Oxidative Stress-induced Apoptotic Cell Death Research Articles

The E3 Ligase RNF157 Inhibits Lens Epithelial Cell Apoptosis by Negatively Regulating p53 in Age-Related Cataracts.

PurposeAge-related cataract (ARC) is a major cause of vision impairment worldwide. The E3 ubiquitin ligase RING finger protein 157 (RNF157) is involved in regulating cell survival and downregulated in human cataractous lens samples. However, the function of RNF157 in cataracts remains unclear. This study aimed to determine the role of RNF157 in ARC.MethodsReal-time polymerase chain reaction (PCR) and Western blotting were used to analyze the expression of RNF157 in clinical lens capsules, rat cataract models, and oxidative stress cell models. Western blot analysis and flow cytometry were used to evaluate cell apoptosis. Co-IP assay, protein stability assay, and ubiquitination assay were used to detect the interaction between RNF157 and its substrate p53.ResultsThe expression of RNF157 was downregulated in human cataract samples, UVB-induced rat cataract model, and H2O2-treated human lens epithelial cells (LECs). Ectopic expression of RNF157 protected LECs from H2O2-induced apoptosis. In contrast, knockdown of RNF157 enhanced oxidative stress-induced apoptotic cell death. Moreover, silence of RNF157 in the rat ex vivo lens model exacerbated lens opacity. Mechanistically, RNF157 causes ubiquitination and degradation of the tumor antigen p53. Overexpression of p53 eliminated the antiapoptotic effects of RNF157, whereas p53 knockdown rescued RNF157 silencing-induced cell death.ConclusionsOur findings revealed that reduced RNF157 expression promoted LEC apoptosis by upregulating p53 in cataracts, suggesting that the regulation of RNF157 expression may serve as a potential therapeutic strategy for cataracts.

Connexin hemichannels regulate redox potential via metabolite exchange and protect lens against cellular oxidative damage

Increased oxidative stress contributes to cataract formation during aging. Anterior epithelial cells are a frontline antioxidant defense system with powerful capacities to maintain redox homeostasis and lens transparency. In this study, we report a new molecular mechanism of connexin (Cx) hemichannels (HCs) in lens epithelial cells to protect lens against oxidative stress. Our results showed haploinsufficiency of Cx43 elevated oxidative stress and susceptibility to cataracts in the mouse lens. Cx43 HCs opened in response to hydrogen peroxide (H2O2) or ultraviolet radiation (UVR) in human lens epithelium HLE-B3 cells, and this activation contributed to a cellular protective mechanism against oxidative stress-induced apoptotic cell death. Furthermore, we found that Cx43 HCs mediated the exchange of oxidants and antioxidants in lens epithelial cells undergoing oxidative stress. These transporting activities facilitated a reduction of intracellular reactive oxygen species (ROS) accumulation and maintained the intracellular glutathione (GSH) level through the exchange of redox metabolites and change of anti-oxidative gene expression. In addition, we show that Cx43 HCs can be regulated by the intracellular redox state and this regulation is mediated by residue Cys260 located at the Cx43 C-terminus. Together, our results demonstrate that Cx43 HCs activated by oxidative stress in the lens epithelial cells play a key role in maintaining redox homeostasis in lens under oxidative stress. Our findings contribute to advancing our understanding of oxidative stress induced lens disorders, such as age-related non-congenital cataracts.

Sirt3-dependent deacetylation of COX-1 counteracts oxidative stress-induced cell apoptosis.

