Sphingosine Kinases at the Intersection of Pro-Inflammatory LPS and Anti-Inflammatory Endocannabinoid Signaling in BV2 Mouse Microglia Cells.

Microglia, the resident immune cells of the central nervous system, play important roles in brain homeostasis as well as in neuroinflammation, neurodegeneration, neurovascular diseases, and traumatic brain injury. In this context, components of the endocannabinoid (eCB) system have been shown to shift microglia towards an anti-inflammatory activation state. Instead, much less is known about the functional role of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) system in microglia biology. In the present study, we addressed potential crosstalk of the eCB and the S1P systems in BV2 mouse microglia cells challenged with lipopolysaccharide (LPS). We show that URB597, the selective inhibitor of fatty acid amide hydrolase (FAAH)-the main degradative enzyme of the eCB anandamide-prevented LPS-induced production of tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß), and caused the accumulation of anandamide itself and eCB-like molecules such as oleic acid and cis-vaccenic acid ethanolamide, palmitoylethanolamide, and docosahexaenoyl ethanolamide. Furthermore, treatment with JWH133, a selective agonist of the eCB-binding cannabinoid 2 (CB2) receptor, mimicked the anti-inflammatory effects of URB597. Interestingly, LPS induced transcription of both SphK1 and SphK2, and the selective inhibitors of SphK1 (SLP7111228) and SphK2 (SLM6031434) strongly reduced LPS-induced TNFα and IL-1ß production. Thus, the two SphKs were pro-inflammatory in BV2 cells in a non-redundant manner. Most importantly, the inhibition of FAAH by URB597, as well as the activation of CB2 by JWH133, prevented LPS-stimulated transcription of SphK1 and SphK2. These results present SphK1 and SphK2 at the intersection of pro-inflammatory LPS and anti-inflammatory eCB signaling, and suggest the further development of inhibitors of FAAH or SphKs for the treatment of neuroinflammatory diseases.

Rare Phytocannabinoids Exert Anti-Inflammatory Effects on Human Keratinocytes via the Endocannabinoid System and MAPK Signaling Pathway.

Increasing evidence supports the therapeutic potential of rare cannabis-derived phytocannabinoids (pCBs) in skin disorders such as atopic dermatitis, psoriasis, pruritus, and acne. However, the molecular mechanisms of the biological action of these pCBs remain poorly investigated. In this study, an experimental model of inflamed human keratinocytes (HaCaT cells) was set up by using lipopolysaccharide (LPS) in order to investigate the anti-inflammatory effects of the rare pCBs cannabigerol (CBG), cannabichromene (CBC), Δ9-tetrahydrocannabivarin (THCV) and cannabigerolic acid (CBGA). To this aim, pro-inflammatory interleukins (IL)-1ß, IL-8, IL-12, IL-31, tumor necrosis factor (TNF-ß) and anti-inflammatory IL-10 levels were measured through ELISA quantification. In addition, IL-12 and IL-31 levels were measured after treatment of HaCaT cells with THCV and CBGA in the presence of selected modulators of endocannabinoid (eCB) signaling. In the latter cells, the activation of 17 distinct proteins along the mitogen-activated protein kinase (MAPK) pathway was also investigated via Human Phosphorylation Array. Our results demonstrate that rare pCBs significantly blocked inflammation by reducing the release of all pro-inflammatory ILs tested, except for TNF-ß. Moreover, the reduction of IL-31 expression by THCV and CBGA was significantly reverted by blocking the eCB-binding TRPV1 receptor and by inhibiting the eCB-hydrolase MAGL. Remarkably, THCV and CBGA modulated the expression of the phosphorylated forms (and hence of the activity) of the MAPK-related proteins GSK3ß, MEK1, MKK6 and CREB also by engaging eCB hydrolases MAGL and FAAH. Taken together, the ability of rare pCBs to exert an anti-inflammatory effect in human keratinocytes through modifications of eCB and MAPK signaling opens new perspectives for the treatment of inflammation-related skin pathologies.

The TRPV1 Receptor Is Up-Regulated by Sphingosine 1-Phosphate and Is Implicated in the Anandamide-Dependent Regulation of Mitochondrial Activity in C2C12 Myoblasts.

