Cannabinoids ameliorate disease progression in a model of multiple sclerosis in mice, acting preferentially through CB₁ receptor-mediated anti-inflammatory effects

Abstract

Multiple sclerosis (MS) is an autoimmune disease that affects the CNS and it is characterized by inflammation, demyelination, remyelination, gliosis and axonal damage that occur mainly in the spinal cord. Cannabinoids have been proposed as promising therapeutic agents in MS given their capability to alleviate specific MS symptoms (e.g., spasticity, pain). Although MS has been considered mainly an inflammatory disorder, recent evidence, however, revealed the importance of neurodegenerative events, opening the possibility that cannabinoid agonists, given their cytoprotective properties, may also serve to reduce oligodendrocyte death and axonal damage in MS. Thus, the treatment with WIN55,512-2, a potent CB₁ and CB₂ agonist, was reported to be effective to ameliorate tremor and spasticity in mice with chronic relapsing experimental autoimmune encephalomyelitis, a murine model of MS, but also to delay disease progression in this and other murine models of MS. The purpose of this investigation was to further explore the mechanism(s) underlying the amelioration in disease progression caused by WIN55,212-2. We have particularly focused on anti-glutamatergic and anti-inflammatory effects of this cannabinoid agonist. In this study, we used mice treated with myelin oligodendrocyte glycoprotein (MOG) that induces a progressive pattern of EAE and conducted the pharmacological experiments in early stages of the disease. As expected, the administration of WIN55,512-2 (5 mg/kg, i.p) had a positive effect in reducing neurological disability and improving motor coordination of EAE mice. Levels of glutamate and GABA in the spinal cord and also in the brainstem of EAE mice were similar to control animals, and, accordingly, they were not altered by the treatment with WIN55,212-2. However, EAE mice showed some subtle alterations in mRNA levels for the glutamate transporter GLT1 and, to a lesser extent, GLAST too, changes that were altered by the treatment with WIN55,212-2 in the spinal cord, but not in the brainstem. Regarding to inflammatory responses, EAE mice showed a marked up-regulation in mRNA levels for COX-2, inducible NOS and TNF-α in the spinal cord and the brainstem, these responses being attenuated after the treatment with WIN55,212-2. We also observed the presence of cell aggregates in the spinal cord of EAE mice that were significantly attenuated by the treatment with WIN55,212-2. Immunohistochemical analysis (with Iba-1 and Cd11b) of these aggregates indicated that they corresponded to microglia (resident macrophages) and peripheral macrophages. Lastly, experiments conducted with selective antagonists for the CB₁ (e.g. rimonabant) or CB₂ (e.g. AM-630) receptors revealed that WIN55,212-2 effects in EAE mice were mediated by the activation of CB₁ but not CB₂ receptors, as reflected the reversion of positive effects of this cannabinoid on neurological decline, TNF-α generation and accumulation of cell aggregates in the spinal cord with rimonabant, but not with AM-630. This was concordant with the lack of positive effects on neurological decline observed in EAE mice when they received HU-308, a selective CB₂ receptor agonist, instead WIN55,212-2. In summary, the treatment of EAE mice with the cannabinoid agonist WIN55,512-2 reduced their neurological disability and the progression of the disease. This effect was exerted through the activation of CB₁ receptors, which would exert a positive influence in the reduction of inflammatory events linked to the pathogenesis of this disease.

