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Region-specific neuroprotective effect of ZM 241385 towards glutamate uptake inhibition in cultured neurons
European Journal of Pharmacology 617 (2009) 28–32
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Region-specific neuroprotective effect of ZM 241385 towards glutamate uptake inhibition in cultured neurons Rita Pepponi, Antonella Ferrante, Roberta Ferretti, Alberto Martire, Patrizia Popoli ⁎ Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Roma, Italy
a r t i c l e
i n f o
Article history: Received 8 April 2009 Received in revised form 25 June 2009 Accepted 9 July 2009 Available online 18 July 2009 Keywords: Adenosine A2A receptor Glutamate uptake Cell culture LDH Cortex Striatum
a b s t r a c t Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Adenosine A2A receptors regulate extracellular glutamate levels by acting on both the release and the uptake of glutamate. The aim of this study was to evaluate whether the inhibition of the effects of glutamate uptake blockers by adenosine A2A receptor antagonists resulted in neuroprotection. In cortical and striatal neuronal cultures, the application of L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), induced a dose-dependent increase in lactate dehydrogenase (LDH) levels, an index of cytotoxicity. Such an effect of PDC was significantly reduced by pretreatment with the adenosine A2A receptor antagonist ZM 241385 (50 nM) in striatal, but not cortical, cultures. The protective effects of ZM 241385 were specifically due to a counteraction of PDC effects, since ZM 241385 was totally ineffective in preventing the cytotoxicity induced by direct application of glutamate to cultures. These results indicate that adenosine A2A receptor antagonists prevent the toxic effects induced by a transportable competitive inhibitor of glutamate uptake, that such an effect specifically occurs in the striatum and that it does not depend on a direct blockade of glutamate-induced toxicity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Glutamate is the main excitatory transmitter in the central nervous system (CNS). While a physiological glutamatergic transmission ensures numerous CNS processes, such as learning and memory, excessive glutamate concentrations may result in neuronal toxicity (excitotoxicity) and eventual neuronal cell death (Choi, 1988, 1992). Glutamate homeostasis is accomplished by the activity of glutamate transporters (excitatory amino-acid transporters, EAATs) expressed by neurons and glial cells (see Danbolt, 2001, for review), which represent the primary mechanism for maintaining low, non-toxic extracellular concentrations of glutamate. Indeed, an impairment of the glutamate transporter system is thought to play a pathogenetic role in conditions such as cerebral ischemia (Seki et al., 1999; Rossi et al., 2000) and chronic neurodegenerative diseases (Harris et al., 1995; Lin et al., 1998; Behrens et al., 2002). Adenosine A2A receptors are being regarded as promising targets for the development of neuroprotective strategies, in particular for those neurodegenerative diseases in which excitotoxicity plays a critical pathogenetic role. Adenosine A2A receptor antagonists blocked high-K+- (Corsi et al., 2000; Pintor et al., 2001), ischemia- (Melani et al., 2006; Marcoli et al., 2003) or quinolinic acid (QA)-stimulated glutamate outflow (Popoli et al., 2002, 2003), an effect most probably due to the blockade of adenosine A2A receptors on pre-synaptic
⁎ Corresponding author. Tel.: +39 06 49902482; fax: +39 06 49902014. E-mail address: [email protected] (P. Popoli). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.07.016
glutamatergic terminals (Hettinger et al., 2001). In addition, A2A receptors have been reported to negatively modulate glutamate transport (Nishizaki et al., 2002). In agreement, in a striatal microdialysis study, the selective adenosine A2A receptor antagonists SCH 58261 and ZM 241385 prevented the raise in extracellular glutamate induced by glutamate uptake inhibitors (Pintor et al., 2004). Whether the inhibition of the effects of glutamate uptake blockers by A2A receptor antagonists may result in neuroprotection has never been investigated. The aim of the present work was to evaluate the influence of the selective adenosine A2A receptor antagonist ZM 241385 on the cytotoxicity induced by glutamate uptake inhibition in primary neuronal cultures. To better mimic reverse transport occurring in some disease states such as ischemia (Nafia et al., 2008 and references therein; Rossi et al., 2000), L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a transportable competitive inhibitor that also induces glutamate release through heteroexchange (Volterra et al., 1996; Koch et al., 1999) was used. Two brain areas characterized by a very different expression of adenosine A2A receptors (namely the cortex and the striatum) were compared. 2. Materials and methods 2.1. Western blotting Since adenosine A2A receptors regulate the glial glutamate transporter-1 (GLT-1) (Nishizaki et al., 2002; Pintor et al., 2004), and since a certain
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degree of neuronal GLT-1 expression has been reported in hippocampal neurons (Mennerick et al., 1998), we wanted to verify first the content of GLT-1 in our primary cortical and striatal neurons (see below) by Western blot analysis. In the same samples we verified also the presence of excitatory amino-acid carrier-1 (EAAC 1). Proteins extracted from cortex of C57BL/6 mice were used as positive control for GLT-1. After 15 days of culture, cells were lysed in Ripa buffer (PBS containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml PMSF, leupeptin, aprotinin, pepastatin A and sodium orthovanadate) for 30 min on ice. The lysates were centrifuged at 12,000 rcf for 20 min at 4 °C and the pellets were discarded. Protein analysis was conducted by the Biorad Protein Assay (BioRad Laboratories, Milan, Italy) using BSA as standard. Fifty micrograms of protein was separated onto 8% acrylamide gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). Membranes were blocked with 5% non-fat dry milk in TBST buffer (0.1 M Tris base, 0.15 M NaCl, 0.05% Tween 20, pH 7.4) and then incubated with anti-GLT-1 antibody (1:2000) or with anti-EAAC 1 antibody (1:500) overnight at 4 °C. Following incubation with HRP-linked anti-rabbit antibody (1:5000), visualization of the bound antibodies was performed with the Enhanced ChemiLuminescence (ECL) system (Pierce, S.I.A.L., Rome, Italy). The blots were exposed to X-ray film and then scanned. Antiglutamate transporter GLT-1 (AB1783), anti-EAAC 1 (MAB 1587) and HRPlinked anti-rabbit antibodies were purchased from Chemicon International (Milan, Italy). Supersignal West Pico Chemiluminescent substrate was purchased from Pierce (S.I.A.L., Rome, Italy).
