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 (10 mM) 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.