Role of protein phosphatases and mitochondria in the neuroprotective effects of estrogens

Abstract

In the present treatise, we provide evidence that the neuroprotective and mito-protective effects of estrogens are inexorably linked and involve the ability of estrogens to maintain mitochondrial function during neurotoxic stress. This is achieved by the induction of nuclear and mitochondrial gene expression, the maintenance of protein phosphatases levels in a manner that likely involves modulation of the phosphorylation state of signaling kinases and mitochondrial pro- and anti-apoptotic proteins, and the potent redox/antioxidant activity of estrogens. These estrogen actions are mediated through a combination of estrogens receptor (ER)-mediated effects on nuclear and mitochondrial transcription of protein vital to mitochondrial function, ER-mediated, non-genomic signaling and non-ER-mediated effects of estrogens on signaling and oxidative stress. Collectively, these multifaceted, coordinated action of estrogens leads to their potency in protecting neurons from a wide variety of acute insults as well as chronic neurodegenerative processes.

Fig. 1

Effects of calyculin A on estrogen-mediated neuroprotection from glutamate neurotoxicity. HT-22 cells or C6-glioma cells were seeded into 96-well plates at a density of 3500 cells/well. (A) HT-22 cells were treated simultaneously with 10 μM 17β-estradiol, varying concentrations of calyculin A and/or 10 mM glutamate. (B) C6-glioma cells were treated simultaneously with 10 μM 17β-estradiol, varying concentrations of calyculin A and/or 20 mM glutamate. Cell viability was determined by calcein AM assay after 24 h exposure to the various compounds. All data were normalized to percentage survival of vehicle control. Data are represented as mean ± SEM for n = 10. *P < 0.05 vs. control; †P < 0.05 vs. glutamate treated group.

Fig. 2

Effects of PPI2, endothall, or cyclosporine A on 17β-estradiol mediated neuroprotection in HT-22 cells and C6-glioma. HT-22 (A–C) and C6-glioma (D–F) cells were seeded into 96-well plates at a density of 3500 cells/well. (A and D) Cells were treated simultaneously with with 200 nM PPI2, 10 mM glutamate, and/or 10 μM 17β-estradiol. (B and E) Cells were treated simultaneously with 9 μM endothall, 10 mM glutamate, and/or 10 μ 17β-estradiol. (C and F) Cells were treated simultaneously with 500 nM CsA, 10 mM glutamate, and/or 10 μ 17β-estradiol. Cell viability was determined by calcein AM assay (Molecular Probes, Eugene, OR) after 24 h exposure to the various compounds. All data were normalized to % survival of non-treated control. Depicted are mean ± SEM for 10 independent experiments with two replicates per experiment. *P < 0.05 vs. vehicle control; †P < 0.05 vs. glutamate treated group.

Fig. 3

Time course of the effects 17β-estradiol, glutamate, and their combination on PP1 protein levels in HT-22. HT-22 cells were treated with either 10 mM glutamate (A) or 10 μM 17β-estradiol (B) and simultaneously with 10 mM glutamate and 10 μM 17β-estradiol (C). Cells were harvested at the times indicated for Western blot analysis of PP1. The graphs represent relative OD as a percentage of time 0 control and were normalized to β-actin (not shown). Data are represented as mean ± SEM for n = 3. *P < 0.05 versus time 0 control.

Fig. 4

PP2A activity in HT-22 cells following treatment with glutamate and/or 17β-estradiol in the presence of specific inhibitors of PP1, PP2A, or calcineurin. HT-22 cells were seeded in 100 mm dishes at a density of 250,000 cells/ml. (A) Cells were treated simultaneously with 100 nM okadaic acid, 10 mM glutamate, and/or 10 μM 17β-estradiol. (B) Cells were treated simultaneously with 200 nM PPI2, 10 mM glutamate, and/or 10 μM 17β-estradiol. (C) Cells were treated simultaneously with 9 μM endothall, 10 mM glutamate, and/or 10 μM 17β-estradiol. (D) Cells were treated simultaneously with 500 nM CsA, 10 mM glutamate, and/or 10 μM 17β-estradiol. PP2A activity was determined using a serine/threonine phosphatase activity assay (Promega, Madison, WI) after 24 h exposure to the various compounds. All data were normalized to % survival of vehicle treated control. Depicted are mean ± SEM for six independent experiments with triplicates per experiment. *P < 0.05 vs. glutamate treated group.

Fig. 5

Relationship between neuroprotective and mitoprotective potency of estrogens. Eleven estrogens were selected from our library of compounds and tested for potency in protection from glutamate-induced cells death (x-axis) as well as their potency in protection from H₂O₂-induced collapse of mitochondrial member potential (y-axis). Depicted are the EC₅₀ values for each assay. Denoted with arrows are a potent synthetic estrogen (ZYC-33), 17βb-estradiol (17β-E2) and an inactive estrogen (E2550).