Leyre Mestre

Decreased endocannabinoid levels in the brain and beneficial effects of agents activating cannabinoid and/or vanilloid receptors in a rat model of multiple sclerosis

Recent studies have addressed the changes in endocannabinoid ligands and receptors that occur in multiple sclerosis, as a way to explain the efficacy of cannabinoid compounds to alleviate spasticity, pain, tremor, and other signs of this autoimmune disease. Using Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, we recently found a decrease in cannabinoid CB1 receptors mainly circumscribed to the basal ganglia, which could be related to the motor disturbances characteristic of these rats. In the present study, using the same model, we explored the potential changes in several neurotransmitters in the basal ganglia that might be associated with the motor disturbances described in these rats, but we only found a small increase in glutamate contents in the globus pallidus. We also examined whether the motor disturbances and the changes of CB1 receptors found in the basal ganglia of EAE rats disappear after the treatment with rolipram, an inhibitor of type IV phosphodiesterase able to supress EAE in different species. Rolipram attenuated clinical decline, reduced motor inhibition, and normalized CB1 receptor gene expression in the basal ganglia. As a third objective, we examined whether EAE rats also exhibited changes in endocannabinoid levels as shown for CB1 receptors. Anandamide and 2-arachidonoylglycerol levels decreased in motor related regions (striatum, midbrain) but also in other brain regions, although the pattern of changes for each endocannabinoid was different. Finally, we hypothesized that the elevation of the endocannabinoid activity, following inhibition of endocannabinoid uptake, might be beneficial in EAE rats. AM404, arvanil, and OMDM2 were effective to reduce the magnitude of the neurological impairment in EAE rats, whereas VDM11 did not produce any effect. The beneficial effects of AM404 were reversed by blocking TRPV1 receptors with capsazepine, but not by blocking CB1 receptors with SR141716, thus indicating the involvement of endovanilloid mechanisms in these effects. However, a role for CB1 receptors is supported by additional data showing that CP55,940 delayed EAE progression. In summary, our data suggest that reduction of endocannabinoid signaling is associated with the development of EAE in rats. We have also proved that the reduction of CB1 receptors observed in these rats is corrected following treatment with a compound used in EAE such as rolipram. In addition, the direct or indirect activation of vanilloid or cannabinoid receptors may reduce the neurological impairment experienced by EAE rats, although the efficacy of the different compounds examined seems to be determined by their particular pharmacodynamic and pharmacokinetic characteristics.

Pharmacological modulation of the endocannabinoid system in a viral model of multiple sclerosis

Theilers virus infection of the central nervous system (CNS) induces an immune‐mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Cannabinoids have been shown to exert beneficial effects on animal models of MS and evidence suggests that the endocannabinoid system plays a role in the tonic control of spasticity. In this study we show that OMDM1 [(R)‐N‐oleoyl‐(1′‐hydroxybenzyl)‐2′‐ethanolamine] and OMDM2 [(S)‐N‐oleoyl‐(1′‐hydroxybenzyl)‐2′‐ethanolamine], two selective inhibitors of the putative endocannabinoid transporter and hence of endocannabinoid inactivation, provide an effective therapy for Theiler murine encephalomyelitis virus‐induced demyelinating disease (TMEV‐IDD). Treatment of TMEV‐infected mice with OMDM1 and OMDM2 enhanced anandamide levels in the spinal cord and ameliorated motor symptoms. This was associated with a down‐regulation of inflammatory responses in the spinal cord. In addition we show that OMDM1 and OMDM2 down‐regulate macrophage function by (i) decreasing the surface expression of major histocompatibility complex (MHC) class II molecules, (ii) inhibiting nitric oxide synthase‐2 (NOS‐2) expression and (iii) reducing the production of the pro‐inflammatory cytokines interleukin‐1beta (IL‐1β) and interleukin‐12 (IL‐12p40). Taken together, these results point to the manipulation of the endocannabinoid system as a possible strategy to develop future MS therapeutic drugs.

Activation of cannabinoid CB2 receptor negatively regulates IL-12p40 production in murine macrophages: role of IL-10 and ERK1/2 kinase signaling

