Expression level of P2X7 receptor is a determinant of ATP-induced death of mouse cultured neurons

Neuroscience 319 (2016) 35–45

EXPRESSION LEVEL OF P2X7 RECEPTOR IS A DETERMINANT OF ATP-INDUCED DEATH OF MOUSE CULTURED NEURONS A. OHISHI, Y. KENO, A. MARUMIYA, Y. SUDO, Y. UDA, K. MATSUDA, Y. MORITA, T. FURUTA, K. NISHIDA AND K. NAGASAWA *

cells. Of P2Rs, P2X7Rs have unique characteristics and are activated by high concentrations (ca. mM) of ATP under pathophysiological conditions such as ischemia, resulting in the formation of a non-selective cationic channel/pore, through which large molecules of up to 900 Da can pass (North, 2002; Skaper et al., 2009). There is controversy on functional expression of P2X7R in brain neuronal cells, especially neurons and astrocytes (Pedata et al., 2015 and references therein). However, recent findings have demonstrated that functional expression of P2X7Rs is found in neurons and astrocytes not only in vitro, but also in situ and in vivo conditions (Arbeloa et al., 2012; Kamatsuka et al., 2014; Hirayama et al., 2015). Among neuronal cells, P2X7Rs are known to exhibit different functionality (Duan and Neary, 2004; Franke and Illes, 2006). Exposure of neurons to high concentrations of ATP activates P2X7Rs, resulting in their death (Nishida et al., 2012). In microglia, a typical immune cell in the brain, activation of P2X7Rs leads to generation of reactive oxygen species (ROS) and pro-inflammatory cytokines, and contributes to exacerbation of brain injury (North, 2002; Sim et al., 2004; Anderson and Nedergaard, 2006; Duan and Neary, 2004; Jabs et al., 2007; Surprenant and North, 2009). In addition, microglial P2X7Rs play a role in the regulation of their migration (Higashi et al., 2011). On the other hand, P2X7Rs expressed by astrocytes are constitutively activated by ATP released by themselves in an autocrine/paracrine manner under exogenous ligand-free resting conditions (Nagasawa et al., 2009; Kamatsuka et al., 2014). As one of the roles of their constitutive activation in astrocytes, we recently revealed that P2X7Rs regulate their engulfing activity (Yamamoto et al., 2013). In two strains of mice, which have the identical nucleotide sequence of cDNA of P2X7R, P2X7R channel/pore activity was greater in the astrocytes obtained from SJL-strain mice, their engulfing activity also being greater, than in astrocytes from ddYstrain mice (Kido et al., 2014). Furthermore, we demonstrated that the difference in channel/pore activity of P2X7Rs between the two mouse strain-derived astrocytes was due, at least in part, to the different expression profiles of their splice variants, but not the total cellular expression level of P2X7Rs (Kido et al., 2014). Recent accumulating evidence indicates that functional alteration of P2X7Rs induced by SNPs is associated with mood disorders such as bipolar disorders, major depressive disorders, etc. (Bartlett et al., 2014; Caseley et al., 2014; Sperlagh and Illes, 2014). In addition, Cao et al. demonstrated that a

Department of Environmental Biochemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan

Abstract—Activation of P2X7 receptor (P2X7R), a purinergic receptor, expressed by neurons is well-known to induce their death, but whether or not their sensitivity to ATP depends on its expression levels remains unclear. Here, we examined the effect of the expression level of P2X7Rs on cell viability using pure neuron cultures, co-cultures with astrocytes derived from SJL- and ddY-strain mice, and mouse P2X7Rexpressing HEK293T cell systems. Treatment of pure neuron cultures with 5 mM ATP for 2 h, followed by 3-h incubation in fresh medium, resulted in death of both types of neurons, and their death was prevented by administration of P2X7Rspecific antagonists. In both SJL- and ddY-neurons, ATP-induced neuronal death was inhibited by a mitochondrial permeability transition pore inhibitor cyclosporine A, mitochondrial dysfunction being involved in their death. The ATP-induced neuronal death was greater for SJL-neurons than for ddY-ones, this being correlated with the expression level of P2X7R in them, and the same results were obtained for the HEK293T cell systems. Co-culture of neurons with astrocytes increased the ATP-induced neuronal death compared to the case of pure neuron cultures. Overall, we reveal that neuronal vulnerability to ATP depends on the expression level of P2X7R, and co-existence of astrocytes exacerbates ATP-induced neuronal death. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: ATP, P2X7 receptor, neuron, astrocyte, cell death.

