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Role of N-Methyl-d-aspartate Receptors in the Neuroprotective Activation of Extracellular Signal-regulated Kinase 1/2 by Cisplatin

2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês

10.1074/jbc.m301554200

ISSN

1083-351X

Autores

Agata Góźdź, Agata Habas, Jacek Jaworski, Magdalena Zielińska, Jan Albrecht, Marcin Chlystun, Ahmad Jalili, Michal Hetman,

Tópico(s)

Cell death mechanisms and regulation

Resumo

Neurons are exposed to damaging stimuli that can trigger cell death and subsequently cause serious neurological disorders. Therefore, it is important to define defense mechanisms that can be activated in response to damage to reduce neuronal loss. Here we report that cisplatin (CPDD), a neurotoxic anticancer drug that damages DNA, triggered apoptosis and activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in cultured rat cortical neurons. Inhibition of ERK1/2 activation using either pharmacological inhibitors or a dominant-negative mutant of the ERK1/2 activator, mitogen-activated protein kinase kinase 1, increased the toxicity of CPDD. Interestingly, N-methyl-d-aspartate (NMDA) receptor (NMDAR) antagonists reduced the ERK1/2 activation and exacerbated apoptosis in CPDD-treated neurons. Pre-treatment with CPDD increased ERK1/2 activation triggered by exogenous NMDA, suggesting that CPDD augmented NMDAR responsiveness. CPDD-enhanced response of NMDAR and CPDD-mediated ERK1/2 activation were both decreased by inhibition of poly(ADP-ribose) polymerase (PARP). Interestingly, PARP activation did not produce ATP depletion, suggesting involvement of a non-energetic mechanism in NMDAR regulation by PARP. Finally, CPDD toxicity was reduced by brain-derived neurotrophic factor, and this protection required ERK1/2. In summary, our data identify a novel compensatory circuit in central nervous system neurons that couples the DNA injury, through PARP and NMDAR, to the defensive ERK1/2 activation. Neurons are exposed to damaging stimuli that can trigger cell death and subsequently cause serious neurological disorders. Therefore, it is important to define defense mechanisms that can be activated in response to damage to reduce neuronal loss. Here we report that cisplatin (CPDD), a neurotoxic anticancer drug that damages DNA, triggered apoptosis and activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in cultured rat cortical neurons. Inhibition of ERK1/2 activation using either pharmacological inhibitors or a dominant-negative mutant of the ERK1/2 activator, mitogen-activated protein kinase kinase 1, increased the toxicity of CPDD. Interestingly, N-methyl-d-aspartate (NMDA) receptor (NMDAR) antagonists reduced the ERK1/2 activation and exacerbated apoptosis in CPDD-treated neurons. Pre-treatment with CPDD increased ERK1/2 activation triggered by exogenous NMDA, suggesting that CPDD augmented NMDAR responsiveness. CPDD-enhanced response of NMDAR and CPDD-mediated ERK1/2 activation were both decreased by inhibition of poly(ADP-ribose) polymerase (PARP). Interestingly, PARP activation did not produce ATP depletion, suggesting involvement of a non-energetic mechanism in NMDAR regulation by PARP. Finally, CPDD toxicity was reduced by brain-derived neurotrophic factor, and this protection required ERK1/2. In summary, our data identify a novel compensatory circuit in central nervous system neurons that couples the DNA injury, through PARP and NMDAR, to the defensive ERK1/2 activation. The central nervous system is exposed to damaging stimuli that may trigger neuronal death and cause serious neurological diseases (1Yuan J. Yankner B.A. Nature. 2000; 407: 802-809Crossref PubMed Scopus (1596) Google Scholar). However, most neurons survive minor damages with which they are challenged during the life span of the organism. Therefore, one can expect the existence of defense mechanisms that help neurons to survive initial insult and resume proper functions after damage. Neurons receive multiple signals inhibiting cell death (2Miller F.D. Kaplan D.R. Cell. Mol. Life Sci. 2001; 58: 1045-1053Crossref PubMed Scopus (285) Google Scholar). For example, neuronal survival during development is promoted by neurotrophins and neurotransmitters. The effects of these agents on survival are mediated through several signaling molecules, including extracellular signal-regulated kinase 1/2 (ERK1/2) 1The abbreviations used are: ERK1/2, extracellular signal-regulated kinase 1/2; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; KA, kynurenic acid; NMDA, N-methyl-d-aspartate; NMDAR, NMDA receptor; CPDD, cis-diaminodichloroplatinum; PARP, poly(ADP-ribose) polymerase; MKK1/2, mitogen-activated protein kinase kinase 1/2; 3-ABA, 3-aminobenzamide; PHEN, 6(5H)-phenanthridinone; BDNF, brain-derived neurotrophic factor; APV, dl-2-amino-5-phosphonovaleric acid; NBQX, 