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Trabajo presentado en la 19ª Reunión Anual de la Sociedad Española de Investigacion sobre Cannabinoides, celebrada en Madrid del 22 al 24 de noviembre de 2018.

Figure S1. VCE-004.8 inhibits COX-2 expression and PGE2 release in LPS-stimulated rat primary microglia cells. (A) PGE2 released was analyzed in the supernatants from primary microglia cells treated as in A. Data represent the mean ± SD (n = 3). *p

Recent studies have begun to point out the contribution of microbiota to multiple sclerosis (MS) pathogenesis. Theiler’s murine encephalomyelitis virus induced demyelinating disease (TMEV-IDD) is a model of progressive MS. Here, we first analyze the effect of intracerebral infection with TMEV on commensal microbiota and secondly, whether the early microbiota depletion influences the immune responses to TMEV on the acute phase (14 dpi) and its impact on the chronic phase (85 dpi). The intracranial inoculation of TMEV was associated with a moderate dysbiosis. The oral administration of antibiotics (ABX) of broad spectrum modified neuroimmune responses to TMEV dampening brain CD4+ and CD8+ T infiltration during the acute phase. The expression of cytokines, chemokines and VP2 capsid protein was enhanced and accompanied by clusters of activated microglia disseminated throughout the brain. Furthermore, ABX treated mice displayed lower levels of CD4+ and CD8+T cells in cervical and mesenteric lymph nodes. Increased mortality to TMEV was observed after ABX cessation at day 28pi. On the chronic phase, mice that survived after ABX withdrawal and recovered microbiota diversity showed subtle changes in brain cell infiltrates, microglia and gene expression of cytokines. Accordingly, the surviving mice of the group ABX-TMEV displayed similar disease severity than TMEV mice., This study was supported by the “Red Española de Esclerosis Múltiple” (REEM, RD 12/0008, RD16/0015/0021, F.J.C.S., L.M., M.M., A.F., C.G.; RD12/0032, RD16/0015/0001 L.M.V., J.C.A.C.; RD12/0017, RD16/0015/0004 C.E., X.M.), sponsored by the Fondo de Investigación Sanitaria (FIS) of the Instituto Nacional de Salud Carlos III, and the Spanish Ministry of Economy and Competitiveness and by the FIS PI12-00239, PI13/205 and SAF2013-42784R. CE and RdC are supported by the “Miguel Servet” program (CP07/00146; CP13/00028; CB05/00137) of the FIS of the Ministry of Economy and Competitiveness of Spain.

The failure to undergo remyelination is a critical impediment to recovery in multiple sclerosis. Chondroitin sulfate proteoglycans (CSPGs) accumulate at demyelinating lesions creating a nonpermissive environment that impairs axon regeneration and remyelination. Here, we reveal a new role for 2-arachidonoylglycerol (2-AG), the major CNS endocannabinoid, in the modulation of CSPGs deposition in a progressive model of multiple sclerosis, the Theiler's murine encephalomyelitis virus-induced demyelinating disease. Treatment with a potent reversible inhibitor of the enzyme monoacylglycerol lipase, which accounts for 85% of the 2-AG degradation in the mouse CNS, modulates neuroinflammation and reduces CSPGs accumulation and astrogliosis around demyelinated lesions in the spinal cord of Theiler's murine encephalomyelitis virus-infected mice. Inhibition of 2-AG hydrolysis augments the number of mature oligodendrocytes and increases MBP, leading to remyelination and functional recovery of mice. Our findings establish a mechanism for 2-AG promotion of remyelination with implications in axonal repair in CNS demyelinating pathologies.SIGNIFICANCE STATEMENT The deposition of chondroitin sulfate proteoglycans contributes to the failure in remyelination associated with multiple sclerosis. Here we unveil a new role for 2-arachidonoylglycerol, the major CNS endocannabinoid, in the modulation of chondroitin sulfate proteoglycan accumulation in Theiler's murine encephalomyelitis virus-induced demyelinating disease. The treatment during the chronic phase with a potent reversible inhibitor of the enzyme monoacylglycerol lipase, which accounts for 85% of the 2-arachidonoylglycerol degradation in the mouse CNS, modulates neuroinflammation and reduces chondroitin sulfate proteoglycan deposition around demyelinated lesions in the spinal cord of Theiler's murine encephalomyelitis virus-infected mice. The increased 2-arachidonoylglycerol tone promotes remyelination in a model of progressive multiple sclerosis ameliorating motor dysfunction.

Tesis doctoral inédita, leída en la Universidad Autónoma de Madrid, Facultad de Medicina,Departamento de Anatomía, Histología y Neurociencia. Fecha de lectura: 15 de abril de 2016, La esclerosis múltiple, la causa más común de discapacidad neurológica en adultos jóvenes, es una enfermedad autoinmune compleja con componente neuroinflamatorio, caracterizada por la infiltración de leucocitos en el sistema nervioso central, la desmielinización y el daño axonal. A pesar de ser la enfermedad desmielinizante más común del sistema nervioso central, aún no existe una terapia satisfactoria, especialmente en las formas de EM primaria-progresiva y durante la fase secundaria de la enfermedad. Por ello, existe una clara necesidad de investigación para el desarrollo de agentes terapéuticos capaces de disminuir el desarrollo de este trastorno progresivo. Los fitocannabinoides son compuestos químicos presentes en la planta Cannabis sativa. Muchos de ellos no presentan efectos psicotrópicos, y se consideran de especial interés para el tratamiento de enfermedades del SNC. Entre estos fitocannabinoides sin efectos psicoactivos cabe destacar el cannabidiol y cannabigerol, entre otros. Su acción antiinflamatoria está medidada por los receptores CB clásicos, y se ha demostrado que los receptores PPAR también pueden estar involucrados en estos efectos, presentando un potencial terapéutico para el tratamiento de enfermedades con un componente inflamatorio. Se han desarrollado una serie de nuevos derivados de cannabigerol (VCE-003, VCE-003.2 y VCE-006) y de cannabidiol (VCE-004.8), cuyas modificaciones en su estructura química hacen que presenten mayor afinidad que los compuestos de origen por los receptores CB2 y PPARγ, exhibiendo una actividad dual por los mismos. Los resultados obtenidos en esta tesis doctoral muestran que VCE-003 y VCE-004.8 son compuestos que reducen la muerte neuronal y ejercen efectos antiinflamatorios en cultivos celulares. Asimismo, actúan como agentes inmunomoduladores que activan los receptores CB2 y PPARγ en dos modelos de esclerosis múltiple, tanto en la encefalomielitis murina inducida por el virus de Theiler como en la encefalomielitis autoinmune experimental. Además, estos compuestos reducen la infiltración de linfocitos T CD4 y disminuyen la activación microglial en la médula espinal. Ambos compuestos preservan la estructura de las vainas de mielina y contribuyen a la reducción del daño axonal . Su agonismo por los receptores CB2 y PPARγ hace que se postulen como unos prometedores agentes antiinflamatorios y neuroprotectores para el posible tratamiento de la EM., Multiple sclerosis, the most common cause of neurological disability in young adults, is a complex autoimmune disease characterized by neuroinflammation as reflected by the presence of leukocyte infiltrates into the central nervous system, the loss of myelin and the axonal damage in both the brain and the spinal cord. Despite multiple sclerosis prevalence, no satisfactory therapy has been found for MS, particularly for the variants primary-progressive and the secondary phase. There is a clear need for the development of novel agents able to stop the development of this progressive disorder. Non-psychotropic phytocannabinoids are considered of special interest as novel therapeutic agents in CNS diseases. These include cannabidiol and cannabigerol, among others. Besides their effects through the classical CB receptors, PPARγ receptors have been shown to be involved the attenuation neuroinflammation by these compounds, showing a therapeutic potential for the treatment of inflammatory diseases. We have developed a series of new cannabigerol (VCE-003, VCE-003.2 and VCE-006) and cannabidiol derivatives (VCE-004.8) that show CB2 and PPARγ receptors affinity. The findings of this doctoral thesis show that VCE-003 and VCE-004.8 are compounds that reduce neuronal death and exert anti-inflammatory effects in cell cultures. Moreover, these compounds act as immunomodulatory agents targeting CB2 and PPARγ receptors in two models of multiple sclerosis, Theiler's murine encephalomyelitis virus-induced demyelinating disease and experimental autoimmune encephalomyelitis. Furthermore, these compounds reduce CD4 T cells infiltrates and diminish microglial activation, preserve myelin sheets structure and reduce axonal damage in the spinal cord. Their dual affinity for CB2 and PPARγ receptors is critical for their consideration as a novel anti-inflammatory and neuroprotective compounds for the treatment of several inflammatory diseases, such as multiple sclerosis.