INTRODUCTION ATP plays critical roles in the neuron-glia network as a neuro- and glio-transmitter in the brain (Pankratov et al., 2006; Higashi et al., 2011; Segawa et al., 2014; Rodrigues et al., 2015), and P2 receptors (P2Rs) are involved in ATP-mediated signaling in the brain neuronal *Corresponding author. Tel: +81-75-595-4648; fax: +81-75-5954756. E-mail address: [email protected] (K. Nagasawa). Abbreviations: DIV, days in vitro; GAPDH, glyceraldehyde-3phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; mPTP, mitochondrial permeability transition pore; PARP, poly(ADP-ribose)polymerase. http://dx.doi.org/10.1016/j.neuroscience.2016.01.048 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 35

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A. Ohishi et al. / Neuroscience 319 (2016) 35–45

decrease in ATP release from astrocytes induced depressive-like behavior in mice (Cao et al., 2013). Together, it is suggested that functional alteration of ATP/P2X7R-mediated signaling in the neuron-glia network might be a determinant of the development and/or clinical outcome of mood disorders. Neurons play a central role in brain neuronal activity, and their dysfunction leads not only to cognitive impairment but also mental disorders (Moylan et al., 2014; Sperlagh and Illes, 2014). Since ATP is a neurotransmitter, fine-tuned regulation of its signaling is critical for neuronal function. Nevertheless, to our knowledge, it is unknown whether different expression levels of P2X7Rs in neurons result in difference in the level of ATP-induced neuronal death, and whether the coexistence of astrocytes with different P2X7R activity has a different effect on the neuronal response to ATP. Here, we examined the ATP-sensitivity of neurons in pure cultures and co-cultures with astrocytes obtained from SJL- and ddY-strain mice, and mechanism underlying the difference.

EXPERIMENTAL PROCEDURES Reagents The chemicals and reagents for experiments were purchased from Wako Pure Chemical Ind. (Osaka, Japan) except where otherwise noted.

of comparison of ATP sensitivity of neurons between pure cultures and neuron-astrocyte co-cultures, for which pure neuron cultures were used at 6 DIV. With this procedure, the cultured cells comprised 96% or more neurons, with a negligible amount of glial cells, as judged on immunocytochemistry for microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) (Table 1). In the case of neuron-astrocyte co-cultures, we seeded neurons obtained as above on primary cultured astrocytes. The astrocyte cultures were prepared following the previous reports, and the purity of the cultures of astrocytes was 97% or more, as reported previously (Nagasawa et al., 2009; Kido et al., 2014; Segawa et al., 2015). The neuron suspension in B-27Neurobasal medium after filtration through the mesh was seeded on the cultured astrocytes at 20 or more DIV at the density of 0.3
106 cells/well of 24-well plates. A day after the seeding, the cultured cells were treated with 20 lM cytosine arabinoside to kill the contaminating microglia for 2 days, followed by replacement with fresh culture medium, and then they were cultured at 37 °C under a humidified atmosphere of 5% CO2 in air. A half volume of the culture medium was changed at 3 or 4 DIV. When neurons were co-cultured with astrocytes, they grew very well and reached confluence by 6 or 7 DIV, and then their viability gradually decreased, and thus we used them at 6 or 7 DIV. The cultures of neurons and astrocytes obtained from SJL- and ddY-strain mice were defined as SJL-neurons and SJL-astrocytes, and ddYneurons and ddY-astrocytes, respectively.

Neuron cultures Primary cultured cortical neurons as a pure neuron culture were prepared by the method reported previously with slight modification (Nagasawa et al., 2004; Nishida et al., 2012). All experiments were approved by the Experimental Animal Research Committee of Kyoto Pharmaceutical University and were performed according to the Guidelines for Animal Experimentation of Kyoto Pharmaceutical University. Briefly, E14 embryos of SJL (Japan Charles River, Kanagawa, Japan)- and ddY (Japan SLC, Hamamatsu, Japan)-strain mice were decapitated, and their brains were rapidly removed and placed in petri dishes halffilled with ice-cold HBSS (137 mM NaCl, 5.4 mM KCl, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 5.6 mM glucose, and 2.5 mM HEPES). The dissected cortices were treated with 0.1% trypsin for 5–10 min, followed by centrifugation and then mechanically dissociation with a pipette in 10% fetal bovine serum (FBS) (Biowest, Miami, FL, USA)containing Eagle’s MEM (Nissui, Tokyo, Japan). The cell suspension was filtered through a mesh with a pore size 150 lm, followed by resuspension in an appropriate volume of B-27 SupplementÒ (Invitrogen, Carlsbad, CA, USA)-supplemented NeurobasalÒ medium (GIBCO). The cells obtained were seeded into polyethyleneiminecoated 24-well-plates (Corning, Oneonta, NY, USA) at the density of 0.6
106 cells/well, and then cultured at 37 °C under a humidified atmosphere of 5% CO2 in air. A half volume of the culture medium was changed every 3–4 days, and the cells were used for experiments after culturing in vitro for 11–13 days (DIV), except in the case