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; CNQX, cyano-nitroquinoxaline; TPDD, trans-diaminodichloroplatinum; BAPTA/AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Me2SO, dimethyl sulfoxide; Z-VAD-fmk, benzyloxycarbonyl-VAD-fluoromethyl ketone; HA, hemagglutinin; MK-801, dizocilpine.1The abbreviations used are: ERK1/2, extracellular signal-regulated kinase 1/2; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; KA, kynurenic acid; NMDA, N-methyl-d-aspartate; NMDAR, NMDA receptor; CPDD, cis-diaminodichloroplatinum; PARP, poly(ADP-ribose) polymerase; MKK1/2, mitogen-activated protein kinase kinase 1/2; 3-ABA, 3-aminobenzamide; PHEN, 6(5H)-phenanthridinone; BDNF, brain-derived neurotrophic factor; APV, dl-2-amino-5-phosphonovaleric acid; NBQX, 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; CNQX, cyano-nitroquinoxaline; TPDD, trans-diaminodichloroplatinum; BAPTA/AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Me2SO, dimethyl sulfoxide; Z-VAD-fmk, benzyloxycarbonyl-VAD-fluoromethyl ketone; HA, hemagglutinin; MK-801, dizocilpine. and phoshpatidyloinositol 3-kinase (1Yuan J. Yankner B.A. Nature. 2000; 407: 802-809Crossref PubMed Scopus (1596) Google Scholar, 2Miller F.D. Kaplan D.R. Cell. Mol. Life Sci. 2001; 58: 1045-1053Crossref PubMed Scopus (285) Google Scholar). Consequently, survival signaling pathways are good candidates to contribute to the defense mechanisms in injured neurons. Glutamate is an important neurotransmitter that promotes survival. It acts through several types of receptors, including two families of ionotropic receptors, AMPA and NMDA (3Westbrook G.L. Curr. Opin. Neurobiol. 1994; 4: 337-346Crossref PubMed Scopus (78) Google Scholar). NMDA receptor (NMDAR) is required for neuronal survival during development (4Llado J. Caldero J. Ribera J. Tarabal O. Oppenheim R.W. Esquerda J.E. J. Neurosci. 1999; 19: 10803-10812Crossref PubMed Google Scholar, 5Ikonomidou C. Bosch F. Miksa M. Bittigau P. Vockler J. Dikranian K. Tenkova T.I. Stefovska V. Turski L. Olney J.W. Science. 1999; 283: 70-74Crossref PubMed Scopus (1694) Google Scholar). On the other hand, excessive activation of NMDA signaling produces excitotoxicity (6Olney J.W. Neurobiol. Aging. 1994; 15: 259-260Crossref PubMed Scopus (75) Google Scholar). Therefore, NMDAR inhibitors are used to improve the outcome of several neurological diseases (7Le D.A. Lipton S.A. Drugs Aging. 2001; 18: 717-724Crossref PubMed Scopus (76) Google Scholar). In addition, it has been recently proposed that NMDAR antagonists may be active against malignant tumors and that their combination with anticancer chemotherapy would be a valuable therapeutic approach (8Rzeski W. Turski L. Ikonomidou C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6372-6377Crossref PubMed Scopus (213) Google Scholar). It is intriguing that neurons, which are postmitotic cells, demonstrate high vulnerability to DNA damage (9Morris E.J. Geller H.M. J. Cell Biol. 1996; 134: 757-770Crossref PubMed Scopus (283) Google Scholar, 10Park D.S. Morris E.J. Padmanabhan J. Shelanski M.L. Geller H.M. Greene L.A. J. Cell Biol. 1998; 143: 457-467Crossref PubMed Scopus (241) Google Scholar). Often, genotoxic anticancer agents including cisplatin (CPDD) produce neurological side effects that limit their usage against central nervous system tumors (11Keime-Guibert F. Napolitano M. Delattre J.Y. J. Neurol. 1998; 245: 695-708Crossref PubMed Scopus (205) Google Scholar, 12Stewart D.J. Rottenberg D.A. Neurological Complications of Cancer Treatment. Butterworth-Heinemann, Boston1991: 143-170Google Scholar). DNA damage may also be an important trigger of neuron loss in common neurodegenerative diseases (13Alam Z.I. Jenner A. Daniel S.E. Lees A.J. Cairns N. Marsden C.D. Jenner P. Halliwell B. J. Neurochem. 1997; 69: 1196-1203Crossref PubMed Scopus (699) Google Scholar). DNA damage activates both the reparative response and death signaling (14Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2628) Google Scholar). For example, DNA damage may mobilize poly(ADP-ribose) polymerase (PARP), which ribosylates target proteins to activate DNA repair (15Ha H.C. Snyder S.H. Neurobiol. Dis. 2000; 7: 225-239Crossref PubMed Scopus (192) Google Scholar). The substrate of PARP is a highly energetic molecule, NAD+. In consequence, PARP activation may deplete cellular energy stores, resulting in neuronal membrane depolarization with enhanced NMDAR signaling and, finally, necrotic cell death (15Ha H.C. Snyder S.H. Neurobiol. Dis. 2000; 7: 225-239Crossref PubMed Scopus (192) Google Scholar). Genotoxin-induced neuronal death can be suppressed by neurotrophins (16Zheng J.L. Stewart R.R. Gao W.Q. J. Neurosci. 1995; 15: 5079-5087Crossref PubMed Google Scholar, 17Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 18Anderson C.N. Tolkovsky A.M. J. Neurosci. 