Cannabioids as disease modifiers in multiple sclerosis and amyotrophic lateral sclerosis Los cannabinoides como agentes modificadores de la enfermedad en la esclerosis múltiple y la esclerosis lateral amiotrófica MEMORIA PARA OPTAR AL GRADO DE DOCTORA PRESENTADA POR

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-a 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 WIN55,2122. 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-a generation and accumulation of cell aggregates in the spinal cord with rimonabant, but not with AM-630. This was Abbreviations: CFA, complete Freund’s adjuvant; CNS, Central Nervous System; COX-2, cyclooxygenase-2; CREAE, chronic relapsing experimental autoimmune encephalomyelitis; CSF, cerebrospinal fluid; EAE, experimental autoimmune encephalomyelitis; GLAST, glutamate/asparte transporter; GLT1, glutamate transporter-1; iNOS, inducible nitric oxide synthase; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; PBS, phosphate-buffered saline; TMEV, Theiler’s murine encephalomyelitis virus; TNF-a, tumor necrosis factor-a. * Corresponding author. Department of Biochemistry and Molecular Biology, Faculty of Medicine, Complutense University, Ciudad Universitaria s/n, 28040 Madrid, Spain. Tel. : þ34 913941450; fax: þ34 913941691. E-mail address: jjfr@med.ucm. es (J. Fernández-Ruiz). 1 In memoriam of Ana Cabranes who dreamed the results obtained in these studies, but who unexpectedly died before being all her work finished and published.