The mitochondrial complexes are prone to sirtuin (Sirt)3-mediated deacetylation modification, which may determine cellular response to stimuli, such as oxidative stress. In this study, we show that the cytochrome c oxidase (COX)-1, a core catalytic subunit of mitochondrial complex IV, was acetylated and deactivated both in 2,2'-azobis(2-amidinopropane) dihydrochloride-treated NIH/3T3 cells and hydrogen peroxide-treated primary neuronal cells, correlating with apoptotic cell death induction by oxidative stress. Inhibition of Sirt3 by small interfering RNA or the inhibitor nicotinamide induced accumulation of acetylation of COX-1, reduced mitochondrial membrane potential, and increased cell apoptosis. In contrast, overexpression of Sirt3 enhanced deacetylation of COX-1 and inhibited oxidative stress-induced apoptotic cell death. Significantly, rats treated with ischemia/reperfusion injury, a typical oxidative stress-related disease, presented an inhibition of Sirt3-induced hyperacetylation of COX-1 in the brain tissues. Furthermore, K13, K264, K319, and K481 were identified as the acetylation sits of COX-1 in response to oxidative stress. In conclusion, COX-1 was discovered as a new deacetylation target of Sirt3, indicating that the Sirt3/COX-1 axis is a promising therapy target of stress-related diseases.-Tu, L.-F., Cao, L.-F., Zhang, Y.-H., Guo, Y.-L., Zhou, Y.-F., Lu, W.-Q., Zhang, T.-Z., Zhang, T., Zhang, G.-X., Kurihara, H., Li, Y.-F., He, R.-R. Sirt3-dependent deacetylation of COX-1 counteracts oxidative stress-induced cell apoptosis.

Neuroprotective effects of Momordica charantia extract against hydrogen peroxide-induced cytotoxicity in human neuroblastoma SK-N-MC cells

Purpose: Many studies have suggested that neuronal cells protect against oxidative stress-induced apoptotic cell death by polyphenolic compounds. We investigated the neuroprotective effects and the mechanism of action of Momordica charantia ethanol extract (MCE) against H2O2-induced cell death of hu...

Cytoprotective Mechanism of Cyanidin and Delphinidin against Oxidative Stress-Induced Tenofibroblast Death.

Age-related rotator cuff tendon degeneration is related to tenofibroblast apoptosis. Anthocyanins reduce oxidative stress-induced apoptotic cell death in tenofibroblasts. The current study investigated the presence of cell protective effects in cyanidin and delphinidin, the most common aglycon forms of anthocyanins. We determined whether these anthocyanidins have antiapoptotic and antinecrotic effects in tenofibroblasts exposed to H2O2, and evaluated their biomolecular mechanisms. Both cyanidin and delphinidin inhibited H2O2-induced apoptosis in a dose-dependent manner. However, at concentrations of 100 μg/ml or greater, delphinidin showed cytotoxicity against tenofibroblasts and a decreased antinecrotic effect. Cyanidin and delphinidin both showed inhibitory effects on the H2O2-induced increase in intracellular ROS formation and the activation of ERK1/2 and JNK. In conclusion, both cyanidin and delphinidin have cytoprotective effects on cultured tenofibroblasts exposed to H2O2. These results suggest that cyanidin and delphinidin are both beneficial for the treatment of oxidative stress-mediated tenofibroblast cell death, but their working concentrations are different.

Lysosomal membrane permeabilization is involved in oxidative stress-induced apoptotic cell death in LAMP2-deficient iPSCs-derived cerebral cortical neurons

Patients with Danon disease may suffer from severe cardiomyopathy, skeletal muscle dysfunction as well as varying degrees of mental retardation, in which the primary deficiency of lysosomal membrane-associated protein-2 (LAMP2) is considerably associated. Owing to the scarcity of human neurons, the pathological role of LAMP2 deficiency in neural injury of humans remains largely elusive. However, the application of induced pluripotent stem cells (iPSCs) may shed light on overcoming such scarcity.In this study, we obtained iPSCs derived from a patient carrying a mutated LAMP2 gene that is associated with Danon disease. By differentiating such LAMP2-deficient iPSCs into cerebral cortical neurons and with the aid of various biochemical assays, we demonstrated that the LAMP2-deficient neurons are more susceptible to mild oxidative stress-induced injury.The data from MTT assay and apoptotic analysis demonstrated that there was no notable difference in cellular viability between the normal and LAMP2-deficient neurons under non-stressed condition. When exposed to mild oxidative stress (10μM H2O2), the LAMP2-deficient neurons exhibited a significant increase in apoptosis. Surprisingly, we did not observe any aberrant accumulation of autophagic materials in the LAMP2-deficient neurons under such stress condition.Our results from cellular fractionation and inhibitor blockade experiments further revealed that oxidative stress-induced apoptosis in the LAMP2-deficient cortical neurons was caused by increased abundance of cytosolic cathepsin L. These results suggest the involvement of lysosomal membrane permeabilization in the LAMP2 deficiency associated neural injury.

