STX,a Novel Membrane Estrogen Receptor Ligand,Protects Against Amyloid-β Toxicity

Abstract

Because STX is a selective ligand for membrane estrogen receptors, it may be able to confer the beneficial effects of estrogen without eliciting the deleterious side effects associated with activation of the nuclear estrogen receptors. This study evaluates the neuroprotective properties of STX in the context of amyloid-β (Aβ) exposure. MC65 and SH-SY5Y neuroblastoma cell lines, as well as primary hippocampal neurons from wild type (WT) and Tg2576 mice, were used to investigate the ability of STX to attenuate cell death, mitochondrial dysfunction, dendritic simplification, and synaptic loss induced by Aβ. STX prevented Aβ-induced cell death in both neuroblastoma cell lines; it also normalized the decrease in ATP and mitochondrial gene expression caused by Aβ in these cells. Notably, STX also increased ATP content and mitochondrial gene expression in control neuroblastoma cells (in the absence of Aβ). Likewise in primary neurons, STX increased ATP levels and mitochondrial gene expression in both genotypes. In addition, STX treatment enhanced dendritic arborization and spine densities in WT neurons and prevented the diminished outgrowth of dendrites caused by Aβ exposure in Tg2576 neurons. These data suggest that STX can act as an effective neuroprotective agent in the context of Aβ toxicity, improving mitochondrial function as well as dendritic growth and synaptic differentiation. In addition, since STX also improved these endpoints in the absence of Aβ, this compound may have broader therapeutic value beyond Alzheimer’s disease.

Keywords

Amyloid-β toxicity mitochondrial dysfunction neuroprotection selective estrogen receptor modulator

Introduction

The neuroprotective and cognitive-enhancing effects of natural and synthetic estrogens have been widely demonstrated in humans [1 –4], as well as in numerous experimental model systems [5 –10]. However, the increased risk of thrombosis and hormone-sensitive cancers associated with treatments using the natural estrogen 17β-estradiol (E2) in post-menopausal women has limited the development of estrogen-related compounds for treating neurodegenerative diseases. The recent identification of new selective estrogen receptor modulators (SERMs) may offer a solution to this problem. The diphenylacrylamide compound STX is one example of a SERM that exhibits the neuroprotective effects of estrogen without inducing oncogenic and thrombotic side effects [11, 12]. Unlike E2 and its functional analogs, STX does not bind nuclear estrogen receptors (ER-α and ER-β), which regulate transcriptional responses to E2. Instead, STX binds the G protein-coupled estrogen receptor GqMER, which is located in the plasma membrane [9]. This uncoupling of nuclear and membrane ER activation suggests that STX could provide a novel therapeutic option for treating patients afflicted with neurodegenerative disease and age-related dementias.

Alzheimer’s disease (AD) is the most common form of dementia worldwide [13], and the risk of AD is associated with age-related loss of sex steroid hormones in both men and women [14, 15]. In AD patients, the accumulation of extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles are accompanied by neuronal loss, synaptic dysfunction, and severe cognitive impairment [16 –19]. Mitochondrial dysfunction is thought to contribute to these deleterious physiological changes [20]: Deficits in proteins associated with the electron transport chain (ETC) [21], decreased ATP production, and general impairment of mitochondrial activity have all been observed in the brains of AD patients, as well as in many in vitro and in vivo models of amyloid pathology [22 –27].

Several estrogen-dependent signaling pathways converge on mitochondria to modulate cellular respiration and ATP production [28 –30]. E2 has been shown to enhance brain mitochondrial function [31], increase mitochondrial respiratory capacity [32], and induce the expression of mitochondrial proteins [29, 32]. E2 has likewise been shown to protect against Aβ-induced cell death in vitro [33 –35] and in vivo [36, 37], and to attenuate bioenergetic deficits induced by Aβ in both neuroblastoma cells [38] and mouse models of AD [39, 40].

Despite the growing literature describing the beneficial action of estradiol on brain mitochondrial function in the context of AD, comparatively little is known about the ability of SERMs (acting via extranuclear ERs) to recapitulate these neuroprotective effects. We have now addressed this issue by examining the neuroprotective effects of STX on cell viability, mitochondrial function, and neuronal morphology in several in vitro models of Aβ toxicity.

Methods

STX preparation

STX was produced by AAPharmaSyn, LLC (Ann Arbor, MI) under contract with the authors of the synthetic protocol for STX, published in Tobias et al. [41]. Stock solutions of STX (2 mM) were prepared in 100% anhydrous dimethyl sulfoxide (DMSO), which was then diluted to working concentrations in culture medium, as described below.

MC65 cell culture

MC65 cells were cultured in MEMα supplemented with 10% fetal bovine serum (FBS; GIBCO/Life Technologies), 2 mM L-glutamine (Sigma-Aldrich), and 0.1% tetracycline (Sigma-Aldrich). For each experiment, cells were trypsinized and resuspended in Opti-MEM without phenol red (GIBCO/Life Technologies), then treated with STX or matched DMSO concentrations in the presence and absence of tetracycline. All endpoints were compared to those obtained with tetracycline-treated cells, either with or without the addition of STX. For assays of viability, cells were plated at 10,000 cells/well in 96 well plates and assessed after 72 h of continuous treatment. For assays of gene expression and ATP determination, cells were plated at 60,000 cells/well in 12 well plates and were harvested after 48 h of continuous treatment.

SH-SY5Y cell culture

SH-SY5Y neuroblastoma cells were cultured in DMEM/F12 medium (GIBCO/Life Technologies) supplemented with 10% FBS and 1% penicillin-streptomycin (Sigma-Aldrich). For assays of gene expression and ATP production, cells were plated at 200,000 cells/well in 12-well plates. For assays of cell viability, cells were plated at 15,000 cells/well in 96 well plates. Three days after plating, cells were washed with phosphate-buffered saline (PBS) and switched to serum-free DMEM/F12 medium containing 1% N-2 growth supplement (GIBCO/Life Technologies) plus STX (100 nM). The following day, the cells were treated with 50μM Aβ_25 - 35 (American Peptide Company), a fragment that has been shown to recapitulate the toxic effects of full-length Aβ in vitro [42, 43]. Aβ_25 - 35 solutions were incubated at 37°C for 72 h, prior to addition to the cell cultures. All endpoints were assessed after 48 h of treatment.

