NF-κB transcriptional activation by TNFα requires phospholipase C, extracellular signal-regulated kinase 2 and poly(ADP-ribose) polymerase-1.

BACKGROUND: The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is required for pro-inflammatory effects of TNFα. Our previous studies demonstrated that PARP-1 mediates TNFα-induced NF-κB activation in glia. Here, we evaluated the mechanisms by which TNFα activates PARP-1 and PARP-1 mediates NF-κB activation. METHODS: Primary cultures of mouse cortical astrocytes and microglia were treated with TNFα and suitable signaling pathway modulators (pharmacological and molecular). Outcome measures included calcium imaging, PARP-1 activation status, NF-κB transcriptional activity, DNA damage assesment and cytokine relesease profiling. RESULTS: TNFα induces PARP-1 activation in the absence of detectable DNA strand breaks, as measured by the PANT assay. TNFα-induced transcriptional activation of NF-κB requires PARP-1 enzymatic activity. Enzymatic activation of PARP-1 by TNFα was blocked in Ca(2+)-free medium, by Ca(2+) chelation with BAPTA-AM, and by D609, an inhibitor of phoshatidyl choline-specific phospholipase C (PC-PLC), but not by thapsigargin or by U73112, an inhibitor of phosphatidyl inisitol-specific PLC (PI -PLC). A TNFR1 blocking antibody reduced Ca(2+) influx and PARP-1 activation. TNFα-induced PARP-1 activation was also blocked by siRNA downregulation of ERK2 and by PD98059, an inhibitor of the MEK / ERK protein kinase cascade. Moreover, TNFα-induced NF-κB (p65) transcriptional activation was absent in cells expressing PARP-1 that lacked ERK2 phosphorylation sites, while basal NF-κB transcriptional activation increased in cells expressing PARP-1 with a phosphomimetic substitution at an ERK2 phophorylation site. CONCLUSIONS: These results suggest that TNFα induces PARP-1 activation through a signaling pathway involving TNFR1, Ca(2+) influx, activation of PC-PLC, and activation of the MEK1 / ERK2 protein kinase cascade. TNFα-induced PARP-1 activation is not associated with DNA damage, but ERK2 mediated phosphorylation of PARP-1.

Neuronal Sirt3 protects against excitotoxic injury in mouse cortical neuron culture.

BACKGROUND: Sirtuins (Sirt), a family of nicotinamide adenine nucleotide (NAD) dependent deacetylases, are implicated in energy metabolism and life span. Among the known Sirt isoforms (Sirt1-7), Sirt3 was identified as a stress responsive deacetylase recently shown to play a role in protecting cells under stress conditions. Here, we demonstrated the presence of Sirt3 in neurons, and characterized the role of Sirt3 in neuron survival under NMDA-induced excitotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: To induce excitotoxic injury, we exposed primary cultured mouse cortical neurons to NMDA (30 µM). NMDA induced a rapid decrease of cytoplasmic NAD (but not mitochondrial NAD) in neurons through poly (ADP-ribose) polymerase-1 (PARP-1) activation. Mitochondrial Sirt3 was increased following PARP-1 mediated NAD depletion, which was reversed by either inhibition of PARP-1 or exogenous NAD. We found that massive reactive oxygen species (ROS) produced under this NAD depleted condition mediated the increase in mitochondrial Sirt3. By transfecting primary neurons with a Sirt3 overexpressing plasmid or Sirt3 siRNA, we showed that Sirt3 is required for neuroprotection against excitotoxicity. CONCLUSIONS: This study demonstrated for the first time that mitochondrial Sirt3 acts as a prosurvival factor playing an essential role to protect neurons under excitotoxic injury.

Minocycline protects cardiac myocytes against simulated ischemia­reperfusion injury by inhibiting poly(ADP-ribose) polymerase-1.

