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AKT signaling
www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 28 (2005) 189 – 194
Neuroprotective effects of the synthetic cannabinoid HU-210 in primary cortical neurons are mediated by phosphatidylinositol 3-kinase/AKT signaling Francisco Molina-Holgado,1,2 Emmanuel Pinteaux,2 Laura Heenan, Jonathan D. Moore,3 Nancy J. Rothwell,* and Rosemary M. Gibson School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK Received 25 May 2004; revised 25 August 2004; accepted 1 September 2004
Cannabinoids (CBs) are neuroprotective in vivo and in vitro, but the mechanisms of their actions are unknown. The aim of this study was to elucidate the signaling pathways that mediate the protective effect of CBs on primary cultured neurons. The neurotoxin S-AMPA induced significant death of rat primary cortical neurons, which was inhibited by the CB agonist HU-210. Antagonists selective for CB1 or CB2 receptors (AM 281 or AM 630, respectively) reversed the neuroprotective effect of HU-210 on S-AMPA-induced cell death. HU-210 triggered activation of AKT, but not activation of the ERK1/2, JNK or p38 signaling pathways. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors LY294002 and wortmannin prevented phosphorylation of AKT in response to HU-210, and reversed the neuroprotective effect of HU-210 on S-AMPA-induced excitotoxicity. Thus the PI 3-K/AKT signaling pathway mediates the neuroprotective effect of exogenous cannabinoids such as HU-210 in primary CNS neurons. D 2004 Elsevier Inc. All rights reserved.
Introduction Exogenous and endogenous cannabinoids (CBs) are potent anti-inflammatory and neuroprotective agents (Mechoulam et al., 2002a). CBs suppress inflammatory and immune responses, and are neuroprotective in experimentally induced excitotoxicity,
* Corresponding author. School of Biological Sciences, University of Manchester, 1.124 Stopford Building, Oxford Road, Manchester, M13 9PT, UK. Fax: +44 161 275 5948. E-mail address: [email protected] (N.J. Rothwell). 1 Present address: Guy’s, King’s and St. Thomas’ School of Biomedical Sciences, Wolfson Centre for Age-Related Diseases, King’s College London, London SE1 1UL, UK. 2 These authors contributed equally to this work. 3 Present address: Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK. Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2004.09.004
global and focal ischemia and traumatic brain injury in rodents (reviewed by Mechoulam et al., 2002a). Growing evidence suggests that a major physiological function of the endocannabinoid system is to modulate neuroinflammation, which has significant therapeutic implications (reviewed by Walter and Stella, 2004). However, the mechanisms of CB actions are not known. Cannabinoids exert their effects through two types of receptors, CB1 and CB2 (Matsuda et al., 1990; Munro et al., 1993), although additional receptors may exist (Howlett et al., 2002). CB1 receptors are found predominantly in the central nervous system (CNS), notably in the cerebral cortex, basal ganglia, hippocampus and cerebellum (Herkenham et al., 1991; Mailleux and Vanderhaeghen, 1992). CB1 receptors are expressed on dendritic spines and axon terminals of neurons (Ong and Mackie, 1999), on oligodendrocytes and their progenitors (Molina-Holgado et al., 2002a) and on astrocytes (Molina-Holgado et al., 2002b). Although CB2 receptors are expressed mainly in the immune system (Munro et al., 1993), they have also been found on oligodendrocytes and their progenitors (Molina-Holgado et al., 2002a), cerebellar neurons (Skaper et al., 1996), microglia (Carlisle et al., 2002), human astrocytomas and cultured C6 glioma cells (Galve-Roperh et al., 2000). Furthermore, CB2 receptors are expressed in neuritic plaque-associated astrocytes and microglia in postmortem brains from patients with Alzheimer’s disease (Benito et al., 2003). In addition, the existence of novel CB receptors, tentatively termed CB3 receptors, which are distinct from CB1 or CB2, and sensitive to SR141716A and responsive to (+)-Win 55212-2 and anandamide, has been proposed (Howlett et al., 2002). Cannabinoids are neuroprotective in vivo and in vitro, through a variety of proposed mechanisms including antioxidative actions (Hampson et al., 1998), effects on glucose metabolism or ketone body production (Guzman and Sanchez, 1999), inhibition of nitric oxide release (Molina-Holgado et al., 2002b), inhibition of calcium influx (Mackie and Hille, 1992) and glutamate release (Shen et al., 1996), induction of endogenous interleukin-1 receptor antagonist
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(Molina-Holgado et al., 2003) or brain-derived growth factor (BDNF) (Khaspekov et al., 2004), or by counteracting the endothelin (ET-1)-induced vasoconstriction that aggravates brain damage (Mechoulam et al., 2002b). Agonist binding to the CB1 receptor leads to inhibition of adenylyl cyclase, modulation of calcium and potassium channels, and changes in neurotransmitter release (Porter and Felder, 2001). In addition, CB receptor activation in non-neuronal cells can trigger phosphorylation cascades involving mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated protein kinases (ERK1/2) (Derkinderen et al., 2003), c-Jun N-terminal kinase (JNK), and p38 (Liu et al., 2000; Rueda et al., 2000). The CB1 receptor also stimulates the phosphoinositide 3-kinase (PI 3-K)/AKT signaling pathway (Gomez et al., 2000), which is strongly implicated in survival signaling in many cell types including neurons (reviewed by Brunet et al., 2001). In the present study, the aim was to test the hypothesis that the PI 3-K/AKT pathway is required for CBmediated protection of neurons from excitotoxicity. We demonstrate that treatment of primary cortical neurons with the synthetic cannabinoid HU-210 leads to activation of AKT, which is blocked by either CB1 or CB2 receptor antagonists, and inhibition of PI 3-K and AKT signaling prevents HU-210-mediated neuroprotection from excitotoxic insults. Results The glutamatergic agonist S-AMPA induced death of rat primary cortical neurons (S-AMPA 20 AM: 3.4-fold increase compared to basal cell death), and the cannabinoid agonist HU-210 reduced the cell death significantly (by 33%) (Fig. 1), as we have shown previously (Molina-Holgado et al., 2003). To assess the
Fig. 1. Neuroprotective effect of HU-210 on excitotoxicity in primary rat neuronal cultures is blocked by CB1 and CB2 selective antagonists. Neuronal cultures were pre-treated with HU-210 (1 AM) in the presence or absence of the CB antagonists AM 281 or AM 630 (1 AM) for 1 h, and then exposed to S-AMPA (20 AM). Cell death was analyzed after 24 h by LDH assay. Data are presented as mean percent total LDH release (% of TritonX-100-lysed cells) F SEM of three independent experiments on separate cultures, each carried out in duplicate. Statistical differences (one-way ANOVA and Neuman–Keuls post hoc test): *P b 0.001 vs. control, #P b 0.05 vs. S-AMPA, §P b 0.05 vs. S-AMPA + HU-210.
