels of the other major eCB, 2-arachidonoylglycerol, similar to its biosynthetic enzyme, were higher in the hippocampus of P56-70 compared to P14 rats. In line with these data, extracellular field recordings in CA1 hippocampus showed that enhancement of endogenous AEA and 2-AG significantly counteracted KA-induced epileptiform bursting in P56-70 and P14 rats, respectively. On the contrary, while the CB1R antagonist SR141716 per se did not affect the population spike, it did worsen KA-induced bursts, confirming increased eCB tone upon KA treatment. Altogether these data indicate an age-specific alteration of the eCB system caused by KA and provide insights for the protective mechanism of the cannabinoid system against epileptiform discharges.

Altered Expression of Type-1 and Type-2 Cannabinoid Receptors in Celiac Disease

Anandamide (AEA) is the prominent member of the endocannabinoid family and its biological action is mediated through the binding to both type-1 (CB1) and type-2 (CB2) cannabinoid receptors (CBR). The presence of AEA and CBR in the gastrointestinal tract highlighted their pathophysiological role in several gut diseases, including celiac disease. Here, we aimed to investigate the expression of CBR at transcriptional and translational levels in the duodenal mucosa of untreated celiac patients, celiac patients on a gluten-free diet for at least 12 months and control subjects. Also biopsies from treated celiac patients cultured ex vivo with peptic-tryptic digest of gliadin were investigated. Our data show higher levels of both CB1 and CB2 receptors during active disease and normal CBR levels in treated celiac patients. In conclusion, we demonstrate an up-regulation of CB1 and CB2 mRNA and protein expression, that points to the therapeutic potential of targeting CBR in patients with celiac disease.

Abnormal anandamide metabolism in celiac disease

The endocannabinoid system has been extensively investigated in experimental colitis and inflammatory bowel disease, but not in celiac disease, where only a single study showed increased levels of the major endocannabinoid anandamide in the atrophic mucosa. On this basis, we aimed to investigate anandamide metabolism in celiac disease by analyzing transcript levels (through quantitative real-time reverse transcriptase-polymerase chain reaction), protein concentration (through immunoblotting) and activity (through radioassays) of enzymes responsible for anandamide synthesis (N-acylphosphatidyl-ethanolamine specific phospholipase D, NAPE-PLD) and degradation (fatty acid amide hydrolase, FAAH) in the duodenal mucosa of untreated celiac patients, celiac patients on a gluten-free diet for at least 12 months and control subjects. Also, treated celiac biopsies cultured ex vivo with peptic-tryptic digest of gliadin were investigated. Our in vivo experiments showed that mucosal NAPE-PLD expression and activity are higher in untreated celiac patients than treated celiac patients and controls, with no significant difference between the latter two groups. In keeping with the in vivo data, the ex vivo activity of NAPE-PLD was significantly enhanced by incubation of peptic-tryptic digest of gliadin with treated celiac biopsies. On the contrary, in vivo mucosal FAAH expression and activity did not change in the three groups of patients, and accordingly, mucosal FAAH activity was not influenced by treatment with peptic-tryptic digest of gliadin. In conclusion, our findings provide a possible pathophysiological explanation for the increased anandamide concentration previously shown in active celiac mucosa.