Metabolic substrates prevent PARP1-mediated mitochondrial dysfunction and cell death

Introduction

Poly(ADP-Ribose) Polymerase-1 (PARP1) participates in DNA repair, and consumes NAD+ to form PAR polymers on acceptor proteins ¹ . PARP1 hyperactivation occurs in ischemic stroke and excitotoxicity, and promotes cell death by mechanisms that remain poorly understood. Induction of the mitochondrial permeability transition (MPT) is a causative factor in both apoptotic and necrotic cell death in excitotoxicity and ischemia ² . We previously showed that MPT induction is necessary for PARP1-mediated cytotoxicity, and that NAD+ repletion is protective ³ . Here we show that i) NAD+ protection against PARP1 hyperactivation requires glucose, and ii) tricarboxylic acid (TCA) substrates prevent PARP1-induced mitochondrial dysfunction and cell death. We hypothesize that PARP1 hyperactivation leads to glycolytic blockade through NAD+ depletion, and provide further evidence of NAD+ as a link between PARP1 activation, mitochondrial dysfunction, and cellular demise.

Methods

Mouse cortical astrocytes and neurons were prepared as previously described ³ . DNA damage was performed with the DNA alkylating agent, MNNG (M; 100microM) for 30 min. Pharmacological compounds: PARP inhibitor DPQ (D; 25microM); MPT inhibitor CsA (C; 200 nM); NAD+ (2–5 mM). Cell death was assayed after 24 h by LDH release (astrocytes) and PI-staining (neurons). Mitochondrial membrane potential (MMP) was assayed with the potentiometric indicator TMRM (1nM), and fluorescence (F) was normalized to baseline (Fo). MPT induction was assayed using calcein/mitotracker. Values expressed as mean ± SE (n=3; **p<0.001 compared to control).

Results

MNNG activated PARP1 by inducing DNA damage. PARP1 hyperactivation led to NAD+ depletion, and also promoted MMP depolarization (gray bars), MPT induction, and cell death (black bars). These were prevented in PARP1 deficient cells, by PARP1 inhibition (DPQ), or MPT inhibition (CsA). Restoration of intracellular NAD+ after PARP1 hyperactivation prevented collapse of MMP, MPT induction, and cell death. NAD+ protection against PARP1-mediated MMP depolarization and cell death was dependent on glucose. NAD+ did not inhibit PARP1 activation. Treatment with TCA cycle substrates (glutamine (Gln), pyruvate (Pyr), or alpha-ketoglutarate (aKG)) to bypass glycolysis and directly provide substrate to mitochondria, also prevented MMP depolarization and cell death, further suggesting glycolytic failure with PARP1 hyperactivation.

Conclusion

These results indicate that PARP1 hyperactivation leads to glycolytic inhibition, presumably through NAD+ depletion. This leads to a decrease in substrate availability to mitochondria, which promotes mitochondrial dysfunction and subsequent cell death cascade. Therefore, cellular NAD+ serves as a key molecular signal for MPT induction and subsequent cell death (See Figure 1).

Figure 1