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Fig. 1. GLT-1 is not expressed in neuron-enriched cortical and striatal cultures. The figure shows a representative Western blotting analysis. Panel A: GLT1 is clearly revealed in the positive control (cortex of C57BL/6 mice, lane 3), while no expression is seen in cortical and striatal cultures (lanes 1 and 2). Beta-actin was used as a loading control. Panel B: the presence of EAAC 1 in cortical and striatal cultures (lanes 1 and 2) is confirmed. Beta-actin was used as a loading control.
Results are expressed as percentage of control, which was considered as 100%, and represent means± SEM values of 3–4 independent experiments, assayed in triplicate. 2.3. Drugs L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and 4-(2-[7Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino] ethyl)phenol (ZM 241385) were purchased from Tocris Cookson (Bristol, UK). MK-801 was purchased from RBI (Natick, MA, USA). L-glutamic acid was from Sigma-Aldrich (Milan, Italy).
3. Results 3.1. Western blotting
2.2. Cytotoxicity in primary neuronal cultures To evaluate the potential neuroprotective effects of ZM 241385 towards PDC-induced toxicity, primary neuronal cultures were used. Neuronal cultures were preferred because of their higher vulnerability to uptake blockers with respect to mixed astrocytic/neuronal or astrocyte-enriched cell cultures (Blitzblau et al., 1996). Animal care and use followed the directives of the Council of the European Communities (86/609/EEC). Brain areas were isolated from 17–18 day old embryos. Pregnant rats were ether anaesthetized, decapitated and the foetuses collected and rapidly decapitated. After removal of the meninges, the cortices and the striata were dissected, transferred to Hank's balanced salt solution (HBSS) and mechanically fragmented. The tissue fragments were transferred to 0.025% trypsin solution and incubated for 15 min at 37 °C. Striatal and cortical cells were then washed in HBSS and resuspended in Neurobasal medium supplemented with 0.5 mM L-glutamine, 2% B-27 supplement, 5 U/ml penicillin and 5 μg/ml streptomycin (referred as complete medium). Aliquots of 2–3 × 104 cells were placed in 24-well culture plates coated with poly-L-lysine (5 μg/ml) and maintained at 37 °C in humidified air with 5% CO2. Every 4 days, 0.5 ml of medium was removed and replaced by the same volume of fresh complete medium. Under these experimental conditions, cultures consist predominantly of neurons as previously described (Brewer et al., 1993; Brewer, 1995). In particular, in our cultures the percentage of contaminating astrocytes is normally ≤10%. Assays were done on 15 day old cultures. At the time of the experiment, culture medium was removed and substituted by an appropriate volume of Neurobasal medium supplemented with penicillin (5 U/ml) and streptomycin (5 μg/ml). Cultured cells were then exposed to PDC (12.5–200 μM) or glutamate (12.5–100 μM) for 1 h, preincubated or not for 15 min in ZM 241385 (50 nM), MK-801 (10 μM) or control medium. All drug treatments were performed at 37 °C in humidified air with 5% CO2. Following exposure to the drugs, culture medium was removed and replaced with fresh complete medium. Cultures were then returned to the incubator and cellular damage was evaluated, 24 h later, by measuring the amount of lactate dehydrogenase (LDH) released into the medium using a cytotoxicity detection kit (Roche Diagnostic, Indianapolis, IN). The reaction was run at room temperature with light protection for 30 min.
As shown in Fig. 1A, it is not possible to reveal GLT-1 presence either in cortical (lane 1) or in striatal (lane 2) primary cultures, where, on the contrary, EAAC 1 is clearly expressed (Fig. 1B). GLT-1 can be detected only in the cortex of C57BL/6 mice used as positive control (Fig. 1A, lane 3). 3.2. Influence of ZM 241385 on PDC-induced striatal and cortical cell injury One hour incubation of striatal cultures with PDC (12.5–200 μM, N=4– 12/dose) induced a significant, dose-dependent increase in LDH release with respect to basal levels (Fig. 2A). PDC-induced toxicity was mainly mediated by the NMDA receptors, since it was prevented by MK-801 (MK801 10 μM+PDC 100 μM: LDH release 88±3.8% of basal, N=3, Fig. 2A). Exposure to ZM 241385 (50 nM, N=4–9/group) 15 min before and then together with PDC significantly reduced LDH release with respect to PDC alone (Fig. 2B). When applied alone, neither MK-801 nor ZM 241385 influenced basal LDH release (not shown). PDC (N = 4–9/dose) induced a concentration-dependent cytotoxicity also in cortical cultures, as shown in Fig. 2C, an effect prevented, also in this case, by MK-801 (MK-801 10 μM + PDC 100 μM: LDH release 98.7 ± 5.9% of basal, N = 3, Fig. 2C). Unlike in the striatal cultures, ZM 241385 (50 nM) did not influence LDH release vs. PDC alone (Fig. 2D). In a limited number of experiments ZM 241385 was tested at 100 nM and it was ineffective as well in reducing PDC toxicity in cortical neurons (N = 3, data not shown). These data indicate that the protective effects of ZM 241385 towards PDC are region-specific. 3.3. Influence of ZM 241385 on glutamate-induced neuronal cell injury To verify whether ZM 241385 effects depended upon a specific influence on glutamate transport, this antagonist was tested towards the toxicity induced by direct application of glutamate. One hour incubation of striatal and cortical cells with glutamate (12.5–100 μM, N = 6–9/dose) induced a significant, dose-dependent increase in LDH release with respect to basal levels (Fig. 3A and C). Glutamate-induced toxicity depended on NMDA receptor activation, since it was fully prevented by MK 801 both in striatal and cortical cultures (MK 801 10 μM + Glu 100 μM: LDH release 101.5 ± 6.7% of