Mouse P2X7R-expressing HEK293T cells HEK293T cells were grown in 10% FBS- and 2 mM glutamine-containing Dulbecco’s modified Eagle medium until 70–80% sub-confluence was reached. As described previously (Kido et al., 2014), the plasmid containing the mouse P2X7R (mP2X7R) gene was diluted to 1333 ng/mL with OPTI-MEM medium in the presence of Lipofectamine 2000 (Invitrogen). After standing at room temperature for 20 min, the DNA-liposome complex mixture was added to HEK293T cells. The transfected amount of DNA of the plasmid for mP2X7R was 1600 ng/well of 24-well plates. After 48-h transfection, the cells were re-plated onto 10-cm culture dishes (Corning) and then cultured in fresh culture medium containing Table 1. Purity of neurons in the culture systems % of total cell number SJL-mouse neuron culture

ddY-mouse neuron culture

MAP2-positive cells GFAP-positive cells

96.1 ± 1.5

MAP2-positive cells GFAP-positive cells

97.2 ± 0.7

3.9 ± 1.5

3.3 ± 1.3

The purity of neurons and astrocytes in the culture systems (11–13 DIV) were calculated based on the immunocytochemical detection of MAP2- and GFAPpositive cells, respectively, as to total cell number. Each value represents the mean ± SD (N = 3).

A. Ohishi et al. / Neuroscience 319 (2016) 35–45

0.4 mg/mL of zeocin (Invitrogen). Some colonies obtained were picked up and then cultured in the culture medium containing 0.4 mg/mL zeocin. By repeating this selection step, we finally obtained three colonies (HEK293T/ mP2X7R#1, #5 and #6), which were maintained in the medium containing 0.2 mg/mL zeocin. Western blotting Protein homogenates were obtained from cell cultures, subjected to SDS–polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes, as described previously (Nagasawa et al., 2009; Kido et al., 2014). After blocking, each membrane was incubated overnight at 4 °C with a rabbit anti-P2X7R antibody (1:1000, #APR-004, of which the epitope location is the C-terminus; Alomone Labs, Jerusalem, Israel), a mouse anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (1:5000, #sc-32233; Santa Cruz, CA), or a mouse anti-b-actin antibody (1:1000, #01324553; Wako). After several washes, each membrane was incubated with an anti-rabbit or anti-mouse IgG HRP-linked antibody at 1:10,000 dilution for 1 h at room temperature. The membrane was then washed and the signal was detected with ECL reagent (Perkin Elmer, Boston, MA). As described previously (Nagasawa et al., 2009), the optical density of each protein band was quantified with the Image J software (ver. 1.48; NIH, Bethesda, MD) and normalized as to the corresponding GAPDH or b-actin one. Immunocytochemistry As described previously (Nagasawa et al., 2009; Kido et al., 2014), immunostaining was performed for cells grown on glass cover slips and fixed by 20- or more min incubation in ice-cold 4% paraformaldehyde, followed by 3-min sequential dehydration steps in 70%, 90% and 100% ethanol. Cells were incubated for 1 h at 4 °C in a blocking buffer (0.1 M phosphate-buffered saline (PBS) containing 2% sheep or goat serum, 0.2% Triton X-100 and 0.1% bovine serum albumin), and then with primary antibodies diluted in the same blocking buffer overnight at 4 °C. The following primary antibodies were used: a mouse anti-MAP2 antibody (1:2000, #M4403, Sigma, St. Louis, MO, USA) or a rabbit anti-P2X7R antibody (1:200, #APR-004, Alomone Labs). After six rinses in PBS, cells were incubated with Alexa Fluor 488-conjugated anti-mouse antibodies or Alexa Fluor 546-conjugated donkey anti-rabbit antibodies (Invitrogen) at 1:1000 dilution for 1 h at room temperature, rinsed, mounted on a glass slide, and then photographed under a laser confocal microscope (LSM510 META; Carl Zeiss, Germany) or a fluorescence microscope (IX51; Olympus, Tokyo, Japan) equipped with a digital camera (coolSNAP; Nippon Roper, Tokyo, Japan). The nuclei were counterstained with Hoechst33258 (2 lg/mL).

