title: Cellular NAD replenishment confers marked neuroprotection against ischemic cell death: role of enhanced DNA repair.
abstract: 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.
authors.affiliation: Department of Neurology, S-507 BST, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Suping
authors.initials: S
authors.lastname: Wang
authors.forename: Zili
authors.initials: Z
authors.lastname: Xing
authors.forename: Peter S
authors.initials: PS
authors.lastname: Vosler
authors.forename: Hannah
authors.initials: H
authors.lastname: Yin
authors.forename: Wenjin
authors.initials: W
authors.lastname: Li
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.forename: Armando P
authors.initials: AP
authors.lastname: Signore
authors.forename: R Anne
authors.initials: RA
authors.lastname: Stetler
authors.forename: Yanqin
authors.initials: Y
authors.lastname: Gao
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
mesh_terms: 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
chemical_list: D018696:Neuroprotective Agents; D009243:NAD; D019951:DNA Polymerase beta; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase