Neuroprotective Role And The Mechanism Induced By Rapid Estrogen Signaling

Objective Making four-vessel occlusion (4-VO) global cerebral ischemia (GCI) animal model, to study the neuroprotective and cognitive enhancement role of two estrogen conjugates (E2 dendrimer, EDC, and E2-BSA) induced by membrane estrogen receptor (mER) and further to clarify the possible mechanism following GCI in hippocampal CA1 region of the rats.Methods 1) Adult SD female rats were bilaterally ovariectomized, and 1 week later GCI (10 min) was induced by four-vessel occlusion. 2) Experimental animals were randomly divided into five groups: sham, ischemia-reperfusion, drug-treated (EDC, E2-BSA), inhibitor-treated (ICI182780, LY294002, PD98059) and vehicle groups. EDC and E2-BSA were injected intracerebroventricularly (icv) into the right lateral ventricle 1h before cerebral ischemia and inhibitors were injected icv 10min before EDC and E2-BSA administration. 3) Immunofluorescence was used to observe the location of EDC and E2-BSA and neuron survival. 4) The spatial learning and memory of the rats were evaluated by Morris water maze test. 5) Western blotting and Immunnofluorescence methods were used to detect the level change of protein kinases, such as AKT, ERK1/2 or JNK.Results 1. EDC and E2-BSA major localize to the membrane of hippocampal CA1 cells, as the green fluorescent signal mainly distributed in this region and displayed a punctuate membrane pattern of localization, 1h after injected icv.2. Either EDC or E2-BSA exerts a robust neuroprotective role against GCI. Examination of NeuN-positive cells at 7 d after GCI revealed that Vehicle-treated animals that underwent 10-min GCI displayed a significant loss of NeuN-positive cells in the hippocampal CA1 region as compared to sham controls. Importantly, EDC or E2-BSA administered by icv 60 min prior to GCI can significantly increased the number of survival neuron in the hippocampal CA1 region, about 4 folds compared to Vehicle-treated animals. The EDC and E2-BSA treated animal pretreatment with the ER antagonist, ICI182,780 completely abolished the neuro-protective effect.3. EDC or E2-BSA enhanced cognitive outcome following GCI. Vehicle-treated animals that underwent GCI showed significant higher latencies in finding the submerged platform on days7-9 post stroke as compared to sham controls. In contrast, EDC-treated rats had significantly decreased latencies to find the submerged platform on day 7-9 as compared to the vehicle group, an effect that was significantly reversed by the ER antagonist ICI182,780. Furthermore, vehicle-treated animals spent significantly less time in the quadrant where the submerged platform was located as compared to sham animals on Day 9. In contrast, EDC-treated rats spent significantly greater amount of time in the quadrant where the submerged platform was located as compared to Vehicle group, and this effect was significantly reversed by the ER antagonist, ICI182,780, suggesting ER mediation of the cognitive enhancing/preservation effect of EDC.4. EDC and E2-BSA neuroprotective effects against GCI are mediated by AKT Signaling Pathway. A time course study revealed that p-AKT levels in the CA1 increased slightly but significantly from 10 min - 6h following reperfusion, with a fall back to sham levels at 1d reperfusion. Total AKTandβ-actin levels did not change at any time point. EDC treatment led to a significant elevation of p-AKT levels in the CA1 from 10 min - 3h, with a fall back to sham levels at 1d. E2-BSA similarly enhances p-Akt levels at 10 min after reperfusion following GCI, while total AKT levels were unchanged.5. EDC and E2-BSA neuroprotective effects against GCI are mediated by ERK1/2 signaling pathways. Reperfusion had a biphasic elevation of p-ERK levels in the CA1 after GCI, with an initial 2-fold increase over sham observed at 10 min, followed by a fall back to sham levels from 3h-6h, and a second elevation of p-ERK at 1d. Total ERK1 levels showed no change at any time point. EDC treatment significant increased p-ERK levels in the CA1 region from 10 min - 6h, as compared to the reperfusion or vehicle control. E2-BSA has a similar enhancing effect upon p-ERK levels in the CA1 region at 10 min reperfusion following GCI.6. We next examined EDC and E2-BSA upon activation of JNK, a pro-apoptotic factor. P-JNK levels are increased at all times after reperfusion as compared to sham, except for the 3h time point which was not significantly elevated. Total JNK levels did not change regardless of time or treatment. EDC significantly inhibited p-JNK levels at all time points following GCI, except the 3h time point where p-JNK levels were not elevated following reperfusion. E2-BSA also significantly inhibited the elevation of p-JNK at 10 min after reperfusion, while having no effect upon total JNK levels.7. Inhibition of ERK or PI3K-Akt signaling abolishes EDC and E2-BSA Neuroprotection. We next examined the role of ERK or PI3K-Akt signaling in mediating EDC and E2-BSA neuroprotection following GCI. Pretreatment with the PI3K inhibitor, LY294002 markedly attenuated the ability of EDC and E2-BSA to enhance p-AKT levels in the CA1 region at 10 min reperfusion after GCI .EDC and E2-BSA enhancement of p-ERK levels in the CA1 region at 10 min reperfusion after GCI is abolished by pretreatment with the MEK inhibitor, PD98059. Furthermore, additional work suggests that EDC and E2-BSA regulatory effects upon ERK, AKT and JNK activation after GCI are mediated by estrogen receptors, as pretreatment with the ER antagonist, ICI182,780 abolished EDC and E2-BSA modulation of ERK, AKT and JNK activation in the CA1 region at 10 min reperfusion.8. Activation of CREB and enhanced levels of BDNF by EDC following GCI. EDC rapidly increased pCREB levels in the CA1 region 10min post reperfusion, with a subsequent increase at 3h, 6h and 1d as compared to sham and vehicle controls. E2-BSA also increased pCREB levels similar to EDC at 10min post-reperfusion. The early elevation of pCREB levels by EDC, was followed by elevation of its transcriptional target, BDNF at latter time points (6h and 1d reperfusion) in the hippocampal CA1 region. Administration of the PI3K or MEK inhibitor markedly attenuated EDC-induced pCREB elevation in the CA1 region at 10min and 6h after reperfusion, as well as significantly attenuated the EDC-induced BDNF elevation at 6h after reperfusion, suggesting that PI3K-Akt and MEK-ERK signaling pathways mediate the EDC-induced elevation of pCREB and BDNF in the hippocampus following GCI.Conclusion The study provided important new evidence of a critical role for extranuclear estrogen receptor activation in estrogen-induced neuroprotection and improved functional cognitive outcome following GCI, and suggests that AKT/ERK1/2-CREB-BDNF signaling is an important component mediating extranuclear estrogen receptor beneficial neural effects.