Neuroprotective and neurotoxic effects of endocannabinoid-like compounds, N-arachidonoyl dopamine and N-docosahexaenoyl dopamine in differentiated cultures of induced pluripotent stem cells derived from patients with Parkinson's disease.

The prominent protective effects in diverse neuron injury paradigms exerted by cannabinoids and in particular their endogenously produced species render the endocannabinoid system a promising molecular target in the treatment of neurodegenerative diseases. However, the effects of individual endocannabinoids in human cells remain poorly investigated. Neural derivatives of human induced pluripotent stem cells (iPSC) offer unique opportunities for studying the neuroprotective compounds and development of patient-specific treatment. For the first time the cytotoxic and neuroprotective effects endocannabinoids N-arachidonoyl dopamine (N-ADA) and N-docosahexaenoyl dopamine (N-DDA) were assessed in human neural progenitors and dopamine neurons derived from iPSCs of healthy donors and patients with Parkinson's disease. While the short-term treatment with the investigated compounds in 0.1-10 µM concentration range exerted no toxicity in these cell types, the long-term exposure to 0.1-5 µM N-ADA or N-DDA reduced the survival of human neural progenitors. At the same time, both N-ADA and N-DDA protected neural progenitors and terminally differentiated neurons both from healthy donors and patients with Parkinson's disease against oxidative stress induced by hydrogen peroxide. The observed dramatic difference in the mode of action of N-acyl dopamines points on the possible existence of novel pathogenic mechanism of neurodegeneration induced by prolonged uncompensated production of these substances within neuronal tissue and should also be considered as a precaution in the future development of N-acyl dopamine-based therapeutic drugs.

Biophysical and biomechanical properties of neural progenitor cells as indicators of developmental neurotoxicity.

Conventional in vitro toxicity studies have focused on identifying IC50 and the underlying mechanisms, but how toxicants influence biophysical and biomechanical changes in human cells, especially during developmental stages, remain understudied. Here, using an atomic force microscope, we characterized changes in biophysical (cell area, actin organization) and biomechanical (Young's modulus, force of adhesion, tether force, membrane tension, tether radius) aspects of human fetal brain-derived neural progenitor cells (NPCs) induced by four classes of widely used toxic compounds, including rotenone, digoxin, N-arachidonoylethanolamide (AEA), and chlorpyrifos, under exposure up to 36 h. The sub-cellular mechanisms (apoptosis, mitochondria membrane potential, DNA damage, glutathione levels) by which these toxicants induced biochemical changes in NPCs were assessed. Results suggest a significant compromise in cell viability with increasing toxicant concentration (p < 0.01), and biophysical and biomechanical characteristics with increasing exposure time (p < 0.01) as well as toxicant concentration (p < 0.01). Impairment of mitochondrial membrane potential appears to be the most sensitive mechanism of neurotoxicity for rotenone, AEA and chlorpyrifos exposure, but compromise in plasma membrane integrity for digoxin exposure. The surviving NPCs remarkably retained stemness (SOX2 expression) even at high toxicant concentrations. A negative linear correlation (R2 = 0.92) exists between the elastic modulus of surviving cells and the number of living cells in that environment. We propose that even subtle compromise in cell mechanics could serve as a crucial marker of developmental neurotoxicity (mechanotoxicology) and therefore should be included as part of toxicology assessment repertoire to characterize as well as predict developmental outcomes.

2-Arachidonoylglycerol mobilizes myeloid cells and worsens heart function after acute myocardial infarction.

AIMS: Myocardial infarction (MI) leads to an enhanced release of endocannabinoids and a massive accumulation of neutrophils and monocytes within the ischaemic myocardium. These myeloid cells originate from haematopoietic precursors in the bone marrow and are rapidly mobilized in response to MI. We aimed to determine whether endocannabinoid signalling is involved in myeloid cell mobilization and cardiac recruitment after ischaemia onset. METHODS AND RESULTS: Intravenous administration of endocannabinoid 2-arachidonoylglycerol (2-AG) into wild type (WT) C57BL6 mice induced a rapid increase of blood neutrophil and monocyte counts as measured by flow cytometry. This effect was blunted when using cannabinoid receptor 2 knockout mice. In response to MI induced in WT mice, the lipidomic analysis revealed significantly elevated plasma and cardiac levels of the endocannabinoid 2-AG 24 h after infarction, but no changes in anandamide, palmitoylethanolamide, and oleoylethanolamide. This was a consequence of an increased expression of 2-AG synthesizing enzyme diacylglycerol lipase and a decrease of metabolizing enzyme monoacylglycerol lipase (MAGL) in infarcted hearts, as determined by quantitative RT-PCR analysis. The opposite mRNA expression pattern was observed in bone marrow. Pharmacological blockade of MAGL with JZL184 and thus increased systemic 2-AG levels in WT mice subjected to MI resulted in elevated cardiac CXCL1, CXCL2, and MMP9 protein levels as well as higher cardiac neutrophil and monocyte counts 24 h after infarction compared with vehicle-treated mice. Increased post-MI inflammation in these mice led to an increased infarct size, an impaired ventricular scar formation assessed by histology and a worsened cardiac function in echocardiography evaluations up to 21 days. Likewise, JZL184-administration in a myocardial ischaemia-reperfusion model increased cardiac myeloid cell recruitment and resulted in a larger fibrotic scar size. CONCLUSION: These findings suggest that changes in endocannabinoid gradients due to altered tissue levels contribute to myeloid cell recruitment from the bone marrow to the infarcted heart, with crucial consequences on cardiac healing and function.

