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Endocrinology Vol. 147, No. 11 5303-5313
Copyright © 2006 by The Endocrine Society

Dose and Temporal Pattern of Estrogen Exposure Determines Neuroprotective Outcome in Hippocampal Neurons: Therapeutic Implications

Shuhua Chen, Jon Nilsen and Roberta Diaz Brinton

Department of Molecular Pharmacology and Toxicology, University of Southern California, School of Pharmacy Pharmaceutical Sciences Center, Los Angeles, California 90089-9121

Address all correspondence and requests for reprints to: Roberta Diaz Brinton, Ph.D., Department of Molecular Pharmacology and Toxicology, Norris Foundation Laboratory for Neuroscience Research, Pharmaceutical Sciences Center, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90089-9121. E-mail: rbrinton{at}hsc.usc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To address controversies of estrogen therapy, in vitro models of perimenopause and prevention vs. treatment modes of 17ß-estradiol (E2) exposure were developed and used to assess the neuroprotective efficacy of E2 against ß-amyloid-1–42 (Aß1–42)-induced neurodegeneration in rat primary hippocampal neurons. Low E2 (10 ng/ml) exposure exerted neuroprotection in each of the perimenopausal temporal patterns, acute, continuous, and intermittent. In contrast, high E2 (200 ng/ml) was ineffective at inducing neuroprotection regardless of temporal pattern of exposure. Although high E2 alone was not toxic, neurons treated with high-dose E2 resulted in greater Aß1–42-induced neurodegeneration. In prevention vs. treatment simulations, E2 was most effective when present before and during Aß1–42 insult. In contrast, E2 treatment after Aß1–42 exposure was ineffective in reversing Aß-induced degeneration, and exacerbated Aß1–42-induced cell death when administered after Aß1–42 insult. We sought to determine the mechanism by which high E2 exacerbated Aß1–42-induced neurodegeneration by investigating the impact of low vs. high E2 on Aß1–42-induced dysregulation of calcium homeostasis. Results of these analyses indicated that low E2 significantly prevented Aß1–42-induced rise in intracellular calcium, whereas high E2 significantly increased intracellular calcium and did not prevent Aß1–42-induced calcium dysregulation. Therapeutic benefit resulted only from low-dose E2 exposure before, but not after, Aß1–42-induced neurodegeneration. These data are relevant to impact of perimenopausal E2 exposure on protection against neurodegenerative insults and the use of estrogen therapy to prevent vs. treat Alzheimer’s disease. Furthermore, these data are consistent with a healthy cell bias of estrogen benefit.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PROFOUND DISPARITIES between the largely positive basic science findings of gonadal steroid action in brain and the adverse outcomes of recent hormone therapy clinical trials in women who are either aged postmenopausal or postmenopausal with Alzheimer’s disease, has led to an intense reassessment of gonadal hormone action and the model systems used in basic and clinical science (1). The power of model systems is their predictive validity for a target population. In the case of estrogen or hormone therapy, the target population is menopausal women who must consider the health benefits and risks of hormone therapy (1). Analysis of the model systems used across the basic to clinical research continuum separate into two broad classes, those that use prevention interventions in healthy organisms and those that use hormone interventions in organisms with compromised neurological function (1). Basic science analyses that led to elucidation of the neurotrophic and neuroprotective effects of estrogen and the underlying mechanisms of action typically used a prevention experimental paradigm (1). The prevention paradigm relies on healthy neurons/brains/animals/humans as the starting foundation followed by exposure to estrogen/hormone followed by exposure to neurodegenerative insult. The prevention paradigm of basic science analyses parallels the studies of Sherwin (2), who investigated the cognitive impact of estrogen therapy in women with surgical or pharmacological-induced menopause. Observational, retrospective and prospective, studies are also consistent with the outcomes of basic science analyses (1). For the most part, the epidemiological observational data indicate reduction in risk of Alzheimer’s disease in women who began estrogen or hormone therapy at the time of the menopause (1). The comparable benefit seen in observational studies and basic science analyses suggest that the data within the observational studies were derived from women with healthy neurological status.

