c-Jun N-terminal kinase mediates hydrogen peroxide-induced cell death via sustained poly(ADP-ribose) polymerase-1 activation

Reactive oxygen species (ROS) have been closely associated with both apoptotic and non-apoptotic/necrotic cell death. Our previous study has illustrated that c-Jun-N-terminal kinase 1 (JNK1) is the main executor in hydrogen peroxide (H(2)O(2))-induced nonapoptotic cell death. The main objective of this study is to further elucidate the molecular mechanisms downstream of JNK1 in H(2)O(2)-induced cell death. In this study, poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair protein, was readily activated by H(2)O(2) and inhibition of PARP-1 activation by either a pharmacological or genetic approach offered significant protection against H(2)O(2)-induced cell death. More importantly, H(2)O(2)-mediated PARP-1 activation is subject to regulation by JNK1. Suppression of JNK1 activation by a chemical inhibitor or genetic deletion markedly suppressed the late-phase PARP-1 activation induced by H(2)O(2), suggesting that JNK1 contributes to the sustained activation of PARP-1. Such findings were supported by the temporal pattern of nuclear translocation of activated JNK and a direct protein-protein interaction between JNK1 and PARP-1 in H(2)O(2)-treated cells. Finally, in vitro kinase assay suggests that PARP-1 may serve as the direct phosphorylation target for JNK1. Taken together, data from our study reveal a novel underlying mechanism in H(2)O(2)-induced nonapoptotic cell death: JNK1 promotes a sustained PARP-1 activation via nuclear translocation, protein-protein interaction and PARP-1 phosphorylation.     

Iduna protects the brain from glutamate excitotoxicity and stroke by interfering with poly(ADP-ribose) polymer-induced cell death

Glutamate acting on N-methyl-D-aspartate (NMDA) receptors induces neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 (PARP-1) and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a previously undescribed NMDA receptor-induced survival protein that is neuroprotective against glutamate NMDA receptor-mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer-induced cell death (parthanatos). Iduna's protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer-binding protein, and mutation at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion-induced stroke in mice. To our knowledge, these results define Iduna as the first known endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling could be a new therapeutic strategy for the treatment of neurologic disorders.     

Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction

Background

Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress.

Methods

PARP-1(−/−) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings were suspended in organ chambers for isometric tension recording to analyze vascular function.

Results

PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(−/−), but not PARP-1(+/+) mice (p < 0.0001). Maximal relaxation was 45% in PQ treated PARP-1(−/−) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, l-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(−/−) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(−/−) mice (p < 0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(−/−) mice (p < 0.05 vs. PQ treated PARP-1(+/+).

Conclusion

PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.     

Blockade of SOCE protects HT22 cells from hydrogen peroxide-induced apoptosis

Oxidative stress is an established event in the pathology of neurobiological diseases. Previous studies indicated that store-operated Ca²⁺ entry (SOCE) has been involved in oxidative stress. The present study was carried out to investigate the effects of SOCE inhibition on neuronal oxidative stress injury induced by hydrogen peroxide (H₂O₂) in HT22 cells, a murine hippocampal neuronal model. H₂O₂ insult induced significant intracellular Ca²⁺ overload, mitochondrial dysfunction and cell viability decrease. Inhibition of SOCE by pharmacological inhibitor and STIM1 RNAi significantly alleviated intracellular Ca²⁺ overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and eventually inhibited H₂O₂-induced cell apoptosis. These findings suggest that SOCE inhibition exhibited neuroprotection against oxidative stress induced by H₂O₂ and SOCE might be a useful therapeutic target in neurobiological disorders.     

Protein kinase C protects from DNA damage-induced necrotic cell death by inhibiting poly(ADP-ribose) polymerase-1

The goal of the current study, conducted in freshly isolated thymocytes was (1) to investigate the possibility that the activation of poly(ADP-ribose) polymerase-1 (PARP-1) in an intact cell can be regulated by protein kinase C (PKC) mediated phosphorylation and (2) to examine the consequence of this regulatory mechanism in the context of cell death induced by the genotoxic agent. In cells stimulated by the PKC activating phorbol esters, DNA breakage was unaffected, PARP-1 was phosphorylated, 1-methyl-3-nitro-1-nitrosoguanidine-induced PARP activation and cell necrosis were suppressed, with all these effects attenuated by the PKC inhibitors GF109203X or G?6976. Inhibition of cellular PARP activity by PKC-mediated phosphorylation may provide a plausible mechanism for the previously observed cytoprotective effects of PKC activators.     