Culture of primary hippocampal neurons

Embryonic Tg2576 mice and their wild type (WT) littermates were used to generate primary neuronal cultures. The Tg2576 line expresses the human AβPPswe double mutation (K670N-M671 L) under the control of the hamster prion promoter [44, 45], resulting in an accumulation of Aβ_1 - 42 in the brain and the development of age-dependent Aβ plaques. Previous studies have shown that after several weeks in culture, primary neurons isolated from these animals display metabolic alterations [46] and a dystrophic phenotype that includes impaired dendritic arborization and loss of synaptic spines [19], similar to what is observed in the brains of adult Tg2576 animals [22, 47].

Breeding pairs of Tg2576 mice were raised in an in-house facility at the OHSU. Mice were maintained in a climate-controlled environment with a 12-h light/12-h dark cycle and fed Pico Lab Rodent Diet 5LOD (LabDiets, St. Louis, MO). Diet and water were supplied ad libitum. Litters were weaned and group-housed (4–6/cage) until commencement of breeding. All procedures were conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the institutional Animal Care and Use Committee of OHSU.

Hippocampal neurons were isolated from embryonic mice, based on the methods of Kaech and Banker [48]. Briefly, embryos were harvested at 18 days of gestation from anesthetized females. Hippocampi were dissected, gently minced, and trypsinized to generate suspensions of dispersed neurons, which were then plated in MEM medium (GIBCO /Life Technologies), 5% FBS (Atlanta Biologicals), and 0.6% glucose (Sigma-Aldrich). After 4 h, the medium was removed and replaced with Neurobasal Medium supplemented with 1x GlutaMAX (GIBCO/Life Technologies) and 1x GS21 (MTI-GlobalStem). The animals were genotyped by PCR using DNA extracted from tail samples taken after dissection of the hippocampi. For analyses of gene expression and ATP levels, dissociated hippocampal cells were plated at 200,000 per well in 12-well plates that had been pre-coated with 1 mg/mL poly-L-lysine. After 7 days in vitro (DIV), cells were treated with STX (100 nM) and harvested 48 h later for analysis.

For Sholl analyses of dendritic complexity, we used two different protocols that yielded equivalent results. In the first protocol, 150,000 hippocampal neurons were plated in 60 mm dishes in 1x MEM with 5% FBS, each dish containing four poly-L-lysine-coated glass coverslips with paraffin wax spacers [48]. After 4 h, the coverslips were flipped into 60 mm dishes containing neural stem cell-derived glial cells (provided by Dr. Gary Banker, Jungers Center, OHSU) and maintained in 6 ml Neurobasal media with GlutaMAX and GS21 [48]. Each dish was fed every week by removing 1 ml of the culture medium and adding 1 ml fresh Neurobasal media containing GlutaMAX plus GS21, with the first feed (at 5 DIV) containing 6μM cytosine β-D-arabinofuranoside hydrochloride (AraC; Sigma-Aldrich). The first and second feed (5 DIV and 12 DIV, respectively) also contained STX (100 nM) or DMSO. At 19 DIV, each coverslip was fixed in 4% PFA in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, pH 7.4). Coverslips were stained with Anti-MAP2B (Sigma-Aldrich #M4403; 3.3μg/ml) and Goat anti-mouse IgG1-Cy3 (Jackson ImmunoResearch #115-165-205; 1.5μg/ml). Immunostained neurons were imaged with a Zeiss ApoTome2 microscope. In the second protocol, freshly isolated hippocampal neurons were electroporated with plasmids encoding enhanced Green Fluorescent Protein (eGFP) under the control of the CMV immediate-early enhancer and the chicken β-actin promoter [49]. 300,000 electroporated hippocampal neurons were plated with 150,000 non-electroporated neurons of the same genotype onto poly-L-lysine-coated coverslips, then cultured and processed as described above. For both protocols, blinded Sholl analyses were performed using the Fiji platform [50] with the plug-in created by Ferreira et al. [51]. At least thirty cells were analyzed per treatment condition. Statistical differences between treatment groups were calculated using Student’s unpaired t-tests.

For an analysis of dendritic spines, 150,000 hippocampal neurons were electroporated with plasmids encoding eGFP (as described above) and plated onto dishes with coverslips containing 300,000 cortical neurons of the same genotype (plated 7 days prior to the addition of the hippocampal neurons). This strategy promoted robust synapse formation while maintaining the electroporated hippocampal neurons at a density that permitted the unambiguous visualization of non-intersecting dendritic segments. After 4 h, coverslips were flipped into 60 mm dishes containing stem cell-derived glial cells in 6 ml Neurobasal media with GlutaMAX and GS21. Each dish was fed every week with 1 ml Neurobasal media plus GlutaMAX and GS21, with the first feed (at 7 DIV) containing AraC and the second feed (at 14 DIV) containing STX (100 nM) or DMSO. Coverslips were then fixed in 4% PFA in PHEM buffer at 21 DIV and immunostained with anti-GFP (Life Technologies #A11122; 2μg/ml), detected with Alexa-488-conjugated goat anti-Rabbit secondary antibodies (Life Technologies #A11034; 2μg/ml). The immunostained neurons were then imaged using a Zeiss ApoTome2 microscope and analyzed blind by a separate investigator not involved in the image collection. 16–20 images were collected from different neurons in each treatment group, and spines were quantified on 50–100μm segments of dendrite length per image using FIJI software.

Quantitative real time PCR

Total RNA from neuroblastoma cells and cultured hippocampal neurons was extracted using Tri-Reagent (Molecular Research Center). RNA was reverse transcribed with the Superscript III First Strand Synthesis kit (Invitrogen) to generate cDNA, as per the manufacturer’s instructions. Relative gene expression was determined using TaqMan Gene Expression Master Mix and commercially available TaqMan primers (Life Technologies) for mitochondrially encoded NADH dehydrogenase 1 (Mt-ND1), mitochondrially encoded ATP synthase 6 (Mt-ATP6), mitochondrially encoded cytochrome c oxidase 1 (Mt-CO1), mitochondrially encoded cytochrome B (Mt-CYB), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Quantitative polymerase chain reactions (qPCR) was performed on a StepOne Plus Machine (Applied Biosystems) and analyzed using the delta-delta Ct method.

Cell number determination

Cell number was determined both by DNA quantification as well as MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) assays. For DNA quantification, media was removed from the wells and the plates were frozen at –80°C for 24 h, prior to subsequent analysis. Cell numbers were quantified using the CyQUANT Cell Proliferation Assay kit (Thermo Fisher), as per the manufacturer’s instructions. The MTS assay was performed using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega), as per the manufacturer’s instructions.

