Role of Dickkopf-1, an antagonist of the Wnt/beta-catenin signaling pathway, in estrogen-induced neuroprotection and attenuation of tau phosphorylation

Abstract

17beta-Estradiol (E2) has been implicated to be neuroprotective in a variety of neurodegenerative disorders, although the mechanism remains poorly understood. The current study sheds light on this issue by demonstrating that low physiological levels of E2 protects the hippocampus CA1 against global cerebral ischemia by preventing elevation of dickkopf-1 (Dkk1), an antagonist of the Wnt/beta-catenin signaling pathway, which is a principal mediator of neurodegeneration in cerebral ischemia and Alzheimer's disease. E2 inhibition of Dkk1 elevation correlated with a reduction of phospho-beta-catenin and elevation of nuclear beta-catenin levels, as well as enhancement of Wnt-3, suggesting E2 activation of the Wnt/beta-catenin signaling pathway. In agreement, the beta-catenin downstream prosurvival factor, survivin, was induced by E2 at 24 and 48 h after cerebral ischemia, an effect observed only in surviving neurons because degenerating neurons lacked survivin expression. E2 suppression of Dkk1 elevation was found to be caused by attenuation of upstream c-Jun N-terminal protein kinase (JNK)/c-Jun signaling, as E2 attenuation of JNK/c-Jun activation and a JNK inhibitor significantly blocked Dkk1 induction. Tau hyperphosphorylation has been implicated to have a prodeath role in Alzheimer's disease and cerebral ischemia, and E2 attenuates tau hyperphosphorylation. Our study demonstrates that tau hyperphosphorylation is strongly induced after global cerebral ischemia, and that E2 inhibits tau hyperphosphorylation by suppressing activation of the JNK/c-Jun/Dkk1 signaling pathway. Finally, exogenous Dkk1 replacement via intracerebroventricular administration completely reversed E2-induced neuroprotection, nuclear beta-catenin induction, and phospho-tau attenuation, further suggesting that E2 inhibition of Dkk1 is a critical mechanism underlying its neuroprotective and phospho-tau regulatory effects after cerebral ischemia.

Figures

Figure 1.

Aa–Al, NeuN immunostaining and Fluoro-Jade B staining of representative coronal brain sections at the level of the dorsal hippocampus at 7 d after reperfusion from sham, placebo (Pla)- and E2-treated female ovariectomized rats subjected to global cerebral ischemia. Global ischemia induced significant neuronal degeneration and neuronal cell loss in the CA1 pyramidal cell layer, with little or no cell loss apparent in CA3 or DG. E2 treatment afforded nearly complete protection of the hippocampus CA1 from global cerebral ischemia-induced neuronal degeneration and neuronal cell loss. Boxed areas in the left column are shown at higher magnification in right column (magnification, 40×). Scale bar, 50 μm. B, Neuronal counts of NeuN-positive neurons. Counts refer to CA1 neurons of sham-, placebo-, and 17β-estradiol-treated ovariectomized rats. Note the significant neuroprotection by E2. Values are mean ± SEM of determinations from seven individual rats. #p < 0.01 versus placebo.

Figure 2.

A,B, Effect of 17β-estradiol on Dkk1 protein levels in hippocampus CA1 after global cerebral ischemia. Values are mean ± SEM of determinations from five to six individual rats and expressed as fold change versus sham control. Pla, Placebo; R, reperfusion. *p < 0.05 versus sham control; #p < 0.05 versus placebo treatment group. Ca–Ci, Confocal analysis of NeuN and Dkk1 immunostaining in hippocampus CA1 at 24 h after global cerebral ischemia (magnification, 40×). Scale bar, 50 μm.

Figure 3.

A, B, 17β-Estradiol enhances Wnt3 protein levels in hippocampus CA1 after global cerebral ischemia. Values are mean ± SEM of determinations from five to six individual rats expressed as fold change versus sham control. Pla, Placebo; R, reperfusion. #p < 0.05 versus the Pla group at the same time point. Ca–Cf, DAB immunostaining for Wnt3 in hippocampus CA1 at 24 h after global cerebral ischemia. Results are representative of staining observed in five (n = 5) individual animals per group. Ca–Cc, Magnification is 5×. Scale bar, 200 μm. Cd–Cf, Magnification is 40×. Scale bar, 50 μm.

Figure 4.

A, B, 17β-Estradiol prevents cerebral ischemia-induced elevation of p-β-catenin levels in nuclear fraction of hippocampus CA1 after global cerebral ischemia. Values are mean ± SEM of determinations from five to six individual rats expressed as fold change versus sham control. Pla, Placebo; R, reperfusion. Ca–Cf, DAB immunostaining for p-β-catenin in hippocampus CA1 at 24 h after global cerebral ischemia. Results are representative of staining observed in four individual animals per group. D, E, E2 prevents cerebral ischemia-induced reduction of nuclear β-catenin levels in hippocampus CA1 after global cerebral ischemia. Values are mean ± SEM of determinations from five to six individual rats expressed as fold change versus sham control. *p < 0.05 versus sham control; #p < 0.05 versus the Pla group at the same time point. Fa–Fi, Confocal analysis of NeuN and β-catenin immunostaining in hippocampus CA1 at 24 h after global cerebral ischemia. Results are representative of staining observed in five individual animals per group (magnification, 40×). Scale bars, 50 μm.

