Wwl70-induced ABHD6 inhibition attenuates memory deficits and pathological phenotypes in APPswe/PS1dE9 mice.

Synaptic dysfunction plays a crucial role in the pathogenesis of Alzheimer's disease (AD). α/ß-hydrolase domain-containing 6 (ABHD6) contributes to synaptic dysfunctions, and ABHD6 inhibition has shown potential therapeutic value in neurological disorders. However, the role of ABHD6 in AD has not been fully defined. In this study, we demonstrated that adeno-associated virus (AAV) mediated shRNA targeting ABHD6 in hippocampal neurons attenuated synaptic dysfunction and memory impairment of APPswe/PS1dE9 (APP/PS1) mice, while it didn't affect the amyloid-beta (Aß) levels and neuroinflammation in the brains. In addition, intraperitoneal injection of wwl70, a specific inhibitor of ABHD6, improved synaptic plasticity and memory function in APP/PS1 mice, which might attribute to the activation of endogenous cannabinoid signaling. Furthermore, wwl70 significantly decreased the Aß levels and neuroinflammation in the hippocampus of AD mice, and enhanced Aß phagocytized by microglia. In conclusion, for the first time our data have shown that ABHD6 inhibition might be a promising strategy for AD treatment, and wwl70 is a potential candidate for AD drug development pipeline.

Cannabidiol attenuates behavioral changes in a rodent model of schizophrenia through 5-HT1A, but not CB1 and CB2 receptors.

Preclinical and clinical data indicate that cannabidiol (CBD), a non-psychotomimetic compound from the Cannabis sativa plant, can induce antipsychotic-like effects. In an animal model of schizophrenia based on the antagonism of NMDA receptors, the behavioral and molecular changes induced by repeated treatment with the NMDA receptor antagonist MK-801 were prevented when CBD was co-administered with MK-801. It is unknown, however, if CBD would reverse these changes once they have been established. Thus, in the present study we used male C57BL/6J mice, 6 weeks old, to evaluate whether daily CBD injection for seven days, starting after the end of the repeated treatment with MK-801 for 14 days, would reverse MK-801-induced deficits in the social interaction (SI) and novel object recognition (NOR) tests, which have been used to investigate the negative and cognitive symptoms of schizophrenia, respectively. We also assessed whether CBD effects would be blocked by pretreatment with AM251, a CB1 receptor antagonist, AM630, a CB2 receptor antagonist, or WAY100635, a 5-HT1A receptor antagonist. CBD and the second-generation antipsychotic clozapine, used as a positive control, attenuated the impairments in the SI and NOR tests induced by repeated administered MK-801. CBD effects were blocked by WAY100635, but not by AM251 or AM630. These data suggest that CBD induces antipsychotic-like effects by activating 5-HT1A receptors and indicate that this compound could be an interesting alternative for the treatment of negative and cognitive symptoms of schizophrenia.

Formation of OX-1R/CB1R heteromeric complexes in embryonic mouse hypothalamic cells: Effect on intracellular calcium, 2-arachidonoyl-glycerol biosynthesis and ERK phosphorylation.

Orexin 1 (OX-1R) and cannabinoid receptor (CB1R) belong to the superfamily of G-protein-coupled receptors (GPCRs) and are mostly coupled to Gq and Gi/o proteins, respectively. In vitro studies in host cells over-expressing OX-1R and CB1R revealed a functional interaction between these receptors, through either their ability to form heteromers or the property for OX-1R to trigger the biosynthesis of 2-arachidonoylglycerol (2-AG), an endogenous CB1R ligand. Since: i) OX-1R and CB1R co-espression has been described at postsynaptc sites in hypothalamic circuits involved the regulation of energy homeostasis, and ii) increased orexin-A (OX-A) and 2-AG levels occur in hypothalamic neurons during obesity, we sought here to investigate the OX-1R/CB1R interaction in embryonic mouse hypothalamic NPY/AgRP mHypoE-N41 neurons which express, constitutively, both receptors. Treatment of mHypoE-N41 cells with OX-A (0.1-0.3µM), but not with the selective CB1R agonist, arachidonyl-2-chloroethylamide (ACEA; 0.1-0.3µM), transiently elevated [Ca(2+)]i. Incubation with a subeffective dose of OX-A (0.1µM)+ACEA (0.1µM) led to stronger and longer lasting elevation of [Ca(2+)]i, antagonized by OX-1R or CB1R antagonism with SB-334867 or AM251, respectively. FRET and co-immunoprecipitation experiments showed the formation of OX-1R/CB1R heteromers after incubation with OX-A (0.2µM), or OX-A (0.1µM)+ACEA (0.1µM), but not after ACEA (0.2µM), in a manner antagonized by SB-334867 or AM251. OX-A (0.2µM) or OX-A (0.1µM)+ACEA (0.1µM) also led to 2-AG biosynthesis. Finally, a stronger activation of ERK1/2(Thr202/185) phosphorylation in comparison to basal or each agonist alone (0.1-0.2µM), was induced by incubation with OX-A (0.1µM)+ACEA (0.1µM), again in a manner prevented by OX-1R or CB1R antagonism. We suggest that OX-A, alone at effective concentrations on [Ca(2+)]i, or in combination with ACEA, at subeffective concentrations, triggers intracellular signaling events via the formation of OX-1R/CB1R heteromers and an autocrine loop mediated by 2-AG.