The sphingosine 1-phosphate (S1P) and endocannabinoid (ECS) systems comprehend bioactive lipids widely involved in the regulation of similar biological processes. Interactions between S1P and ECS have not been so far investigated in skeletal muscle, where both systems are active. Here, we used murine C2C12 myoblasts to investigate the effects of S1P on ECS elements by qRT-PCR, Western blotting and UHPLC-MS. In addition, the modulation of the mitochondrial membrane potential (ΔΨm), by JC-1 and Mitotracker Red CMX-Ros fluorescent dyes, as well as levels of protein controlling mitochondrial function, along with the oxygen consumption were assessed, by Western blotting and respirometry, respectively, after cell treatment with methanandamide (mAEA) and in the presence of S1P or antagonists to endocannabinoid-binding receptors. S1P induced a significant increase in TRPV1 expression both at mRNA and protein level, while it reduced the protein content of CB2. A dose-dependent effect of mAEA on ΔΨm, mediated by TRPV1, was evidenced; in particular, low doses were responsible for increased ΔΨm, whereas a high dose negatively modulated ΔΨm and cell survival. Moreover, mAEA-induced hyperpolarization was counteracted by S1P. These findings open new dimension to S1P and endocannabinoids cross-talk in skeletal muscle, identifying TRPV1 as a pivotal target.

Effects of Rare Phytocannabinoids on the Endocannabinoid System of Human Keratinocytes.

The decriminalization and legalization of cannabis has paved the way for investigations into the potential of the use of phytocannabinoids (pCBs) as natural therapeutics for the treatment of human diseases. This growing interest has recently focused on rare (less abundant) pCBs that are non-psychotropic compounds, such as cannabigerol (CBG), cannabichromene (CBC), Δ9-tetrahydrocannabivarin (THCV) and cannabigerolic acid (CBGA). Notably, pCBs can act via the endocannabinoid system (ECS), which is involved in the regulation of key pathophysiological processes, and also in the skin. In this study, we used human keratinocytes (HaCaT cells) as an in vitro model that expresses all major ECS elements in order to systematically investigate the effects of CBG, CBC, THCV and CBGA. To this end, we analyzed the gene and protein expression of ECS components (receptors: CB1, CB2, GPR55, TRPV1 and PPARα/γ/δ; enzymes: NAPE-PLD, FAAH, DAGLα/ß and MAGL) using qRT-PCR and Western blotting, along with assessments of their functionality using radioligand binding and activity assays. In addition, we quantified the content of endocannabinoid(-like) compounds (AEA, 2-AG, PEA, etc.) using UHPLC-MS/MS. Our results demonstrated that rare pCBs modulate the gene and protein expression of distinct ECS elements differently, as well as the content of endocannabinoid(-like) compounds. Notably, they all increased CB1/2 binding, TRPV1 channel stimulation and FAAH and MAGL catalytic activity. These unprecedented observations should be considered when exploring the therapeutic potential of cannabis extracts for the treatment of human skin diseases.

µSPE followed by HPLC-MS/MS for the determination of series D and E resolvins in biological matrices.

The critical role of acute inflammatory processes is recognized in many chronic diseases; a key point in molecular mechanisms of acute inflammation resolution is represented by a new group of pro-resolving lipid mediators that include distinct families of molecules: lipoxins, resolvins, protectins and maresins, collectively termed "specialized pro-resolving mediators" (SPMs). In particular, resolvins are active in the picogram to nanogram dose range, whereby they can directly modulate a plethora of anti-inflammatory responses. The presented method proposes an analytical protocol able to extract and to quantify 6 different resolvins from 3 different matrices (plasma, cells and exudates). The method, validated according to the EMA guideline for bioanalysis, exhibited good precision (1%-20%) and accuracy (2%-20%). In particular, the combination of two different sample preparation techniques, Liquid-Liquid Extraction (LLE) and micro-Solid Phase Extraction (µSPE), applied for the first on this class of molecules, used for the extraction and clean-up respectively, led to high enrichment factor (20 fold) and consequently a high sensitivity (LOQ between 1 and 38 pg mL-1); moreover the validation data proved the versatility of µSPE as clean-up tool as it was capable to manage huge enrichment factor without negatively affect accuracy and precision of analysis.