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Fig. 2. Influence of ZM 241385 towards PDC-induced LDH release in striatal cultures. Panel A: dose–response curve of PDC-induced effects in striatal neurons. LDH release is expressed as percentage of the basal levels. Each bar represents the mean ± S.E.M. from 4–12 experiments. ° = P b 0.05 vs. PDC 12.5; § = P b 0.05 vs. PDC 12.5, 25 and 50 μM; ⁎ = P b 0.05 vs. PDC 100. Panel B: ZM 241385 significantly reduced PDC-induced LDH release. ZM 241385 (50 nM) was applied 15 min before and then together with PDC (N = 4–9/group). ⁎ = P b 0.05 vs. PDC alone (two-tailed Student's t test). Influence of ZM 241385 towards PDC-induced LDH release in cortical cultures. Panel C: dose–response curve of PDC-induced effects in cortical neurons. LDH release is expressed as percentage of the basal levels. Each bar represents the mean ± S.E.M. from 4–9 experiments. ° = P b 0.05 vs. PDC 12.5; § = P b 0.05 vs. PDC 12.5, 25 and 50 μM; ⁎ = P b 0.05 vs. PDC 100. Panel D: ZM 241385 did not influence PDC-induced LDH release in cortical neurons. ZM 241385 (50 nM, N = 4–8/group) was applied 15 min before and then together with PDC.
Fig. 3. Influence of ZM 241385 towards glutamate-induced LDH release in striatal cultures. Panel A: dose–response curve of glutamate-induced effects in cortical neurons. LDH release is expressed as percentage of the basal levels. Each bar represents the mean ± S.E.M. from 6–8 experiments. ° = P b 0.05 vs. GLU 12.5 and 25 μM; ⁎ = P b 0.05 vs. GLU 100. Panel B: ZM 241385 (50 nM, N = 4–6/group) did not influence glutamate-induced LDH release in striatal neurons. Influence of ZM 241385 towards PDC-induced LDH release in cortical cultures. Panel C: dose–response curve of glutamate-induced effects in cortical neurons. LDH release is expressed as percentage of the basal levels. Each bar represents the mean ± S.E.M. from 6–9 experiments. ° = P b 0.05 vs. GLU 12.5 μM; ⁎ = P b 0.05 vs. GLU 100. Panel D: ZM 241385 (50 nM, N = 4–6/group) did not influence glutamate-induced LDH release in cortical neurons.
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basal, N = 3, striatal cultures, Fig. 3A; LDH release 87.8 ± 3.4% of basal, N = 4, cortical cultures, Fig. 3C). Conversely, ZM 241385 (50 nM, N = 4–6/group) was totally ineffective (Fig. 3B and D).
4. Discussion In agreement with previous studies (Blitzblau et al., 1996; Velasco et al., 1996; Velasco and Tapia, 2002), we found that the glutamate transporter inhibitor PDC induces cytotoxicity in primary cultures. In particular, our results are in line with the previously reported frank toxicity of PDC in astrocyte-poor cortical cultures (Amin and Pearce, 1997; Blitzblau et al., 1996). Also in agreement with previous reports, the toxic effects of PDC were completely prevented by the NMDA receptor antagonist MK-801 (Blitzblau et al., 1996, Velasco et al., 1996). Although such a finding might indicate that PDC-induced toxicity depends on a direct NMDA agonistic activity, this possibility has been excluded in previous studies (Blitzblau et al., 1996; Gouix et al., 2009). According to the above studies, PDC-induced cytotoxicity should rather be ascribed to an increase in extracellular glutamate concentration in the bathing medium of cultures. Although PDC is a non-selective uptake blocker, an involvement of transporters other than excitatory amino-acid carrier-1 (EAAC 1), which is the main glutamate transporter expressed by neuronenriched cultures, and which is expressed by neurons all along the development (see Guillet et al., 2002), is very unlikely here. Indeed, no expression of the astrocytic transporter GLT-1 was revealed by Western blotting in our cultures, where, instead, the presence of EAAC 1 is clear. On a functional ground, these findings are also supported by the inability of the preferential GLT-1 blocker dihydrokainic acid (DHK, tested up to 1 mM) to induce cytotoxicity in our cultures (unpublished results). These data indicate that the reported neuronal GLT-1 expression is mainly confined to the hippocampus (Mennerick et al., 1998), while (at least under our conditions) it seems negligible in the cortex and in the striatum. In agreement, only a very weak and transient GLT-1 immunoreactivity (at 7 but not 14 day in vitro) was found in rat cortical neurons (Guillet et al., 2002). A major involvement of the glutamate–aspartate transporter GLAST is very unlikely as well, since GLAST is only expressed by astrocytes (Danbolt, 2001) and, in our experimental conditions, cultures consist predominantly of neurons. Thus, the toxic effects induced by PDC should mainly depend on the neuronal transporter EAAC 1. Interestingly, since PDC is a transportable inhibitor that also induces glutamate release through heteroexchange (Volterra et al., 1996; Koch et al., 1999), it may mimic reverse transport occurring in some disease states such as ischemia (Nafia et al., 2008 and references therein; Rossi et al., 2000). The fact that in our cell cultures PDC should mainly act on the neuronal transporter is even more relevant to the perturbation occurring in disease states, since during ischemia glutamate is released from neurons rather than glia (Rossi et al., 2000). With this in mind, the protective effects of ZM 241385 towards PDC-induced toxicity may indicate that the modulation of the neuronal glutamate transporter is one of the mechanisms of the beneficial effects exerted by A2A receptor antagonists in brain ischemia (Chen and Pedata, 2008; Melani et al., 2006; Monopoli et al., 1998). Anyway, since adenosine A2A receptors regulate also the glial transporter GLT-1, a modulatory effect on this transporter can well contribute to the regulation of glutamate extracellular concentrations during in vivo ischemia. The ability of ZM 241385 to prevent PDC-induced toxicity was only evident in striatal cultures. Whether such a difference (which might depend on the different expression of A2A receptors in striatal vs. cortical cultures) could influence the in vivo neuroprotective potential of A2A receptor antagonists is worth exploring. Interestingly, indeed, the A2A receptor antagonist SCH 58261 reduced damage in the striatum much
