title: Impaired DNA repair via the base-excision repair pathway after focal ischemic brain injury: a protein phosphorylation-dependent mechanism reversed by hypothermic neuroprotection.
abstract: Cerebral ischemia and reperfusion induces rapid accumulation of oxidative DNA lesions in the brain, which, if not repaired promptly, may trigger cell death. The base-excision repair (BER) pathway is the main mechanism employed by neurons to repair various types of oxidative DNA damage. Recent studies have suggested that the cellular activity of BER is highly regulated (up- or down-regulated) after ischemic brain injury, and this regulation may contribute to the outcome of cell injury. The mechanism through which cellular BER is regulated in response to neuronal injury is currently poorly understood. In the present study, we have examined BER regulation in the rat model of focal ischemic brain injury induced by 2 hr of middle cerebral artery occlusion and 0-72 hr of reperfusion. As determined using cerebral nuclear extracts, focal ischemia resulted in a marked reduction in BER activities, including the overall BER activity, AP endonuclease activity and DNA polymerase-beta activity, indicating functional impairment of the BER pathway. BER reduction occurred as early as 0.5 hr after the onset of reperfusion. Thereafter, BER activity failed to recover, and there were persistent accumulations of apurinic/apyrimidinic abasic sites and DNA single-strand breaks in ischemic tissues. The reduction in BER during the early reperfusion phase (less than 6 hr) was not accompanied by any alterations in the levels of essential BER enzymes in brain extracts. However, increased serine- and threonine-specific phosphorylation was detected for both AP endonuclease and DNA polymerase-beta after ischemia, with the time course of serine phosphorylation closely correlated to that of changes in BER activity. Furthermore, dephosphorylation of nuclear extracts with alkaline phosphatase largely restored AP endonuclease and DNA polymerase-beta activities. Taking advantage of the neuroprotective effect of mild hypothermia (33 degrees C), which was induced in the brain during the first 2 hr of reperfusion, we found that the post-ischemic suppression of BER activity is a reversible event. Hypothermic treatment diminished the serine-specific phosphorylation of AP endonuclease and DNA polymerase-beta, promoted BER activities, and attenuated the levels of oxidative DNA lesions after ischemia. These results suggest that the functional impairment of the BER pathway after severe focal cerebral ischemia is due to the loss-of-function post-translational modifications of repair enzymes. Further investigations elucidating the precise mechanism underlying the post-translational regulation of BER enzymes may lead to novel therapeutic strategies for cerebral ischemia.
authors.affiliation: Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Yumin
authors.initials: Y
authors.lastname: Luo
authors.forename: Xunming
authors.initials: X
authors.lastname: Ji
authors.forename: Feng
authors.initials: F
authors.lastname: Ling
authors.forename: Wenjin
authors.initials: W
authors.lastname: Li
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.forename: Guodong
authors.initials: G
authors.lastname: Cao
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000201:enzymology* / Q000235:genetics / Q000628:therapy; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D007036:Hypothermia, Induced; D008297:Male; D018384:Oxidative Stress; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000201:enzymology / Q000235:genetics / Q000628:therapy
chemical_list: D019951:DNA Polymerase beta; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D045643:DNA Repair Enzymes