Tyrosine Phosphorylation Regulates the Proteolytic Activation of Protein Kinase Cδ in Dopaminergic Neuronal Cells

Abstract

Oxidative stress is a key apoptotic stimulus in neuronal cell death and has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson disease (PD). Recently, we demonstrated that protein kinase C-δ (PKCδ) is an oxidative stress-sensitive kinase that can be activated by caspase-3-dependent proteolytic cleavage to induce apoptotic cell death in cell culture models of Parkinson disease (Kaul, S., Kanthasamy, A., Kitazawa, M., Anantharam, V., and Kanthasamy, A. G. (2003) Eur. J. Neurosci. 18, 1387-1401 and Kanthasamy, A. G., Kitazawa, M., Kanthasamy, A., and Anantharam, V. (2003) Antioxid. Redox. Signal. 5, 609-620). Here we showed that the phosphorylation of a tyrosine residue in PKCδ can regulate the proteolytic activation of the kinase during oxidative stress, which consequently influences the apoptotic cell death in dopaminergic neuronal cells. Exposure of a mesencephalic dopaminergic neuronal cell line (N27 cells) to H2O2(0-300 μm) induced a dose-dependent increase in cytotoxicity, caspase-3 activation and PKCδ cleavage. H2O2-induced proteolytic activation of PKC was δ mediated by the activation of caspase-3. Most interestingly, both the general Src tyrosine kinase inhibitor genistein (25 μm) and the p60Src tyrosine-specific kinase inhibitor (TSKI; 5 μm) dramatically inhibited H2O2 and the Parkinsonian toxin 1-methyl-4-phenylpyridinium-induced PKCδ cleavage, kinase activation, and apoptotic cell death. H2O2 treatment also increased phosphorylation of PKCδ at tyrosine site 311, which was effectively blocked by co-treatment with TSKI. Furthermore, N27 cells overexpressing a PKCδY311F mutant protein exhibited resistance to H2O2-induced PKCδ cleavage, caspase activation, and apoptosis. To our knowledge, these data demonstrate for the first time that phosphorylation of Tyr-311 on PKCδ can regulate the proteolytic activation and proapoptotic function of the kinase in dopaminergic neuronal cells. Oxidative stress is a key apoptotic stimulus in neuronal cell death and has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson disease (PD). Recently, we demonstrated that protein kinase C-δ (PKCδ) is an oxidative stress-sensitive kinase that can be activated by caspase-3-dependent proteolytic cleavage to induce apoptotic cell death in cell culture models of Parkinson disease (Kaul, S., Kanthasamy, A., Kitazawa, M., Anantharam, V., and Kanthasamy, A. G. (2003) Eur. J. Neurosci. 18, 1387-1401 and Kanthasamy, A. G., Kitazawa, M., Kanthasamy, A., and Anantharam, V. (2003) Antioxid. Redox. Signal. 5, 609-620). Here we showed that the phosphorylation of a tyrosine residue in PKCδ can regulate the proteolytic activation of the kinase during oxidative stress, which consequently influences the apoptotic cell death in dopaminergic neuronal cells. Exposure of a mesencephalic dopaminergic neuronal cell line (N27 cells) to H2O2(0-300 μm) induced a dose-dependent increase in cytotoxicity, caspase-3 activation and PKCδ cleavage. H2O2-induced proteolytic activation of PKC was δ mediated by the activation of caspase-3. Most interestingly, both the general Src tyrosine kinase inhibitor genistein (25 μm) and the p60Src tyrosine-specific kinase inhibitor (TSKI; 5 μm) dramatically inhibited H2O2 and the Parkinsonian toxin 1-methyl-4-phenylpyridinium-induced PKCδ cleavage, kinase activation, and apoptotic cell death. H2O2 treatment also increased phosphorylation of PKCδ at tyrosine site 311, which was effectively blocked by co-treatment with TSKI. Furthermore, N27 cells overexpressing a PKCδY311F mutant protein exhibited resistance to H2O2-induced PKCδ cleavage, caspase activation, and apoptosis. To our knowledge, these data demonstrate for the first time that phosphorylation of Tyr-311 on PKCδ can regulate