Cannabinoid signalling and effects of cannabis on the male reproductive system.

Marijuana is the most widely consumed recreational drug worldwide, which raises concerns for its potential effects on fertility. Many aspects of human male reproduction can be modulated by cannabis-derived extracts (cannabinoids) and their endogenous counterparts, known as endocannabinoids (eCBs). These latter molecules act as critical signals in a variety of physiological processes through receptors, enzymes and transporters collectively termed the endocannabinoid system (ECS). Increasing evidence suggests a role for eCBs, as well as cannabinoids, in various aspects of male sexual and reproductive health. Although preclinical studies have clearly shown that ECS is involved in negative modulation of testosterone secretion by acting both at central and testicular levels in animal models, the effect of in vivo exposure to cannabinoids on spermatogenesis remains a matter of debate. Furthermore, inconclusive clinical evidence does not seem to support the notion that plant-derived cannabinoids have harmful effects on human sexual and reproductive health. An improved understanding of the complex crosstalk between cannabinoids and eCBs is required before targeting of ECS for modulation of human fertility becomes a reality.

Corrigendum to "Artificial Neural Network to Predict Varicocele Impact on Male Fertility through Testicular Endocannabinoid Gene Expression Profiles".

[This corrects the article DOI: 10.1155/2018/3591086.].

Characterization of Endocannabinoid System and Interleukin Profiles in Ovine AEC: Cannabinoid Receptors Type-1 and Type-2 as Key Effectors of Pro-Inflammatory Response.

Amniotic epithelial cells (AEC) have been proposed as promising clinical candidates for regenerative medicine therapies due to their immunomodulatory capacity. In this context, the endocannabinoid system (ECS) has been identified as mediating the immune-stem cell dialogue, even if no information on AEC is available to date. Therefore, this study was designed to assess whether ECS is involved in tuning the constitutive and lipopolysaccharide (LPS)-induced ovine AEC anti-inflammatory and pro-inflammatory interleukin (IL-10, IL-4, and IL-12) profiles. Firstly, interleukins and ECS expressions were studied at different stages of gestation. Then, the role of cannabinoid receptors 1 and 2 (CB1 and CB2) on interleukin expression and release was investigated in middle stage AEC using selective agonists and antagonists. AEC displayed a degradative more than a synthetic endocannabinoid metabolism during the early and middle stages of gestation. At the middle stage, cannabinoid receptors mediated the balance between pro-inflammatory (IL-12) and anti-inflammatory (IL-4 and IL-10) interleukins. The activation of both receptors mediated an overall pro-inflammatory shift-CB1 reduced the anti-inflammatory and CB2 increased the pro-inflammatory interleukin release, particularly after LPS stimulation. Altogether, these data pave the way for the comprehension of AEC mechanisms tuning immune-modulation, crucial for the development of new AEC-based therapy protocols.

CSF Levels of the Endocannabinoid Anandamide are Reduced in Patients with Untreated Narcolepsy Type 1: A Pilot Study.

BACKGROUND: Endocannabinoids (ECs) modulate both excitatory and inhibitory components in the CNS. There is a growing body of evidence that shows ECs influence both hypothalamic orexinergic and histaminergic neurons involved in narcolepsy physiopathology. Therefore, ECs may influence sleep and sleep-wake cycle. OBJECTIVE: To evaluate EC levels in the CSF of untreated narcoleptic patients to test whether ECs are dysregulated in Narcolepsy Type 1 (NT1) and Type 2 (NT2). METHODS: We compared CSF Anandamide (AEA), 2-Arachidonoylglycerol (2-AG) and orexin in narcoleptic drug-naïve patients and in a sample of healthy subjects. RESULTS: We compared NT1 (n=6), NT2 (n=6), and healthy controls (n=6). We found significantly reduced AEA levels in NT1 patients compared to both NT2 and controls. No differences were found between AEA levels in NT2 versus controls and between 2-AG levels in all groups, although a trend toward a decrease in NT1 was evident. Finally, the CSF AEA level was related to CSF orexin levels in all subjects. CONCLUSION: We demonstrated that the EC system is dysregulated in NT1.

