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

Results

To explore the ECS system in MS, we first evaluated the levels of the two endocannabinoids AEA and 2-AG in the CSF of our patients. We found a 6-fold increase of AEA in these subjects (control: 2.4 ± 1.3 pmol/ml; MS: 20.3 ± 15.7 pmol/ml) (n = 11 for both groups, P < 0.001) while 2-AG concentration was normal (n = 5, P > 0.05). Notably, AEA levels were particularly high in those subjects showing contrast-enhancement lesions at the MRI following standard dose (0.2 ml/kg e.v.) of gadolinium (n = 11, R 2 = 0.87, P < 0.0001). In contrast, lack of correlation with T2 lesion volume was noted (n = 11, R 2 = 0.01, P = 0.74) (Fig. 1).

Figure 1.

Endocannabinoid levels in the CSF of control and MS subjects. (A) Endogenous levels of 2-AG were unchanged in the CSF of MS patients. (B) This scatter plot shows that endogenous levels of AEA were increased in the CSF of MS patients. (C) The correlation plot shows that AEA levels in MS patients were not related with lesion volume calculated in MRI T2 scans; R 2 = 0.01, P = 0.74. (D) The correlation plot shows that AEA levels in MS patients were strongly correlated with the number of active lesions; R 2 = 0.87, P < 0.0001. *P < 0.001.

A significant increase of AEA content was also found in peripheral lymphocytes of MS patients (n = 3, P < 0.001). N-acylphosphatidylethanolamine (NAPE)-specific phospholipase D (NAPE-PLD) is considered a major player in the synthesis of AEA (Okamoto et al., 2004), although NAPE-PLD knockout mice do not have altered levels of this endocannabinoid (Leung et al., 2006), possibly due to additional biosynthetic pathways that have been recently identified and await further characterization (Liu et al., 2006). AEA is conversely degradated by the fatty acid amide hydrolase (FAAH) (Cravatt et al., 2001; Maccarrone, 2006). Thus, to see whether the observed increase of AEA was mediated by increased synthesis and/or reduced degradation of this endocannabinoid, we measured the activity of NAPE-PLD and that of FAAH in lymphocytes from the peripheral blood of MS patients. We found that NAPE-PLD activity was increased in MS patients (n = 6, P < 0.0001), while both FAAH activity (n = 17, P < 0.0001) and FAAH protein content (n = 5, P < 0.001) were reduced compared to healthy controls. In peripheral lymphocytes, the binding of CB receptors was conversely normal (n = 16, P > 0.05) (Fig. 2). Incidentally, it should be stressed that AEA levels in peripheral lymphocytes are in the same picomole range as AEA content in human blood (Monteleone et al., 2005), and that FAAH activity and CB receptor binding in healthy subjects corroborate previous data on a large collection of control human lymphocytes (Maccarrone et al., 2001), as does the level of AEA in healthy CSF (Pisani et al., 2005). Furthermore, mammalian immune cells express both CB1 and CB2 receptors (Klein, 2005), and CP55.940 binds both of them (Pertwee, 2002). In human lymphocytes, we found that ∼40% of bound [3H]CP55.940 (400 pM) was displaced by 1 µM SR141716A, a CB1 antagonist (Pertwee, 2002), whereas 1 µM SR144528, an antagonist of CB2 receptors (Pertwee, 2002), displaced it by ∼60% (data not shown). Therefore, it can be concluded that human peripheral lymphocytes express both receptor subtypes to a comparable extent.

Figure 2.

Endocannabinoid metabolism in peripheral lymphocytes of control and MS subjects. (A) AEA levels were increased in peripheral lymphocytes of MS patients. (B) The activity of NAPE-PLD, key enzyme in the AEA synthesis, was increased in MS patients. The activity (C) and protein expression (D) of the AEA degrading enzyme FAAH were reduced in these patients. (E) The binding of CB receptors was conversely unaltered in peripheral lymphocytes of MS patients. *P < 0.001; **P < 0.0001.

Higher concentrations of AEA (n = 3 mice, P < 0.001), coupled with increased activity of NAPE-PLD (n = 4 mice, P < 0.0001) and reduced activity of FAAH (n = 5 mice, P < 0.001), were also observed in the striatum of EAE mice killed 20 to 25 days after EAE induction, at the peak of their neurological deficits (mean clinical score: 2.8). The activity of NAPE-PLD and that of FAAH were conversely normal in the frontal cortex of these mice (Fig. 3). The observed increase in AEA levels extends previous findings on chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, another animal model of MS (Baker et al., 2001). Instead, decreased endocannabinoid levels were reported in the brain of Lewis rats with EAE (Cabranes et al., 2005), suggestive that the animal model of MS (mouse versus rat) might lead to heterogeneity of ECS alterations observed in previous studies. In this context, it should be stressed that the ECS is well regulated and sensitive, and there is the possibility that a number of factors like soluble mediators of inflammation can influence ECS directly or indirectly. Therefore, it is imperative that all experimental conditions are strictly controlled and kept identical, when handling control and EAE mice, in order to minimize possible pitfalls due to differences in the phase of the disorder, or in its association with inflammation. Incidentally, the picomole amounts of AEA found here corroborate previous data in rat striatum (Di Marzo et al., 2000) and mouse brain (Baker et al., 2001).

Figure 3.

