Cannabinoids promote oligodendrocyte progenitor survival: involvement of cannabinoid receptors and phosphatidylinositol-3 kinase/Akt signaling

Abstract

Cannabinoids exert pleiotropic actions in the CNS, including the inhibition of inflammatory responses and the enhancement of neuronal survival after injury. Although cannabinoid receptors are distributed widely in brain, their presence has not been investigated previously in oligodendrocytes. This study examined the expression of cannabinoid type 1 (CB1) receptors in rat oligodendrocytes in vivo and in culture and explored their biological function. Expression of CB1 receptors by oligodendrocytes was demonstrated immunocytochemically in postnatal and in adult white matter as well as in oligodendrocyte cultures. Reverse transcription-PCR and Western blotting further confirmed the presence of CB1 receptors. Oligodendrocyte progenitors undergo apoptosis with the withdrawal of trophic support, as determined by TUNEL assay and caspase-3 activation, and both the selective CB1 agonist arachidonyl-2'-chloroethylamide/(all Z)-N-(2-cycloethyl)-5,8,11,14-eicosatetraenamide (ACEA) and the nonselective cannabinoid agonists HU210 and (+)-Win-55212-2 enhanced cell survival. To investigate intracellular signaling involved in cannabinoid protection, we focused on the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. HU210, (+)-Win-55212-2, and ACEA elicited a time-dependent phosphorylation of Akt. Pertussis toxin abolished Akt activation, indicating the involvement of G(i)/G(o)-protein-coupled receptors. The CB1 receptor antagonist SR141716A partially inhibited Akt phosphorylation in response to HU210 and (+)-Win-55212-2 and abolished the effects of ACEA. Trophic support deprivation downregulated Akt activity, and cannabinoids recovered phospho-Akt levels. Inhibition of PI3K abrogated the survival action and the recovery of Akt activity in response to cannabinoids. SR141716A prevented only the protection conferred by ACEA. Nevertheless, SR141716A and the selective CB2 receptor antagonist SR144528 in combination inhibited the prosurvival action of HU210, which is in accordance with the finding of CB2 receptor expression by oligodendroglial cells. These data identify oligodendrocytes as potential targets of cannabinoid action in the CNS.

Fig. 1.

Oligodendroglial cells express CB1 receptorsin vivo and in culture. Shown is double immunostaining with the oligodendrocyte monoclonal antibody RIP (A, green) and anti-CB1 receptor (B, red) in P9 rat corpus callosum. Also shown is double immunocytochemistry of cultured oligodendrocyte progenitors with A2B5 (D, green) and anti-CB1 receptor (E, red). Differentiated oligodendrocytes were double labeled with anti-MBP (G, green) and anti-CB1 receptor (H, red). C, F, I, L, O, Overlays of oligodendroglial markers and CB1 receptors. Magnifications show an oligodendrocyte progenitor (J–L) and a differentiated oligodendrocyte (M–O) expressing CB1 receptors in culture. Scale bars, A–C, 50 μm; D–I, 40 μm;J–O, 20 μm.

Fig. 2.

Expression of cannabinoid CB1 receptor in cultured brain glial cells. A, Total RNA was extracted from purified cultures of oligodendrocyte progenitors, differentiated oligodendrocytes, microglial cells, and astrocytes. RT-PCR amplification was performed with specific CB1 primers that used 2 μg of RNA, as described in Materials and Methods. B, The expression of CB1 receptor protein also was demonstrated by Western blot analysis (anti-CB1 diluted to 1:1500) of whole-cell lysates (25 μg of protein). CB1 mRNA and protein levels in progenitors and differentiated oligodendrocytes appeared relatively higher than in microglia and astrocytes. OP, Oligodendrocyte progenitors; OL, differentiated oligodendrocytes;mi, microglia; AST, astrocytes.

Fig. 3.

Cannabinoids protect oligodendrocyte progenitors from apoptosis induced by withdrawal of trophic support.A, Phase-contrast images of live cells showing the effects of HU210 (500 nm) or (+)-Win 55,212-2 (25 nm) on cultures deprived of trophic support.B, Photomicrographs showing the reduction of TUNEL⁺ (green) oligodendrocyte progenitors (A2B5⁺; red) in cultures deprived of trophic support and treated with HU210 or (+)-Win 55,212-2.C, Immunoreactivity of active caspase-3 (red) in oligodendrocyte progenitors (A2B5⁺; green) deprived of trophic support and treated with HU210 and (+)-Win 55,212-2. D,E, Arrows indicate oligodendrocyte progenitors (A2B5⁺; red orgreen) that display condensed chromatin (bis-benzimide⁺; blue), DNA fragmentation (TUNEL⁺; green), or active caspase-3 immunostaining (red). Scale bars:A–C, 50 μm; D, E, 10 μm.

Fig. 4.

Cannabinoids produce a time-dependent phosphorylation of Akt and GSK-3β. Oligodendrocyte progenitors were stimulated with cannabinoid agonists in DMEM containing 1% FCS for different time periods: 5, 30, and 60 min (A) or 10 min (B). Western blot analysis was performed with antibodies specific for phospho-Akt (Ser⁴⁷³) and phospho-GSK-3β (Ser⁹). The antibodies were stripped off, and the membranes were reprobed with antibodies recognizing the total antigen (phosphorylated and unphosphorylated). Immunoblots in A also were analyzed by densitometry (top right); the values are expressed as the means ± SEM of three independent experiments performed in duplicate.

Fig. 5.

