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 ϩ 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 ϩ to restore neuronal NAD ϩ levels after PARP-1 activation. Exogenous NAD ϩ was found to enter neurons through P2X 7 -gated channels. Restoration of cytosolic NAD ϩ 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 ϩ with NAD ϩ glycohydrolase produced a block in glycolysis inhibition, mitochondrial depolarization, AIF translocation, and neuron death, independent of PARP-1 activation. These results establish NAD ϩ depletion as a causal event in PARP-1-mediated cell death and place NAD ϩ depletion and glycolytic failure upstream of mitochondrial AIF release.

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. PARP-1,1 the most abundant of the poly(ADP-ribose) polymerase family members, is rapidly activated by DNA damage. PARP-1 utilizes NADϩ to form poly(ADP-ribose) polymers on specific acceptor proteins. PARP-1 activation appears to facilitate DNA repair under moderate stress conditions (3), but extensive PARP-1 activation promotes cell death under conditions that cause extensive DNA damage such as excitotoxicity and ischemia (2, 4). Both genetic deletion and pharmacological inhibition of PARP-1 activity can markedly reduce cell death under these conditions (5-7).The sequence of events leading from PARP-1 activation to cell death has not been fully established. PARP-1-mediated cell death is caspase-independent, lacks many morphological hallmarks of classical apoptosis, and was long considered a direct result of energy failure (5, 8, 9). Extensive PARP-1 activation causes NAD ϩ depletion, which, in turn, can lead to ATP depletion (5, 8, 10). However, it has also been shown that translocation of the apoptosis-inducing factor (AIF) from mitochondria to the nucleus is required for PARP-1-mediated cell death (11). Thus, a fundamental unanswered question is whether NAD ϩ depletion is a necessary and causal event in PARP-1-mediated cell death, and, if so, what is the link between NAD ϩ depletion and mitochondrial release of AIF.Mitochondria permeability transition (MPT), like PARP-1 activation, is a significant factor leading to cell death after excitotoxicity, ischemia-reperfusion, and other disorders (12, 13). MPT results from the opening of a large conductance channel in the inner mitochondrial membrane (14, 15). Pharmacol...

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 M 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.death ͉ doxycycline ͉ inflammation ͉ neuron M inocycline and other tetracycline derivatives have neuroprotective effects unrelated to their antimicrobial properties (1). Minocycline can reduce neuronal death after excitotoxicity and ionizing radiation in culture (2, 3), and in animal models of stroke (1, 3-5), Parkinson's disease (6, 7), Huntington's disease (8), and amyotrophic lateral sclerosis (9). The neuroprotective effects of minocycline have been attributed both to reduced inflammation and a direct effect on neuronal survival (2, 3, 5, 9, 10).Poly(ADP-ribose) polymerase-1 (PARP-1) plays a key role in neuronal death and survival under stress conditions (11). PARP-1 is the most abundant of several PARP family members, accounting for Ͼ85% of nuclear PARP activity (11). When activated by DNA damage, PARP-1 consumes NAD ϩ to form branched poly(ADP-ribose) on target proteins. Poly(ADPribose) formation on histones and enzymes involved in DNA repair appears to facilitate DNA repair by preventing chromatid exchange and by loosening histone wrapping (12, 13). Poly(ADPribose) formation also has effects on gene transcription through interactions with transcription factors, notably NF-B (14-16), and PARP-1 inhibition or gene deletion attenuates the brain microglial response to cytokines and other triggers (17-19). Extensive PARP-1 activation can, in addition, lead to neuronal death through mechanisms linked to NAD ϩ deplet...

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.

BackgroundSirtuins (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 FindingsTo 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.ConclusionsThis study demonstrated for the first time that mitochondrial Sirt3 acts as a prosurvival factor playing an essential role to protect neurons under excitotoxic injury.