Endocannabinoid metabolism in striatal and cortical slices of control and EAE mice. (A) Endogenous levels of AEA were increased in the striatum of EAE mice. NAPE-PLD activity was increased (B) and FAAH activity was reduced in the striatum of these mice (C). The activity of NAPE-PLD (D) and that of FAAH (E) were conversely normal in the frontal cortex of EAE mice. *P < 0.001, **P < 0.0001.

In EAE mice, we also performed electrophysiological recordings from single neurons to explore the functionality of neuronal ECS. We selected the nucleus striatum for our neurophysiological investigations, since this subcortical area contains among the highest contents of cannabinoid CB1 receptors in the brain (Piomelli, 2003), and it is particularly vulnerable to the neurodegenerative process associated to MS (Bakshi et al., 2002; Bermel et al., 2003).

In the striatum, stimulation of CB1 receptors modulates both excitatory (Gerdeman and Lovinger, 2001; Huang et al., 2001; Gubellini et al., 2002; Centonze et al., 2005) and inhibitory synaptic transmission through a presynaptic action (Szabo et al., 1998; Centonze et al., 2004, 2005). To record glutamate-mediated EPSCs, we performed whole-cell recordings with potassium-based pipettes. At -80 mV HP and in the presence of bicuculline, EPSCs could be detected in striatal neurons following the activation of corticostriatal fibres. Application of the CB1 receptor agonist HU210 dose-dependently reduced EPSCs in control slices (10 min, n = 10 and P < 0.05 compared to pre-drug values for both 0.3 and 1 µM HU210). In striatal neurons from EAE mice, the effects of HU210 were still present and remarkably similar to those recorded in the respective controls (10 min, n = 10 and P < 0.05 for both concentrations). In both experimental groups, the depressant action of HU210 was associated with increased PPR, as expected for a presynaptic action of this compound. Furthermore, pre-incubating the slices with the selective antagonist of CB1 receptors SR141716A failed to change the amplitude of EPSCs, but fully prevented the HU210 effects in both control and EAE mice (for both groups: n = 4 and P > 0.05 for SR141716A and 1 µM HU210) (Fig. 4).

Figure 4.

Effects of CB1 receptor stimulation on evoked glutamate-mediated EPSCs in corticostriatal slices of control and EAE mice. (A) Application of the CB1 receptor agonist HU210 reduced EPSC amplitude in control and EAE striatal neurons. (B) Examples of voltage-clamp recordings showing that evoked EPSCs are reversibly reduced by 1 µM HU210. (C) The depressant effect of HU210 was dose-dependent and similar in control and EAE mice. (D) HU210 application enhanced PPR in control and EAE slices. On the right there are samples of PPR recordings before and during the application of HU210 in EAE mice. (E) The depressant action of HU210 was abolished pre-incubating the slices with the selective antagonist of CB1 receptors SR141716A. *P < 0.05.

We also investigated the sensitivity of GABA synapses to HU210. Following intrastriatal stimulation, GABA-mediated IPSCs were recorded, in the presence of MK-801 plus CNQX, in both control and EAE mice. These currents were fully blocked by the GABAA receptor antagonist bicuculline (n = 5 for both groups) and were detected as inward deflections from the baseline. Application of the cannabinoid CB1 receptor agonist HU210 (10 min, n = 8 for both groups and 0.3 and 1 µM HU210) significantly (P < 0.05) reduced IPSCs in control slices, an effect again associated with increased PPR and prevented by SR141716A, which caused, per se, no effect of basal EPSC amplitude in both groups (n = 5 and P > 0.05 for SR141716A and 1 µM HU210). In striatal neurons from EAE mice, HU210 was ineffective (10 min application, n = 10 and P > 0.05 for both concentrations compared to pre-drug values), indicating the loss of CB1-mediated control of GABA transmission in these mice (Fig. 5).

Figure 5.

Effects of CB1 receptor stimulation on evoked GABA-mediated IPSCs in corticostriatal slices of control and EAE mice. (A) Application of the CB1 receptor agonist HU210 reduced IPSC amplitude in control but not in EAE striatal neurons. (B) Examples of voltage-clamp recordings showing the lack of effect of HU210 on evoked IPSCs recorded from EAE mice. (C) The depressant effect of HU210 was dose-dependent in control mice. In EAE mice, conversely, the depressant action of HU210 on GABA transmission was lost for both concentrations. (D) HU210 application enhanced PPR in control but not in EAE slices. On the right there are samples of PPR recordings before and during the application of HU210 in control (upper traces) and EAE mice (lower traces). (E) The depressant action of HU210 on IPSC recorded in control mice was abolished pre-incubating the slices with the selective antagonist of CB1 receptors SR141716A. *P < 0.05.

To further explore the possible rearrangements of cannabinoid receptor expression and function in EAE, we measured the binding of the CB1 receptor agonist [3H]CP55,940 to striatal and cortical membranes from EAE and control mice. We found that the binding properties of CB1 receptors were markedly decreased in the striatum of EAE mice (n = 4, P < 0.0001), while this parameter was normal in the cortex (Fig. 6).

Figure 6.

Cannabinoid receptor binding in striatal and frontal cortical slices of control and EAE mice. (A) Cannabinoid CB1 receptor binding was reduced in the striatum of EAE mice. (B) Cannabinoid CB1 receptor binding was conversely normal in the cortex of these animals. **P < 0.0001.

Comments

3090D553-9492-4563-8681-AD288FA52ACE
Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.
Post as:

processing....