1999; 19: 664-673Crossref PubMed Google Scholar). The protection has been reported to require activation of the ERK1/2 pathway (17Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 18Anderson C.N. Tolkovsky A.M. J. Neurosci. 1999; 19: 664-673Crossref PubMed Google Scholar). Therefore, we hypothesized that DNA damage by CPDD can activate the ERK1/2 pathway as a defense response to support neuronal survival. Our results indicate that the CPDD-activated ERK1/2 pathway attenuates cortical neuron death. Interestingly, activation of ERK1/2 by CPDD depended on PARP and NMDAR activity and was not accompanied by decreased ATP levels. Our observations identify a novel protective pathway that couples DNA damage, through PARP and NMDAR, to ERK1/2. Our results also suggest the possibility of toxic interactions between NMDAR antagonists and genotoxic anticancer agents that reach the central nervous system. Materials—The following plasmids have been described elsewhere: pON260 (19Cherrington J.M. Mocarski E.S. J. Virol. 1989; 63: 1435-1440Crossref PubMed Google Scholar), HA-tagged expression vectors for wild type, constitutive active MKK1 (ΔN3-S218E/S222D), and dominant-negative MKK1 (K97M) (20Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1258) Google Scholar). A polyclonal anti-phospho-ERK1/2 antibody (anti-ACTIVE™ mitogen-activated protein kinase polyclonal antibody) was purchased from Promega; anti-ERK2 antibody was obtained from Santa Cruz Biotechnology; monoclonal anti-ERK1/2 antibody was obtained from Cell Signaling; polyclonal antibody to β-galactosidase was obtained from 5 Prime → 3 Prime, Inc. (Boulder, CO), and anti-HA monoclonal antibody (12CA5) was obtained from Roche Applied Science. Polyclonal antibody to poly(ADP)ribose and all secondary antibodies were obtained from Calbiochem. PD98059 and LY294002 were purchased from Calbiochem. 3-aminobenzamide (ABA), 6(5H)-phenanthridinone (PHEN), BDNF, cycloheximide, dizocilpine (MK-801), dl-2-amino-5-phosphonovaleric acid (APV), NBQX, CNQX, CPDD, and transplatin (TPDD) were obtained from Sigma. BAPTA/AM was purchased from Molecular Probes. Cell Culture and Transfection—Cortical neurons were prepared from newborn rats (Sprague-Dawley) and kept in basal medium Eagle supplemented with 10% heat-inactivated bovine calf serum (HyClone), as described (17Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). 1-(β-d-Arabinofuranosyl)cytosine (2.5 μm) was added at day 2 in vitro to inhibit proliferation of non-neuronal cells. Cortical neurons were transiently transfected at days 3 or 4 in vitro by using a modified calcium-phosphate co-precipitation protocol (17Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Cortical neurons cultured on poly-d-lysine/laminin-coated (Sigma) glass coverslips in 35-mm plates were transfected with 4 μg/plate expression plasmid DNA for wild-type MKK1 (MKK1wt), dominant-negative MKK1 (MKK1dn), constitutive-active MKK1 (MKK1ca), or empty cloning vector, pCEP4. In addition, neurons were co-transfected with 2 μg/plate pON260 plasmid DNA, which contains an expression cassette for bacterial β-galactosidase. Expression of recombinant forms of MKK1 was confirmed by immunostaining for HA epitope tag attached to MKK1 proteins. In addition, cell nuclei were counterstained with Hoechst 33258 to reveal apoptotic alterations in chromatin structure. β-Galactosidase was used as a marker to identify transfected cells. Because β-galactosidase remains stable in dying cells (21Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R.J. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2215) Google Scholar), we were able to score for apoptosis in transfected neurons on the single cell level without a bias to exclude apoptotic cells. Drug Treatment—At days 5 or 6 in vitro, cortical neurons were treated with CPDD or TPDD. These drugs were dissolved in dimethyl sulfoxide (Me2SO). The final concentration of Me2SO in the media was 0.2%. PD98059, SL327, MK-801, NBQX, CNQX, 3-ABA, PHEN, and BAPTA/AM were also dissolved in Me2SO. When cultures were co-treated with CPDD and one of these drugs, the final concentration of Me2SO was below 0.4%. BDNF was diluted in phosphate-buffered saline containing 0.1% bovine serum albumin before addition to the cells. SL327 or PD98059 was added 30 min before BDNF in co-treatment experiments. Quantitation of Apoptosis by Nuclear Morphological Changes—To visualize nuclear morphology, cells were fixed in 4% paraformaldehyde and stained with 2.5 μg/ml of the DNA dye Hoechst 33258 (Sigma) (17Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Apoptosis was quantitated by scoring the percentage of cells with apoptotic nuclear morphology at the single cell level after Hoechst staining. Uniformly stained nuclei were scored as healthy, viable neurons. Condensed or fragmented nuclei were scored as apoptotic. To obtain unbiased counting, samples were coded, and cells were scored blind without knowledge of their prior treatment. Quantitation of Neuronal