Poly(ADP-ribose) polymerase-1 protects neurons against apoptosis induced by oxidative stress

In neurons, DNA is prone to free radical damage, although repair mechanisms preserve the genomic integrity. However, activation of the DNA repair system, poly(ADP-ribose) polymerase (PARP-1), is thought to cause neuronal death through NAD+ depletion and mitochondrial membrane potential (delta psi(m)) depolarization. Here, we show that abolishing PARP-1 activity in primary cortical neurons can either enhance or prevent apoptotic death, depending on the intensity of an oxidative stress. Only in severe oxidative stress does PARP-1 activation result in NAD+ and ATP depletion and neuronal death. To investigate the role of PARP-1 in an endogenous model of oxidative stress, we used an RNA interference (RNAi) strategy to specifically knock down glutamate-cysteine ligase (GCL), the rate-limiting enzyme of glutathione biosynthesis. GCL RNAi spontaneously elicited a mild type of oxidative stress that was enough to stimulate PARP-1 in a Ca2+-calmodulin kinase II-dependent manner. GCL RNAi-mediated PARP-1 activation facilitated DNA repair, although neurons underwent delta psi(m) loss followed by some apoptotic death. PARP-1 inhibition did not prevent delta psi(m) loss, but enhanced the vulnerability of neurons to apoptosis upon GCL silencing. Conversely, mild expression of PARP-1 partially prevented to GCL RNAi-dependent apoptosis. Thus, in the mild progressive damage likely occur in neurodegenerative diseases, PARP-1 activation plays a neuroprotective role that should be taken into account when considering therapeutic strategies.     

Deleterious activation of poly(ADP-ribose)polymerase-1 in brain after in vivo oxidative stress

Oxidative stress has been shown to be implicated in the pathogenesis of central nervous system injuries such as cerebral ischemia and trauma, and chronic neurodegenerative diseases. In vitro studies show that oxidative stress, particularly peroxynitrite, could trigger DNA strand breaks, which lead to the activation of repairing enzymes including Poly(ADP-ribose) Polymerase-1 (PARP-1). As excessive activation of this enzyme induces cell death, we examined whether such a cascade also occurs in vivo in a model of oxidative stress in rat brain. For this purpose, the mitochondrial toxin malonate, which promotes free radical production, was infused into the left striatum of rats. Immunohistochemistry showed that 3-nitrotyrosine, an indicator of nitrosative stress, and poly(ADP-ribose), a marker of poly(ADP-ribose)polymerase-1 activation, were present as early as 1 h after malonate, and that they persisted for 24 h. The PARP inhibitor, 3-aminobenzamide, significantly reduced the lesion and inhibited PARP-1 activation induced by malonate. These results demonstrate that oxidative stress induced in vivo in the central nervous system leads to the activation of poly(ADP-ribose)polymerase-1, which contributes to neuronal cell death.     

Novel poly(ADP-ribose) polymerase-1 inhibitors

Synthesis and activity of a series of 3-aroyl-derived analogs of novel pyrrolocarbazole 1 as poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors are disclosed.     

Novel poly(ADP-ribose) polymerase-1 inhibitors

Synthesis and activity of a series of 4-thiazol-yl substituted analogs of novel pyrrolocarbazole 1 as poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been disclosed.     

Neuroprotective effect of Capsicum annuum var. abbreviatum against hydrogen peroxide-induced oxidative stress in HT22 hippocampus cells

ABSTRACT

Phenolic compounds isolated from pepper (Capsicum annum) have been demonstrated to have neuroprotective effects, whereas the physiological properties of Capsicum annuum var. abbreviatum (CAA) have not been studied. Thus, we investigate the chemical composition and neuroprotective activity of CAA extract (CAAE) in HT22 hippocampus cells against H₂O₂-induced neurotoxicity. CAAE treatment resulted in a significant protection of H₂O₂-exposed HT22, this protection ultimately occurred through an inhibition of MDA and ROS levels and an induction of SOD activity. Furthermore, CAAE treatment reduced H₂0₂-induced apoptosis though decreasing the expression of pro-apoptotic factors (Bax, cytochrome c, and cleaved caspases-3) while increasing the expression of the anti-apoptotic factors (Bcl-2), as well as the accumulation of nucleus-Nrf2-mediated HO-1 signaling. Interestingly, CAAE has a high concentration of unique phenolic compositions (chlrogenic acid, tangeretin, etc.) than other capsicum annum extracts. Altogether, these findings suggest that CAAE can be a useful natural resource for alleviating neurodegenerative diseases.     