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according to the manufacturers’ instruction manuals. As reported previously (Kido et al., 2014), real-time quantitative PCR was conducted with an ABI PRISM 7500 Real Time PCR System using SYBR Premix Ex Taq (Takara). PCR amplification was performed using the gene-specific primer sets shown in Tables 2 and 3. All reactions were carried out with the following cycling parameters: 94 °C for 5 min, and 30 cycles of 94 °C for 30 s, 60 °C for 60 s, and 72 °C for 15 s. Treatment conditions and assessment of neuronal death Following the previously reported protocol (Nishida et al., 2012), experiments were initiated by replacing the culture medium with a balanced salt solution (BSS). The BSS comprised (in mM): 3.1 KCl, 134 NaCl, 1.2 CaCl2, 1.2 MgSO4, 0.25 KH2PO4, and 15.7 NaHCO3, with 2 glucose, and the pH was adjusted to 7.2 while the solution was equilibrated with 5% CO2 at 37 °C. After pre-incubation of neurons in BSS with or without 5 mM ATP in the presence or absence of an inhibitor/antagonist for 2 h, followed by two washes with warmed BSS, they were incubated in fresh BSS for 3 h. Then, to assess neuronal death, the cells were subjected to immunocytochemistry for MAP2 with counterstaining of nuclei with Hoechst33258 as mentioned above. Using the photomicrographs, we judged a neuron exhibiting MAP2-immuno-reactivity, and an integrated cell body and dendrites as a live one, and cell viability was calculated as the percentage of live neurons among the total. To count neurons, we prepared 3 wells for each sample, and randomly selected 3 fields/ well, 40 or more neurons existing in each field. The same experimental procedure was repeated at least 3 times. In this study, we compared the neuronal viability between pure neuron cultures and neuron-astrocyte co-cultures, and thus we adopted this immunocytochemical method for quantification of neuronal death, this method being confirm to give almost the same results obtained by measuring LDH levels released from dead neurons in pure neuron culture system (data not shown). Intracellular Ca2+ level Following the previous report (Nishida et al., 2012), after neurons had been loaded with 2 lM fura-2/AM for 30 min, they were washed twice with BSS, followed by de-esterification in fresh BSS with or without 10 lM A438079. Thereafter, neurons were treated with 5 mM ATP. Sequential monitoring of the intracellular Ca2+ level was performed using a microplate reader (ARVOTM-MX; PerkinElmer, Wellesley, MA) at excitation wavelengths of 340 and 380 nm, and an emission wavelength of 510 nm, and the ratio (F340/F380) was taken as the relative index of the intracellular Ca2+ level. At the end of each experiment, neurons were exposed to 4 lM ionomycin to calibrate complete Ca2+ influx.

Reverse transcription (RT) and real-time quantitative PCR

Cell viability assay

Total RNA was extracted and reverse transcribed with a GenEluteTM Mammalian Total RNA kit (Sigma) and a PrimeScriptTM RT reagent kit (Takara, Shiga, Japan)

In the HEK293T cell system, cell viability was assessed by means of the MTT assay, as reported previously (Nagasawa et al., 2004).

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Table 2. Specific primer sets for RT-PCR amplification of P2Rs Primer sequences

Product size (bp)

P2X1R

Forward Reverse

50 -GGTCTACGTCATTGGGTGGGTGTTTGT-30 50 -CCTTGGGCTTTCCTTTCTGCTTTTCCT-30

306

P2X2R

Forward Reverse

50 -TGTGGGACGTGGAGGAATAC-30 50 -CTTGGAGAACTTGAACTTGG-30

362

P2X3R

Forward Reverse

50 -AGGGCACTTCTGTCTTTGTCATC-30 50 -CTCGCTGCCATTCTCCATCTTGT-30

626

P2X4R

Forward Reverse

50 -AGAGATTCCTGATAAGACCAGCATT-30 50 -GTCCCGGTAGTAGTATCTCTTCTTCA-30

750

P2X5R

Forward Reverse

50 -CCCGGATGGCGAGTGTTCAGAGG-30 50 -AAGATGGGGCAGTAGAGATTGGT-30

324

P2X7R

Forward Reverse

50 -ACACCGTGCTTACAGGTGCTATG-30 50 -GCAACAGCTGGGCAGAATG-30

82

P2Y1R

Forward Reverse

50 -TTTTAGTGTTCATCATAGGCTTCC-30 50 -TTTTGTTTTTCCGAGTCCCAGTGC-30

420

P2Y2R

Forward Reverse

50 -CCGTGTCCTATGGCGTGGTGT-30 50 -TAGGCTCCGTGGGTGGCTTGG-30

870

P2Y4R

Forward Reverse

50 -TGCCCACCCTCGTCTACTACTATG-30 50 -CGCCCACCTGCTGATGCTTTCTTC-30

785

P2Y6R

Forward Reverse

50 -CGGACCTGATGTATGCCTGTT-30 50 -GACTCTCTGCCTCTGCCACTT-30

773

P2Y12R

Forward Reverse

50 -CACCGTCCTGTTCTTTGCTGGGCTCAT-30 50 -CTTCTTGTCCTTTCTTCTTGTTTGTCC-30

899

P2Y13R

Forward Reverse

50 -ATTTCCGTCTGGTCCCTGATGTTCTTC-30 50 -CCGCTTGTGCCTGCTGTCCTTACTCCT-30

258

Table 3. Specific primer sets for real-time PCR amplification of P2X7R and its splice variants Primer sequences 0

