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. 2018 Jan 19;24(6):495–507. doi: 10.1111/cns.12806

Nuclear and membrane estrogen receptor antagonists induce similar mTORC2 activation‐reversible changes in synaptic protein expression and actin polymerization in the mouse hippocampus

Fang‐Zhou Xing 1, Yan‐Gang Zhao 2, Yuan‐Yuan Zhang 2, Li He 3, Ji‐Kai Zhao 2, Meng‐Ying Liu 2, Yan Liu 1,✉, Ji‐Qiang Zhang 2,✉
PMCID: PMC6490049  PMID: 29352507

Summary

Aims

Estrogens play pivotal roles in hippocampal synaptic plasticity through nuclear receptors (nERs; including ERα and ERβ) and the membrane receptor (mER; also called GPR30), but the underlying mechanism and the contributions of nERs and mER remain unclear. Mammalian target of rapamycin complex 2 (mTORC2) is involved in actin cytoskeleton polymerization and long‐term memory, but whether mTORC2 is involved in the regulation of hippocampal synaptic plasticity by ERs is unclear.

Methods

We treated animals with nER antagonists (MPP/PHTPP) or the mER antagonist (G15) alone or in combination with A‐443654, an activator of mTORC2. Then, we examined the changes in hippocampal SRC‐1 expression, mTORC2 signaling (rictor and phospho‐AKTSer473), actin polymerization (phospho‐cofilin and profilin‐1), synaptic protein expression (GluR1, PSD95, spinophilin, and synaptophysin), CA1 spine density, and synapse density.

Results

All of the examined parameters except synaptophysin expression were significantly decreased by MPP/PHTPP and G15 treatment. MPP/PHTPP and G15 induced a similar decrease in most parameters except p‐cofilin, GluR1, and spinophilin expression. The ER antagonist‐induced decreases in these parameters were significantly reversed by mTORC2 activation, except for the change in SRC‐1, rictor, and synaptophysin expression.

Conclusions

nERs and mER contribute similarly to the changes in proteins and structures associated with synaptic plasticity, and mTORC2 may be a novel target of hippocampal‐dependent dementia such as Alzheimer's disease as proposed by previous studies.

Keywords: actin polymerization, estrogen receptors, hippocampus, mTORC2, synaptic plasticity

1. INTRODUCTION

Estrogens, especially 17‐β estradiol (E2), have been shown to regulate hippocampal structure and function, including synaptic plasticity such as synaptic protein levels, dendritic spine density, and synapse density. E2 also plays pivotal roles in the regulation of long‐term potentiation (LTP) and long‐term depression (LTD), learning and memory behavior, and cognition.1, 2, 3, 4 Studies have demonstrated that E2 functions via two classical nuclear receptors (nERs; ERα and ERβ) and/or one membrane receptor (mER, also known as GPR30 or GPER‐1). Numerous studies have indicated the critical roles of these receptors in the regulation of hippocampal synaptic plasticity, spatial learning, and memory performance.5, 6, 7, 8, 9, 10 For example, an ERβ agonist facilitated actin polymerization in adult rat hippocampal slices.11 An ERα antagonist impaired the expression of synapsins, a family of proteins that have long been implicated in the regulation of neurotransmitter release at synapses and, thus, affect the strength of synaptic transmission.12 An ERα selective antagonist MPP (1,3‐Bis(4‐hydroxyphenyl)‐4methyl‐5‐[4‐(2‐piperidinylethoxy) phenol]‐1H‐pyrazole dihydrochloride) and an ERβ selective antagonist PHTPP (4‐[2‐Phenyl‐5,7‐bis(trifluoromethyl)pyrazolo [1,5‐a]pyrimidin‐3‐yl]phenol) have been shown to block the protective effect of E2 against neuronal death in cultured rat hippocampal neurons13 and modulate the reorganization of the actin cytoskeleton in rat hippocampal slices.14 Meanwhile, mER (GPR30) has been shown to participate in rapid, nonclassical E2 actions;15, 16 its agonist G1 (1‐(4‐(6‐Bromobenzo[1,3]dioxol‐5‐yl)‐3a,4,5,9b‐tetrahydro‐3H‐cyclopenta[c]quinolin‐8‐yl)‐ethanone) or antagonist G15 ((3aS,4R,9bR)‐4‐(6‐bromo‐1,3‐benzodioxol‐5‐yl)‐3a,4,5,9b‐tetrahydro‐3H‐cyclopenta[c]‐quinolone) modulate synaptic protein expression and spatial recognition acquisition.7, 8, 10 However, the related mechanisms and whether there are any differences between the contributions of nERs and mER to E2 regulation of hippocampal synaptic structural plasticity are not clarified.