ATP and protein quantification

ATP was quantified using the ATP determination kit (Life Technologies), as per the manufacturer’s instructions. Cells were lysed with 0.1% Triton X 100 in PBS and incubated with the reaction solution for 15 min at room temperature, prior to measurement. Values were normalized to total protein content, as determined by the BCA method (Thermo Fisher). Briefly, this colorimetric assay combines the reduction and chelation of cuprous copper (Cu¹⁺) to peptides under alkaline conditions in the presence of sodium potassium tartrate. Reduced Cu¹⁺ is then reacted with bicinchoninic acid (BCA) to produce a concentration-dependent reaction product detectable in a spectrophotometer at 562 nm. Protein concentrations of cell lysates were analyzed in triplicate by comparison to a standard curve generated with serial dilutions of BSA (bovine serum albumin; 0.03–2 mg/ml) in a microplate reader, as per the manufacturer’s instructions. Protein concentrations were then calculated in Excel.

Statistics

Statistical significance was determined using Bonferroni post-hoc tests and one-way analysis of variance with appropriate t-tests. Significance was defined as p≤0.05. Analyses were performed using Excel or GraphPad Prism 6.

Results

STX protects against Aβ-induced cytotoxicity in neuroblastoma models

In previous work, we used two different neuroblastoma models of amyloid toxicity to investigate the protective effects of candidate therapeutic compounds. MC65 neuroblastoma cells conditionally express AβPP-C99 under the control of a tetracycline-responsive promoter, which in turn is cleaved by endogenous secretases to generate Aβ peptides (predominantly Aβ_1 - 40/₄₂) [52 –54]. Following tetracycline withdrawal (Tet^–), Aβ levels progressively accumulate in these cultures, resulting in extensive cell death by 72 h [52]. Alternatively, SH-SY5Y neuroblastoma cells have been used in many studies to test the effects of exogenously introduced Aβ (including Aβ_25 - 35 and Aβ_1 - 42), both of which have been shown to induce progressive cell death in a concentration-dependent manner [43 , 55–57]. Accordingly, we used these two cell culture models to investigate the protective effects of STX. As shown in Fig. 1A, STX treatment protected MC65 cells from Aβ-induced cell death in a dose-dependent manner (between 1–100 nM). When analyzed with CyQUANT assays of cell proliferation, these dosages had no detectable effect in the absence of Aβ (Tet⁺ condition). The highest dose, 1000 nM, was toxic in both the Tet⁺ and Tet^– conditions. By comparison, analyzing replicate sets of MC65 cells using the MTS assay (a measure of cellular metabolic activity) revealed that treatment with 100 nM STX significantly improved the viability of both control cultures (STX+Tet⁺) and cultures exposed to toxic Aβ levels (STX+Tet^–). Accordingly, we used 100 nM STX for our subsequent experiments.

To test whether STX also confers beneficial effects in cells exposed to exogenous amyloid peptides, we pre-treated SH-SY5Y cells with 100 nM STX for 24 h before the addition of 50μM Aβ_25 - 35, based on previous evidence that treatment with Aβ_25 - 35 recapitulates the toxic effects of full-length Aβ [42 , 59]. As shown in Fig. 1C, Aβ_25 - 35 treatment resulted in a significant reduction in viability (based on CyQUANT assays of proliferation), whereas pre-treatment with STX prevented this effect. In control cells (not treated with Aβ), STX treatment alone did not alter cell viability. As in our MC65 cell assays, we found that STX pre-treatment significantly improved the metabolic activity of control SH-SY5Y cultures (as measured with MTS assays), as well as partially rescuing the viability of cells treated with Aβ_25 - 35 (Fig. 1D). In combination, these results indicate that STX can protect cells against the deleterious effects of multiple toxic forms of Aβ, whether they are generated endogenously or introduced exogenously.

STX increases ATP production and induces mitochondrial gene expression in neuroblastoma cells

We also used our neuroblastoma cell assays to examine whether STX could protect against the reduction in ATP levels caused by Aβ exposure, as an assay of mitochondrial function. As shown in Fig. 2A, ATP levels were significantly reduced in MC65 cells by 48 h after tetracycline removal (Tet^–), coincident with the accumulation of Aβ in these cultures (similar to our previous results; [60]). In contrast, STX treatment attenuated this decrease (Fig. 2A), while STX treatment also significantly increased ATP production in MC65 cells that were maintained in the presence of tetracycline (and therefore not expressing Aβ). Similarly, treating SH-SY5Y cells with exogenous Aβ caused a decrease in ATP levels that was prevented by STX pre-treatment (Fig. 2B), while treating control SH-SY5Y cells with STX increased intracellular ATP levels (in the absence of Aβ).

We also evaluated whether STX prevented the down-regulation of mitochondrial DNA-derived genes by Aβ; specifically, we examined the expression of Mt-ND1, Mt-CYB, Mt-CO1, and Mt-ATP, which encode proteins in complexes I, III, IV, and V of the ETC, respectively. All four ETC genes were repressed in MC65 cells expressing Aβ (under Tet^– conditions), whereas STX treatment attenuated this effect (Fig. 2C). Consistent with our analysis of ATP levels, control MC65 treated with STX (under Tet⁺ conditions) also coordinately increased the expression of all the mitochondrial genes, independent of Aβ expression. Likewise in SH-SY5Y cells, Aβ treatment significantly repressed the expression of the mitochondrial genes, while STX treatment prevented this decrease (Fig. 2D), restoring their expression to control levels. Once again, treating control SH-SY5Y cells with STX (in the absence of Aβ) also enhanced the basal expression of all four ETC genes to a similar degree. In combination, these results provide additional evidence that STX bolsters mitochondrial function while protecting against the deleterious effects of Aβ exposure, suggesting that this compound might also have beneficial effects in the context of other disease-associated insults that affect cellular metabolism and viability.

STX increases ATP production and mitochondrial gene expression in primary hippocampal neurons

Because the metabolism of neuroblastoma cells differs substantially from that of neurons [61], we also evaluated the effects of STX on the mitochondrial parameters of primary neurons, to confirm that similar mechanistic pathways could be regulated by this compound in a more physiologically relevant cellular context. As shown in Fig. 2E, STX treatment significantly increased ATP content in cultured hippocampal neurons isolated either from WT littermate control mice or from Tg2576 mice, which overexpress the human AβPPswe double mutation and accumulate Aβ_1 - 42 [44, 45]. Of note is that there was no difference in ATP content between the control-treated WT and Tg2576 neurons in this experiment (analyzed using neurons at 9 DIV). STX treatment also significantly increased the expression of ETC genes in hippocampal neurons from both WT and Tg2576 animals to a similar degree (Fig. 2F). As observed with our analysis of ATP levels, no differences between the two genotypes were detected in the basal expression of any these genes.

