Cannabinoids have been shown to modulate the immune system in vitro and in animal models. A major area of interest is how cannabinoids impact the brain. A whole variety of neuropathies or brain disorders, such as AIDS dementia, Parkinson’s disease, Multiple Sclerosis and Alzheimer’s disease, are associated with a hyperinflammatory response within the brain. Microglia, the resident macrophages of the brain, are the major cell type responsible for the persistent elicitation of pro-inflammatory cytokines (IL-1a, IL-1b, IL-6, TNFa) and other mediators. In vitro experiments have demonstrated that the partial exogenous cannabinoid agonist delta-9-tetrahydrocannabinol (D9-THC) and the potent synthetic exogenous cannabinoid agonist CP55940 down-regulate the robust production of pro-inflammatory cytokines elicited in response to bacterial lipopolysaccharide (LPS) at the mRNA level. These observations suggest that cannabinoids, devoid of psychotropic properties, have the potential to betherapeutic agents. These highly lipophilic compounds can pass through the blood brain barrier and act through specific cannabinoid receptors, cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). CB1 and CB2 are expressed in the brain and the periphery, respectively, and may serve as molecular targets for ablating chronic brain inflammation. Electrophoretic mobility shift assays (EMSA) were used to assess the effects of D9-THC and CP55940 on the LPS-induced binding interactions of the universal transcription factor NFkB to its cognate promoter binding site in BV-2 microglial-like cells. EMSA analyses demonstrated that the D9-THC and CP55940 down-regulated LPS-induced NFkB binding in BV-2 cells in a biphasic manner. Furthermore, reporter activity assays determined that D9-THC and CP55940 attenuated LPS-induced, NFkB transcriptional activity in the same biphasic manner. We then determined the specificity in which cannabinoids inhibit NFkB function. Real-Time RT-PCR analysis demonstrated that BV-2 cells did not express CB1 mRNA, but they do express CB2 mRNA when untreated and stimulated with IFN-g or LPS. We performed specificity studies using CB1 and CB2 selective agonists and antagonists with our reporter activity assays. The CB1-selective agonist ACEA did not affect NFkB transcriptional activity but the CB2-selective agonist O-2137 exerted a significant decrease in activity. Furthermore, the CB1 antagonist SR141716A could not reverse the inhibitory effects of CP55490 but those effects were blocked by the CB2 antagonist SR144528. Lastly, we determined the site of action in which cannabinoids inhibit NFkB function by assessing the effects of D9-THC and CP55940 on NFkB’s inhibitor protein IkBa. IkBa retains NFkB in the cytoplasm until stimulus-induced cell activation. Neither cannabinoid compound was able to inhibit the phosphorylation of IkBa, which initiates its degradation. However both cannabinoids inhibited the complete degradation of IkBa. Western immunoblot analysis also demonstrated that comparable levels of endogenous and phosphorylated p65, the transactivation subunit of the NFkB protein (p65/p50), were detected in the nucleus of LPS-stimulated BV-2 cells pre-treated with or without D9-THC. These results suggest that, in addition to inhibiting the proteolytic degradation of IkBa, there is also a mechanism of action in the nucleus that prevents the proper binding and subsequent transcriptional activity of NFkB. Collectively, these results suggest that cannabinoids suppress pro-inflammatory cytokine gene expression at the transcriptional level, but it is likely that there is more than one signal transduction pathway involved in the cannabinoid-mediated inhibition of NFkB function.

