Cellular NAD replenishment confers marked neuroprotection against ischemic cell death: role of enhanced DNA repair.
OBJECTIVE
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.
Wang
Suping
S
Department of Neurology, S-507 BST, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
Xing
Zili
Z
Vosler
Peter S
PS
Yin
Hannah
H
Li
Wenjin
W
Zhang
Feng
F
Signore
Armando P
AP
Stetler
R Anne
RA
Gao
Yanqin
Y
Chen
Jun
J
D000818:Animals; D002545:Brain Ischemia / Q000188:drug therapy / Q000378:metabolism* / Q000503:physiopathology; D016923:Cell Death; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D019610:Cytoprotection / Q000187:drug effects; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D009243:NAD / Q000378:metabolism* / Q000494:pharmacology; D009410:Nerve Degeneration / Q000188:drug therapy / Q000378:metabolism* / Q000503:physiopathology; D018696:Neuroprotective Agents / Q000378:metabolism / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects
D018696:Neuroprotective Agents; D009243:NAD; D019951:DNA Polymerase beta; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase