Acetylation of the Pro-Apoptotic Factor, p53 in the Hippocampus following Cerebral Ischemia and Modulation by Estrogen Limor Raz., Quan-guang Zhang . , Dong Han, Yan Dong, Liesl De Sevilla, Darrell W. Brann* Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, Georgia, United States of America Abstract Background: Recent studies demonstrate that acetylation of the transcription factor, p53 on lysine373 leads to its enhanced stabilization/activity and increased susceptibility of cells to stress. However, it is not known whether acetylation of p53 is altered in the hippocampus following global cerebral ischemia (GCI) or is regulated by the hormone, 17b-estradiol (17b2E2), and thus, this study examined these issues. Methodology/Principal Findings: The study revealed that Acetyl p53-Lysine373 levels were markedly increased in the hippocampal CA1 region after GCI at 3 h, 6 h and 24 h after reperfusion, an effect strongly attenuated by 17b2E2. 17b2E2 also enhanced interaction of p53 with the ubiquitin ligase, Mdm2, increased ubiquitination of p53, and induced its down- regulation, as well as attenuated elevation of the p53 transcriptional target, Puma. We also observed enhanced acetylation of p53 at a different lysine (Lys382) at 3 h after reperfusion, and 17b2E2 also markedly attenuated this effect. Furthermore, administration of an inhibitor of CBP/p300 acetyltransferase, which acetylates p53, was strongly neuroprotective of the CA1 region following GCI. In long-term estrogen deprived (LTED) animals, the ability of 17b2E2 to attenuate p53 acetylation was lost, and intriguingly, Acetyl p53-Lysine373 levels were markedly elevated in sham (non-ischemic) LTED animals. Finally, intracerebroventricular injections of Gp91ds-Tat, a specific NADPH oxidase (NOX2) inhibitor, but not the scrambled tat peptide control (Sc-Tat), attenuated acetylation of p53 and reduced levels of Puma following GCI. Conclusions/Significance: The studies demonstrate that p53 undergoes enhanced acetylation in the hippocampal CA1 region following global cerebral ischemia, and that the neuroprotective agent, 17b2E2, markedly attenuates the ischemia- induced p53 acetylation. Furthermore, following LTED, the suppressive effect of 17b2E2 on p53 acetylation is lost, and p53 acetylation increases in the hippocampus, which may explain previous reports of increased sensitivity of the hippocampus to ischemic stress following LTED. Citation: Raz L, Zhang Q-g, Han D, Dong Y, De Sevilla L, et al. (2011) Acetylation of the Pro-Apoptotic Factor, p53 in the Hippocampus following Cerebral Ischemia and Modulation by Estrogen. PLoS ONE 6(10): e27039. doi:10.1371/journal.pone.0027039 Editor: Huaibin Cai, National Institute of Health, United States of America Received April 8, 2011; Accepted October 9, 2011; Published October 26, 2011 Copyright: 2011 Raz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research was supported by a research grant (NS050730) to DWB from the National Institute of Neurological Disorders and Stroke, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dbrann@georgiahealth.edu . These authors contributed equally to this work. Introduction Stroke is the third leading cause of death and the number one cause of disability in the United States [1–3]. In order to help understand the molecular mechanisms and processes that underlie neuronal cell death following ischemic stroke, animal models of focal and global cerebral ischemia (GCI) have been developed [4,5] [6,7]. The hippocampal CA1 region, an area critical for learning and memory [8–10], is highly sensitive to damage following GCI. Along these lines, neurons in the hippocampal CA1 region have been shown to undergo delayed apoptotic neuronal cell death following GCI [8,9,11]. Apoptotic neuronal cell death has also been shown to occur in the penumbra region of the cerebral cortex after focal cerebral ischemia [12]. P53, a pro-apoptotic factor, has been implicated to play a major role in apoptotic neuronal cell death following cerebral ischemia [13]. Evidence from p53 knock-out mice studies revealed reduced neuronal cell death after GCI as compared to WT-controls [14]. In addition, pifithrin-alpha (PFTa), a p53 specific pharmacological inhibitor, attenuated p53 nuclear transport and DNA binding, while increasing the number of surviving neurons [15] and promoting functional recovery following stroke [16]. In non- neuronal cells, p53 has been implicated to induce cell death following cell stress via both a transcriptional-dependent nuclear mechanism, as well as a transcription-independent mechanism involving its direct action with a subset of Bcl-2 family member proteins in the cytosol and mitochondria. However, recent work suggests that the apoptotic activity of p53 in neurons does not rely on its direct action at the cytosol/mitochondria and appears to be mediated exclusively through its transcription-dependent nuclear functions to induce the p53 pro-apototic BH3 family gene, Puma (p53 upregulated modulator of apoptosis) [17]. PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e27039