Two decades ago, McEwen and colleagues provided primary evidence showing that E2 could increase hippocampal CA1 dendritic spine and synapse density.17, 18 Dendritic spines are mushroom‐shaped protrusions of the postsynaptic membrane; their number and shape are regulated by actin cytoskeleton polymerization, which is determined by the shift between soluble G‐actin monomers and insoluble F‐actin filaments.19, 20 Cofilin has been shown to be responsible for the depolymerization of F‐actin, and profilin‐1 has been shown to be responsible for the polymerization of G‐actin.21, 22 Interestingly, recent studies have shown that in the hippocampus, actin cytoskeleton polymerization is controlled by mammalian target of rapamycin complex 2 (mTORC2). Using mTORC2‐deficient mice in which rictor, the regulatory component of mTORC2 was conditionally deleted, and mTORC2 has been demonstrated to affect neuronal morphology, synaptic function, and long‐term memory formation through regulation of actin cytoskeleton polymerization.23, 24 Additional evidence has shown that in the brain of aged mice and flies, loss of mTORC2‐mediated actin polymerization contributed to age‐associated memory loss, which could be reversed by mTORC2 activation with A‐443654, the specific mTORC2 activator.25 Sun et al reported that in the hippocampus of both wild‐type and Angelman syndrome mice, LTP and actin polymerization were significantly increased by A‐443654 treatment.26 Furthermore, several pieces of evidence have shown that mTORC2 could be stimulated by E2 administration in breast cancer27 and that mTORC2 activation could be modulated by E2 in female cardiomyocytes.28 Meanwhile, our previous studies revealed that in the hippocampus of mice, mTORC2 signaling and actin polymerization could be regulated by letrozole, the E2 synthase inhibitor, or E2 administration.29 However, whether mTORC2 activation is involved in estrogen receptor regulation of hippocampal synaptic structural plasticity is not clearly elucidated.

To address the above issues, in this study, we first examined the expression of steroid receptor coactivator‐1 (SRC‐1), mTORC2 signaling molecules, actin polymerization regulatory proteins, and some key synaptic proteins after injection of G15 or a combination of MPP and PHTPP alone or combined with A‐443654 in vivo. Then, we examined the changes in CA1 dendritic spine and synapse density under MPP/PHTPP, G15, and A‐443654 treatment.

2. MATERIALS AND METHODS

2.1. Animals and drug administration

Adult (12 weeks old, 20‐25 g) female C57/BL6 mice were purchased from the Experimental Animal Center of the Third Military Medical University. All experiments were performed in accordance with approved Institutional Animal Care and Use protocols of this university. Before mice were sacrificed, vaginal smears were prepared, and the cycling of all the animals was examined with Tar purple staining; only the diestrus mice were used for further experiments.

For treatment with nER antagonists and the mTORC2 activator, the animals were randomly divided into 3 groups: a control group (40% DMSO + 60% sterile saline solution; 100 μL/mouse), a group that received coadministration of the ERα antagonist MPP (200 μg/kg; sc‐204098, Santa Cruz Biotechnology, Shanghai, China) and the ERβ antagonist PHTPP (100 μg/kg; sc‐204191, Santa Cruz) (the MPP/PHTPP group), and a group that received a combination of MPP/PHTPP and the mTORC2 specific activator A‐443654 (2.5 mg/kg, HY‐10425, MedChem Express, Shanghai, China; the MPP/PHTPP/A‐443654 group). For treatment with the mER antagonist and mTORC2 activator, the animals were randomly divided into an additional 3 groups: a control group (40% DMSO + 60% sterile saline solution; 100 μL/mouse), a group treated with the mER antagonist G15 (20 μg/mouse; 14673, Cayman Chemical Company, Michigan, USA) (the G15 group), and a group treated with a combination of G15 and A‐443654 (2.5 mg/kg; HY‐10425, MedChem Express) (the G15/A‐443654 group). MPP and PHTPP were prepared as one injection (coadministration), and G15 was prepared as another single injection. For A‐443654 treatment, a solution containing MPP, PHTPP, and A‐443654 or a solution containing G15 and A‐443654 was prepared.

In our preliminary time‐dependent (1‐day, 3‐days, or 7‐days) tests, we found that G15, MPP, and PHTPP treatment induced a significant decrease in rictor, p‐AKT(Ser473)/AKT, p‐cofilin(Ser3), and profilin‐1 expression, mostly seen after 3 days (P < 0.05) and especially after 7 days (P < 0.01). Furthermore, we observed that G15 treatment induced a learning and memory impairment at day 4 (Y. Zhang, M. Liu, Y. Zhao, L. He, J. Zhao, F. Xing and J. Zhang, unpublished data). Similar behavior impairments were also detected after MPP and PHTPP treatment.30 Thus, a 7‐days treatment duration was used in this study. Mice were anaesthetized with 100 mg/kg sodium pentobarbital before injection; the drugs were given intraperitoneally every morning (from 09:00 to 11:00) for 1 week.

2.2. Tissue preparation and immunohistochemistry

Tissue preparation and immunohistochemistry were performed as described previously.31 Briefly, mice were perfused with saline and 4% paraformaldehyde under deep anesthesia. The brains were dissected and cut into 20‐μm‐thick sections with a cryostat (CM1900, Leica Microsystems, Heidelberger, Germany). The sections were washed with PBS and quenched for 15 minutes with 3% H2O2. Then, the slices were incubated overnight at 4°C with the primary antibodies against ERα (1:200, sc‐542, Santa Cruz), ERβ (1:100, sc‐6821, Santa Cruz), GPR30 (1:200, sc‐48525‐R, Santa Cruz), SRC‐1 (1:200, sc‐8995, Santa Cruz), or profilin‐1 (1:200, GTX64356, GeneTex, Texas, USA) prepared with the antibody diluent (ZLI‐9028, Zhongshan Biotech, Beijing, China). Sections were rinsed in PBS and incubated for 1 hour with the corresponding secondary antibody (1:200, ZB‐2010, Zhongshan Biotech, Beijing, China). Sections were washed in PBS and incubated with the HRP‐labeled streptavidin reagent (1:200, ZB‐2404, Zhongshan Biotech) for 1 hour and then visualized using a DAB‐nickel chromogen solution (SK‐4100, Vector Laboratories Inc., Burlingame, USA) for 5 minutes at room temperature. Finally, slides were dehydrated, cleared with xylene, and mounted.

