The Endocannabinoid System Is Dysregulated in Multiple Sclerosis and in Experimental Autoimmune Encephalomyelitis

Diego Centonze; Monica Bari; Silvia Rossi; Chiara Prosperetti; Roberto Furlan; Filomena Fezza; Valentina De Chiara; Luca Battistini; Giorgio Bernardi; Sergio Bernardini; Gianvito Martino; Mauro Maccarrone

Disclosures

Brain. 2007;130(10):2543-2553. 

In This Article

Discussion

The results of the present study show that MS and EAE are associated with significant alterations of the ECS. We found that AEA, but not 2-AG, is increased in the CSF of MS patients, indicating that the two endocannabinoids are differentially engaged during CNS inflammatory diseases. These results are consistent with the emerging concept that AEA and 2-AG, although sharing many pharmacological actions, have differential regulatory mechanisms, and that AEA but not 2-AG is preferentially involved during pathological events (Chevaleyre et al., 2006). We also found increased synthesis, reduced degradation and increased levels of AEA in lymphocytes of MS patients. These data might suggest that an important source of AEA in the CNS of MS subjects are inflammatory cells invading the brain from the periphery. In line with this hypothesis, a previous study reported significantly higher concentrations of AEA in active lesions than in quiescent lesions in MS patients (Eljaschewitsch et al., 2006), and we have observed higher AEA levels in patients with inflammatory activity at the MRI. It should be recalled, however, that because of the special cellular and cytokine microenvironment in the CNS, it is not possible to draw direct conclusions from peripheral lymphocytes to lymphocytes inside the CNS. Furthermore, it should be also noted that only AEA synthesis catalysed by NAPE-PLD was measured in this investigation, through a specific radiochromatographic method that we have recently developed (Fezza et al., 2005) to assay the authentic NAPE-PLD described by Okamoto et al. (2004).

Increased synthesis, reduced degradation and increased levels of AEA have also been detected in the brains of EAE mice in the acute phase of the disease, paralleling the finding in acute MS subjects. In this respect, the normal (Witting et al., 2006) or even low levels of AEA (Cabranes et al., 2005) found in other studies is a likely expression of a different phase of the disease or of a different model. In the study by Witting et al. (2006), in fact, AEA levels have been measured in EAE mice with scarce disease activity (clinical score between 0 and 2), while in the study by Cabranes et al. (2005) EAE has been induced in rats. Furthermore, the consistency of the biochemical data obtained in striatal slices from EAE mice with those seen in patients with MS confirms the validity of our animal model of MS to study critical aspects of the human pathology, and lends support to the conclusion that striatal brain slice preparations do not perturb significantly ECS. In line with this idea, it has been previously observed that the alterations of AEA metabolism in corticostriatal slices from a rat model of Parkinson's disease (Gubellini et al., 2002) were indeed similar to those seen in the CSF of Parkinson's disease patients (Pisani et al., 2005).

In EAE mice, we have also provided direct physiological evidence that neuronal responsiveness to cannabinoid CB1 receptor activation is altered. The selective loss of cannabinoid-mediated control of GABA transmission seen in this model of MS correlates with reduced expression of CB1 receptors, and indicates a fine rearrangement of neuronal ECS, which spares the regulation of glutamate transmission and involves the nucleus striatum, a subcortical area particularly vulnerable to the neurodegenerative process associated to MS (Bakshi et al., 2002; Bermel et al., 2003). This brain region is also a preferential site of action of the ECS in physiological and pathological conditions (Piomelli, 2003). Based on the assumption that the activation of the ECS system in MS and in EAE aims at limiting neuronal damage, it can be hypothesized that the down-regulation of cannabinod receptors inhibiting GABA synapses takes part in this protective action by contrasting neuronal excitotoxic damage.