1 Cannabinoid (CB) receptor agonists have potential utility as anti‐inflammatory drugs for the treatment of many disease conditions. In the present study, we investigated the effects of the synthetic CB2 ligand, JWH‐133 on the production of interleukins (ILs), IL‐12 and IL‐10 by lipopolyssacharide (LPS) or Theilers virus (TMEV)‐activated macrophages. 2 JWH‐133 evoked a concentration‐related inhibition (10 nM–5 μM) of LPS/IFN‐γ induced IL‐12p40 release. The effect of JWH‐133 (100 nM) was significantly blocked by the CB2 antagonist SR‐144528 (1 μM). Macrophages infected with TMEV increased IL‐12p40 production and activation of CB2 receptors by JWH‐133 (100 nM) inhibited it. 3 The inhibitory effect of JWH‐133 (100 nM) on IL‐12p40 production may involve extracellular‐regulated kinase (ERK1/2) signaling: (i) JWH‐133 induced a greater and sustained activation of ERK1/2 kinase in comparison with the level of activation observed following LPS; (ii) the inhibition of ERK1/2 by the specific inhibitor PD98059 increased LPS‐induced IL‐12p40 production in the presence or absence of JWH‐133 suggesting a negative regulation of ERK pathway on IL‐12p40 biosynthesis. 4 Activation of CB2 receptors by JWH‐133 (10 nM–5 μM) enhanced IL‐10 release by LPS/IFN‐γ‐activated macrophages and addition of SR144558 (1 μM) totally blocked the effect of JWH (100 nM). 5 Inhibition of ERK by PD98059 significantly suppressed IL‐10 production by LPS‐activated macrophages. Endogenous IL‐10 plays a modulatory role in IL‐12 production. Blocking IL‐10 with neutralizing antibody resulted in increased IL‐12p40 secretion by LPS‐activated macrophages in the absence or presence of JWH‐133. In contrast, the addition of exogenous mIL‐10 reduced the secretion of IL‐12p40 in response to LPS.

Excitotoxicity in a chronic model of multiple sclerosis: Neuroprotective effects of cannabinoids through CB1 and CB2 receptor activation

Inflammation, autoimmune response, demyelination and axonal damage are thought to participate in the pathogenesis of multiple sclerosis (MS). Understanding whether axonal damage causes or originates from demyelination is a crucial issue. Excitotoxic processes may be responsible for white matter and axonal damage. Experimental and clinical studies indicate that cannabinoids could prove efficient in the treatment of MS. Using a chronic model of MS in mice, we show here that clinical signs and axonal damage in the spinal cord were reduced by the AMPA antagonist, NBQX. Amelioration of symptomatology by the synthetic cannabinoid HU210 was also accompanied by a reduction of axonal damage in this model. Moreover, HU210 reduced AMPA-induced excitotoxicity both in vivo and in vitro through the obligatory activation of both CB1 and CB2 cannabinoid receptors. Together, these data underline the implication of excitotoxic processes in demyelinating pathologies such as MS and the potential therapeutic properties of cannabinoids.

A cannabinoid agonist interferes with the progression of a chronic model of multiple sclerosis by downregulating adhesion molecules.

Adhesion molecules are critical players in the regulation of transmigration of blood leukocytes across the blood-brain barrier in multiple sclerosis (MS). Cannabinoids (CBs) are potential therapeutic agents in the treatment of MS, but the mechanisms involved are only partially known. Using a viral model of MS we observed that the cannabinoid agonist WIN55,212-2 administered at the time of virus infection suppresses intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in brain endothelium, together with a reduction in perivascular CD4+ T lymphocytes infiltrates and microglial responses. WIN55,212-2 also interferes with later progression of the disease by reducing symptomatology and neuroinflammation. In vitro data from brain endothelial cell cultures, provide the first evidence of a role of peroxisome proliferator-activated receptors gamma (PPARgamma) in WIN55,212-2-induced downregulation of VCAM-1. This study highlights that inhibition of brain adhesion molecules by WIN55,212-2 might underline its therapeutic effects in MS models by targeting PPAR-gamma receptors.

Anandamide enhances IL‐10 production in activated microglia by targeting CB2 receptors: Roles of ERK1/2, JNK, and NF‐κB

The endocannabinoid system exhibits anti‐inflammatory properties by regulating cytokine production. Anandamide (AEA) down‐regulates proinflammatory cytokines in a viral model of multiple sclerosis (MS). However, little is known about the mechanisms by which AEA exerts these effects. Microglial cells are the main source of cytokines within the brain and the first barrier of defense against pathogens by acting as antigen presenting cells. IL‐10 is a key physiological negative regulator of microglial activation. In this study we show that AEA enhances LPS/IFNγ‐induced IL‐10 production in microglia by targeting CB2 receptors through the activation of ERK1/2 and JNK MAPKs. AEA also inhibits NF‐κB activation by interfering with the phosphorylation of IκBα, which may result in an increase of IL‐10 production. Moreover, endogenously produced IL‐10 negatively regulates IL‐12 and IL‐23 cytokines, which in its turn modify the pattern of expression of transcription factorsinvolved in Th commitment of splenocytes. This suggeststhat by altering the cytokine network, AEA could indirectly modify the type of immune responses within the central nervous system (CNS). Accordingly, pharmacological modulation of AEA uptake and degradation might be a useful tool for treating neuroinflammatory diseases.

Study of the regulation of the endocannabinoid system in a virus model of multiple sclerosis reveals a therapeutic effect of palmitoylethanolamide

Cannabinoids have recently been approved as a treatment for pain in multiple sclerosis (MS). Increasing evidence from animal studies suggests that this class of compounds could also prove efficient to fight neurodegeneration, demyelination, inflammation and autoimmune processes occurring in this pathology. However, the use of cannabinoids is limited by their psychoactive effects. In this context, potentiation of the endogenous cannabinoid signalling could represent a substitute to the use of exogenously administrated cannabinoid ligands. Here, we studied the expression of different elements of the endocannabinoid system in a chronic model of MS in mice. We first studied the expression of the two cannabinoid receptors, CB1 and CB2, as well as the putative intracellular cannabinoid receptor peroxisome proliferator‐activated receptor‐α. We observed an upregulation of CB2, correlated to the production of proinflammatory cytokines, at 60 days after the onset of the MS model. At this time, the levels of the endocannabinoid, 2‐arachidonoylglycerol, and of the anti‐inflammatory anandamide congener, palmithoylethanolamide, were enhanced, without changes in the levels of anandamide. These changes were not due to differences in the expression of the degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase, or of biosynthetic enzymes, diacylglycerol lipase‐α and N‐acylphosphatidylethanolamine phospholipase‐D at this time (60 days). Finally, the exogenous administration of palmitoylethanolamide resulted in a reduction of motor disability in the animals subjected to this model of MS, accompanied by an anti‐inflammatory effect. This study overall highlights the potential therapeutic effects of endocannabinoids in MS.

Cannabidiol provides long-lasting protection against the deleterious effects of inflammation in a viral model of multiple sclerosis: A role for A2A receptors

Inflammation in the central nervous system (CNS) is a complex process that involves a multitude of molecules and effectors, and it requires the transmigration of blood leukocytes across the blood-brain barrier (BBB) and the activation of resident immune cells. Cannabidiol (CBD), a non-psychotropic cannabinoid constituent of Cannabis sativa, has potent anti-inflammatory and immunosuppressive properties. Yet, how this compound modifies the deleterious effects of inflammation in TMEV-induced demyelinating disease (TMEV-IDD) remains unknown. Using this viral model of multiple sclerosis (MS), we demonstrate that CBD decreases the transmigration of blood leukocytes by downregulating the expression of vascular cell adhesion molecule-1 (VCAM-1), chemokines (CCL2 and CCL5) and the proinflammatory cytokine IL-1β, as well as by attenuating the activation of microglia. Moreover, CBD administration at the time of viral infection exerts long-lasting effects, ameliorating motor deficits in the chronic phase of the disease in conjunction with reduced microglial activation and pro-inflammatory cytokine production. Adenosine A2A receptors participate in some of the anti-inflammatory effects of CBD, as the A2A antagonist ZM241385 partially blocks the protective effects of CBD in the initial stages of inflammation. Together, our findings highlight the anti-inflammatory effects of CBD in this viral model of MS and demonstrate the significant therapeutic potential of this compound for the treatment of pathologies with an inflammatory component.

A role for CB2 receptors in anandamide signalling pathways involved in the regulation of IL-12 and IL-23 in microglial cells

The endocannabinoid system represents a novel therapeutic target for autoimmune and chronic inflammatory diseases. IL-12 and IL-23 are functionally related heterodimeric cytokines that play a crucial role in the pathogenesis of multiple sclerosis (MS). In the present study we investigated the effects of the endocannabinoid anandamide (AEA) on the inducible expression of the biologically active cytokines IL-12p70 and IL-23, and their forming subunits, in activated microglial cells. We also studied the signalling pathways involved in the regulation of IL-12p70/IL-23 expression and addressed the possible interactions of AEA with these pathways. Here, we show that AEA was capable to inhibit the production of biologically active IL-12p70 and IL-23, and their subunits, by activated human and murine microglial cultures. Treatment of activated microglial cells with inhibitors of several mitogen-activated protein kinase (MAPK) reveals that AEA acts through the ERK1/2 and JNK pathways to down-regulate IL-12p70 and IL-23. These effects were partially mediated by CB2 receptor activation. Together, our results provide the first demonstration of a role of AEA in inhibiting IL-12p70/IL-23 axis in human and murine microglial cells via the CB2 receptor and suggest that the pharmacological manipulation of the endocannabinoid system is a potential tool for treating brain inflammatory and autoimmune diseases, like MS.

An endocannabinoid tone limits excitotoxicity in vitro and in a model of multiple sclerosis

The aim of this study was to evaluate how endocannabinoids interact with excitotoxic processes both in vitro, using primary neural cell cultures, and in vivo, in the TMEV-IDD model of multiple sclerosis. First, we observed that neuronal cells respond to excitotoxic challenges by the production of endocannabinoid molecules which in turn exerted neuroprotective effects against excitotoxicity. The inhibitor of endocannabinoid uptake, UCM707, protected specifically against AMPA-induced excitotoxicity, by activating CB(1) and CB(2) cannabinoid receptors, as well as the nuclear factor, PPARgamma. This neuroprotective effect was reverted by blocking the glial glutamate transporter, GLT-1. Mice subjected to the model of multiple sclerosis showed a decrease in the expression of GLT-1. UCM707 reversed this loss of GLT-1 and induced a therapeutic effect. Our data indicate that the enhancement of the endocannabinoid tone leads to neuroprotection against AMPA-induced excitotoxicity and provides therapeutic effects in this model of multiple sclerosis.