Extranuclear estrogen receptors mediate the neuroprotective effects of estrogen in the rat hippocampus

Abstract

Background: 17beta-estradiol (E2) has been implicated to exert neuroprotective effects in the brain following cerebral ischemia. Classically, E2 is thought to exert its effects via genomic signaling mediated by interaction with nuclear estrogen receptors. However, the role and contribution of extranuclear estrogen receptors (ER) is unclear and was the subject of the current study.

Methodology/principal findings: To accomplish this goal, we employed two E2 conjugates (E2 dendrimer, EDC, and E2-BSA) that can interact with extranuclear ER and exert rapid nongenomic signaling, but lack the ability to interact with nuclear ER due to their inability to enter the nucleus. EDC or E2-BSA (10 microM) was injected icv 60 min prior to global cerebral ischemia (GCI). FITC-tagged EDC or E2-BSA revealed high uptake in the hippocampal CA1 region after icv injection, with a membrane (extranuclear) localization pattern in cells. Both EDC and E2-BSA exerted robust neuroprotection in the CA1 against GCI, and the effect was blocked by the ER antagonist, ICI182,780. EDC and E2-BSA both rapidly enhanced activation of the prosurvival kinases, ERK and Akt, while attenuating activation of the proapoptotic kinase, JNK following GCI, effects that were blocked by ICI182,780. Administration of an MEK or PI3K inhibitor blocked the neuroprotective effects of EDC and E2-BSA. Further studies showed that EDC increased p-CREB and BDNF in the CA1 region in an ERK- and Akt-dependent manner, and that cognitive outcome after GCI was preserved by EDC in an ER-dependent manner.

Conclusions/significance: In conclusion, the current study demonstrates that activation of extranuclear ER results in induction of ERK-Akt-CREB-BDNF signaling in the hippocampal CA1 region, which significantly reduces ischemic neuronal injury and preserves cognitive function following GCI. The study adds to a growing literature that suggests that extranuclear ER can have important actions in the brain.

Figure 1. (A-a) Extranuclear localization of EDC and E2-BSA in neurons in hippocampal CA1 region of the rats.

FITC-tagged EDC and E2-BSA were injected into the lateral ventricles, and 1h later, the rats were underwent transcardial perfusion and cut into 25µm coronal brain sections with a cryostat. Confocal analysis showed that EDC and E2-BSA entered neurons in the hippocampal CA1 region but are incapable of penetrating into the nucleus. (A-b,c) EDC and E2-BSA2 protect neurons of the hippocampus CA1 region from injury induced by GCI. NeuN immunostaining of representative hippocampus sections from sham, vehicle, EDC, EDC+ICI, E2-BSA and E2-BSA+ICI-treated female ovariectomized rats subjected to 10min GCI followed by 7d reperfusion. Global ischemia induced significant neuronal cell loss in the CA1 pyramidal cell layer. EDC or E2-BSA treatment afforded nearly complete protection from global cerebral ischemia-induced neuronal cell loss. ICI182,780 (ICI) abrogated the neuroprotection induced by EDC or E2-BSA. NeuN-positive neurons in per 1 mm length of CA1 region was counted as surviving neurons. F(5,30) = 105,^* p<0.001 vs. vehicle and EDC+ICI groups; F(5,30) = 105, ^# p<0.001 vs. vehicle and E2-BSA+ICI groups. Magnification 40×; Scale bar 50µm. (B) Effects of EDC on spatial learning and memory ability in the Morris water maze. (a) Latency to find the submerged platform. F(11,48) = 77,^* p<0.001 vs. sham; F(11,48) = 77, ^# p<0.001 vs. Vehicle and EDC+ICI. (b) Time spent in the quadrant, which initially contains the platform. F(3,16) = 37, ^* p<0.001 vs. sham; F(3, 16) = 37, ^# p<0.01 vs. Vehicle and EDC+ICI. (c) Representative sample paths from the maze trials (i–iv) and the probe trials (v–viii) at 9 days after reperfusion. (i,v: sham; ii,vi: vehicle; iii,vii: EDC; iv,viii: EDC+ICI). R: reperfusion. ICI: ICI182,780.

Figure 2. Effects of EDC and E2-BSA on activation of Akt following cerebral ischemia in the hippocampal CA1 region.

(A) Time courses of p-Akt and Akt, as well as the effects of EDC. Rats were subjected to 10min ischemia followed by 10min, 30min, 3h, 6h and 1d reperfusion. Bands corresponding to p-Akt were scanned and the intensities represented as values were shown in (B). F(11,36) = 19, ^* p<0.05 vs. sham; F(11,36) = 19, ^# p<0.05, significant difference vs. Vehicle. (C) Effect of E2-BSA on Akt activation at 10min of reperfusion. F(3,12) = 20, ^* p<0.01 vs. sham; F(3,12) = 20, ^# p<0.05 significant difference vs. Vehicle. (D) Confocal analysis of NeuN and p-Akt immunostaining in hippocampus CA1 at 10min reperfusion after GCI. Sham(a,b,c); vehicle(d,e,f); R10min+EDC(g,h,i); R10min+E2-BSA(j,k,l). Magnification 40×; Scale bar 50µm.

