P2X7 Receptor Blockade Prevents ATP Excitotoxicity in Oligodendrocytes and Ameliorates Experimental Autoimmune Encephalomyelitis

YNBDI-02584; No. of pages: 8; 4C: 7 Neurobiology of Disease xxx (2012) xxx–xxx

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P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia Joana Arbeloa a, 1, Alberto Pérez-Samartín a, 1, Miroslav Gottlieb b, Carlos Matute a,⁎ a b

CIBERNED and Laboratory of Neurobiology, Department of Neurosciences, University of the Basque Country, 48940-Leioa, Spain Institute of Neurobiology, Slovak Academy of Sciences, 04001-Kosice, Slovak Republic

a r t i c l e

i n f o

Article history: Received 13 July 2011 Revised 9 November 2011 Accepted 4 December 2011 Available online xxxx Keywords: Excitotoxicity Ischemia Neuronal damage P2X7

a b s t r a c t Overactivation of subtype P2X7 receptors can induce excitotoxic neuronal death by calcium (Ca 2 +) overload. In this study, we characterize the functional properties of P2X7 receptors using electrophysiology and Ca 2 + monitoring in primary cortical neuron cultures and in brain slices. Both electrical responses and Ca2 + influx induced by ATP and benzoyl-ATP were reduced by Brilliant Blue G (BBG) at concentrations which specifically inhibit P2X7 receptors. In turn, oxygen-glucose deprivation (OGD) caused neuronal death that was reduced with BBG application. OGD in neuron cultures and brain slices generated an inward current, which was delayed and reduced by BBG. To assess the relevance of these in vitro findings, we used middle cerebral artery occlusion in rats as a model of transient focal cerebral ischemia to study the neuroprotective effect of BBG in vivo. Treatment with BBG (twice per day, 30 mg/kg) produced a 60% reduction in the extent of brain damage compared to treatment with vehicle alone. These results show that P2X7 purinergic receptors mediate tissue damage after OGD in neurons and following transient brain ischemia. Therefore, these receptors are a relevant molecular target for the development of new treatments to attenuate brain damage following stroke. © 2011 Elsevier Inc. All rights reserved.

Introduction Cell surface purine/pyrimidine nucleotide receptors, termed P2 receptors (Ralevic and Burnstock, 1998), are activated by adenosine triphosphate (ATP) and subdivided into two major groups: metabotropic receptors, P2Y, which are G-protein coupled [P2Y1, 2,4,6,11–14], and ionotropic receptors, P2X, which are ligandgated ion channels [P2X1–7] (Burnstock, 2007). P2X receptors are cation-selective channels with almost equal permeability to Na + and K +, significant permeability to Ca 2 +, and, at least concerning P2X7 receptors, permeable to molecules up to 700 Da in size (Surprenant et al., 1996). P2X receptors are expressed throughout the central and peripheral nervous systems (Burnstock and Knight, 2004; Gever et al., 2006) and are involved in a wide range of physiologic and pathologic processes (Khakh and North, 2006). Specifically, P2X7 receptors in peripheral tissues mediate inflammation, cell proliferation, and apoptosis (Burnstock, 2007) while in the nervous system they are involved in modulation of neurotransmitter release, as well as microglial and astroglial activation (Sperlágh et al., 2006). During and after stressful events and pathological conditions such as ischemia, damaged neurons and nonneuronal cells release ⁎ Corresponding author. Fax: +34 94 6015055. E-mail address: [email protected] (C. Matute). 1 Both authors contributed equally to this study. Available online on ScienceDirect (www.sciencedirect.com).

ATP into the extracellular space (Braun et al., 1998; Juranyi et al., 1999; Melani et al., 2005; for recent reviews, see also Rossi et al., 2007 and Yenari et al., 2010). Previous studies reported conflicting data concerning the participation of P2X7 receptors in neuronal death after excitotoxicity. Deletion of P2X7 receptors did not affect cell death induced by transient cerebral ischemia and P2X antagonists did not affect ischemic or excitotoxic death (Le Feuvre et al., 2002). However, more recent studies found that traumatic damage to the spinal cord caused the release of ATP and lethal overactivation of P2X7 receptors in neurons (Wang et al., 2004). In addition, blockage of P2X7 receptors is protective in white matter injury (Domercq et al., 2010). In this study we reanalyzed the idea that P2X7 receptors mediate ischemic brain damage. Here, we provide evidence that these receptors are indeed crucial in triggering neuronal death after ischemia both in vitro and in vivo.

