Miriam Hernangómez

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.

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.

CD200-CD200R1 interaction contributes to neuroprotective effects of anandamide on experimentally induced inflammation.

The endocannabinoid anandamide (AEA) is released by macrophages and microglia on pathological neuroinflammatory conditions such as multiple sclerosis (MS). CD200 is a membrane glycoprotein expressed in neurons that suppresses immune activity via its receptor (CD200R) mainly located in macrophages/microglia. CD200‐CD200R interactions contribute to the brain immune privileged status. In this study, we show that AEA protects neurons from microglia‐induced neurotoxicity via CD200‐CD200R interaction. AEA increases the expression of CD200R1 in LPS/IFN‐γ activated microglia through the activation of CB2 receptors. The neuroprotective effect of AEA disappears when microglial cells derive from CD200R1−/− mice. We also show that engagement of CD200R1 by CD200Fc decreased the production of the proinflammatory cytokines IL‐1β and IL‐6, but increased IL‐10 in activated microglia. In the chronic phases of Theilers virus‐induced demyelinating disease (TMEV‐IDD) the expression of CD200 and CD200R1 was reduced in the spinal cord. AEA‐treated animals up‐regulated the expression of CD200 and CD200R1, restoring levels found in sham animals together with increased expression of IL‐10 and reduced expression of IL‐1β and IL‐6. Treated animals also improved their motor behavior. Because AEA up‐regulated the expression of CD200R1 in microglia, but failed to enhance CD200 in neurons we suggest that AEA‐induced up‐regulation of CD200 in TMEV‐IDD is likely due to IL‐10 as this cytokine increases CD200 in neurons. Our findings provide a new mechanism of action of AEA to limit immune response in the inflamed brain.

Therapeutic potential of CB2 targeting in multiple sclerosis.

Background: Cannabinoids have been shown to exert beneficial actions in different animal models of multiple sclerosis (MS). However, the use of cannabinoids compounds in human therapy is greatly limited by their psychoactivity. Thus, new hopes in MS therapy have arisen from the evidence for a cannabinoid receptor, termed CB2, which is devoid of psychoactive effects in animal models. Objective: This review discusses the different mechanisms by which CB2 activation could induce therapeutic actions in MS. Methods: Particular focus is given to the potential effects on inflammation/autoimmunity, remyelination and neuroprotection. Conclusion: This review discusses the importance of glial cells in sustaining these effects, as well as the putative secondary effects that would limit the use of CB2 agonists in the treatment of MS.

Brain Innate Immunity in the Regulation of Neuroinflammation: Therapeutic Strategies by Modulating CD200-CD200R Interaction Involve the Cannabinoid System

The central nervous system (CNS) innate immune response includes an arsenal of molecules and receptors expressed by professional phagocytes, glial cells and neurons that is involved in host defence and clearance of toxic and dangerous cell debris. However, any uncontrolled innate immune responses within the CNS are widely recognized as playing a major role in the development of autoimmune disorders and neurodegeneration, with multiple sclerosis (MS) Alzheimers disease (AD) being primary examples. Hence, it is important to identify the key regulatory mechanisms involved in the control of CNS innate immunity and which could be harnessed to explore novel therapeutic avenues. Neuroimmune regulatory proteins (NIReg) such as CD95L, CD200, CD47, sialic acid, complement regulatory proteins (CD55, CD46, fH, C3a), HMGB1, may control the adverse immune responses in health and diseases. In the absence of these regulators, when neurons die by apoptosis, become infected or damaged, microglia and infiltrating immune cells are free to cause injury as well as an adverse inflammatory response in acute and chronic settings. We will herein provide new emphasis on the role of the pair CD200-CD200R in MS and its experimental models: experimental autoimmune encephalomyelitis (EAE) and Theiler’s virus induced demyelinating disease (TMEV-IDD). The interest of the cannabinoid system as inhibitor of inflammation prompt us to introduce our findings about the role of endocannabinoids (eCBs) in promoting CD200-CD200 receptor (CD200R) interaction and the benefits caused in TMEV-IDD. Finally, we also review the current data on CD200-CD200R interaction in AD, as well as, in the aging brain.

Cannabinoid system and neuroinflammation: implications for multiple sclerosis.

There is a growing amount of evidence suggesting that cannabinoids may be neuroprotective in central nervous system inflammatory conditions. Advances in the understanding of the physiology and pharmacology of the cannabinoid system have potentiated the interest in cannabinoids as potential therapeutic targets. Here our aim was to update the actions of cannabinoids on immune system and glial cells and their implications on multiple sclerosis. We also show our results on the modulation of cytokines of the IL-12 family by cannabinoids in macrophages and brain microglia. We used murine primary cultures of macrophage and microglia activated by lipopolysaccharide/IFN-γ and Theiler’s virus to study the effects of cannabinoids on the regulation of IL-12 and IL-23 mRNA and protein IL-12p40, evaluated by RT-PCR and ELISA, respectively. Cannabinoids negatively regulate the production of these cytokines by microglial cells in part due to the activation of CB2 receptors. The effects of cannabinoids on cytokine brain work and on the regulation of neuroinflammatory processes may affect chronic inflammatory demyelinating diseases such as multiple sclerosis.