Fig. 2. HU-210 induces phosphorylation of AKT in primary rat neuronal cultures: inhibition by CB1 and CB2 selective antagonists. Neuronal cultures were pre-treated with HU-210 (1 AM) in the presence or absence of the CB antagonists AM 281 or AM 630 (1 AM) for 15 min, and then exposed to S-AMPA (20 AM) for 30 min. Western blot and densitometry analysis of total and phosphorylated AKT (S473) were performed in three independent experiments on separate cultures. Data are presented as mean F SEM. Statistical differences (one-way ANOVA and Neuman–Keuls post hoc test): *P b 0.001 vs. control, #P b 0.01 vs. HU-210.
contribution of the CB1 and CB2 receptors to these neuroprotective effects, neurons were pre-treated with HU-210 in the presence of the CB1-selective antagonist AM 281 (1 AM) or the CB2-selective antagonist AM 630 (1 AM), before incubation with S-AMPA for 24 h. The CB1 and CB2 receptor antagonists had no effects alone (data not shown), but significantly decreased cell survival after HU-210 and S-AMPA treatment (Fig. 1), suggesting that the neuroprotective effects of HU-210 on S-AMPA-induced neuronal death are mediated by both CB receptors. To characterize the intracellular signaling pathways that mediate the neuroprotective actions of cannabinoids, changes in phosphorylation of key signaling proteins were analyzed by immunoblots with phospho-specific antibodies. The kinase AKT plays a key role in neuronal survival signaling (Brunet et al., 2001). Immunoblotting showed that phosphorylation of AKT on serine 473, which is a key step in activation of the kinase (Scheid and Woodgett, 2003), increased in response to treatment with HU-210 (15 min) (Fig. 2). Both the CB1 and CB2 antagonists (AM 281 and AM 630, respectively) prevented the phosphorylation of AKT S473 induced by HU-210 (Fig. 2). However, brief exposure to SAMPA (15 min) did not alter phosphorylation of AKT S473. Both cannabinoids and glutamatergic agonists can activate the mitogen-activated protein kinase (MAPK) ERK1/2 (Derkinderen et al., 2003; Sweatt, 2001). However, there was no detectable change in phosphorylation of ERK1/2 in response to HU-210 or S-AMPA (Fig. 3A), nor were changes detected in phosphorylation of the other MAPKs, p38 or JNK (Figs. 3B,C). Taken together, these results suggest that the major signaling enzyme activated in response to HU-210 in primary cortical neurons is AKT. Activation of AKT requires not only phosphorylation, but also interaction with phospholipids, generated by phosphatidylinositol 3-kinase (PI 3-K) (Scheid and Woodgett, 2003). To investigate the role of PI 3-K in HU-210-mediated neuroprotection, the reversible
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excitotoxic damage remain elusive. We now report for the first time that the cannabinoid-induced neuroprotection against excitotoxic death of primary cortical neurons is mediated via the PI 3-K/ AKT signaling pathway. The nature of the cannabinoid receptors that mediate the neuroprotective effects of CBs has not been resolved. Activation of the CB1 receptor has been reported to be either protective (van der Stelt et al., 2001) or neurotoxic (Hansen et al., 2002), whilst other reports exclude either the CB1 receptor (Marsicano et al., 2002), or both the known CB1 and CB2 receptors (Sinor et al., 2000) from a role in cannabinoid-induced neuroprotection. The synthetic cannabinoid HU-210 binds to both the known CB receptors with similar affinities (Howlett et al., 2002), and we found that the neuroprotective effects and activation of AKT by HU-210 were blocked by both CB1 and CB2 receptor antagonists. The CB1 receptor is expressed predominantly in the CNS, and expression by primary cortical neurons is consistent with its localization to the cerebral cortex (Mailleux and Vanderhaeghen, 1992). CB2 receptors are expressed predominantly in the periphery, but have been detected in the brain, specifically in cerebellar neurons, oligodendrocytes and the retina (Skaper et al., 1996; Molina-Holgado et al., 2002a; Lu et al., 2000). The involvement of the CB1 and CB2 receptors was assessed using the selective antagonists AM 281 (Gifford et al., 2000) and AM 630 (Hosohata et al., 1997), respectively. Although AM 630 has some inverse
Fig. 3. HU-210 does not activate ERK1/2, p38 or JNK in primary rat neuronal cultures. Neuronal cultures were pre-treated with HU-210 (1 AM) in the presence or absence of the CB antagonists AM 281 or AM 630 (1 AM) for 15 min, and then exposed to S-AMPA (20 AM) for 30 min. Whole cell lysates were analyzed by immunoblot using antibodies specific for the total and phosphorylated forms of ERK1/2 (p42/p44), p38 and JNK (p46/ p54). The Western blots shown are representative of three independent experiments on separate cultures.