Survival by MTT Assay—The MTT assay was performed in 96-well plates as described (17Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). DNA Ladder Assay—To examine DNA cleavage, soluble cytoplasmic DNA was isolated from 4 × 106 cells and subjected to 1.8% agarose gel electrophoresis (17Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Caspase Assay—Caspase assay was performed with a colorimetric caspase assay kit purchased from Promega. For each measurement, protein lysate from 1 × 106 cells was used. To reveal the caspase-dependent activity, each sample was incubated with the caspase substrate in the absence or presence of 10 μm Z-VAD-fmk, a specific caspase inhibitor. Western Analysis and Immunostaining—Western blot analysis with anti-phospho-ERK1/2 or anti-ERK1/2 antibodies was performed as described (17Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Briefly, 10 μg of total protein was used in each lane. In addition, blots were re-probed with antibodies specific for total ERK1/2 or ERK2 to ensure equal protein loading of the blots. Quantification of phospho-ERK1/2 was performed by densitometric analysis and normalized against total ERK1/2. Western blot analysis with anti-poly(ADP-ribose) antibody was performed using 4 μg of nuclear proteins per sample. Nuclear proteins were extracted as described (22Lukasiuk K. Kaczmarek L. Condorelli D.F. Neurochem. Int. 1995; 26: 173-178Crossref PubMed Scopus (13) Google Scholar). Transfected cells were detected by immunostaining with an antibody against β-galactosidase and Texas Red-conjugated goat antibody to rabbit immunoglobulin. Cells transfected with the HA epitope-tagged constructs were also immunostained with an antibody to HA followed by fluorescein-conjugated goat antibody to mouse immunoglobulin. Determination of Glutamate and Glycine Concentration—Amino acids were extracted from culture media with 0.6 n perchloric acid, followed by centrifugation and neutralization with potassium hydroxide. The analysis was performed as described (23Hilgier W. Zielinska M. Borkowska H.D. Gadamski R. Walski M. Oja S.S. Saransaari P. Albrecht J. J. Neurosci. Res. 1999; 56: 76-84Crossref PubMed Scopus (58) Google Scholar). NAD+ and ATP Assays—To extract nucleotides, 2 × 106 cells were treated with 3.5% perchloric acid as described (24Komatsu N. Nakagawa M. Oda T. Muramatsu T. J. Biochem. (Tokyo). 2000; 128: 463-470Crossref PubMed Scopus (52) Google Scholar). NAD+ level was measured in a reaction catalyzed by alcohol dehydrogenase as described (25Williamson J.R. Corkey B.E. Methods Enzymol. 1964; 13: 481-483Google Scholar). ATP measurement was performed according to the two-step procedure of Williamson and Corkey (26Williamson J.R. Corkey B.E. Methods Enzymol. 1964; 13: 488-491Google Scholar). Statistical Analysis—Statistical analysis of the data was performed by using one- or two-way analysis of variance followed by post hoc tests. CPDD-induced Apoptosis in Cortical Neurons—To test the hypothesis that DNA damage can activate the defensive ERK1/2 pathway, we treated cortical neurons with CPDD. CPDD applied for 48 h reduced neuronal survival (Fig. 1A). Interestingly, cells exposed to 10 μg/ml CPDD showed higher survival rates than cells treated with 5 μg/ml (56.4 versus 28%, Fig. 1A). Neurons treated with CPDD showed an apoptotic pattern of DNA fragmentation (Fig. 1B) and activation of proapoptotic caspases (Fig. 1C). In addition, cells dying in response to CPDD displayed morphological features of apoptosis including fragmentation and condensation of nuclear chromatin (Fig. 1, D–J). 10 μg/ml CPDD induced significantly less apoptosis than 5 μg/ml (at 24 h, 28 versus 51.2%, respectively; p < 0.01) (Fig. 1J). A translation inhibitor, cycloheximide, protected against CPDD-induced death (Fig. 1K), suggesting that protein synthesis may be involved in this process. This finding is also consistent with the apoptotic character of CPDD-induced death in cortical neurons. In addition, TPDD, an isomer of CPDD that is unable to induce DNA strand breaks (27Trimmer E.E. Essigmann J.M. Essays Biochem. 1999; 34: 191-211Crossref PubMed Scopus (121) Google Scholar), did not produce neuronal apoptosis (Fig. 1L), indicating that CPDD-induced neuronal apoptosis is triggered by DNA damage. Activation of ERK1/2 Pathway in CPDD-treated Cortical Neurons—Phosphorylation of ERK1/2 residues Thr183 and Tyr185 (position numbers as in human ERK2) by MKK1/2 controls ERK1/2 activation (28Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (758) Google Scholar). Therefore, we determined the activity of the ERK1/2 pathway by immunoblotting for phosphorylated ERK1/2 (Fig. 2). In cells exposed to 10 μg/ml CPDD, activation peaked at 24 h after treatment (Fig. 2, A and B). At that time point, the extent of CPDD-mediated activation of the ERK1/2 pathway was directly proportional to the concentration of CPDD (Fig. 2 C and D), with