the proteolytic activation and proapoptotic function of the kinase in dopaminergic neuronal cells. Oxidative stress and apoptosis are key mediators of numerous neurodegenerative processes in the nervous system, including Alzheimer (4.Veurink G. Fuller S.J. Atwood C.S. Martins R.N. Ann. Hum. Biol. 2003; 30: 639-667Crossref PubMed Scopus (38) Google Scholar, 5.Pratico D. Sung S. J. Alzheimers Dis. 2004; 6: 171-175Crossref PubMed Scopus (182) Google Scholar) and Huntington disease (6.Borlongan C.V. Kanning K. Poulos S.G. Freeman T.B. Cahill D.W. Sanberg P.R. J. Fla. Med. Assoc. 1996; 83: 335-341PubMed Google Scholar, 7.Schapira A.H. Curr. Opin. 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Recently, we showed that Parkinsonian toxin MPP+ 1The abbreviations used are: MPP+, 1-methyl-4-phenylpyridinium; PD, Parkinson disease; PKC, protein kinase C; ROS, reactive oxygen species; ANOVA, analysis of variance; DAG, diacylglycerol; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; Z, benzyloxy-carbonyl; FMK, fluoromethyl ketone; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein. 1The abbreviations used are: MPP+, 1-methyl-4-phenylpyridinium; PD, Parkinson disease; PKC, protein kinase C; ROS, reactive oxygen species; ANOVA, analysis of variance; DAG, diacylglycerol; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; Z, benzyloxy-carbonyl; FMK, fluoromethyl ketone; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein.-induced ROS generation promotes apoptotic cell death in dopaminergic neurons via caspase-3-mediated proteolytic cleavage of protein kinase C-δ (PKCδ) (1.Kaul S. Kanthasamy A. Kitazawa M. Anantharam V. Kanthasamy A.G. Eur. J. Neurosci. 2003; 18: 1387-1401Crossref PubMed Scopus (142) Google Scholar). PKCδ is a member of the PKC serine-threonine protein kinase family classified into three groups, namely the classical (α, β, and γ activated by DAG and Ca2+), the atypical (ζ and λ/ι DAG and Ca2+-independent), and the novel (δ, ϵ, η, and [phis] activated by DAG but Ca2+-independent). PKCδ activation requires either the phosphorylation of its activation loop residues, leading to enzyme translocation, or the proteolytic cleavage of the kinase to yield catalytically active fragments. In the cellular models of Parkinson disease, we observed a caspase-3 mediated activation of PKCδ without any evidence of membrane translocation (1.Kaul S. Kanthasamy A. Kitazawa M. Anantharam V. Kanthasamy A.G. Eur. J. Neurosci. 2003; 18: 1387-1401Crossref PubMed Scopus (142) Google Scholar). PKCδ is known to be phosphorylated at tyrosine residues Tyr-52, Tyr-155, Tyr-187, Tyr-311, Tyr-332, and Tyr-565 when activated in response to certain stimuli, particularly to the known oxidative stress-inducing agent hydrogen peroxide (H2O2) (19.Denning M.F. Dlugosz A.A. Threadgill D.W. Magnuson T. Yuspa S.H. J. Biol. Chem. 1996; 271: 5325-5331Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 20.Szallasi Z. Denning M.F. Chang E.Y. Rivera J. Yuspa S.H. Lehel C. Olah Z. Anderson W.B. Blumberg P.M. Biochem. Biophys. Res. Commun. 1995; 214: 888-894Crossref PubMed Scopus (70) Google Scholar, 21.Kikkawa U. Matsuzaki H. Yamamoto T. J. Biochem. (Tokyo). 2002; 132: 831-839Crossref PubMed Scopus (193) Google Scholar). Src kinase, a member of the nonreceptor protein-tyrosine kinase family, variably modulates PKCδ activity by increasing tyrosine phosphorylation, depending on the cell type and the insult (22.Blake R.A. Garcia-Paramio P. Parker P.J. Courtneidge S.A. 