There is an increase in reactive oxygen and nitrogen species in cardiomyocytes during myocardial ischemia/reperfusion injury. This leads to oxidative DNA damage and activation of nuclear repair enzymes such as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 activation promotes DNA repair under normal conditions. However, excessive activation of PARP-1 leads to cell death. We report that PARP-1 enzymatic activity is directly inhibited by minocycline, and we propose that one mechanism of minocycline cardioprotection is the result of PARP-1 inhibition. Using cultured adult rat cardiac myocytes, we evaluated the mechanism of minocycline protection in which PARP-1 activation was induced by simulated ischemia/reperfusion injury using oxygen­glucose deprivation.We found an increase in reactive oxygen species production, PARP-1 activation, and PARP-1-mediated cell death after simulated ischemia/reperfusion. Cell death was significantly reduced by the PARP inhibitors 3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (10 µM) and PJ-34 (500 nM) or by minocycline (500 nM). Cellular NAD(+) depletion and poly(ADP-ribose) formation, which are biochemical markers of PARP-1 activation, were also blocked by minocycline. Finally, simulated ischemia/reperfusion led to induction of the mitochondrial permeability transition, which was prevented by minocycline. Therefore, we propose that the protective effect of minocycline on cardiac myocyte survival is the result of inhibition of PARP-1 activity.

Vincristine attenuates N-methyl-N'-nitro-N-nitrosoguanidine-induced poly-(ADP) ribose polymerase activity in cardiomyocytes.

The DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) causes cardiomyocyte death as a result of energy loss from excessive activation of poly-(ADP) ribose polymerase-1 (PARP-1) resulting in depletion of its substrates nicotinamide adenine dinucleotide (NAD) and ATP. Previously we showed that the chemotherapeutic agent vincristine (VCR) is cardioprotective. Here we tested the hypothesis that VCR inhibits MNNG-induced PARP activation. Adult mouse cardiomyocytes were incubated with 100 micromol/L MNNG with or without concurrent VCR (20 micromol/L) for 2 to 4 hours. Cardiomyocyte survival was measured using the trypan blue exclusion assay. Western blots were used to measure signaling responses. MNNG-induced cardiomyocyte damage was time- and concentration-dependent. MNNG activated PARP-1 and depleted NAD and ATP. VCR completely protected cardiomyocytes from MNNG-induced cell damage and maintained intracellular levels of NAD and ATP. VCR increased phosphorylation of the prosurvival signals Akt, GSK-3beta, Erk1/2, and p70S6 kinase. VCR delayed PARP activation as evidenced by Western blot and by immunofluorescence staining of poly (ADP)-ribose, but without directly inhibiting PARP-1 itself. Known PARP-1 inhibitors also protected cardiomyocytes from MNNG-induced death. Repletion of ATP, NAD, pyruvate, and glutamine had effects similar to PARP-1 inhibitors. We conclude that VCR protects cardiomyocytes from MNNG toxicity by regulating PARP-1 activation, intracellular energy metabolism, and prosurvival signaling.

NAD+ depletion is necessary and sufficient for poly(ADP-ribose) polymerase-1-mediated neuronal death.

Poly(ADP-ribose)-1 (PARP-1) is a key mediator of cell death in excitotoxicity, ischemia, and oxidative stress. PARP-1 activation leads to cytosolic NAD(+) depletion and mitochondrial release of apoptosis-inducing factor (AIF), but the causal relationships between these two events have been difficult to resolve. Here, we examined this issue by using extracellular NAD(+) to restore neuronal NAD(+) levels after PARP-1 activation. Exogenous NAD(+) was found to enter neurons through P2X(7)-gated channels. Restoration of cytosolic NAD(+) by this means prevented the glycolytic inhibition, mitochondrial failure, AIF translocation, and neuron death that otherwise results from extensive PARP-1 activation. Bypassing the glycolytic inhibition with the metabolic substrates pyruvate, acetoacetate, or hydroxybutyrate also prevented mitochondrial failure and neuron death. Conversely, depletion of cytosolic NAD(+) with NAD(+) glycohydrolase produced a block in glycolysis inhibition, mitochondrial depolarization, AIF translocation, and neuron death, independent of PARP-1 activation. These results establish NAD(+) depletion as a causal event in PARP-1-mediated cell death and place NAD(+) depletion and glycolytic failure upstream of mitochondrial AIF release.

High-density lipoprotein determines adult mouse cardiomyocyte fate after hypoxia-reoxygenation through lipoprotein-associated sphingosine 1-phosphate.