the maximum stimulation by 10 μg/ml (5.9-fold above controls). Maximal ERK1/2 activation by 10 μg/ml CPDD correlated with the decreased toxicity of CPDD at 10 μg/ml, as compared with 5 μg/ml (Fig. 1). This finding suggests ERK1/2 involvement in the defensive reaction to damage. Importantly, TPDD (10 μg/ml) did not increase ERK1/2 activity (Fig. 2E). ERK1/2 activation by CPDD was not affected by cycloheximide (1 μg/ml) (Fig. 2F). These data suggest that the ERK1/2 response is triggered by DNA damage and does not involve protein synthesis. CPDD-mediated Activation of ERK1/2 Supports Neuronal Survival—Pharmacological inhibitors of the ERK1/2 pathway, PD98059 or SL327, effectively abolished ERK1/2 activation by CPDD (Fig. 3, A and D). Therefore, we used these compounds to determine the effect of the ERK1/2 response on CPDD-induced cell death. Consistent with our previous observations (17Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar), neither SL327 (50 μm) nor PD98059 (40 μm) significantly affect basal apoptosis in cortical neurons (Fig. 3, B and E). By 24 h, cells exposed to either 5 or 10 μg/ml CPDD showed a significant increase of apoptosis upon co-treatment with SL327 (28.3% at 10 μg/ml CPDD versus 57.8% at SL327 + 10 μg/ml of CPDD; p < 0.001) (Fig. 3B). Interestingly, the CPDD concentration dependence of the SL327 effect on apoptosis correlated with the concentration dependence of CPDD-mediated ERK1/2 activation (Fig. 2, C and D). PD98059 also increased apoptosis induced by CPDD (Fig. 3E, p < 0.001). In addition, SL327 (50 μm) or PD98059 (40 μm) further reduced neuronal viability after CPDD treatment (Fig. 3, C and F). To complement the pharmacological approach, we studied the effects of a dominantnegative mutant form of the ERK1/2 activator, MKK1 (MKK1dn) (20Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1258) Google Scholar), on CPDD-induced apoptosis. Cortical neurons were transfected with expression plasmids for either wild type MKK1 (MKK1wt), MKK1dn, or empty cloning vector, pCEP4 (Fig. 3, G and H). Forty-eight hours after transfection, neurons were treated for 24 h with either vehicle (0.2% Me2SO) or CPDD (10 μg/ml). In vehicle-treated cells, apoptosis was unaffected by any of the transfected plasmids (average of 15.5%, Fig. 3I). In contrast, CPDD caused apoptosis in more cells expressing MKK1dn (46.5 versus 31% in pCEP4 or 26% in MKK1wt-transfected neurons; p < 0.001, Fig. 3I). Therefore, inhibition of ERK1/2 increased apoptotic cell death induced by CPDD. Finally, we studied the effects of PD98059 on CPDD-mediated caspase activation, a marker of apoptotic cell death. 5 but not 10 μg/ml CPDD significantly activated caspases at 12 h (1.9-fold above control, Fig. 3J). When PD98059 was combined with 10 μg/ml CPDD, caspase activation occurred (1.7-fold above control, Fig. 3J). This result confirms that ERK1/2 activation by CPDD suppresses neuronal apoptosis. CPDD Activates ERK1/2 through NMDA Receptors—ERK1/2 can be activated in cortical neurons by the glutamate-triggered influx of Ca2+ ions through an open NMDAR channel (29Bading H. Greenberg M.E. Science. 1991; 253: 912-914Crossref PubMed Scopus (413) Google Scholar). Because moderate activity of NMDAR is implicated in anti-apoptotic signaling (5Ikonomidou C. Bosch F. Miksa M. Bittigau P. Vockler J. Dikranian K. Tenkova T.I. Stefovska V. Turski L. Olney J.W. Science. 1999; 283: 70-74Crossref PubMed Scopus (1694) Google Scholar, 30Hetman M. Cavanaugh J.E. Kimelman D. Xia Z. J. Neurosci. 2000; 20: 2567-2574Crossref PubMed Google Scholar), we tested for NMDAR involvement in the protective ERK1/2 activation by CPDD. The CPDD-mediated increase of ERK1/2 activity (6.1-fold above controls) was reduced by the NMDAR antagonists kynurenic acid plus Mg2+, MK-801, or APV (1.4-, 0.5-, or 0.6-fold above controls, respectively; Fig. 4A). Also, co-treatment with the cytosolic Ca2+ chelator BAPTA/AM significantly reduced ERK1/2 activation by CPDD (7.1-versus 3.7-fold above controls; Fig. 4A). Furthermore, NMDA receptor antagonists increased apoptosis induced by 24-h treatment with CPDD (30.3% at 10 μg/ml of CPDD versus 51.4% at 10 μg/ml CPDD plus 10 μm MK-801; p < 0.01) (Fig. 4, B and C). Inhibitors of AMPA/KA receptors, CNQX or NBQX, did not affect the ERK1/2 response to CPDD (Fig. 4D). Collectively, the data suggest that the defensive ERK1/2 activation by CPDD is mediated through NMDAR. CPDD Increases NMDAR Signaling—NMDAR can be activated by increased concentration of its ligands, glutamate or NMDA (Fig. 5A). If increased concentrations of NMDAR ligands are responsible for CPDD activation of ERK1/2, one would expect that conditioned media from cells that are exposed to CPDD would produce ERK1/2 activation in untreated neurons. However, ERK1/2 was not activated by conditioned media collected from cells treated for 24 h with 10 μg/ml CPDD (Fig. 5B). Consistently, media concentrations of the NMDAR ligand, glutamate, and its co-ligand, glycine (3Westbrook G.L. Curr. Opin. Neurobiol. 