STX attenuates Aβ-induced impairments in dendritic morphology in hippocampal neurons

As previously reported, cultured neurons isolated from the brains of Tg2576 mice progressively develop neurodegenerative phenotypes (compared to WT control neurons), including substantially reduced dendritic complexity and lower spine densities caused by their chronic production of Aβ_1 - 42 [19, 62]. Consistent with these reports, we observed that Tg2576 hippocampal neurons exhibited substantially reduced patterns of dendritic arborization after 19 days in culture, compared to neurons from WT littermate controls (Fig. 3A). Sholl analyses revealed that STX treatment restored the dendritic complexity of Tg576 neurons to WT levels (Figs. 3B, C). We obtained equivalent results when we used cultures containing non-electroporated neurons or cultures containing neurons expressing eGFP. These results are consistent with previous evidence that inducing the expression of eGFP in cultured hippocampal neurons does not significantly alter their overall development or survival rates [48]. Using this protocol, we also observed a similar protective effect of STX on the elaboration of dendritic spines in neurons expressing eGFP. As shown in Fig. 4A and 4B, spine densities in Tg2576 neurons were significantly reduced compared to WT neurons after 3 weeks in culture, recapitulating previous findings by other investigators [19]. In contrast, treatment with STX treatment rescued this deficit (Fig. 4B), restoring Tg2576 spine densities to control levels. In addition, STX also resulted in a significant increase in the spine densities of WT neurons, compared to vehicle-control treated neurons. These results demonstrate that STX treatments prevented the deleterious effects of Aβ on both neuronal metabolism and dendritic morphology.

Discussion

SERMs are promising therapeutic compounds because of their ability to provide the neuroprotective effects of natural estrogen without its negative side effects. STX is one such compound that has been shown to confer beneficial neurological effects by binding selectively to a G protein-coupled membrane estrogen receptor (GqMER), rather than the classical nuclear estrogen receptors [9, 63]. Notably, STX does not induce the deleterious oncogenic and thrombotic effects seen with E2 treatments, indicating that it might provide an improved strategy for hormone replacement therapy [11, 12]. In this study, we report that STX is also an effective neuroprotective compound, mitigating the toxic effects of several different forms of Aβ in a variety of different assays.

STX treatment protected against Aβ-induced cell death and mitochondrial dysfunction in neuroblastoma cells, and it attenuated Aβ-induced dendritic simplification and spine loss in primary hippocampal neurons. In both our MC65 and SH-SY5Y models of Aβ toxicity, STX treatment significantly reduced Aβ-induced cell death. It is notable that we obtained similar results in the MC65 cells exposed to full-length Aβ peptides (Aβ_1 - 40/42) and SH-SY5Y cells exposed to the shorter peptides (Aβ_25 - 35) that have been shown to recapitulate the cytotoxic effects of Aβ oligomers [42 , 59]. Likewise, the coherence of our observations in both neuroblastoma lines and cultured neurons suggests that the protective effects of STX are not limited to a particular set of in vitro conditions [64]. In addition, our results are consistent with previous reports that STX is neuroprotective in whole-animal models of ischemic stroke [9, 63], supporting the hypothesis that this compound might be beneficial in the context of a variety of neurodegenerative conditions. However the molecular mechanisms underlying this protective effect have yet to be determined. Both STX and E2 can rapidly activate multiple signaling pathways independent of altered gene expression, demonstrating the involvement of non-nuclear estrogen receptors [65, 66]. In rats, STX-dependent stimulation of GqMER results in the activation of both the ERK/MAPK [67] and PLC/PKCδ/PKA signaling pathways [67, 68]. Alternatively, experiments using neuroblastoma cells have shown that activation of GqMER can also initiate PI3K signaling [69]. Each of these pathways has been shown to contribute to neuroprotective responses in a variety of model systems [70 –72] and have been linked with E2-dependent neuronal growth responses, including dendritic branching and spine elaboration [73 –77]. These observations suggest that one or more of these signaling pathways might also mediate the protective effects of STX in our neuroblastoma and neuronal culture assays (a topic for future investigations).

Reduced dendritic arborization, diminished spine numbers, synaptic loss, and neuronal death have been widely reported in AD patients and in animal models of this disease [17 , 78–82]. Loss of dendritic complexity and reduced spine densities are also prominent features of AD and correlate significantly with cognitive decline [16]. These same morphological changes occur in neurons isolated from Tg2576 mice that overexpress human AβPPswe, a mutant form of AβPP that causes the accumulation of Aβ_1 - 42 and early-onset AD in human patients [19]. In this study, we found that STX treatment reversed the deleterious effects of Aβ on dendritic arborization and spine density in hippocampal neurons isolated from Tg2576 mice. Notably, STX also improved spine densities in cultures of WT neurons. These results are consistent with the well-documented beneficial effects of E2 treatment on dendritic arborization and spine density [73 , 83–85], supporting other evidence that STX can confer the neuroprotective responses of natural estrogen in a variety of contexts [9, 63]. Whether these effects of STX on neuronal morphology involve the same mechanisms underlying its cytoprotective actions remain to be defined.

We also found that STX improved mitochondrial function in two different neuroblastoma models of Aβ toxicity. STX treatment attenuated both the decrease in ATP content and reduced ETC gene expression caused by Aβ exposure, while increasing these parameters in control cultures (not treated with Aβ). In contrast, we found that STX did not significantly reduce the more general effects of H₂O₂ on viability (unpublished data), supporting the hypothesis that STX mediates its protective actions on cellular metabolism via one or more specific signaling pathways (as summarized above). We were able to recapitulate many of the same effects on mitochondrial endpoints in primary neurons: STX increased ATP content and ETC gene expression in both WT and Tg2576 neurons. In contrast to our analyses of neuronal morphology, however, we detected no significant differences in baseline ATP levels or mitochondrial function between the genotypes. This result is likely due to the fact that the neurons used these assays were only grown for 9 days in culture before mitochondrial gene expression and ATP levels were measured, whereas other investigators reported that phenotypic changes between the genotypes were only detectable after 19 DIV [19]. Previous studies have also shown that E2 can increase the expression of ETC genes in cultured spinal cord neurons [86] and can modulate mitochondrial dynamics and bioenergetics in the brain [87]. Likewise, estrogen treatments can reverse deficits in ATP production caused by either Aβ or tau accumulation in SH-SY5Y cells [38]. To our knowledge, however, this is the first report that the protective effects of STX include enhanced mitochondrial activity, providing additional evidence that this compound provides the beneficial actions of natural estrogens without their adverse side effects.