In contrast, studies that fall within the second class, hormone intervention in women with compromised neurological function, i.e. a treatment paradigm, exhibited a mixed profile (1). In a randomized double-blind clinical trial of estrogen therapy in a cohort in aged women, 72 yr of age or older, diagnosed with Alzheimer’s disease, estrogen therapy resulted in a modest benefit of estrogen therapy in the short-term (2 months) and adverse progression of disease in the long-term (12 months) (3). In the Women’s Health Initiative Memory Study (WHIMS) cohort of women, 65 yr of age or older, with no indicators of neurological disease but with variable health status, there was no statistically significant increase in Alzheimer’s disease risk in the conjugated equine estrogen (CEE) arm of the trial—statistical significance on all causes of dementia occurred only when results of the CEE and the CEE plus medroxyprogesterone acetate (MPA) were combined, and hormone therapy for 5 yr increased the risk of developing Alzheimer’s disease (4).

Collectively, these data suggest that as the continuum of neurological health progresses from healthy to unhealthy so too do the benefits of estrogen or hormone therapy (1). If neurons are healthy at the time of estrogen exposure, their response to estrogen is beneficial for both neurological function and survival. In contrast, when neurological health is compromised, estrogen exposure over time exacerbates neurological demise. Based on these and other data, we sought to test the healthy cell bias of gonadal hormone action hypothesis.

To pursue the roots of the disparity between results of basic science analyses and observation studies and those of recent clinical trials of estrogen therapy, we developed in vitro models simulating perimenopause and prevention vs. treatment modes of estrogen exposure. In these model systems, we tested whether the disparities may be due, in part, to the complex dose and temporal requirements of estrogen therapy and or due to neurological status of neurons at the time of E2 exposure. To address these issues, we determined the protective efficacy of 17ß-estradiol (E2) against ß-amyloid 1–42 (Aß1–42)-induced neurodegeneration under two dose and three temporal paradigms of E2 exposure. First, the neuroprotective efficacy of acute, continuous or intermittent E2 exposure at 10 or 200 ng/ml to simulate the low to high fluctuations of E2 characteristic of perimenopause transition was determined. Second, hippocampal neurons were treated with 10 ng/ml of E2 before or after Aß1–42-induced insult to simulate either a prevention or treatment paradigm. Third, we determined the duration of neuronal protection after E2 withdrawal.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The use of animals for the study was approved by the Institutional Animal Care and Use Committee at the University of Southern California (protocol no. 10256). Pregnant Sprague-Dawley rats were purchased from Harlan, Inc. (Indianapolis, IN). They were kept individually in separate cages under controlled conditions of temperature (18–24 C), humidity (30–70%), and light (12-h light/12-h dark) with free access to food and water. Animals were maintained and used according to the Institutional Animal Care and Use Committee guidelines.

Chemicals
All culture materials were purchased from Life Technologies (Carlsbad, CA). E2 was purchased from Steraloids (Newport, RI). Aß1–42 was purchased from American Peptide (Sunnyvale, CA). E2 was dissolved in analytically pure ethanol at 1 or 20 mg/ml and diluted in culture medium to the final concentrations. Thus, all experimental conditions received the same dose of ethanol vehicle. Aß1–42 was preaggregated by initially dissolving it in 10 mM HCl at a concentration of 1 mM as a stock solution and stored at –20 C. ßA1–42 stock (1 mM) was then diluted in 0.1 M PBS at a concentration of 100 µM for 3 d at room temperature before use. This aggregated Aß1–42 was diluted in neural basal medium at 1.5 µM just before use and applied for treating cells.

