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Cellular NAD replenishment confers marked neuroprotection against ischemic cell death: role of enhanced DNA repair - PubMed

Cellular NAD replenishment confers marked neuroprotection against ischemic cell death: role of enhanced DNA repair

Suping Wang et al. Stroke. 2008 Sep.

Abstract

Background and purpose: NAD(+) is an essential cofactor for cellular energy production and participates in various signaling pathways that have an impact on cell survival. After cerebral ischemia, oxidative DNA lesions accumulate in neurons because of increased attacks by ROS and diminished DNA repair activity, leading to PARP-1 activation, NAD(+) depletion, and cell death. The objective of this study was to determine the neuroprotective effects of NAD(+) repletion against ischemic injury and the underlying mechanism.

Methods: In vitro ischemic injury was induced in rat primary neuronal cultures by oxygen-glucose deprivation (OGD) for 1 to 2 hours. NAD(+) was replenished by adding NAD(+) directly to the culture medium before or after OGD. Cell viability, oxidative DNA damage, and DNA base-excision repair (BER) activity were measured quantitatively up to 72 hours after OGD with or without NAD(+) repletion. Knockdown of BER enzymes was achieved in cultures using AAV-mediated transfection of shRNA.

Results: Direct NAD(+) repletion in neurons either before or after OGD markedly reduced cell death and OGD-induced accumulation of DNA damage (AP sites, single and double strand breaks) in a concentration- and time-dependent manner. NAD(+) repletion restored nDNA repair activity by inhibiting serine-specific phosphorylation of the essential BER enzymes AP endonuclease and DNA polymerase-beta. Knocking down AP endonuclease expression significantly reduced the prosurvival effect of NAD(+) repletion.

Conclusions: Cellular NAD(+) replenishment is a novel and potent approach to reduce ischemic injury in neuronal cultures. Restoration of DNA repair activity via the BER pathway is a key signaling event mediating the neuroprotective effect of NAD(+) replenishment.

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Figures

Figure 1
Figure 1

NAD replenishment protects neurons against OGD. NAD+ treatment reduces LDH release (A, C) and increases cell viability (B, D) after OGD in a concentration- and time-dependent manner. A and B, NAD+ was added to cultures for 3 hours at 30 minutes before OGD. C and D, 15 mmol/L of NAD+ was added for 3 hour at the indicated time points before or after 2 hours of OGD. LDH and cell viability were measured 24 hours after OGD. E, Percentages of condensed nuclei based on DAPI staining at 24 hours after OGD; NAD+ was added at the indicated time points before or after 2 hours of OGD. **P<0.01; ***P<0.001 vs no NAD+ treatment. n=12 per data point from 4 independent experiments. F, Phase-contrast photomicrographs show preservation of neuronal morphology in NAD+-treated neurons 24 hours after 2 hours of OGD (Post, NAD+ added immediately or 2 hours after the completion of OGD). G, Nuclear staining shows reduced nuclear condensation in NAD+-treated neurons 24 hours after 2 hours of OGD.

Figure 2
Figure 2

NAD replenishment attenuates oxidative DNA damage after OGD. AP sites (A) and DNA single strand breaks (B) were quantitatively measured in neurons at various time points after 2 hours of OGD. NAD+ (15 mmol/L) was added to cultures either immediately before (Pre) or after (Post) OGD. *P<0.05; ***P<0.001 vs vehicle treatment at the same time point. n=9 per data point from 3 independent experiments. C, Representative triple-label fluorescent images for PANT (green, detecting SSB), PARS (red), and DAPI (blue) in control neurons or at 0.5 hour or 2 hours after 2 hours of OGD. NAD+ (15 mmol/L) or 3-AB (5 mmol/L) was added to cultures immediately before OGD. D, Representative Comet assay showing increased DNA comet tails in neurons 2 hours after OGD, which were absent in neurons treated with NAD+. E, Quantitative analysis of the comet tails in neurons at various time points after 2 hours of OGD. NAD+ (15 mmol/L) was added to cultures at 0 hour or 0.5 hour after the completion of OGD. *P<0.05; ***P<0.001 vs vehicle treatment at the same time point. Each data point was derived from the analysis of at least 100 neurons, based on four independent experiments.

Figure 3
Figure 3

NAD+ replenishment restores DNA repair activity after OGD. Representative autoradiographs show changes in APE activity (A), βpol activity (B), and total BER activity (C) in nuclear extracts from control non-OGD neurons or at 0.5, 1, 2, and 6 hours after 2 hours of OGD. Vehicle or NAD+ (15 mmol/L) was added to cultures immediately before OGD. The mature repair products for APE and βpol migrated at 25 bp and 50 bp, respectively. D, Quantitative analysis for APE activity, βpol activity, and total BER activity, based on 4 independent experiments. Vehicle or NAD+ (15 mmol/L) was added to cultures either immediately before (Pre) OGD or 0 hour after the completion of OGD (Post). ***P<0.001 versus vehicle treatment.

Figure 4
Figure 4

NAD+ replenishment attenuates the serine phosphorylation of BER enzymes. A, Western blots show the expression levels of major BER enzymes in nuclear extracts (20 μg protein per lane) after 2 hour of OGD. B, Quantitative analysis of relative abundance of BER enzymes after OGD, based on 4 independent experiments. *P<0.05 vs non-OGD controls. C and F, Autoradiograph shows the complete restoration of APE activity (C) and βpol activity (F) in nuclear extracts (0.5 or 2 hours after 2 hours of OGD) treated with alkaline phosphatase (Ap). D and G, Increased phosphorylation of APE1 (D) and βpol (G) on serine residues after 2 hours of OGD, which was reduced in neurons treated with NAD+ (15 mmol/L, added at 0 hour after OGD). Nuclear extracts were immunoprecipitated using the anti-APE1 and anti-βpol antibodies, respectively, and then immunoblotted with an antibody against phosphorylated serine (p-Ser). E and H, Semiquantitative analysis of serine phosphorylation of APE1 (E) and βpol (H) after OGD, based on 4 experiments. Vehicle or NAD+ (15 mmol/L) was added to cultures at 0 hour after the completion of OGD. **P<0.0; **P<0.01 vs vehicle treatment.

Figure 5
Figure 5

APE1 knockdown impairs the neuroprotective effect of NAD+ replenishment. AAV-mediated transfection for 3 days of the shRNA targeting sequence (shRNAt), but not the scramble sequence (shRNAs), reduced the protein expression of APE1 (A, shRNAt had no effect on the expression of other BER enzymes), APE activity (B, the repair product is indicated as 25 bp), and total BER activity (C). The graphs in the right panel illustrate the results from 4 experiments. ***P<0.001 vs nontransfected neurons. D, Quantitative measurement of AP sites in neurons at 2 hours after 2 hours of OGD with or without NAD+ treatment (15 mmol/L). Note that AP sites were significantly increased in shRNAt-transfected neurons in the presence of NAD+. E and F, Quantitative measurement of cell viability and LDH release at 24 hours after 2 hours of OGD with or without NAD+ treatment (15 mmol/L). The prosurvival effect of NAD+ was significantly reduced in shRNAt-transfected neurons, as compared to controls. *P<0.05; **P<0.01; ***P<0.001 vs the indicated conditions, based on 4 independent experiments.

Figure 6
Figure 6

Working hypothesis on the mechanism underlying NAD+-mediated neuroprotection.

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