Product size (bp) 0

82

P2X7R

Forward Reverse

5 -ACACCGTGCTTACAGGTGCTATG-3 50 -GCAACAGCTGGGCAGAATG-30

P2X7R-v2

Forward Reverse

50 -TCAAAGGCCAAGAAGTTCCAGTA-30 50 -TAGATCCGACCCCTTCCTTCTG-30

94

P2X7R-v3

Forward Reverse

50 -AAGTCTGCAAGTTGTCAAAGG-30 50 -TAGAGTCAGTCAAAGCATCTC-30

99

P2X7R-v4

Forward Reverse

50 -TTCCAACCTCCAGGAGAGTA-30 50 -AAGCCTTCTTCCTTCTTGGC-30

75

b-Actin

Forward Reverse

50 -AGGTCATCACTATTGGCAACGA-30 50 -CACTTCATGATGGAATTGAATGTAGTT-30

171

Statistical analysis The data are expressed as mean ± SD. Based on the hypothesis that the data population obtained here shows a normal distribution and the population variance is equal, as reported by Gouix et al. (2014), comparisons between two or more groups were performed by means of Student’s t-test or an analysis of variance (ANOVA, followed by Fisher’s PLSD), respectively, differences with a p-value of 0.05 or less being considered statistically significant.

RESULTS ATP-induced neuronal death First, we examined whether or not treatment of neurons in pure cultures at 11–13 DIV with ATP induced their death. As shown in Fig. 1a, the treatment decreased the number of MAP2-immuno-reactive neurons for both SJL- and ddY-neurons. On comparing neuronal viability between the two types of neurons, the viability was found to be significantly less in SJL-neurons than in ddY-neurons

A. Ohishi et al. / Neuroscience 319 (2016) 35–45

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Fig. 1. ATP induced P2X7R-mediated neuronal death in SJL- and ddY-neurons. After neurons had been incubated in BSS with or without 5 mM ATP in the presence or absence of 10 lM A438079 or KN-62 for 2 h in a CO2 incubator at 37 °C, followed by two washes with warmed BSS, they were incubated in fresh BSS for 3 h. Thereafter, the neurons were subjected to immunocytochemistry for MAP2 with counterstaining with Hoechst 33258 to determine their viability. Panel a shows representative photomicrographs of SJL- and ddY-neurons treated with or without ATP. Red signals indicate the MAP2-immunoreactivity. Bar = 100 lm. Panels b, c and d show the alteration of neuronal viability on treatment of neurons with ATP in the presence or absence of an antagonist for P2X7R. Each column represents the mean ± SD (N = 3). *p < 0.01 (vs. control group in SJLneurons). yp < 0.01 (vs. ATP group in SJL-neurons). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Fig. 1b). This decreased viability of both types of neurons was almost completely restored to the control level on administration of P2X7R-specific antagonists A438079 and KN-62 (Fig. 1c, d), suggesting the involvement of P2X7Rs in the ATP-induced neuronal death. Expression of P2Rs in neurons At the transcript levels, expression of P2XR isoforms 3, 4, 5 and 7, and P2YR isoforms 1, 2, 4, 6, 12, 13 and 14 in both SJL- and ddY-neurons was detected (Fig. 2a, b). By Western blot analysis for P2X7Rs (Fig. 2c), both SJL- and ddY-neurons expressed P2X7Rs, as revealed by the findings on immunocytochemical analysis (Fig. 2e), while the expression levels in the former were significantly greater than those in the latter (Fig. 2d), this being comparable with the results of quantitative PCR analysis shown in Fig. 2f. We detected expression of mRNAs for all of the P2X7R splice variants, and the expression levels of

P2X7R-v2, -v3 and -v4 were significantly lower than those of full-length P2X7R in the two types of neurons, but their levels in ddY-neurons were significantly lower than those in SJL-neurons (Fig. 2f). The relative expression of P2X7R-v2, -v3 and -v4 to that of fulllength P2X7R was calculated to be 8.6 ± 2.6%, 15.2 ± 2.1% and 43.9 ± 13.1% in SJL-neurons, and 6.6 ± 2.8%, 17.4 ± 4.2% and 48.6 ± 9.4% in ddY-neurons, respectively, there being no apparent difference in the relative expression levels. To confirm the functionality of P2X7Rs, we evaluated Ca2+-influx in ATP-treated neurons. In both types of neurons, as shown in Fig. 2g, the intracellular Ca2+ level was significantly increased by the ATP treatment, and it was almost completely suppressed to the control level on treatment with A438079. The ATP-induced increase in the intracellular Ca2+ level disappeared under the condition of chelation of the extracellular Ca2+ with EGTA (data not shown). Thus, P2X7Rs were suggested to be expressed by SJL- and ddY-neurons.

