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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 7, 2005 as doi:10.1096/fj.04-2464fje. |
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* Neuroimmunology Group, Neural Plasticity Department, Cajal Institute, CSIC, Madrid,
Departamento de Química Orgánica I, Universidad Complutense, Madrid,
Neuroinflammation Group, Hospital Nacional de Parapléjicos, Toledo;
Instituto de Química Orgánica General, CSIC, Madrid, Spain; and
|| Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei Pozzuoli (NA), Italy
1Correspondence: Avda Dr Arce, 37, Madrid, Spain 28002. E-mail: cgjb{at}cajal.csic.es
SPECIFIC AIMS
Recent studies have described the involvement of the cannabinoid system in the progression of multiple sclerosis (MS) and the benefits associated to the administration of cannabinoid agonists.
The aim of this study was to establish whether enhancement of the endogenous cannabinoid tone by administration of the anandamide uptake inhibitor UCM707 could induce functional recovery in the Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) model, a well-characterized murine model of MS that closely resembles the chronic and progressive clinical form of the disease and to get deeper insights about the possible pathways involved in this effect.
Here we have investigated whether the use of the selective anandamide uptake inhibitor UCM707 1) improves motor function on TMEV-IDD infected mice, 2) promotes changes in the inflammatory state of the spinal cords of the diseased mice, and 3) the possible cellular mechanisms involved targeting microglial cells.
PRINCIPAL FINDINGS
1. Treatment with a selective endocannabinoid uptake inhibitor improves motor function on established neurological symptomatology
Several studies correlate poor ability to perform rotarod test with demyelination induced by TMEV infection. TMEV-infected mice exhibited reduced ability in rotarod performance before treatment compared with sham mice (Fig. 1
A) but administration of UCM707 or methanandamide induces a significant effect (P < 0.05) in the ability of TMEV-infected mice to perform the rotarod assay 1 day after the end of the treatment (Fig. 1B
). Other motor parameters as vertical and horizontal activity are also ameliorated by treatment with UCM707 or methanandamide (Fig. 1C, D, E
). In none of the cases, administration of UCM707 to sham mice induced any significant behavioral effects by itself.
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The activation of the ECS with exogenous agonists has been involved in the attenuation of the motor deficits in different models of MS. Besides, it has been indicated that the endocannabinoid tone could provide a protective physiological mechanism based on the enhancement in the levels of endogenous cannabinoids that follows the progress of the disease. All these effects have been justified based on the influence of the ECS on the spasticity together with an anti-inflammatory action. Consistent with this hypothesis, administration of UCM707, indirect agonist of the ECS, clearly induced motor recovery in the TMEV-IDD multiple sclerosis model. This improvement runs in parallel to a decrease in the microglial reactivity and almost a complete disappearance of the microglial MHC class II antigen expression within the spinal cord of the TMEV-infected mice. These results are compatible with the proposed immunomodulatory mechanism in which activation of ECS induces a decrease in the inflammatory environment.
2. Administration of the anandamide uptake inhibitor reduces microglial activation in TMEV-infected mice
Microglial cells are one of the first cell types to respond to neuroinflammation. Microglia in the spinal cord of TMEV-infected mice showed a reactive morphology in white and gray matter (Fig. 2
A, C); 12 day treatment with UCM707 markedly switched their morphology toward a resting one (Fig. 2B, D
).
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Macrophage/microglial cells can process and present myelin epitopes in association with MHC class II molecules to CD4+ T cells within the CNS of TMEV-infected mice. Additionally, double immunostaining for Mac-1 and H-2A (SJL/J MHC class II haplotype) revealed a predominant colocalization of MHC class II antigen expression on Mac-1+ cells in the white and gray mater of TMEV-infected mice spinal cords. One day after treatment, positive immunostaining for MHC class II almost disappeared in microglial cells.