Then, the slides were imaged using an Olympus microscope (BX60, Olympus, Tokyo, Japan), and the images were captured using a digital camera (DP70, Leica Microsystems, Germany). The average optical density was analyzed using Image‐Pro Plus software 6.0 (Media Cybernetics, Rockville, USA) according to the user manual, and the mean value of the entire hippocampus (including CAs and dentate gyrus) was used to represent the regional expression level for each group. To prevent the detection of false changes between groups due to possible differences in the localization of similar anatomical areas along the anterior to posterior axis, only stained sections between Bregma −1.70 and Bregma −2.18 were used for data analysis, ensuring the highest possible consistency between groups. The mean optical density from 3 to 5 sections from each hippocampus was used to represent the regional expression level for each animal.

2.3. Western blot analysis

To examine the expression of the hippocampal synaptic proteins, mTORC2 signaling proteins, and actin remodeling proteins before and after drug treatments, mice were sacrificed, and the hippocampi were dissected and lysed with RIPA buffer (P0013B, Beyotime Biotech, Beijing, China) containing protease inhibitor (04693132001, Roche, Shanghai, China) and phosphorylase inhibitor (4906845001, Roche). Protein concentrations were measured using a BCA Assay Kit (P0010, Beyotime Biotech). An equal amount of proteins was denatured at 95°C for 5 minutes in protein‐loading buffer, separated by SDS‐polyacrylamide gel electrophoresis (PAGE), and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Millipore, Darmstadt, Germany). After the PVDF membranes were blocked with 5% freshly prepared milk‐TBST for 2 hours at room temperature, they were incubated with primary antibodies overnight at 4°C. Finally, the membranes were washed with TBST and incubated with an HRP‐conjugated goat anti‐rabbit secondary antibody (1:2000, ZB‐2301, Zhongshan Biotech), goat anti‐mouse secondary antibody (1:2000, ZB‐2305, Zhongshan Biotech), or rabbit anti‐goat (1:2000, ZB‐2306, Zhongshan Biotech) for 1.5 hours at room temperature. To measure the phospho‐AKTSer473/total AKT or phospho‐cofilinSer3/total cofilin ratios, the phospho‐antibodies were first used in the protocol described above. Then, the membranes were washed with a specific stripping buffer (P0023A, Beyotime Biotech), blocked with 5% freshly prepared milk‐TBST, and incubated with the primary antibodies against AKT or cofilin, followed by the next steps described above. Finally, the blots were developed with a chemiluminescent HRP substrate (WBKLS0100, MerkMillipore, Massachusetts, USA) and visualized with Western Lighting‐ECL (Bio‐Rad, Hercules, USA). The absorbance values of target proteins were analyzed with Quantity One software (Bio‐Rad). The levels of β‐actin, total AKT, or total cofilin were also examined and used as an internal standard to normalize the relative absorbance values of target proteins.

The primary antibodies used in the Western blot analysis were as follows: rabbit polyclonal anti‐SRC‐1 (1:1000, sc‐8995, Santa Cruz), rabbit mAb‐phospho‐AKTSer473 (1:2000, 9271, Cell Signaling, Shanghai, China), rabbit mAb‐AKT (1:2000, 9272, Cell Signaling), rabbit mAb‐rictor (1:600, 2140, Cell Signaling), rabbit mAb‐phospho‐cofilinSer3 (1:2000, 3311, Cell Signaling), rabbit mAb‐cofilin (1:2000, 3312, Cell Signaling), rabbit mAb‐GluR1 (1:600, 2452486, Millipore), rabbit mAb‐PSD95 (1:800, 3409, Cell Signaling), goat polyclonal antispinophilin (1:600, sc‐14774, Santa Cruz), rabbit mAb‐synaptophysin (1:20000, 04‐1019, Millipore), and mouse mAb‐β‐actin (1:1000, AA128, Beyotime Biotech).

2.4. Golgi‐Cox staining and dendritic spine measurement

To assess the effects of the drug treatments on the spine density in hippocampal neurons, Golgi staining was carried out using the FD Rapid Golgi Stain Kit (PK401, FD NeuroTechnologies, Columbia, USA) according to the manufacturer's instructions. In short, mice were anesthetized, and the whole brain was dissected, rinsed in distilled water, and then immersed in the Golgi‐Cox solution (Solution A:B = 1:1), which was changed to fresh solution the next day. The brains were kept in darkness for 2 weeks at room temperature, followed by immersion in Solution C for 3 days at 4°C in darkness. The brains were then cut into 200‐μm‐thick slices with a vibratome (Microslicer DTK‐600, Dosaka EM, Tokyo, Japan), and the slices were mounted on gelatin‐coated slides and air‐dried in a dark place. The slices were then stained with a mixture of Solution D, Solution E, and distilled water (1:1:2) for 10 minutes. Then, the slides were dehydrated in 50%, 75%, 95%, and 100% alcohol. Finally, the sections were cleaned, mounted, and photographed with an Olympus microscope (100× oil immersion lens). Then, the number of dendritic spines along the secondary branching of the dendrites of CA1 pyramidal neurons was measured with Image‐Pro Plus software (Media Cybernetics), and the number of dendritic spines per 10 μm was counted and used for further analysis.