Introduction

After many years of continuous research, it is becoming more evident that cannabinoid compounds and the endocannabinoid signaling system offer a promising novel form of therapy for multiple sclerosis (MS) that already covers the treatment of specific symptoms (e.g. spasticity, pain; see Rog, 2010, for review), but that might be extended in the future to the control of disease progression (reviewed in de Lago et al., 2010). The first link between MS and cannabinoids derives from anecdotal, uncontrolled or preclinical data generated in the early 90s, which indicated that some MS patients frequently self-medicated with cannabis to alleviate spasticity, dystonia, tremor, ataxia, pain and other MS symptoms (for review, see Consroe et al., 1997; British Medical Association Report, 1997; Pertwee, 2002). Trying to reproduce these beneficial effects in experimental models of MS, Baker and coworkers developed during the present decade a series of pharmacological studies with different types of cannabinoid agonists that always resulted highly effective in reducing spasticity in the chronic relapsing form of experimental autoimmune encephalomyelitis (CREAE) in mice (Baker et al., 2000, 2001; Brooks et al., 2002; de Lago et al., 2004, 2006; Pryce and Baker, 2007; reviewed in Baker and Pryce, 2008). Parallel studies by other authors in this and other MS models also demonstrated efficacy of cannabinoid agonists on other symptoms (reviewed in Pertwee, 2007) or proved immunomodulatory/anti-inflammatory properties for these compounds in MS (Cabranes et al., 2005; Baker et al., 2007; Kubajewska and Constantinescu, 2010). This solid pharmacological evidence has sustained the development of various clinical studies aimed at determining the efficacy of different cannabinoid-based medicines for the treatment of MS symptoms (e.g. spasticity, pain, sleep disturbances) in patients, in general with positive results (Zajicek et al., 2003, 2005; Vaney et al., 2004; Rog et al., 2005; Barnes, 2006; Wade et al., 2006; Collin et al., 2007, 2010; reviewed in Lakhan and Rowland, 2009; Rog, 2010), facilitating the recent approval of the oromucosal spray Sativex^®, which contains equimolecular combinations of botanical extracts enriched in Δ⁹-THC and cannabidiol (Wright, 2007), for the treatment of spasticity in MS in different countries (Kmietowicz, 2010).

Relief of symptoms in MS by cannabinoids has been reported to be mostly mediated by the activation of CB₁ receptors (Pryce and Baker, 2007), which have been reported to be altered in some CNS structures in CREAE mice (Cabranes et al., 2006) or in postmortem tissue from MS patients (Benito et al., 2007). Anandamide has been also found to be elevated in active MS lesions (Eljaschewitsch et al., 2006), lymphocytes and CSF samples (Centonze et al., 2007), or in plasma (Jean-Gilles et al., 2009) of MS patients, in concordance with the increase found in the spinal cord and, to a lesser extent, in the brain, of CREAE mice during the spastic phase of disease (Baker et al., 2001). In contrast, other authors found lowered endocannabinoid levels in the CSF of MS patients compared to control subjects, although elevations were found during relapsing periods (Di Filippo et al., 2008). In general, changes reported for the cannabinoid signaling system in patients and different MS models have been interpreted as adaptative responses aimed at limiting neuronal damage (reviewed by de Lago et al., 2010). On one hand, the evidence of higher levels of endocannabinoids or cannabinoid receptors suggests a contribution in limiting excessive excitatory neurotransmission and the ongoing inflammatory process that have potential neuroprotective implications (Centonze et al., 2007). On the other hand, lower endocannabinoid levels, mainly 2-arachidonoylglycerol, occurring during a neuroimmunological attack have been related to the molecular mechanisms that control endocannabinoid generation by microglia (Witting et al., 2006). Irrespective of this interpretation, the changes elicited in the cannabinoid signaling system in CNS structures during the progression of MS support the idea that the pharmacological management of this system may be beneficial in this disease, not only for specific symptoms, but also for the control of disease progression, a fact based on the well-recognized properties of cannabinoid compounds as cytoprotective agents (reviewed in Fernández-Ruiz et al., 2007, 2010). In this respect, a new clinical trial, named Cannabinoid Use in Progressive Inflammatory Brain Disease (CUPID; see http://sites.pcmd.ac.uk/cnrg/cupid.php) is presently being developed with 493 patients with progressive MS to validate the possibility that cannabinoids may be also used as a disease-modifying agent to slowdown MS progression. The marked anti-inflammatory properties of cannabinoid compounds demonstrated in preclinical studies support this possibility (reviewed in Fernández-Ruiz et al., 2005, 2010). These properties have been related to those compounds that selectively target the CB₂ receptor located in glial cells that are recruited and activated in response to inflammatory but also infectious, traumatic or excitotoxic stimuli and that, upon activation, express CB₂ receptors (reviewed in Fernández-Ruiz et al., 2005, 2007 and 2010). Anti-inflammatory/neuroprotective effects can be also reached with inhibitors of the cellular uptake of endocannabinoids, for example UCM707, OMDM1 and OMDM2, which diminished neuroinflammation in parallel to decreased microglial reactivity in the model of MS generated by infection with Theiler's murine encephalomyelitis virus (TMEV; Ortega-Gutiérrez et al., 2005; Mestre et al., 2005). It appears reasonable that these uptake inhibitors are enhancing the action of endocannabinoids at the CB₂ receptor, so that this receptor might represent a new and promising therapeutic target in MS (reviewed in Docagne et al., 2008) able to control inflammation and then providing protection against glial-derived cytotoxic mediators.