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more clearly than in the cortex in rats subjected to middle cerebral artery occlusion (MCAO, Melani et al., 2006). Finally, the protective effects of ZM 241385 do not depend on a direct blockade of glutamate-induced toxicity. This finding is in line with previous observations showing the inability of adenosine A2A receptor antagonists to prevent neuronal death due to direct application of NMDA or other excitotoxins (Popoli et al., 2002; Rebola et al., 2005; Tebano et al., 2004). This further strengthens the concept that adenosine A2A receptor blockade influences glutamate outflow (acting both on the pre-synaptic release and on the uptake) but it becomes ineffective once a large amount of glutamate has been made available to neurons. These data are only apparently in contrast with the results obtained from Boeck et al. (2005), who reported neuroprotective effects of ZM 241385 against glutamate-induced toxicity. In fact, in the above study ZM 241385 was protective only after a long lasting pretreatment (24 h), while it became ineffective after acute application. Furthermore, the experiments were performed in a different cell population (cerebellar granule cells vs. striatal and cortical neurons). Our study confirms the hypothesis that A2ARs play a major role in modulating the glutamate transporter functioning. Since the pharmacological blockade of the A2A receptor abolished the PDC effects, adenosine A2A receptors are supposed to be endogenously activated by adenosine under these experimental conditions. This is perfectly conceivable indeed, since PDC-induced glutamate release causes the activation of NMDA receptors which, in turn, can increase extracellular adenosine levels (Craig and White, 1993; Melani et al., 1999) thus leading to the activation of A2A receptors. In conclusion the present data show, for the first time, that the adenosine A2A receptor antagonist ZM 241385 prevents the toxic effects induced by a transportable inhibitor of glutamate uptake, that such an effect specifically occurs in the striatum and that it does not depend on a direct blockade of glutamate-induced toxicity. Since an impairment in glutamate transporter function plays an important role in neurodegenerative disorders, the present results further support the neuroprotective potential of adenosine A2A receptor antagonists. References Amin, N., Pearce, B., 1997. Glutamate toxicity in neuron-enriched and neuron-astrocyte co-cultures: effect of the glutamate inhibitorL-trans-pyrrolidine-2,4-dicarboxylate. Neurochem. Int. 30, 271–276. Behrens, P.F., Franz, P., Woodman, B., Lindenberg, K.S., Landwehrmeyer, G.B., 2002. Impaired glutamate transport and glutamate–glutamine cycling: downstream effects of the Huntington mutation. Brain 125, 1908–1922. Blitzblau, R., Shalini, G., Djali, S., Robinson, M.B., Rosenberg, P.A., 1996. The glutamate transport inhibitorL-trans-pyrrolidine-2,4-dicarboxilate indirectly evokes NMDA receptor mediated neurotoxicity in rat cortical cultures. Eur. J. Neurosci. 8, 1840–1852. Boeck, C.R., Kroth, E.H., Bronzatto, M.J., Vendite, D., 2005. Adenosine receptors co-operate with NMDA preconditioning to protect cerebellar granule cells against glutamate neurotoxicity. Neuropharmacology 49, 17–24. Brewer, G.J., 1995. Serum-free B27/neurobasal medium support differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J. Neurosci. Res. 42, 674–683. Brewer, G.J., Torricelli, J.R., Evege, E.K., Price, P.J., 1993. Optimized survival of hippocampal neurons in B27-supplemented neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576. Chen, J.F., Pedata, F., 2008. Modulation of ischemic brain injury and neuroinflammation by adenosine A2A receptors. Curr. Pharm. Des. 14, 1490–1499. Choi, D.W., 1988. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1, 623–634. Choi, D.W., 1992. Excitotoxic cell death. J. Neurobiol. 23, 1261–1276. Corsi, C., Melani, A., Bianchi, L., Pedata, F., 2000. Striatal A2A adenosine receptor antagonism differentially modifies striatal glutamate outflow in vivo in young and aged rats. Neuroreport 11, 2591–2595. Craig, C.G., White, T.D., 1993. NMDA-evoked adenosine release from rat cortex does not require the intermediate formation of nitric oxide. Neurosci. Lett. 158, 167–169. Danbolt, N.C., 2001. Glutamate uptake. Prog. Neurobiol. 65, 1–105. Gouix, E., Léveillé, F., Nicole, O., Melon, C., Had-Aissouni, L., Buisson, A., 2009. Reverse glial glutamate uptake triggers neuronal cell death through extrasynaptic NMDA receptor activation. Mol. Cell. Neurosci. 40, 463–473. Guillet, B., Lortet, S., Masmejean, F., Samuel, D., Nieoullon, A., Pisano, P., 2002. Developmental expression and activity of high affinity glutamate transporters in rat cortical primary cultures. Neurochem. Int. 40, 661–671.