and the irreversible inhibitors of PI 3-K, LY294002 and wortmannin, respectively, were used. Pretreatment of neurons with these inhibitors abolished phosphorylation of AKT S473 in response to HU-210 (Fig. 4A), showing that at the doses used, the inhibitors effectively inhibited PI 3-K and consequently the downstream kinase, AKT. These agents were then used to assess directly the contribution of PI 3-K and AKT to the HU-210-mediated protection against S-AMPA. Pretreatment with LY294002 and wortmannin significantly reduced the neuroprotective effect of HU-210 on S-AMPA-induced cell death (Fig. 4B), implicating PI 3-K and AKT as key enzymes mediating neuroprotection by this exogenous synthetic cannabinoid. Discussion Cannabinoids are neuroprotective in experimentally induced excitotoxicity in vivo (van der Stelt et al., 2001) and in vitro (Shen and Thayer, 1998). Recent evidence implicate interleukin-1 receptor antagonist (IL-1 ra) and brain-derived neurotropic factor (BDNF) in CB1-receptor-dependent protection against excitotoxicity (Khaspekov et al., 2004; Molina-Holgado et al., 2003), and deletion of CB1 receptors increases susceptibility to excitotoxicity (Khaspekov et al., 2004). However, the signaling pathways that mediate the neuroprotective actions of cannabinoids against
Fig. 4. PI 3-K inhibitors block AKT activation and HU-210-induced neuroprotection in primary rat neuronal cultures. (A) Cultures were pretreated with LY294002 (10 AM) and wortmannin (0.1 AM) for 15 min, and then HU-210 (1 AM) for 15 min, prior to exposure to S-AMPA (20 AM) for 30 min. Western blot analysis for the total and phosphorylated AKT (S473) was carried out in three independent experiments on separate cultures. (B) Cultures were pre-treated with LY294002 (10 AM) and wortmannin (0.1 AM) for 15 min, and then HU-210 (1 AM) for 15 min, prior to exposure to S-AMPA (20 AM). Cell death was analyzed after 24 h by LDH assay. Data are presented as mean percent total LDH release (% of Triton-X-100-lysed cells) F SEM of three independent experiments on separate cultures, each carried out in duplicate. Statistical differences (one-way ANOVA and Neuman–Keuls post hoc test): *P b 0.001 vs. S-AMPA, #P b 0.05 vs. SAMPA + HU-210.
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agonistic activity at human CB1 and CB2 receptors (Landsman et al., 1998; Ross et al., 1999), recent in vivo studies showed that the effects of AM 1241, a potent and selective CB2 agonist, were reversed by AM 630 but not by the CB1 receptor-selective antagonist AM 251 (Quartilho et al., 2004), suggesting that AM 630 is indeed a selective antagonist for CB2 receptors. The results reported here therefore implicate both receptors in neuroprotection. Although unexpected from the reported peripheral location of CB2 receptors, these observations agree with studies demonstrating expression of CB2 receptors in glial cells: cultured microglia express CB2 receptors (Carlisle et al., 2002); astrocytes are known to possess G-protein-coupled receptors that are activated by cannabinoids but distinct from the CB1 receptor (Berrendero et al., 1998; Sagan et al., 1999), and CB2 receptors has been detected in human astrocytoma and C6 glioma cells (Galve-Roperh et al., 2000). Furthermore, our data do not exclude a role for novel CB receptor(s). Ligand binding to CB receptors can activate several mitogenactivated protein kinases (MAPK) including ERK1/2 (Derkinderen et al., 2003), JNK and p38 (Galve-Roperh et al., 2000; Rueda et al., 2000). However, in the primary cortical neurons analyzed in this report, no activation of ERK1/2, JNK or p38 was detected, suggesting that primary cortical neurons may not activate MAPK in response to HU210. In support of this conclusion, inhibitors of ERK1/2 or p38 did not reverse the neuroprotective effects of this CB (data not shown). In contrast, delta-9tetrahydrocannabinol (THC) induces apoptosis of cultured cortical neurons, via the CB1 receptor and activation of JNK (Downer et al., 2003). Cannabinoids may therefore have diverse effects on neuronal survival/death (Guzman, 2003), and the outcome may depend on a variety of influences, including the maturity of the cultures, nature and time of exposure to the insult and the particular cannabinoid used. The major signaling response to HU-210 reported here was phosphorylation of AKT on serine 473 (Fig. 2). AKT is a critical downstream kinase that regulates growth factor-induced survival of neurons including cerebellar granule neurons (Dudek et al., 1997) and sympathetic neurons (Crowder and Freeman, 1998; Philpott et al., 1997). Activation of AKT requires phosphorylation of the enzyme on T308 and S473, and phosphorylation of both sites is required for full activity (reviewed by Scheid and Woodgett, 2003). We have used phosphorylation of S473 as a marker of AKT activity, and report here that in primary cortical neurons, the CB HU-210 increased activity of AKT, and that this effect was reduced by both CB1 and CB2 receptor antagonists. Activation of AKT requires not only phosphorylation, but also interaction with phospholipids, generated by phosphatidylinositol 3-kinase (PI 3-K), that bind to its pleckstrin homology domain. Indeed, plasma membrane localization is required for S473 phosphorylation (Scheid et al., 2002). Inhibition of PI 3-K by the inhibitors LY294002 and wortmannin blocked phosphorylation of AKT S473 in response to HU-210, and prevented the neuroprotective effects of the CB. These results indicate that PI 3-K and the downstream kinase, AKT, play key roles in mediating the neuroprotective effects of HU-210. AKT is critical for neuronal survival, and a variety of targets of AKT have been implicated in its pro-survival effects, including Bad, Bim, transcription factors of the Forkhead family, and GSK-3h (reviewed by Brunet et al., 2001). The identity of the AKT substrate that mediates the neuroprotective effects of HU-210 in cortical neurons has yet to be identified, but in oligodendrocytes,
CBs lead to phosphorylation of GSK-3h downstream of AKT (Molina-Holgado et al., 2002a). In conclusion, the CNS effects of CBs have been known for decades, and it has been realized recently that CBs also have significant neuroprotective potential, that could be exploited for therapeutic benefit. The results presented here demonstrate for the first time that exogenous CBs such as HU210 can protect cultured neurons from the neurotoxic effects of the excitotoxin S-AMPA through phosphorylation of the critical pro-survival enzyme AKT.