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Furthermore, recent studies have demonstrated that PKCδ, when phosphorylated on the tyrosine residue Tyr-311, exhibits an increased catalytic activity in H2O2-treated cells (28.Rybin V.O. Guo J. Sabri A. Elouardighi H. Schaefer E. Steinberg S.F. J. Biol. Chem. 2004; 279: 19350-19361Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 29.Konishi H. Yamauchi E. Taniguchi H. Yamamoto T. Matsuzaki H. Takemura Y. Ohmae K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6587-6592Crossref PubMed Scopus (215) Google Scholar). However, the relationship between PKCδ tyrosine phosphorylation and its proteolytic cleavage has never been explored, particularly whether PKCδ tyrosine phosphorylation can regulate its proteolytic activation and proapoptotic function. Here we demonstrate that phosphorylation of the tyrosine residue Tyr-311 in PKCδ is essential for proteolytic activation, and that inhibition of tyrosine phosphorylation can attenuate oxidative stress-induced apoptotic cell death in dopaminergic neuronal cells. Chemicals—Hydrogen peroxide (H2O2), β-actin antibody (mouse monoclonal), histone H1, β-glycerophosphate, ATP, and protein A-Sepharose were purchased from Sigma. PKCδ rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin was obtained from Bachem Biosciences (King of Prussia, PA); and FITC-VAD-FMK was purchased from Promega (Madison, WI). Cell death detection ELISA Plus assay kit (DNA Fragmentation kit) was purchased from Roche Applied Science. Anti-phosphotyrosine (4G10)-agarose conjugate was obtained from Upstate Biotechnology, Inc. (Charlottesville, VA). [γ-32P]ATP was obtained from New England Biolabs. Genistein and TSKI (tyrosine-specific kinase inhibitor) were obtained from Calbiochem. ECL detection kit and [35S]methionine were purchased from Amersham Biosciences. PKCδY311-phospho-specific antibody was obtained from Cell Signaling Technology (Beverly, MA). RPMI 1640, fetal bovine serum, l-glutamine, penicillin, and streptomycin were purchased from Invitrogen. Plasmids encoding PKCδ-CF-GFP and PKCδY311F proteins were kindly provided by Drs. D. Kufe (Dana-Farber Cancer Institute, Harvard Medical School, Boston) (30.Bharti A. Kraeft S.K. Gounder M. Pandey P. Jin S. Yuan Z.M. Lees-Miller S.P. Weichselbaum R. Weaver D. Chen L.B. Kufe D. Kharbanda S. Mol. Cell. Biol. 1998; 18: 6719-6728Crossref PubMed Scopus (201) Google Scholar) and U. Kikkawa (Biosignal Research Center, Kobe University, Kobe, Japan), respectively (29.Konishi H. Yamauchi E. Taniguchi H. Yamamoto T. Matsuzaki H. Takemura Y. Ohmae K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6587-6592Crossref PubMed Scopus (215) Google Scholar). Cell Culture—The immortalized rat mesencephalic dopaminergic neuronal cell line 1RB3AN27, normally referred to as N27 cells, was a kind gift from Dr. Kedar N. Prasad (University of Colorado Health Sciences Center, Denver, CO). N27 cells represent a homogeneous population of tyrosine hydroxylase-positive dopaminergic cells. The cell line is a widely used cell culture model of Parkinson disease (1.Kaul S. Kanthasamy A. Kitazawa M. Anantharam V. Kanthasamy A.G. Eur. J. Neurosci. 2003; 18: 1387-1401Crossref PubMed Scopus (142) Google Scholar, 2.Yang Y. Kaul S. Zhang D. Anantharam V. Kanthasamy A.G. Mol. Cell. Neurosci. 2004; 25: 406-421Crossref PubMed Scopus (59) Google Scholar, 31.Prasad K.N. Clarkson E.D. La Rosa F.G. Edwards-Prasad J. Freed C.R. Mol. Genet. Metab. 1998; 65: 1-9Crossref PubMed Scopus (35) Google Scholar, 32.Anantharam V. Kitazawa M. Wagner J. Kaul S. Kanthasamy A.G. J. Neurosci. 2002; 22: 1738-1751Crossref PubMed Google Scholar, 34.Latchoumycandane C. Anantharam V. Kitazawa M. Yang Y. Kanthasamy A. Kanthasamy A.G. J. Pharmacol. Exp. Ther. 2005; 313: 46-55Crossref PubMed Scopus (130) Google Scholar, 35.Zhou W. Hurlbert M.S. Schaack J. Prasad K.N. Freed C.R. Brain Res. 2000; 866: 33-43Crossref PubMed Scopus (192) Google Scholar). The cells were grown in RPMI 1640 medium containing 10% fetal bovine serum, 2 mm l-glutamine, 50 units of penicillin, and 50 μg/ml streptomycin. Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C as described previously (31.Prasad K.N. Clarkson E.D. La Rosa F.G. Edwards-Prasad J. Freed C.R. Mol. Genet. Metab. 