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Region-specific neuroprotective effect of ZM 241385 towards glutamate uptake inhibition in cultured neurons Rita Pepponi, Antonella Ferrante, Roberta Ferretti, Alberto Martire, Patrizia Popoli ⁎ Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Roma, Italy
a r t i c l e
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Article history: Received 8 April 2009 Received in revised form 25 June 2009 Accepted 9 July 2009 Available online 18 July 2009 Keywords: Adenosine A2A receptor Glutamate uptake Cell culture LDH Cortex Striatum
a b s t r a c t Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Adenosine A2A receptors regulate extracellular glutamate levels by acting on both the release and the uptake of glutamate. The aim of this study was to evaluate whether the inhibition of the effects of glutamate uptake blockers by adenosine A2A receptor antagonists resulted in neuroprotection. In cortical and striatal neuronal cultures, the application of L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), induced a dose-dependent increase in lactate dehydrogenase (LDH) levels, an index of cytotoxicity. Such an effect of PDC was significantly reduced by pretreatment with the adenosine A2A receptor antagonist ZM 241385 (50 nM) in striatal, but not cortical, cultures. The protective effects of ZM 241385 were specifically due to a counteraction of PDC effects, since ZM 241385 was totally ineffective in preventing the cytotoxicity induced by direct application of glutamate to cultures. These results indicate that adenosine A2A receptor antagonists prevent the toxic effects induced by a transportable competitive inhibitor of glutamate uptake, that such an effect specifically occurs in the striatum and that it does not depend on a direct blockade of glutamate-induced toxicity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Glutamate is the main excitatory transmitter in the central nervous system (CNS). While a physiological glutamatergic transmission ensures numerous CNS processes, such as learning and memory, excessive glutamate concentrations may result in neuronal toxicity (excitotoxicity) and eventual neuronal cell death (Choi, 1988, 1992). Glutamate homeostasis is accomplished by the activity of glutamate transporters (excitatory amino-acid transporters, EAATs) expressed by neurons and glial cells (see Danbolt, 2001, for review), which represent the primary mechanism for maintaining low, non-toxic extracellular concentrations of glutamate. Indeed, an impairment of the glutamate transporter system is thought to play a pathogenetic role in conditions such as cerebral ischemia (Seki et al., 1999; Rossi et al., 2000) and chronic neurodegenerative diseases (Harris et al., 1995; Lin et al., 1998; Behrens et al., 2002). Adenosine A2A receptors are being regarded as promising targets for the development of neuroprotective strategies, in particular for those neurodegenerative diseases in which excitotoxicity plays a critical pathogenetic role. Adenosine A2A receptor antagonists blocked high-K+- (Corsi et al., 2000; Pintor et al., 2001), ischemia- (Melani et al., 2006; Marcoli et al., 2003) or quinolinic acid (QA)-stimulated glutamate outflow (Popoli et al., 2002, 2003), an effect most probably due to the blockade of adenosine A2A receptors on pre-synaptic
⁎ Corresponding author. Tel.: +39 06 49902482; fax: +39 06 49902014. E-mail address: [email protected] (P. Popoli). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.07.016
glutamatergic terminals (Hettinger et al., 2001). In addition, A2A receptors have been reported to negatively modulate glutamate transport (Nishizaki et al., 2002). In agreement, in a striatal microdialysis study, the selective adenosine A2A receptor antagonists SCH 58261 and ZM 241385 prevented the raise in extracellular glutamate induced by glutamate uptake inhibitors (Pintor et al., 2004). Whether the inhibition of the effects of glutamate uptake blockers by A2A receptor antagonists may result in neuroprotection has never been investigated. The aim of the present work was to evaluate the influence of the selective adenosine A2A receptor antagonist ZM 241385 on the cytotoxicity induced by glutamate uptake inhibition in primary neuronal cultures. To better mimic reverse transport occurring in some disease states such as ischemia (Nafia et al., 2008 and references therein; Rossi et al., 2000), L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a transportable competitive inhibitor that also induces glutamate release through heteroexchange (Volterra et al., 1996; Koch et al., 1999) was used. Two brain areas characterized by a very different expression of adenosine A2A receptors (namely the cortex and the striatum) were compared. 2. Materials and methods 2.1. Western blotting Since adenosine A2A receptors regulate the glial glutamate transporter-1 (GLT-1) (Nishizaki et al., 2002; Pintor et al., 2004), and since a certain
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degree of neuronal GLT-1 expression has been reported in hippocampal neurons (Mennerick et al., 1998), we wanted to verify first the content of GLT-1 in our primary cortical and striatal neurons (see below) by Western blot analysis. In the same samples we verified also the presence of excitatory amino-acid carrier-1 (EAAC 1). Proteins extracted from cortex of C57BL/6 mice were used as positive control for GLT-1. After 15 days of culture, cells were lysed in Ripa buffer (PBS containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml PMSF, leupeptin, aprotinin, pepastatin A and sodium orthovanadate) for 30 min on ice. The lysates were centrifuged at 12,000 rcf for 20 min at 4 °C and the pellets were discarded. Protein analysis was conducted by the Biorad Protein Assay (BioRad Laboratories, Milan, Italy) using BSA as standard. Fifty micrograms of protein was separated onto 8% acrylamide gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). Membranes were blocked with 5% non-fat dry milk in TBST buffer (0.1 M Tris base, 0.15 M NaCl, 0.05% Tween 20, pH 7.4) and then incubated with anti-GLT-1 antibody (1:2000) or with anti-EAAC 1 antibody (1:500) overnight at 4 °C. Following incubation with HRP-linked anti-rabbit antibody (1:5000), visualization of the bound antibodies was performed with the Enhanced ChemiLuminescence (ECL) system (Pierce, S.I.A.L., Rome, Italy). The blots were exposed to X-ray film and then scanned. Antiglutamate transporter GLT-1 (AB1783), anti-EAAC 1 (MAB 1587) and HRPlinked anti-rabbit antibodies were purchased from Chemicon International (Milan, Italy). Supersignal West Pico Chemiluminescent substrate was purchased from Pierce (S.I.A.L., Rome, Italy).