The (endo)cannabinoid signaling in female reproduction: What are the latest advances?

Cannabis extracts like marijuana have the highest consumption rate worldwide. Yet, their societal acceptance as recreational and therapeutic drugs could represent a serious hazard to female human reproduction, because cannabis ingredients [termed (phyto)cannabinoids] can perturb an endogenous system of lipid signals known as endocannabinoids. Accumulated evidence on animal models and humans has demonstrated a crucial role of these endogenous signals on different aspects of female reproduction, where they act through an ensamble of proteins that synthesize, transport, degrade and traffic them. Several reports have recently evidenced the potential role of endocannabinoids as biomarkers of female infertility for disease treatment and prevention, as well as their possible epigenetic effects on pregnancy. The purpose of this review is to provide an update of data collected in the last decade on the effects of cannabinoids and endocannabinoids on female reproductive events, from development and maturation of follicles and oocytes, to fertilization, oviductal transport, implantation and labor. In this context, a particular attention has been devoted to the ovary and the production of fertilizable oocytes, because recent studies have addressed this hot topic with conflicting results among species.

The endocannabinoid system is affected by cholesterol dyshomeostasis: Insights from a murine model of Niemann Pick type C disease.

The dyshomeostasis of intracellular cholesterol trafficking is typical of the Niemann-Pick type C (NPC) disease, a fatal inherited lysosomal storage disorder presenting with progressive neurodegeneration and visceral organ involvement. In light of the well-established relevance of cholesterol in regulating the endocannabinoid (eCB) system expression and activity, this study was aimed at elucidating whether NPC disease-related cholesterol dyshomeostasis affects the functional status of the brain eCB system. To this end, we exploited a murine model of NPC deficiency for determining changes in the expression and activity of the major molecular components of the eCB signaling, including cannabinoid type-1 and type-2 (CB1 and CB2) receptors, their ligands, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), along with their main synthesizing/inactivating enzymes. We found a robust alteration of distinct components of the eCB system in various brain regions, including the cortex, hippocampus, striatum and cerebellum, of Npc1-deficient compared to wild-type pre-symptomatic mice. Changes of the eCB component expression and activity differ from one brain structure to another, although 2-AG and AEA are consistently found to decrease and increase in each structure, respectively. The thorough biochemical characterization of the eCB system was accompanied by a behavioral characterization of Npc1-deficient mice using a number of paradigms evaluating anxiety, locomotor activity, spatial learning/memory abilities, and coping response to stressful experience. Our findings provide the first description of an early and region-specific alteration of the brain eCB system in NPC and suggest that defective eCB signaling could contribute at producing and/or worsening the neurological symptoms of this disorder.

Modulation of Endocannabinoid-Binding Receptors in Human Neuroblastoma Cells by Tunicamycin.

Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB1 and CB2) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB1 and CB2, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB1 (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB1 with GlcNAc residues, and showed that tunicamycin reduced CB1 membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors' expression and localization, in particular for CB1.

Artificial Neural Network to Predict Varicocele Impact on Male Fertility through Testicular Endocannabinoid Gene Expression Profiles.

The relationship between varicocele and fertility has always been a matter of debate because of the absence of predictive clinical indicators or molecular markers able to define the severity of this disease. Even though accumulated evidence demonstrated that the endocannabinoid system (ECS) plays a central role in male reproductive biology, particularly in the testicular compartment, to date no data point to a role for ECS in the etiopathogenesis of varicocele. Therefore, the present research has been designed to investigate the relationship between testicular ECS gene expression and fertility, using a validated animal model of experimental varicocele (VAR), taking advantage of traditional statistical approaches and artificial neural network (ANN). Experimental induction of VAR led to a clear reduction of spermatogenesis in left testes ranging from a mild (Johnsen score 7: 21%) to a severe (Johnsen score 4: 58%) damage of the germinal epithelium. However, the mean number of new-borns recorded after two sequential matings was quite variable and independent of the Johnsen score. While the gene expression of biosynthetic and degrading enzymes of AEA (NAPE-PLD and FAAH, respectively) and of 2-AG (DAGLα and MAGL, respectively), as well as their binding cannabinoid receptors (CB1 and CB2), did not change between testes and among groups, a significant downregulation of vanilloid (TRPV1) expression was recorded in left testes of VAR rats and positively correlated with animal fertility. Interestingly, an ANN trained by inserting the left and right testicular ECS gene expression profiles (inputs) was able to predict varicocele impact on male fertility in terms of mean number of new-borns delivered (outputs), with a very high accuracy (average prediction error of 1%). The present study provides unprecedented information on testicular ECS gene expression patterns during varicocele, by developing a freely available predictive ANN model that may open new perspectives in the diagnosis of varicocele-associated infertility.

