Poly(ADP-ribose) Polymerase-1-mediated Cell Death in Astrocytes Requires NAD+ Depletion and Mitochondrial Permeability Transition

Abstract

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...

Prevention of Acute/Severe Hypoglycemia-Induced Neuron Death by Lactate Administration

“…Interestingly, repletion of NAD + after PARP-1 activation has been shown to restore glycolytic capacity and prevent cell death (Ying et al, 2003(Ying et al, , 2005 in primary neuronal cultures. Similarly, providing nonglucose substrates such as pyruvate and a-ketoglutarate that can be metabolized without the need for cytosolic NAD + also preserves cell viability, even when delivered hours after PARP-1 activation (Alano et al, 2004;Ying et al, 2002). However, to be used oxidatively, lactate must be first converted to pyruvate by lactate dehydrogenase, which also requires NAD + and thus competes with glycolysis for NAD + .…”

Cerebral Endothelial Cell Apoptosis after Ischemia—Reperfusion: Role of PARP Activation and AIF Translocation

“…Studies in PARP-1 knockout mice and PARP-1-null neuronal cultures indicate that PARP-1 activation may signal this translocation step secondary to the poly(ADP-ribosyl)ation of unidentified nuclear proteins that translocate from the nucleus to the mitochondria (Hong et al, 2004;Yu et al, 2003). Studies in cultured astrocytes and other cells indicate that it is also possible that depletion of ATP and/or NAD þ in mitochondria, secondary to PARP-1 activation, signals mitochondrial AIF release (Alano et al, 2004). In any event, it is now well established that pharmacologic or genetic interruption of PARP-1 activation is cytoprotective in a wide variety of in vivo and in vitro models in response to an equally wide variety of injurious stimuli.…”

Combination treatment with arsenic trioxide and phytosphingosine enhances apoptotic cell death in arsenic trioxide–resistant cancer cells

“…Recently, it has been shown that ROS-mediated DNA damage triggers activation of the PARP-1 and subsequent cell death (30,31). Although PARP-1 -mediated cell death is thought to be necrotic (32,33), recent reports have shown that PARP-1 -mediated cell death also has many features in common with the apoptotic forms of cell death (30,31,34,35).…”