1998; 65: 1-9Crossref PubMed Scopus (35) Google Scholar, 32.Anantharam V. Kitazawa M. Wagner J. Kaul S. Kanthasamy A.G. J. Neurosci. 2002; 22: 1738-1751Crossref PubMed Google Scholar). Treatment Paradigm—H2O2 (0-300 μm) or MPP+ (300 μm) was added to the cells for the duration of the experiment. The cells were removed from the flask by using a cell scraper and centrifuged at 200 × g for 5 min, washed with PBS twice, and homogenized as described previously (1.Kaul S. Kanthasamy A. Kitazawa M. Anantharam V. Kanthasamy A.G. Eur. J. Neurosci. 2003; 18: 1387-1401Crossref PubMed Scopus (142) Google Scholar). Cell lysates, collected by spinning down the cell fragments at 20,000 × g for 45 min at 4 °C, were used for immunoprecipitation studies to determine caspase-3 enzyme activity, DNA fragmentation, and PKCδ cleavage. Untreated cells were grown in the complete medium and used as control samples. For real time fluorescence imaging, the cells were grown in 24-well plates and viewed in the culture wells. Cytotoxicity Assays—Cell death was determined after exposing the N27 cells to H2O2 (100 μm) using the Sytox® green cytotoxicity assay and the LIVE/DEAD® viability/cytotoxicity kit (Molecular Probes, Eugene, OR). The Sytox® green cytotoxicity assay is based on the principle that Sytox® green cannot enter cells with intact membranes (live cells) but permeates cells with compromised plasma membranes and intercalates with DNA to produce green fluorescence (36.Roth B.L. Poot M. Yue S.T. Millard P.J. Appl. Environ. Microbiol. 1997; 63: 2421-2431Crossref PubMed Google Scholar, 37.Sherer T.B. Betarbet R. Stout A.K. Lund S. Baptista M. Panov A.V. Cookson M.R. Greenamyre J.T. J. Neurosci. 2002; 22: 7006-7015Crossref PubMed Google Scholar). Briefly, N27 cells were grown in 24-well cell culture plates at equal densities and treated with H2O2 (0-300 μm) and 1 μm Sytox® green fluorescent dye for a period of 4 h. The Sytox® green assay allows dead cells to be viewed directly under the fluorescence microscope as well as quantitatively measured with a fluorescence microplate reader (excitation 485 nm; emission 538 nm) (SpectraMax Gemini XS model, Molecular Devices, Sunnyvale, CA). The LIVE/DEAD® kit consists of a combination of two dyes: SYTO 10 (green fluorescence), a highly cell permeable cell dye which stains all cells, and an ethidium homodimer (DEAD Red; red fluorescence), a membrane impermeant dye that only binds to nucleic acids in cells with compromised cell membranes. Fluorescent images were taken after exposure to H2O2 with a NIKON TE2000 microscope, and pictures were captured with a SPOT digital camera. In Situ Fluorometric Analysis of Caspase Activity—FITC-VAD-FMK, a cell-permeable fluorescent probe that binds to active caspase-3, was used as an in situ marker for caspase activity. The entire procedure was performed according to Promega's CaspACE® kit, as described previously (1.Kaul S. Kanthasamy A. Kitazawa M. Anantharam V. Kanthasamy A.G. Eur. J. Neurosci. 2003; 18: 1387-1401Crossref PubMed Scopus (142) Google Scholar). Fluorescent images were captured using a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI). Enzymatic Assay for Caspases—Caspase-3 activity was measured as described previously (32.Anantharam V. Kitazawa M. Wagner J. Kaul S. Kanthasamy A.G. J. Neurosci. 2002; 22: 1738-1751Crossref PubMed Google Scholar, 38.Yoshimura S. Banno Y. Nakashima S. Takenaka K. Sakai H. Nishimura Y. Sakai N. Shimizu S. Eguchi Y. Tsujimoto Y. Nozawa Y. J. Biol. Chem. 1998; 273: 6921-6927Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Acetyl-DEVD-amino-4-methylcoumarin (50 μm) was the fluorometric caspase-3 substrate used for the reaction. Enzymatic activity, measured using a Spectramax microplate reader at 405 nm, was represented as fluorescence units/mg of protein. Western Blot Analysis—Cells were collected after exposure to 100 μm H2O2, resuspended in 300 μl of homogenization buffer (20 mm Tris-HCl (pH 8.0), 2 mm EDTA, 10 mm EGTA, 2 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 25 μg/ml aprotinin, and 10 μg/ml leupeptin), sonicated, and then centrifuged at 10,000 × g for 1 h at 4 °C (1.Kaul S. Kanthasamy A. Kitazawa M. Anantharam V. Kanthasamy A.G. Eur. J. Neurosci. 