Anandamide reduces the migration of lymphocytes to the intestine by CB2 activation and reduces TNF-α in the target organs, protecting mice from graft-versus-host disease.

Graft-versus-host disease (GVHD) is a serious inflammatory illness that often occurs as a secondary complication of bone marrow transplantation. Current therapies have limited effectiveness and fail to achieve a balance between inflammation and the graft-versus-tumor effect. In this study, we investigate the effects of the endocannabinoid anandamide on the complex pathology of GVHD. We assess the effects of an irreversible inhibitor of fatty acid amine hydrolase or exogenous anandamide and find that they increase survival and reduce clinical signs in GVHD mice. In the intestine of GVHD mice, treatment with exogenous anandamide also leads to a reduction in the number of CD3+, CD3+CD4+, and CD3+CD8+ cells, which reduces the activation of CD3+CD4+ and CD3+CD8+ cells, as assessed by enhanced CD28 expression, a T cell co-stimulatory molecule. Exogenous AEA was also able to reduce TNF-α and increase IL-10 in the intestine of GVHD mice. In the liver, exogenous AEA reduces injury, TNF-α levels, and the number of CD3+CD8+ cells. Interestingly, anandamide reduces Mac-1α, which lowers the adhesion of transplanted cells in mesenteric veins. These effects are mimicked by JWH133-a CB2 selective agonist-and abolished by treatment with a CB2 antagonist. Furthermore, the effects caused by anandamide treatment on survival were related to the CB2 receptor, as the CB2 antagonist abolished it. This study shows the critical role of the CB2 receptor in the modulation of the inflammatory response of GVHD by treatment with anandamide, the most prominent endocannabinoid.

Experimental ischemia/reperfusion model impairs endocannabinoid signaling and Na+/K+ ATPase expression and activity in kidney proximal tubule cells.

LLC-PK1 cells, an immortalized epithelial cell line derived from pig renal proximal tubules, express all the major players of the endocannabinoid system (ECS) such as CB1, CB2 and TRPV1 receptors, as well as the main enzymes involved in the biosynthesis and degradation of the major endocannabinoids named 2-arachidonoylglycerol, 2-AG and anandamide, AEA. Here we investigated whether the damages caused by ischemic insults either in vitro using LLC-PK1 cells exposed to antimycin A (an inductor of ATP-depletion) or in vivo using Wistar rats in a classic renal ischemia and reperfusion (IR) protocol, lead to changes in AEA and 2-AG levels, as well as altered expression of genes from the main enzymes involved in the regulation of the ECS. Our data show that the mRNA levels of the CB1 receptor gene were downregulated, while the transcript levels of monoacylglycerol lipase (MAGL), the main 2-AG degradative enzyme, were upregulated in LLC-PK1 cells after IR model. Accordingly, IR was accompanied by a significant reduction in the levels of 2-AG and AEA, as well as of the two endocannabinoid related molecules, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in LLC-PK1 cells. In kidney cortex homogenates, only AEA levels were significantly decreased. In addition, we found that in both the in vitro and in vivo model IR caused a reduction in the expression and activity of the Na+/K+ ATPase. These changes were reversed by the CB1/CB2 agonist WIN55,212, in a CB1-receptor dependent manner in the LLC-PK1 IR model. In conclusion, the ECS and Na+/K+ ATPase are down-regulated following IR in LLC-PK1 cells and rat kidney. We suggest that CB1 agonists might represent a potential strategy to reverse the consequences of IR injury in kidney tissues.