1994; 4: 337-346Crossref PubMed Scopus (78) Google Scholar), did not significantly increase after CPDD treatment (Fig. 5C). Therefore, it seems unlikely that activation of NMDAR by CPDD is caused by an elevated release of the NMDAR ligands. An alternative possibility is that CPDD increases the neuronal responses to basal levels of NMDAR stimulation. Indeed, we found that neurons that were pretreated with 5 μg/ml CPDD for 24 h demonstrated increased ERK1/2 responses to NMDA (Fig. 5, D and E). In cells that were not exposed to CPDD, a 5-min treatment with NMDA evoked a concentration-dependent ERK1/2 activation that appeared at 20 μm (6.1-fold above control) and declined at 50 or 100 μm (3.6- or 2.3-fold above control, respectively; Fig. 5, D and E). Neurons that were pretreated with 5 μg/ml CPDD for 24 h responded with significantly enhanced ERK1/2 activation (p < 0.01) which was present at 10 μm NMDA (5.3-fold above control), reached maximal levels at 50 μm (7.6-fold above control), and slightly declined at 100 μm (4.9-fold above control; Fig. 5, D and E). Therefore, it appears that CPDD decreases the threshold of NMDAR stimulation that is required to activate ERK1/2 and inhibits desensitization of the ERK1/2 response following more intense NMDAR stimulation. These data indicate that CPDD enhances intracellular signaling by NMDAR. PARP Activity Contributes to CPDD-mediated ERK1/2 Activation—ERK1/2 activation apparently was caused by CPDD-induced DNA strand breaks (Fig. 2E) and was dependent upon NMDAR (Figs. 4 and 5). Thus, augmentation of NMDAR signaling in CPDD-treated neurons may be secondary to DNA damage. It has been proposed that a DNA damage-response enzyme, PARP, may regulate NMDAR in neurons (15Ha H.C. Snyder S.H. Neurobiol. Dis. 2000; 7: 225-239Crossref PubMed Scopus (192) Google Scholar). Therefore, we have evaluated the possibility that the CPDD-mediated increase in NMDAR signaling is mediated by PARP. Indeed, we observed increased PARP activity after CPDD treatment, indicated by the elevated polyribosylation of neuronal nuclear proteins (Fig. 6A). Also, cellular NAD+ content that is reduced during PARP activation (31D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar) significantly decreased after CPDD (Fig. 6B). The NAD+ decrease preceded the CPDD-induced reduction of neuronal survival (Fig. 6C). These data indicate that CPDD activates PARP. To evaluate whether PARP activation can contribute to the protective ERK1/2 activation by CPDD, we studied the effects of the PARP inhibitors, 3-ABA (5 mm) or PHEN (50 μm), on CPDD-induced ERK1/2 activation or cell death. 3-ABA or PHEN reduced ERK1/2 activation in response to CPDD (Fig. 6D) and increased neuronal apoptosis triggered by CPDD (Fig. 6E). Neuronal apoptosis in basal conditions was not affected by either 3-ABA or PHEN (data not shown). Also, the CPDD-mediated reduction in neuronal survival was enhanced in the presence of 3-ABA (Fig. 6F). Therefore, our data suggest that PARP contributes to protective ERK1/2 activation by CPDD. To further support the idea that PARP activation contributes to the increased responsiveness of NMDAR in CPDD-treated neurons, we evaluated the effects of 3-ABA on ERK1/2 activation by NMDA in cells that were pretreated with CPDD (5 μg/ml). Interestingly, neurons pretreated with CPDD in the presence of 3-ABA did not show enhanced ERK1/2 signaling after stimulation with NMDA (Fig. 6G). PARP inhibition abolished ERK1/2 activation at 10 μm NMDA (5.9-versus 0.7-fold above control; Fig. 6G), decreased the activation level at 20 μm (10.2-versus 1.8-fold above control; Fig. 6G), and increased desensitization of ERK1/2 response at 100 μm NMDA (7.4-versus 1.3-fold above control; Fig. 6G). These data suggest that PARP enhances NMDAR-mediated ERK1/2 activation in CPDD-treated neurons. The PARP-mediated increase in NMADR signaling has been suggested to result from ATP depletion after increased NAD+ re-synthesis (31D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). However, we did not find a significant reduction of ATP levels until at least 12 h after CPDD addition (Fig. 6H). A moderate decrease of ATP content occurred at 24 h (79.1% of control values; p < 0.001). This alteration correlates with 82.4% cell survival found at 24 h after 10 μg/ml CPDD treatment (Fig. 6C). Therefore, the reduced ATP content most likely reflects cell loss rather than the energetic deprivation of living neurons. In summary, these data suggest that PARP regulates NMDAR-mediated ERK1/2 activation through an ATP depletion-independent mechanism. BDNF Reduces CPDD Toxicity by Activation of ERK1/2 Pathway—A question can be raised whether neuroprotective agents that activate ERK1/2 would be able to reduce CPDD-mediated cell death. A 6-h treatment with 10 ng/ml