2003; 18: 1387-1401Crossref PubMed Scopus (142) Google Scholar). Proteins were separated by 10-12% SDS-PAGE. PKCδ polyclonal (1: 2000), PKCδ-Tyr-311 (1:500), and β-actin (1:5000) antibodies were used to blot the membranes. Secondary horseradish peroxidase-conjugated anti-rabbit (1:2000) and anti-mouse (1:2000) were used for antibody detection with an ECL detection kit (Amersham Biosciences). Immunoprecipitation and Kinase Assay—Immunoprecipitation studies were conducted to determine the phosphorylative changes in the PKCδ protein obtained from H2O2-treated N27 cells. Briefly, cells were washed once with 1× PBS and resuspended in 500 μl of PKC lysis buffer (25 mm HEPES (pH 7.5), 20 mm β-glycerophosphate, 0.1 mm sodium orthovanadate, 0.1% Triton X-100, 0.3 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, 10 mm NaF, and 4 μg/ml each of aprotinin and leupeptin) (1.Kaul S. Kanthasamy A. Kitazawa M. Anantharam V. Kanthasamy A.G. Eur. J. Neurosci. 2003; 18: 1387-1401Crossref PubMed Scopus (142) Google Scholar). The cell suspension was kept on ice for 30 min and then centrifuged at 13,000 × g for 5 min. The resultant supernatant was collected as the cytosolic fraction. Cytosolic protein (∼200 μg total) was immunoprecipitated overnight at 4 °C using 20-40 μl of anti-phosphotyrosine (4G10)-agarose conjugated antibody. The Sepharose-bound antigen-antibody complexes were washed three times with buffer. Samples were then mixed with 2× SDS-PAGE loading buffer, boiled for 5 min, and then separated on 10-12% SDS-PAGE. For the kinase assay, the immunoprecipitation was done by using a polyclonal PKCδ rabbit antibody and protein-Sepharose A and washed three times with kinase buffer (40 mm Tris (pH 7.4), 20 mm MgCl2, 20 μm ATP, 2.5 mm CaCl2). The reaction was started by adding 20 μl of buffer containing 0.4 mg of histone and 5 μCi of [γ-32P]ATP (4,500 Ci/mm). After incubation for 10 min at 30 °C, SDS loading buffer (2×) was added to the samples to terminate the reaction. The reaction products were separated on SDS-PAGE (12.5%), and the H1-phosphorylated bands were detected using a Personal Molecular Imager (FX model, Bio-Rad Labs) and quantified with Quantity One 4.2.0 software. DNA Fragmentation Assay—DNA fragmentation was measured using a recently developed Cell Death Detection ELISA Plus assay kit, a fast, highly sensitive, and reliable assay for the detection of early apoptotic death (32.Anantharam V. Kitazawa M. Wagner J. Kaul S. Kanthasamy A.G. J. Neurosci. 2002; 22: 1738-1751Crossref PubMed Google Scholar, 39.Reyland M.E. Anderson S.M. Matassa A.A. Barzen K.A. Quissell D.O. J. Biol. Chem. 1999; 274: 19115-19123Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Briefly, after treatment with 100 μm H2O2, the cells were spun down at 200 × g for 5 min and washed once with PBS. Cells were then lysed in 450 μl of lysis buffer (provided with the kit) and spun down again at 5,000 rpm for 10 min to collect the supernatant, which was used to measure DNA fragmentation as per the manufacturer's protocol. Readings were taken in a Spectramax multiwell plate reader at 405 nm, with 490 nm as a reference reading. Measurement of ROS Generation—The ROS generation in N27 cells was measured using the fluorescence probe dihydroethidine, as described previously (40.Bindokas V.P. Jordan J. Lee C.C. Miller R.J. J. Neurosci. 