N-oleoyldopamine modulates activity of midbrain dopaminergic neurons through multiple mechanisms.

N-oleoyl-dopamine (OLDA) is an amide of dopamine and oleic acid, synthesized in catecholaminergic neurons. The present study investigates OLDA targets in midbrain dopaminergic (DA) neurons. Substantia Nigra compacta (SNc) DA neurons recorded in brain slices were excited by OLDA in wild type mice. In transient receptor potential vanilloid 1 (TRPV1) knockout (KO) mice, however, SNc DA neurons displayed sustained inhibition of firing. In the presence of the dopamine type 2 receptor (D2R) antagonist sulpiride or the dopamine transporter blocker nomifensine no such inhibition was observed. Under sulpiride OLDA slightly excited SNc DA neurons, an action abolished upon combined application of the cannabinoid1 and 2 receptor antagonists AM251 and AM630. In ventral tegmental area (VTA) DA neurons from TRPV1 KO mice a transient inhibition of firing by OLDA was observed. Thus OLDA modulates the firing of nigrostriatal DA neurons through interactions with TRPV1, cannabinoid receptors and dopamine uptake. These findings suggest further development of OLDA-like tandem molecules for the treatment of movement disorders including Parkinson's disease.

Memantine alters striatal plasticity inducing a shift of synaptic responses toward long-term depression.

Memantine is an open channel blocker that antagonizes NMDA receptors reducing the inappropriate calcium (Ca(2+)) influx occurring in presence of moderately increased glutamate levels. At the same time, memantine has the ability to preserve the transient physiological activation of NMDA receptor, essential for learning and memory formation at synaptic level. In the present study we investigated the effects exerted by memantine on striatal synaptic plasticity in rat striatal spiny projection neurons (SPNs). In vitro application of memantine in striatal slices elicited a disruption of long-term potentiation (LTP) induction and maintenance, and revealed, in the majority of the recorded neurons, a long-term depression (LTD), whose amplitude was concentration-dependent (0.3-10 µM). Interestingly, preincubation with the dopamine (DA) D2 receptor antagonist sulpiride (10 µM) prevented memantine-induced LTD and restored LTP. Moreover, the DA D2 agonist quinpirole (10 µM), similarly to memantine, induced LTD in a subgroup of SPNs. In addition, memantine-induced LTD was also prevented by the CB1 endocannabinoid receptor antagonist AM 251 (1 µM). These results suggest that the actions exerted by memantine on striatal synaptic plasticity, and in particular the induction of LTD observed in SPNs, could be attributed to its ability to activate DA D2 receptors. By contrast, blockade of NMDA receptor is not involved in memantine-induced LTD since APV (30 µM) and MK801 (10 µM), two NMDA receptor antagonists, failed to induce this form of synaptic plasticity. Our data indicate that memantine could be used as treatment of neurological disorders in which DA D2 receptor represents a possible therapeutic target.

Cannabidiol induces rapid-acting antidepressant-like effects and enhances cortical 5-HT/glutamate neurotransmission: role of 5-HT1A receptors.

Cannabidiol (CBD), the main non-psychotomimetic component of marihuana, exhibits anxiolytic-like properties in many behavioural tests, although its potential for treating major depression has been poorly explored. Moreover, the mechanism of action of CBD remains unclear. Herein, we have evaluated the effects of CBD following acute and chronic administration in the olfactory bulbectomy mouse model of depression (OBX), and investigated the underlying mechanism. For this purpose, we conducted behavioural (open field and sucrose preference tests) and neurochemical (microdialysis and autoradiography of 5-HT1A receptor functionality) studies following treatment with CBD. We also assayed the pharmacological antagonism of the effects of CBD to dissect out the mechanism of action. Our results demonstrate that CBD exerts fast and maintained antidepressant-like effects as evidenced by the reversal of the OBX-induced hyperactivity and anhedonia. In vivo microdialysis revealed that the administration of CBD significantly enhanced serotonin and glutamate levels in vmPFCx in a different manner depending on the emotional state and the duration of the treatment. The potentiating effect upon neurotransmitters levels occurring immediately after the first injection of CBD might underlie the fast antidepressant-like actions in OBX mice. Both antidepressant-like effect and enhanced cortical 5-HT/glutamate neurotransmission induced by CBD were prevented by 5-HT1A receptor blockade. Moreover, adaptive changes in pre- and post-synaptic 5-HT1A receptor functionality were also found after chronic CBD. In conclusion, our findings indicate that CBD could represent a novel fast antidepressant drug, via enhancing both serotonergic and glutamate cortical signalling through a 5-HT1A receptor-dependent mechanism.