Oleoylethanolamide-induced anorexia in rats is associated with locomotor impairment.

The endogenous peroxisome proliferator-activated receptor alpha (PPAR-α) agonist Oleoylethanolamide (OEA) inhibits eating in rodents, mainly by delaying the onset of meals. The underlying mechanisms of OEA-induced anorexia, however, remain unclear. Animals treated with high OEA doses were shown to display signs of discomfort and impaired locomotion. Therefore, we first examined whether the impaired locomotion may contribute to OEA's anorectic effect. Second, it is controversial whether abdominal vagal afferents are necessary for OEA's anorectic effect. Thus, we explored alternative peripheral neural pathways mediating IP OEA's anorectic effect by performing a celiac-superior mesenteric ganglionectomy (CGX) or a subdiaphragmatic vagal deafferentation (SDA) alone or in combination. Exogenously administered OEA at a commonly used dose (10 mg/kg BW, IP) concurrently reduced food intake and compromised locomotor activity. Attempts to dissociate both phenomena using the dopamine D2/D3 receptor agonist Quinpirole (1 mg/kg BW, SC) failed because Quinpirole antagonized both, OEA-induced locomotor impairment and delay in eating onset. CGX attenuated the prolongation of the latency to eat by IP OEA, but neither SDA nor CGX prevented IP OEA-induced locomotor impairment. Our results indicate that IP OEA's anorectic effect may be secondary to impaired locomotion rather than due to physiological satiety. They further confirm that vagal afferents do not mediate exogenous OEA's anorectic effects, but suggest a role for spinal afferents in addition to an alternative, nonneuronal signaling route.

Detrimental effects of 2-arachidonoylglycerol on whole blood platelet aggregation and on cerebral blood flow after a focal ischemic insult in rats.

2-Arachidonoylglycerol (2-AG) is a major modulator of blood flow and platelet aggregation and a potential neuroprotectant. The present study investigated, for the first time, the effects of 2-AG on cerebral blood flow (CBF) in the first critical hours during middle cerebral artery occlusion (MCAO) and on platelet aggregation in rats. Adult male Sprague-Dawley rats ( n = 30) underwent permanent MCAO under isoflurane anesthesia and were randomly assigned to receive either 2-AG (6 mg/kg iv), monoacylglycerol lipase inhibitor JZL-184 (10 mg/kg iv), or vehicle ( n = 6 rats/group) treatment. CBF and cardiovascular responses were measured, by a blinded investigator, for up to 4 h. In separate experiments, platelet aggregation by 2-AG (19-300 µM) was assessed by whole blood aggregometry ( n = 40). 2-AG and JZL-184 significantly increased the severity of the CBF deficit versus vehicle (20.2 ± 8.8% and 22.7 ± 6.4% vs. 56.4 ± 12.1% of pre-MCAO baseline, respectively, P < 0.05) but had no effect on blood pressure or heart rate. While JZL-184 significantly increased the number of thrombi after MCAO, this did not reach significance by 2-AG. 2-AG induced platelet aggregation in rat whole blood in a similar manner to arachidonic acid and was significantly reduced by the cyclooxygenase inhibitors indomethacin and flurbiprofen and the thromboxane receptor antagonist ICI 192,605 ( P < 0.05). This is the first study showing that 2-AG increases the severity of the CBF deficit during MCAO, and further interrogation confirmed 2-AG-induced platelet aggregation in rats. These findings are important because 2-AG had previously been shown to exert neuroprotective actions and therefore force us to reevaluate the circumstances under which 2-AG is beneficial. NEW & NOTEWORTHY 2-Arachidonoylglycerol (2-AG) has neuroprotective properties; however, the present study revealed that 2-AG increases the severity of the cerebral blood flow deficit during middle cerebral artery occlusion in rats. Further interrogation showed that 2-AG induces platelet aggregation in rats. These findings force us to reevaluate the circumstances under which 2-AG is beneficial.

