Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1μM CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFNγ through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPARγ receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2α, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2α induced by LPS/IFNγ. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the ‘oligoprotective’ effects of CBD during inflammation.

Dose–response studies of the effects of CBD on OPCs

As CBD has different effects on distinct cell types, we evaluated its effects on primary OPC cultures under basal conditions. In dose–response experiments (0.1, 1, 2.5 and 5μM) CBD failed to induce cell death at low concentrations (0.1 and 1μM: Figure 1a), although some cytotoxicity was observed at higher concentrations, resulting in the death of 23.38±3.98% of cells (P0.01) after a 24-h exposure to 2.5μM CBD, and of 33.13±5.6% (P0.001) at 5μM. Based on these findings, a dose of 1μM was selected for the subsequent experiments.

Figure 1

Effects of CBD on OPC cytotoxicity. (a) Although CBD did not induce cell death at low concentrations (0.1 and 1μM), cytotoxic effects were observed at 2.5 and 5μM. OPCs were exposed to CBD and cell death was quantified …

We next investigated the effect of CBD (1μM) on the cell cycle by evaluating OPC proliferation. Quantification of BrdU⁺ cells in OPC primary cultures (n=10000 cells, Figure 1b) revealed that CBD did not increase the proliferation of OPCs in vitro (47.65±8.61%) with respect to the controls (48.21±10.5%). These results were confirmed by subsequently analyzing the cell cycle by flow cytometry (Figure 1c), in which no differences were observed between G0/G1, S and G2/M phases.

At doses of 0.1 and 1μM, CBD has been reported to increase intracellular Ca²⁺and to induce oligodendrocyte death in the optic nerve.⁸ Thus, we investigated whether 1μM CBD-induced Ca²⁺ release from intracellular stores in cultured OPCs loaded with the Ca²⁺-sensitive dye, Fluo-4. There was no difference in the fluorescence signal obtained under basal conditions and in the presence of CBD (1μM: Figures 1d and e), indicating that intracellular Ca²⁺ levels were not significantly modified by CBD.

CBD protects OPCs from inflammatory stimuli and from apoptosis in a cannabinoid receptor-independent manner

As we previously reported that the LPS/IFNγ inflammatory stimuli induced cytotoxicity in OPCs,²² it was noteworthy that CBD (1μM) protected OPCs from the noxious effects of inflammation (P0.001, Figure 2a), decreasing cell death from 37.84±4.95% to 10.41±4.41%. Indeed, the induction of the apoptotic effector caspase 3 (Figure 2b) by LPS/IFNγ was reversed by the treatment with CBD (P0.05), and the number of TUNEL⁺ cells (24.35±1.54%) fell to control levels in the presence of CBD (3.34±0.96%, P0.001) when quantified 24h post treatment (Figures 2c and d). To investigate the mechanisms underlying the oligoprotection offered by CBD, antagonists of CB1, CB2, TRPV1 and PPARγreceptors were administered before the LPS/IFNγ insult in the presence and absence of CBD. None of these antagonists reversed the protective effects of CBD observed in inflammatory conditions, suggesting that the effects of CBD are not mediated by these receptors (Figure 2a).

Figure 2

CBD protects against inflammatory damage by diminishing apoptosis and decreases the number of TUNEL⁺ OPCs through a mechanism that does not involve CB1, CB2, TRPV1 or PPARγreceptors. (a) LPS/IFNγ-induced cytotoxicity in OPCs was …

CBD rescues OPCs from H₂O₂-induced death by decreasing ROS production

CBD is a resorcinol-based compound with direct and potent antioxidant properties;²³ hence, we investigated whether CBD acts as an antioxidant in OPCs subjected to oxidative stress by evaluating its effects on the cell death and ROS production induced by hydrogen peroxide (100μM). CBD prevented the detachment and swelling of OPCs induced by direct oxidative stress (Figure 3a), and decreased cell death from 84.89±6.93% to 39.12±1.82% (P0.01, Figure 3b). Moreover, this effect was accompanied by a decrease in ROS production, as determined in DCF-DA assays 1h post treatment (H₂O₂+vehicle, 216.63±15.7% H₂O₂+CBD, 151.59±5.10%: P0.001, Figure 3c). To separate the antioxidant properties of CBD of its possible cytoprotective effects on OPCs during inflammation, we also evaluated the effects of CBD on NO production in response to LPS/IFN-γ by measuring nitrites and by western blot to assess NOS-2 expression, but CBD was not able to modify NO production (data not shown).