Systemic poly(ADP-ribose) polymerase-1 activation,chronic inflammation,and oxidative stress in COPD patients

Oxidative stress and systemic inflammation in chronic obstructive pulmonary disease (COPD) strongly suggest a role for the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1, E.C.2.4.2.30) in the disease pathophysiology. PARP-1 is highly activated by reactive oxygen species-induced DNA strand breaks, upon which it forms extensive poly(ADP-ribose) (PAR) polymers from its substrate NAD(+). We hypothesized that in COPD, chronic inflammation and oxidative stress would lead to systemic PARP-1 activation and to a reduced NAD(+) status. In a patient-control study, systemic PARP-1 activation was assessed by immunofluorescent detection of PAR polymers in peripheral blood lymphocytes. The percentage of PAR polymer-positive lymphocytes appeared to be higher in COPD patients (27 +/- 3%) than in healthy age-matched controls (17 +/- 2%, p <.05). Trolox equivalent antioxidant capacity (TEAC) of deproteinized plasma (p <.001), plasma uric acid (p <.05), as well as blood NAD(+) (p <.01) of stable COPD patients were significantly reduced when compared to controls. In addition, levels of proinflammatory cytokines IL-6, IL-8, and sICAM-1 were increased (p <.005) in COPD patients. In this study, evidence was found for the presence of systemic inflammation, chronic oxidative stress, and systemic PARP-1 activation in stable COPD patients. These data support a contribution of oxidative stress-induced PARP-1 activation to the pathophysiology of COPD.     

Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADP-ribose)polymerase-1 inhibition in oxidative stress

According to the classical view, the cytoprotective effect of inhibitors of poly(ADP-ribose)polymerase (PARP) in oxidative stress was based on the prevention of NAD+ and ATP depletion, thus the attenuation of necrosis. Our previous data on PARP inhibitors in an inflammatory model suggested that PARP-catalyzed ADP-ribosylations may affect signaling pathways, which can play a significant role in cell survival. To clarify the molecular mechanism of cytoprotection, PARP activity was inhibited pharmacologically by suppressing PARP-1 expression by a small interfering RNA (siRNA) technique or by transdominantly expressing the N-terminal DNA-binding domain of PARP-1 (PARP-DBD) in cultured cells. Cell survival, activation of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt system, and the preservation of mitochondrial membrane potential were studied in hydrogen peroxide-treated WRL-68 cells. Our data showed that suppression of the single-stranded DNA break-induced PARP-1 activation by pharmacological inhibitor, siRNA, or by the transdominant expression of PARP-DBD protected cells from oxidative stress and induced the phosphorylation and activation of Akt. Furthermore, prevention of Akt activation by inhibiting PI3-kinase counteracted the cytoprotective effect of PARP inhibition. Microscopy data showed that PARP inhibition-induced Akt activation was responsible for protection of mitochondria in oxidative stress because PI3-kinase inhibitors diminished the protective effect of PARP inhibition. Similarly, Src kinase inhibitors, which decrease Akt phosphorylation, also counteracted the protection of mitochondrial membrane potential supporting the pivotal role of Akt in cytoprotection. These data together with the finding that PARP inhibition in the absence of oxidative stress induced the phosphorylation and activation of Akt indicate that PARP inhibition-induced Akt activation is dominantly responsible for the cytoprotection in oxidative stress.     

A novel function of poly(ADP-ribose) polymerase-1 in modulation of autophagy and necrosis under oxidative stress

Under oxidative stress, poly(ADP-ribose) polymerase-1 (PARP-1) is activated and contributes to necrotic cell death through ATP depletion. On the other hand, oxidative stress is known to stimulate autophagy, and autophagy may act as either a cell death or cell survival mechanism. This study aims to explore the regulatory role of PARP-1 in oxidative stress-mediated autophagy and necrotic cell death. Here, we first show that hydrogen peroxide (H(2)O(2)) induces necrotic cell death in Bax-/- Bak-/- mouse embryonic fibroblasts through a mechanism involving PARP-1 activation and ATP depletion. Next, we provide evidence that autophagy is activated in cells exposed to H(2)O(2). More importantly, we identify a novel autophagy signaling mechanism linking PARP-1 to the serine/threonine protein kinase LKB1-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway, leading to stimulation of autophagy. Finally, we demonstrate that autophagy plays a cytoprotective role in H(2)O(2)-induced necrotic cell death, as suppression of autophagy by knockdown of autophagy-related gene ATG5 or ATG7 greatly sensitizes H(2)O(2)-induced cell death. Taken together, these findings demonstrate a novel function of PARP-1: promotion of autophagy through the LKB1-AMPK-mTOR pathway to enhance cell survival in cells under oxidative stress.     

