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. 2011 Aug;163(7):1520-32.
doi: 10.1111/j.1476-5381.2011.01414.x.

Cannabinoid Receptor Agonists Modulate Oligodendrocyte Differentiation by Activating PI3K/Akt and the Mammalian Target of Rapamycin (mTOR) Pathways

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Cannabinoid Receptor Agonists Modulate Oligodendrocyte Differentiation by Activating PI3K/Akt and the Mammalian Target of Rapamycin (mTOR) Pathways

O Gomez et al. Br J Pharmacol. .
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Abstract

Background and purpose: The endogenous cannabinoid system participates in oligodendrocyte progenitor differentiation in vitro. To determine the effect of synthetic cannabinoids on oligodendrocyte differentiation, we exposed differentiating cultures of oligodendrocytes with cannabinoid CB(1), CB(2) and CB(1)/CB(2) receptor agonists and antagonists. The response of the PI3K/Akt and the mammalian target of rapamycin (mTOR) signalling pathways were studied as effectors of cannabinoid activity.

Experimental approach: Purified oligodendrocyte progenitor cells (OPC) obtained from primary mixed glial cell cultures were treated for 48 h with CB(1), CB(2) and CB(1) /CB(2) receptor agonists (ACEA, JWH133 and HU210, respectively) in the presence or absence of the antagonists AM281 (CB(1) receptor) and AM630 (CB(2) receptor). Moreover, inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR pathways (LY294002 and rapamycin, respectively) were used to study the involvement of these pathways on cannabinoid-induced OPC maturation.

Key results: ACEA, JWH133 and HU-210 enhanced OPC differentiation as assessed by the expression of stage specific antigens and myelin basic protein (MBP). Moreover, this effect was blocked by the CB receptor antagonists. ACEA, JWH133 and HU210 induced a time-dependent phosphorylation of Akt and mTOR, whereas the inhibitors of PI3K/Akt (LY294002) or of mTOR (rapamycin) reversed the effects of HU-210 on oligodendrocyte differentiation and kinase activation.

Conclusions and implications: Activation of cannabinoid CB(1) or CB(2) receptors with selective agonists accelerated oligodendrocyte differentiation through the mTOR and Akt signalling pathways.