Despite the fact that many of the mitochondrial effects of estrogens appear to be modulated by non-nuclear ERs [88, 89], research to date has focused on hormone analogs and other compounds that act primarily via the classical nuclear ERs. Future experiments will be needed to investigate the specific role of membrane estrogen receptors (including GqMER) in transducing the protective effects of STX on mitochondrial function. Although the mechanisms underlying these effects have not been fully elucidated, manipulations that target PKA signaling have been reported to increase ETC activity in rat hippocampal neurons and to reverse the bioenergetic impairment caused by Aβ treatment [90, 91], while ERK and PI3K activation have been shown to restore ATP production in a neuronal model of mitochondrial injury [92]. As noted above, these same signaling pathways have been implicated in regulating STX-responses in other contexts [67 –69]. In light of recent evidence that mitochondrial abnormalities accompany impairments in dendritic arborization and spine density [16], our studies will provide a framework for investigating whether the protective effects of STX on neuronal morphology are functionally linked to improved mitochondrial activity.

The experiments described in this study demonstrate the neuroprotective action of STX in several models of Aβ toxicity and support the potential value of this compound as a novel therapeutic agent for treating AD. Interestingly, many of the beneficial effects of STX were also observed in control cultures and thus appear to be independent of Aβ exposure. These observations suggest that STX might be suitable for treating a variety of conditions that have been linked with mitochondrial dysfunction, including other neurodegenerative diseases and age-related deficits in cognitivefunction.

Footnotes

ACKNOWLEDGMENTS

This work was funded by a Department of Veterans Affairs Merit Review grant awarded to J. Quinn, and by NIH grants NS078363 and AG025525 awarded to P. Copenhaver. The authors thank Dr. Martin Kelly for providing his expertise and the STX used in this study, and Dr. Gary Banker and Ms. Barbara Smoody for their support of our experiments with primary hippocampal neurons. We also wish to thank Ms. Jenna Fisk for her assistance with image acquisition in some of our studies. Dr. Doris Kretzschmar provided critical input on this manuscript. We gratefully acknowledge Dr. Stefanie Kaech and Ms. Aurelie Snyder for their invaluable assistance with the confocal imaging analysis that was performed in the Advanced Light Microscopy Core, Jungers Center, OHSU. Confocal imaging was supported in part by NIH P30 NS061800, and by the OHSU School of Medicine Faculty Innovation Fund (to P. Copenhaver). MC65 cells were generously provided by Randy Woltjer, MD, PhD.

Authors’ disclosures available online ().

References

Henderson

, St John

, Hodis

, McCleary

, Stanczyk

, Karim

, Shoupe

, Kono

, Dustin

, Allayee

, Mack

(2013) Cognition, mood, and physiological concentrations of sex hormones in the early and late postmenopause. Proc Natl Acad Sci U S A 110, 20290–20295.

Gorenstein

, Rennó

Jr , Vieira Filho

, Gianfaldoni

, Gonçcalves

, Halbe

, Fernandes

, Demétrio

(2011) Estrogen replacement therapy and cognitive functions in healthy postmenopausal women: A randomized trial. Arch Womens Ment Health 14, 367–373.

Ryan

, Scali

, Carriere

, Ritchie

, Ancelin

(2008) Hormonal treatment, mild cognitive impairment and Alzheimer’s disease. Int Psychogeriatr 20, 47–56.

Acosta

, Mayer

, Talboom

, Zay

, Scheldrup

, Castillo

, Demers

, Enders

, Bimonte-Nelson

(2009) Premarin improves memory, prevents scopolamine-induced amnesia and increases number of basal forebrain choline acetyltransferase positive cells in middle-aged surgically menopausal rats. Horm Behav 55, 454–464.

Brown

, Suzuki

, Jelks

, Wise

(2009) Estradiol is a potent protective, restorative, and trophic factor after brain injury. Semin Reprod Med 27, 240–249.

DonCarlos

, Azcoitia

, Garcia-Segura

(2009) Neuroprotective actions of selective estrogen receptor modulators. Psychoneuroendocrinology 34, S113–S122.

Gröger

, Plesnila

(2014) The neuroprotective effect of 17β-estradiol is independent of its antioxidative properties. Brain Res 1589C, 61–67.

Han

, Scott

, Dong

, Raz

, Wang

, Zhang

(2015) Attenuation of mitochondrial and nuclear p38α signaling: A novel mechanism of estrogen neuroprotection in cerebral ischemia. Mol Cell Endocrinol 400, 21–31.

Lebesgue

, Traub

, De Butte-Smith

, Chen

, Zukin

, Kelly

, Etgen

(2010) Acute administration of non-classical estrogen receptor agonists attenuates ischemia-induced hippocampal neuron loss in middle-aged female rats.. PLoS One 5, e8642

10.

Wang

, Song

, Gao

, Zhou

(2015) Estradiol prevents Aβ 25-35-inhibited long-term potentiation induction through enhancing survival of newborn neurons in the dentate gyrus. Int J Neurosci 22, 1–9.

11.

Qiu

JRO

, Kelly

(2008) Modulation of hypothalamic neuronal activity through a novel G-protein-coupled estrogen membrane receptor. Steroids 783, 985–991.

12.

Roepke

, Bosch

, Rick

, Lee

, Wagner

, Seidlova-Wuttke

, Wuttke

, Scanlan

, Ronnekleiv

, Kelly

(2010) Contribution of a membrane estrogen receptor to the estrogenic regulation of body temperature and energy homeostasis. Endocrinology 151, 4926–4937.

13.

Brookmeyer

, Johnson

, Ziegler-Graham

, Arrighi

(2007) Forecasting the global burden of Alzheimer’s disease. Alzheimers Dement 3, 186–191.

14.

Barron

, Pike

(2012) Sex hormones, aging, and Alzheimer’s disease. Front Biosci (Elite Ed) 4, 976–997.

15.

Pike

, Carroll

, Rosario

, Barron

(2009) Protective actions of sex steroid hormones in Alzheimer’s disease. Front Neuroendocrinol 30, 239–258.

16.

Baloyannis

(2009) Dendritic pathology in Alzheimer’s disease. J Neurol Sci 283, 153–157.

17.

Dumanis

, Tesoriero

, Babus

, Nguyen

, Trotter

, Ladu

, Weeber

, Turner

, Xu

, Rebeck

, Hoe

(2009) ApoE4 decreases spine density and dendritic complexity in cortical neurons in vivo. J Neurosci 29, 15317–15322.