Investigation of changes in endocannabinoids and N-acylethanolamides in biofluids, and their correlations with female infertility.

Female infertility is a worldwide medical problem, and the scarcity of infertility biomarkers has hindered the ability to launch preventive and therapeutic measures in a timely manner. Intriguingly, alterations in endocannabinoids (eCBs) and N-acylethanolamides (NAEs) have been observed in the biofluids of infertile females. Therefore, a hypothesis of using eCB and NAEs in biofluids as infertility biomarkers was proposed by several researchers; however, little evidence exists to verify the hypothesis. To investigate their correlations with female infertility, we developed a magnetic liquid microextraction-chemical derivatization (MLME-CD) method coupled with liquid chromatography-tandem mass spectrometry for the quantification of eCBs and NAEs in biofluids. The target compounds were first purified with magnetic toluene as sorbents, and then labeled with 4-(N,N-dimethyamino)benzoyl chloride (4-DMABC). The MLME-CD method offered several advantages, including reliable quantification results by preventing the isomerization of eCB, high throughput by requiring 20min for sample preparation, and good sensitivity with limits of detection at 3.0-54.3 fmol. The intra-day and inter-day relative standard deviations were below 14.5%, and the recoveries were 87.4%-117.9%. Concentrations of eCBs and NAEs in the serum of 49 infertile women and 53 fertile women (controls), and in the ovarian follicular fluid of 21 infertile women and 20 controls were then quantified. Using unpaired t test analysis indicated significant differences in AEA and PEA in serum, and OEA in follicular fluid between infertile women and healthy controls, and the areas under the curve were in the range of 0.605-0.707.

Human sperm motility, capacitation and acrosome reaction are impaired by 2-arachidonoylglycerol endocannabinoid.

The endocannabinoids are cannabinoids synthesized by mammalian tissues. These compounds are closely related to the regulation of the male reproductive system. However, little is known about the effects produced by 2-arachidonoylglycerol (2AG) on in vitro human sperm functions. This study was undertaken to determine the effects produced by 2AG on fresh human sperm and in the capacitation technique. Semen samples from healthy young men were exposed to different concentrations of 2AG before and during capacitation technique. In this work, we have demonstrated that 2AG induces the spontaneous acrosome reaction and reduces progressive motility in fresh human sperm. During the capacitation technique, sperm becomes more sensitive to low concentrations of 2AG, triggering the acrosome reaction and inhibiting protein phosphorylation. In summary, 2AG affects the in vitro functionality of human sperm by reducing motility, inhibiting capacitation and triggering the acrosome reaction.

Cannabis: A Treasure Trove or Pandora's Box?

BACKGROUND & OBJECTIVE: Cannabis is one of the earliest cultivated plants. Cannabis of industrial utility and culinary value is generally termed as hemp. Conversely, cannabis that is bred for medical, spiritual and recreational purposes is called marijuana. The female marijuana plant produces a significant quantity of bio- and psychoactive phytocannabinoids, which regained the spotlight with the discovery of the endocannabinoid system of the animals in the early 90's. Nevertheless, marijuana is surrounded by controversies, debates and misconceptions related to its taxonomic classification, forensic identification, medical potential, legalization and its long-term health consequences. METHOD: In the first part, we provide an in-depth review of the botany and taxonomy of Cannabis. We then overview the biosynthesis of phytocannabinoids within the glandular trichomes with emphasis on the role of peculiar plastids in the production of the secreted material. We also compile the analytical methods used to determine the phytocannabinoid composition of glandular trichomes. In the second part, we revisit the psychobiology and molecular medicine of marijuana. RESULTS & CONCLUSION: We summarize our current knowledge on the recreational use of cannabis with respect to the modes of consumption, short-term effects, chronic health consequences and cannabis use disorder. Next, we overview the molecular targets of a dozen major and minor bioactive cannabinoids in the body. This helps us introduce the endocannabinoid system in an unprecedented detail: its up-todate molecular biology, pharmacology, physiology and medical significance, and beyond. In conclusion, we offer an unbiased survey about cannabis to help better weigh its medical value versus the associated risks.

Modulation of anxiety-like behavior by the endocannabinoid 2-arachidonoylglycerol (2-AG) in the dorsolateral periaqueductal gray.