Figure 5.

A, B, 17β-Estradiol enhances expression of the antiapoptotic protein survivin in hippocampus CA1 after global cerebral ischemia. Values are mean ± SEM of determinations from five to six individual rats expressed as fold change versus sham control. Pla, Placebo; R, reperfusion. *p < 0.05 versus sham control; #p < 0.05 versus the Pla group at the same time point. C, DAB and confocal analysis shows that survivin is induced in NeuN-positive neurons by E2 in hippocampus CA1 at 24 h after global cerebral ischemia. Results are representative of staining observed in five individual animals per group (magnifications, 40×). Scale bars, 50 μm.

Figure 6.

A, B, 17β-Estradiol prevents cerebral ischemia-induced elevation of p-JNK and p-c-Jun levels in hippocampus CA1 after global cerebral ischemia. Values are mean ± SEM of determinations from five to six individual rats, corrected by total JNK or c-Jun, and expressed as fold change versus sham control. Pla, Placebo; R, reperfusion. Ca–Cl, Confocal analysis demonstrates that p-c-Jun and Dkk1 are induced in the same neurons at 24 h after global cerebral ischemia. Results are representative of staining observed in five individual animals per group (magnification, 40×). Scale bar, 50 μm. D, E, Administration of the JNK inhibitor SP600125 (10 mg/kg, i.v.) prevents activation of JNK and c-Jun and prevents cerebral ischemia induction of Dkk1 in hippocampus CA1 at 24 h after global cerebral ischemia. Values are mean ± SEM of determinations from five to six individual rats expressed as fold change versus sham control. *p < 0.05 versus sham control; #p < 0.05 versus Pla group at the same time point.

Figure 7.

A, C, 17β-Estradiol reduces tau hyperphosphorylation after global cerebral ischemia as determined by Western blot analysis with PHF-1 antibodies. Values are mean ± SEM of determinations from five to six individual rats, and expressed as fold change versus sham control. Pla, Placebo; R, reperfusion. Ba–Bf, DAB immunostaining showing tau hyperphosphorylation at 24 h after global cerebral ischemia in hippocampus CA1 and attenuation by E2. Results are representative of staining observed in five individual animals per group (magnification, 40×). Scale bar, 50 μm. D, E, Administration of the JNK inhibitor SP600125 abolishes global cerebral ischemia-induced tau hyperphosphorylation at 24 h after global cerebral ischemia. Values are means ± SEM of determinations from five to six individual rats expressed as fold change versus sham control. *p < 0.05 versus sham control, #p < 0.05 versus Pla group at the same time point. F, Administration of the JNK inhibitor SP600125 significantly attenuated hippocampal CA1 neuronal cell death as measured by counting of NeuN-positive cells at 7 d after global cerebral ischemia. Representative NeuN staining in hippocampus CA1 from vehicle- and SP600125-treated rats is shown in the inset. Values are mean ± SEM of determinations from five to six individual rats. #p < 0.01 versus vehicle and placebo.

Figure 8.

Exogenous Dkk1 administration reverses 17β-estradiol-induced neuroprotection and phospho-tau regulation after global cerebral ischemia. Dkk1 (5 μg/5 μl) was administered via intracerebroventricular injection into both lateral ventricles at 12 h after global cerebral ischemia. For a control, separate animals received vehicle into both lateral cerebral ventricles. Additionally, a nonischemic sham control also received Dkk1 into the lateral ventricles. Aa–Ad, High-power magnification of NeuN staining in hippocampus CA1 at 7 d in sham animals and after 7 d reperfusion after global cerebral ischemia in E2-treated rats that received vehicle or exogenous Dkk1 in both lateral ventricles. Magnification is 40×. Scale bar, 50 μm. Ae–Ah, Low-power magnifications of representative whole hippocampus sections showing NeuN staining in hippocampus CA1 at 7 d in sham animals and after 7 d reperfusion after global cerebral ischemia in E2-treated rats that received vehicle or exogenous Dkk1 in both lateral ventricles. Magnification is 5×. Scale bar, 200 μm. B, CA1 cell counts of NeuN-positive neurons in all animals show that exogenous Dkk1 had no significant effect on CA1 neuronal cell survival in nonischemic sham controls, although it significantly reversed E2 neuroprotection in ischemic animals. Values are mean ± SEM of determinations from five to six individual rats. #p < 0.01 versus vehicle. C, D, Exogenous Dkk1 injection also reversed E2 reduction of phospho-tau and elevation of nuclear β-catenin in the hippocampus CA1 at 24 h after global cerebral ischemia. Values are mean ± SEM of determinations from five to six individual rats expressed as fold change versus sham control. #p < 0.05 versus vehicle treatment group.

Figure 9.

Summary diagram of E2 regulation of Wnt/β-catenin signaling pathway after cerebral ischemia and proposed role in E2 ischemic neuroprotection. E2 acts to inhibit cerebral ischemia activation of JNK/Jun/Dkk1 signaling, which prevents subsequent phosphorylation of tau and β-catenin and cell death, while it acts to enhances expression of Wnt3, leading to accumulation of nuclear β-catenin and enhanced expression of survivin, which inhibits apoptosis and enhances neuronal cell survival.