BDNF activated ERK1/2 pathway in the absence or presence of CPDD (5 μg/ml) (Fig. 7A). BDNF also decreased CPDD-induced apoptosis from 48.5 to 22.0% (Fig. 7B, p < 0.001). This effect was abolished by 50 μm SL327 indicating that ERK1/2 is required for BDNF-mediated protection of CPDD-treated neurons. To determine whether ERK1/2 activation is sufficient for BDNF-mediated protection, we used a constitutively active mutant form of the ERK1/2 activator, MKK1 (MKK1ca) (20Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1258) Google Scholar). Cortical neurons were transfected with either MKK1ca or empty cloning vector, pCEP4. Forty-eight hours after transfection, neurons were treated for 24 h with 5 μg/ml CPDD. Neurons receiving MKK1ca were protected against CPDD-induced apoptosis (50.3% in pCEP4 versus 19.0% in MKK1ca-transfected cells; p < 0.001) (Fig. 7C). These results suggest that activation of ERK1/2 is both required and sufficient for BDNF to reduce CPDD-induced apoptosis in neurons. In this study, we tested the possibility that ERK1/2 is activated by DNA damage to support survival of stressed neurons. Indeed, we observed that CPDD-induced apoptosis in rat primary cortical neurons was accompanied by anti-apoptotic ERK1/2 activation. We also identified PARP and NMDAR as mediators of CPDD-induced ERK1/2 response (Fig. 8). Finally, we showed that ERK1/2 activation was both necessary and sufficient for BDNF-mediated protection against CPDD-induced apoptosis. In our hands, CPDD induced cortical neuron apoptosis. CPDD was also shown to induce apoptosis in peripheral nervous system neurons of sensory and auditory systems (32Gill J.S. Windebank A.J. J. Clin. Investig. 1998; 101: 2842-2850Crossref PubMed Scopus (245) Google Scholar, 33Liu W. Staecker H. Stupak H. Malgrange B. Lefebvre P. Van De Water T.R. Neuroreport. 1998; 9: 2609-2614Crossref PubMed Scopus (138) Google Scholar). Apoptotic death of these neuronal populations has been suggested as a mechanism of peripheral neurotoxicities of CPDD. Similarly, CPDD-induced cortical neuron apoptosis may underlie the robust central nervous system neurotoxicity observed after local delivery of CPDD to treat intracranial tumors (12Stewart D.J. Rottenberg D.A. Neurological Complications of Cancer Treatment. Butterworth-Heinemann, Boston1991: 143-170Google Scholar). CPDD activated ERK1/2 by increasing NMDAR signaling. CPDD increased NMDAR sensitivity to low concentrations of NMDA and also inhibited desensitization of ERK1/2 response after intense receptor stimulation. Furthermore, our results indicate that the mechanism of enhanced NMDAR signaling involves PARP activation. It has been suggested that the PARP-mediated increase of NMADR signaling results from ATP depletion after increased NAD+ resynthesis (15Ha H.C. Snyder S.H. Neurobiol. Dis. 2000; 7: 225-239Crossref PubMed Scopus (192) Google Scholar, 31D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). However, we did not find a significant reduction of ATP levels in neurons that showed robust activation of ERK1/2. Consequently, the possible mechanisms of PARP-mediated NMDAR regulation in CPDD-treated cells may include posttranscriptional modifications, differential expression of NMDAR sub-units, depolarization of the membrane through activity of other glutamate receptors, and, finally, enhanced coupling of NMDAR to ERK1/2. In hippocampus, enhancement of the NMDAR response is produced by the primary increase of AMPA/KA receptor activity (3Westbrook G.L. Curr. Opin. Neurobiol. 1994; 4: 337-346Crossref PubMed Scopus (78) Google Scholar). This mechanism is unlikely to explain increased NMDAR signaling in CPDD-treated neurons because AMPA/KA receptor blockers did not affect ERK1/2 activation by CPDD. The alternative mechanism producing enhanced NMDAR signaling during development is increased expression of NMDAR subunits (34Sheng M. Cummings J. Roldan L.A. Jan Y.N. Jan L.Y. Nature. 1994; 368: 144-147Crossref PubMed Scopus (1099) Google Scholar). However, we found no CPDD-induced increases in expression of the two NMDAR subunits, NR1 or NR2B, whose expression is detectable in cultured cortical neurons. 2A. Gozdz and M. Hetman, unpublished data. Therefore, it remains to be resolved which mechanism contributes to CPDD/PARP-mediated regulation of NMDAR. It is well established that excessive stimulation of NMDARs results in excitotoxic neuronal death (6Olney J.W. Neurobiol. Aging. 1994; 15: 259-260Crossref PubMed Scopus (75) Google Scholar). However, the toxic abilities of NMDARs are not directly proportional to their activity. In fact, some NMDAR activity is required for optimal survival of various populations of central nervous system nerve cells, including cortical neurons (5Ikonomidou C. Bosch F. Miksa M. Bittigau P. Vockler J. Dikranian K. Tenkova T.I. Stefovska V. Turski L. Olney J.W. Science. 