2.5. Transmission electron microscopy

Transmission electron microscopy was used to evaluate the changes in synapse density in the CA1 region of the hippocampus after drug treatment. Briefly, the hippocampi were dissected, rapidly transferred into 2.5% glutaraldehyde, cut into slices containing the upper and the middle of the CA1 stratum radiatum, and fixed in glutaraldehyde for 3 days. After the sections were washed with PBS, they were postfixed with 1% osmium tetroxide and stained with an aqueous solution of 2% uranyl acetate. After the sections were dehydrated with gradient ethyl alcohols, they were permeabilized and embedded with an embedding medium. Then, the sections were incubated in an oven, cut into ultrathin sections, stained with 4% uranyl acetate and citrate, and then observed with a transmission electron microscope (JEM‐1400 Plus, JEOL, Japan) under 30 000 ×  magnification. Image‐Pro Plus software was used to analyze the density of spine synapses. The postsynaptic density (PSD) was measured by the Calibration Tools option of the Image‐Pro Plus software according to Guldner et al32 The density of spine synapses was calculated using quantitative analysis of three‐dimensional metrology. The number of synapses per unit volume of tissue (Nv) was calculated from the number of synapses per unit of area (Na) according to the formula Nv=8ENa/π2 (E is the mean of the reciprocal of the PSD length for each synaptic profile category for each of the six groups of animals) as described in a previous study.33

2.6. Statistical analysis

All statistics were analyzed using SPSS software, and the data were shown as the mean ± SE. For multiple‐group comparisons, a one‐way ANOVA and post hoc test were used. To analyze the contribution of nERs or mER to the hippocampal synaptic plasticity‐related parameters, a ratio (%) describing the decrease in expression of specific proteins after MPP/PHTPP or G15 treatment was compared using an independent‐sample t‐test. In both conditions, P < 0.05 was considered to be statistically significant.

3. RESULTS

We first used immunohistochemistry to verify the subcellular localization of nERs (ERα and ERβ) and mER in the hippocampus of adult mice. As shown in Figure 1A‐C, the nER‐immunopositive material was predominantly detected in the cell nuclei, while mER‐immunopositive staining was predominantly detected in the plasma membrane.

Figure 1.

Figure 1

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Localization of estrogen receptors and the effects of MPP/PHTPP, G15, and A‐443654 on the expression of SRC‐1 in the hippocampus of adult female mice. A,B, Immunopositive materials of nERs (ERα and ERβ) were predominantly localized in the nuclei. C, mER (GPR30 or GPER1)‐immunopositive materials were most likely detected in the cell membrane. A1, B1, and C1 are the corresponding magnifications of the inserts of A, B, and C. D,E, Western blot results showed that the MPP/PHTPP‐ and G15‐induced dramatic decrease in SRC‐1 was not reversed by A‐443654. F,G, Immunohistochemical results showed that the MPP/PHTPP‐ and G15‐induced dramatic decrease in SRC‐1 was not reversed by A‐443654. Bar = 200 μm (A‐C and F‐G) or 20 μm (A1‐C1). **P < 0.01 when compared to the control (DMSO; one‐way ANOVA and post hoc test)

3.1. The MPP/PHTPP‐ and G15‐induced decrease in SRC‐1 was not reversed by A‐443654

SRC‐1 has been shown to enhance the transcriptional activity of nuclear steroid receptors.34, 35 A one‐way ANOVA and post hoc test revealed that there were differences among the control, MPP/PHTPP, and MPP/PHTPP/A‐443654 treatments in the expression of hippocampal SRC‐1 (F (2,15) = 6.717, P = 0.008 for Western blot and F (2,15) = 6.648, P = 0.009 for immunohistochemistry). Similar results were also detected among the groups treated with control, G15, and G15/A‐443654 solutions (F (2,15) = 12.490, P = 0.001 for Western blot and F (2,15) = 8.252, P = 0.004 for immunohistochemistry). In both Western blot and immunohistochemistry analyses, SRC‐1 was significantly downregulated by MPP/PHTPP and G15 treatment when compared to SRC‐1 expression in the control group (P < 0.01). However, there were no differences in expression between the MPP/PHTPP/A‐443654 and MPP/PHTPP groups or between the G15/A‐443654 and G15 groups (P > 0.05) as shown in Figure 1D‐G. Therefore, the ER antagonists induced a decrease in SRC‐1 expression that was not restored by A‐443654 treatment.

3.2. The MPP/PHTPP‐ and G15‐induced decrease in p‐AKTSer473 but not rictor was reversed by A‐443654

Rictor is the regulatory component of mTORC2, and p‐AKTSer473 (p‐AKT) is the direct target of the mTORC2 cascade, which is activated by A‐443654. As shown in Figure 2A,B, the levels of total AKT were unchanged after MPP/PHTPP, MPP/PHTPP/A‐443654, G15, and G15/A‐443654 treatment when compared with those of the control (P > 0.05, one‐way ANOVA and post hoc test). However, there were general differences in the levels of p‐AKT among control, MPP/PHTPP, and MPP/PHTPP/A‐443654 treatments (F (2,15) = 7.535, P = 0.005) as well as among control, G15, and G15/A‐443654 treatments (F (2,15) = 7.350, P = 0.006). Specifically, p‐AKT was significantly downregulated by MPP/PHTPP and G15 treatment when compared to p‐AKT expression in the control group (P < 0.01). Furthermore, the MPP/PHTPP‐ and G15‐induced decrease in p‐AKT was significantly reversed by A‐443654 treatment (P < 0.01 when compared to p‐AKT expression in the respective MPP/PHTPP and G15 groups) and reached the control level (P > 0.05 when compared to the expression in the control group). Therefore, the p‐AKT/AKT ratio was significantly downregulated by MPP/PHTPP and G15 treatment (P < 0.01); this decrease was rescued by A‐443654 treatment (P < 0.01) and reached the control level (P > 0.05 when the ratio was compared to the ratio in the control group).

