ERβ agonist alters RNA splicing factor expression and has a longer window of antidepressant effectiveness than estradiol after long-term ovariectomy

Abstract

Background: Estrogen therapy (ET), an effective treatment for perimenopausal depression, often fails to ameliorate symptoms when initiated late after the onset of menopause. Our previous work has suggested that alternative splicing of RNA might mediate these differential effects of ET.

Methods: Female Sprague–Dawley rats were treated with estradiol (E2) or vehicle 6 days (early ET) or 180 days (late ET) after ovariectomy (OVX). We investigated the differential expression of RNA splicing factors and tryptophan hydroxylase 2 (TPH2) protein using a customized RT2 Profiler PCR Array, reverse-transcription polymerase chain reaction, immunoprecipitation and behaviour changes in clinically relevant early and late ET.

Results: Early ET, but not late ET, prolonged swimming time in the forced swim test and reduced anxiety-like behaviours in the elevated plus maze. It reversed OVX-increased (SFRS7 and SFRS16) or OVX-decreased (ZRSR2 and CTNNB1) mRNA levels of splicing factors and ERβ splicing changes in the brains of OVX rats. Early ET, but not late ET, also increased the expression of TPH2 and decreased monoamine oxidase A levels in the dorsal raphe in the brains of OVX rats. In late ET, only diarylpropionitrile (an ERβ-specific agonist) achieved similar results — not E2 (an ERα and ERβ agonist) or propylpyrazoletriol (an ERα-specific agonist).

Limitations: Our experimental paradigm mimicked early and late ET in the clinical setting, but the contribution of age and OVX might be difficult to distinguish.

Conclusion: These findings suggest that ERβ alternative splicing and altered responses in the regulatory system for serotonin may mediate the antidepressant efficacy of ET associated with the timing of therapy initiation. It is likely that ERβ-specific ligands would be effective estrogen-based antidepressants late after the onset of menopause.

Fig. 1

E2 and ER-specific agonists regulate mobility and anxiety-like behaviours in OVX rats. (A) Experimental regimen. Female Sprague–Dawley rats were ovariectomized at day 0 (9 mo of age), when irregular estrous cycles usually begin in laboratory rodents. They were then separated into 2 treatment groups: early and late ET, with different durations of ovarian hormone deprivation (6 d and 180 d). This experimental paradigm mimicked a common clinical setting, in which perimenopausal women (about 50 yr of age) receive ET at different points postmenopause. In the early ET group, OVX rats were treated with either E2 or vehicle (corn oil) at day 7 (equal to 4 mo in humans). In the late ET group, OVX rats were treated with E2, DPN, PPT or vehicle at day 181 (equal to age > 11 yr in humans). After 2 days of treatment, rats were subjected to a forced swim test, and samples were collected on the following day. (B) Behavioural tests. The forced swim test (a, c) as an assessment of antidepressant activity and the elevated plus maze (b, d) as an assessment of anxiety-like behaviour were performed in rats treated with vehicle or E2 6 days post-OVX (a, b) or vehicle, E2, DPN or PPT 180 days post-OVX (c, d). Data were analyzed using 1-way ANOVA and a subsequent Bonferroni post hoc test, and are presented as mean ± SEM, n = 6 for early ET, n = 5 for late ET. #p < 0.05 v. sham + V; *p < 0.05 v. OVX + V. ANOVA = analysis of variance; DPN = diarylpropionitrile; E2 = estradiol; ER = estrogen receptor; ET = estrogen therapy; FST = forced swim test; OVX = ovariectomy; PPT = propylpyrazoletriol; Sac = sacrifice; SEM = standard error of the mean; V = vehicle.

Fig. 2

E2 and ER-specific agonists regulate mRNA expression of splicing factors. We measured SFRS7, SFRS16, ZRSR2 and CTNNB1 gene expression in frontal cortex using real-time qPCR with 18S rRNA expression as an internal control from rats receiving vehicle or E2 6 days post-OVX (A, C, E, G) or from rats receiving vehicle, E2, DPN or PPT 180 days post-OVX (B, D, F, H). Data were analyzed using 1-way ANOVA and a subsequent Bonferroni post hoc test, and are presented as mean ± SEM. #p < 0.05 v. sham + V; *p < 0.05 v. OVX + V. ANOVA = analysis of variance; DPN = diarylpropionitrile; E2 = estradiol; ER = estrogen receptor; ET = estrogen therapy; OVX = ovariectomy; PPT = propylpyrazoletriol; qPCR = quantitative polymerase chain reaction; SEM = standard error of the mean; V = vehicle.

Fig. 3

E2 and ER-specific agonists differentially regulate protein expression of 2 main ERβ isoforms in leukocytes of OVX rats. Leukocytes were extracted from whole blood, and proteins were extracted by RIPA buffer and sonication; total protein was separated by SDS–PAGE. We detected ERβ and ERβ2 protein expression using specific antibodies, with β-actin as the internal control. We compared protein levels of ERβ and ERβ2 in leukocytes from rats receiving vehicle or E2 6 days after OVX (A, C) and from rats receiving vehicle, E2, DPN or PPT 180 days after OVX (B, D). We compared ERβ2:ERβ protein expression ratio in early ET (E) and late ET (F) groups. Inserts in D and E clearly show the differences between each group. Data were analyzed using 1-way ANOVA and a subsequent Bonferroni post hoc test, and are presented as mean ± SEM of ROD; n = 5–6 for early ET, n = 4–9 for late ET. #p < 0.05 v. sham + V; *p < 0.05 v. OVX + V. ANOVA = analysis of variance; DPN = diarylpropionitrile; E2 = estradiol; ER = estrogen receptor; ET = estrogen therapy; OVX = ovariectomy; PPT = propylpyrazoletriol; RIPA = radioimmunoprecipitation assay; ROD = relative optical density; SDS–PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEM = standard error of the mean; V = vehicle.