Figure 3

Oxidative stress induced by H₂O₂ induces OPC detachment and death, an effect that was attenuated by CBD by decreasing ROS production. (a) Phase contrast images ( × 10) show that treatment with H₂O₂ induced the swelling and detachment of OPCs, …

ER stress induces OPC death, an effect that is attenuated by CBD via decreased eiF2α phosphorylation

As ER stress is implicated in several diseases with inflammatory components, such as MS,²¹ we analyzed primary OPC death in response to activation of this cellular program. Tunicamycin-induced ER stress led to OPC death 24h post treatment (45.81±4.7%: Figure 4a), which was attenuated by treatment with CBD (20.39±2.54%, P0.01). Exposure to CBD also decreased the phosphorylation of the eiF2α protein (P0.05), an initiator of the apoptotic pathway induced by ER stress (Figure 4b). Interestingly, tunicamycin-induced ER stress was not mediated by the phosphorylation of PKR, a potential activators of eiF2α in the ER (data not shown).

Figure 4

OPC death is mediated by ER stress, an effect that is attenuated by CBD through decreased PKR and eiF2α phosphorylation in conditions of inflammation. (a) CBD attenuated tunicamycin-induced OPC death. OPCs were incubated with tunicamycin (1 …

CBD attenuates the activation of the ER apoptotic pathway in inflammatory conditions

Given the link between the mechanisms underlying inflammation and ER stress recently reported,²⁴ we investigated the ER stress response induced by LPS/IFNγ. When we examined the effect of LPS/IFN-γ on PKR and eiF2αphosphorylation in OPCs, an increase in the phosphorylation of these proteins was evident in Western blots (Figures 4c and d). Interestingly, CBD impaired this increase in PKR and eiF2α phosphorylation, which remained at control levels (P0.05), suggesting that one of the protective effects of CBD in OPCs involves the reduction of ER stress during neuroinflammation.

To confirm the effects of CBD in combating the ER stress associated with inflammation, we analyzed ER mediators of the apoptotic pathway. CBD significantly dampened the induction of ER pro-apoptotic molecules by LPS/IFNγ, such as the initiator CHOP, a major ER stress marker, and the effector caspase 12, as evident by real-time (RT)-PCR (P0.01: Figure 5). In addition, the balance of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins, which is deregulated by LPS/IFNγ treatment, returned to the basal state in the presence of CBD (P0.05). Furthermore, the expression of GADD34, a negative feedback protein in this pathway augmented when OPCs were treated with CBD (P0.001), suggesting that this cannabinoid represses this apoptotic program.

Figure 5

Inflammation activates the ER apoptotic pathway in OPCs, an effect that was attenuated by CBD. (a) CBD attenuated the inflammation-induced increase in CHOP expression. (b) The upregulation of caspase 12 expression in conditions of inflammation was attenuated …

Animals

Animal handling and care was performed in compliance with European Union guidelines (86/609/EEC) and Spanish regulations (BOE67/8509-12; BOE1201/2005) regarding the use and care of laboratory animals, and all the protocols were approved by the local Animal Care and Ethics Committee of the CSIC.

Reagents

Griess reagent (sulfanilamide, N-(1-naphtyl)ethylenediamine), BrdU, DCFDA, hydrogen peroxide, LPS and tunicamycin were purchased from Sigma-Aldrich (Madrid, Spain). IFNγ was acquired from Peprotech (London, UK), CPZ from Alexis Biochemicals (Lausen, Sweden), CBD, AM630 and GW9662 from Tocris (Bristol, UK) and SR1 was kindly provided by Sanofi-Aventis (Montpellier, France).

Culture of oligodendrocyte progenitors

Primary cultures of OPCs derived from P0–P2 Wistar rats were prepared as described previously,^22, 36 with some modifications i; doi:10.1038/protex.2011.218 (Open Nature exchange protocols only on line). Cells were plated on poly--lysine-coated plates at 50000 cells/cm² for western blotting or 25000 cells/cm² for other assays, and they were maintained for 3 days at 37°C and 5% CO₂ in serum-free defined medium containing 5ng/ml of growth factors (bFGF and PDGF-AA).