Neuroprotection by (endo)Cannabinoids in Glaucoma and Retinal Neurodegenerative Diseases.

BACKGROUND: Emerging neuroprotective strategies are being explored to preserve the retina from degeneration, that occurs in eye pathologies like glaucoma, diabetic retinopathy, age-related macular degeneration, and retinitis pigmentosa. Incidentally, neuroprotection of retina is a defending mechanism designed to prevent or delay neuronal cell death, and to maintain neural function following an initial insult, thus avoiding loss of vision. METHODS: Numerous studies have investigated potential neuroprotective properties of plant-derived phytocannabinoids, as well as of their endogenous counterparts collectively termed endocannabinoids (eCBs), in several degenerative diseases of the retina. eCBs are a group of neuromodulators that, mainly by activating G protein-coupled type-1 and type-2 cannabinoid (CB1 and CB2) receptors, trigger multiple signal transduction cascades that modulate central and peripheral cell functions. A fine balance between biosynthetic and degrading enzymes that control the right concentration of eCBs has been shown to provide neuroprotection in traumatic, ischemic, inflammatory and neurotoxic damage of the brain. RESULTS: Since the existence of eCBs and their binding receptors was documented in the retina of numerous species (from fishes to primates), their involvement in the visual processing has been demonstrated, more recently with a focus on retinal neurodegeneration and neuroprotection. CONCLUSION: The aim of this review is to present a modern view of the endocannabinoid system, in order to discuss in a better perspective available data from preclinical studies on the use of eCBs as new neuroprotective agents, potentially useful to prevent glaucoma and retinal neurodegenerative diseases.

Modulation of Type-1 and Type-2 Cannabinoid Receptors by Saffron in a Rat Model of Retinal Neurodegeneration.

Experimental studies demonstrated that saffron (Crocus sativus) given as a dietary supplement counteracts the effects of bright continuous light (BCL) exposure in the albino rat retina, preserving both morphology and function and probably acting as a regulator of programmed cell death [1]. The purpose of this study was to ascertain whether the neuroprotective effect of saffron on rat retina exposed to BCL is associated with a modulation of the endocannabinoid system (ECS). To this aim, we used eight experimental groups of Sprague-Dawley rats, of which six were exposed to BCL for 24 hours. Following retinal function evaluation, retinas were quickly removed for biochemical and morphological analyses. Rats were either saffron-prefed or intravitreally injected with selective type-1 (CB1) or type-2 (CB2) cannabinoid receptor antagonists before BCL. Prefeeding and intravitreally injections were combined in two experimental groups before BCL. BCL exposure led to enhanced gene and protein expression of retinal CB1 and CB2 without affecting the other ECS elements. This effect of BCL on CB1 and CB2 was reversed by saffron treatment. Selective CB1 and CB2 antagonists reduced photoreceptor death, preserved morphology and visual function of retina, and mitigated the outer nuclear layer (ONL) damage due to BCL. Of interest, CB2-dependent neuroprotection was more pronounced than that conferred by CB1. These data suggest that BCL modulates only distinct ECS elements like CB1 and CB2, and that saffron and cannabinoid receptors could share the same mechanism in order to afford retinal protection.

Anandamide Suppresses Proinflammatory T Cell Responses In Vitro through Type-1 Cannabinoid Receptor-Mediated mTOR Inhibition in Human Keratinocytes.