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Fig. 1. GLT-1 is not expressed in neuron-enriched cortical and striatal cultures. The figure shows a representative Western blotting analysis. Panel A: GLT1 is clearly revealed in the positive control (cortex of C57BL/6 mice, lane 3), while no expression is seen in cortical and striatal cultures (lanes 1 and 2). Beta-actin was used as a loading control. Panel B: the presence of EAAC 1 in cortical and striatal cultures (lanes 1 and 2) is confirmed. Beta-actin was used as a loading control.
Results are expressed as percentage of control, which was considered as 100%, and represent means± SEM values of 3–4 independent experiments, assayed in triplicate. 2.3. Drugs L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and 4-(2-[7Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino] ethyl)phenol (ZM 241385) were purchased from Tocris Cookson (Bristol, UK). MK-801 was purchased from RBI (Natick, MA, USA). L-glutamic acid was from Sigma-Aldrich (Milan, Italy).
3. Results 3.1. Western blotting
2.2. Cytotoxicity in primary neuronal cultures To evaluate the potential neuroprotective effects of ZM 241385 towards PDC-induced toxicity, primary neuronal cultures were used. Neuronal cultures were preferred because of their higher vulnerability to uptake blockers with respect to mixed astrocytic/neuronal or astrocyte-enriched cell cultures (Blitzblau et al., 1996). Animal care and use followed the directives of the Council of the European Communities (86/609/EEC). Brain areas were isolated from 17–18 day old embryos. Pregnant rats were ether anaesthetized, decapitated and the foetuses collected and rapidly decapitated. After removal of the meninges, the cortices and the striata were dissected, transferred to Hank's balanced salt solution (HBSS) and mechanically fragmented. The tissue fragments were transferred to 0.025% trypsin solution and incubated for 15 min at 37 °C. Striatal and cortical cells were then washed in HBSS and resuspended in Neurobasal medium supplemented with 0.5 mM L-glutamine, 2% B-27 supplement, 5 U/ml penicillin and 5 μg/ml streptomycin (referred as complete medium). Aliquots of 2–3 × 104 cells were placed in 24-well culture plates coated with poly-L-lysine (5 μg/ml) and maintained at 37 °C in humidified air with 5% CO2. Every 4 days, 0.5 ml of medium was removed and replaced by the same volume of fresh complete medium. Under these experimental conditions, cultures consist predominantly of neurons as previously described (Brewer et al., 1993; Brewer, 1995). In particular, in our cultures the percentage of contaminating astrocytes is normally ≤10%. Assays were done on 15 day old cultures. At the time of the experiment, culture medium was removed and substituted by an appropriate volume of Neurobasal medium supplemented with penicillin (5 U/ml) and streptomycin (5 μg/ml). Cultured cells were then exposed to PDC (12.5–200 μM) or glutamate (12.5–100 μM) for 1 h, preincubated or not for 15 min in ZM 241385 (50 nM), MK-801 (10 μM) or control medium. All drug treatments were performed at 37 °C in humidified air with 5% CO2. Following exposure to the drugs, culture medium was removed and replaced with fresh complete medium. Cultures were then returned to the incubator and cellular damage was evaluated, 24 h later, by measuring the amount of lactate dehydrogenase (LDH) released into the medium using a cytotoxicity detection kit (Roche Diagnostic, Indianapolis, IN). The reaction was run at room temperature with light protection for 30 min.
As shown in Fig. 1A, it is not possible to reveal GLT-1 presence either in cortical (lane 1) or in striatal (lane 2) primary cultures, where, on the contrary, EAAC 1 is clearly expressed (Fig. 1B). GLT-1 can be detected only in the cortex of C57BL/6 mice used as positive control (Fig. 1A, lane 3). 3.2. Influence of ZM 241385 on PDC-induced striatal and cortical cell injury One hour incubation of striatal cultures with PDC (12.5–200 μM, N=4– 12/dose) induced a significant, dose-dependent increase in LDH release with respect to basal levels (Fig. 2A). PDC-induced toxicity was mainly mediated by the NMDA receptors, since it was prevented by MK-801 (MK801 10 μM+PDC 100 μM: LDH release 88±3.8% of basal, N=3, Fig. 2A). Exposure to ZM 241385 (50 nM, N=4–9/group) 15 min before and then together with PDC significantly reduced LDH release with respect to PDC alone (Fig. 2B). When applied alone, neither MK-801 nor ZM 241385 influenced basal LDH release (not shown). PDC (N = 4–9/dose) induced a concentration-dependent cytotoxicity also in cortical cultures, as shown in Fig. 2C, an effect prevented, also in this case, by MK-801 (MK-801 10 μM + PDC 100 μM: LDH release 98.7 ± 5.9% of basal, N = 3, Fig. 2C). Unlike in the striatal cultures, ZM 241385 (50 nM) did not influence LDH release vs. PDC alone (Fig. 2D). In a limited number of experiments ZM 241385 was tested at 100 nM and it was ineffective as well in reducing PDC toxicity in cortical neurons (N = 3, data not shown). These data indicate that the protective effects of ZM 241385 towards PDC are region-specific. 3.3. Influence of ZM 241385 on glutamate-induced neuronal cell injury To verify whether ZM 241385 effects depended upon a specific influence on glutamate transport, this antagonist was tested towards the toxicity induced by direct application of glutamate. One hour incubation of striatal and cortical cells with glutamate (12.5–100 μM, N = 6–9/dose) induced a significant, dose-dependent increase in LDH release with respect to basal levels (Fig. 3A and C). Glutamate-induced toxicity depended on NMDA receptor activation, since it was fully prevented by MK 801 both in striatal and cortical cultures (MK 801 10 μM + Glu 100 μM: LDH release 101.5 ± 6.7% of