Neuronal cultures
Primary cultures of hippocampal neurons were prepared as described previously (5). Briefly, hippocampi were dissected from the brains of embryonic d 18 rat fetuses. The hippocampi were treated with 0.02% trypsin in Hanks’ balanced salt solution (137 mM NaCl, 5.4 mM KCl, 0.4 mM KH2PO4, 0.34 mM Na2HPO47H2O, 10 mM glucose, and 10 mM HEPES) for 5 min at 37 C and dissociated by repeated passage through a series of fire-polished constricted Pasteur pipettes. Neuronal cells were plated onto poly-D-lysine-coated solid black and clear-bottom 96-well plates for cell viability analyses. Cells were grown in phenol-red-free neurobasal medium (NBM; Invitrogen, Carlsbad, CA) supplemented with 2% B27, 10 U/ml penicillin, 10 µg/ml streptomycin, 0.5 mM glutamine, and 25 µM glutamate, incubated at 37 C in a humidified 5% CO2 atmosphere. Cultures growing in serum-free NBM yield approximately 99.5% neurons and 0.5% glial cells. Microscopically, glial cells were not apparent in hippocampal cultures when these cultures were used for experimental analyses. The culture media were changed with glutamate-free NBM 3 d after cell culture, and the hippocampal neurons were fed with glutamate-free NBM twice weekly.

Treatment
The temporal patterns of exposure were selected to reflect the continuous or intermittent exposure to E2 that characterizes the erratic pattern of hyperestrogenic blood levels that can occur during the perimenopause transition (6, 7). Blood levels of E2 during the perimenopause can range from 25–550 pg/ml (8). Our previous in vitro findings have demonstrated that E2 at 10 ng/ml reliably induces a neuroprotective response in cultured hippocampal neurons. Although neuroprotective responses could be observed with lower concentrations of E2, the 10 ng/ml allows for reliable throughput experimentation. Based on the 10 ng/ml of E2 dose requirement for induction of neuroprotection, we simulated the 20-fold range in E2 levels during the perimenopause reflective of the relative magnitude of fluctuations that can occur during the perimenopausal transition (8). Thus, the 20-fold difference between low and high E2 level was simulated in vitro using 10 ng/ml for the E2 low dose and a 20-fold higher dose of 200 ng/ml E2 for the high dose (Fig. 1AGo).


Figure 1
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  		FIG. 1. Effects of different temporal pattern and dose of E2 treatment on hippocampal neuronal viability. Primary hippocampal neurons were treated with vehicle (control) or different temporal and dose of E2 and then exposed to 1.5 µM Aß1–42 for 3 d. After Aß exposure, cell viability was measured using a two-color fluorescence calcein AM/ethidium homodimer assay, in which viable cells fluoresce green and dead cells fluoresce red. Cells subjected to Aß1–42 alone treatment (black bar) showed viable cell loss (C) and dead cell increase (D). When pretreatment with 10 ng/ml E2 (gray bar) for 2 d, 7 d, or three times during 7 d, E2 significantly increased the number of live cells (C) and decreased the dead cell number (D). In contrast, high E2 concentration, 200 ng/ml (dark gray), lost neuroprotection and showed greater Aß1–42-induced neurodegeneration. Results are presented as the percentage of vehicle-treated control cultures (light gray) and expressed as mean ± SEM. Data are derived from three separate experiments. #, P < 0.05 and ##, P < 0.01 compared with vehicle-treated control cultures; **, P < 0.01 compared with Aß1–42 alone-treated cultures.

 
Experiment 1
Four-day-old primary hippocampal neurons were grown on the solid black and clear bottom 96-well plate were respectively treated with 0.001% ethanol as vehicle control, 10 or 200 ng/ml E2 for 7 d, as continuous exposure. E2 (10 or 200 ng/ml) was readministered with each requisite media change every 4 d. Ten or 200 ng/ml E2 were treated for 3 d, followed by a 1 d E2 withdraw, and then retreated with the same E2 concentration every other day as a simulation of perimenopause. Nine-day-old neurons were respectively treated with 10 or 200 ng/ml E2 for 2 d as acute exposure (Fig. 1AGo). Eleven-day-old pretreated neurons were exposed to Aß1–42 at 1.5 µM for 3–4 d simultaneously with E2 or 0.001% ethanol. After Aß1–42 exposure, analysis of cell viability was conducted.