Experimental methods Reagents All tissue culture reagents were obtained from Invitrogen (Paisley, UK). Antibodies that recognize signaling enzymes were obtained from Cell Signaling (Hitchin, UK) (total AKT, AKT phosphorylated on S473, ERK1/2 phosphorylated on T202 and Y204, respectively, total p38), Santa Cruz (Santa Cruz, USA) (total JNK, total ERK1/2) and Promega (Southampton, UK) (JNK phosphorylated on T183 and Y185, p38 phosphorylated on T180 and Y182). The horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies were from Dako (Cambridge, UK). The PI 3-K inhibitors LY 294002 and wortmannin, the CB agonist HU-210, and the CB antagonists AM 281 and AM 630 were obtained from Tocris (Bristol, UK). All other reagents were obtained from Sigma (Poole, UK) unless stated otherwise. Neuronal cell cultures Primary cerebrocortical neurons (CCN) were prepared from embryonic (day 18) Sprague–Dawley rat pups as previously described (Moore et al., 2002). Cells were seeded (7.5 104 viable cells/cm2) onto plates coated with poly-d-lysine, in plating media (neurobasal medium with B27 supplement plus antioxidants, 2 mM glutamine, 25 AM glutamate, 100 IU/ml penicillin and 100 Ag/ml streptomycin). After 2 DIV, cytosine arabinoside (20 AM) was added to the cultures to eliminate the majority of proliferating non-neuronal cells. The cultures were used after 13–14 DIV, and consisted of N98% neurons as analzed by immunocytochemistry (not shown). To study excitotoxicity, neurons grown in 24-well plates were pre-treated with HU-210 (1 AM) in the presence or absence of the CB antagonists AM 281 or AM 630 (1 AM), or a mixture of LY294002 (10 AM) and wortmannin (0.1 AM) for 1 h at 378C, and then exposed to S-AMPA (20 AM) for 24 h at 378C. To study kinase activation, the culture medium of the neurons was changed to DMEM without B27 or serum for 2 h, and then the cells were pre-treated with a mixture of LY294002 (10 AM) and wortmannin (0.1 AM) for 15 min, and then HU-210 (1 AM) for 15 min, prior to exposure to S-AMPA (20 AM) for 30 min at 378C. Cells were then harvested for Western blot analysis. Assessment of cell death Cytotoxicity was evaluated by release of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium by dead and dying cells (CytoTox-96 LDH assay, Promega). Total LDH release was calculated by incubating untreated cells with 0.1% Triton X-
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100 for 10 min (378C, 5% CO2, 95% air) to induce maximal cell lysis. Treatment values were then expressed as a percent of the total LDH release. Background LDH release (media alone) was subtracted from the experimental values. Western blot analysis Immunoblotting was carried out as previously described (Parker et al., 2002). The membranes were incubated at 48C overnight in primary antibodies, diluted as follows: anti-phospho-AKT (1:1000), anti-AKT (1:1000), anti-phospho-ERK1/2 (1:5000), anti-phospho-JNK (1:5000), anti-phospho-p38 (1:1000), anti-total ERK1/2 (1:60000), anti-JNK (1:30000), and anti-total p38 (1:1000). After washing in TBS with 0.1% Tween 20 (TBS-T), blots were incubated with horseradish peroxidase-conjugated antirabbit or anti-mouse secondary antibodies (1:2000, in 5% dried skimmed milk in TBS-T) for 1–2 h at RT. Finally, the protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences, UK), and analyzed by densitometry using Northern Eclipse software (Empix Imaging Inc., Canada). Statistical analysis Results are presented as the mean F SE of at least three independent experiments performed on separate cell preparations; duplicate or triplicate determinations were performed for each experiment. One-way analysis of variance was followed by posteriori Newman–Keuls multiple comparison. Statistical significance was established at P b 0.05. Acknowledgments This work was supported by the Medical Research Council, UK (EP, LH, NJR, RMG), and The Wellcome Trust (FM-H). References Benito, C., Nunez, E., Tolon, R.M., Carrier, E.J., Rabano, A., Hillard, C.J., Romero, J., 2003. Cannabinoid CB2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaqueassociated glia in Alzheimer’s disease brains. J. Neurosci. 23, 11136 – 11141. Berrendero, F., Garcı´a-Gil, L., Herna´ndez, M.L., Romero, J., Cebeira, M., de Miguel, R., Ramos, J.A., Ferna´ndez-Ruiz, J.J., 1998. Localization of mRNA expression and activation of signal transduction mechanisms for cannabinoid receptor in rat brain during fetal development. Development 125, 3179 – 3188. Brunet, A., Datta, S.R., Greenberg, M.E., 2001. Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr. Opin. Neurobiol. 11, 297 – 305. Carlisle, S.J., Marciano-Cabral, F., Staab, A., Ludwick, C., Cabral, G.A., 2002. Differential expression of the CB2 cannabinoid receptor by rodent macrophages and macrophage-like cells in relation to cell activation. Int. Immunopharmacol. 2, 69 – 82. Crowder, R.J., Freeman, R.S., 1998. Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons. J. Neurosci. 18, 2933 – 2943. Downer, E.J., Fogarty, M.P., Campbell, V.A., 2003. Tetrahydrocannabinolinduced neurotoxicity depends on CB1 receptor-mediated c-Jun Nterminal kinase activation in cultured cortical neurons. Br. J. Pharmacol. 140, 547 – 557.
