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J. Biol. Chem., Vol. 279, Issue 18, 18895-18902, April 30, 2004
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From the Department of Neurology, University of California, San Francisco and the Veterans Affairs Medical Center, San Francisco, California 94121
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.
Received for publication, December 5, 2003 , and in revised form, February 6, 2004.
* This work was supported by National Institutes of Health Grants NS11048 (to W. Y.) and NS14543 (to R. A. S.) and a grant from the Department of Veterans Affairs (to R. A. S. and C. C. A). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work
To whom correspondence may be addressed: Dept. of Neurology (127), Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-750-2011; Fax: 415-750-2273; E-mail: calano{at}itsa.ucsf.edu.
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