Material and methods Animals All experiments were conducted under the supervision and with the approval of our internal animal ethics committee (University of the Basque Country, UPV/EHU). Animals were handled in accordance with the European Communities Council Directive. All possible efforts were made to minimize animal suffering and the number of animals used.

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Please cite this article as: Arbeloa, J., et al., P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia, Neurobiol. Dis. (2012), doi:10.1016/j.nbd.2011.12.014

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Cortical neuron culture Cortical neurons were obtained from the E18 Sprague–Dawley rat embryos according to previously described procedures (Cheung et al., 1998; Larm et al., 1996). Neurons were resuspended in B27 Neurobasal medium plus 10% fetal bovine serum (FBS) and then seeded onto poly-L-ornithine-coated plates or glass coverslips at 1.5 × 10 5 cells per well. The medium was replaced by serum-free, B27-supplemented Neurobasal medium 24 h later. The cultures were essentially free of astrocytes and microglia and were maintained at 37 °C and 5% CO2. Cultures were used at 8–10 days in vitro (div). Oxygen and glucose deprivation (OGD) in neuronal cultures In vitro ischemia (1 h) was achieved by replacing O2 with N2 and external glucose (10 mM) with sucrose and adding iodoacetate (IAA; 20, 50, and 100 μM) to block glycolysis in an extracellular solution containing (in mM) NaCl (130), KCl (5.4), CaCl2 (1.8), NaHCO3 (26), MgCl2 (0.8), and NaH2PO4 (1.18) (pH 7.4). Cell death was determined 24 h later using calcein-AM (Invitrogen, Barcelona, Spain) as previously described (Matute et al., 2007) using a Synergy-HT fluorimeter (BioTek Instruments). The antagonist was applied during OGD and during the next 24 h incubation period. Data were expressed as percentage of cell death versus the respective control with or without antagonist, which did not alter cell survival (data not shown).

cell voltage-clamp recordings of cortical neurons were performed using a Multiclamp 700B amplifier (Molecular Devices, U.S.A.). Pipettes (4–7 MΩ) were filled with the same internal solution used above. To simulate ischemia, glucose was replaced by 7 mM sucrose and 95% O2/5% CO2 was replaced with 95% N2/5% CO2. Transient middle cerebral artery occlusion Transient focal ischemia was induced by middle cerebral artery occlusion (MCAO) in male Wistar rats (270–300 g) using the intraluminal filament technique (Longa et al., 1989). Rats were anesthetized with 4% halothane in an anesthetic chamber and maintained during surgery with 1.5% halothane using a rodent mask. Body temperature was maintained at 37 °C with a heat pad. MCAO was carried out for 90 min by inserting a 4-0 nylon monofilament via the right external carotid artery into the internal carotid artery to block the origin of the middle cerebral artery (MCA). The P2X7 receptor antagonist BBG (30 mg/kg body weight per day), which crosses the blood– brain-barrier (Peng et al., 2009), was administrated intraperitoneally every 12 h beginning 30 min after the onset of ischemia. Animals were euthanized 3 days after ischemia, the brain was removed and infarct volume and number of degenerated neurons were calculated as described below. Sham-operated controls were surgically treated as the ischemic group, but the middle cerebral artery was not occluded. Neurological examination