Cell toxicity

Oligodendrocyte death was quantified by measuring the release of lactate dehydrogenase (LDH) from damaged cells into the bathing medium 18, 24 and 48h after exposure to hydrogen peroxide, tunicamycin and LPS/IFNγ, respectively, according to the manufacturer’s instructions (cytotoxicity detection kit, Roche, Manhein, Germany). All experiments compared the cell death induced with the basal levels of cell death in the cultures.

TUNEL assay

OPCs were seeded on poly--lysine-coated coverslips and maintained for 24h with LPS/IFNγ and/or CBD. After they were fixed in 4% PFA, post-fixed in EtOH/acetic acid for 5min at −20°C and maintained in equilibration buffer for 1min, they were incubated with the TdT enzyme in a reaction buffer for 1h at 37°C (Apoptosis detection kit, Chemicon, Millipore Ibérica, Madrid, Spain). After washing, the coverslips were incubated with the anti-DIG antibody in blocking solution and counterstained with DAPI. The data represent TUNEL⁺ cells as a percentage of the total number of cells.

Immunocytochemistry

For BrdU analysis, OPCs were seeded on poly--lysine-coated coverslips and incubated with BrdU (10μM) for 24h in the presence or absence of CBD. Cells were then fixed in 4% PFA for 20min, treated with 2N HCl for 10min, blocked and incubated with anti-BrdU antibody (DSHB, 1:1000). After washing, the cells were incubated with an anti-mouse secondary antibody (1:1000) and counterstained with DAPI. For A2B5 staining, the cells were treated for 24h with LPS/IFNγ in the presence or absence of CBD, incubated in vivo with anti-A2B5 marker (R&D Systems, Mineapolis, MN, USA, 1:200), fixed in 4% PFA and incubated with the secondary antibody (1:1000).

Flow cytometry

Freshly isolated OPCs were maintained on uncoated plates for 24h with vehicle or CBD. The cells were then collected, fixed in cold 70% ethanol for 30min, resuspended in phosphate buffer saline (PBS) and incubated with 100μg/ml Ribonuclease A (Sigma-Aldrich) and 50μg/ml propidium iodide (PI; Sigma-Aldrich) for 30min. Argon laser excitation at 488nm was used to measure PI fluorescence through a band-pass 616/23nm filter using a FACSAria flow cytometer (BD Biosciences, San Diego, CA, USA). Debris and duplets were excluded from the analysis and a minimum of 10000 cells were acquired in each experiment. FACSDiva analysis software (BD Biosciences) was used to define the cell cycle stages.

Measurement of intracellular Ca²⁺

Cells were seeded on coverslips and incubated for 20–30min at 37°C in PBS containing Fluo4-AM (0.3μM, Invitrogen, Barcelona, Spain) and pluronic F-127 (0.04%, Sigma-Aldrich). The coverslips were then transferred to the microscope chamber and bathed in an extracellular medium containing (in mM): NaCl 140, KCl 5, MgCl₂ 4, HEPES 10, glucose 10 and sucrose 6 (pH 7.35). All experiments were performed at room temperature. Cells were imaged with an Olympus (Barcelona, Spain) FV3 scanning confocal microscope equipped with a × 40 objective (NA, 0.8) and laser emission at 488nm was used to excite Fluo-4. The fluorescent signal recorded is displayed as a pseudocolor image and the look-up-table used is indicated in each figure. The time frame acquisition was 1.12s.

ROS determination

OPCs were loaded with dichlorodihydrofluorescein diacetate acetyl ester (DCFDA, 2μM) for 30min, washed and stimulated with hydrogen peroxide for 2h or with LPS/IFNγ for 24h in the presence or absence of CBD. Fluorescence was measured at 485/530nm in a microplate reader and normalized to the negative control.

Western blot analysis

Western blot analysis was performed as previously described ⁷ using 30–40μg of OPC protein extract and an anti-cleaved caspase3 (Asp¹⁷⁵; 1:1000, Cell Signaling Technology, Denvers, MA, USA), anti-phospho (Ser⁵¹) eIF2α (1:1000; Cell Signaling Technology), anti-total eIF2α (1:1000; Cell Signaling Technology), anti-phospho (Thr⁴⁴⁶) PKR (1:1000, Upstate, Millipore Ibérica, Madrid, Spain) and anti-total PKR (1:1000, Millipore, Madrid, Spain). Anti-α-tubulin (1:40000; Sigma-Aldrich) was used as a loading control.