Experiment 2
As illustrated by Fig. 2AGo, 7-d-old neurons were pretreated with 10 ng/ml E2 or 0.001% ethanol for 2 d followed by 3 d Aß1–42 (1.5 µM) exposure simultaneously with E2 or 0.001% ethanol. Nine-day-old neurons were exposed to 1.5 µM Aß1–42 for 3 d or one of the following conditions: 1) 10 ng/ml E2 simultaneously with 3 d Aß1–42; 2) 10 ng/ml E2 addition 1 d after Aß1–42 exposure; or 3) 10 ng/ml E2 addition 2 d after Aß1–42 exposure, as the window of opportunity for estrogen neuroprotective efficacy against Aß1–42 insult. For the treatment model, 7-d-old neurons were treated with 1.5 µM Aß1–42 for 3 d followed by removal of Aß1–42 and replacement with 10 ng/ml E2 or 0.001% ethanol for 2 d or continuous Aß1–42 exposure for 2 additional days with 10 ng/ml E2 or 0.001% ethanol (Fig. 3AGo). After Aß1–42 exposure, analysis of cell viability was conducted.


Figure 2
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  		FIG. 2. A window of opportunity for estrogen neuroprotective efficacy against Aß insult. Neurons were pretreated with 10 ng/ml E2 at 2 d before ßA exposure, at the same time as Aß exposure, or 1 d and 2 d after Aß exposure. After treatment, cell viability was measured using a two-color fluorescence calcien AM/ethidium homedimer assay. As shown in B, 10 ng/ml E2 pretreatment (gray bar) significantly increased neuron survival compared with neurons subjected to ßA alone treatment (black bar). E2 treatment at the same time exposed to Aß, without pretreatment, slightly increased neuron survival. Although E2 treatment at 1 or 2 d after Aß exposure, could not improve cell damage (dark gray bar). Results are presented as the percent of vehicle-treated control cultures and expressed as mean ± SEM. Data are derived from three separate experiments. ##, P < 0.01 compared with vehicle-treated control cultures; **, P < 0.01 compared with Aß1–42 alone-treated cultures; *, P < 0.05 compared with Aß1–42 alone-treated cultures.

 

Figure 3
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  		FIG. 3. Efficacy of estrogen for treatment to reverse Aß1–42-induced degeneration. Neurons were treated with E2 3 or 5 d after Aß1–42 exposure. Cell viability was evaluated using calcien AM/ethidium homodimer two-color fluorescence assay. As shown in B and C, E2 treatment after Aß insult was ineffective to reverse ßA-induced neurodegeneration, and even exacerbates Aß-induced cell damage. Results are presented as the percent of vehicle-treated control cultures and expressed as mean ± SEM. Data are derived from three separate experiments; ##, P < 0.01 compared with vehicle-treated control cultures; *, P < 0.05 compared with Aß1–42 alone-treated cultures.

 
Experiment 3
For the prevention paradigm, 9-d-old neurons were pretreated with 10 ng/ml E2 for 2 d followed by 3 d Aß1–42 exposure simultaneously with E2, or during 3 d Aß1–42 exposure no more E2 (Fig. 4AGo). Ten-day-old neurons were pretreated with 10 ng/ml E2 for 1 d followed by 3 d of Aß1–42 exposure with 1 additional day of E2. Eleven-day-old neurons were treated with 10 ng/ml E2 for the first 2 d of a 3-d Aß1–42 exposure. After Aß1–42 exposure, analysis of cell viability was conducted. As illustrated by Fig. 5AGo, 7-, 8-, and 9-d-old neurons were treated with 10 ng/ml E2 for 2 d followed by E2 withdrawal for 5, 4, and 3 d. During the last 3 d, neurons were exposed to 1.5 µM ßA1–42, which was followed by the analysis of cell viability to determine the duration of estrogen neuroprotection against ßA1–42.