Electrophysiology and Ca 2 + imaging in cultured cells Whole-cell voltage-clamp recordings of cortical neurons were performed at room temperature using the EPC-7 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany). Currents were recorded at a holding membrane potential of − 70 mV. Extracellular bath solution with a pH of 7.3 contained the following (in mM): NaCl (150), KCl (5), CaCl2 (2.5), MgCl2 (1), HEPES (10), and glucose (10). Divalent cation-free extracellular solutions were obtained by omitting Ca 2 + and Mg 2 +. Patch-clamp pipettes (5–7 MΩ) were filled with internal solution at a pH of 7.3 containing the following (in mM): potassium gluconate (140), CaCl2 (1), MgCl2 (2), HEPES (10), EGTA (10), NaGTP (0.2), and Mg-ATP (2). For electrophysiology monitoring, ischemia was induced chemically using the glycolytic blocker IAA (1 mM), the oxidative phosphorylation inhibitor antimycin (0.25 μM), and replacing glucose with sucrose. For Ca 2 + recording, cells were loaded with fura-2 AM (5 μM; Invitrogen, Barcelona, Spain) in culture medium for 30 min at 37 °C. Experiments were carried out in a chamber perfused with a buffer containing (in mM) NaCl (150), KCl (5), CaCl2 (2.5), MgCl2 (1), HEPES (10), and glucose (10) at 1 ml/min. Cells were visualized with a high-resolution digital B/W CCD camera (ORCA; Hamamatsu Photonics Iberica, Barcelona, Spain). Image acquisition (acquisition rate 1/300 ms) and data analysis were carried out using the AquaCosmos software program (Hamamatsu Photonics Iberica). [Ca 2 +]i was estimated by the 340/380 ratio method, using a Kd value of 224 nM. Electrophysiology in brain slices Brain slice recordings were carried out using 12–13 day old Sprague Dawley rats. Animals were anesthetized with isofluorane. The brain was rapidly dissected and tissue block was cut in horizontal slices (300 μm thick) on a vibratome (Pelco 100, Pelco, Clovis, CA, USA) in ice cold artificial cerebrospinal fluid containing (in mM): NaCl (126), NaHCO3 (24), NaH2PO4 (1), KCl (2.5), CaCl2 (2.5), MgCl2 (2), and D-glucose (10) (bubbled with 95% O2/5% CO2) at pH 7.4. Slices were allowed to recover at least 1 h and were then transferred to a chamber with continuous flow (1.5 ml/min). Cells were visualized using upright microscopy (Leica DMLFSA, Germany). Whole

Neurological deficit was assessed in each animal on a numerical scale of 0–4 at the end of ischemic insult and 60 min after MCAO and, later, at intervals of 24 h. The scoring system based on Bederson et al. (1986) was used: 0, no detectable deficits; 1, forelimb flexion and torso turning to the contralateral side when lifted by tail; 2, same behavior as grade 1 and decreased resistance to lateral push; 3, same behavior as grade 2 with unilateral circling; 4, no spontaneous walking and a depressed level of consciousness. Rats with a neurological deficit lower than 2 were excluded from the study. Determination of brain infarct and histological analysis Analysis of cerebral ischemic damage was carried out using 2,3,5triphenyl tetrazolium chloride (TTC), FluoroJade C staining, and NeuN immunohistochemistry. TTC stains dehydrogenases and its absence allows quantification of the infarct area, whereas FluoroJade C is a marker for degenerating neurons (Schmued et al., 2005). The animals were euthanized after reperfusion, under chloral hydrate anesthesia followed by decapitation. The brains were rapidly dissected out and the forebrains cut into seven coronal sections, 2 mm thick, using a rat brain matrix (Activational Systems, MI, USA). Analysis of cerebral ischemic damage was carried out using 2,3,5-triphenyltetrazolium chloride (TTC, Sigma). The sections were stained by incubating them in a 1% solution at 37 °C for 15 min and fixed in 10% buffered formalin. Anterior and posterior sides of all sections were scanned using a high-resolution scanner (Hewlett Packard Scanjet). The non-ischemic hemisphere, ischemic hemisphere, and infarct area of each brain section was measured in a blinded manner, using Image J software (National Institutes of Health, Bethesda, Maryland, USA). The average infarct area (mm 2) in each section was calculated by the following formula: (infarct area on the anterior surface + infarct area on the posterior surface)/2. The corrected infarct area in each slice was calculated to compensate for brain edema (Callaway et al., 2000). Corrected infarct volumes (mm 3) were calculated by multiplying the corrected area by the slice thickness and summing the volume. Coronal sections after TTC staining were cryostat cut at 10 μm, sections mounted onto gelatinized microscope slides, and stored at

Please cite this article as: Arbeloa, J., et al., P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia, Neurobiol. Dis. (2012), doi:10.1016/j.nbd.2011.12.014

