NAD+ repletion prevents PARP-1-induced glycolytic blockade and cell death in cultured mouse astrocytes

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that is involved in DNA repair and activated by DNA damage. When activated, PARP-1 consumes NAD(+) to form ADP-ribose polymers on acceptor proteins. Extensive activation of PARP-1 leads to glycolytic blockade, energy failure, and cell death. These events have been postulated to result from NAD(+) depletion. Here, we used primary astrocyte cultures to directly test this proposal, utilizing the endogenous expression of connexin-43 hemichannels by astrocytes to manipulate intracellular NAD(+) concentrations. Activation of PARP-1 with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) produced NAD(+) depletion, glycolytic blockade, and cell death. Cultures incubated in high (10mM) extracellular concentrations of NAD(+) after MNNG exposure showed normalization of intracellular NAD(+) concentrations. Repletion of intracellular NAD(+) in this manner completely restored glycolytic capacity and prevented cell death. These results suggest that NAD(+) depletion is the cause of glycolytic failure after PARP-1 activation.     

Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition

Extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1) by DNA damage is a major cause of caspase-independent cell death in ischemia and inflammation. Here we show that NAD(+) depletion and mitochondrial permeability transition (MPT) are sequential and necessary steps in PARP-1-mediated cell death. Cultured mouse astrocytes were treated with the cytotoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine or 3-morpholinosydnonimine to induce DNA damage and PARP-1 activation. The resulting cell death was preceded by NAD(+) depletion, mitochondrial membrane depolarization, and MPT. Sub-micromolar concentrations of cyclosporin A blocked MPT and cell death, suggesting that MPT is a necessary step linking PARP-1 activation to cell death. In astrocytes, extracellular NAD(+) can raise intracellular NAD(+) concentrations. To determine whether NAD(+) depletion is necessary for PARP-1-induced MPT, NAD(+) was restored to near-normal levels after PARP-1 activation. Restoration of NAD(+) enabled the recovery of mitochondrial membrane potential and blocked both MPT and cell death. Furthermore, both cyclosporin A and NAD(+) blocked translocation of the apoptosis-inducing factor from mitochondria to nuclei, a step previously shown necessary for PARP-1-induced cell death. These results suggest that NAD(+) depletion and MPT are necessary intermediary steps linking PARP-1 activation to AIF translocation and cell death.     

Calcium-dependent modulation of poly(ADP-ribose) polymerase-1 alters cellular metabolism and DNA repair

After genotoxic stress poly(ADP-ribose) polymerase-1 (PARP-1) can be hyperactivated, causing (ADP-ribosyl)ation of nuclear proteins (including itself), resulting in NAD(+) and ATP depletion and cell death. Mechanisms of PARP-1-mediated cell death and downstream proteolysis remain enigmatic. beta-lapachone (beta-lap) is the first chemotherapeutic agent to elicit a Ca(2+)-mediated cell death by PARP-1 hyperactivation at clinically relevant doses in cancer cells expressing elevated NAD(P)H:quinone oxidoreductase 1 (NQO1) levels. Beta-lap induces the generation of NQO1-dependent reactive oxygen species (ROS), DNA breaks, and triggers Ca(2+)-dependent gamma-H2AX formation and PARP-1 hyperactivation. Subsequent NAD(+) and ATP losses suppress DNA repair and cause cell death. Reduction of PARP-1 activity or Ca(2+) chelation protects cells. Interestingly, Ca(2+) chelation abrogates hydrogen peroxide (H(2)O(2)), but not N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PARP-1 hyperactivation and cell death. Thus, Ca(2+) appears to be an important co-factor in PARP-1 hyperactivation after ROS-induced DNA damage, which alters cellular metabolism and DNA repair.     

Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart

The opening of the mitochondrial permeability transition pore (PTP) has been suggested to play a key role in various forms of cell death, but direct evidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD(+) glycohydrolase activity is associated with mitochondria. This activity was not modified by the addition of Triton X-100, although it was abolished by mild treatment with the protease Nagarse, a condition that did not affect the energy-linked properties of mitochondria. The addition of Ca(2+) to isolated rat heart mitochondria resulted in a profound decrease in their NAD(+) content, which followed mitochondrial swelling. Cyclosporin A(CsA), a PTP inhibitor, completely prevented NAD(+) depletion but had no effect on the glycohydrolase activity. Thus, in isolated mitochondria PTP opening makes NAD(+) available for its enzymatic hydrolysis. Perfused rat hearts subjected to global ischemia for 30 min displayed a 30% decrease in tissue NAD(+) content, which was not modified by extending the duration of ischemia. Reperfusion resulted in a more severe reduction of both total and mitochondrial contents of NAD(+), which could be measured in the coronary effluent together with lactate dehydrogenase. The addition of 0.2 microm CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained higher NAD(+) contents, especially in mitochondria, and significantly protected the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD(+), which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP opening is a causative event in reperfusion damage of the heart.     

