Acute estradiol protects CA1 neurons from ischemia-induced apoptotic cell death via the PI3K/Akt pathway

Abstract

Global ischemia arising during cardiac arrest or cardiac surgery causes highly selective, delayed death of hippocampal CA1 neurons. Exogenous estradiol ameliorates global ischemia-induced neuronal death and cognitive impairment in male and female rodents. However, the molecular mechanisms by which a single acute injection of estradiol administered after the ischemic event intervenes in global ischemia-induced apoptotic cell death are unclear. Here we show that acute estradiol acts via the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling cascade to protect CA1 neurons in ovariectomized female rats. We demonstrate that global ischemia promotes early activation of glycogen synthase kinase-3beta (GSK3beta) and forkhead transcription factor of the O class (FOXO)3A, known Akt targets that are related to cell survival, and activation of caspase-3. Estradiol prevents ischemia-induced dephosphorylation and activation of GSK3beta and FOXO3A, and the caspase death cascade. These findings support a model whereby estradiol acts by activation of PI3K/Akt signaling to promote neuronal survival in the face of global ischemia.

Fig. 1. The PI3K inhibitor LY294002 attenuates estradiol protection

Ovariectomized female rats were subjected to global ischemia (white and black bars) or sham operation (grey bars) and treated with estradiol or vehicle immediately upon reperfusion. Animals also received LY294002 or vehicle icv at 0 and 12 h after vehicle or estradiol injection (n = 3-12 animals per group). Global ischemia induced extensive death of pyramidal cells in the hippocampal CA1 at 7 days post-ischemia (i, m, j, n, q). LY294002 greatly reduced estradiol protection, assessed at 7 days after surgery (l, p, q). Scale bars: lower magnification, 400 μm; higher magnification, 40 μm. so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. ***, P < 0.001 vs. all sham groups; ^##, P < 0.01 ischemia+estradiol vs. ischemia and ^###, P < 0.001 vs. ischemia+estradiol+LY294002).

Fig. 2. The PI3K inhibitor LY294002 attenuates global ischemia- induced increase in Akt phosphorylation in CA1

Representative Western blots (a) and relative abundance of p-Akt (b) in CA1 whole-cell lysates from rats subjected to sham surgery or ischemia, infused icv with vehicle or LY294002 immediately upon reperfusion. Westerns were probed with antibodies to p-Ser473-Akt and Akt. Ischemia markedly increased phosphorylation of Akt (a,b). LY294002 significantly attenuated the ischemia-induced increase in p-Akt (a,b). Data are representative of 3-6 animals per group. There was no effect on total protein when expressed relative to β-actin in the same sample (data not shown). Values for ischemic rats were normalized to the corresponding values for control (sham-operated, vehicle-infused) animals. ***, P < 0.001 vs. control and ^##, P < 0.01 ischemia vs. ischemia+LY294002).

Fig. 3. Estradiol and global ischemia transiently increase Akt phosphorylation in CA1

Representative Western blots (a) and relative abundance of p-Akt (b) in CA1 whole-cell lysates from rats subjected to sham surgery or ischemia, infused icv with vehicle or estradiol and killed at 1, 3, or 24 h after surgery. Ischemia transiently increased p-Akt at 1 h (a,b). Estradiol significantly enhanced Akt (b) phosphorylation in sham-operated females but did not significantly alter Akt phosphorylation in ischemic rats (b). Neither ischemia nor estradiol treatment altered levels of p-Akt in CA1 at 3 or 24 h after ischemia. Data are representative of 3-11 animals per group. Values for ischemic rats were normalized to the corresponding values for control (sham-operated, vehicle-infused) animals. *, P < 0.05; ***, P < 0.001 vs. sham-operated, vehicle-infused rats.

Fig. 4. Estradiol prevents ischemia-induced dephosphorylation of ERK2 in CA1

Representative Western blots (a) and relative abundance of p-ERK1 (b) and p-ERK2 (c) in CA1 whole-cell lysates from rats subjected to sham operation or global ischemia and vehicle- and estradiol-treated at 1 and 3 h after surgery. Westerns were probed with antibodies to p-ERK1/2 and ERK1/2. Ischemia induced dephosphorylation of ERK1 (a,b), and ERK2 (a,c). Estradiol did not significantly change ERK1 (b) and ERK2 (c) phosphorylation in shams but maintained levels of p-ERK2 (c) in ischemic rats and prevented the effect of ischemia on p-ERK1 (b). Data are representative of 6-12 animals per group. Values for ischemic rats were normalized to the corresponding sham value. *, P < 0.05; ***, P < 0.001, vs. all sham groups; ^#, P < 0.01 ischemia+estradiol vs. ischemia+vehicle-infused rats.

Fig. 5. Estradiol prevents ischemia-induced inhibition of GSK-3β phosphorylation in hippocampal CA1

Representative Western blots (a) and relative abundance of p-GSK-3β (b) in the cytosolic fraction of CA1 from rats subjected to sham operation or global ischemia, infused icv with vehicle or estradiol and killed at 1 and 3 h after surgery. Westerns were probed with antibodies to p-Ser9-GSK-3β and GSK-3β. Global ischemia did not change the levels of p-GSK3β at any times examined (a,b). Estradiol significantly increased GSK-3β phosphorylation 3 h after global ischemia (a,b). Data are representative of 6-12 animals per group. Band densities for experimental animals were normalized to the corresponding sham value. *, P < 0.05.

Fig. 6. Estradiol prevents ischemia-induced FOXO3A dephosphorylation

Representative Western blots (a) and relative p-FOXO3A abundance (b) in the cytosolic fraction of CA1 of rats subjected to sham operation or ischemia, infused icv with vehicle or estradiol and killed at 3 h after surgery. Westerns were probed with antibodies to p-FOXO3A and FOXO3A. Ischemia promoted dephosphorylation of FOXO3A in the cytosolic fraction of CA1 neurons. Estradiol maintained the phosphorylated, inactivated state of FOXO3A in the CA1 of ischemic rats. Data are representative of 6-9 animals per group. Values for experimental animals were normalized to the corresponding sham value. *, P < 0.05 vs. sham-operated, vehicle-infused rats; ^##, P < 0.01 ischemia+estradiol vs. ischemia+vehicle-infused rats.

Fig. 7. Estradiol blocks ischemia-induced caspase-3-like activity in CA1

Representative brain sections at the level of the dorsal hippocampus from control (sham-operated) (a,b) and experimental animals subjected to global ischemia (c-f) labeled with FAM-DEVD-FMK, a cell-permeant, irreversible inhibitor of caspases and calpains. In control brain caspase activity was undetectable (a,b,g). Global ischemia activated caspase in CA1 neurons, evident at 24 h (c,d,g). Estradiol prevented ischemia-induced caspase-3 activity (e,f,g). Data are representative of 3-5 animals per group. Abbreviations as in Fig. 1. Scale bars: lower magnification, 400 μm; higher magnification, 50 μm. *, P < 0.05 vs. sham and ^#, P < 0.05 vs. ischemia+estradiol.