Anti-apoptotic effect of taxodione on serum/glucose deprivation-induced PC12 cells death.

Taxodione, a diterpenoid from the roots of Salvia chorassanica Bunge, possesses cytotoxic, apoptotic, and antimicrobial activity. This study was designed to investigate the protective effects of taxodione on serum/glucose deprivation-induced ischemic injury in PC12 cells and related mechanisms. In an in vitro model of ischemia, PC12 cells were exposed to serum and glucose deprivation for 6 and 18h. The protective effects of the methanol extract of S. chorassanica and taxodione were assessed using alamarBlue(®) assay. Intracellular ROS production was measured by fluorimetry using 2',7'-dichlorofluorescin diacetate (DCFH-DA). The levels of PARP, Bcl-2, and Bax proteins were detected after western blot analysis. It was shown that taxodione (0.2-1.5μM) significantly increased cell viability in a dose-dependent manner after ischemic insult. Taxodione has antioxidant activity and protects PC12 cells against oxidative stress-induced apoptotic cell death. Meanwhile, pretreatment with taxodione significantly induced an increase in Bcl-2 and a decrease in Bax protein level. The results of this study confirmed the protective effect of taxodione in serum/glucose deprivation-induced ischemic injury and the putative role of apoptosis as a underling mechanisms. Thus, it would be fair to consider taxodione as a promising ingredient of S. chorassanica for the expansion on novel class of anti-ischemic agents.

Carboxypeptidase E/NFα1: A New Neurotrophic Factor against Oxidative Stress-Induced Apoptotic Cell Death Mediated by ERK and PI3-K/AKT Pathways

Mice lacking Carboxypeptidase E (CPE) exhibit degeneration of hippocampal neurons caused by stress at weaning while over-expression of CPE in hippocampal neurons protect them against hydrogen peroxide-induced cell death. Here we demonstrate that CPE acts as an extracellular trophic factor to protect neurons. Rat hippocampal neurons pretreated with purified CPE protected the cells against hydrogen peroxide-, staurosporine- and glutamate-induced cell death. This protection was observed even when hippocampal neurons were treated with an enzymatically inactive mutant CPE or with CPE in the presence of its inhibitor, GEMSA. Purified CPE added to the culture medium rescued CPE knock-out hippocampal neurons from cell death. Both ERK and AKT were phosphorylated within 15 min after CPE treatment of hippocampal neurons and, using specific inhibitors, both signaling pathways were shown to be required for the neuroprotective effect. The expression of the anti-apoptotic protein, B-cell lymphoma 2 (BCL-2), was up-regulated after hippocampal neurons were treated with CPE. Furthermore, hydrogen peroxide induced down-regulation of BCL-2 protein and subsequent activation of caspase-3 were inhibited by CPE treatment. Thus, this study has identified CPE as a new neurotrophic factor that can protect neurons against degeneration through the activation of ERK and AKT signaling pathways to up-regulate expression of BCL-2.

TDAG51 deficiency promotes oxidative stress-induced apoptosis through the generation of reactive oxygen species in mouse embryonic fibroblasts

Apoptosis has an important role in maintaining tissue homeostasis in cellular stress responses such as inflammation, endoplasmic reticulum stress, and oxidative stress. T-cell death-associated gene 51 (TDAG51) is a member of the pleckstrin homology-like domain family and was first identified as a pro-apoptotic gene in T-cell receptor-mediated cell death. However, its pro-apoptotic function remains controversial. In this study, we investigated the role of TDAG51 in oxidative stress-induced apoptotic cell death in mouse embryonic fibroblasts (MEFs). TDAG51 expression was highly increased by oxidative stress responses. In response to oxidative stress, the production of intracellular reactive oxygen species was significantly enhanced in TDAG51-deficient MEFs, resulting in the activation of caspase-3. Thus, TDAG51 deficiency promotes apoptotic cell death in MEFs, and these results indicate that TDAG51 has a protective role in oxidative stress-induced cell death in MEFs.