Fig. 4

E2 and ER-specific agonists regulate TPH2 expression in the dorsal raphe of OVX rats. We measured TPH2 mRNA expression in rat dorsal raphe using real-time qPCR with 18S rRNA expression as an internal control from rats receiving ET 6 days post-OVX (A) and 180 days post-OVX (B). We detected the expression of TPH2 protein in dorsal raphe using SDS–PAGE probed for TPH2 after immunoprecipitation (C). We used whole cell lysate from TPH2-expressing SH-SY5Y neuroblastoma cells for positive controls. We used no primary antibody for the negative control in immunoprecipitation. We compared the optical density of TPH2 in rats receiving E2 6 d after OVX (D) or rats receiving E2, DPN or PPT 180 d after OVX (E). The data were analyzed using 1-way ANOVA and a subsequent Bonferroni post hoc test, and are presented as mean ± SEM; n = 6 for early ET, n = 4–8 for late ET. #p < 0.05 v. sham + V; *p < 0.05 v. OVX + V. Ab = antibody; ANOVA = analysis of variance; DPN = diarylpropionitrile; E2 = estradiol; ER = estrogen receptor; ET = estrogen therapy; OD = optical density; OVX = ovariectomy; PC = positive control; PPT = propylpyrazoletriol; qPCR = quantitative polymerase chain reaction; SDS–PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEM = standard error of the mean; TPH2 = tryptophan hydroxylase 2; V = vehicle.

Fig. 5

E2 reduces OVX-induced oxidative stress and glial cell activation in rats (6 d post-OVX). (A) Representative immunofluorescence images of dorsal raphe of OVX female rats treated with vehicle, OVX + vehicle and OVX + E2. Nuclei were counterstained with DAPI (blue). Note the nuclear staining of NeuN (green), cytosol punctate staining of MAO-A (magenta) and glial cell–specific staining of GFAP (red). Also note the even distribution of the NeuN staining in the 3 conditions, while both MAO-A and GFAP expressions were increased in OVX rats. Scale bar = 50 μm. Arrows and arrowheads indicate the representative cells expressing MAO-A + NeuN and MAO-A + GFAP, respectively. (B) The immunoreactive intensities of MAO-A, GFAP and NeuN in the dorsal raphe of sham + vehicle, OVX + vehicle and OVX + E2 female rats, analyzed using 1-way ANOVA and a subsequent Bonferroni post hoc test. Note the significant increases of MAO-A (F_2,3110 = 8.80, p < 0.001) and GFAP (F_2,8082 = 11.71, p < 0.001) expression in the dorsal raphe of OVX rats, but OVX did not change the expression of NeuN (F_2,2655 = 1.73, p = 0.18); E2 significantly reduced OVX-induced MAO-A and GFAP expression. Although OVX did not change NeuN expression, E2 increased NeuN expression in OVX rats v. sham + vehicle rats. ANOVA = analysis of variance; E2 = estradiol; MAO-A = monoamine oxidase A; OVX = ovariectomy; V = vehicle.

Fig. 6

E2 and ERα-specific agonist increased MAO-A and GFAP expression, and reduced NeuN expression in the dorsal raphe of OVX rats. ERβ-specific agonist ameliorated GFAP expression and maintained NeuN expression in the dorsal raphe of OVX rats (180 d post-OVX). (A) Representative immunofluorescence images of the dorsal raphe of OVX female rats treated with vehicle (sham + vehicle), OVX + vehicle, OVX + E2, OVX + DPN and OVX + PPT. Nuclei were counterstained with DAPI (blue). Note the nuclear staining of NeuN (green), cytosol punctate staining of MAO-A (magenta) and glial cell–specific staining of GFAP (red). Scale bar = 50 μm. Arrows and arrowheads indicate the representative cells expressing MAO-A + NeuN and MAO-A + GFAP, respectively. (B) We analyzed the immunoreactive intensities of MAO-A, GFAP and NeuN in the dorsal raphe of rats using 1-way ANOVA and a subsequent Bonferroni post hoc test. Note the significant increases of MAO-A (F_4,5248 = 33.66, p < 0.001) and GFAP (F_4,19808 = 11.88, p < 0.001) expression in OVX rats; however, OVX did not alter the expression of NeuN (F_4,6953 = 4.95, p = 0.26). Both E2 and PPT increased MAO-A and GFAP (p < 0.001 and p < 0.05, respectively, for E2; p < 0.001 and p < 0.05, respectively, for PPT) v. OVX, but reduced NeuN expression (p < 0.001 for E2 and p < 0.06 for PPT); DPN ameliorated MAO-A and GFAP expression (p = 0.86 for MAO-A and p = 0.09 for GFAP) and maintained NeuN expression (p = 0.9) in rats 180 d after OVX. ANOVA = analysis of variance; DPN = diarylpropionitrile; E2 = estradiol; MAO-A = monoamine oxidase A; OVX = ovariectomy; PPT = propylpyrazoletriol.