18.

Mavroudis

, Fotiou

, Manani

, Njaou

, Frangou

, Costa

, Baloyannis

(2011) Dendritic pathology and spinal loss in the visual cortex in Alzheimer’s disease: A Golgi study in pathology. Int J Neurosci 121, 347–354.

19.

, Hudry

, Hashimoto

, Kuchibhotla

, Rozkalne

, Fan

, Spires-Jones

, Xie

, Arbel-Ornath

, Grosskreutz

, Bacskai

, Hyman

(2010) Amyloid beta induces the morphological neurodegenerative triad of spine loss, dendritic simplification, and neuritic dystrophies through calcineurin activation. J Neurosci 30, 2636–2649.

20.

Leuner

, Müller

, Reichert

(2012) From mitochondrial dysfunction to amyloid beta formation: Novel insights into the pathogenesis of Alzheimer’s disease. Mol Neurobiol 46, 186–193.

21.

Manczak

, Park

, Jung

, Reddy

(2004) Differential expression of oxidative phosphorylation genes in patients with Alzheimer’s disease: Implications for early mitochondrial dysfunction and oxidative damage. Neuromolecular Med 5, 147–162.

22.

Cuadrado-Tejedor

, Cabodevilla

, Zamarbide

, Gómez-Isla

, Franco

, Perez-Mediavilla

(2013) Age-related mitochondrial alterations without neuronal loss in the hippocampus of a transgenic model of Alzheimer’s disease. Curr Alzheimer Res 10, 390–405.

23.

, Guo

, Yan

, Sosunov

, McKhann

, Yan

(2010) Early deficits in synaptic mitochondria in an Alzheimer’s disease mouse model. Proc Natl Acad Sci U S A 107, 18670–18675.

24.

Manczak

, Calkins

, Reddy

(2011) Impaired mitochondrial dynamics and abnormal interaction of amyloid beta with mitochondrial protein Drp1 in neurons from patients with Alzheimer’s disease: Implications for neuronal damage. Hum Mol Genet 20, 2495–2509.

25.

McManus

, Murphy

, Franklin

(2011) The mitochondria-targeted antioxidant MitoQ prevents loss of spatial memory retention and early neuropathology in a transgenic mouse model of Alzheimer’s disease. J Neurosci 31, 15703–15715.

26.

Rhein

, Baysang

, Rao

, Meier

, Bonert

, Müller-Spahn

, Eckert

(2009) Amyloid-beta leads to impaired cellular respiration, energy production and mitochondrial electron chain complex activities in human neuroblastoma cells. Cell Mol Neurobiol 29, 1063–1071.

27.

Saraiva

, Borges

, Madeira

, Tavares

, Paula-Barbosa

(1985) Mitochondrial abnormalities in cortical dendrites from patients with Alzheimer’s disease. J Submicrosc Cytol 17, 459–464.

28.

Nilsen

, Diaz Brinton

(2006) Mechanism of estrogen-mediated neuroprotection: Regulation of mitochondrial calcium and Bcl-2 expression. Proc Natl Acad Sci U S A 100, 2842–2847.

29.

Nilsen

, Irwin

, Gallaher

, Brinton

(2007) Estradiol in vivo regulation of brain mitochondrial proteome. J Neurosci 27, 14069–14077.

30.

Olsen

, Johnson

, Zuloaga

, Limoli

, Raber

(2013) Enhanced hippocampus-dependent memory and reduced anxiety in mice over-expressing human catalase in mitochondria. J Neurochem 25, 303–313.

31.

Brinton

(2008) The healthy cell bias of estrogen action: Mitochondrial bioenergetics and neurological implications. Trends Neurosci 31, 529–537.

32.

Irwin

, Yao

, To

, Hamilton

, Cadenas

, Brinton

(2012) Selective oestrogen receptor modulators differentially potentiate brain mitochondrial function. J Neuroendocrinol 24, 236–248.

33.

Hosoda

, Nakajima

, Honjo

(2001) Estrogen protects neuronal cells from amyloid beta-induced apoptotic cell death. Neuroreport 12, 1965–1970.

34.

Napolitano

, Costa

, Piacentini

, Grassi

, Lanzone

, Gulino

(2014) 17β-estradiol protects cerebellar granule cells against β-amyloid-induced toxicity via the apoptoticmitochondrial pathway. Neurosci Lett 21, 134–139.

35.

Yao

, Nguyen

, Pike

(2007) Estrogen regulates Bcl-w and Bim expression: Role in protection against beta-amyloid peptide-induced neuronal death. J Neurosci 27, 1422–1433.

36.

Szego

, Csorba

, Janáky

, Kékesi

, Abrahám

, Mórotz

, Penke

, Palkovits

, Murvai

, Kellermayer

, Kardos

, Juhász

(2011) Effects of estrogen on beta-amyloid-induced cholinergic cell death in the nucleus basalis magnocellularis. Neuroendocrinology 93, 90–105.

37.

Yenki

, Khodagholi

, Shaerzadeh

(2013) Inhibition of phosphorylation of JNK suppresses Aβ-induced ER stress and upregulates prosurvivalmitochondrial proteins in rat hippocampus. J Mol Neurosci 49, 262–269.

38.

Grimm

, Biliouris

, Lang

, Götz

, Mensah-Nyagan

, Eckert

(2016) Sex hormone-related neurosteroids differentially rescue bioenergetic deficits induced by amyloid-β or hyperphosphorylated tau protein. Cell Mol Life Sci 73, 201–215.

39.

Yao

, Irwin

, Zhao

, Nilsen

, Hamilton

, Brinton

(2009) Mitochondrial bioenergetic deficit precedes Alzheimer’s pathology in female mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A 106, 14670–14675.

40.

Yao

, Irwin

, Chen

, Hamilton

, Cadenas

, Brinton

(2012) Ovarian hormone loss induces bioenergetic deficits and mitochondrial β-amyloid. Neurobiol Aging 33, 1507–1521.

41.

Tobias

, Qiu

, Kelly

, Scanlan

(2006) Synthesis and biological evaluation of SERMs with potent nongenomic estrogenic activity. ChemMedChem 1, 565–571.

42.

Yankner

, Duffy

, Kirschner

(1990) Neurotrophic and neurotoxic effects of amyloid beta protein: Reversal by tachykinin neuropeptides. Science 250, 279–282.

43.

