Figure 4. NADPH oxidase inhibition attenuates Acetyl p53- Lysine373 levels and Puma levels following GCI. Inhibition of NADPH oxidase using the competitive NOX2 inhibitor, Gp91ds-Tat (Gp91), reduces Acetyl p53-Lysine373 levels, while diminishing Puma protein levels, as compared to the scrambled tat (Sc-Tat) control group. CA1 total lysates were collected at 3 h upon reperfusion onset and probed with appropriate antibodies to detect protein expression in Western blot. P53 protein expression remained unchanged. Actin was used as a loading control. Data is represented as fold differences in comparison to sham from four to five animals. *p,0.05 vs. Sham, #p,0.05 vs. Sc-Tat; S = Sham. doi:10.1371/journal.pone.0027039.g004 hippocampal CA1 region at 6 h and 1 d after GCI using real time (RT)-PCR. As shown in Fig. 3A, neither ischemia (Pla) nor 17b2E2 (E2) treatment significantly affected p53 gene expression in the hippocampal CA1 region. Actin, used as a loading experimental control, revealed equal loading on the RT-PCR gel (data not shown). We next examined ubiquitination of p53 by co-immuno- precipitated (Co-IP) of p53 and immunoblotting for ubiquitin at the 24 h time-point after reperfusion using total hippocampal CA1 region samples. The results showed that 17b2E2 treatment significantly increases p53 ubiquitination as compared to placebo- treated or sham control animals (Fig. 3B). Co-IPs were also performed examining p53-Mdm2 interaction in the hippocampal CA1 region after GCI (Fig. 3C). As shown in Fig. 3C, the Pla group had a pattern for a decrease of p53-Mdm2 interaction that correlated with an increase of p53 protein levels. In contrast, 17b2E2-treated animals had a significant enhancement of p53- Mdm2 interaction in the CA1 region, which correlated with a significant decrease in p53 protein levels (Fig. 3C). NOX2 NADPH Oxidase Inhibition Attenuates Acetyl p53/ p53 Ratio and Puma Levels after GCI Previous work by our laboratory revealed that 17b2E2 can attenuate NOX2 NADPH oxidase-induced superoxide (O22) production, and that administration of a competitive NOX2 inhibitor, Gp91ds-Tat, is strongly neuroprotective against GCI- induced neuronal damage in the hippocampal CA1 region [36]. We therefore hypothesized that NADPH oxidase-induced ROS may regulate p53 and its acetylation following GCI. To address this possibility, we used icv injections of the specific NOX2 inhibitor, Gp91ds-Tat. As a control, we used a scrambled tat peptide (Sc-Tat) that does not exert inhibitory effects on NADPH oxidase. Pro-apoptotic p53 levels remained unchanged in all of the treatment groups examined, further validating our previous Acetylation of p53 observations (Figure 4). When compared to scrambled-tat control peptide-treated animals, Gp91ds-Tat-treated animals had a significant attenuation of GCI-induced elevation of Acetyl p53 (expressed as an acetyl p53/p53 ratio) and a significant decrease in Puma protein levels in the hippocampal CA1 region at 3 h after GCI reperfusion, a time of peak NADPH oxidase activation following GCI [36]. This finding suggests that NADPH oxidase activation potentially contributes to cerebral ischemia-induced elevation of acetyl p53 and downstream Puma expression following GCI (Figure 4). Long-term Estrogen Deprivation Promotes p53 Acetylation and Activation in the Nucleus of Hippocampal CA1 Cells after Global Cerebral Ischemia Finally, our group recently demonstrated that 17b2E2 neuroprotection against GCI is lost if it is preceded by a prolonged period of 17b2E2 deprivation (e.g. 10 weeks ovariectomy) [36]. We thus examined whether long-term estrogen deprivation leads to a loss of 17b2E2 ability to attenuate Acetyl p53-lysine373 levels in the hippocampal CA1 region after GCI. We therefore compared animals treated immediately with 17b2E2 (Imm) to those in which 17b2E2 treatment was initiated after a long term period of 17b2E2 deprivation (e.g. treated 10 weeks after ovariectomy) (10 W) (Figure 5A, 5B). Representative photomi- crographs of the hippocampal CA region from the 24 h time-point after GCI from the various groups are presented in Fig. 5A, and semi-quantitative analysis of number of Acetyl p53-lysine373 positive cells are presented in Fig. 5B. As shown in Fig 5A, 5B, in the immediate (Imm) treatment paradigm, Pla-treated animals had robust staining and number of Acetyl p53-lysine373 positive cells in the CA1 region as compared to sham controls, while immediate 17b2E2-treatment caused a profound reduction in the staining and number of Acetyl p53-lysine373 positive cells. In contrast, in long-term E2 deprived (10 W) sham animals, there was robust Acetyl p53 staining and a high number of Acetyl p53- lysine373-positive cells (Fig. 5A, 5B). In addition, long-term E2 deprived (10 W) Pla animals also showed robust Acetyl p53 staining and a high number of Acetyl p53-lysine373 positive cells in the hippocampal CA1 region, which was not significantly different from the 10 W sham controls. Interestingly, the ability of 17b2E2 to attenuate Acetyl p53 staining and number of Acetyl p53- lysine373-positive cells was completely lost in the long-term E2 deprived animals (Fig. 5A, 5B). Inhibition of CBP/p300 acetyltransferase is neuro- protective of the hippocampus CA1 following global cerebral ischemia. We next examined whether inhibition of CBP/p300 acetyltranferases, which acetylate p53, would exert neuroprotection against GCI. To examine this question, we utilized the selective CBP/p300 acetyltransferase inhibitor, curcumin [31,32]. As shown in Figure 6A and 6B, administration of curcumin 20 minutes prior to GCI was strongly neuroprotective in the hippocampal CA1 region, as evidenced by preservation of NeuN-stained surviving cells and a decrease in TUNEL-positive cells in the CA1 region at 7 days after GCI. Discussion The current study advances the field by demonstrating that p53 undergoes enhanced acetylation on Lysine373 and Lysine382 in the hippocampal CA1 region following global cerebral ischemia. Acetylation of p53 at Lysine373 and Lysine382 has been shown to enhance stability and pro-apoptotic activity of p53, while increasing sensitivity to cell stress [27–30,41,46]. Thus, our study suggests that post-translational modification of p53 by acetylation PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e27039