Nuclear poly(ADP-ribose) polymerase-1 rapidly triggers mitochondrial dysfunction

To obtain further information on time course and mechanisms of cell death after poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, we used HeLa cells exposed for 1 h to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. This treatment activated PARP-1 and caused a rapid drop of cellular NAD(H) and ATP contents, culminating 8-12 h later in cell death. PARP-1 antagonists fully prevented nucleotide depletion and death. Interestingly, in the early 60 min after challenge with N-methyl-N'-nitro-N-nitrosoguanidine, mitochondrial membrane potential and superoxide production significantly increased, whereas cellular ADP contents decreased. Again, these events were prevented by PARP-1 inhibitors, suggesting that PARP-1 hyperactivity leads to mitochondrial state 4 respiration. Mitochondrial membrane potential collapsed at later time points (3 h), when mitochondria released apoptosis-inducing factor and cytochrome c. Using immunocytochemistry and targeted luciferase transfection, we found that, despite an exclusive localization of PARP-1 and poly(ADP-ribose) in the nucleus, ATP levels first decreased in mitochondria and then in the cytoplasm of cells undergoing PARP-1 activation. PARP-1 inhibitors rescued ATP (but not NAD(H) levels) in cells undergoing hyper-poly(ADP-ribosyl)ation. Glycolysis played a central role in the energy recovery, whereas mitochondria consumed ATP in the early recovery phase and produced ATP in the late phase after PARP-1 inhibition, further indicating that nuclear poly(ADP-ribosyl)ation rapidly modulates mitochondrial functioning. Together, our data provide evidence for rapid nucleus-mitochondria cross-talk during hyper-poly(ADP-ribosyl)ation-dependent cell death.     

The path from mitochondrial ROS to aging runs through the mitochondrial permeability transition pore

Excessive production of mitochondrial reactive oxygen species (mROS) is strongly associated with mitochondrial and cellular oxidative damage, aging, and degenerative diseases. However, mROS also induces pathways of protection of mitochondria that slow aging, inhibit cell death, and increase lifespan. Recent studies show that the activation of the mitochondrial permeability transition pore (mPTP), which is triggered by mROS and mitochondrial calcium overloading, is enhanced in aged animals and humans and in aging‐related degenerative diseases. mPTP opening initiates further production and release of mROS that damage both mitochondrial and nuclear DNA, proteins, and phospholipids, and also releases matrix NAD that is hydrolyzed in the intermembrane space, thus contributing to the depletion of cellular NAD that accelerates aging. Oxidative damage to calcium transporters leads to calcium overload and more frequent opening of mPTP. Because aging enhances the opening of the mPTP and mPTP opening accelerates aging, we suggest that mPTP opening drives the progression of aging. Activation of the mPTP is regulated, directly and indirectly, not only by the mitochondrial protection pathways that are induced by mROS, but also by pro‐apoptotic signals that are induced by DNA damage. We suggest that the integration of these contrasting signals by the mPTP largely determines the rate of cell aging and the initiation of cell death, and thus animal lifespan. The suggestion that the control of mPTP activation is critical for the progression of aging can explain the conflicting and confusing evidence regarding the beneficial and deleterious effects of mROS on health and lifespan.     

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.     

