PARP‐1 activation causes mitochondrial dysfunction

When activated by DNA damage, PARP1 consumes NAD+ to form ADP-ribose polymers on acceptor proteins. Excessive PARP1 activation during oxidative DNA damage is known to cause NAD+ depletion and cell death, but the intervening steps are not well understood. Sustained inhibition of the NAD+-dependent step in glycolysis is a potential consequence of NAD+ depletion. If this occurs, it should be possible to rescue cells from PARP1-mediated cell death by supplying alternative substrates for the mitochondrial tricarboxylic acid cycle. PARP1 was activated in 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 a sustained reduction in glycolytic flux, and studies with the JC-1 and TMRM indicate mitochondrial membrane depolarization. The addition of 1 mm of a-ketoglutarate or 1 mm pyruvate after washout of MNNG reduced cell death from 60 to 80% to near control levels, while addition of PARP inhibitors or excess glucose after MNNG washout had negligible effect. Cytoprotective effects remained significant with substrate delivery delayed up to 3 h after MNNG washout. The findings suggest that impaired glycolytic flux is a major factor in PARP1-mediated cell death, and suggest that supply of alternative mitochondrial substrates may be a promising strategy for delayed treatment of PARP1-mediated cell death in ischemia and other disorders.