Anandamide and 2-arachidonoylglycerol (2-AG) are the two main endocannabinoids, exerting their effects by activating type 1 (CB1r) and type 2 (CB2r) cannabinoid receptors. Anandamide inhibits anxiety-like responses through the activation of CB1r in certain brain regions, including the dorsolateral periaqueductal gray (dlPAG). 2-AG also attenuates anxiety-like responses, although the neuroanatomical sites for these effects remained unclear. Here, we tested the hypothesis that enhancing 2-AG signaling in the dlPAG would induce anxiolytic-like effects. The mechanisms involved were also investigated. Male Wistar rats received intra-dlPAG injections of 2-AG, URB602 (inhibitor of the 2-AG hydrolyzing enzyme, mono-acylglycerol lipase--MGL), AM251 (CB1r antagonist) and AM630 (CB2r antagonist). The behavior was analyzed in the elevated plus maze after the following treatments. Exp. 1: vehicle (veh) or 2-AG (5 pmol, 50 pmol, and 500 pmol). Exp. 2: veh or URB602 (30 pmol, 100 pmol or 300 pmol). Exp. 3: veh or AM251 (100 pmol) followed by veh or 2-AG (50 pmol). Exp. 4: veh or AM630 (1000 pmol) followed by veh or 2-AG. Exp. 5: veh or AM251 followed by veh or URB602 (100 pmol). Exp. 6: veh or AM630 followed by veh or URB602. 2-AG (50 pmol) and URB602 (100 pmol) significantly increased the exploration of the open arms of the apparatus, indicating an anxiolytic-like effect. These behavioral responses were prevented by CB1r (AM251) or CB2r (AM630) antagonists. Our results showed that the augmentation of 2-AG levels in the dlPAG induces anxiolytic-like effects. The mechanism seems to involve both CB1r and CB2r receptors.

Influence of serum and albumin on the in vitro anandamide cytotoxicity toward C6 glioma cells assessed by the MTT cell viability assay: implications for the methodology of the MTT tests.

Anandamide (AEA), an endogenous ligand of cannabinoid CB1 and CB2 receptors, which also binds transient receptor potential vanilloid type 1 receptor (TRPV1), has been shown to display substantial selective cytotoxicity toward some cancer cell lines in vitro, although the relevant data are not consistent. In the present study, we employed the MTT test to assess short-term cytotoxicity of AEA on C6 rat glioma cell culture. When anandamide was administered to the culture medium with foetal bovine serum (FBS), no cytotoxic effect was observed following 24 h exposure of the glioma cells to micromolar concentrations of AEA. However, if no serum was present in the medium, micro-to-submicromolar concentrations of AEA induced dose-dependent cytotoxicity clearly detectable after 24 h. Control experiments made it possible to exclude significant interference of serum with the MTT test per se. Bovine serum albumin mimicked the effect of FBS. We conclude that the apparent inhibition of short-term cytotoxicity of AEA toward C6 rat glioma cells in vitro is caused by binding AEA to serum proteins such as albumin. Taking into account that blood serum or albumin is practically always present in cell culture media, we discuss implications of binding substances to serum proteins for methodology and interpretation of in vitro cytotoxicity testing.

Involvement of transient receptor potential vanilloid type 1 channels in the pro-convulsant effect of anandamide in pentylenetetrazole-induced seizures.

Anandamide, an endogenous agonist of CB(1) receptors, also activates TRPV1 but at a higher concentration. Studies demonstrate the anticonvulsant activity of anandamide via CB(1) receptors, while its action through TRPV1 is still ambiguous. Thus, the present study investigated the influence of anandamide on pentylenetetrazole-induced seizures in mice pretreated with TRPV1 or CB(1) receptor antagonists. Acute intracerebroventricular administration of low doses of anandamide (10, 20, or 40µg/mouse) produced anticonvulsant effect, while the pro-convulsant effect was evident at high doses (80 or 100µg/mouse). Interestingly, AM251 (2µg/mouse), a CB(1) antagonist pretreatment blocked the anticonvulsant effect, but augmented the pro-convulsant effect. Conversely, in the presence of inactive dose of capsazepine (1µg/mouse), a TRPV1 antagonist, anandamide exhibited significant anticonvulsant effect even at high doses with no change in its anticonvulsant effect. Moreover, mice treated with capsaicin, a TRPV1 agonist (10, or 100µg/mouse) exhibited pro-convulsant activity that was blocked by capsazepine pretreatment. However, capsazepine, per se at doses 10 or 100µg/mouse exhibited anticonvulsant effect. Like anandamide, the agents (AM404 and URB597), which increase its synaptic concentrations produced similar biphasic effects. Thus, these results indicate that anandamide exhibits both pro- and anticonvulsant activities by activating TRPV1 and CB(1) receptor respectively.