1996; 16: 1324-1336Crossref PubMed Google Scholar). Briefly, N27 cells were plated in 24-well plates at a density of 2 × 106 cells per well for a period of 24 h prior to treatment. After 24 h, the RPMI culture medium was removed from the wells and replaced with clear HBSS medium supplemented with 2 mm CaCl2. 1 μm dihydroethidine (final concentration) was added to the HBSS for a period of 15 min. The cells were then treated with either H2O2 (100 μm) alone or along with the tyrosine kinase inhibitors genistein (25 μm) and TSKI (5 μm) for a period of 1 h. After the 1-h incubation period, fluorescence was measured by using a microplate reader (Beckman and Coulter). The data were quantified using SpectraMax spectrophotometer analysis software. Transient Transfections—cDNA encoding PKCδ catalytic fragment (PKCδ-CF) from the pEGFPN1 vector was subcloned into the lentiviral expression vector plenti6/V5-d-TOPO (herein referred to as plenti/PKCδ-CF) by PCR using standard cloning procedures. PKCδY311F encoded in pcDNA3 vector encodes a protein in which tyrosine residue at position 311 is mutated to phenylalanine. The expression of PKCδ-CF in mammalian cells can be monitored by using an antibody directed against V5 epitope. Transfections of PKCδ-CF and PKCδY311F mutants were done by using an AMAXA® Nucleofector™ kit for cell lines (AMAXA GmbH, Germany). Plasmid pCDNA3.1 was used as vector control. Briefly, N27 cells were grown in T-175 flasks at a density of 3 × 106 per ml and harvested for the transfection procedure. The Nucleofector™ solution V was primed by adding a supplement solution (provided by manufacturer) and plasmid DNA. The cells were then resuspended in this DNA-containing Nucleofector solution at an optimal density of 3 million cells per 100 μl of solution. Electroporation was carried out with AMAXA® Nucleofector™ transfector instrument as per the manufacturer's protocol. The procedure was repeated for each subsequent sample. 100 μl of cell suspension containing 5 μg of pmax GFP DNA (provided with the kit) was used to determine the transfection efficiency. The transfected cells were then transferred to T-75 flasks or 6-well plates as desired and allowed to grow for a 24-h period before being used for the treatment paradigm. Transfection efficiency was determined to be >75% as determined by GFP expression. Statistical Analysis—Data were analyzed with Prism 3.0 software (GraphPad Software, San Diego, CA). Bonferroni's and Dunnett's multiple comparison testing were used in order to delineate significant differences between the MPP+-treated groups and the control (untreated) and the inhibitor-treated samples. Differences with p < 0.05, p < 0.01, and p < 0.001 were considered to be statistically significant and are indicated by asterisks in the figures. Low Dose H2O2 Exposure Induces Dose-dependent Cell Death in Dopaminergic Neuronal Cells—H2O2 is an oxidative insult commonly used to investigate oxidative stress-induced apoptotic signaling in many cell types including dopaminergic neuronal cells (41.Chang H. Oehrl W. Elsner P. Thiele J.J. Free Radic. Res. 2003; 37: 655-663Crossref PubMed Scopus (66) Google Scholar, 42.Kumar S. Bharti A. Mishra N.C. Raina D. Kharbanda S. Saxena S. Kufe D. J. Biol. Chem. 2001; 276: 17281-17285Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). To determine the effects of low dose oxidative stress on the dopaminergic system, we treated mesencephalic dopaminergic clonal cells (N27) with low doses of H2O2 (0-300 μm) for a period of 4 h, and cytotoxicity was assayed at 1-h intervals over the entire treatment period. Fig. 1A demonstrates a fluorescent image from a group of N27 cells treated with H2O2 (100 μm) for 4 h and assayed for cell death by a LIVE/DEAD® viability/cytotoxicity kit (Molecular Probes). H2O2 treatment induced a clear increase in the number of dead cells, as evident from the increase in red fluorescence-labeled cells (dead