• Mechanisms of Estrogen Neuroprotection in Stroke

      Raz, Limor; Department of Neurology (2011-04)
      17-β estradiol (17-β-E2) has been implicated to be neuroprotective, yet the mechanisms underlying 17-β-E2-mediated protection against stroke remain unclear. The purpose of the current study was to elucidate the role of 17-β-E2 in NADPH oxidase (NOX2) activation during ischemic reperfusion induction of superoxide (O2 -) in the hippocampus CA1 region following global cerebral ischemia (GCI) and to investigate the post-translational deacetylation of downstream pro-apoptotic factors by 17-β-E2. Using a 4-vessel occlusion model to induce GCI, we showed that neuronal NOX2 localizes to the membrane and that NADPH oxidase activity and O2 - production were rapidly and markedly attenuated by 17-β-E2 following reperfusion, in an estrogen receptor-dependent manner. Inhibition of NADPH oxidase activation via icv administration of a NOX2 competitive inhibitor, gp91ds-tat, strongly attenuated O2 - production and was neuroprotective. The increase of neuronal NOX2 and O2 - following cerebral ischemia was shown to require Rac1 activation, as administration of a Rac1 inhibitor (NSC23766) significantly attenuated these factors following stroke. Interestingly, we found that 17-β-E2 antioxidant ability to diminish neuronal NOX2-induced O2 - generation involves the attenuation of Rac1 activation. We also provide evidence for 17-β-E2 post-translational deacetylation of downstream pro-apoptotic p53 and a reduction of p53 transcriptional target, Puma. Our results revealed that p53 acetylation (activation) is markedly increased in ischemic animals 24h after reperfusion and that 17-β-E2 strongly attenuated that elevation, as well as total p53 protein levels. In support of this suggestion, we also found 17-β-E2 to strongly attenuate ischemia-mediated Puma upregulation, thus interfering with its transcription-dependent function. We further propose that 17-β-E2-induced attenuation of p53 levels may involve an upregulation in p53-Mdm2 interactions and p53 mediated degradation via the ubiquitination pathway. Lastly, we provide evidence showing that treatment with Gp91ds-tat, but not the scrambled tat peptide control, attenuated acetylation of downstream p53 and reduced levels of Puma, thus supporting O2 —p53 crosstalk signaling after stroke. Altogether, our studies reveal a novel, membrane-mediated antioxidant mechanism of 17-β-E2-induced neuroprotection via reduction of neuronal NOX2 activation and O2 - production, while providing evidence for 17-β-E2–mediated deacetylation and inactivation of p53, thereby protecting the hippocampus CA1 against cerebral ischemia.