P2X(7) nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons

Abstract

Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X(7) nucleotide receptor (P2X(7)R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation in rPCNs induced by the P2X(7)R agonist BzATP were inhibited by the P2X(7)R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X(7)R antisense oligonucleotide indicating a direct involvement of the P2X(7)R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X(7)R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate that extracellular nucleotides mediate neuronal apoptosis through activation of P2X(7)Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3.

Figure 1

Purity of rat primary cortical neurons (rPCNs) and P2X₇R expression. (A) Rat PCNs were cultured on coverslips in 6-well plates and neurons were labeled with mouse NeuN monoclonal antibody (Panel a) and cell nuclei with TO-PRO-3 (Panel b); merged picture shown in Panel c. (B) RNA was isolated from rPCNs maintained in culture for 7–10 days and RT-PCR was used to amplify mRNAs to P2X_1–7Rs, as described in the Materials and methods. The amplified PCR products were resolved by gel electrophoresis, and data shown are representative of results from three independent experiments. Results are shown of PCRs performed in the presence (+) or absence (−) of reverse transcriptase (RT). G3PDH primers were used to amplify G3PDH mRNA as a positive control. (C) P2X₇R protein expression was detected by Western blot analysis, as described in the Materials and methods. Human 1321N1 cells expressing the recombinant human P2X₇R or the empty expression vector pLXSN were used as positive (+) or negative (−) controls, respectively. Precision Plus Protein Standards are indicated as ‘75KD’ and ‘100KD.’

Figure 2

ATP- and BzATP-induced increases in [Ca²⁺]_i in rat PCNs. (A) Single cell calcium assays were performed on rPCNs that were cultured for 7–10 days on poly-d-lysine-coated coverslips. Fura-2 loaded cells were incubated in PSS with the indicated concentration of BzATP or ATP and the maximum increase in [Ca²⁺]_i was determined, as described in the Materials and methods. (B) The percentage of cells responding to BzATP or ATP is shown as the mean ± S.E.M. of results from three experiments.

Figure 3

ATP/BzATP-induced nuclear condensation mediated by the P2X₇R is dependent on caspase-3 activation. (A) Rat PCNs were cultured for 7–10 days, incubated in serum-free HGGMEM for 6 h and stimulated with BzATP (300 µM) or ATP (100 µM) for 16 h or with H₂O₂ (1 µM) for 2 h. When indicated, 500 mM oATP was added 2 h prior to addition of BzATP. Cells cultured in B27-AO Neurobasal medium (NB) or in serumfree HGGMEM (−) overnight were used as controls. Then, nuclear condensation was determined by DAPI staining and detected by fluorescence microscopy, as described in the Materials and methods. (B) Cells were treated as in (A) except that the data were expressed as a percentage of cells that exhibited DAPI stained nuclei. Data are the means ± S.E.M. of results from at least four experiments, where *P < 0.05, and ***P < 0.001 indicate significant differences from the serum-starved control (−), and where ^###P < 0.001 indicates a significant difference from BzATP treatment. (C) rPCNs were treated as in (A) except that 10 µM ZDEVD-FMK, a caspase-3 inhibitor, was added for 1 h prior to BzATP, when indicated.

Figure 4

BzATP stimulates DNA fragmentation in rat PCNs. Rat PCNs were incubated in serum-free HGGMEM for 6 h and then stimulated with BzATP (300 µM) for 16 h or H₂O₂ (1 mM) for 2 h. Cells cultured in serumfree HGGMEM (−) overnight were used as the control. Then, cells were lysed, total DNA was purified, the DNA concentration was determined, and 3 µg of purified DNA were loaded onto each lane of a 2% (w/v) agarose gel and electrophoresed, as described in the Materials and methods. DNAfragmentation was visualized after electrophoresis of DNA. Apoptotic U937 cells (+++; provided in the Apoptotic DNA-ladder kit) were used as a positive control and ‘M’ indicates the 100 bp DNA ladder marker.