Salvia macilenta exhibits antiglycating activity and protects PC12 cells against H2O2-induced apoptosis

Salvia macilenta is a member of the genus Salvia (Laminaceae) whose antioxidant activity and neuroprotective effect has been shown previously. The present study aimed to examine the antiglycating and antiapoptotic abilities of methanolic extract of this plant. Moreover, the effect of S. macilenta on neurite outgrowth and complexity after exposure to H2O2 has been studied. Base on our results, S. macilenta has antiglycating activity and protects PC12 cells against oxidative stress-induced apoptotic cell death, as examined by Hoechst staining and Western blot analysis of caspase-3, Bax, Bcl-2 and PARP. We further showed that S. macilenta decreased neurite growth and complexity impairment in differentiated PC12 cells exposed to oxidative stress. It caused a decrease in cell body area, neurite width, and the proportion of bipolar cells, while significantly increasing neurite length, the number of primary neurites per cell and the ratio of nodes to primary neuritis. All around, the mentioned results open a new horizon for future works to use this plant as a potential neuroprotective agent.

Nuclear Factor-κB (NF-κB) Inhibitory Protein IκBβ Determines Apoptotic Cell Death following Exposure to Oxidative Stress

The transcription factor NF-κB regulates the cellular response to inflammatory and oxidant stress. Although many studies have evaluated NF-κB activity following exposure to oxidative stress, the role of the IκB family of inhibitory proteins in modulating this activity remains unclear. Specifically, the function of IκBβ in mediating the cellular response to oxidative stress has not been evaluated. We hypothesized that blocking oxidative stress-induced NF-κB signaling through IκBβ would prevent apoptotic cell death. Using IκBβ knock-in mice (AKBI), in which the IκBα gene is replaced with the IκBβ cDNA, we show that IκBβ overexpression prevented oxidative stress-induced apoptotic cell death. This was associated with retention of NF-κB subunits in the nucleus and maintenance of NF-κB activity. Furthermore, the up-regulation of pro-apoptotic genes in WT murine embryonic fibroblasts (MEFs) exposed to serum starvation was abrogated in AKBI MEFs. Inhibition of apoptosis was observed in WT MEFs overexpressing IκBβ with simultaneous IκBα knockdown, whereas IκBβ overexpression alone did not produce this effect. These findings represent a necessary but not sufficient role of IκBβ in preventing oxidant stress-induced cell death.

Protective effect of the octadecaneuropeptide on hydrogen peroxide‐induced oxidative stress and cell death in cultured rat astrocytes

Oxidative stress, resulting from accumulation of reactive oxygen species (ROS), plays a critical role on astrocyte death associated with neurodegenerative diseases. Astroglial cells produce endozepines, a family of biologically active peptides that have been implicated in cell protection. Thus, the purpose of the present study was to investigate the potential protective effect of one of the endozepines, the octadecaneuropeptide ODN, on hydrogen peroxide (H(2) O(2) )-induced oxidative stress and cell death in rat astrocytes. Incubation of cultured astrocytes with graded concentrations of H(2) O(2) for 1 h provoked a dose-dependent reduction of the number of living cells as evaluated by lactate dehydrogenase assay. The cytotoxic effect of H(2) O(2) was associated with morphological modifications that were characteristic of apoptotic cell death. H(2) O(2) -treated cells exhibited high level of ROS associated with a reduction of both superoxide dismutases (SOD) and catalase activities. Pre-treatment of astrocytes with low concentrations of ODN dose-dependently prevented cell death induced by H(2) O(2) . This effect was accompanied by a marked attenuation of ROS accumulation, reduction of mitochondrial membrane potential and activation of caspase 3 activity. ODN stimulated SOD and catalase activities in a concentration-dependent manner, and blocked H(2) O(2) -evoked inhibition of SOD and catalase activities. Blockers of SOD and catalase suppressed the effect of ODN on cell survival. Taken together, these data demonstrate for the first time that ODN is a potent protective agent that prevents oxidative stress-induced apoptotic cell death.