Figures

Figure 1
Figure 1
Treatment of differentiating OPC with selective CB receptor agonists stimulates MBP expression in a dose-dependent manner. Cultures were maintained for 48 h in the presence of ACEA and JWH133 (0, 0.5 and 1 µM) in SFM + T3. The levels of MBP after ACEA (A–B) or JWH133 (C–D) treatment relative to α-tubulin were assessed in Western blots by densitometry, and they are shown as the percentage of untreated controls. Values were obtained as the means ± SEM from three independent experiments (**P < 0.01, control vs. 0.5 or 1 µM ACEA or JWH133, one-way anova followed by Tukey's post hoc test).
Figure 4
Figure 4
The selective CB1 and CB2 receptor agonists ACEA and JWH133 enhance oligodendrocyte maturation. (A–O) OPC cultures grown for 48 h in the presence of SFM plus PDGF-AA and b-FGF were switched to SFM without growth factors but supplemented with T3 (SFM + T3), and they were treated for 48 h with 0.5 µM ACEA or JWH133 with or without the CB receptor antagonists AM281 or AM630 (both at 1 µM). ACEA and JWH133 significantly increase the branching of cell processes as shown by O4 (green) and α-tubulin (red) staining (D–F and J–L), an effect that was reversed in the presence of AM281 (G–I) or AM630 (M–O). (P) After live staining with O4 (green), cells were fixed and processed for α-tubulin (red) labelling and then classified into the following categories according to Marin-Husstege et al. (2002): type A, cells with simple morphology and only a few short primary branches; type B, cells with an intermediate morphology (O4+) with abundant primary and secondary branches; type C, cells with a complex morphology (O4+) and profuse tertiary branches. (Q) The activation of cannabinoid receptors by ACEA or JWH133 is accompanied by an increase in cells classified as types B and C with a parallel decrease in the type A population, an effect that was reversed by the selective antagonists (*P < 0.05; **P < 0.01; ***P < 0.001 vs. control, one-way anova followed by Tukey's post hoc test).
Figure 2
Figure 2
Treatment of differentiating OPC with the selective CB1 and CB2 receptor agonists, ACEA and JWH133, increases MBP levels. (A) Regulation of CNPase and MBP expression in cultures treated for 24 or 48 h in the presence of ACEA and JWH133 (0.5 µM) in SFM + T3. Molecular weights of marker proteins are indicated on the right. Western blots were quantified by densitometry, and the expression of MBP after ACEA (B–C) or JWH133 (D–E) treatment was calculated as the percentage of untreated controls relative to α-tubulin. Values were obtained as means ± SEM from three independent experiments (*P < 0.05, control vs. ACEA or ACEA vs. ACEA + AM281; ***P < 0.001, control vs. JWH133 or JWH133 vs. JWH133 + AM630, one-way anova followed by Tukey's post hoc test).
Figure 3
Figure 3
Effects of AM281 or AM630 on OPC differentiation. (A) In Western blots, there were no significant changes in MAG levels in cultures exposed to AM281 or AM630 (1 µM) in SFM + T3 for 48 h. Blots were quantified by densitometry, and the expression of MAG relative to α-tubulin was calculated as the percentage of the untreated controls. The values obtained (means ± SEM from three independent experiments run in duplicate) are shown in the corresponding text. Molecular weights of marker proteins are indicated on the right. (B) In accordance with the Western blots, immunocytochemical staining with O4 (green) did not reveal changes in the proportion of O4 + pre-oligodendrocytes or in the branching of cell processes after a 48 h exposure to AM281, AM630 or both antagonists. Scale bar: 25 µm.
Figure 5
Figure 5
The CB1/CB2 receptor agonist HU210 augments oligodendrocyte maturation. (A–O) OPC cultures grown for 48 h in the presence of SFM plus PDGF-AA and b-FGF were switched to SFM + T3 and treated for 48 h HU210 (0.5 µM) with or without the CB receptor antagonists AM281, AM630 or both (1 µM). O4 (green) and α-tubulin (red) staining revealed a significant arborization of the cell processes after exposure to HU210 (D–F) with an increase in types B and C cells paralleled by a decrease in the type A population (*P < 0.05; **P < 0.01 vs. control, one-way anova followed by Tukey's post hoc test). These effects were abolished in the presence of AM281, AM630 or both (P). HU210 increased the expression of MBP (Q) in relation to the untreated control (R), as seen in Western blots quantified by densitometry and relative to α-tubulin. The values were obtained as the means ± SEM from three independent experiments (***P < 0.001, control vs. HU210; HU210 vs. HU210 + AM281; HU210 vs. HU210 + AM630 or HU210 vs. HU210 + AM281/AM630, one-way anova followed by Tukey's post hoc test). Scale bar: 25 µm.
Figure 6
Figure 6
The CB receptor agonists ACEA, JWH133 and HU210 induce a time-dependent phosphorylation of Akt and mTOR. Cultures of OPC grown for 48 h in the presence of SFM plus PDGF-AA and b-FGF were switched to HBSS containing 1% FCS, and they were then stimulated with 0.5 µM of HU210, ACEA or JWH133 for different times. (A, C, E) Western blots probed with antibodies specific for phospho-Akt (Ser473) and phospho-mTOR (Ser2448). (B, D, F) Immunoblots were assessed by densitometry, and the values were expressed as the means ± SEM of three independent experiments.
Figure 7
Figure 7
Inhibitors of PI3K and mTOR decrease the HU210-induced phosphorylation of Akt, mTOR and ERK, as well as MBP expression. (A) Cultures of OPC were treated for 30 min with either 0.75 nM rapamycin or 2.5 µM LY290042 and then stimulated for 10 min with HU210 (0.5 µM). (B–D) The densitometric data represent the mean ± SEM of three independent experiments (P-ERK: *P < 0.05 control vs. HU210; P-mTOR: ***P < 0.001, control vs. HU210; HU-210 vs. HU-210 + Rap or HU-210 + LY; P-Akt: *P < 0.05 control vs. HU210, ***P < 0.001 HU210 vs. HU-210 + Rap or HU-210 + LY, one-way anova followed by Tukey's post hoc test). (E–F) Western blot analysis of MBP expression 48 h after differentiation in the presence of HU210 showed a 2.5-fold increase in MBP levels that diminished significantly in the presence of LY294002 (2.5 µM: ***P < 0.001 control vs. HU210 or HU-210 vs. HU210 + LY294002, one-way anova followed by Tukey's post hoc test). After HU210 incubation, a partial reduction in the levels of MBP was observed with rapamycin (0.75 or 1.5 nM: ***P < 0.001 control vs. HU210 or HU210 vs. HU210 + rapamycin, one-way anova followed by Tukey's post hoc test, G–H).
Figure 8
Figure 8
Inhibitors of PI3K and mTOR decrease HU210-mediated oligodendrocyte maturation. (A–I) OPC cultures grown for 48 h in the presence of SFM plus PDGF-AA and b-FGF were switched to SFM + T3 and treated for 48 h HU210 (0.5 µM) in the presence or absence of LY294002 (2.5 µM) or rapamycin (1.5 nM). O4 (green) and α-tubulin (red) staining revealed significant arborization of cell processes after HU210 (A–C) that was reversed after LY294002 or rapamycin (D–I). Inhibitors augmented the type A cells, while the mature types B and C populations diminished (**P < 0.01 vs. HU210; *P < 0.05 vs. HU210, one-way anova followed by Tukey's post hoc test) (J).

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