Inhibition of PI3K results in the blockade of cannabinoid-induced phosphorylation of Akt and GSK-3β.A, Whole-cell lysates were prepared and immunoblotted as described in Materials and Methods with antibodies that recognize phospho-Akt (Ser⁴⁷³), phospho-GSK-3β (Ser⁹), and total Akt and GSK-3β.B, Oligodendrocyte progenitors were treated for 30 min with either 100 nm wortmannin or 10 μmLY294002 and then stimulated for 10 min with the nonselective cannabinoids HU210 (500 nm) and (+)-Win 55,212-2 (25 nm). The densitometric data represent the means ± SEM of three independent experiments performed in duplicate. *p < 0.001 versus control untreated cells; #p < 0.001 versus HU210- or (+)-Win 55,212-2-stimulated cultures.

Fig. 6.

The phosphorylation of Akt in response to cannabinoids is PTX-sensitive and partially dependent on CB1 receptors. Oligodendrocyte progenitors were incubated for 12 hr with 100 ng/ml pertussis toxin (A) or were pretreated for 50 min with 1 μm of the CB1 receptor antagonist SR141716A, followed by 10 min of stimulation with the selective CB1 agonist ACEA (20 nm) or with the nonselective cannabinoids HU210 (500 nm) and (+)-Win 55,212-2 (25 nm) (b). Whole-cell lysates were prepared and immunoblotted as described in Materials and Methods with antibodies that recognize phospho-Akt (Ser⁴⁷³) and total Akt protein. The densitometric data represent the means ± SEM of three independent experiments performed in duplicate.A, *p < 0.001 versus control untreated cells; #p < 0.001 versus HU210- or (+)-Win 55,212-2-stimulated cultures. B, *p < 0.001 versus control untreated cells; #p < 0.001 versus HU210-, (+)-Win 55,212-2-, or ACEA-stimulated cultures.

Fig. 7.

The prosurvival action of cannabinoids in oligodendrocyte progenitors requires PI3K. Oligodendrocyte progenitors were cultured in serum-free defined medium plus 5 ng/ml PDGF/bFGF (controls, CTL), or cells were switched overnight (12 hr) to DMEM/F12 with or without HU210 (500 nm), (+)-Win 55,212-2 (25 nm), and the PI3K inhibitor LY294002 (10 μm). A, The effect of cannabinoids on Akt and GSK-3β phosphorylation was examined in the presence of LY294002 by Western blot. Whole-cell lysates were prepared and immunoblotted as described in Materials and Methods with antibodies that recognize phospho-Akt (Ser⁴⁷³), phospho-GSK-3β (Ser⁹), and total GSK-3β. The densitometric data of the ratio P-GSK-3β/GSK-3β represent the means ± SEM of three independent experiments performed in duplicate. B, The effect of cannabinoids on oligodendrocyte progenitor survival was established by the MTT assay and by a count of A2B5-positive progenitors. Values are expressed as a percentage of control. The MTT data are the means ± SEM of four independent experiments performed in triplicate. Quantification of A2B5-positive oligodendrocyte progenitors was obtained from eight coverslips (5 microscopic fields/coverslip), and results are the means ± SEM of four independent cultures. *p < 0.001 versus control cells; #p < 0.001 versus cultures deprived of trophic support (DMEM/F12); Δp < 0.001 versus cultures treated with HU210 or (+)-Win 55,212-2.

Fig. 8.

The prosurvival action of cannabinoids is blocked by coincubation with CB1 and CB2 receptor antagonists. Oligodendrocyte progenitors were cultured in serum-free defined medium plus 5 ng/ml PDGF/bFGF (control, CTL), or cells were switched to DMEM/F12 with or without cannabinoids for 12 hr. Cell viability was monitored by MTT assay and by quantification of A2B5-positive oligodendrocyte progenitors. A, Effect of the CB1 receptor antagonist SR141716A (1 μm) on the protective action of HU210 (500 nm), (+)-Win 55,212-2 (25 nm), or ACEA (20 nm). B, Effects of coincubation of SR141716A (1 μm) and the selective CB2 receptor antagonist SR144528 (1 μm) on the prosurvival action of HU210 (500 nm). Values are expressed as a percentage of control. The MTT data are the means ± SEM of four independent experiments performed in triplicate. Quantification of A2B5-positive oligodendrocyte progenitors was obtained from eight coverslips (5 microscopic fields/coverslip), and results are the means ± SEM of four inde pendent experiments. A, *p < 0.001 versus control cells; #p < 0.001 versus cultures deprived of trophic support (DMEM/F12); Δp < 0.001 versus cultures treated with ACEA. B, *p < 0.001 versus control cells; #p < 0.01 and ##p < 0.001 HU210-treated cells versus cultures deprived of trophic support (DMEM/F12); Δp < 0.01 and ΔΔp < 0.001 cultures coincubated with CB1 and CB2 antagonists versus HU210-treated cells.

Fig. 9.

Expression of cannabinoid CB2 receptors in cultured brain glial cells. A, The expression of CB2 receptor protein was demonstrated by Western blot analysis (anti-CB2 diluted to 1:2000) of whole-cell lysates (25 μg of protein).OP, Oligodendrocyte progenitors; OL, differentiated oligodendrocytes. B, The presence of CB2 receptors in cultured progenitors (A2B5⁺) and differentiated oligodendrocytes (MBP⁺) also was evidenced by immunocytochemistry. Scale bars: A2B5/CB2 (top), 15 μm; MBP/CB2 (bottom), 30 μm.