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Fig. 2. Expression of P2Rs in SJL- and ddY-neurons. Total RNA, which was reverse transcribed into cDNA, and protein were extracted from SJLand ddY-neuron cultures, and then subjected to RT-PCR or real-time PCR, and Western blotting or immunocytochemistry, respectively. Panels a and b show the expression profiles of mRNAs for P2XRs and P2YRs in SJL- and ddY-neurons, respectively. Protein expression of P2X7Rs is shown in panels c, d and e. In panel c, HEK293T/mP2X7R#1 is a positive control. Panel d shows the quantitative results for panel c. #1, #2 and #3 mean the different lots of cultures. Panel e shows the immunocytochemistry for P2X7Rs in SJL- and ddY-neurons. Quantitative analysis of the expression of mRNAs for P2X7R and its splice variants is shown in panel f. Panel g shows alteration of the intracellular Ca2+ level in SJL- and ddYneurons. Sequential monitoring of the intracellular Ca2+ level was performed with fura-2/AM, and the F340/F380 ratio was taken as the relative index of the intracellular Ca2+ level. Each column represents the mean ± SD (N = 3–4). Representative images of three independent experiments are shown in panels a, b and e. Bar = 20 lm. *p < 0.05 (vs. SJL-neurons). yp < 0.05 (vs. the corresponding mRNA expression in SJL-neurons).

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Fig. 3. Mitochondrial dysfunction and PARP activation are involved in ATP-induced neuronal death in SJL- and ddY-neurons. Neurons were preincubated in BSS with or without 50 or 200 nM CsA, 20 or 70 nM FK, 1 mM Apo, 50 lM AEBSF, 5 mM AB or 100 nM MC for 30 min, and then ATP was added to the cultures to the final concentration of 5 mM, followed by 2-h incubation in a CO2 incubator at 37 °C. After two washes, the neurons were incubated in fresh BSS for 3 h, and then subjected to immunocytochemistry for MAP2 with counterstaining with Hoechst 33258 to determine their viability. Each column represents the mean ± SD (N = 3–4). *p < 0.01 (vs. control group in SJL-neurons). yp < 0.01 (vs. ATP group in SJLneurons).

Involvement of mitochondrial dysfunction and poly (ADP-ribose)polymerase (PARP) activation, but not NADPH oxidase activation, in neuronal death We previously reported that ATP-induced death of rat neurons is mediated by mitochondrial dysfunction (Nishida et al., 2012). Thus, we examined its possible involvement in ATP-induced death of mouse neurons. As shown in Fig. 3a, b, a mitochondrial permeability transition pore (mPTP) inhibitor cyclosporine A (CsA), but not FK506 (FK) as a negative control, completely inhibited the neuronal death in both types of neurons, indicating mitochondrial dysfunction was involved in the ATP-induced neuronal death. Although representative NADPH oxidase assembly inhibitors apocynin (Apo) and 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF) had no effect on the decrease of neuronal viability (Fig. 3c), PARP inhibitors 3-aminobenzamide (AB) and minocycline (MC) clearly inhibited the ATP-induced neuronal death (Fig. 3d). Thus, it was suggested that ATP-induced neuronal death in both types of neurons was mediated by mitochondrial dysfunction, leading to PARP activation, but NADPH oxidase had no or only a negligible role in the death.

Effect of expression level of P2X7Rs on ATP-induced cell death As aforementioned, it is considered that the difference in the level of ATP-induced neuronal death between SJLand ddY-neurons might be due to their different expression levels of P2X7Rs. To clarify whether or not expression levels of P2X7Rs are a determinant of ATPinduced cell death, we used three types of P2X7Rexpressing cells, HEK293T/mP2X7R#1, #5 and #6, in which the expression levels of P2X7Rs are different, being in the order of #1  #6 > #5 (Fig. 4a). Treatment of these cells with ATP under the same experimental protocol as that for primary cultured neurons decreased the cell viability of HEK293T/mP2X7R#1 and #6, while there was no apparent decrease in the viability of HEK293T/mP2X7R#5 (Fig. 4b). Thus, it is suggested that the ATP-induced neuronal death corresponds to the expression levels of P2X7Rs. Effect of co-culture of neurons with astrocytes on ATP-induced neuronal death First, we examined whether or not ATP can kill young (6–7 DIV) neurons like aged (11–13 DIV) ones. Treatment

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our aforementioned idea that the ATP-induced decrease of cell viability depends on the expression levels of P2X7Rs. In the co-culture system of SJL- and ddY-neurons with the respective astrocytes, treatment with ATP significantly decreased the number of MAP2-immuno-reactive cells for both SJL- and ddY-neurons (Fig. 5d), and the neuronal viability of them in the co-culture system was 59.4 ± 12.3% and 75.4 ± 12.6%, respectively, these values being smaller than those in the pure neuron cultures (72.6 ± 11.7% and 97.2 ± 22.0%, respectively) (Fig. 5a). When SJL- and ddY neurons were co-cultured with ddY- and SJL-astrocytes, respectively, the neuronal viability also decreased with ATP treatment, and the levels were approximately equal (Fig. 5d). Thus, it is suggested that co-cultured astrocytes might increase ATP-induced neuronal death in the both types of neurons, but SJL-neurons cultured with SJL-astrocytes exhibited a much greater decrease in viability.