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Neuroprotective effects of the synthetic cannabinoid HU-210 in primary cortical neurons are mediated by phosphatidylinositol 3-kinase/AKT signaling Francisco Molina-Holgado,1,2 Emmanuel Pinteaux,2 Laura Heenan, Jonathan D. Moore,3 Nancy J. Rothwell,* and Rosemary M. Gibson School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK Received 25 May 2004; revised 25 August 2004; accepted 1 September 2004
Cannabinoids (CBs) are neuroprotective in vivo and in vitro, but the mechanisms of their actions are unknown. The aim of this study was to elucidate the signaling pathways that mediate the protective effect of CBs on primary cultured neurons. The neurotoxin S-AMPA induced significant death of rat primary cortical neurons, which was inhibited by the CB agonist HU-210. Antagonists selective for CB1 or CB2 receptors (AM 281 or AM 630, respectively) reversed the neuroprotective effect of HU-210 on S-AMPA-induced cell death. HU-210 triggered activation of AKT, but not activation of the ERK1/2, JNK or p38 signaling pathways. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors LY294002 and wortmannin prevented phosphorylation of AKT in response to HU-210, and reversed the neuroprotective effect of HU-210 on S-AMPA-induced excitotoxicity. Thus the PI 3-K/AKT signaling pathway mediates the neuroprotective effect of exogenous cannabinoids such as HU-210 in primary CNS neurons. D 2004 Elsevier Inc. All rights reserved.
Introduction Exogenous and endogenous cannabinoids (CBs) are potent anti-inflammatory and neuroprotective agents (Mechoulam et al., 2002a). CBs suppress inflammatory and immune responses, and are neuroprotective in experimentally induced excitotoxicity,
* Corresponding author. School of Biological Sciences, University of Manchester, 1.124 Stopford Building, Oxford Road, Manchester, M13 9PT, UK. Fax: +44 161 275 5948. E-mail address: [email protected] (N.J. Rothwell). 1 Present address: Guy’s, King’s and St. Thomas’ School of Biomedical Sciences, Wolfson Centre for Age-Related Diseases, King’s College London, London SE1 1UL, UK. 2 These authors contributed equally to this work. 3 Present address: Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK. Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2004.09.004
global and focal ischemia and traumatic brain injury in rodents (reviewed by Mechoulam et al., 2002a). Growing evidence suggests that a major physiological function of the endocannabinoid system is to modulate neuroinflammation, which has significant therapeutic implications (reviewed by Walter and Stella, 2004). However, the mechanisms of CB actions are not known. Cannabinoids exert their effects through two types of receptors, CB1 and CB2 (Matsuda et al., 1990; Munro et al., 1993), although additional receptors may exist (Howlett et al., 2002). CB1 receptors are found predominantly in the central nervous system (CNS), notably in the cerebral cortex, basal ganglia, hippocampus and cerebellum (Herkenham et al., 1991; Mailleux and Vanderhaeghen, 1992). CB1 receptors are expressed on dendritic spines and axon terminals of neurons (Ong and Mackie, 1999), on oligodendrocytes and their progenitors (Molina-Holgado et al., 2002a) and on astrocytes (Molina-Holgado et al., 2002b). Although CB2 receptors are expressed mainly in the immune system (Munro et al., 1993), they have also been found on oligodendrocytes and their progenitors (Molina-Holgado et al., 2002a), cerebellar neurons (Skaper et al., 1996), microglia (Carlisle et al., 2002), human astrocytomas and cultured C6 glioma cells (Galve-Roperh et al., 2000). Furthermore, CB2 receptors are expressed in neuritic plaque-associated astrocytes and microglia in postmortem brains from patients with Alzheimer’s disease (Benito et al., 2003). In addition, the existence of novel CB receptors, tentatively termed CB3 receptors, which are distinct from CB1 or CB2, and sensitive to SR141716A and responsive to (+)-Win 55212-2 and anandamide, has been proposed (Howlett et al., 2002). Cannabinoids are neuroprotective in vivo and in vitro, through a variety of proposed mechanisms including antioxidative actions (Hampson et al., 1998), effects on glucose metabolism or ketone body production (Guzman and Sanchez, 1999), inhibition of nitric oxide release (Molina-Holgado et al., 2002b), inhibition of calcium influx (Mackie and Hille, 1992) and glutamate release (Shen et al., 1996), induction of endogenous interleukin-1 receptor antagonist
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(Molina-Holgado et al., 2003) or brain-derived growth factor (BDNF) (Khaspekov et al., 2004), or by counteracting the endothelin (ET-1)-induced vasoconstriction that aggravates brain damage (Mechoulam et al., 2002b). Agonist binding to the CB1 receptor leads to inhibition of adenylyl cyclase, modulation of calcium and potassium channels, and changes in neurotransmitter release (Porter and Felder, 2001). In addition, CB receptor activation in non-neuronal cells can trigger phosphorylation cascades involving mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated protein kinases (ERK1/2) (Derkinderen et al., 2003), c-Jun N-terminal kinase (JNK), and p38 (Liu et al., 2000; Rueda et al., 2000). The CB1 receptor also stimulates the phosphoinositide 3-kinase (PI 3-K)/AKT signaling pathway (Gomez et al., 2000), which is strongly implicated in survival signaling in many cell types including neurons (reviewed by Brunet et al., 2001). In the present study, the aim was to test the hypothesis that the PI 3-K/AKT pathway is required for CBmediated protection of neurons from excitotoxicity. We demonstrate that treatment of primary cortical neurons with the synthetic cannabinoid HU-210 leads to activation of AKT, which is blocked by either CB1 or CB2 receptor antagonists, and inhibition of PI 3-K and AKT signaling prevents HU-210-mediated neuroprotection from excitotoxic insults. Results The glutamatergic agonist S-AMPA induced death of rat primary cortical neurons (S-AMPA 20 AM: 3.4-fold increase compared to basal cell death), and the cannabinoid agonist HU-210 reduced the cell death significantly (by 33%) (Fig. 1), as we have shown previously (Molina-Holgado et al., 2003). To assess the
Fig. 1. Neuroprotective effect of HU-210 on excitotoxicity in primary rat neuronal cultures is blocked by CB1 and CB2 selective antagonists. Neuronal cultures were pre-treated with HU-210 (1 AM) in the presence or absence of the CB antagonists AM 281 or AM 630 (1 AM) for 1 h, and then exposed to S-AMPA (20 AM). Cell death was analyzed after 24 h by LDH assay. Data are presented as mean percent total LDH release (% of TritonX-100-lysed cells) F SEM of three independent experiments on separate cultures, each carried out in duplicate. Statistical differences (one-way ANOVA and Neuman–Keuls post hoc test): *P b 0.001 vs. control, #P b 0.05 vs. S-AMPA, §P b 0.05 vs. S-AMPA + HU-210.