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−20 °C until staining. Some sections were air dried and stained with 0.0001% Fluoro Jade C for 20 min, washed, and coverslipped with DPX (Schmued et al., 2005). The slides were examined using Zeiss Axoplan microscope and images acquired using a digital camera. Fluoro-Jade C positive cells were counted. For immunohistochemistry, sections were incubated with primary antibodies (mouse monoclonal anti-NeuN (1:200; Millipore, Madrid, Spain) overnight at 4 °C and subsequently with fluorescent secondary Alexa 594 goat anti-mouse (1:200; Molecular Probes) antibodies for 2 h at room temperature, and then coverslipped for image analysis. Microphotographs were taken from the ipsilateral and contralateral sides of the cerebral cortex with a 20 × objective and NeuN positive cells were counted.

Statistical analysis All data are reported as mean ± SEM. p values are from Student's t-tests and were considered significant when less than 0.05. For more than two groups, ANOVA and Bonferroni post hoc test were done. The electrophysiological dose–response experiment curves were obtained by non linear dose–response sigmoidal regression. The effect of BBG on the neurological score was examined by Kruskal– Wallis analysis and the difference between the two groups was analyzed with Mann–Whitney U test. All data were analyzed using GraphPad Prism v. 4 software.

Results Cortical neurons express functional P2X7 receptors We initially studied the properties of P2X7 receptors in primary cortical neuron cultures using electrophysiological and Ca 2 + monitoring techniques. In normal extracellular Ringer bath solution, ATP (1 mM) induced a non-desensitizing inward current (20.13 ± 4.75 pA, n = 10; Figs. 1A, D). This current was strongly potentiated (173.68 ± 16.04 pA, n = 130; Figs. 1A, D) when the extracellular solution was free of divalent cations Ca 2 + and Mg 2 +. Although the responses had the same kinetics the EC50 changed from 4.2 mM to 153.4 μM in the presence or absence of Ca 2 + and Mg 2 +, respectively (Fig. 1B). The ATP analog BzATP (100 μM) also induced an inward current (110.8 ± 7.6 pA, n = 25) and the dose–response analysis indicated that BzATP was an order of magnitude more potent than ATP, a specific feature of the P2X7 receptors (Moatassim and Dubyak, 1992). Its EC50 value in Ca 2 +- and Mg 2 +- free medium was 9.7 μM (Fig. 1B). BBG is a potent P2X7 antagonist and at 50 nM is selective for the P2X7 subtype (Jiang et al., 2000; Anderson and Nedergaard, 2006). BBG at 50 nM substantially reduced ATP (1 mM) responses (68.2 ± 8.3 pA, n = 40; Figs. 1A, C, and D). A further slight reduction of the responses to ATP (1 mM) was observed when BBG was applied at 5 μM (56.3 ± 7.8 pA, n = 40; Fig. 1D), which supports the idea that other BBG sensitive P2X receptors are involved in ATP responses in cortical neurons in culture, but only marginally (Jiang et al., 2000). A similar blockade of the ATP (1 mM) responses was observed with the more recently developed P2X7 antagonist A438079 (100 μM; 65.3 ± 5.38% reduction). To further characterize the functional properties of P2X7 receptors, we next monitored the concentration of intracellular Ca 2 + in cultured neurons after application of ATP. Application of ATP 1 mM caused a sustained increase in cytosolic Ca 2 + that was completely blocked by BBG (50 nM) (Fig. 1E). Together, this pharmacological profile of responses to ATP indicates that P2X7 receptors are the major P2X receptor in cortical neurons, and that other P2X receptors contribute only marginally to electrophysiologic and Ca 2 + responses to ATP in these cells.