The lipid mediator sphingosine 1-phosphate (S1P) confers survival benefits in cardiomyocytes and isolated hearts subjected to oxidative stress. High-density lipoprotein (HDL) is a major carrier of S1P in the serum, but whether HDL-associated S1P directly mediates survival in a preparation composed exclusively of cardiomyocytes has not been demonstrated. Accordingly, we tested the hypothesis that signal activation and survival during simulated ischemia-reperfusion injury in response to HDL require lipoprotein-associated S1P. As a model, we used adult mouse cardiomyocytes subjected to hypoxia-reoxygenation. Cells were treated or not with autologous mouse HDL, which significantly increased myocyte viability as measured by trypan blue exclusion. This survival effect was abrogated by the S1P(1) and SIP(3) receptor antagonist VPC 23019. The selective S1P(3) antagonist CAY10444, the G(i) antagonist pertussis toxin, the MEK (MAPK/ERK) kinase inhibitor PD-98059, and the phosphoinositide-3 kinase inhibitor wortmannin also inhibited the prosurvival effect of HDL. We observed that HDL activated both Akt (protein kinase B) and the MEK1/2-ERK1/2 pathway and also stimulated phosphorylation of glycogen synthase kinase-3beta. ERK1/2 activation was through an S1P(1) subtype receptor-G(i) protein-dependent pathway, whereas the activation of Akt was inhibited by CAY10444, indicating mediation by S1P(3) subtype receptors. We conclude that HDL, via its cargo of S1P, can directly protect cardiomyocytes against simulated oxidative injury in the absence of vascular effects and that prosurvival signal activation is dependent on both S1P(1) and S1P(3) subtype receptors.

Astrocytic poly(ADP-ribose) polymerase-1 activation leads to bioenergetic depletion and inhibition of glutamate uptake capacity.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a ubiquitous nuclear enzyme involved in genomic stability. Excessive oxidative DNA strand breaks lead to PARP-1-induced depletion of cellular NAD(+), glycolytic rate, ATP levels, and eventual cell death. Glutamate neurotransmission is tightly controlled by ATP-dependent astrocytic glutamate transporters, and thus we hypothesized that astrocytic PARP-1 activation by DNA damage leads to bioenergetic depletion and compromised glutamate uptake. PARP-1 activation by the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), caused a significant reduction of cultured cortical astrocyte survival (EC(50) = 78.2 +/- 2.7 microM). HPLC revealed MNNG-induced time-dependent reductions in NAD(+) (98%, 4 h), ATP (71%, 4 h), ADP (63%, 4 h), and AMP (66%, 4 h). The maximal [(3)H]glutamate uptake rate (V(max)) also declined in a manner that corresponded temporally with ATP depletion, falling from 19.3 +/- 2.8 in control cells to 2.1 +/- 0.8 nmol/min/mg protein 4 h post-MNNG. Both bioenergetic depletion and loss of glutamate uptake capacity were attenuated by genetic deletion of PARP-1, directly indicating PARP-1 involvement, and by adding exogenous NAD(+) (10 mM). In mixed neurons/astrocyte cultures, MNNG neurotoxicity was partially mediated by extracellular glutamate and was reduced by co-culture with PARP-1(-/-) astrocytes, suggesting that impairment of astrocytic glutamate uptake by PARP-1 can raise glutamate levels sufficiently to have receptor-mediated effects at neighboring neurons. Taken together, these experiments showed that PARP-1 activation leads to depletion of the total adenine nucleotide pool in astrocytes and severe reduction in neuroprotective glutamate uptake capacity.

Cardiomyocyte S1P1 receptor-mediated extracellular signal-related kinase signaling and desensitization.