Fig. 2. HU-210 induces phosphorylation of AKT in primary rat neuronal cultures: inhibition by CB1 and CB2 selective antagonists. Neuronal cultures were pre-treated with HU-210 (1 AM) in the presence or absence of the CB antagonists AM 281 or AM 630 (1 AM) for 15 min, and then exposed to S-AMPA (20 AM) for 30 min. Western blot and densitometry analysis of total and phosphorylated AKT (S473) were performed in three independent experiments on separate cultures. Data are presented as mean F SEM. Statistical differences (one-way ANOVA and Neuman–Keuls post hoc test): *P b 0.001 vs. control, #P b 0.01 vs. HU-210.
contribution of the CB1 and CB2 receptors to these neuroprotective effects, neurons were pre-treated with HU-210 in the presence of the CB1-selective antagonist AM 281 (1 AM) or the CB2-selective antagonist AM 630 (1 AM), before incubation with S-AMPA for 24 h. The CB1 and CB2 receptor antagonists had no effects alone (data not shown), but significantly decreased cell survival after HU-210 and S-AMPA treatment (Fig. 1), suggesting that the neuroprotective effects of HU-210 on S-AMPA-induced neuronal death are mediated by both CB receptors. To characterize the intracellular signaling pathways that mediate the neuroprotective actions of cannabinoids, changes in phosphorylation of key signaling proteins were analyzed by immunoblots with phospho-specific antibodies. The kinase AKT plays a key role in neuronal survival signaling (Brunet et al., 2001). Immunoblotting showed that phosphorylation of AKT on serine 473, which is a key step in activation of the kinase (Scheid and Woodgett, 2003), increased in response to treatment with HU-210 (15 min) (Fig. 2). Both the CB1 and CB2 antagonists (AM 281 and AM 630, respectively) prevented the phosphorylation of AKT S473 induced by HU-210 (Fig. 2). However, brief exposure to SAMPA (15 min) did not alter phosphorylation of AKT S473. Both cannabinoids and glutamatergic agonists can activate the mitogen-activated protein kinase (MAPK) ERK1/2 (Derkinderen et al., 2003; Sweatt, 2001). However, there was no detectable change in phosphorylation of ERK1/2 in response to HU-210 or S-AMPA (Fig. 3A), nor were changes detected in phosphorylation of the other MAPKs, p38 or JNK (Figs. 3B,C). Taken together, these results suggest that the major signaling enzyme activated in response to HU-210 in primary cortical neurons is AKT. Activation of AKT requires not only phosphorylation, but also interaction with phospholipids, generated by phosphatidylinositol 3-kinase (PI 3-K) (Scheid and Woodgett, 2003). To investigate the role of PI 3-K in HU-210-mediated neuroprotection, the reversible
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excitotoxic damage remain elusive. We now report for the first time that the cannabinoid-induced neuroprotection against excitotoxic death of primary cortical neurons is mediated via the PI 3-K/ AKT signaling pathway. The nature of the cannabinoid receptors that mediate the neuroprotective effects of CBs has not been resolved. Activation of the CB1 receptor has been reported to be either protective (van der Stelt et al., 2001) or neurotoxic (Hansen et al., 2002), whilst other reports exclude either the CB1 receptor (Marsicano et al., 2002), or both the known CB1 and CB2 receptors (Sinor et al., 2000) from a role in cannabinoid-induced neuroprotection. The synthetic cannabinoid HU-210 binds to both the known CB receptors with similar affinities (Howlett et al., 2002), and we found that the neuroprotective effects and activation of AKT by HU-210 were blocked by both CB1 and CB2 receptor antagonists. The CB1 receptor is expressed predominantly in the CNS, and expression by primary cortical neurons is consistent with its localization to the cerebral cortex (Mailleux and Vanderhaeghen, 1992). CB2 receptors are expressed predominantly in the periphery, but have been detected in the brain, specifically in cerebellar neurons, oligodendrocytes and the retina (Skaper et al., 1996; Molina-Holgado et al., 2002a; Lu et al., 2000). The involvement of the CB1 and CB2 receptors was assessed using the selective antagonists AM 281 (Gifford et al., 2000) and AM 630 (Hosohata et al., 1997), respectively. Although AM 630 has some inverse
Fig. 3. HU-210 does not activate ERK1/2, p38 or JNK in primary rat neuronal cultures. Neuronal cultures were pre-treated with HU-210 (1 AM) in the presence or absence of the CB antagonists AM 281 or AM 630 (1 AM) for 15 min, and then exposed to S-AMPA (20 AM) for 30 min. Whole cell lysates were analyzed by immunoblot using antibodies specific for the total and phosphorylated forms of ERK1/2 (p42/p44), p38 and JNK (p46/ p54). The Western blots shown are representative of three independent experiments on separate cultures.