cells) compared with the number of cells exhibiting green fluorescence (live cells). In untreated cells, only a very few red fluorescence-positive cells were observed. We also measured the cytotoxicity both qualitatively and quantitatively by using another fluorescence dye Sytox® green, which stains only dead/dying cells. Fig. 1B is representative of untreated N27 cells (top row) and cells treated with H2O2 (100 μm) at the end of a 4-h treatment period (bottom row) in both phase contrast and FITC fluorescence imaging. An increase in green fluorescence indicates an increase in cell death in H2O2-treated cells, because the Sytox® green dye only permeates compromised cell membranes to stain the nuclear chromatin. Fig. 1C depicts the quantitative measurement by a microplate reader of Sytox® green fluorescence and demonstrates a dose-dependent increase of cell death in N27 cells treated with varying doses of H2O2 (0-300 μm). H2O2 increased cytotoxicity by 216, 380, 468, and 638% over untreated controls at 10, 30, 100, and 300 μm concentrations 4 h after exposure, demonstrating dose-dependent oxidative stress-induced cell death in N27 cells. Because 100 μm H2O2 consistently induced significant oxidative damage in these cells, we used this concentration for all subsequent experimental analyses in this study. H2O2 Induces a Time-dependent Increase in Caspase-3-mediated Cellular Apoptosis in N27 Dopaminergic Cells—To determine whether apoptotic cell death occurs during H2O2 treatment, we measured the activity of the key apoptotic cellular enzyme caspase-3 as well as the extent of DNA fragmentation in the treated cells. H2O2 (100 μm) induced a time-dependent increase in caspase-3 activity over a 4-h treatment period (Fig. 2A). Treatment with 100 μm H2O2 induced 260, 438, 1055, and 1369% increases in caspase-3 activity in N27 cells at 90, 120, 150, and 240 min post-exposure. We further confirmed the activation of caspases during oxidative insult by labeling the activated caspase enzyme with the fluorescent substrate Z-VAD-FITC followed by observation under a fluorescence microscope (Fig. 2A, inset). H2O2 treatment induced a significant increase in the fluorescent labeling of the N27 cells after the 4-h post-exposure as compared with the untreated cells. Cellular apoptosis is often marked by the final precipitating events of chromatin breakdown and DNA fragmentation, which are considered hallmarks of programmed cell death. Therefore, we determined the extent of DNA fragmentation following H2O2 treatment in N27 cells using a DNA ELISA technique. H2O2 (100 μm) induced time-dependent increases in DNA fragmentation of 109, 154, and 392% at 1, 2, and 4 h, respectively, as compared with the untreated cells (Fig. 2B). Together, these data clearly indicate that N27 dopaminergic cells are highly sensitive to low dose oxidative stress and can undergo activation of caspase-3 and subsequent DNA fragmentation. Oxidative Stress Induces Caspase-3-mediated Proteolytic Cleavage of PKCδ—We showed previously (1.Kaul S. Kanthasamy A. Kitazawa M. Anantharam V. Kanthasamy A.G. Eur. J. Neurosci. 2003; 18: 1387-1401Crossref PubMed Scopus (142) Google Scholar, 32.Anantharam V. Kitazawa M. Wagner J. Kaul S. Kanthasamy A.G. J. Neurosci. 2002; 22: 1738-1751Crossref PubMed Google Scholar) that apoptotic cell death is mediated by caspase-3-dependent proteolytic activation in Parkinson disease models. To determine whether oxidative stress induces the proteolytic activation of PKCδ in dopaminergic neuronal cells, we examined the effect of H2O2 on PKCδ cleavage in N27 cells. H2O2 (100 μm) induced a time-dependent proteolytic cleavage of the full-length PKCδ (72-74 kDa) to cleaved catalytically active fragments migrating at 42 and 38 kDa, respectively (Fig. 3A). Additionally, H2O2-induced PKCδ cleavage was completely inhibited by co-treatment with 50