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Fig. 2. Influence of ZM 241385 towards PDC-induced LDH release in striatal cultures. Panel A: dose–response curve of PDC-induced effects in striatal neurons. LDH release is expressed as percentage of the basal levels. Each bar represents the mean ± S.E.M. from 4–12 experiments. ° = P b 0.05 vs. PDC 12.5; § = P b 0.05 vs. PDC 12.5, 25 and 50 μM; ⁎ = P b 0.05 vs. PDC 100. Panel B: ZM 241385 significantly reduced PDC-induced LDH release. ZM 241385 (50 nM) was applied 15 min before and then together with PDC (N = 4–9/group). ⁎ = P b 0.05 vs. PDC alone (two-tailed Student's t test). Influence of ZM 241385 towards PDC-induced LDH release in cortical cultures. Panel C: dose–response curve of PDC-induced effects in cortical neurons. LDH release is expressed as percentage of the basal levels. Each bar represents the mean ± S.E.M. from 4–9 experiments. ° = P b 0.05 vs. PDC 12.5; § = P b 0.05 vs. PDC 12.5, 25 and 50 μM; ⁎ = P b 0.05 vs. PDC 100. Panel D: ZM 241385 did not influence PDC-induced LDH release in cortical neurons. ZM 241385 (50 nM, N = 4–8/group) was applied 15 min before and then together with PDC.
Fig. 3. Influence of ZM 241385 towards glutamate-induced LDH release in striatal cultures. Panel A: dose–response curve of glutamate-induced effects in cortical neurons. LDH release is expressed as percentage of the basal levels. Each bar represents the mean ± S.E.M. from 6–8 experiments. ° = P b 0.05 vs. GLU 12.5 and 25 μM; ⁎ = P b 0.05 vs. GLU 100. Panel B: ZM 241385 (50 nM, N = 4–6/group) did not influence glutamate-induced LDH release in striatal neurons. Influence of ZM 241385 towards PDC-induced LDH release in cortical cultures. Panel C: dose–response curve of glutamate-induced effects in cortical neurons. LDH release is expressed as percentage of the basal levels. Each bar represents the mean ± S.E.M. from 6–9 experiments. ° = P b 0.05 vs. GLU 12.5 μM; ⁎ = P b 0.05 vs. GLU 100. Panel D: ZM 241385 (50 nM, N = 4–6/group) did not influence glutamate-induced LDH release in cortical neurons.
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basal, N = 3, striatal cultures, Fig. 3A; LDH release 87.8 ± 3.4% of basal, N = 4, cortical cultures, Fig. 3C). Conversely, ZM 241385 (50 nM, N = 4–6/group) was totally ineffective (Fig. 3B and D).
4. Discussion In agreement with previous studies (Blitzblau et al., 1996; Velasco et al., 1996; Velasco and Tapia, 2002), we found that the glutamate transporter inhibitor PDC induces cytotoxicity in primary cultures. In particular, our results are in line with the previously reported frank toxicity of PDC in astrocyte-poor cortical cultures (Amin and Pearce, 1997; Blitzblau et al., 1996). Also in agreement with previous reports, the toxic effects of PDC were completely prevented by the NMDA receptor antagonist MK-801 (Blitzblau et al., 1996, Velasco et al., 1996). Although such a finding might indicate that PDC-induced toxicity depends on a direct NMDA agonistic activity, this possibility has been excluded in previous studies (Blitzblau et al., 1996; Gouix et al., 2009). According to the above studies, PDC-induced cytotoxicity should rather be ascribed to an increase in extracellular glutamate concentration in the bathing medium of cultures. Although PDC is a non-selective uptake blocker, an involvement of transporters other than excitatory amino-acid carrier-1 (EAAC 1), which is the main glutamate transporter expressed by neuronenriched cultures, and which is expressed by neurons all along the development (see Guillet et al., 2002), is very unlikely here. Indeed, no expression of the astrocytic transporter GLT-1 was revealed by Western blotting in our cultures, where, instead, the presence of EAAC 1 is clear. On a functional ground, these findings are also supported by the inability of the preferential GLT-1 blocker dihydrokainic acid (DHK, tested up to 1 mM) to induce cytotoxicity in our cultures (unpublished results). These data indicate that the reported neuronal GLT-1 expression is mainly confined to the hippocampus (Mennerick et al., 1998), while (at least under our conditions) it seems negligible in the cortex and in the striatum. In agreement, only a very weak and transient GLT-1 immunoreactivity (at 7 but not 14 day in vitro) was found in rat cortical neurons (Guillet et al., 2002). A major involvement of the glutamate–aspartate transporter GLAST is very unlikely as well, since GLAST is only expressed by astrocytes (Danbolt, 2001) and, in our experimental conditions, cultures consist predominantly of neurons. Thus, the toxic effects induced by PDC should mainly depend on the neuronal transporter EAAC 1. Interestingly, since PDC is a transportable inhibitor that also induces glutamate release through heteroexchange (Volterra et al., 1996; Koch et al., 1999), it may mimic reverse transport occurring in some disease states such as ischemia (Nafia et al., 2008 and references therein; Rossi et al., 2000). The fact that in our cell cultures PDC should mainly act on the neuronal transporter is even more relevant to the perturbation occurring in disease states, since during ischemia glutamate is released from neurons rather than glia (Rossi et al., 2000). With this in mind, the protective effects of ZM 241385 towards PDC-induced toxicity may indicate that the modulation of the neuronal glutamate transporter is one of the mechanisms of the beneficial effects exerted by A2A receptor antagonists in brain ischemia (Chen and Pedata, 2008; Melani et al., 2006; Monopoli et al., 1998). Anyway, since adenosine A2A receptors regulate also the glial transporter GLT-1, a modulatory effect on this transporter can well contribute to the regulation of glutamate extracellular concentrations during in vivo ischemia. The ability of ZM 241385 to prevent PDC-induced toxicity was only evident in striatal cultures. Whether such a difference (which might depend on the different expression of A2A receptors in striatal vs. cortical cultures) could influence the in vivo neuroprotective potential of A2A receptor antagonists is worth exploring. Interestingly, indeed, the A2A receptor antagonist SCH 58261 reduced damage in the striatum much