Figure 5

BzATP or ATP induces DNA strand breakage in rat PCNs. Rat PCNs were cultured for 7–10 days, incubated in serum-free HGGMEM for 6 h, and stimulated with BzATP (300 µM) or ATP (100 µM) for 16 h or with H₂O₂ for 2 h. Cells cultured in serum-free HGGMEM (−) overnight were used as the control. Then, cells were fixed and DNA strand breakage was determined by TUNEL assay, as described in the Materials and methods. DNA strand breakage was observed as green fluorescence using fluorescence microscopy.

Figure 6

P2X₇Rs mediate caspase-3 activation in rat PCNs. (A) Rat PCNs were incubated in serum-free HGGMEM for 6 h and then the indicated concentration of ATP or BzATP was added for 16 h or the time indicated. Cells cultured in serum-free HGGMEM (−) overnight were used as controls. (B) Cells were treated as in (A) except that 500 µM oATP was added for 2 h or the indicated concentration (1, 10 or 100 µM) of Z-DEVD-FMK was added for 1 h prior to addition of 300 µM BzATP for 16 h. (C) Cells were treated as in (A) except that P2X₇ antisense or sense oligonucleotide was added at the indicated concentration in mg/ml for 8 h prior to addition of 300 µM BzATP for 16 h or 1 mM H₂O₂ for 2 h. Western analysis was performed as described in Materials and methods to determine the relative amounts of full length pan caspase-3, cleaved caspase-3 or P2X₇R protein. Data are the means ± S.E.M. of results from at least three experiments, where *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences from BzATP-treated (B, C) cells. (D) Rat PCNs were serum-starved overnight in the presence (Panels B, D and F ) or absence (Panels A, C, and E) of 300 mM BzATP and then immunofluorescence of the neuron-specific marker NeuN (green; Panels A, B), cleaved caspase-3 (red; Panels C and D), and the nuclear stain TO-PRO (blue; Panels E and F ) were detected as described in the Materials and methods. Arrows (pink or white) indicate apoptotic neurons that are both caspase-3 and NeuN positive, whereas yellow arrows indicate apoptosis in a NeuN-negative cell. Similar results were obtained in three individually isolated groups of cells.

Figure 7

Role of caspase-8 and caspase-9 in P2X₇R-mediated apoptosis in rPCNs. (A) Rat PCNs were incubated in serum-free HGGMEM and BzATP (300 µM) was added for 16 h. Cells cultured in B27-AO Neurobasal medium (NB) or in serum-free HGGMEM (−) overnight were used as controls. (B) Cells were treated as in (A) except that the indicated concentration (µM) of a specific caspase-8 inhibitor, Ac-IETDCHO was added for 1 h prior to addition of 300 µM BzATP. (C) Cells were treated as in (B) except that the indicated concentration of a specific caspase-9 inhibitor, Z-LEHD-FMK, was added. ‘Pan’ ( full-length) and cleaved caspase-8 were detected by Western analysis, as described in the Materials and methods. Data are the means ± S.E.M. of results from at least three experiments, where *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences from either serum-starved (A) or BzATPtreated (B, C) cells.

Figure 8

BzATP or ATP induces ERK1/2 and JNK1 phosphorylation in rPCNs. Rat PCNs were incubated in serum-free HGGMEM for 6 h and treated with the indicated concentration (µM) of BzATP or ATP for 5 min or with 100 µM ATP or 300 µM BzATP for the indicated time period. Cells cultured in serum-free HGGMEM (−) overnight were used as the control. Then, cell lysates were prepared and (A) ERK1/2 or (B) JNK1 phosphorylation was detected by Western analysis, as described in the Materials and methods. Total ERK1/2 and actin were used as protein loading controls for (A) and (B), respectively.

Figure 9

Inhibitors of MEK and JNK decrease BzATP-induced caspase-3 cleavage in rat PCNs. Rat PCNs were incubated in serum-free HGGMEM for 6 h and treated for 30 min with the indicated concentration (µM) of (A) the MEK inhibitor U0126, (B) the JNK inhibitor SP600125 or (A, B) 0.1% (v/v) DMSO as a vehicle control. Then, the cells were incubated with 300 mM BzATP for 16 h, and Western analysis was used to detect cleaved or pan caspase-3, as described in the Materials and methods. Cells cultured in serum-free HGGMEM (−) overnight were used as the control. Data are the means ± S.E.M. of results from at least three experiments, where *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences from BzATP-treated cells.