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P2X7 antagonist prevents neurons from death after OGD To examine the role of P2X receptors in cell death after ischemia, we mimicked ischemic conditions by depriving neurons of oxygen and glucose in the presence of various concentrations of the glycolytic inhibitor IAA. Under these conditions, neuronal death was dependent on IAA concentration (20 to 100 μM) (Fig. 2). The effects of ischemia were greatly reduced by BBG at 50 nM and 5 μM when the ischemic conditions were less severe (IAA at 20 or 50 μM), and the protective effect of BBG was comparable to that of the NMDA blocker AP5 (50 μM; Fig. 2). However, and in contrast to AP5, BBG was not effective in attenuating ischemic neuronal death at more stringent conditions (IAA at 100 μM; Fig. 2). As the protective effect on neuronal death observed with lower and higher concentrations of BBG were similar, these findings indicate that P2X7 receptors mediate ischemic damage to neurons. OGD induces an inward current in neuron cultures which is modulated by BBG We next studied whether P2X7 receptor activation contributes to post-anoxic depolarization after the onset of ischemia OGD. Ischemic conditions were simulated by oxygen and glucose deprivation plus the addition of IAA 1 mM and antimycin 0.25 μM to the cultures. These ischemic conditions activated an inward current in neurons within 5.65 ± 0.5 min (Fig. 3A). Ischemia-induced currents had a purinergic receptor-mediated component as their onset was delayed to 11 ± 1.5 min in the presence of BBG (Figs. 3A and B) and their slope was significantly lower (m = −0.0085 ± 0.0018 in control ischemia versus m = − 0.0046 ± 0.0009 in ischemia during exposure to BBG). We then tested the effect of BBG after the onset of post-anoxic current. To that end, we measured the current slopes before and after applying BBG; results were m = − 0.0085 ± 0.0022 and m = 0.0037 ± 0.0017, respectively (Figs. 3C and D). This change in the current slope from negative to positive indicates that P2X7 receptors opened during ischemia are blocked by BBG. In addition, we tested the pannexin 1 hemichannel blocker probenecid (1 mM) and found that its application after the onset of the post-anoxic current had a similar effect than BBG (slope m = 0.0029 ± 0,001085, p b 0.01 as compared to ischemia control). These findings suggest that during ischemia, P2X7 receptors are activated and that their activation contributes to post-anoxic depolarization in neurons, and that the pannexin 1 hemichannel opening also makes a substantial contribution. Blockade of P2X7 receptors suppresses the post-anoxic ischemic current in neurons in brain slices We next examined if activation of P2X7 receptors during anoxic depolarization also occurs in neurons in brain slices following acute ischemia, a more integral preparation than neuronal cultures. As in cultures of cortical neurons, ischemia activated an inward current that in slices showed a latency of 18.1 ± 2.3 min (n = 14; Fig. 4A). Addition of BBG (50 nM) to the perfusion solution after the onset of the post-anoxic depolarization effectively reversed the inward current (Figs. 4A, B). Thus, the slope of the current changed from − 0.035 ± 0.013 to 0.013 ± 0.051 (Figs. 4A, B). Basal membrane current also recovered substantially (61% ± 9.5). However, the efficacy of BBG decayed with the progression of the ischemic insult (Figs. 4A, B). Blockade of P2X7 receptor reduces brain damage after MCAO The findings above indicate that activation of P2X7 receptors contributes to the onset of the post-anoxic depolarization current leading to neuronal demise in neuronal cultures and in acute brain slices, and that blockage of P2X7 receptors attenuates the post-ischemic current

Please cite this article as: Arbeloa, J., et al., P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia, Neurobiol. Dis. (2012), doi:10.1016/j.nbd.2011.12.014

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Time (min) Fig. 1. Neurons express functional P2X7 receptors. (A) Cortical neurons respond to ATP (1 mM) with an inward non-desensitizing current that is strongly potentiated in the absence of Ca2 + and Mg2 +. ATP currents are reduced in the presence of P2X7 antagonist BBG (50 nM). (B) P2X receptor agonists dose–response curves show that Ca2 + and Mg2 + reduce ATP and BzATP responses (n = 8). (C) Dose–response curve of the response to ATP in the absence and presence of the P2X7 antagonist BBG (50 nM) (n = 6). (D) Histogram showing the reduction in ATP response by BBG (50 nM and 5 μM) (n = 10, 130, 46 and 24 respectively). (E) ATP induces an increase in [Ca2 +]i which is prevented in the presence of BBG (50 nM) (n = 90 cells). Arrow shows the beginning of drugs' application.