RNA extraction, reverse transcription and RT-PCR

Total OPC RNA was extracted using the RNeasy mini columns kit (Qiagen, Crawley, UK) and treated with DNaseI (Qiagen), and the RNA concentration and purity were determined in a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Total RNA (1μg) was reverse transcribed using the Promega reverse transcription kit (Promega, Madrid, Spain) and RT-PCR was performed on 1μl of cDNA (corresponding to 50ng RNA input) with 200nM of the primers listed below (Applied Biosystems, Warrington, UK), quantifying expression using SYBR Green (Applied Biosystems). The amplification cycles involved an initial activation step at 50°C for 2min and a denaturation step at 95°C for 10min, followed by 40 cycles of denaturation at 95°C for 15s and annealing/extension at 60°C for 1min. PCR assays were carried out in 96-well plates using a 7500 Real-Time PCR system (Applied Biosystems). Each sample was assayed in duplicate and a 6-point standard curve was run in parallel. The ratio between the values obtained for each gene and the 18S house-keeping gene provided a relative quantification of expression. The 5′-3′primer secuences used were the following: CHOP (forward 5′-CCAAAATAACAGCCGGAACCT-3′ reverse 5′-CAAAGGCGAAAGGCAGAGACT-3′), GADD34 (forward 5′-AGCATGGACACGCCTTAGAAA-3′; reverse 5′-AGGCTGGGAGGAGGGATTT-3′), Bax (forward 5′-GGGTGGCAGCTGACATGTTT-3′; reverse 5′-TGATCAGCTCGGGCACTTTA-3′), Bcl-2 (forward 5′-TGAGAGCAACCGAACGCCCG-3′; reverse 5′-CCGTGGCAAAGCGTCCCCTC-3′), caspase 12 (forward 5′-GAAGGAAGGCCGAACCCGCC-3′; reverse 5′-TGCTCTGGACGGCCAGCAAAC-3′) and 18S (forward 5′-ATGCTCTTAGCTGAGTGTCCCG-3′; reverse 5′-ATTCCTAGCTGCGGTATCCAGG-3′)

Data analysis

All data are expressed as the mean±S.E.M. (n), where n refers to the number of cultures assayed, each obtained from a different group of animals and evaluated in triplicate. One-way ANOVA followed by the Bonferroni post-hoc test, or Kruskal—Wallis ANOVA followed by Mann–Whitney U test was used to determine the statistical significance in all cases. The level of significance was set at P<0.05.

We acknowledge the financial support of the Ministry of Science and Innovation (MICINN, project SAF-2010/17501), Instituto de Salud Carlos III (project RD07/0060/0010 RETICS program, Red Española de Esclerosis Múltiple, REEM). Mecha M is supported by REEM. We thank to Alfonso Araque and Eduardo D Martín for their help with electrophysiological studies. We are also grateful to Joaquín Sancho and Elisa Baides Rosell for their excelent technical assistance.

ATF-6

activation transcription factor 6

BrdU

5-bromo-2′-deoxyuridine

CB1

cannabinoid receptor type 1

CB2

cannabinoid receptor type 2

CBD

cannabidiol

DCF-DA

2′,7′-dichlorofluorescein-diacetate

eiF2α

translation initiation factor 2α

ER

endoplasmic reticulum

IFNγ

interferon-gamma

IRE1α

inositol-requiring enzyme 1α

ROS

reactive oxygen species

LPS

lipopolysaccharide

MS

multiple sclerosis

NO

nitric oxide

NOS-2

nitric oxide syntase-2

OPCs

oligodendrocyte precursor cells

PKR

double-stranded RNA-activated serine/threonine kinase

PPARγ

peroxisome proliferator-activated receptor gamma

RT-PCR

reverse transcription PCR

TRPV1

transient receptor potential cation channel subfamily V member 1

TUNEL

terminal deoxynucleotidyl transferase dUTP nick end labeling

UPR

unfolded protein response

Edited by D Bano

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