1999; 283: 70-74Crossref PubMed Scopus (1694) Google Scholar, 30Hetman M. Cavanaugh J.E. Kimelman D. Xia Z. J. Neurosci. 2000; 20: 2567-2574Crossref PubMed Google Scholar). Also, moderate concentrations of NMDA can protect cortical neurons from trophic deprivation-induced death (35Terro F. Esclaire F. Yardin C. Hugon J. Neurosci. Lett. 2000; 278: 149-152Crossref PubMed Scopus (25) Google Scholar, 36Lafon-Cazal M. Perez V. Bockaert J. Marin P. Eur. J. Neurosci. 2002; 16: 575-583Crossref PubMed Scopus (59) Google Scholar). Therefore, NMDAR signaling plays an important role to support neuronal survival during development. Our results identify a new role for the anti-apoptotic activity of NMDAR in protecting neurons exposed to genotoxic stress. Our data suggest that the activation of PARP in neurons is protective against CPDD-induced cell death. Protective properties of PARP were also reported in various other cell types including cancer cells, embryonic mouse tissue, and rat hippocampal neurons (37Bowman K.J. Newell D.R. Calvert A.H. Curtin N.J. Br. J. Cancer. 2001; 84: 106-112Crossref PubMed Scopus (126) Google Scholar, 38Nagayama T. Simon R.P. Chen D. Henshall D.C. Pei W. Stetler R.A. Chen J. J. Neurochem. 2000; 74: 1636-1645Crossref PubMed Scopus (106) Google Scholar, 39de Murcia J.M. Niedergang C. Trucco C. Ricoul M. Dutrillaux B. Mark M. Oliver F.J. Masson M. Dierich A. LeMeur M. Walztinger C. Chambon P. de Murcia G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7303-7307Crossref PubMed Scopus (959) Google Scholar). For instance, in a rat global ischemia model, CA1 neuron loss was enhanced by the PARP inhibitor 3-ABA (38Nagayama T. Simon R.P. Chen D. Henshall D.C. Pei W. Stetler R.A. Chen J. J. Neurochem. 2000; 74: 1636-1645Crossref PubMed Scopus (106) Google Scholar). On the other hand, other reports showed that intense PARP activation depletes cellular energy stores and induces necrosis (15Ha H.C. Snyder S.H. Neurobiol. Dis. 2000; 7: 225-239Crossref PubMed Scopus (192) Google Scholar). The discrepancies between observations suggesting that PARP mediated protection and those indicating that deleterious PARP effects can be attributed to the differences in the intensity of PARP activation. For example, PARP activation after CPDD resulted in a 20% reduction of NAD+ levels at 12 h after treatment and undetectable changes in ATP levels. Similarly, protective PARP activation by global ischemia did not significantly reduce NAD+ levels (38Nagayama T. Simon R.P. Chen D. Henshall D.C. Pei W. Stetler R.A. Chen J. J. Neurochem. 2000; 74: 1636-1645Crossref PubMed Scopus (106) Google Scholar). In contrast, deleterious PARP activation after treatment with an alkylating drug, N-methyl-N′-nitro-N-nitrosoguanidine, decreased NAD+ levels by at least 80% within 60 min (40Yu S.W. Wang H. Poitras M.F. Coombs C. Bowers W.J. Federoff H.J. Poirier G.G. Dawson T.M. Dawson V.L. Science. 2002; 297: 259-263Crossref PubMed Scopus (1560) Google Scholar). Therefore, PARP activation by CPDD may be insufficient to produce pro-necrotic energy depletion. In conclusion, data presented here suggest that, as in the case of NMDAR signaling, moderate PARP activation also promotes neuronal survival. Defensive activation of ERK1/2 by CPDD is not a unique stress-activated compensatory response in neurons. In fact, it has been revealed that NF-κB activated by various forms of stress protects neurons from death (41Mattson M.P. Goodman Y. Luo H. Fu W. Furukawa K. J. Neurosci. Res. 1997; 49: 681-697Crossref PubMed Scopus (523) Google Scholar). Interestingly, Gonzalez-Zulueta et al. (42Gonzalez-Zulueta M. Feldman A.B. Klesse L.J. Kalb R.G. Dillman J.F. Parada L.F. Dawson T.M. Dawson V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 436-441Crossref PubMed Scopus (300) Google Scholar) reported that transient ischemic stimulation activated ERK1/2 by NMDAR. Inhibition of this signaling increased sensitivity to a subsequent ischemic insult. Therefore, ERK1/2 activation by NMDAR may be used by neurons to resist various forms of injury. In summary, CPDD-induced genotoxic stress activated an anti-apoptotic ERK1/2 response. This effect was mediated by PARP, which enhanced NMDAR signaling in CPDD-treated neurons. Therefore, our results identify a novel compensatory circuit to defend central nervous system neurons against genotoxic apoptosis. This defensive pathway couples DNA damage through PARP and NMDAR to ERK1/2 activation. Moreover, our data suggest that PARP regulates NMDAR signaling using a novel, ATP decline-independent mechanism. Our data also indicate the possibility of potentially toxic interactions between clinically used NMDAR antagonists, including ketamine or memantine, and genotoxic therapies that target tumors. We thank Drs. Scott Whittemore, Theo Hagg, Richard Benton, Jennifer Glick, and Jane Cavanaugh for critical reading of this manuscript.

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