NAD⁺ Depletion Is Necessary and Sufficient forPoly(ADP-Ribose) Polymerase-1-Mediated Neuronal Death

NAD⁺ repletion prevents PARP-1-mediated neuron death. A, B, Neurons were incubated with the designated concentrations of either MNNG (A) for 30 min or SIN1 (B) for 60 min, and NAD⁺ (2.5–10 mm) was added after washout of the MNNG. Neuron death was assayed at 24 h. n = 3; *p < 0.05, **p < 0.01 versus no added NAD⁺. Error bars indicate SEM. C, Immunostaining for PAR at 60 min after MNNG washout showed no inhibition of PARP-1 activity by 5 mm NAD⁺. The PARP inhibitor PJ34 serves as a positive control. Images are representative of four independent experiments. Scale bar, 40 μm. D, Western blot analysis of PAR formation after MNNG exposure (75 μm for 30 min). NAD⁺ was used at 2 and 5 mm and PJ34 at 200 nm. Cells were lysed 30 min after MNNG washout.

Effects of NAD⁺ on PARP-1-induced neuronal death. A–I, Neuronal death was induced by MNNG (A, D, G), NMDA (B, E, H), and chemical ischemia (C, F, I). Incubation times were 30 min for MNNG, 10 min for NMDA, and variable with CI. A–C, Dose–response curves are shown for each condition. NMDA-induced neuronal death was blocked by 10 μm MK801 as a positive control. Neuronal death in all three conditions was reduced by coincubation with the PARP inhibitor, DPQ (10 μm). **p < 0.01 versus control (wash only); n = 3–4. D–F show the effects of posttreatment with 1–10 mm NAD⁺ on neuronal death under each of the conditions shown in A–C. The NAD⁺ was added after washout of MNNG, CI, or NMDA. **p < 0.01 versus no added NAD⁺; n = 3–4. G–I show the effects of 5 mm NAD⁺ or 10 μm DPQ on neuronal survival when added to the culture medium at increasing time intervals after washout of MNNG, CI, or NMDA. **p < 0.01 versus no added NAD⁺; n = 3–4. Error bars indicate SEM.

PARP-1 activation causes a block in neuronal glycolysis that is reversed by NAD⁺. Neurons were treated with 75 μm MNNG for 30 min or 2.5 mm SIN1 for 60 min. Where used, PJ34 (200 nm) and DPQ (10 μm) were added with MNNG or SIN1. A, B, Glycolytic rate and pyruvate content were measured 1 h after MNNG or SIN1 washout. n = 3; **p < 0.001 versus control. C, D, Five millimolar NAD⁺ or 2.5 mm pyruvate was added to the after washout of MNNG or SIN1. Glycolytic rate was restored by NAD⁺, but not pyruvate, whereas intracellular pyruvate concentrations were restored by both NAD⁺, but not pyruvate. PJ34 or DPQ were given simultaneously with MNNG or SIN1. n = 3; **p < 0.001 versus no posttreatment (white bar). E, F, ATP and AMP were measured 1 h after MNNG or SIN1 washout. The effects of MNNG on ATP and AMP levels were blocked by cotreatment with PJ34 or DPQ, and also blocked by posttreatment with NAD⁺ or pyruvate. Error bars indicate SEM.

PARP-1-induced neuronal death is prevented by nonglycolytic substrates. A, B, Neurons were provided with pyruvate (Pyr) (2.5 mm) or α-ketoglutarate (αKG) (2.5 mm) after washout of MNNG or SIN1. The reduction in cell death was comparable with that achieved by coincubation with the PARP inhibitors, DPQ (10 μm) and PJ34 (200 nm). n = 3; **p < 0.001 versus MNNG or SIN1 alone. Error bars indicate SEM. C, Representative images of neurons 24 h after the designated treatments. Cell fields were photographed with phase contrast optics and with calcein fluorescence to identify live cells and PI fluorescence to identify dead cells.

PARP-1-induced mitochondrial depolarization is prevented by NAD⁺ and pyruvate. A, Representative images of TMRM fluorescence in neurons after 30 min incubation with 2 mm SIN1. The loss of TMRM fluorescence, indicating loss of mitochondrial membrane potential, was prevented by coincubation with 200 nm PJ34. B, Mitochondrial depolarization over time after PARP-1 activation (red traces) with either MNNG (75 μm) or SIN1 (2 mm). The decrease in TMRM fluorescence was prevented by coincubation with the PARP inhibitor PJ34 (200 nm) or by postincubation with either NAD⁺ or pyruvate (2.5 mm). Neurons were exposed to FCCP (1 μm) at the end of each experiment to calibrate complete mitochondrial depolarization. C, Quantified TMRM fluorescence 2 h after MNNG or SIN1 treatment. n = 3; *p < 0.01, **p < 0.001 versus SIN1 or MNNG alone. Error bars indicate SEM.

Depletion of cytosolic NAD⁺ with NADase kills neurons independent of PARP-1 activation. A, NADase transfection (10, 30, 100 μg/ml) produced a dose-dependent decrease in neuronal NAD⁺ content in both wild-type and PARP-1^−/− neurons, as measured 3 h after BioPORTER transfection (BP). This decrease was blocked by the NADase inhibitor NAM (200 μm) and not observed in cultures treated with the BioPORTER vehicle alone (far right bar). B, NADase transfection also produced a dose-dependent neuronal death in both wild-type and PARP-1^−/− neurons, evaluated 24 h later. This decrease was blocked by the NADase inhibitor NAM (200 μm) and not observed in cultures treated with the BioPORTER vehicle alone. C, Neuron death caused by NADase transfection (30 μg/ml) was significantly prevented by exogenous NAD⁺ treatment (2.5, 5, and 10 mm) in both wild-type and PARP-1^−/− neurons. Error bars indicate SEM. *p < 0.01, **p < 0.001 (in A and B, versus control; in C, versus NADase alone).

AIF translocation induced by PARP-1 or NADase is prevented by pyruvate. A, Immunostaining for AIF (green) in cultures fixed 3 h after incubation with MNNG alone (75 μm for 30 min), MNNG with 200 nm PJ34, MNNG followed by 5 mm NAD⁺, MNNG followed by 2.5 mm pyruvate, or medium exchanges only (control). Nuclei are counterstained with PI (red). Merged images show that DPQ, NAD⁺, and pyruvate block AIF translocation to the nucleus. B, C, Neurons transfected with NADase showed AIF translocation to the nucleus in both wild-type and PARP-1^−/− neurons fixed 4 h after BioPORTER transfection. The AIF translocation was blocked by 2.5 mm pyruvate. Images are representative of four independent experiments. Scale bar, 40 μm. D, E, Western blots show both total and mitochondrial AIF content at designated time points (hours) after transfection with NADase, with or without pyruvate. The Western blots were quantified after normalizing to either β-actin for total AIF, or the mitochondrial protein VDAC for mitochondrial AIF. Mitochondrial AIF release occurred in both wild-type and PARP-1^−/− neurons and was blocked by 2.5 mm pyruvate. F, G, Western blots show both total and mitochondrial AIF content at designated time points (hours) after placement in glucose-free medium, with or without pyruvate. Total AIF was quantified after normalizing to β-actin, and mitochondrial AIF was quantified after normalizing to the mitochondrial marker, VDAC. Mitochondrial AIF release occurred in both wild-type and PARP-1^−/− neurons and was blocked by 2.5 mm pyruvate. n = 3; *p < 0.01, **p < 0.001. Error bars indicate SEM.