The endocannabinoid system comprises cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, anandamide (AEA) and 2-arachidonoylglycerol, and metabolic enzymes of these ligands. The endocannabinoid system has recently been implicated in the regulation of various pathophysiological processes of the skin that include immune competence and/or tolerance of keratinocytes, the disruption of which might promote the development of skin diseases. Recent evidence showed that CB1 in keratinocytes limits the secretion of proinflammatory chemokines, suggesting that this receptor might also regulate T cell dependent inflammatory diseases of the skin. In this article, we sought to investigate the cytokine profile of IFN-γ-activated keratinocytes, and found that CB1 activation by AEA suppressed production and release of signature TH1- and TH17-polarizing cytokines, IL-12 and IL-23, respectively. We also set up cocultures between a conditioned medium of treated keratinocytes and naive T cells to disclose the molecular details that regulate the activation of highly proinflammatory TH1 and TH17 cells. AEA-treated keratinocytes showed reduced an induction of IFN-γ-producing TH1 and IL-17-producing TH17 cells, and these effects were reverted by pharmacological inhibition of CB1 Further analyses identified mammalian target of rapamycin as a proinflammatory signaling pathway regulated by CB1, able to promote either IL-12 and IL-23 release from keratinocytes or TH1 and TH17 polarization. Taken together, these findings demonstrate that AEA suppresses highly pathogenic T cell subsets through CB1-mediated mammalian target of rapamycin inhibition in human keratinocytes. Thus, it can be speculated that the latter pathway might be beneficial to the physiological function of the skin, and can be targeted toward inflammation-related skin diseases.

Potential Therapeutic Value of a Novel FAAH Inhibitor for the Treatment of Anxiety.

Anxiety disorders are among the most prevalent psychiatric diseases with high personal costs and a remarkable socio-economic burden. However, current treatment of anxiety is far from satisfactory. Novel pharmacological targets have emerged in the recent years, and attention has focused on the endocannabinoid (eCB) system, given the increasing evidence that supports its central role in emotion, coping with stress and anxiety. In the management of anxiety disorders, drug development strategies have left apart the direct activation of type-1 cannabinoid receptors to indirectly enhance eCB signalling through the inhibition of eCB deactivation, that is, the inhibition of the fatty acid amide hydrolase (FAAH) enzyme. In the present study, we provide evidence for the anxiolytic-like properties of a novel, potent and selective reversible inhibitor of FAAH, ST4070, orally administered to rodents. ST4070 (3 to 30 mg/kg per os) administered to CD1 male mice induced an increase of time spent in the exploration of the open arms of the elevated-plus maze. A partial reduction of anxiety-related behaviour by ST4070 was also obtained in Wistar male rats, which moderately intensified the time spent in the illuminated compartment of the light-dark box. ST4070 clearly inhibited FAAH activity and augmented the levels of two of its substrates, N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine, in anxiety-relevant brain regions. Altogether, ST4070 offers a promising anxiolytic-like profile in preclinical studies, although further studies are warranted to clearly demonstrate its efficacy in the clinic management of anxiety disorders.

Truffles contain endocannabinoid metabolic enzymes and anandamide.

Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors.

A functional interplay between 5-lipoxygenase and µ-calpain affects survival and cytokine profile of human Jurkat T lymphocyte exposed to simulated microgravity.

A growing body of evidence strongly indicates that both simulated and authentic weightlessness exert a broad range of effects on mammalian tissues and cells, including impairment of immune cell function and increased apoptotic death. We previously reported that microgravity-dependent activation of 5-lipoxygenase (5-LOX) might play a central role in the initiation of apoptosis in human T lymphocytes, suggesting that the upregulation of this enzyme might be (at least in part) responsible for immunodepression observed in astronauts during space flights. Herein, we supplement novel information about the molecular mechanisms underlying microgravity-triggered apoptotic cell death and immune system deregulation, demonstrating that under simulated microgravity human Jurkat T cells increase the content of cytosolic DNA fragments and cytochrome c (typical hallmarks of apoptosis) and have an upregulated expression and activity of µ-calpain. These events were paralleled by the unbalance of interleukin- (IL-) 2 and interferon- (INF-) γ, anti- and proapoptotic cytokines, respectively, that seemed to be dependent on the functional interplay between 5-LOX and µ-calpain. Indeed, we report unprecedented evidence that 5-LOX inhibition reduced apoptotic death, restored the initial IL-2/INF-γ ratio, and more importantly reverted µ-calpain activation induced by simulated microgravity.

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.