Figure 2.

Figure 2

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The MPP/PHTPP‐ and G15‐induced decreases in p‐AKT, AKT, and rictor expression were differently affected by mTORC2 activation with A‐443654 in the adult female hippocampus. A,B, The MPP/PHTPP‐ and G15‐induced dramatic decrease in p‐AKT was significantly rescued by A‐443654, while the levels of AKT were not affected by any of the treatments. Therefore, the p‐AKT/AKT ratio showed a significant decrease after MPP/PHTPP and G15 treatment, and this decrease was reversed by A‐443654 treatment. **P < 0.01 when compared to other groups (one‐way ANOVA and post hoc test). C,D, Neither the MPP/PHTPP‐ nor G15‐induced decrease in rictor expression was reversed by A‐443654. **P < 0.01 when compared to the control (DMSO; one‐way ANOVA and post hoc test)

For the expression of rictor, the one‐way ANOVA and post hoc test showed that there were general differences in its expression among the control, MPP/PHTPP, and MPP/PHTPP/A‐443654 groups (F (2,15) = 14.734, P = 0.000) as well as among the control, G15, and G15/A‐443654 treatment groups (F (2,15) = 8.477, P = 0.003). Rictor expression was significantly decreased by MPP/PHTPP and G15 treatment when compared to its expression in the control groups (P < 0.01), but this decrease was not reversed by A‐443654 (P > 0.05 when compared to rictor expression in the respective MPP/PHTPP and G15 groups). These results are shown in Figure 2C,D.

3.3. The MPP/PHTPP‐ and G15‐induced decreases in actin cytoskeleton remodeling proteins were reversed by A‐443654

Cofilin is an actin cytoskeleton polymerization disruptor, and its phosphorylation on serine 3 (p‐cofilin) is known to block this activity. As shown in Figure 3A,B, the one‐way ANOVA and post hoc test on the expression of cofilin showed that there were no differences among the control, MPP/PHTPP, and MPP/PHTPP/A‐443654 treatment groups (F (2,15) = 0.019, P = 0.981) or among the control, G15, and G15/A‐443654 treatment groups (F (2,15) = 0.192, P = 0.827). These results indicated that hippocampal cofilin expression was not affected by ER antagonist or A‐443654 treatment. However, the one‐way ANOVA and post hoc test on the levels of p‐cofilin showed that there were significant differences among the control, MPP/PHTPP, and MPP/PHTPP/A‐443654 treatment groups (F (2,15) = 10.144, P = 0.002) and among the control, G15, and G15/A‐443654 treatment groups (F (2,15) = 11.944, P = 0.001). MPP/PHTPP and G15 treatment induced a significant decrease in p‐cofilin when compared to the control (P < 0.01); this decrease was significantly reversed by A‐443654 (P < 0.01 when compared to p‐cofilin expression in the respective MPP/PHTPP and G15 groups) and reached the control level (P > 0.05 when compared to the expression in the control). Therefore, the ratio of p‐cofilin/cofilin was significantly downregulated by MPP/PHTPP and G15 treatment (P < 0.01 when compared to the ratio in the control), and this decrease was rescued after A‐443654 treatment (P < 0.01 when the ratio was compared to that of the respective MPP/PHTPP and G15 groups). Furthermore, there were no significant differences in the p‐cofilin/cofilin ratio between the control and MPP/PHTPP/A‐443654 or between the G15 and G15/A‐443654 groups (P > 0.05).

Figure 3.

Figure 3

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The MPP/PHTPP‐ and G15‐induced decreases in the expression of actin cytoskeleton regulator proteins were reversed by mTORC2 activation with A‐443654 in the adult female hippocampus. A,B, Western blot results showed that both the MPP/PHTPP‐ and G15‐induced dramatic decrease in p‐cofilin were significantly reversed by A‐443654, but these treatments did not affect the expression of cofilin. Therefore, the p‐cofilin/cofilin ratio showed a significant decrease after MPP/PHTPP and G15 treatment, and this decrease was reversed by A‐443654 treatment. C,D, Immunohistochemistry results showed that the MPP/PHTPP‐ and G15‐induced dramatic decrease in profilin‐1 expression was significantly reversed by A‐443654. **P < 0.01 when compared to other groups (one‐way ANOVA and post hoc test). Bar=200 μm

Immunohistochemistry was employed to examine the changes in the expression of profilin‐1, the actin polymerization stabilizer, after treatment with the ER antagonists by themselves or in combination with A‐443654. Immunopositive staining was predominantly detected in the CA3 and dentate gyrus as shown in Figure 3C,D. The one‐way ANOVA and post hoc test showed that there were significant differences in profilin‐1 expression among the control, MPP/PHTPP, and MPP/PHTPP/A‐443654 treatment groups (F (2,15) = 7.151, P = 0.007) and among the control, G15, and G15/A‐443654 treatment groups (F (2,15) = 8.708, P = 0.003). MPP/PHTPP and G15 treatment induced a significant decrease in profilin‐1 when compared to that in the control (P < 0.01). This decrease was significantly rescued by A‐443654 treatment when profilin‐1 expression was compared to that detected in the MPP/PHTPP and G15 groups (P < 0.01) and reached the control level (P > 0.05 when compared to that in the control).