Synthetic cannabinoid JWH-018 and its halogenated derivatives JWH-018-Cl and JWH-018-Br impair Novel Object Recognition in mice: Behavioral, electrophysiological and neurochemical evidence.

It is well known that an impairment of learning and memory function is one of the major physiological effects caused by natural or synthetic cannabinoid consumption in rodents, nonhuman primates and in humans. JWH-018 and its halogenated derivatives (JWH-018-Cl and JWH-018-Br) are synthetic CB1/CB2 cannabinoid agonists, illegally marketed as "Spice" and "herbal blend" for their Cannabis-like psychoactive effects. In the present study the effects of acute exposure to JWH-018, JWH-018-Cl, JWH-018-Br (JWH-018-R compounds) and Δ(9)-THC (for comparison) on Novel Object Recognition test (NOR) has been investigated in mice. Moreover, to better characterize the effects of JWH-018-R compounds on memory function, in vitro electrophysiological and neurochemical studies in hippocampal preparations have been performed. JWH-018, JWH-018-Cl and JWH-018-Br dose-dependently impaired both short- and long-memory retention in mice (respectively 2 and 24 h after training session). Their effects resulted more potent respect to that evoked by Δ(9)-THC. Moreover, in vitro studies showed as JWH-018-R compounds negatively affected electrically evoked synaptic transmission, LTP and aminoacid (glutamate and GABA) release in hippocampal slices. Behavioral, electrophysiological and neurochemical effects were fully prevented by CB1 receptor antagonist AM251 pretreatment, suggesting a CB1 receptor involvement. These data support the hypothesis that synthetic JWH-018-R compounds, as Δ(9)-THC, impair cognitive function in mice by interfering with hippocampal synaptic transmission and memory mechanisms. This data outline the danger that the use and/or abuse of these synthetic cannabinoids may represent for the cognitive process in human consumer.

Δ9-Tetrahydrocannabinol reverses TNFα-induced increase in airway epithelial cell permeability through CB2 receptors.

Despite pharmacological treatment, bronchial hyperresponsiveness continues to deteriorate as airway remodelling persists in airway inflammation. Previous studies have demonstrated that the phytocannabinoid Δ9-tetrahydrocannabinol (THC) reverses bronchoconstriction with an anti-inflammatory action. The aim of this study was to investigate the effects of THC on bronchial epithelial cell permeability after exposure to the pro-inflammatory cytokine, TNFα. Calu-3 bronchial epithelial cells were cultured at air-liquid interface. Changes in epithelial permeability were measured using Transepithelial Electrical Resistance (TEER), then confirmed with a paracellular permeability assay and expression of tight junction proteins by Western blotting. Treatment with THC prevented the TNFα-induced decrease in TEER and increase in paracellular permeability. Cannabinoid CB1 and CB2 receptor-like immunoreactivity was found in Calu-3 cells. Subsequent experiments revealed that pharmacological blockade of CB2, but not CB1 receptor inhibited the THC effect. Selective stimulation of CB2 receptors displayed a similar effect to that of THC. TNFα decreased expression of the tight junction proteins occludin and ZO-1, which was prevented by pre-incubation with THC. These data indicate that THC prevents cytokine-induced increase in airway epithelial permeability through CB2 receptor activation. This highlights that THC, or other cannabinoid receptor ligands, could be beneficial in the prevention of inflammation-induced changes in airway epithelial cell permeability, an important feature of airways diseases.

Stress induces analgesia via orexin 1 receptor-initiated endocannabinoid/CB1 signaling in the mouse periaqueductal gray.