Lee

, Park

, Lee

, Jang

(2013) Attenuation of beta-amyloid-induced oxidative cell death by sulforaphane via activation of NF-E2-related factor 2. Oxid Med Cell Longev 2013, 313510.

44.

Hsiao

, Chapman

, Nilsen

, Eckman

, Harigaya

, Younkin

, Yang

, Cole

(1996) Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 274, 99–102.

45.

King

, Arendash

, Crawford

, Sterk

, Menendez

, Mullan

(1999) Progressive and gender-dependent cognitive impairment in the APP(SW) transgenic mouse model for Alzheimer’s disease. Behav Brain Res 103, 145–162.

46.

Calkins

, Manczak

, Mao

, Shirendeb

, Reddy

(2011) Impaired mitochondrial biogenesis, defective axonal transport of mitochondria, abnormal mitochondrial dynamics and synaptic degeneration in a mouse model of Alzheimer’s disease. Hum Mol Genet 20, 4515–4529.

47.

Masliah

, Sisk

, Mallory

, Mucke

, Schenk

, Games

(1996) Comparison of neurodegenerative pathology in transgenic mice overexpressing V717F β-amyloid precursor protein and Alzheimer’s disease. J Neurosci 16, 5795–5811.

48.

Kaech

, Banker

(2006) Culturing hippocampal neurons. Nat Protoc 1, 2406–2415.

49.

Niwa

, Yamamura

, Miyazaki

(1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199.

50.

Schindelin

, Arganda-Carreras

, Frise

, Kaynig

, Longair

, Pietzsch

, Preibisch

, Rueden

, Saalfeld

, Schmid

, Tinevez

, White

, Hartenstein

, Eliceiri

, Tomancak

, Cardona

(2012) Fiji: An open-source platform for biological-image analysis. Nat Methods 9, 676–682.

51.

Ferreira

, Blackman

, Oyrer

, Jayabal

, Chung

, Watt

, Sjostrom

, van Meyel

(2014) Neuronal morphometry directly from bitmap images. Nat Methods 11, 982–984.

52.

Sopher

, Kavanagh

, Furlong

, Martin

, GM (1996) Neurodegenerative mechanisms in Alzheimer disease. A role for oxidative damage in amyloid beta protein precursor-mediated cell death. Mol Chem Neuropathol 29, 153–168.

53.

Maezawa

, Hong

, Wu

, Battina

, Rana

, Iwamoto

, Radke

, Pettersson

, Martin

, Hua

, Jin

(2006) A novel tricyclic pyrone compound ameliorates cell death associated with intracellular amyloid-beta oligomeric complexes. J Neurochem 98, 57–67.

54.

Woltjer

, McMahan

, Milatovic

, Kjerulf

, Shie

, Rung

, Montine

(2007) Effects of chemical chaperones on oxidative stress and detergent-insoluble species formation following conditional expression of amyloid precursor protein carboxy-terminal fragment. Neurobiol Dis 25, 427–437.

55.

Wei

, Wang

, Kusiak

(2002) Signaling events in amyloid beta-peptide-induced neuronal death and insulin-like growth factor I protection. J Biol Chem 277, 17649–17656.

56.

Petratos

, Li

, George

, Hou

, Kerr

, Unabia

, Hatzinisiriou

, Maksel

, Aguilar

, Small

(2008) The beta-amyloid protein of Alzheimer’s disease increases neuronal CRMP-2 phosphorylation by a Rho-GTP mechanism. Brain 131, 90–108.

57.

Gray

, Morre

, Kelley

, Maier

, Stevens

, Quinn

, Soumyanath

(2014) Caffeoylquinic acids in Centella asiatica protect against amyloid-beta toxicity. J Alzheimers Dis 40, 359–373.

58.

Pike

, Walencewicz-Wasserman

, Kosmoski

, Cribbs

, Glabe

, Cotman

(1995) Structure-activity analyses of beta-amyloid peptides: Contributions of the beta 25-35 region to aggregation and neurotoxicity. J Neurochem 64, 253–265.

59.

Murvai

, Somkuti

, Smeller

, Penke

, Kellermayer

(2015) Structural and nanomechanical comparison of epitaxially and solution-grown amyloid beta25-35 fibrils. Biochim Biophys Acta 1854, 327–332.

60.

Gray

, Sampath

, Zweig

, Quinn

, Soumyanath

(2015) Centella asiatica attenuates amyloid-β-induced oxidative stress and mitochondrial dysfunction. J Alzheimers Dis 45, 933–946.

61.

Schneider

, Giordano

, Zelickson

, Johnson

, Benavides

, Ouyang

, Fineberg

, Darley-Usmar

, Zhang

VMJ

(2011) Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress. Free Radic Biol Med 51, 2007–2017.

62.

, Jung

, Jeong

, Kim

, Jung

, Lee

, Kim

, Jeong

, Son

, Kim

(2014) Neuritin can normalize neural deficits of Alzheimer’s disease. Cell Death Dis 5, e1523.

63.

Etgen

, Jover-Mengual

, Zukin

(2011) Neuroprotective actions of estradiol and novel estrogen analogs in ischemia: Translational implications. Front Neuroendocrinol 32, 336–352.

64.

Alasia

, Aimar

, Merighi

, Lossi

(2012) Context-dependent toxicity of amyloid-beta; peptides on mouse cerebellar cells. J Alzheimers Dis 30, 41–51.

65.

Kelly

, Levin

(2001) Rapid actions of plasma membrane estrogen receptors. Trends Endocrinol Metab 12, 152–156.

66.

Rønnekleiv

, Malyala

, Kelly

(2007) Membrane-initiated signaling of estrogen in the brain. Semin Reprod Med 25, 165–177.

67.

Nag

, Mokha

(2014) Activation of a Gq-coupled membrane estrogen receptor rapidly attenuates α2-adrenoceptor-induced antinociception via an ERK I/II-dependent, non-genomic mechanism in the female rat. Neuroscience 16, 122–134.

68.

Qiu

, Bosch

, Tobias

, Grandy

, Scanlan

, Ronnekleiv

, Kelly

(2003) Rapid signaling of estrogen in hypothalamic neurons involves a novel G-protein-coupled estrogen receptor that activates protein kinase C. J Neurosci 23, 9529–9540.

69.

Clark

, Rainville

, Zhao

, Katzenellenbogen

, Pfaff

, Vasudevan

(2014) Estrogen receptor-mediated transcription involves the activation of multiple kinase pathways in neuroblastoma cells. J Steroid Biochem Mol Biol 139, 45–53.

70.