3.4. Hippocampal synaptic proteins were differentially regulated by MPP/PHTPP, G15, and A‐443654

Western blot analysis was used to examine the expression of several synaptic proteins including postsynaptic GluR1, PSD95, spinophilin, and presynaptic synaptophysin. There were significant differences in GluR1 expression among the control, MPP/PHTPP, and MPP/PHTPP/A‐443654 treatment groups (F (2,15) = 8.138, P = 0.004) and among the control, G15, and G15/A‐443654 treatment groups (F (2,15) = 9.826, P = 0.002). GluR1 levels were significantly decreased by MPP/PHTPP and G15 treatment when compared with those detected in the control (P < 0.01), and this decrease was significantly rescued by A‐443654 when GluR1 expression was compared to that in the MPP/PHTPP and G15 groups (P < 0.01). However, there were no differences in GluR1 expression between the control and MPP/PHTPP/A‐443654 groups (P > 0.05) or between the control and G15/A‐443654 groups (P > 0.05). These results are shown in Figure 4A,B.

Figure 4.

Figure 4

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The MPP/PHTPP‐ and G15‐induced changes in synaptic protein expression were differently affected by mTORC2 activation with A‐443654 in the adult female hippocampus. A‐F, Western blot results showed that the MPP/PHTPP‐ and G15‐induced dramatic decreases in GluR1 (A,B), PSD95 (C,D), and spinophilin (E,F) were significantly reversed by A‐443654. G,H, MPP/PHTPP or G15 treatment and mTORC2 activation did not affect the expression of synaptophysin. Spino: spinophilin. SYN: synaptophysin. **P < 0.01 when compared to other groups (one‐way ANOVA and post hoc test)

For PSD95, there were significant differences among the control, MPP/PHTPP, and MPP/PHTPP/A‐443654 treatment groups (F (2,15) = 14.396, P = 0.000) and among the control, G15, and G15/A‐443654 treatment groups (F (2,15) = 9.514, P = 0.002). PSD95 expression was significantly inhibited by MPP/PHTPP and G15 when compared to its expression in the control (P < 0.01). Additionally, these decreases were significantly reversed by A‐443654 administration (P < 0.01 when compared to PSD95 expression in the MPP/PHTPP and G15 groups). However, there were no differences in PSD95 expression between the control and MPP/PHTPP/A‐443654 (P > 0.05) or between the control and G15/A‐443654 (P > 0.05) groups. These results are shown in Figure 4C,D.

There were also significant differences in the expression of spinophilin among the control, MPP/PHTPP, and MPP/PHTPP/A‐443654 treatment groups (F (2,15) = 7.858, P = 0.005) and among the control, G15, and G15/A‐443654 treatment groups (F (2,15) = 11.517, P = 0.001). Its expression was significantly decreased by MPP/PHTPP and G15 treatment when compared to the expression in the control groups (P < 0.01). A‐443654 treatment significantly reversed these decreases when spinophilin expression in these groups was compared to that detected in the MPP/PHTPP and G15 treatment groups (P < 0.01), and its expression reached the control level (P > 0.05 when compared to that in the control), as shown in Figure 4E,F.

There were no differences in synaptophysin expression among the control, MPP/PHTPP and MPP/PHTPP/A‐443654 treatment groups (F (2,15) = 0.870, P = 0.439) and among the control, G15, and G15/A‐443654 treatment groups (F (2,15) = 1.893 P = 0.185). Therefore, neither the ER antagonists nor A‐443654 affected the expression of hippocampal synaptophysin. These results are shown in Figure 4G,H.

3.5. The MPP/PHTPP‐ and G15‐induced decreases in spine density and synapse density were reversed by A‐443654

Dendritic spine density and synapse density are important parameters for synaptic plasticity. We used Golgi‐Cox staining to examine the CA1 spine density and transmission electronic microscopy to examine the CA1 synapse density. For the changes in the spine density, a one‐way ANOVA and post hoc test revealed that there were significant differences among the control, MPP/PHTPP, and MPP/PHTPP/A‐443654 treatment groups (F (2,42) = 111.601, P = 0.000) and among the control, G15, and G15/A‐443654 treatment groups (F (2,42) = 16.422, P = 0.000). There were also significant differences in synapse density among the control, MPP/PHTPP, and MPP/PHTPP/A‐443654 treatment groups (F (2,27) = 33.515, P = 0.000) and among the control, G15, and G15/A‐443654 treatment groups (F (2,27) = 11.710, P = 0.000). Specifically, CA1 spine density and synapse density were significantly decreased by MPP/PHTPP and G15 treatment when compared to those in the control group (P < 0.01). A‐443654 administration significantly reversed these decreases when the CA1 spine and synapse densities were compared to those of the MPP/PHTPP and G15 treatment groups (P < 0.01), and the spine and synapse densities reached the control level (P > 0.05 when compared to those in the control group), as shown in Figure 5A‐D.