The orexin system consists of orexin A/hypocretin 1 and orexin B/hypocretin 2, and OX1 and OX2 receptors. Our previous electrophysiological study showed that orexin A in the rat ventrolateral periaqueductal gray (vlPAG) induced antinociception via an OX1 receptor-initiated and endocannabinoid-mediated disinhibition mechanism. Here, we further characterized antinociceptive effects of orexins in the mouse vlPAG and investigated whether this mechanism in the vlPAG can contribute to stress-induced analgesia (SIA) in mice. Intra-vlPAG (i.pag.) microinjection of orexin A in the mouse vlPAG increased the hot-plate latency. This effect was mimicked by i.pag. injection of WIN 55,212-2, a CB1 agonist, and antagonized by i.pag. injection of the antagonist of OX1 (SB 334867) or CB1 (AM 251), but not OX2 (TCS-OX2-29) or opioid (naloxone), receptors. [Ala(11), D-Leu(15)]-orexin B (i.pag.), an OX2 selective agonist, also induced antinociception in a manner blocked by i.pag. injection of TCS-OX2-29, but not SB 334867 or AM 251. Mice receiving restraint stress for 30 min showed significantly longer hot-plate latency, more c-Fos-expressing orexin neurons in the lateral hypothalamus and higher orexin levels in the vlPAG than unrestrained mice. Restraint SIA in mice was prevented by i.pag. or intraperitoneal injection of SB 334867 or AM 251, but not TCS-OX2-29 or naloxone. These results suggest that during stress, hypothalamic orexin neurons are activated, releasing orexins into the vlPAG to induce analgesia, possibly via the OX1 receptor-initiated, endocannabinoid-mediated disinhibition mechanism previously reported. Although activating either OX1 or OX2 receptors in the vlPAG can lead to antinociception, only OX1 receptor-initiated antinociception is endocannabinoid-dependent.

Novel halogenated derivates of JWH-018: Behavioral and binding studies in mice.

JWH-018 is a synthetic CB1 and CB2 agonist illegally marketed as products named "Spice" or "herbal blend" for its psychoactive effects which are much higher than those produced by cannabis. In the last year, the European Monitoring Centre for Drugs and Drug Addiction reported to the Italian National Early Warning System the seizure of plant material containing new halogenated derivatives of JWH-018 (JWH-018 Cl and JWH-018 Br). The present study aimed to investigate the in vitro and in vivo activity of these two new synthetic cannabinoids in mice. In vitro competition binding experiments performed on mouse and human CB1 receptors revealed a high affinity and potency of the halogenated compounds. Synthetic cannabinoids (0.01-6 mg/kg i.p.) impaired motor activity and induced catalepsy in mice and their effects were more severe with respect to those evoked by Δ(9)-THC. Moreover, they increased the mechanical and thermal pain threshold and induced a marked hypothermia. It is interesting to note that whereas high doses of JWH-018 cause seizures, myoclonia and hyperreflexia, the halogenated compounds, in particular JWH-018Br, were less effective. Behavioral and neurological changes were prevented by the selective CB1 receptor antagonist AM 251. These data demonstrate for the first time that JWH-018 Cl and JWH-018 Br act similarly to JWH-018 while inducing less convulsive episodes and myoclonias. These data support the hypothesis that the halogenated compounds may have been introduced onto market to produce similar intoxicating effects as JWH-018 while causing less side effects.

Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA-salt hypertensive rats.

AIMS: This study was performed to examine whether hypertension affects the sympathetic transmission to resistance vessels of pithed rats via inhibitory presynaptic cannabinoid CB1 receptors and whether endocannabinoids are involved in this response. MATERIALS AND METHODS: We compared uninephrectomised rats rendered hypertensive by high salt diet and deoxycorticosterone acetate (DOCA) injections with normotensive animals (uninephrectomy only). Experiments were performed on vagotomised and pithed animals. Increases in diastolic blood pressure (DBP) were induced four times (S1-S4) by electrical stimulation or phenylephrine injection. KEY FINDINGS: Electrical stimulation (0.75Hz, 1ms, 50V, 5 impulses) of the preganglionic sympathetic nerve fibres innervating the blood vessels more strongly increased DBP in normotensive than in DOCA-salt rats. Phenylephrine (0.01µmol/kg) induced similar increases in DBP in both groups. The cannabinoid receptor agonist CP55940 (0.01-1µmol/kg) did not modify the rises in DBP induced by phenylephrine. However, it inhibited the electrically stimulated increases in DBP, more strongly in DOCA-salt than in normotensive animals (maximally by 50 and 30%, respectively). The effect of CP55940 was attenuated by the CB1 antagonist AM251 (3µmol/kg). AM251 enhanced the neurogenic vasopressor response during S4 by itself in hypertensive rats only. URB597 (3µmol/kg), which inhibits degradation of the endocannabinoid anandamide, did not modify the electrically stimulated increases in DBP. SIGNIFICANCE: The function of inhibitory presynaptic CB1 receptors on sympathetic nerves is enhanced in DOCA-salt hypertensive rats. Thus, the CB1 receptor-mediated inhibition of noradrenaline release from the sympathetic nerve fibres innervating the resistance vessels might play a protective role in hypertension.