We examined the ability of sphingosine-1-phosphate (S1P) to desensitize extracellular signal-related kinase (ERK), a mitogen-activated protein kinase linked to antiapoptotic responses in the heart. In isolated adult mouse cardiomyocytes, S1P (10 nM-5 microM) induced ERK phosphorylation in a time- and dose-dependent manner. S1P stimulation of ERK was completely inhibited by an S1P1/3 subtype receptor antagonist (VPC23019), by a Gi protein inhibitor (pertussis toxin) and by a mitogen-activated protein kinase/ERK kinase inhibitor (PD98059). A selective S1P3 receptor antagonist (CAY10444) had no effect on S1P-induced ERK activation. The selective S1P1 agonist SEW2871 also induced ERK phosphorylation. Activation of ERK by restimulation with 100 nM S1P was suppressed after 1 hour of preincubation with 100 nM S1P but recovered fully the next day, suggesting receptor recycling. Similar results were obtained in protein kinase C epsilon-null cardiomyocytes. Treatment with the nonselective S1P receptor agonist FTY720 for 1 hour also reduced phospho-ERK expression in response to subsequent S1P stimulation. In contrast to S1P, some desensitization to FTY720 persisted after overnight exposure. Cell death induced by hypoxia/reoxygenation was reduced by pretreatment with exogenous S1P. This enhanced survival was abrogated by pretreatment with PD98059, VPC23019, or pertussis toxin. Thus, exogenous S1P induces rapid and reversible S1P1-mediated ERK phosphorylation. S1P-induced adult mouse cardiomyocyte survival requires ERK activation mediated via an S1P1-Gi pathway.

Pyrroloquinoline quinone preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes.

We investigated the ability of pyrroloquinoline quinone (PQQ) to confer resistance to acute oxidative stress in freshly isolated adult male rat cardiomyocytes. Fluorescence microscopy was used to detect generation of reactive oxygen species (ROS) and mitochondrial membrane potential (Deltapsi(m)) depolarization induced by hydrogen peroxide. H(2)O(2) caused substantial cell death, which was significantly reduced by preincubation with PQQ. H(2)O(2) also caused an increase in cellular ROS levels as detected by the fluorescent indicators CM-H2XRos and dihydroethidium. ROS levels were significantly reduced by a superoxide dismutase mimetic Mn (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) or by PQQ treatment. Cyclosporine-A, which inhibits mitochondrial permeability transition, prevented H(2)O(2)-induced Deltapsi(m) depolarization, as did PQQ and MnTBAP. Our results provide direct evidence that PQQ reduces oxidative stress, mitochondrial dysfunction, and cell death in isolated adult rat cardiomyocytes. These findings provide new insight into the mechanisms of PQQ action in the heart.

Differences among cell types in NAD(+) compartmentalization: a comparison of neurons, astrocytes, and cardiac myocytes.

Activation of the nuclear enzyme poly(ADP-ribose)-1 leads to the death of neurons and other types of cells by a mechanism involving NAD(+) depletion and mitochondrial permeability transition. It has been proposed that the mitochondrial permeability transition (MPT) is required for NAD(+) to be released from mitochondria and subsequently consumed by PARP-1. In the present study we used the MPT inhibitor cyclosporine-A (CsA) to preserve mitochondrial NAD(+) pools during PARP-1 activation and thereby provide an estimate of mitochondrial NAD(+) pool size in different cell types. Rat cardiac myocytes, mouse cardiac myocytes, mouse cortical neurons, and mouse cortical astrocytes were incubated with the genotoxin N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in order to activate PARP-1. In all four cell types MNNG caused a reduction in total NAD(+) content that was blocked by the PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone. Inhibition of the mitochondrial permeability transition with cyclosporine-A (CsA) prevented PARP-1-induced NAD(+) depletion to a varying degree in the four cell types tested. CsA preserved 83.5% +/- 5.2% of total cellular NAD(+) in rat cardiac myocytes, 85.7% +/- 8.9% in mouse cardiac myocytes, 55.9% +/- 12.9% in mouse neurons, and 22.4% +/- 7.3% in mouse astrocytes. CsA preserved nearly 100% of NAD(+) content in mitochondria isolated from these cells. These results confirm that it is the cytosolic NAD(+) pool that is consumed by PARP-1 and that the mitochondrial NAD(+) pool is consumed only after MPT permits mitochondrial NAD(+) to exit into the cytosol. These results also suggest large differences in the mitochondrial and cytosolic compartmentalization of NAD(+) in these cell types.

Zinc inhibits astrocyte glutamate uptake by activation of poly(ADP-ribose) polymerase-1.