Figure 5.

Figure 5

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The effects of MPP/PHTPP, G15, and A‐443654 on the spine and synapse density as well as the contributions of nERs and mER to changes in hippocampal synaptic plasticity‐related protein expression and morphology. A,B, Golgi‐Cox staining results showed that the MPP/PHTPP‐ and G15‐induced dramatic decreases in the CA1 dendritic spines density were significantly reversed by A‐443654 treatment. Bar = 10 μm. C,D, Transmission electronic microscopy results showed that the MPP/PHTPP‐ and G15‐induced dramatic decreases in CA1 synapse density were significantly reversed by A‐443654. The white arrows in C and D indicate detected synapses. Bar = 1 μm. **P < 0.01 when compared to other groups (one‐way ANOVA and post hoc test). E, The contributions of nERs and mER to the regulation of hippocampal synaptic plasticity‐related protein expression and morphology. The results showed that nERs and mER contributed similarly to most of the examined parameters (P > 0.05, independent‐sample t‐test). However, G15 induced a greater decrease in p‐cofilin, GluR1, and spinophilin expression than MPP. *P < 0.05 (independent‐sample t‐test). Spino: spinophilin. SYN: synaptophysin

3.6. MPP/PHTPP and G15 induced similar changes in synaptic protein expression and actin polymerization

Finally, to evaluate the contributions of nERs and mER in E2 actions on hippocampal synaptic plasticity‐related changes in synaptic proteins and actin polymerization, we compared the decreased level of the above‐mentioned parameters, including SRC‐1, p‐AKT, rictor, and actin remodeling proteins (p‐cofilin and profilin‐1), postsynaptic proteins (GluR1, PSD95, and spinophilin), as well as the density of dendritic spines and synapses, between the MPP/PHTPP and G15 treatment groups. As shown in Figure 5E, there were no significant differences for most of the examined parameters (P > 0.05); the expression levels of only three of the proteins (p‐cofilin, GluR1, and spinophilin) were significantly lower after G15 treatment than after MPP/PHTPP treatment (P < 0.05). Thus, nERs and mER might play similar roles and be similarly effective in the regulation of synaptic protein expression and actin polymerization dynamics.

4. DISCUSSION

E2 is well established to regulate hippocampal structure and function including actin cytoskeleton polymerization,29 expression of synaptic proteins,36 induction of LTP37, and behavior.38 Although mTORC2 has been shown to regulate actin cytoskeleton polymerization23 and be regulated by inhibition of E2 synthesis,29 whether mTORC2 is involved in the action of nERs and mER is not clear. Our recent data showed that coadministration of MPP and PHTPP did not induce further changes in the expression of some actin cytoskeleton remodeling proteins and synaptic proteins when compared to that of MPP or PHTPP alone,30 which raised an interesting question on whether there is any difference in the roles of nERs and mER in the mediation of the action of E2 on hippocampal synaptic plasticity. Therefore, in this study, we first verified the abundant expression of nERs and mER in the hippocampus, which was in general agreement with previous studies showing that nERs were primarily detected in cell nuclei39, 40, 41 and that mER was present in the plasma membrane.42 As high levels of SRC‐1 have been detected in the hippocampus43, 44, 45, 46, 47 and have been shown to be regulated by postnatal development and aging46, 48, 49 and mediate the effects of E2 on hippocampal PSD9550 as well as actin depolymerization,29 we then examined the changes in SRC‐1 expression after nER and mER antagonist treatment and mTORC2 activation. We observed that SRC‐1 expression was suppressed by nER and mER antagonists, strongly indicating its central role in mediating the actions of both nERs and mER. We also found that the levels of hippocampal SRC‐1 were not regulated by mTORC2 activation with A‐443654, indicating that SRC‐1 might not be downstream of mTORC2.

The actin cytoskeleton is the structural element of dendritic spines,20 and its polymerization within the dendritic spine has been shown to play pivotal roles in hippocampal synaptic plasticity and hippocampus‐based learning and memory.51 We therefore examined the expression of some actin cytoskeleton remodeling proteins (rictor, p‐AKT/AKT, p‐cofilin/cofilin, and profilin‐1) after treatment with nER or mER antagonists. In our preliminary tests, we found that G15, MPP, and PHTPP treatment induced significant changes in mTORC2 signals and actin polymerization‐related proteins, mostly seen after 3 days and especially after 7 days of treatment, and the behavioral changes induced by these antagonists were detected after 4 days of treatment.30 These time‐dependent effects led us to choose a 7‐days treatment duration in the present studies. The results showed that the expression of all of the proteins examined, except AKT and cofilin, was significantly downregulated by MPP/PHTPP and G15 administration. These results were in accordance with previous studies showing that nER and mER antagonists blocked actin polymerization, while their agonists promoted actin polymerization.11, 14, 30, 52 Furthermore, these results indicated a parallel role of nERs and mER in mediating E2 regulation in actin cytoskeleton reorganization. We further observed that the decrease in the expression of p‐AKT, p‐cofilin, and profilin‐1 induced by MPP/PHTPP and G15 was significantly rescued by activation of mTORC2, indicating that the effects of nERs and mER on actin polymerization required the involvement of mTORC2. Currently, only sparse studies have reported the effects of mTORC2 activation on actin polymerization. For example, in hippocampal slices, mTORC2 activation by A‐443654 increased actin polymerization,26 and in the brain of fruit flies and aged mice, A‐443654 treatment reversed the loss of mTORC2‐mediated actin polymerization.25 Thus, our current studies provided novel insights into the understanding of the mechanisms of ER regulation of actin polymerization.

