We examined the protein expression of apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, in mice after transient focal cerebral ischemia. Immunohistochemistry showed nuclear expression of this enzyme in the entire region of the control brains. Immunoreactivity and Western analysis showed an early and rapid decrease in APE/Ref-1 in the entire middle cerebral artery. A similar level was detected in the control brains of both transgenic mice that overexpress human CuZn-superoxide dismutase (SOD1) and wild-type littermates. While APE/Ref-1 was significantly reduced 1 hour after transient focal cerebral ischemia in both groups, the SOD1 transgenic mice has less reduction than the wild-type mice. The delayed occurrence of DNA laddering was also significantly reduced in the transgenic mice. Double staining with APE/Ref-1 and TUNEL showed that neurons that lost APE/Ref-1 immunoreactivity became TUNEL-positive. Finally, treatment with the antioxidant 21-aminosteroid also prevented the early decrease of APE/Ref-1 expression after photothrombotic cortical cerebral ischemia in mice. These results suggest that reactive oxygen species contribute to the early decrease of APE/Ref-1 and thereby exacerbate DNA fragmentation after transient focal cerebral ischemia in mice. The DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/Ref-1) is a multifunctional protein in the DNA base excision repair pathway, which is responsible for repairing apurinic/apyrimidinic sites in DNA (Bennett et al. 1997). DNA base excision repair is known to require two types of enzymes, such as DNA glycosylases and APEs (Sancar and Sancar 1988; Doetsch and Cunningham 1990; Lindahl 1990; Demple and Harrison 1994). DNA glycosylases remove a damaged base, which could be caused by various kinds of insults, such as oxidative stress in particular, creating an apurinic/apyrimidinic site in the DNA that is then acted on by an APE (Doetsch and Cunningham 1990; Demple and Harrison 1994). The DNA repair is completed by abasic residue followed by synthesis of a new base by DNA polymerase and ligation. Incomplete repair of apurinic/apyrimidinic sites is reported to cause mutagenesis and genetic instability (Loeb and Preston 1986). DNA damage and repair is drawing more attention in the field of central nervous system injuries, including cerebral ischemia and brain trauma (Chopp et al. 1996). Liu and colleagues (1996)have suggested that free radicals could attack the nuclear genes and cause genetic instability after mouse forebrain ischemia. As for the relationship of the DNA base excision repair pathway to necrosis and/or apoptosis, recent evidence suggests that down-regulation of APE expression is associated with apoptosis in cells of the myeloid lineage (Robertson et al. 1997). We have shown that the loss of APE/Ref-1 is closely associated with the occurrence of DNA fragmentation in hippocampal CAI neurons after transient global ischemia (Kawase et al. 1999). However, the mechanism by which these early modifications of APE/Ref-1 expression after focal cerebral ischemia/reperfusion injury is regulated in vivo is unknown. Antioxidant enzymes and DNA repair proteins are thought to be two major mechanisms by which cells counteract the deleterious effects of reactive oxygen species (ROS), however, little is known about the interaction between them. Moreover, there is no report that indicates the direct correlation between ROS and the expression of DNA repair enzymes including APE/Ref-1, in vivo. To address this critical issue in vivo, we examined APE/Ref-1 expression after transient focal cerebral ischemia (FCI) in both wild-type mice and transgenic (Tg) mice that overexpress human CuZn-superoxide dismutase (SOD1), and with the treatment of a known antioxidant, 21-aminosteroid, in mice after photothrombotic cerebral ischemia.