3. Enhancement of the endocannabinoid tone downregulates the production of proinflammatory factors mediators in microglial cells
The inflammatory environment promoted by the reactive microglia together with the infiltrated peripheral macrophages may contribute to hamper the remyelination process. Microglial cells acquire a reactive phenotype, retracting their processes and migrating toward the site of injury, where they release proinflammatory cytokines such as TNF
, IL-1ß or IL-6. Therefore, we sought whether administration of UCM707 was able to diminish some of the proinflammatory factors which increase during the time course of the disease, such as NO production, iNOS expression and proinflammatory cytokines such as IL-1ß and IL-6. Stimulation of microglial cells in culture with 50 ng/mL lipopolysaccharide (LPS) leads to an increase in the reactivity of the cells that is accompanied by an enhancement in the proinflammatory cytokines TNF, IL-1ß and IL-6. Exposure to UCM707 (5 µM) induces a remarkable decrease in the concentration of TNF, IL-1ß and a reduction in the levels of IL-6 that is dose dependent. The decrease on the levels of the proinflammatory cytokines TNF, IL-1ß or IL-6 produced by administration of UCM707 are consistent with the neuroimmunomodulatory role proposed for ECS. In additional support of this, exposure of LPS-stimulated microglial cells to other structurally unrelated uptake inhibitor, OMDM1, prevented the expression of mRNAs for IL-1ß and TNF- as well as of MHC class II antigen.
NO production by microglial cells is associated with immune-mediated cellular cytotoxicity and pathogenesis of MS by contributing to the cytotoxicity of oligodendrocytes. Treatment with UCM707 decreases NO production in a dose dependent manner. This effect is partially blocked by coincubation with 1 µM of the CB1 and CB2 cannabinoid antagonists SR141716A and SR144528 and mimicked by administration of anandamide (10 µM). Besides, UCM707 is able to potentiate the action of a subeffective dose of anandamide (1 µM). The effects on NO levels are due to a direct effect on iNOS expression, main enzyme involved in NO production under inflammatory conditions. In different models of MS, iNOS expression in the spinal cords correlates with the severity of the disease.
According to the role of UCM707 as an indirect agonist, anandamide mimics its effects. UCM707 is also able to potentiate the effect of a subeffective dose of anandamide, reinforcing the notion that UCM707 enhances the endocannabinoid signaling. Since microglial cells express both type of cannabinoid receptors, we evaluated their contribution to the effects induced by UCM707 and anandamide by blocking with the CB1 and CB2 antagonists SR141716A and SR144528. Coadministration of both antagonists partially reversed the observed effects, which confirms the involvement of cannabinoid receptors but also indicates the contribution of independent CB1 or CB2 actions. However, this is not unusual considering that anandamide exerts its effects by acting on a number of receptors different from the cannabinoids such as vanilloids as well as by receptor-independent mechanisms. All these data, considered together, suggest that the endocannabinoid signaling system has a regulatory function in the inflammatory response, a notion consistent with the fact that microglial cells, important mediators of inflammation events, have the receptors and the molecular machinery to produce and degrade endocannabinoids. Therefore, our results confirm that the pharmacological potentiation of the ECS-induced signaling by administration of an anandamide uptake inhibitor such as UCM707 can constitute a promising therapeutic approach for the treatment of motor deficits associated to the progression of MS.
CONCLUSIONS
In conclusion, the present study describes that enhancement of the endocannabinoid tone by using a selective anandamide uptake inhibitor produces remarkable motor improvements in the chronic TMEV-IDD MS model. These therapeutic effects may be due to the role played by the ECS at the level of immunomodulation, since they run in parallel to decreases in the microglial reactivity and MHC II expression. Besides, administration of the anandamide uptake inhibitor, remarkably diminishes the production of TNF, IL-1ß, and IL-6 proinflammatory cytokines, NO synthesis and iNOS expression by microglial cells, providing for the first time a molecular pathway that underlies the effects observed.
Our results indicate that anandamide uptake inhibitors can be used to efficaciously treat MS confirming the role played by ECS in this pathology. This treatment has the advantage of avoiding the undesirable side effects induced by direct activation of cannabinoid central receptors.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2464fje;
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