Hippocampal synaptic protein levels, dendritic spine density, and synapse density are important components of synaptic plasticity. Our current results showed that levels of postsynaptic GluR1 and PSD95 as well as the dendritic spine marker spinophilin were significantly downregulated by MPP/PHTPP and G15 treatment. Additionally, the spine density and synapse density were also significantly downregulated by MPP/PHTPP and G15 treatment. These results were in agreement with previous findings showing that nER activation improved the levels of hippocampal synaptic proteins (GluR1 and PSD95) and synapse loss induced by ovariectomy6, 53 and that mER activation increased the expression of PSD95 in the hippocampus.54 These observations were also in agreement with the studies shown that the levels of PSD95 and spinophilin and the spine density and synapse density were upregulated by E2 treatment.17, 18, 55. Meanwhile, we found that the expression of the presynaptic protein synaptophysin was not regulated by MPP/PHTPP or G15 treatment. To some extent, this result was in general agreement with a previous study showing that hippocampal synaptophysin was not affected by castration or ovariectomy.56 Furthermore, we noticed that the MPP/PHTPP‐ and G15‐induced decreases in the above parameters were significantly rescued by mTORC2 activation with A‐443654. To the best of our knowledge, this is the first report on the effects of mTORC2 activation on the expression of these synaptic proteins and changes in spine and synapse density.

A few studies have compared the contributions and differences between nER and mER in the mediation of estrogenic action. Romano et al proposed that nERs and mER might influence parallel pathways to achieve similar effects.57 Our recent studies revealed that treatment with MPP, PHTPP, or an MPP/PHTPP combination induced similar changes in selected synaptic protein expression as well as the F‐actin/G‐actin ratio.30 In the present study, we further examined the contributions of MPP/PHTPP and G15 to the changes in the expression of these synaptic proteins and actin polymerization. We found that most of the parameters were not significantly different. Thus, these data strongly indicated that nERs and mER might contribute similarly to the regulation of hippocampal synaptic plasticity, similar to that proposed by Romano et al57 However, the underlying mechanisms are far from clear.

5. CONCLUSIONS

Previously, we found that the upregulation of mTORC2 signals, actin polymerization, and expression of synaptic proteins by nER agonists was blocked by SRC‐1 RNA interference.30 We also found that the mER agonist G1‐induced increase in mTORC2 signals was blocked by the SRC‐1 inhibitor bufalin (Y. Zhang, M. Liu, Y. Zhao, L. He, J. Zhao, F. Xing and J. Zhang, unpublished data). In this study, we demonstrated that inactivation of estrogen receptors induced significant decreases in the expression of SRC‐1, core mTORC2 proteins, actin polymerization remodeling proteins, and postsynaptic proteins as well as in the spine and synapse density. Moreover, the ER antagonist‐induced decreases in actin polymerization, postsynaptic proteins, spine density, and synapse density were rescued by activation of mTORC2 with A‐443654. Thus, we summarized these results in Figure 6. Additionally, we demonstrated that both nERs and mER showed similar effects on hippocampal synaptic plasticity‐related protein expression and morphology. These results provided a novel insight into the mechanisms underlying E2 regulation of actin cytoskeleton polymerization‐based structural plasticity as well as hippocampus‐based learning and memory. Furthermore, because of the role of the hippocampus in neurodegenerative diseases such as Alzheimer's disease (AD) and the controversial effects of E2 replacement against AD,58, 59 these preliminary results may also provide a novel, alternative drug target for the prevention and treatment of AD as proposed in previous studies.23, 25, 30, 60

Figure 6.

Figure 6

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Schematic illustration of the regulation of hippocampal synaptic plasticity‐associated protein expression and morphology by ERs and the role of mTORC2. The activation of E2 was inhibited by its receptor antagonists. Coadministration of nER antagonists (MPP/PHTPP) or the mER antagonist (G15) decreased SRC‐1 expression, mTORC2 signaling (rictor and p‐AKTSer473), actin cytoskeleton remodeling (p‐cofilin/cofilin and profilin‐1), and postsynaptic protein expression (GluR1, PSD95, and spinophilin) and finally affected the dendritic spine and synapse density. The impaired actin polymerization and decreased postsynaptic protein expression, spine density, and synapse density were significantly reversed by activation of mTORC2 with A‐443654. Spino: spinophilin

DISCLOSURE

The authors declare that they have no conflict of interests.

ACKNOWLEDGMENTS

This work was supported by the National Science Foundation of China (NSFC, No. 81571059) and the grant from the Development and Regeneration Key Laboratory of Sichuan Province (SYS15001).

Xing F‐Z, Zhao Y‐G, Zhang Y‐Y, et al. Nuclear and membrane estrogen receptor antagonists induce similar mTORC2 activation‐reversible changes in synaptic protein expression and actin polymerization in the mouse hippocampus. CNS Neurosci Ther. 2018;24:495–507. 10.1111/cns.12806

Contributor Information

Yan Liu, Email: liuyan@swu.edu.cn.

Ji‐Qiang Zhang, Email: zhangjqtmmu@yahoo.com.

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