Increased expression of poly(ADP-ribose) polymerase-1 contributes to caspase-independent myocyte cell death during heart failure

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a pivotal role in regulating genome stability, cell cycle progression, and cell survival. However, overactivation of PARP has been shown to contribute to cell death and organ failure in various stress-related disease conditions. In this study, we examined the role of PARP in the development and progression of cardiac hypertrophy. We measured the expression of PARP in mouse hearts with physiological (swimming exercise) and pathological (aortic banding) cardiac hypertrophy as well as in human heart samples taken at the time of transplantation. PARP levels were elevated both in swimming and banded mice hearts and demonstrated a linear positive correlation with the degree of cardiac hypertrophy. A dramatic increase (4-fold) of PARP occurred in 6-wk banded mice, accompanied by apparent signs of ventricular dilation and myocyte cell death. PARP levels were also elevated (2- to 3-fold) in human hearts with end-stage heart failure compared with controls. However, we found no evidence of caspase-mediated PARP cleavage in either mouse or human failing hearts. Overexpression of PARP in primary cultures of cardiac myocytes led to suppression of gene expression and robust myocyte cell death. Furthermore, data obtained from the analysis of PARP knockout mice revealed that these hearts produce an attenuated hypertrophic response to aortic banding compared with controls. Together, these results demonstrate a role for PARP in the onset and progression of cardiac hypertrophy and suggest that some events related to cardiac hypertrophy growth and progression to heart failure are mediated by a PARP-dependent mechanism.     

Effect of aging and oxidative/genotoxic stress on poly(ADP-ribose) polymerase-1 activity in rat brain

Poly(ADP-ribose) polymerase-1 (PARP-1, EC 2.4.2.30), a DNA-bound enzyme, plays a key role in genome stability, but after overactivation can also be responsible for cell death. The aim of the present study was to investigate PARP-1 activity in the hippocampus, brain cortex, striatum and cerebellum in adult (4 months) and aged (24 months) specific pathogen free Wistar rats and to correlate it with PARP-1 protein level and p53 expression. Moreover, the response of PARP-1 in adult and aged hippocampus to oxidative/genotoxic stress was evaluated. Our data indicated a statistically significant enhancement of PARP-1 activity in aged hippocampus and cerebral cortex comparing to adults without statistically significant changes in PARP-1 protein level. The expression of p53 mRNA was elevated in all aged brain parts with the exception of the cerebral cortex. Our data suggest that enhancement of PARP-1 activity and p53 expression in aged brain may indicate higher DNA damage. Our data also indicate that during excessive oxidative/genotoxic stress there is no response of PARP-1 activity in aged hippocampus in contrast to a significant enhancement of PARP-1 activity in adults which may have important consequences for the physiology and pathology of the brain.     

Proteasome inhibition protects HT22 neuronal cells from oxidative glutamate toxicity

Oxidative stress caused by glutathione depletion after prolonged exposure to extracellular glutamate leads to a form of neuronal cell death that exhibits morphologically mixed features of both apoptosis and necrosis. However, specific downstream executioners involved in this form of cell death have yet to be identified. We report here that glutamate exposure does not activate caspase-3 in the HT22 neuronal cell line. Furthermore, no cytoprotection was achieved with either the pan-caspase inhibitor Z-VAD-fmk or the caspase-3-specific inhibitor DEVD-CHO. In contrast, inhibition of the proteasome by lactacystin protected both HT22 cells and rat primary neuronal cells against cell lysis. In parallel, oxidatively altered and ubiquitinated proteins accumulated in the mitochondrial fraction of cells after proteasome inhibition. These findings suggest that caspases can be decoupled from oxidative stress under some conditions, and implicate the ubiquitin/proteasome pathway in neuronal cell death caused by oxidative glutamate toxicity.     

Poly(ADP-ribose) polymerase-1 mediated caspase-independent cell death after ischemia/reperfusion

In ischemia/reperfusion (I/R) injury increased intracellular Ca(2+) and production of reactive oxygen species (ROS) may cause cell death by intrinsic apoptotic pathways or by necrosis. In this review, an alternative intrinsic cell death pathway, mediated by poly(ADP-ribose) polymerase-1 (PARP-1) and apoptosis-inducing factor (AIF), is described. ROS-induced DNA strand breaks lead to overactivation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30), causing excessive use of energetic substrates such as NAD(+) and ATP, inducing cell death either by apoptosis or by necrosis. Recently, it was demonstrated that activation of PARP-1 induces translocation of apoptosis-inducing factor from the mitochondria to the nucleus, causing DNA condensation and fragmentation, and subsequent cell death. This pathway seems to be triggered by depletion of NAD(+) and appears to be caspase independent. Several lines of evidence suggest that this pathway plays a role in I/R injury, although some studies indicate that mitochondrial dysfunction may also trigger AIF translocation and cell death. At present, the exact mechanisms linking PARP-1 and AIF in the induction of the ROS-induced cell death are still unclear. Therefore, it appears that further investigations will yield valuable information on underlying mechanisms and potential interventions to reduce caspase-independent cell death during ischemia-reperfusion.