and subsequent neuronal damage. To evaluate the relevance of these observations to stroke, we explored the effects of the P2X7 receptor antagonist BBG in transient MCAO in rats. The extent of brain damage after transient MCA occlusion was greatly reduced, as assessed using TTC, in rats treated with BBG (30 mg/kg twice per day; n = 7; Fig. 5A). Thus, the extent of the damaged brain area calculated from the tissue stained with TTC revealed that it was reduced as compared with control, vehicle-treated, and rats after 3 days of reperfusion. In control animals, the volume of the damaged area was 222.1 ± 18.1 mm³ 3 days after 90 min MCAO,

while in the animals treated with BBG it was reduced to 108.7 ± 32.8 mm³ (51.1% reduction; Fig. 5B). The neurological score at 1 h after initiation of reperfusion was similar in both BBG and vehicletreated rats (Fig. 5C). Notably, after 3 days of reperfusion the symptoms were greatly ameliorated in rats treated with BBG (Fig. 5C). To quantify the number of degenerated neurons, cryostat sections of the area with the most extended infarct lesion (slice 3 of TTC in Fig. 5A) were stained with Fluoro Jade C and immunohistochemically for NeuN (Fig. 5D; black area in inset). Fluoro Jade C staining of dying cells showed a large number of stained cells in the cerebral cortex of

Please cite this article as: Arbeloa, J., et al., P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia, Neurobiol. Dis. (2012), doi:10.1016/j.nbd.2011.12.014

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Discussion

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IAA 100 µM Fig. 2. P2X7 receptor blockage prevents neuronal death after ischemia. Ischemia was induced for 1 h by OGD with the addition of the glycolytic blocker IAA at increasing concentrations (20, 50, and 100 μM). Neuron viability was measured using the calcein assay after 24 h of ischemia. BBG (50 nM) is protective in milder ischemic conditions (A and B), whereas it is ineffective at IAA 100 μM (C). NMDA receptor antagonist AP5 (50 μM) was used as a positive control, and was protective in all ischemic conditions assayed. *p b 0.05, **p b 0.01, ***p b 0.001 (n = 20).

the ischemic core as compared with the contralateral side in vehicletreated animals at 3 days after ischemia (567.1 ± 35.0 cells/mm 2; Fig. 5D, left column). In contrast, rats subjected to stroke and subsequently treated with BBG displayed a lower number of positive stained cells (362.8 ± 31.4 cells/mm 2) following 3 days of recirculation (Fig. 5D, left column). Conversely, immunostaining for the neuronal marker NeuN displayed the morphology and localization of some surviving neurons in the territory affected by the occluded artery (Fig. 5D, right column). Comparison of injured cerebral cortex with the contralateral side in vehicle-treated animals showed a marked reduction in positive cells (150.6 ± 18.5 vs. 584 ± 19.2 cells/ mm 2). This reduction was much smaller in the lesional side of BBGtreated rats where the number of NeuN + cells rose to 303.5 ± 31.4 cells/mm 2. Together, these data indicate that blocking purinergic receptors with BBG after the onset of transient focal ischemia in rats ameliorates symptoms and reduces tissue damage.