Le potassium (K+) est impliqué dans la régulation de l’excitabilité cellulaire, la régulation du cycle cellulaire, la viabilité cellulaire, la neuroprotection et le maintien des fonctions microgliales et oligodendrocytaires. Le dysfonctionnement des canaux potassiques, décrit dans plusieurs maladies neurodégénératives comme la Maladie d’Alzheimer (MA), la sclérose en plaques (SEP), la maladie de Parkinson et la maladie de Huntington, pourrait être une potentiel cible thérapeutique. Les mécanismes toxiques sous-jacents de ces pathologies neurodégénératives impliquent des oxystérols, dérivés oxydés du cholestérol, et des acides gras en relation avec le métabolisme peroxysomal. Le 7-cétocholestérol (7KC), le 24S-hydroxycholestérol (24S-OHC) et l'acide tétracosanoïque (C24: 0), souvent trouvés à des taux élevés au niveau du cerveau et dans le plasma de patients atteints de maladies neurodégénératives (MA, maladie de Nieman-Pick, SEP, maladie de Parkinson, maladie de Huntington et X-ALD conduisent une rupture de l’équilibre Redox qui aboutirait à la neurodégénérescence. Dans ce contexte, il est intéressant de déterminer l’éventuelle connexion entre environnement lipidique et homéostasie potassique. L’étude in vitro a été réalisée sur des olygodendrocytes murins 158N et les cellules microgliale BV-2. Nous avons montré que la lipotoxicité du 7KC, 24S-OHC et C24:0 implique une rétention du K+ faisant intervenir les canaux potassium voltage dépendant (Kv). Ces résultats ont montré que l'inhibition des canaux Kv conduisant à une augmentation la [K+]i contribue à la cytotoxicité du 7KC, 24S-OHC et C24:0. Nous nous sommes focalisés sur le canal Kv3.1b. La retention du K+ induite par les oxystérols (7KC et 24S-OHC) serait sous le contrôle de Kv3.1b. L’étude clinique réalisée sur du plasma de MA a révélé une corrélation négative entre le taux d’acide docosahexaénoïque (DHA) et la concentration de K+. Chez les souris transgéniques J20, modèle de la MA, l’étude de la topographie d’expression de Kv3.1b et d’Abcd3, au niveau de l’hippocampe et du cortex, a montré une baisse de l’expression de ces deux marqueurs. Dans leur ensemble, les résultats obtenus ont établi des relations entre lipotoxicité, métabolisme peroxysomal et altération de l’homéostasie potassique dans la neurodégénérescence et suggèrent une possible modulation de l’expression et de l’activité de kv3.1b dans la physiopathologie des maladies neurodégénératives. / Potassium (K+) is involved in the regulation of cellular excitability, cell cycle regulation, cell viability, neuroprotection and maintenance of microglial and oligodendrocytic functions. Potassium dysfunction, described in several neurodegenerative diseases such as Alzheimer's Disease (AD), multiple sclerosis (MS), Parkinson's disease and Huntington's disease, may be a potential therapeutic target. The underlying toxic mechanisms of these neurodegenerative pathologies involve oxysterols, which are oxidized cholesterol derivatives, and fatty acids including those associated with peroxisomal metabolism. 7-ketocholesterol (7KC), 24S-hydroxycholesterol (24S-OHC) and tetracosanoic acid (C24:0), often found at increased levels in the brain and plasma of patients with neurodegenerative diseases (Nieman-Pick disease, MS, Parkinson's disease, Huntington's disease and X-ALD) lead to a breakdown of the redox equilibrium leading to neurodegeneration. In this context, it is interesting to determine the possible connection between the lipid environment and potassium homeostasis The in vitro study was carried out on 158N murine oligodendrocytes and microglial BV-2 cells. We have shown that the lipotoxicity of 7KC, 24S-OHC and C24:0 implies retention of K+ involving the voltage dependent potassium channels (Kv). These results have shown that inhibition of Kv channels lead to an increase in [K +] i contributing to the cytotoxicity of 7KC, 24S-OHC and C24:0. The retention of K+ induced by oxysterols (7KC and 24S-OHC) would be under the control of Kv3.1b. A clinical study, on plasma of patients with Alzheimer’s disease, revealed a negative correlation between docosahexaenoic acid (DHA) and K+ concentration. In the J20 mice, a transgenic model of Alzheimer’s disease, the expression of Kv3.1b and Abcd3 was decreased in the hippocampus and cortex. Overall, the results obtained established relationships between lipotoxicity, peroxisomal metabolism and potassium homeostasis in neurodegeneration and suggest a possible modulation of the expression and activity of kv3.1b in the pathophysiology of neurodegenerative diseases. So, modulation of Kv3.1 could constitute a new therapeuthic approach against some neurodegenerative diseases.