Several processes by which astrocytes protect neurons during ischemia are now well established. However, less is known about how neurons themselves may influence these processes. Neurons release zinc (Zn2+) from presynaptic terminals during ischemia, seizure, head trauma, and hypoglycemia, and modulate postsynaptic neuronal function. Peak extracellular zinc may reach concentrations as high as 400 microM. Excessive levels of free, ionic zinc can initiate DNA damage and the subsequent activation of poly(ADP-ribose) polymerase 1 (PARP-1), which in turn lead to NAD+ and ATP depletion when DNA damage is extensive. In this study, cultured cortical astrocytes were used to explore the effects of zinc on astrocyte glutamate uptake, an energy-dependent process that is critical for neuron survival. Astrocytes incubated with 100 or 400 microM of zinc for 30 min showed significant decreases in ATP levels and glutamate uptake capacity. These changes were prevented by the PARP inhibitors benzamide or DPQ (3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone) or PARP-1 gene deletion (PARP-1 KO). These findings suggest that release of Zn2+ from neurons during brain insults could induce PARP-1 activation in astrocytes, leading to impaired glutamate uptake and exacerbation of neuronal injury.

Minocycline inhibits poly(ADP-ribose) polymerase-1 at nanomolar concentrations.

Poly(ADP-ribose) polymerase-1 (PARP-1), when activated by DNA damage, promotes both cell death and inflammation. Here we report that PARP-1 enzymatic activity is directly inhibited by minocycline and other tetracycline derivatives that have previously been shown to have neuroprotective and anti-inflammatory actions. These agents were evaluated by using cortical neuron cultures in which PARP-1 activation was induced by the genotoxic agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 3-morpholinosydnonimine (SIN-1). In both conditions, neuronal death was reduced by >80% either by 10 muM 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, an established PARP inhibitor, or by 100 nM minocycline. Neuronal NAD(+) depletion and poly(ADP-ribose) formation, which are biochemical markers of PARP-1 activation, were also blocked by 100 nM minocycline. A direct, competitive inhibition of PARP-1 by minocycline (K(i) = 13.8 +/- 1.5 nM) was confirmed by using recombinant PARP-1 in a cell-free assay. Comparison of several tetracycline derivatives showed a strong correlation (r(2) = 0.87) between potency as a PARP-1 inhibitor and potency as a neuroprotective agent during MNNG incubations, with the rank order of potency being minocycline > doxycycline > demeclocycline > chlortetracycline. These compounds are known to have other actions that could contribute their neuroprotective effects, but at far higher concentrations than shown here to inhibit PARP-1. The neuroprotective and antiinflammatory effects of minocycline and other tetracycline derivatives may be attributable to PARP-1 inhibition in some settings.

Players in the PARP-1 cell-death pathway: JNK1 joins the cast.

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) triggers a cell-death pathway in which mitochondria play an integral part, but it remains uncertain how PARP-1 activation in the nucleus is signaled to the mitochondria. A recent report by Xu and colleagues suggests that Jun kinase-1, a member of the mitogen-activated protein kinase family, might have a crucial role in this signaling pathway.

NAD+ as a metabolic link between DNA damage and cell death.

DNA damage occurs in ischemia, excitotoxicity, inflammation, and other disorders that affect the central nervous system (CNS). Extensive DNA damage triggers cell death and in the mature CNS, this occurs primarily through activation of the poly(ADP-ribose) polymerase-1 (PARP-1) cell death pathway. PARP-1 is an abundant nuclear enzyme that, when activated by DNA damage, consumes nicotinamide adenine dinucleotide (NAD)+ to form poly(ADP-ribose) on acceptor proteins. The mechanisms by which PARP-1 activation leads to cell death are not understood fully. We used mouse astrocyte cultures to explore the bioenergetic effects of NAD+ depletion by PARP-1 and the role of NAD+ depletion in this cell death program. PARP-1 activation was induced by the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), using medium in which glucose was the only exogenous energy substrate. PARP-1 activation led to a rapid but incomplete depletion of astrocyte NAD+, a near-complete block in glycolysis, and eventual cell death. Repletion of intracellular NAD+ restored glycolytic function and prevented cell death. The addition of non-glucose substrates to the medium, pyruvate, glutamate, or glutamine, also prevented astrocyte death after PARP-1 activation. These studies suggest PARP-1 activation leads to rapid depletion of the cytosolic but not the mitochondrial NAD+ pool. Depletion of the cytosolic NAD+ pool renders the cells unable to utilize glucose as a metabolic substrate. Under conditions where glucose is the only available metabolic substrate, this leads to cell death. This cell death pathway is particularly germane to brain because glucose is normally the only metabolic substrate that is transported rapidly across the blood-brain barrier.