Figure 3. Effects of EDC and E2-BSA on phosphorylation level and protein expression of ERK1/2 following cerebral ischemia in the hippocampal CA1 region.

The time-course of p-ERK1/2 and the effect of EDC were shown in Fig. 3A. Rats were subjected to 10min ischemia followed by 10min, 30min, 3h, 6h and 1d reperfusion. (B) Bands corresponding to p-ERK1/2 were scanned and the intensities represented as values. F(11,36) = 41, ^* p<0.001 vs. sham; F(11,36) = 41, ^# p<0.05 vs. Vehicle. (C) Effect of E2-BSA on p-ERK1/2 at 10min of reperfusion. F(3,12) = 22, ^* p<0.05 vs. sham; F(3,12) = 22, ^# p<0.001 vs. Vehicle. (D) Confocal analysis of NeuN and p-ERK1/2 immunostaining in hippocampus CA1 at 10min reperfusion after global cerebral ischemia. Sham(a,b,c); vehicle(d,e,f); R10min+EDC(g,h,i); R10min+E2-BSA(j,k,l). Magnification 40×; Scale bar 50µm.

Figure 4. Effects of EDC and E2-BSA on phosphorylation level of JNK following cerebral ischemia in the hippocampal CA1 region.

(A) Time courses of p-JNK and JNK, as well as the effects of EDC. Rats were subjected to 10min ischemia followed by 10min, 30min, 3h, 6h and 1d reperfusion. Bands corresponding to p-JNK were scanned and the intensities represented as values are showed in (B). F(11,36) = 24, ^* p<0.05 vs. sham; F(11,36) = 24, ^# p<0.05 vs. Vehicle. (C) Effect of E2-BSA on p-JNK at 10min of reperfusion. F(3,12) = 9, ^* p<0.05 vs. sham; F(3,12) = 9, ^# p<0.05 vs. Vehicle. (D) Confocal analysis of NeuN and p-JNK immunostaining in hippocampus CA1 at 10min reperfusion after global cerebral ischemia. Sham(a,b,c); vehicle(d,e,f); R10min+EDC(g,h,i); R10min+E2-BSA(j,k,l). Magnification 40× and Scale bar 50µm.

Figure 5. Effects of PD98059 and LY294002 on neuroprotective role by E2-BSA and EDC following cerebral ischemia in hippocampal CA1 region.

(A) Effects of PD on p-ERK1/2 level at 10min of reperfusion in rats pre-treated with EDC or E2-BSA. F(4,15) = 121, ^* p<0.001 vs. sham; F(4,15) = 121, ^* p<0.001 vs. Vehicle. (B) Effects of LY on p-Akt level at 10min of reperfusion in rats pre-treated with EDC or E2-BSA. F(4,17) = 54, ^* p<0.001 vs. sham; F(4,17) = 54, ^# p<0.05 vs. Vehicle. (C) NeuN immunostaining showed the effects of PD or LY on surviving neurons of the hippocampal CA1 region in rats treated with EDC or E2-BSA and subjected to 10min ischemia followed by 7d reperfusion (Fig. 5C, panel a). NeuN-positive neurons in per 1mm length of CA1 region was counted as vital survival neurons as shown in Fig. 5C, panel b. F(7,32) = 68, ^* p<0.001 vs. vehicle group; F(7,32) = 68, ^# p<0.001 vs. groups pre-treated with EDC or E2-BSA. PD: PD98059, LY: LY294002, Veh: vehicle.

Figure 6. Effects of ICI 182,780 on phosphorylation of Akt, ERK1/2 and JNK induced by E2-BSA or EDC at 10min of reperfusion in hippocampal CA1 region.

(A) Western blot analysis showed phosphorylation levels and protein expressions of Akt, ERK1/2 and JNK. Samples were obtained from sham, vehicle, E2-BSA, E2-BSA+ICI, EDC and EDC+ICI treated animals. Bands corresponding to p-Akt, p-ERK1/2 and p-JNK were scanned and the intensities represented as fold increase vs. sham as shown in (B). F(5,18) = 106, p<0.01 for p-Akt; F(5,18) = 38, p<0.01 for p-ERK1/2; and F(5,18) = 23 for p-JNK. ^* indicates vs. E2-BSA groups without ICI, ^# indicates vs. EDC groups without ICI. ICI: ICI 182,780.

Figure 7. Effects of EDC on protein levels of CREB, p-CREB and BDNF following ischemic reperfusion in the hippocampal CA1 region.

(A) Western blot analysis showed the time-courses of p-CREB, CREB and BDNF protein expression, as well as the effects of EDC or E2-BSA. F(3,16) = 31, ^* p<0.001 vs. vehicle at the same timepoint. (B–C) Effects of PD or LY on CREB/p-CREB level and BDNF levels at 10min of reperfusion or 6h reperfusion in rats pre-treated with EDC. B: F(3,16) = 23, ^* p<0.001 vs. sham; F(3,16) = 23, ^# p<0.001 vs. EDC group. C: F(3,12) = 64, ^* p<0.001 vs. sham; F(3,15) = 21, ^# p<0.001 vs. EDC group. R: reperfusion, PD: PD98059, LY: LY294002.