DISCUSSION

Fig. 4. ATP-induced cell death depends on the expression level of P2X7Rs in mP2X7R- stable cell lines. Panel a shows a representative Western blot for comparison of the P2X7R expression level among mock, and HEK293T/mP2X7R#1, #5 and #6 cells (N = 3). In panel b, after mock, and HEK293T/mP2X7R#1, #5 and #6 cells had been incubated in BSS with or without 5 mM ATP for 2 h in a CO2 incubator at 37 °C, followed by two washes with warmed BSS, they were incubated in fresh BSS for 3 h. Thereafter, the MTT assay was performed to evaluate cell viability. Each column represents the mean ± SD (N = 3–4). *p < 0.01 (vs. each control group).

of young SJL-, but not ddY-, neurons with 5 mM ATP caused their death (Fig. 5a), but the level of the decrease was less than for aged ones, as shown in Fig. 1b. Western blot analysis (Fig. 5b) showed that there was no difference in the expression levels of P2X7Rs between young SJL- and ddY-neurons. On comparison of P2X7R expression between young and aged neurons, the young SJL-neurons exhibited approximately a third less expression level of P2X7Rs than aged SJL-neurons did, and the same tendency was observed for ddY-neurons, but the difference was not significant (Fig. 5c). These results strongly support

In this study, we found that (1) treatment of cultured neurons with ATP induced their death via mitochondrial dysfunction and PARP activation, (2) the ATP-induced neuronal death depended on the expression level of P2X7Rs, and (3) co-culture of neurons with astrocytes increased the ATP-induced their death. Overall, it is suggested that functional expression of P2X7Rs in neurons is one of the critical factors that determine their vulnerability to ATP, and the co-existence of astrocytes exacerbated the ATP-induced neuronal injury. Astrocytic expression of P2X7Rs appears to be very low in normal brain (Sim et al., 2004; Anderson and Nedergaard, 2006; Jabs et al., 2007), but the expression is increased in response to brain injury such as ischemia (Franke et al., 2004; Lovatt et al., 2007; Hirayama et al., 2015). In this study, we reveal that the expression level of P2X7Rs is a critical factor to determine the level of ATP-induced neuronal death, and thus the findings obtained here strongly support the idea that antagonism of P2X7Rs is an effective approach to attenuate ischemia-induced neuronal death (Pedata et al., 2015). Recent accumulating evidence indicates that functional alteration of P2X7Rs is involved in psychiatric diseases such as mood disorders, especially depression (Caseley et al., 2014 and references therein). In addition, Cao et al. reported that the ATP level in the extracellular compartment of brain neuronal cells was decreased in a mouse depression model (Cao et al., 2013). Interestingly, although there were no differences in the expression levels of P2X7Rs between young SJL- and ddYneurons, in aged neurons, the expression levels were greater in the former than in the latter. This finding indicates that aging is critical for functional expression of P2X7R in cultured neurons as in the case of cultured astrocytes (Nagasawa et al., 2009). In the study by Seo et al., they used aged SJL male mice as a social target animal in a sociability test for assessment of depressive behavior in mice because they exhibited aggression characteristics (Seo et al., 2012). Thus, we suggest a

A. Ohishi et al. / Neuroscience 319 (2016) 35–45

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Fig. 5. Effect of co-culturing with astrocytes on neuronal viability in the ATP-treated neuron-astrocyte co-culture system. In panel a, after pure cultures (6 DIV) of SJL- and ddY-neurons had been incubated in BSS with or without 5 mM ATP for 2 h in a CO2 incubator at 37 °C, followed by two washes with warmed BSS, they were incubated in fresh BSS for 3 h. Thereafter, the neurons were subjected to immunocytochemistry for MAP2 with counterstaining with Hoechst 33258 to determine their viability. Comparison of expression of P2X7Rs between young SJL- and ddY-neurons, and between young (Y) and aged (A) neurons of SJL and ddY mice were given in panels b and c, respectively. #1, #2 and #3 mean the different lots of cultures. In panel d, SJL- and ddY-neurons were co-cultured with the respective astrocytes, or ddY- and SJL-ones, respectively, for 6–7 DIV. (In the panel, SJL-neurons and -astrocytes, and ddY-neurons and -astrocytes are indicated as SJL-N and SJL-A, and ddY-N and ddY-A, respectively.) After neuron-astrocyte co-cultures had been incubated in BSS with or without 5 mM ATP for 2 h in a CO2 incubator at 37 °C, followed by two washes with warmed BSS, they were incubated in fresh BSS for 3 h. Thereafter, the neurons were subjected to immunocytochemistry for MAP2 with counterstaining with Hoechst 33258 to determine their viability. Each column represents the mean ± SD (N = 3–4). *p < 0.01 (vs. each control group or respective young neurons). yp < 0.01 (vs. ATP group in SJL-neurons/SJL-astrocytes).

possibility that the aging-dependent increase in functional expression of P2X7R in SJL-neurons and -astrocytes might result in alteration of neuron-astrocyte communication, and this might explain expression of psychiatric phenotypes such as aggression characteristics. Differing from the case of astrocytes (Kido et al., 2014), the difference in functional expression of P2X7Rs between aged SJL- and ddY-neurons could not be explained by the expression profiles of their splice variants, and thus further detailed investigations are needed to clarify the underlying mechanism.