Excitotoxicity, in fact, plays a crucial role in MS- and in EAE-associated neuronal degeneration, as suggested by several lines of evidence. For example, glutamate clearance is altered in EAE (Hardin-Pouzet et al., 1997; Ohgoh et al., 2002) as well as in MS (Werner et al., 2001; Vallejo-Illarramendi et al., 2006), and glutamate levels are significantly higher in the CSF (Stover et al., 1997; Sarchielli et al., 2003) and in the brains of MS patients (Srinivasan et al., 2005). Finally, glutamate receptor antagonists exert beneficial effects in EAE (Wallstrom et al., 1996; Bolton and Paul, 1997; Pitt et al., 2000; Smith et al., 2000) and in MS (Plaut, 1987), by limiting not only oligodendrocyte but also neuronal damage (Pitt et al., 2000; Smith et al., 2000). In EAE, the neuroprotective effects of cannabinoids are largely dependent on their ability to limit glutamate release and excitotoxic damage through the activation of CB1 receptors (Pryce et al., 2003), implying that the dramatic increase of AEA levels seen in MS patients serves this biological action by stimulating CB1 receptors located on presynaptic glutamatergic nerve terminals. A concomitant stimulation of CB1 receptors regulating GABA release might be detrimental for this anti-glutamatergic action, by reducing in parallel synaptic inhibition. Thus, the selective insensitivity of GABA synapses to cannabinoid-mediated transmitter release seen in acute EAE might be important to prevent this unwanted effect of AEA, and to maximize its anti-glutamatergic action. However, the increase of AEA tone in the brains of EAE mice do not seem to cause maximal neuroprotection, since it has been shown that manipulations that increase further AEA concentrations in EAE result in additional neuroprotective effects (Mestre et al., 2005; Ortega-Gutierrez et al., 2005; Bari et al., 2006; Ligresti et al., 2006). Soluble mediators released during inflammation have been found to dampen AEA elevation in EAE (Witting et al., 2006), possibly explaining why the concentrations of AEA, although elevated, are sub-optimal for this action.

In this study, we have not explored the effects of cannabinoid receptor stimulation on the inflammatory response in MS or EAE. This issue might be relevant to add further information on the biological significance of the observed rearrangement of AEA levels and metabolism in these disorders. However, the existing literature is highly supportive of a beneficial immunomodulatory action mediated by ECS activation during both MS and EAE (Centonze et al., 2007). Accordingly, virtually all immune cells involved in the pathophysiology of MS and EAE express functional cannabinoid receptors (Galiegue et al., 1995; Yiangou et al., 2006), and evidence exists that stimulation of these receptors mediates complex inhibitory actions in these cells, accounting, at least in part, for the protective effects of direct and indirect cannabinoid agonists in MS and animal models of this disease (Lyman et al., 1989; Wirguin et al., 1994; Arevalo-Martin et al., 2003; Croxford and Miller, 2003; Mestre et al., 2005; Ortega-Gutierrez et al., 2005; Ligresti et al., 2006). Accordingly, activation of CB2 receptors inhibits the production of several inflammatory cytokines, inhibits cell proliferation and chemiotaxis, and limits nitric oxide and arachidonic acid release. All these factors are critical determinants for the maintenance of inflammation in MS and EAE (Pertwee, 2002; Walter and Stella, 2004; Ehrhart et al., 2005; Eljaschewitsch et al., 2006; Centonze et al., 2007).

The fact that activated microglia, macrophages and lymphocytes express not only CB2 but also CB1 receptors suggests the possible involvement of both receptor subtypes in the anti-inflammatory effects of endocannabinoids in MS (Facchinetti et al., 2003; Walter and Stella, 2004; present study). In this context, it should be recalled that AEA is a much better agonist of CB1 than CB2 receptors (Sugiura et al., 2000; Pertwee, 2002), therefore it can be suggested that CB1 receptors are more involved than CB2 receptors in inflammatory neurodegenerative disorders like MS, where only the endogenous tone of AEA is modulated. Furthermore, a recent report allowed to extend to non-CB1 and non-CB2 receptors the inhibitory action of cannabinoids on immune cells. It has been demonstrated, in fact, that the synthetic cannabinoid agonist R(+)WIN55,212-2 inhibits the IL-1 signalling pathway in human astrocytes through a pathway which does not involve characterized cannabinoid receptor subtypes (Curran et al., 2005).

In conclusion, together with the previous experimental findings, our results suggest that during immuno-mediated attack of the CNS, the activation of ECS represents a protective mechanism aimed at reducing both neurodegenerative and inflammatory damage through various and partially converging mechanisms that involve neuronal and immune cells.

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