Here we showed that cortical neurons in dissociated cultures and in slices express P2X7 receptors that are activated during post-anoxic depolarization subsequent to conditions mimicking ischemia. In addition, we provide evidence that blockade of P2X7 receptors attenuates post-ischemic neuronal death and tissue damage. The presence of P2X7 receptors in cortical neurons has been already documented in previous studies using techniques which include mRNA analysis (Yu et al., 2008), as well as calcium imaging, western blot and immunohistochemistry (Díaz-Hernández et al., 2009). However, no electrophysiological responses to Bz-ATP were observed in cultures of cortical neurons (Wirkner et al., 2005). This apparent discrepancy with the findings of the current report may be due to the different procedures employed in that study which include a higher density of plated cells, the use of an alternative attachment substrate and longer time of culture in serum-containing medium. ATP may cause CNS neurodegenerative events, since it is released from virtually all neural cells during pathophysiological responses to mechanical stress, hypoxia, inflammation, and traumatic injury (Amadio et al., 2002; Burnstock, 2006). However, the mechanisms of ATP release relevant to the development of neuronal damage are not yet understood. ATP can be released by exocytosis in a Ca 2 +-dependent manner from synaptic vesicles (Pankratov et al., 2007), and through hemichannels formed by connexins and pannexins (Bao et al., 2004; Kang et al., 2008; Domercq et al., 2010). In addition, ATP can also be released by mechanisms involving other ion channels such as the P2X7 receptor itself (Dale and Frenguelli, 2009; Pellegatti et al., 2005). ATP may act as an excitotoxin in certain pathophysiological conditions when released into the extracellular space by high frequency neuronal stimulation (Wieraszko et al., 1989), ischemia (Braun et al., 1998; Lutz and Kabler, 1997), and mechanical stress resulting in tissue injury (Petruzzi et al., 1994). Moreover, application of exogenous ATP in vitro can be toxic to primary neuronal cultures and cause necrosis and apoptosis (Amadio et al., 2002). On the other hand, pathological conditions can alter the sensitivity of ATP receptors to ATP. Thus, P2X7 receptor density is upregulated in neurons and other cells following ischemia (Cavaliere et al., 2002, 2004; Franke et al., 2004; Milius et al., 2008). Likewise, sustained application of ATP and agonists activating P2X7 receptors switches P2X7 receptors action from a “typical” ion channel selective for small cations including Ca2 + to a mode consistent with a large pore that allows passage of molecules up to 700 Da (Surprenant et al., 1996; North, 2002). This results in increased membrane permeability that promotes actin disaggregation and rapid cytoskeletal rearrangements such as membrane blebbing (Pubill et al., 2001), as well as cell lysis (Kim et al., 2001), cytokine release, and apoptosis (Surprenant et al., 1996). Expression of the P2X7 receptor in the CNS has not been fully characterized. In the current study, we used a pharmacological fingerprint of the P2X7 receptor to prove its expression in primary cortical neuron culture by the voltage clamp technique and Ca 2 + imaging. We observed that neurons respond much more potently to BzATP than to ATP and that divalent cations diminished the ATP response amplitude, features which are characteristic of P2X7 receptors (Khakh and North, 2006). In addition, low concentrations of BBG greatly attenuated ATP responses, further suggesting they were mediated in part by P2X7 receptors, as shown in earlier studies (Anderson and Nedergaard, 2006). In turn, ATP dose–response curves in the presence or absence of BBG provide an estimate of the substantial contribution of P2X7 receptor to the overall response to ATP. During cerebral ischemia intracellular levels of ATP fall while extracellular ATP is elevated as a consequence of secondary anoxic depolarization (Juranyi et al., 1999; Melani et al., 2005; Frenguelli et al., 2007). We hypothesized that the rise in ATP concentration during ischemia might be sufficient to activate P2X7 receptors and kill

Please cite this article as: Arbeloa, J., et al., P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia, Neurobiol. Dis. (2012), doi:10.1016/j.nbd.2011.12.014

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Fig. 3. P2X7 receptor activation contributes to post-anoxic depolarization induced by ischemia in cultured neurons. Ischemia was induced by OGD and the application of IAA (1 mM) and antimycin (0.25 μM). (A) Representative traces of post-anoxic depolarization after OGD in the absence and presence of BBG. OGD causes an inward current that is delayed by BBG (50 nM) applied at the onset of ischemia. (B) Histogram illustrating the time of onset of the post-anoxic current after inducing ischemia and its delay when applying BBG at the time of ischemia initiation. ***p b 0.001. (C) Representative trace of post-anoxic depolarization after OGD and the transient reversing effect during application of BBG. (D) Histogram illustrating the slope of post-anoxic current before and after application of BBG (50 nM). ***p b 0.001 (ischemia n = 20, ischemia + BBG n = 18).

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Fig. 4. P2X7 receptor activation contributes to post-anoxic depolarization induced by ischemia in acute brain slices. (A) Blockage of P2X7 receptors with BBG (50 nM) reverts the post-anoxic depolarization soon after its onset, but it is less effective at later stages. (B) Histogram illustrating the slope of post-anoxic current before and after application of BBG (50 nM). The negative slope of the current is reverted into positive values after applying BBG, and again turns into negative values when BBG is removed. **p b 0.05.

