The importance of postmenopausal estrogen replacement therapy in affording protection against the delayed neuronal death associated with cardiac arrest or cardiac surgery in women is a topic of intense research. Studies by this laboratory have shown that long-term treatment with estrogen at physiological levels ameliorates ischemia-induced hippocampal injury and prevents activation of apoptotic signaling cascades in the hippocampal CA1 (Jover et al., J. NeuroscL 22:2115-2124, 2002). The objectives of the proposed research are to identify the cellular site(s) of action and the molecular mechanisms by which estrogen acts to block apoptotic cell death. Estrogens exert their genomic actions by interaction with the ER(estrogen receptor)alpha and ERbeta. The central hypothesis driving the proposed research is that estrogen acts via ERalpha and coordinate activation of IGF(insulin-like growth factor)-I to block onset of global schema-induced apoptotic cell death. Specific Aims are: 1) Identify the cellular targets that mediate the neuroprotective actions of estrogen on hippocampal neurons. Pharmacological studies will examine the ability of ER subtype-specific agonists to elicit protection and of broad-spectrum ER antagonists to abrogate estrogen protection. Molecular genetic studies will examine the ability of estrogen to afford protection against hippocampal injury in mice with targeted deletions of the ERalpha or ERbeta gene. 2) Determine whether estrogen acts via coordinate activation of ERalpha and IGF-IR to protect hippocampal neurons, and if so, whether protection is mediated by the PI3K (phosphotidylinositol 3-kinase)lAkt/protein kinase B (PKB) and/or the MAPK (mitogen-activated protein kinase) signaling cascades. Experiments will examine 1) ability of IGF-I receptor antagonists to abrogate protection by estrogen; 2) ability of IGF-I to mimic protection by estrogen; 3) ability of IGF-I to rescue neurons in ER-beta-knockout, but not ERalpha-knockout, mice; 4) ability of Akt inhibitors to block estrogen/IGF-I protection; and 5) ability of MAPK inhibitors to block estrogen/IGF-I protection. 3) Examine apoptotic death cascades triggered by global ischemia including the Fas/Forkhead and caspase death cascades, and identify the site at which estrogen intervenes in these cascades. Experiments will examine: 1) ischemia-induced activation and nuclear translocation of FKHRL1 at early times after ischemia; 2) estrogen-induced inactivation of FKHRL1 and ability of ER/IGF-I receptor antagonists to block FKHRL1 inactivation; 3) ability of PI3K inhibitors to block FKHRL1 inactivation; 4) ability of MAPK inhibitors to block FKHRL1 inactivation; and 5) ability of estrogen to activate the anti-apoptotic gene Bcl-2. It is well-established that the risk of cardiac arrest in women increases after menopause, a state characterized by hypoestrogenemia. Premenopausal women show a reduced incidence of cerebral stroke compared with men of equivalent ages. These studies are expected to shed light on the role of estrogen in affording protection against global ischemia arising after cardiac arrest or cardiac surgery in women. [unreadable] [unreadable]