, Xu

, Zhu

, Chen

, Sokabe

, Chen

(2010) DHEA prevents Aβ25-35-impaired survival of newborn neurons in the dentate gyrus through a modulation of PI3K-Akt-mTOR signaling. Neuropharmacology 59, 323–333.

71.

Wang

, Xu

, Zhu

(2012) Upregulation of heme oxygenase-1 by acteoside through ERK and PI3 K/Akt pathway confer neuroprotection against beta-amyloid-induced neurotoxicity. Neurotox Res 21, 368–378.

72.

Xing

, Dong

, Li

, Zou

, Fan

, Wang

, Cai

, Li

, Zhou

, Liu

, Niu

(2011) Neuroprotective effects of puerarin against beta-amyloid-induced neurotoxicity in PC12 cells via aPI3K-dependent signaling pathway. Brain Res Bull 85, 212–218.

73.

Hasegawa

, Hojo

, Kojima

, Ikeda

, Hotta

, Sato

, Ooishi

, Yoshiya

, Chung

, Yamazaki

, Kawato

(2015) Estradiol rapidly modulates synaptic plasticity of hippocampal neurons: Involvement of kinase networks. Brain Res 1621, 147–161.

74.

Murakami

, Tsurugizawa

, Hatanaka

, Komatsuzaki

, Tanabe

, Mukai

, Hojo

, Kominami

, Yamazaki

, Kimoto

, Kawato

(2006) Comparison between basal and apical dendritic spines in estrogen-induced rapid spinogenesis of CA1 principal neurons in the adult hippocampus. Biochem Biophys Res Commun 351, 553–558.

75.

Beyer

, Raab

(1998) Nongenomic effects of oestrogen: Embryonic mouse midbrain neurones respond with a rapid release of calcium from intracellular stores. Eur J Neurosci 10, 255–262.

76.

Beyer

, Karolczak

(2000) Estrogenic stimulation of neurite growth in midbrain dopaminergic neurons depends on cAMP/protein kinase A signalling. J Neurosci Res 59, 107–116.

77.

Ishii

, Tsurugizawa

, Ogiue-Ikeda

, Asashima

, Mukai

, Murakami

, Hojo

, Kimoto

, Kawato

(2007) Local production of sex hormones and their modulation of hippocampal synaptic plasticity. Neuroscientist 13, 323–334.

78.

Dickstein

, Brautigam

, Stockton

Jr , Schmeidler

, Hof

(2010) Changes in dendritic complexity and spine morphology in transgenic mice expressing human wild-type tau. Brain Struct Funct 214, 161–179.

79.

Games

, Adams

, Alessandrini

, Barbour

, Berthelette

, Blackwell

, Carr

, Clemes

, Donaldson

, Gillespie

, Guido

, Hagopian

, Johnson-Wood

, Khan

, Lee

, Leibowitz

, Lieberburg

, Little

, Masliah

, McConlogue

, Montoya-Zavala

, Mucke

, Paganini

, Penniman

, Power

, Schenk

, Seubert

, Snyder

, Soriano

, Tan

, Vitale

, Wadsworth

, Wolozin

, Zhao

(1995) Alzheimer-type neuropathology in transgenic mice overexpressing V717F b-amyloid precursor protein. Nature 373, 523–527.

80.

Kazim

, Blanchard

, Dai

, Tung

, LaFerla

, Iqbal

(2014) Disease modifying effect of chronic oral treatment with a neurotrophic peptidergic compound in a triple transgenic mouse model of Alzheimer’s disease. Neurobiol Dis 71C, 110–130.

81.

Mavroudis

, Manani

, Petrides

, Petsoglou

, Njau

, Costa

, Baloyannis

(2013) Dendritic and spinal pathology of the Purkinje cells from the human cerebellar vermis in Alzheimer’s disease. Psychiatr Danub 25, 221–226.

82.

Mavroudis

, Manani

, Petrides

, Petsoglou

, Njau

, Costa

, Baloyannis

(2014) Dendritic and spinal alterations of neurons from Edinger-Westphal nucleus in Alzheimer’s disease. Folia Neuropathol 52, 197–204.

83.

Brocca

, Pietranera

, Beauquis

, De

Nicola AF

(2013) Estradiol increases dendritic length and spine density in CA1 neurons of the hippocampus of spontaneously hypertensive rats: A Golgi impregnation study. Exp Neurol 247, 158–164.

84.

Dominguez

, Jalali

, de Lacalle

(2004) Morphological effects of estrogen on cholinergic neurons in vitro involves activation of extracellular signal-regulated kinases. J Neurosci 24, 982–990.

85.

Mukai

, Takata

, Ishii

, Tanabe

, Hojo

, Furukawa

, Kimoto

, Kawato

(2006) Hippocampal synthesis of estrogens and androgens which are paracrine modulators of synaptic plasticity: Synaptocrinology. Neuroscience 138, 757–764.

86.

Razmara

, Sunday

, Stirone

, Wang

, Krause

, Duckles

, Procaccio

(2008) Mitochondrial effects of estrogen are mediated by estrogen receptor alpha in brain endothelial cells. J Pharmacol Ex Ther 325, 782–790.

87.

Yao

, Hamilton

, Cadenas

, Brinton

(2010) Decline in mitochondrial bioenergetics and shift to ketogenic profile in brain during reproductive senescence. Biochim Biophys Acta 1800, 1121–1126.

88.

Felty

, Roy

(2005) Estrogen, mitochondria, and growth of cancer and non-cancer cells. J Carcinog 4, 1.

89.

Mannella

, Brinton

(2006) Estrogen receptor protein interaction with phosphatidylinositol 3-kinase leads to activation of phosphorylated Akt and extracellular signal-regulated kinase 1/2 in the same population of cortical neurons: A unified mechanism of estrogen action. J Neurosci 26, 9439–9447.

90.

Arnold

, Victor

, Beyer

(2012) Estrogen and the regulation of mitochondrial structure and function in the brain. J Steroid Biochem Mol Biol 131, 2–9.

91.

Sarkar

, Jun

, Simpkins

JW2

(2015) Estrogen amelioration of Aβ-induced defects in mitochondria is mediated by mitochondrial signaling pathway involving ERβ, AKAP and Drp1. Brain Res 1616, 101–111.

92.

Zhao

, Yang

, Gao

, Wang

, Zuo

, Dong

, Xu

, Su

, Zhou

, Zhu

, Peng

(2014) Upregulation of HIF-1α via activation of ERK and PI3K pathway mediated protective response to microwave-induced mitochondrial injury in neuron-like cells. Mol Neurobiol 50, 1024–1034.