Novel cell death signaling pathways in neurotoxicity models of dopaminergic degeneration: Relevance to oxidative stress and neuroinflammation in Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative movement disorder characterized by extensive degeneration of dopaminergic neurons in the nigrostriatal system. Neurochemical and neuropathological analyses clearly indicate that oxidative stress, mitochondrial dysfunction, neuroinflammation and impairment of the ubiquitin-proteasome system (UPS) are major mechanisms of dopaminergic degeneration. Evidence from experimental models and postmortem PD brain tissues demonstrates that apoptotic cell death is the common final pathway responsible for selective and irreversible loss of nigral dopaminergic neurons. Epidemiological studies imply both environmental neurotoxicants and genetic predisposition are risk factors for PD, though the cellular mechanisms underlying selective dopaminergic degeneration remain unclear. Recent progress in signal transduction research is beginning to unravel the complex mechanisms governing dopaminergic degeneration. During the 12th International Neurotoxicology meeting, discussion at one symposium focused on several key signaling pathways of dopaminergic degeneration. This review summarizes two novel signaling pathways of nigral dopaminergic degeneration that have been elucidated using neurotoxicity models of PD. Dr. Anumantha Kanthasamy described a cell death pathway involving the novel protein kinase C delta isoform (PKCδ) in oxidative stress-induced apoptotic cell death in experimental models of PD. Dr. Ajay Rana presented his recent work on the role of mixed lineage kinase-3 (MLK3) in neuroinflammatory processes in neurotoxic cell death. Collectively, PKCδ and MLK3 signaling pathways provide new understanding of neurodegenerative processes in PD, and further exploration of these pathways may translate into effective neuroprotective drugs for the treatment of PD.

PACAP ameliorates oxidative stress in the chicken inner ear: An in vitro study

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide. Numerous studies prove that PACAP has neuroprotective effects in diverse neuronal systems in vitro and in vivo. The involvement of PACAP in visual and olfactory sensory processing has also been documented, but little is known about its effects in the auditory system. The presence of PACAP and its receptor, the specific PAC1 receptor, has been shown in the cochlea and in brain structures involved in auditory pathways. The aim of the present study was to investigate whether PACAP is protective in cochlear oxidative stress-induced cell death, which is known to play a role in several ototoxic insults. Chicken cochlear cells were exposed to 1 mM H 2O 2, which resulted in a marked reduction of cell viability and a parallel increase of apoptotic and necrotic cells assessed by MTT test, annexin V/propidium iodide flow cytometry and JC-1 apoptosis assay. Co-incubation with 100 nM PACAP increased cell viability and reduced the percentage of apoptotic cells. Furthermore, oxidative stress increased the activation of caspase-3, while simultaneous PACAP treatment reduced it. In summary, our present results demonstrate that PACAP effectively protects cochlear cells against oxidative stress-induced apoptotic cell death.

A new perspective for the role of octadecaneuropeptide ODN in neurodegenerative diseases

