Okadaic acid induces tau phosphorylation in SH-SY5Y cells in an estrogen-preventable manner

Abstract

One of the pathological hallmarks of Alzheimer's disease (AD) is neurofibrillary tangles (NFTs), which are composed of abnormally hyperphosphorylated tau, but the mechanism of tau hyperphosphorylation in AD is still unclear. To investigate the effects of estrogens on tau phosphorylation, SH-SY5Y cells were treated with okadaic acid (OA), a serine/threonine phosphatase inhibitor, to induce tau phosphorylation and the effects of estrogen were observed by co-treatment with 17beta-estradiol (E2). We found that OA induced in vitro tau hyperphosphorylation, which was prevented by E2 in a dose-dependent manner. This effect of E2 was partially blocked by an estrogen receptor (ER) antagonist, ICI 182,780. In addition to tau hyperphosphorylation, inhibition of serine/threonine phosphorylation induced upregulation of cdk5 levels, which was attenuated by E2 in a manner that was counteracted by ICI 182,780. Our results show that cdk5 is involved in OA-induced tau hyperphosphorylation, and estrogens ameliorate the tau hyperphosphorylation, which may be mediated in part by ER.

Figure 1. The dose-dependent effects of 17β-estradiol on tau phosphorylation in OA treated SH-SY5Y cells

SH-SY5Y cells were co-treated with OA (100 nM) and 17β-estradiol (10 nM, 100 nM, 1 μM, 10 μM) for three hours. Phospho-tau (Thr 205) was assessed via western blot analysis. OA and E2 were dissolved in 0.1% DMSO. Vehicle control was treated with 0.1% DMSO. All data are normalized to total tau and are expressed as a percentage of control. Data are presented as mean ± SEM for n=4. ## means p<0.01 compared to control and ** <0.01 compared to OA only. Groups connected by the line were different at *p<0.05 and **p<0.01.

Figure 2. The effect of 17β-estradiol and ICI 182,780 on tau phosphorylation in OA treated SH-SY5Y cells

SH-SY5Y cells were treated with OA (100 nM) in the presence of absence of 17β-estradiol (E2, 10 μM) and/or ICI 182,780 (1 μM) for three hours. Phospho-tau (Thr 205) was assessed via western blot analysis. OA, E2 and ICI 182,780 were dissolved in 0.1% DMSO. Vehicle control was treated with 0.1% DMSO as vehicle. All data are normalized to total tau and are expressed as a percentage of control. Data are presented as mean ± SEM for n=4. ## means p<0.01 compared to control and ** <0.01 compared to OA only. Groups connected by the line were different at *p<0.05 and **p<0.01.

Figure 3. Effects of 17β-estradiol and ICI 182,780 on cdk5 and p25 in OA treated SH-SY5Y cells

SH-SY5Y cells were treated with OA (100 nM) in the presence of absence of 17β-estradiol (E2, 10 μM) and ICI 182,780 (1 μM) for three hours. cdk5 (A) and p25 (B) were assessed via western blot analysis. OA, E2 and/or ICI 182,780 were dissolved in 0.1% DMSO. Vehicle control was treated with 0.1% DMSO as vehicle. All data are normalized to β-actin and are expressed as a percentage of control. Data are presented as mean ± SEM for n=5. # means p<0.05 compared to control and * <0.05, ** <0.01 compared to OA only. Groups connected by the line were different at p<0.05.

Figure 3. Effects of 17β-estradiol and ICI 182,780 on cdk5 and p25 in OA treated SH-SY5Y cells

SH-SY5Y cells were treated with OA (100 nM) in the presence of absence of 17β-estradiol (E2, 10 μM) and ICI 182,780 (1 μM) for three hours. cdk5 (A) and p25 (B) were assessed via western blot analysis. OA, E2 and/or ICI 182,780 were dissolved in 0.1% DMSO. Vehicle control was treated with 0.1% DMSO as vehicle. All data are normalized to β-actin and are expressed as a percentage of control. Data are presented as mean ± SEM for n=5. # means p<0.05 compared to control and * <0.05, ** <0.01 compared to OA only. Groups connected by the line were different at p<0.05.

Figure 4. Effects of 17β-estradiol and ICI 182,780 on GSK3β in OA treated SH-SY5Y cells

Human neuroblastoma SH-SY5Y cells were treated with OA (100 nM) in the presence of absence of 17β-estradiol (E2, 10 μM) and ICI 182,780 (1 μM) for three hours. Total GSK3β and p-GSK3β (Ser 9) were assessed via western blot analysis. OA, E2 and/or ICI 182,780 were dissolved in 0.1% DMSO. Vehicle control was treated with 0.1% DMSO as vehicle. All data are normalized to total GSK3β and are expressed as a percentage of control. Data are presented in mean ± SEM for n=4. # <0.05 compared to control and * <0.05 compared to OA only. Groups connected by the line were different at p<0.05.