Even though there were no differences in the expression levels of P2X7Rs between young SJL- and ddY-neurons in pure cultures, ATP-induced neuronal death was observed only in the former (Fig. 5a, b). We think that this might be explained by undetectable differences in the lower expression levels of P2X7Rs between young SJL- and ddY-neurons, because the detectable, but low, expression of it did not cause an apparent ATP-induced decrease of cell viability in the HEK293T cell system (Fig. 4a, b). In our previous study, although the total cellular expression levels of P2X7Rs

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A. Ohishi et al. / Neuroscience 319 (2016) 35–45

were almost the same in SJL- and ddY-astrocytes, the channel/pore activity of them was greater in the former than in the latter, and this was due, at least in part, to the different expression profiles of P2X7R splice variants (Kido et al., 2014). Thus, there is a possibility that the differences in cellular localization of P2X7Rs between young SJL- and ddY-neurons might result in different ATP sensitivity of young SJL- and ddY-neurons. The underlying mechanism will be demonstrated by us in the near future. When neurons were co-cultured with the respective astrocytes, the astrocytes increased the neuronal sensitivity to ATP (Fig. 5d). We previously demonstrated that astrocytic P2X7R channel/pore activity decreased on administration of ligands of P2X7R (Yamamoto et al., 2013). Astrocytic P2X7Rs can uptake metabolic products of signaling molecules such as adenosine (Okuda et al., 2010). It has been reported that adenosine is generated through ATP metabolism by ecto-enzymes in the brain (Cunha, 2001; Zimmermann et al., 2012). As P2Rs, during ischemia, adenosine P1 receptors (P1Rs) exert important roles, but there is the controversy on roles of them in ischemic insult, because of their complex interplay (Pedata et al., 2015). Astrocytes express ectonucleotidases, of which inhibition by an inhibitor ARL67156 increases the extracellular ATP level (Nagasawa et al., 2009). Based on these findings, it is suggested that in our neuron-astrocyte co-culture system, exogenously administered ATP inhibits astrocytic P2X7Rs, decreases their adenosine uptake, increases the extracellular adenosine level, induces dysregulation of adenosine signaling via P1Rs expressed by not only neurons but also astrocytes, and the neuronal death is deteriorated. On the other hand, we found that exacerbation of neuronal death was greater in the co-culture of SJL-neurons and -astrocytes than that of ddY-neurons and -astrocytes (Fig. 5d). Although we have no reasonable explanation why SJL-astrocytes did not increase the ATP-induced decrease of viability of co-cultured ddY-neurons, compared with the case of co-culturing ddY-neurons with ddY-astrocytes, there is the possibility that the neuronal death might be increased by a decreased release of neurotrophic factor(s) from cocultured astrocytes via spontaneously activated P2X7Rs. Detailed investigations are necessary to clarify the interaction between neurons and astrocytes. In the ATP-P2X7R signaling cascade related to neuronal death, Ca2+ influx via P2X7R channels induces mitochondrial dysfunction and extensive PARP activation, while NADPH oxidase might have no or only a negligible role (Fig. 3). This profile is common in both SJL- and ddY-neurons. The activation of microglia, brain immune cells, is reported to occur as follows: ATPinduced P2X7R activation activates NADPH oxidase, and generated ROS induces extensive activation of PARP-1 (Kauppinen et al., 2008; Higashi et al., 2011). The difference in the ATP-P2X7R signaling cascade between neurons and microglia might be due to their different roles in maintenance of brain homeostasis. In fact, the expression level of NADPH oxidase is suggested to be greater in phagocytic microglia than in non-phagocytic

neurons (Noh and Koh, 2000), and this is considered to be reasonable because microglia have to kill pathogens with ROS, while neurons use ROS as a signaling molecule. Thus, this might result in the difference in the role of NADPH oxidase in the ATP-P2X7R signaling cascade between neurons and microglia.

CONCLUSION We have demonstrated that treatment of neurons with a high concentration of ATP induces their death, the expression level of P2X7Rs is a critical determinant for their death, and the co-existence of astrocytes increases the vulnerability of neurons to ATP.

CONFLICTS OF INTEREST The authors declare that there are no conflicts of interest, financial or otherwise. Acknowledgement—A part of this study was financially supported by a Grant-in-Aid for Scientific Research (C) (24590128) from the Japan Society for the Promotion of Science (JSPS).

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(Accepted 20 January 2016) (Available online 23 January 2016)