The Cannabinoid CB1/CB2 Agonist WIN55212.2 Promotes Oligodendrocyte Differentiation In Vitro and Neuroprotection During the Cuprizone-Induced Central Nervous System Demyelination

Abstract

Aim and methods: Different types of insults to the CNS lead to axon demyelination. Remyelination occurs when the CNS attempts to recover from myelin loss and requires the activation of oligodendrocyte precursor cells. With the rationale that CB1 receptor is expressed in oligodendrocytes and marijuana consumption alters CNS myelination, we study the effects of the cannabinoid agonist WIN55212.2 in (1) an in vitro model of oligodendrocyte differentiation and (2) the cuprizone model for demyelination.

Results: The synthetic cannabinoid agonist WIN55212.2 at 1 μM increased the myelin basic protein mRNA and protein expression in vitro. During cuprizone-induced acute demyelination, the administration of 0.5 mg/kg WIN55212.2 confers more myelinated axons, increased the expression of retinoid X receptor alpha, and declined nogo receptor expression. Controversially, 1 mg/kg of the drug increased the number of demyelinated axons and reduced the expression of nerve growth factor inducible, calreticulin and myelin-related genes coupling specifically with a decrease in 2',3'-cyclic nucleotide 3' phosphodiesterase expression.

Conclusion: The cannabinoid agonist WIN55212.2 promotes oligodendrocyte differentiation in vitro. Moreover, 0.5 mg/kg of the drug confers neuroprotection during cuprizone-induced demyelination, while 1 mg/kg aggravates the demyelination process.

Keywords: Cuprizone; Demyelination; Endocannabinoid system; Neuroprotection; Oligodendrocyte.

Figure 1

Schematic drawing of the experiment. (A) The human oligodendroglial cell line HOG16 was cultured in standard medium, and when they reached 50% confluency, cells were subjected to differentiation medium according to Bologov et al., 15. The effect of 1 μM cannabinoid agonist WIN was tested under both conditions. (B) C57Bl6/J male mice at 8 weeks of age were divided into control groups fed with a regular diet for 0, 3, and 12 weeks (controls) or those mice that received a diet supplemented with 0.2% CPZ for 3 and 6 weeks (treated). In parallel, mice fed with CPZ were single daily injected with WIN (drug) or phosphate‐buffered saline (vehicle). After 0, 3, and 12 weeks, mice were evaluated by behavioral testing, and finally, they were sacrificed as indicated vertical arrows.

Figure 2

HOG16 oligodendrocyte in vitro model. Cells were differentiated from immature oligodendrocytes to a more mature phenotype, that is, cells that were able to synthesize myelin proteins. These cells were grown in both standard medium (SM) and differentiation medium (DM) supplemented with 1 μM of the cannabinoid CB1/CB2 agonist WIN. (A) Fluorescent immunocytochemistry showed a morphological change associated with the differentiation process. Cells changed from a round shape (i‐ii, v‐vi) to a stellated shape (iii‐iv, vii‐viii). The cannabinoid agonist WIN increased myelin basic protein expression under both SM and DM (v, vii). The CB1 receptor protein expression increased in those cells cultured with standard medium supplemented with WIN (vi). (B) The cannabinoid agonist WIN promoted the expression of Mbp under both SM and DM (P < 0.05; P < 0.001, respectively). Cells cultured in differentiation medium supplemented with WIN increased Mbp when compared with those cells grown in standard medium enriched with WIN (P < 0.01). Finally, CB1 receptor mRNA increased in SM supplemented with WIN (P < 0.05). Data are expressed as mean ± SEM. P values were set as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. N = 3. SM, standard medium; DM, differentiation medium; Mbp, myelin basic protein; Cnr1, cannabinoid receptor 1.

Figure 3

Startle response and prepulse inhibition (PPI) during the acute demyelination. We measured an increase in PPI 70 dB in CPZ‐fed mice treated with 0.5 mg/kg of WIN when compared with either control or CPZ alone (P < 0.05) (ii). In addition, CPZ‐fed mice treated with 0.5 mg/kg of WIN displayed higher PPI 75 dB than CPZ alone (P < 0.05) (iii). Mice exposed to CPZ alone declined PPI 80 dB in contrast to control and CPZ‐fed mice subjected to either 0.5 or 1 mg/kg of WIN (P < 0.05) (iv). Data are expressed as mean ± SEM. P values were set as follows: * P < 0.05, ** P < 0.01, and *** P < 0.001. N = 10. CTR, control animals fed with standard diet and treated with phosphate‐buffered saline; CPZ, cuprizone‐fed animals; WIN, WIN55212.2.

Figure 4

The corpus callosum cytoarchitecture. Ultrastructural analysis of the corpus callosum revealed a clear demyelination after 3 weeks of CPZ alone (P < 0.001) (A‐ii, B‐ii, C), while CPZ‐fed mice subjected to 0.5 mg/kg of WIN showed a lower g ratio than those fed with CPZ and acutely treated with vehicle buffer (P < 0.001) (B‐iii, C). In contrast, the administration of 1 mg/kg of WIN in CPZ‐fed mice induced the maximum demyelination as indicated the g ratio when compared with either controls (P < 0.001), CPZ alone (P < 0.001), or CPZ treated with 0.5 mg/kg of WIN (P < 0.001) (B‐iv, C). However, no significant differences concerning the g ratio are reported in the recovery group (C). Data are expressed as mean ± SEM. An § indicates significant differences between CPZ‐fed groups and their respective control group. CPZ intragroup comparisons between vehicle and drug‐treated mice are indicated by an +. Otherwise underlined * indicated comparisons between CPZ‐fed treated with 0.5 and those treated with 1 mg/kg. P values were set at P < 0.001. N = 5. CTR, control animals fed with standard diet and treated with phosphate‐buffered saline; CPZ, cuprizone‐fed animals; WIN, WIN55212.2.