and the irreversible inhibitors of PI 3-K, LY294002 and wortmannin, respectively, were used. Pretreatment of neurons with these inhibitors abolished phosphorylation of AKT S473 in response to HU-210 (Fig. 4A), showing that at the doses used, the inhibitors effectively inhibited PI 3-K and consequently the downstream kinase, AKT. These agents were then used to assess directly the contribution of PI 3-K and AKT to the HU-210-mediated protection against S-AMPA. Pretreatment with LY294002 and wortmannin significantly reduced the neuroprotective effect of HU-210 on S-AMPA-induced cell death (Fig. 4B), implicating PI 3-K and AKT as key enzymes mediating neuroprotection by this exogenous synthetic cannabinoid. Discussion Cannabinoids are neuroprotective in experimentally induced excitotoxicity in vivo (van der Stelt et al., 2001) and in vitro (Shen and Thayer, 1998). Recent evidence implicate interleukin-1 receptor antagonist (IL-1 ra) and brain-derived neurotropic factor (BDNF) in CB1-receptor-dependent protection against excitotoxicity (Khaspekov et al., 2004; Molina-Holgado et al., 2003), and deletion of CB1 receptors increases susceptibility to excitotoxicity (Khaspekov et al., 2004). However, the signaling pathways that mediate the neuroprotective actions of cannabinoids against
Fig. 4. PI 3-K inhibitors block AKT activation and HU-210-induced neuroprotection in primary rat neuronal cultures. (A) Cultures were pretreated with LY294002 (10 AM) and wortmannin (0.1 AM) for 15 min, and then HU-210 (1 AM) for 15 min, prior to exposure to S-AMPA (20 AM) for 30 min. Western blot analysis for the total and phosphorylated AKT (S473) was carried out in three independent experiments on separate cultures. (B) Cultures were pre-treated with LY294002 (10 AM) and wortmannin (0.1 AM) for 15 min, and then HU-210 (1 AM) for 15 min, prior to exposure to S-AMPA (20 AM). Cell death was analyzed after 24 h by LDH assay. Data are presented as mean percent total LDH release (% of Triton-X-100-lysed cells) F SEM of three independent experiments on separate cultures, each carried out in duplicate. Statistical differences (one-way ANOVA and Neuman–Keuls post hoc test): *P b 0.001 vs. S-AMPA, #P b 0.05 vs. SAMPA + HU-210.
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agonistic activity at human CB1 and CB2 receptors (Landsman et al., 1998; Ross et al., 1999), recent in vivo studies showed that the effects of AM 1241, a potent and selective CB2 agonist, were reversed by AM 630 but not by the CB1 receptor-selective antagonist AM 251 (Quartilho et al., 2004), suggesting that AM 630 is indeed a selective antagonist for CB2 receptors. The results reported here therefore implicate both receptors in neuroprotection. Although unexpected from the reported peripheral location of CB2 receptors, these observations agree with studies demonstrating expression of CB2 receptors in glial cells: cultured microglia express CB2 receptors (Carlisle et al., 2002); astrocytes are known to possess G-protein-coupled receptors that are activated by cannabinoids but distinct from the CB1 receptor (Berrendero et al., 1998; Sagan et al., 1999), and CB2 receptors has been detected in human astrocytoma and C6 glioma cells (Galve-Roperh et al., 2000). Furthermore, our data do not exclude a role for novel CB receptor(s). Ligand binding to CB receptors can activate several mitogenactivated protein kinases (MAPK) including ERK1/2 (Derkinderen et al., 2003), JNK and p38 (Galve-Roperh et al., 2000; Rueda et al., 2000). However, in the primary cortical neurons analyzed in this report, no activation of ERK1/2, JNK or p38 was detected, suggesting that primary cortical neurons may not activate MAPK in response to HU210. In support of this conclusion, inhibitors of ERK1/2 or p38 did not reverse the neuroprotective effects of this CB (data not shown). In contrast, delta-9tetrahydrocannabinol (THC) induces apoptosis of cultured cortical neurons, via the CB1 receptor and activation of JNK (Downer et al., 2003). Cannabinoids may therefore have diverse effects on neuronal survival/death (Guzman, 2003), and the outcome may depend on a variety of influences, including the maturity of the cultures, nature and time of exposure to the insult and the particular cannabinoid used. The major signaling response to HU-210 reported here was phosphorylation of AKT on serine 473 (Fig. 2). AKT is a critical downstream kinase that regulates growth factor-induced survival of neurons including cerebellar granule neurons (Dudek et al., 1997) and sympathetic neurons (Crowder and Freeman, 1998; Philpott et al., 1997). Activation of AKT requires phosphorylation of the enzyme on T308 and S473, and phosphorylation of both sites is required for full activity (reviewed by Scheid and Woodgett, 2003). We have used phosphorylation of S473 as a marker of AKT activity, and report here that in primary cortical neurons, the CB HU-210 increased activity of AKT, and that this effect was reduced by both CB1 and CB2 receptor antagonists. Activation of AKT requires not only phosphorylation, but also interaction with phospholipids, generated by phosphatidylinositol 3-kinase (PI 3-K), that bind to its pleckstrin homology domain. Indeed, plasma membrane localization is required for S473 phosphorylation (Scheid et al., 2002). Inhibition of PI 3-K by the inhibitors LY294002 and wortmannin blocked phosphorylation of AKT S473 in response to HU-210, and prevented the neuroprotective effects of the CB. These results indicate that PI 3-K and the downstream kinase, AKT, play key roles in mediating the neuroprotective effects of HU-210. AKT is critical for neuronal survival, and a variety of targets of AKT have been implicated in its pro-survival effects, including Bad, Bim, transcription factors of the Forkhead family, and GSK-3h (reviewed by Brunet et al., 2001). The identity of the AKT substrate that mediates the neuroprotective effects of HU-210 in cortical neurons has yet to be identified, but in oligodendrocytes,
CBs lead to phosphorylation of GSK-3h downstream of AKT (Molina-Holgado et al., 2002a). In conclusion, the CNS effects of CBs have been known for decades, and it has been realized recently that CBs also have significant neuroprotective potential, that could be exploited for therapeutic benefit. The results presented here demonstrate for the first time that exogenous CBs such as HU210 can protect cultured neurons from the neurotoxic effects of the excitotoxin S-AMPA through phosphorylation of the critical pro-survival enzyme AKT.