Event Abstract Back to Event A new perspective for the role of octadecaneuropeptide ODN in neurodegenerative diseases Olfa Masmoudi-Kouki1*, Yosra Hamdi1, Hadhemi Kaddour1, David Vaudry2, 3, Faouzi Jenhani4, Jerome Leprince2, 3, Marie-Christine Tonon2, 3 and Mohamed Amri1 1 Faculty of Science of Tunis, University Tunis El Manar, Laboratory of Functional Neurophysiology and Pathology, 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, Tunisia 2 Inserm U413-EA4310, Inserm U413-EA4310, France 3 Laboratory of Neuronal and Neuroendocrine Communication and differentiation, European Institute for Peptide Research (IFRMP 23), Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN) , France 4 National Centre of Blood Transfusion, El Manar II 2092, Laboratory of Cellular Immunology and Cytometry, Tunisia Oxidative stress, resulting from accumulation of reactive oxygen species, plays a critical role on astroglial cell death associated with neurodegenerative diseases and stroke. Astroglial cells synthesize and release endozepines, a family of biologically active peptides that have been implicated in cellular protection. Thus, the purpose of the present study was to investigate the potential protective effect of one of the endozepines, the octadecaneuropeptide ODN, on H2O2-induced oxidative stress and cell death in rat astrocytes. Incubation of cultured astrocytes with graded concentrations of H2O2 for 1 h provoked a dose-dependent reduction of the number of living cells as evaluated by flow cytometric analysis and lactate dehydrogenase assay. The cytotoxic effect of H2O2 was associated with morphological modifications that are characteristic of apoptotic cell death. H2O2-treated cells exhibited high level of reactive oxygen species associated with a reduction of both superoxide dismutases (SOD) and catalase antioxidant enzymatic activities. Pre-treatment of astrocytes with very low concentrations of ODN (0.01 pM – 0.1 nM), dose-dependently prevented cell death induced by H2O2, and the effect of ODN was accompanied by a marked attenuation of ROS accumulation. ODN stimulated SOD and catalase activities in a concentration-dependent manner, and blocked H2O2-evoked inhibition of SOD and catalase activities. In addition, ODN totally suppressed H2O2-induced reduction of mitochondrial membrane potential. Taken together, these data demonstrate for the first time that the endozepine ODN is a potent protective agent that prevents oxidative stress-induced apoptotic cell death. Endozepines are thus potential candidates for development into therapeutically valuable agents for the treatment of cerebral injuries. Keywords: Apoptosis, Astrocytes, Catalase, Superoxide Dismutase Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Poster Presentation Topic: CNS Diseases Citation: Masmoudi-Kouki O, Hamdi Y, Kaddour H, Vaudry D, Jenhani F, Leprince J, Tonon M and Amri M (2009). A new perspective for the role of octadecaneuropeptide ODN in neurodegenerative diseases. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.156 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Nov 2009; Published Online: 25 Nov 2009. * Correspondence: Olfa Masmoudi-Kouki, Faculty of Science of Tunis, University Tunis El Manar, Laboratory of Functional Neurophysiology and Pathology, 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, 2092 Tunis, Tunisia, olfa.masmoudi@fst.utm.tn Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Olfa Masmoudi-Kouki Yosra Hamdi Hadhemi Kaddour David Vaudry Faouzi Jenhani Jerome Leprince Marie-Christine Tonon Mohamed Amri Google Olfa Masmoudi-Kouki Yosra Hamdi Hadhemi Kaddour David Vaudry Faouzi Jenhani Jerome Leprince Marie-Christine Tonon Mohamed Amri Google Scholar Olfa Masmoudi-Kouki Yosra Hamdi Hadhemi Kaddour David Vaudry Faouzi Jenhani Jerome Leprince Marie-Christine Tonon Mohamed Amri PubMed Olfa Masmoudi-Kouki Yosra Hamdi Hadhemi Kaddour David Vaudry Faouzi Jenhani Jerome Leprince Marie-Christine Tonon Mohamed Amri Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Chronic Low‐Dose Oxidative Stress Induces Caspase‐3‐Dependent PKCδ Proteolytic Activation and Apoptosis in a Cell Culture Model of Dopaminergic Neurodegeneration

Oxidative stress has been implicated as a key event in the degenerative process of dopaminergic neurons; however, the cellular mechanisms underlying chronic oxidative stress-induced neurodegeneration remain to be established. In this study, N27 cells, a dopaminergic neuronal cell line derived from rat mesencephalon, exposed to low doses of H(2)O(2) (0-30 muM for 12-24 hr) exhibited dose- and time-dependent increases in cytotoxicity and ROS generation. In addition, the H(2)O(2)-induced neurotoxicity was accompanied by increased caspase-3 activity and PKCdelta cleavage. Notably, treatment with antioxidants Trolox and MnTBAP or PKCdelta cleavage inhibitor z-DIPD-fmk significantly protected against oxidative stress-induced apoptotic cell death. These results demonstrate that the N27 cell line is a useful model for the study of the chronic low-dose oxidative stress-induced apoptotic cell death cascade and that caspase-3-dependent PKCdelta proteolytic activation may be important in the apoptotic process in dopaminergic neurons undergoing chronic oxidative insult.