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more clearly than in the cortex in rats subjected to middle cerebral artery occlusion (MCAO, Melani et al., 2006). Finally, the protective effects of ZM 241385 do not depend on a direct blockade of glutamate-induced toxicity. This finding is in line with previous observations showing the inability of adenosine A2A receptor antagonists to prevent neuronal death due to direct application of NMDA or other excitotoxins (Popoli et al., 2002; Rebola et al., 2005; Tebano et al., 2004). This further strengthens the concept that adenosine A2A receptor blockade influences glutamate outflow (acting both on the pre-synaptic release and on the uptake) but it becomes ineffective once a large amount of glutamate has been made available to neurons. These data are only apparently in contrast with the results obtained from Boeck et al. (2005), who reported neuroprotective effects of ZM 241385 against glutamate-induced toxicity. In fact, in the above study ZM 241385 was protective only after a long lasting pretreatment (24 h), while it became ineffective after acute application. Furthermore, the experiments were performed in a different cell population (cerebellar granule cells vs. striatal and cortical neurons). Our study confirms the hypothesis that A2ARs play a major role in modulating the glutamate transporter functioning. Since the pharmacological blockade of the A2A receptor abolished the PDC effects, adenosine A2A receptors are supposed to be endogenously activated by adenosine under these experimental conditions. This is perfectly conceivable indeed, since PDC-induced glutamate release causes the activation of NMDA receptors which, in turn, can increase extracellular adenosine levels (Craig and White, 1993; Melani et al., 1999) thus leading to the activation of A2A receptors. In conclusion the present data show, for the first time, that the adenosine A2A receptor antagonist ZM 241385 prevents the toxic effects induced by a transportable inhibitor of glutamate uptake, that such an effect specifically occurs in the striatum and that it does not depend on a direct blockade of glutamate-induced toxicity. Since an impairment in glutamate transporter function plays an important role in neurodegenerative disorders, the present results further support the neuroprotective potential of adenosine A2A receptor antagonists. References Amin, N., Pearce, B., 1997. Glutamate toxicity in neuron-enriched and neuron-astrocyte co-cultures: effect of the glutamate inhibitorL-trans-pyrrolidine-2,4-dicarboxylate. Neurochem. Int. 30, 271–276. Behrens, P.F., Franz, P., Woodman, B., Lindenberg, K.S., Landwehrmeyer, G.B., 2002. Impaired glutamate transport and glutamate–glutamine cycling: downstream effects of the Huntington mutation. Brain 125, 1908–1922. Blitzblau, R., Shalini, G., Djali, S., Robinson, M.B., Rosenberg, P.A., 1996. The glutamate transport inhibitorL-trans-pyrrolidine-2,4-dicarboxilate indirectly evokes NMDA receptor mediated neurotoxicity in rat cortical cultures. Eur. J. Neurosci. 8, 1840–1852. Boeck, C.R., Kroth, E.H., Bronzatto, M.J., Vendite, D., 2005. Adenosine receptors co-operate with NMDA preconditioning to protect cerebellar granule cells against glutamate neurotoxicity. Neuropharmacology 49, 17–24. Brewer, G.J., 1995. Serum-free B27/neurobasal medium support differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J. Neurosci. Res. 42, 674–683. Brewer, G.J., Torricelli, J.R., Evege, E.K., Price, P.J., 1993. Optimized survival of hippocampal neurons in B27-supplemented neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576. Chen, J.F., Pedata, F., 2008. Modulation of ischemic brain injury and neuroinflammation by adenosine A2A receptors. Curr. Pharm. Des. 14, 1490–1499. Choi, D.W., 1988. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1, 623–634. Choi, D.W., 1992. Excitotoxic cell death. J. Neurobiol. 23, 1261–1276. Corsi, C., Melani, A., Bianchi, L., Pedata, F., 2000. Striatal A2A adenosine receptor antagonism differentially modifies striatal glutamate outflow in vivo in young and aged rats. Neuroreport 11, 2591–2595. Craig, C.G., White, T.D., 1993. NMDA-evoked adenosine release from rat cortex does not require the intermediate formation of nitric oxide. Neurosci. Lett. 158, 167–169. Danbolt, N.C., 2001. Glutamate uptake. Prog. Neurobiol. 65, 1–105. Gouix, E., Léveillé, F., Nicole, O., Melon, C., Had-Aissouni, L., Buisson, A., 2009. Reverse glial glutamate uptake triggers neuronal cell death through extrasynaptic NMDA receptor activation. Mol. Cell. Neurosci. 40, 463–473. Guillet, B., Lortet, S., Masmejean, F., Samuel, D., Nieoullon, A., Pisano, P., 2002. Developmental expression and activity of high affinity glutamate transporters in rat cortical primary cultures. Neurochem. Int. 40, 661–671.
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