Experimental methods Reagents All tissue culture reagents were obtained from Invitrogen (Paisley, UK). Antibodies that recognize signaling enzymes were obtained from Cell Signaling (Hitchin, UK) (total AKT, AKT phosphorylated on S473, ERK1/2 phosphorylated on T202 and Y204, respectively, total p38), Santa Cruz (Santa Cruz, USA) (total JNK, total ERK1/2) and Promega (Southampton, UK) (JNK phosphorylated on T183 and Y185, p38 phosphorylated on T180 and Y182). The horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies were from Dako (Cambridge, UK). The PI 3-K inhibitors LY 294002 and wortmannin, the CB agonist HU-210, and the CB antagonists AM 281 and AM 630 were obtained from Tocris (Bristol, UK). All other reagents were obtained from Sigma (Poole, UK) unless stated otherwise. Neuronal cell cultures Primary cerebrocortical neurons (CCN) were prepared from embryonic (day 18) Sprague–Dawley rat pups as previously described (Moore et al., 2002). Cells were seeded (7.5 104 viable cells/cm2) onto plates coated with poly-d-lysine, in plating media (neurobasal medium with B27 supplement plus antioxidants, 2 mM glutamine, 25 AM glutamate, 100 IU/ml penicillin and 100 Ag/ml streptomycin). After 2 DIV, cytosine arabinoside (20 AM) was added to the cultures to eliminate the majority of proliferating non-neuronal cells. The cultures were used after 13–14 DIV, and consisted of N98% neurons as analzed by immunocytochemistry (not shown). To study excitotoxicity, neurons grown in 24-well plates were pre-treated with HU-210 (1 AM) in the presence or absence of the CB antagonists AM 281 or AM 630 (1 AM), or a mixture of LY294002 (10 AM) and wortmannin (0.1 AM) for 1 h at 378C, and then exposed to S-AMPA (20 AM) for 24 h at 378C. To study kinase activation, the culture medium of the neurons was changed to DMEM without B27 or serum for 2 h, and then the cells were pre-treated with a mixture of LY294002 (10 AM) and wortmannin (0.1 AM) for 15 min, and then HU-210 (1 AM) for 15 min, prior to exposure to S-AMPA (20 AM) for 30 min at 378C. Cells were then harvested for Western blot analysis. Assessment of cell death Cytotoxicity was evaluated by release of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium by dead and dying cells (CytoTox-96 LDH assay, Promega). Total LDH release was calculated by incubating untreated cells with 0.1% Triton X-
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100 for 10 min (378C, 5% CO2, 95% air) to induce maximal cell lysis. Treatment values were then expressed as a percent of the total LDH release. Background LDH release (media alone) was subtracted from the experimental values. Western blot analysis Immunoblotting was carried out as previously described (Parker et al., 2002). The membranes were incubated at 48C overnight in primary antibodies, diluted as follows: anti-phospho-AKT (1:1000), anti-AKT (1:1000), anti-phospho-ERK1/2 (1:5000), anti-phospho-JNK (1:5000), anti-phospho-p38 (1:1000), anti-total ERK1/2 (1:60000), anti-JNK (1:30000), and anti-total p38 (1:1000). After washing in TBS with 0.1% Tween 20 (TBS-T), blots were incubated with horseradish peroxidase-conjugated antirabbit or anti-mouse secondary antibodies (1:2000, in 5% dried skimmed milk in TBS-T) for 1–2 h at RT. Finally, the protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences, UK), and analyzed by densitometry using Northern Eclipse software (Empix Imaging Inc., Canada). Statistical analysis Results are presented as the mean F SE of at least three independent experiments performed on separate cell preparations; duplicate or triplicate determinations were performed for each experiment. One-way analysis of variance was followed by posteriori Newman–Keuls multiple comparison. Statistical significance was established at P b 0.05. Acknowledgments This work was supported by the Medical Research Council, UK (EP, LH, NJR, RMG), and The Wellcome Trust (FM-H). References Benito, C., Nunez, E., Tolon, R.M., Carrier, E.J., Rabano, A., Hillard, C.J., Romero, J., 2003. Cannabinoid CB2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaqueassociated glia in Alzheimer’s disease brains. J. Neurosci. 23, 11136 – 11141. Berrendero, F., Garcı´a-Gil, L., Herna´ndez, M.L., Romero, J., Cebeira, M., de Miguel, R., Ramos, J.A., Ferna´ndez-Ruiz, J.J., 1998. Localization of mRNA expression and activation of signal transduction mechanisms for cannabinoid receptor in rat brain during fetal development. Development 125, 3179 – 3188. Brunet, A., Datta, S.R., Greenberg, M.E., 2001. Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr. Opin. Neurobiol. 11, 297 – 305. Carlisle, S.J., Marciano-Cabral, F., Staab, A., Ludwick, C., Cabral, G.A., 2002. Differential expression of the CB2 cannabinoid receptor by rodent macrophages and macrophage-like cells in relation to cell activation. Int. Immunopharmacol. 2, 69 – 82. Crowder, R.J., Freeman, R.S., 1998. Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons. J. Neurosci. 18, 2933 – 2943. Downer, E.J., Fogarty, M.P., Campbell, V.A., 2003. Tetrahydrocannabinolinduced neurotoxicity depends on CB1 receptor-mediated c-Jun Nterminal kinase activation in cultured cortical neurons. Br. J. Pharmacol. 140, 547 – 557.
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