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.

Expression and activity of poly(ADP-ribose) glycohydrolase in cultured astrocytes, neurons, and C6 glioma cells.

Poly(ADP-ribose) metabolism plays a major role in DNA repair, transcription, replication, and recombination. Poly(ADP-ribose) polymerases are localized primarily to the nucleus, whereas significant levels of poly(ADP-ribose) glycohydrolase (PARG) are believed to be located in the cytoplasm. Only one PARG gene has been identified, but prior studies have reported multiple products of this gene. Here we studied PARG activity and PARG gene expression in several CNS cell types that span the cell growth spectrum: rapidly dividing C6 glioma tumor cells, dividing astrocytes, non-dividing astrocytes (due to contact inhibition), and post-mitotic neurons. Activity assays showed no overall differences between these cell types, but the nuclear to cytoplasmic ratio of PARG activity was highest in C6 glioma cells and lowest in neurons. Western blotting revealed full-length PARG as well as lower molecular weight PARG species in all four cell types.

Tricarboxylic acid cycle substrates prevent PARP-mediated death of neurons and astrocytes.

The DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP1), contributes to cell death during ischemia/reperfusion when extensively activated by DNA damage. The cell death resulting from PARP1 activation is linked to NAD+ depletion and energy failure, but the intervening steps are not well understood. Because glycolysis requires cytosolic NAD+, the authors tested whether PARP1 activation impairs glycolytic flux and whether substrates that bypass glycolysis can rescue cells after PARP1 activation. PARP1 was activated in mouse cortical astrocyte and astrocyte-neuron cocultures with the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Studies using the 2-deoxyglucose method confirmed that glycolytic flux was reduced by more than 90% in MNNG-treated cultures. The addition of 5 mmol/L of alpha-ketoglutarate, 5 mmol/L pyruvate, or other mitochondrial substrates to the cultures after MNNG treatment reduced cell death from approximately 70% to near basal levels, while PARP inhibitors and excess glucose had negligible effects. The mitochondrial substrates significantly reduced cell death, with delivery delayed up to 2 hours after MNNG washout. The findings suggest that impaired glycolytic flux is an important factor contributing to PARP1-mediated cell death. Delivery of alternative substrates may be a promising strategy for delayed treatment of PARP1-mediated cell death in ischemia and other disorders.

Mitochondrial permeability transition and calcium dynamics in striatal neurons upon intense NMDA receptor activation.

Deregulation of the intracellular Ca2+ homeostasis by NMDA receptor activation leads to neuronal cell death. Induction of the mitochondrial permeability transition pore (MPT) by Ca2+ is a critical event in mediating cell death. In this study, we used fluorescent Ca2+ indicators to investigate the effect of high concentrations of NMDA on cytosolic and mitochondrial Ca2+ concentrations ([Ca2+]c and [Ca2+]m, respectively) in cultured striatal neurons. Exposure to NMDA resulted in an immediate, sustained increase in [Ca2+]c followed by a secondary increase in [Ca2+]c. This second increase of [Ca2+]c was prevented by pretreatment with N-methyl-valine-4-cyclosporin (NMV-Cys). Exposure of neurons to NMDA also resulted in an increase in [Ca2+]m that was followed by a precipitous decrease in the rhod-2 signal. This decrease followed the time frame of the secondary increase in [Ca2+]c. Preincubation of the neurons with NMV-Cys prevented the decrease in rhod-2 fluorescence. These dynamic changes in the rhod-2 signal and [Ca2+]m in response to NMDA were confirmed by using confocal microscopy. The presented results indicate that MPT can be detected in living neurons using fluorescent Ca2+ indicators, which would allow the study of the physiological role of MPT in cell death.