However, cannabinoids may also exert significant neuroprotective effects in MS independently from the CB₂ receptor-mediated immunomodulatory and/or anti-inflammatory actions (Croxford et al., 2008). This possibility was described for the first time in 2003 by Baker's group who found that induction of CREAE was more intense in CB₁ knockout mice compared to wild-type animals (Pryce et al., 2003). In a further study (Jackson et al., 2005), the same authors showed greater neuronal/axonal loss and demyelination when CREAE was induced in CB₁ knockout animals and how these responses were accompanied by an intense activation of caspase 3. In vitro experiments have also confirmed that neuronal damage is greater in absence of the CB₁ receptor (Jackson et al., 2004). These data suggest that the CB₁ receptor represents another key target for providing neuroprotection with cannabinoids in the CREAE model of MS. Other authors also described benefits for CB₁ receptor agonists against the neurological deficits in other MS models (Arévalo-Martin et al., 2003; Croxford and Miller, 2003). It is important to remark that this neuroprotective effect appears to be exerted independently of the other properties of cannabinoids, since low doses of WIN55,512, which have no anti-inflammatory potential, were able to reduce axonal loss in the spinal cord and improve the clinical score in the EAE model (Croxford et al., 2008). In this context, the greater vulnerability of CB₁ knockout mice to the induction of CREAE (Pryce et al., 2003; Jackson et al., 2005) has been related to the role played by this receptor in regulating glutamate homeostasis, as suggest the fact that CREAE-CB₁ knockout mice were more vulnerable to excitotoxic insults (Pryce et al., 2003). It is well-known that glutamate is a key mediator in neuronal and oligodendrocyte damage in MS, as has been found in studies with patients and also in experimental models (reviewed in Gonsette, 2008), and that CB₁ receptor agonists exert direct neuroprotective effects by limiting glutamate release and excitotoxic damage in several neurodegenerative disorders (reviewed in Fernández-Ruiz et al., 2010). Therefore, CB₁ receptor agonists might also be useful against excitotoxic death of oligodendrocytes and, subsequently, neurons in MS, as it has been shown in various in vitro and in vivo studies (Docagne et al., 2007; Loría et al., 2010; reviewed in Fernández-Ruiz et al., 2010). This means that, in addition to the potential of CB₂ receptors in MS described before, CB₁ receptors may also represent an important protective target able to improve glutamate homeostasis and its influence in the progression of MS. These antiexcitotoxic properties of cannabinoids in MS should be investigated more extensively in years to come.

In the present study, we wanted to explore whether a chronic treatment with the non-selective cannabinoid agonist WIN55,212-2, which already showed neuroprotective effects in MS models (see Croxford et al., 2008; de Lago et al., 2010, for recent reviews), may be acting through a CB₁ receptor-mediated improvement of glutamate anomalies described in various CNS structures in MS. We used the model of chronic progressive EAE induced in mice by inoculation of myelin oligodendrocyte glycoprotein (MOG). However, our experiments proved that beneficial effects of WIN55,212-2 treatment on neurological decline in these animals were not related, in general, to changes in glutamate transmission, so that we conducted additional experimentation centered in the anti-inflammatory/neuroprotective effects of WIN55,212-2 that proved positive results. However, contrarily to the expectations, these effects were mediated by CB₁ but not by CB₂ receptors, as reflected in experiments with selective antagonists for both receptor types, or with a selective CB₂ receptor agonist.