Delocalization of nucleolar poly(ADP-ribose) polymerase-1 to the nucleoplasm and its novel link to cellular sensitivity to DNA damage

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme activated by binding to DNA breaks, which causes PARP-1 automodification. PARP-1 activation is required for regulating various cellular processes, including DNA repair and cell death induction. PARP-1 involved in these regulations is localized in the nucleoplasm, but approximately 40% of PARP-1 can be found in the nucleolus. Previously, we have reported that nucleolar PARP-1 is delocalized to the nucleoplasm in cells exposed to DNA-damaging agents. However, the functional roles of this delocalization in cellular response to DNA damage is not well understood, since this approach simultaneously induces the delocalization of PARP-1 and its automodification. We therefore devised an approach for separating these processes. Unmodified PARP-1 was first delocalized from the nucleolus using camptothecin. Then, PARP-1 was activated by exposure of cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In contrast to treatment with MNNG alone, delocalization of PARP-1 by CPT, prior to its activation by MNNG, induced extensive automodification of PARP-1. DNA repair activity and consumption of intracellular NAD⁺ were not affected by this activation. On the other hand, activation led to an increased formation of apoptotic cells, and this effect was suppressed by inhibition of PARP-1 activity. These results suggest that delocalization of PARP-1 from the nucleolus to the nucleoplasm sensitizes cells to DNA damage-induced apoptosis. As it has been suggested that the nucleolus has a role in stress sensing, nucleolar PARP-1 could participate in a process involved in nucleolus-mediated stress sensing.     

Genistein induces apoptosis of RPE-J cells by opening mitochondrial PTP

Although previous studies demonstrated that genistein-induced apoptosis of various cell types including RPE-J cells, the involvement of mitochondrial events in such types of apoptosis has not been demonstrated to date. In this investigation of genistein-induced apoptosis of RPE-J cells, genistein induced the reduction of the mitochondrial membrane potential and the release of cytochrome c to cytosol. A mitochondrial permeability transition pore (PTP) blocker bongkrekic acid prevented the reduction of the mitochondrial membrane potential and cytochrome c release, and consequently abolished caspase-3 activation, nuclear condensation, and DNA fragmentation. On the other hand, zVAD-fmk did not inhibit the mitochondrial event such as the reduction of the mitochondrial membrane potential and cytochrome c release although it prevented caspase-3 activation, nuclear condensation, and DNA fragmentation. Taken together, genistein induces apoptosis of RPE-J cells by opening the mitochondrial PTP, and the mitochondrial event in this type of apoptosis is caused independently of caspase.     

Protein tyrosine phosphatase alpha regulates cell detachment and cell death profiles induced by nitric oxide donors in the A431 human carcinoma cell line

We investigated the role of protein tyrosine phosphatase-alpha (PTPα) expression in the cell death profile of the A431 human carcinoma cell line that was induced by cytotoxic concentrations of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18). Both NO donors promoted extensive cell detachment in A431 parental cells as compared to the detachment observed for A431 cells that ectopically expressed PTPα (A431 (A27B(PTPα)) cells). The NO-induced cell death characteristics for both cell lines were examined. After incubation for 10 hours with 2.0 mM SNP, attached or detached A431 cells underwent apoptosis. Cells were highly positive for Annexin-V, featured increased cleavage of procaspase-8, activation of downstream caspase-3, and activation of poly-ADP-ribose polymerase 1 (PARP-1). In contrast, exposure of A431 (A27B(PTPα)) cells to 2.0 mM SNP produced an increase in the release of lactate dehydrogenase and enhanced incorporation of propidium iodide. In addition, A431 (A27B(PTPα)) cells showed partial inhibition of the activities of caspase-8, caspase-3, and PARP-1 upon detachment and cell death induced by SNP treatment. Results indicate that necrotic cell damage was induced, characterized by cellular swelling and lysis. We conclude from these results that PTPα regulates the A431 tumor cell death profile mediated by NO donors. Expression of PTPα or its absence may determine the occurrence of NO-induced cell death with necrotic or apoptotic features, respectively.     

Theophylline prevents NAD+ depletion via PARP-1 inhibition in human pulmonary epithelial cells

Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD+, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD+ pool, and of NAD+-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD+ levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzyme inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.     

Induction of mitochondrial dysfunction by poly(ADP-ribose) polymer: Implication for neuronal cell death

Poly(ADP-ribose) polymerase-1 (PARP-1) mediates neuronal cell death in a variety of pathological conditions involving severe DNA damage. Poly(ADP-ribose) (PAR) polymer is a product synthesized by PARP-1. Previous studies suggest that PAR polymer heralds mitochondrial apoptosis-inducing factor (AIF) release and thereby, signals neuronal cell death. However, the details of the effects of PAR polymer on mitochondria remain to be elucidated. Here we report the effects of PAR polymer on mitochondria in cells in situ and isolated brain mitochondria in vitro. We found that PAR polymer causes depolarization of mitochondrial membrane potential and opening of the mitochondrial permeability transition pore early after injury. Furthermore, PAR polymer specifically induces AIF release, but not cytochrome c from isolated brain mitochondria. These data suggest PAR polymer as an endogenous mitochondrial toxin and will further our understanding of the PARP-1-dependent neuronal cell death paradigm.     

Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-Delta2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-Delta2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-Delta2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain.     

Sequential opening of mitochondrial ion channels as a function of glutathione redox thiol status

Mitochondrial membrane potential (DeltaPsi(m)) depolarization contributes to cell death and electrical and contractile dysfunction in the post-ischemic heart. An imbalance between mitochondrial reactive oxygen species production and scavenging was previously implicated in the activation of an inner membrane anion channel (IMAC), distinct from the permeability transition pore (PTP), as the first response to metabolic stress in cardiomyocytes. The glutathione redox couple, GSH/GSSG, oscillated in parallel with DeltaPsi(m) and the NADH/NAD(+) redox state. Here we show that depletion of reduced glutathione is an alternative trigger of synchronized mitochondrial oscillation in cardiomyocytes and that intermediate GSH/GSSG ratios cause reversible DeltaPsi(m) depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in DeltaPsi(m) to sustained depolarization, in association with depletion of GSH. Mitochondrial oscillations were abrogated by 4'-chlorodiazepam, an IMAC inhibitor, whereas cyclosporin A was ineffective. In saponin-permeabilized cardiomyocytes, the thiol redox status was systematically clamped at GSH/GSSG ratios ranging from 300:1 to 20:1. At ratios of 150:1-100:1, DeltaPsi(m) depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized DeltaPsi(m) and induced maximal rates of reactive oxygen species production, NAD(P)H oxidation, and loss of matrix constituents. Mitochondrial GSH sensitivity was altered by inhibiting either GSH uptake, the NADPH-dependent glutathione reductase, or the NADH/NADPH transhydrogenase, indicating that matrix GSH regeneration or replenishment was crucial. The results indicate that GSH/GSSG redox status governs the sequential opening of mitochondrial ion channels (IMAC before PTP) triggered by thiol oxidation in cardiomyocytes.     

Apoptosis driven by IP(3)-linked mitochondrial calcium signals

Increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) evoked by calcium mobilizing agonists play a fundamental role in the physiological control of cellular energy metabolism. Here, we report that apoptotic stimuli induce a switch in mitochondrial calcium signalling at the beginning of the apoptotic process by facilitating Ca(2+)-induced opening of the mitochondrial permeability transition pore (PTP). Thus [Ca(2+)](m) signals evoked by addition of large Ca(2+) pulses or, unexpectedly, by IP(3)-mediated cytosolic [Ca(2+)] spikes trigger mitochondrial permeability transition and, in turn, cytochrome c release. IP(3)-induced opening of PTP is dependent on a privileged Ca(2+) signal transmission from IP(3) receptors to mitochondria. After the decay of Ca(2+) spikes, resealing of PTP occurs allowing mitochondrial metabolism to recover, whereas activation of caspases is triggered by cytochrome c released to the cytosol. This organization provides an efficient mechanism to establish caspase activation while mitochondrial metabolism is maintained to meet ATP requirements of apoptotic cell death.     

Are poly(ADP‐ribosyl)ation by PARP‐1 and deacetylation by Sir2 linked?

Poly(ADP-ribose) polymerase-1 (PARP-1) safeguards genomic integrity by limiting sister chromatid exchanges. Overstimulation of PARP-1 by extensive DNA damage, however, can result in cell death, as prolonged PARP-1 activation depletes NAD(+), a substrate, and elevates nicotinamide, a product. The decline of NAD(+) and the rise of nicotinamide may downregulate the activity of Sir2, the NAD(+)-dependent deacetylases, because deacetylation by Sir2 is dependent on high concentration of NAD(+) and inhibited by physiologic level of nicotinamide. The Sir2 deacetylase family has been implicated in mediating gene silencing, longevity and genome stability. It is conceivable that poly(ADP-ribosyl)ation by PARP-1, which is induced by DNA damage, could modulate protein deacetylation by Sir2 via the NAD(+)/nicotinamide connection. The possible linkage of the two ancient pathways that mediate broad biological activities may spell profound evolutionary roles for the conserved PARP-1 and Sir2 gene families in multicellular eukaryotes.     

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.