neurons. Indeed, we found that blockage of P2X7 receptors was protective under mild to moderate ischemic conditions. Consistent with previous reports, we also found that AP5, an antagonist of ionotropic NMDA glutamate receptors, was neuroprotective even under severe ischemia. These findings indicate that activation of P2X7 receptors contribute to post-anoxic depolarization and neuronal demise following ischemia, and that their contribution to both parameters parallels that observed for NMDA receptors. There are several features that render P2X7 receptors relevant to neuropathology. First, they have an exceptionally high Ca 2 + permeability, which is comparable to the Ca 2 + permeability of N-methylD-aspartate (NMDA) receptors (Abbracchio et al., 2009). Secondly, P2X7 receptors do not desensitize and, after prolonged activation, they form a pore that causes cytolytic cell death (Surprenant et al., 1996). Neuronal excitotoxicity during stroke is caused by activation of large conductance channels, leading to swelling and Ca 2 + dysregulation. Simulated ischemic conditions result in the opening of connexin (pannexin) hemichannels that contribute to post-anoxic depolarization (Thompson et al., 2006), a finding which was also observed in the current study. In addition, our electrophysiological data indicate that blockage of P2X7 receptors reduces the post-anoxic current, indicating that P2X7 receptors are activated by ATP released immediately after anoxic depolarization, as suggested in earlier studies (Frenguelli et al., 2007). In addition, we showed that activation of P2X7 receptors contribute to the profound ionic dysregulation during ischemic neuronal death. This feature is further supported by the fact that P2X7 receptor blockage was also effective in reducing brain tissue damage and attenuating neurological symptoms following transient MCAO. Activation of P2X7 receptors has also been observed in other experimental paradigms relevant to CNS diseases. Thus, ATP is released from neurons in sufficient quantities to activate P2X7 receptors upon metabolic or hypotonic stress (Huang et al., 2007; Locovei et al., 2006; Reigada et al., 2008), trauma (Wang et al., 2004), and ischemia (Cavaliere et al., 2004; Melani et al., 2006). In addition to neurons, oligodendrocytes can also undergo ATP excitotoxicity by overactivation

Please cite this article as: Arbeloa, J., et al., P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia, Neurobiol. Dis. (2012), doi:10.1016/j.nbd.2011.12.014

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reperfusion Fig. 5. Treatment with BBG reduces brain damage after transient MCA occlusion. (A) Representative TTC stained sections of vehicle and BBG treated animals (30 mg/kg/day, i.p.) 3 days after transient focal ischemia. Note a reduction of infarct area in the brain slices of BBG treated animals. (B) Histogram showing the infarct volume calculated from TTC stained slices in vehicle- and BBG-treated rats (n = 8 and 7 in each group). Note the reduction of volume after BBG treatment. (C) The neurological score was significantly decreased in the BBG treated animals 3 days after stroke. (D) Representative microphotographs of infarct core in the cerebral cortex stained with Fluoro Jade C (left column) and immunostained for NeuN (right column). Diagrams below the microphotograph show cell quantification. Drawing in the superior corner of the Fluoro Jade C image shows the area where stained cells were counted. Bar = 50 μm. **p b 0.01, ***p b 0.001 (n = 14).

of P2X7 receptors following experimental autoimmune encephalomyelitis, a model of multiple sclerosis (Matute et al., 2007), and in white matter ischemia (Domercq et al., 2010). In the latter, oligodendrocytes subjected to ischemia release ATP through pannexin hemichannels, leading to P2X7 receptor activation, myelin damage, and axon dysfunction after white matter ischemia (Domercq et al., 2010). After an ischemic episode, the penumbra area is damaged but cells within this area can potentially be saved; consequently, the functional loss associated with stroke can be limited (Lo, 2008). Ischemia-

induced CNS cell death is partially related to glutamate excitotoxicity, and NMDA receptors are considered the main target responsible for Ca 2 + overload in the ischemic brain. However, clinical trials with NMDA receptor antagonists to limit stroke damage failed because neuroprotective doses of antagonists have serious side effects due to the blocking of normal synaptic activity. To overcome this, alternative therapeutic strategies are based on the principle that drugs interact with their target only during states of pathological activation but do not interfere with normal function (Lipton, 2007).

Please cite this article as: Arbeloa, J., et al., P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia, Neurobiol. Dis. (2012), doi:10.1016/j.nbd.2011.12.014

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Please cite this article as: Arbeloa, J., et al., P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia, Neurobiol. Dis. (2012), doi:10.1016/j.nbd.2011.12.014