Tyrosine Phosphorylation Regulates the Proteolytic Activation of Protein Kinase Cδ 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 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. Cell Growth & Differ. 1999; 10: 231-241PubMed Google Scholar, 23.Zhong M. Lu Z. Foster D.A. Oncogene. 2002; 21: 1071-1078Crossref PubMed Scopus (40) Google Scholar, 24.Gschwendt M. Kielbassa K. Kittstein W. Marks F. FEBS Lett. 1994; 347: 85-89Crossref PubMed Scopus (97) Google Scholar, 25.Joseloff E. Cataisson C. Aamodt H. Ocheni H. Blumberg P. Kraker A.J. Yuspa S.H. J. Biol. Chem. 2002; 277: 12318-12323Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Other members of the Src family of kinases that influence PKCδ activity via phosphorylative changes are Fyn and c-Abl kinase (26.Sun X. Wu F. Datta R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 7470-7473Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 27.Crosby D. Poole A.W. J. Biol. Chem. 2003; 278: 24533-24541Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). 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

Roles of Ras-Erk in Apoptosis of PC12 Cells Induced by Trophic Factor Withdrawal or Oxidative Stress

To understand the role of Ras-MAPK (mitogen-activated protein kinase) in trophic factor withdrawal- and oxidative stress-induced apoptotic cell death processes, undifferentiated rat pheochromocytoma PC12 cells and a PC12 variant cell line stably expressing the Ras dominant-negative mutant (M-M17-26) were subjected to serum withdrawal in the absence or presence of H2O2 treatment. The extent of cell death was analyzed by lactate dehydrogenase release, internucleosomal DNA fragmentation, and caspase-3 assays. Both serum withdrawal and H2O2 treatment induced apoptotic cell death in PC12 cells, and the extent of cell death was greatly enhanced in M-M17-26 cells. DNA fragmentation induced by serum withdrawal or H2O2 treatment was blocked completely by a general caspase inhibitor, Z-VAD-FMK. A selective MAPK kinase inhibitor, U0126, blocked the H2O2-induced phosphorylation of Erk1/2 (extracellular signal-regulated kinase) in PC12 cells and increased the levels of active caspase-3 in M-M17-26 under serum withdrawal or H2O2 treatment. In addition, the short-term H2O2 treatment (5-30 min) was sufficient to cause DNA fragmentation in M-M17-26 cells even though H2O2 was removed and cells were incubated in regular growth medium with complete serum for 24 h. However, similar, short-term H2O2 treatment of PC12 cells did not induce DNA fragmentation 24 h later. These results suggest that the Ras-Erk pathway is critical in mediating protection against apoptotic cell death induced by either trophic factor withdrawal or increased oxidative stress.

Inhibition by oestradiol of oxidative stress-induced apoptosis in pig ovarian tissues.

Oxidative stress-induced apoptotic cell death has been implicated in the mechanisms of corpus luteum regression and follicular atresia. The objective of this investigation was to determine whether the antioxidant, oestradiol influenced apoptosis in pig luteal and follicular tissues exposed in vitro to hydrogen peroxide. Immunolocalization of fragmented DNA (a biochemical marker of apoptosis) in large steroidogenic luteal and granulosa cells was inhibited by oestradiol. Protection against apoptosis required relatively high concentrations of oestradiol (> or = 40 pg ml-1 incubation media) and was largely unmitigated by actinomycin D (suggesting a predominantly receptor-independent nongenomic mode of action). Ovarian cells were not protected by other (nonaromatizable) steroid hormones. It is suggested that oestradiol functions at the ovary as a reactive oxygen scavenger during pregnancy-mediated luteal rescue and folliculogenesis.