Figure 4
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  		FIG. 4. Effects of estrogen on prevention of Aß1–42-induced degeneration. Neurons were pretreated with 10 ng/ml E2 for 2 and 3 d during Aß exposure or 2 d treated only. After Aß exposure, cell viability was measured using a two-color fluorescence calcien AM/ethidium homodimer assay. As shown in B, estrogen is most effective to protect neurons against Aß-induced degeneration (black bar) when present before and during Aß insult (gray bar). C is a representative of microscopic images, confirmed estrogen prevented Aß-induced neurodegeneration. Results are presented as the percentage of vehicle-treated control cultures and expressed as mean ± SEM. Data are derived from three separate experiments. ##, P < 0.01 compared with vehicle-treated control cultures; *, P < 0.05; and **, P < 0.01 compared with Aß1–42 alone-treated cultures.

 

Figure 5
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  		FIG. 5. Duration of estrogen neuroprotection against Aß-induced degeneration. Neurons were pretreated with 10 ng/ml E2 for 2 d followed by E2 withdrawal for 5, 4, or 3 d, during the last 3 d neurons were exposed to 1.5 µM Aß1–42. After treatment, cell viability was evaluated using a two-color fluorescence cell viability assay. Results shown in B revealed that estrogen-induced improvement of cell viability persisted for 3 d after pretreatment with E2. Results are presented as the percent of vehicle-treated control cultures and expressed as mean ± SEM. Data are derived from three separate experiments. ##, P < 0.01 compared with vehicle-treated control cultures; *, P < 0.05; and **, P < 0.01 compared with Aß1–42 alone-treated cultures.

 
Cell viability
Cell viability was assessed by using dual staining with calcein AM and ethidium homodimer-1 (Molecular Probes, Eugene, OR). The combined use of calcein AM and ethidium homodimer provides a two-color fluorescence analysis that allows simultaneous determination of live and dead cells with two probes that measure recognized parameters of cell viability, intracellular esterase activity, and plasma membrane integrity, respectively. The fluorescent intensities of calcein and ethidium homodimer are markers of cell vitality and damage, respectively, and direct indicators of cell survival and cell death. Thus we report both values for increased confidence in the reported effects. Loading of neurons with 1 µM of calcein AM and 2 µM of ethidium homodimer for 30 min at room temperature was found to be the optimal experimental condition (9). After ßA1–42 exposure, cultures were rinsed with PBS and incubated with the calcein AM and ethidium homodimer mixture at 100 µl/well. The fluorescence intensity was measured on a dual-wavelength-scanning microplate spectrofluorometer (Molecular Devices Corp., Sunnyvale, CA) using appropriate excitation and emission filter combinations (485–530 nm for calcein AM and 530–645 nm for ethidium homodimer). For microscopic analyses, the green cytoplasmic fluorescence and red nuclear fluorescence were observed, and images were captured under Axiovert 200M Marianas Digital Microscopy Workstation (Intelligent Imaging Innovations, Denver, CO). For quantification, the fluorescence intensities were measured on a SpectraMax GEMINI EM dual wavelength scanning microplate spectrafluometer (Molecular Devices Corp.) using appropriate excitation and emission wavelength combinations (485/530 for calcein and 530/645 for ethidium homodimer). Data were normalized against the values obtained from vehicle-treated control cultures. Data were presented as the mean ± SEM from three separate experiments with eight wells per condition per experiment. The therapeutic neuroprotective efficacy (TNE) is derived from the percentage of the toxin-induced cell death prevented by the intervention and was calculated by the equation TNE = [(S – Tave)/(Cave – Tave)] x 100; where S is the individual sample value, Tave is the mean value for toxin treated cultures, and Cave is the mean value for control cultures.

Measurement of cytoplasmic Ca2+ using fura 2-AM
Hippocampal neurons were treated with E2 (10 ng/ml) or vehicle control for 48 h before loading in the dark with fura 2-AM (2 µM) in HBS (45 min; 37 C). Intracellular Ca2+ concentration ([Ca2+]i) was determined by comparing the 340/380 ratio to a standard curve as previously described (10). Data are presented as representative traces averaged from at least 10 cells per coverslip. Responses to steroids were quantified as the difference between the average [Ca2+]i for 1 min during glutamate exposure (30–90 sec after glutamate exposure) and the average [Ca2+]i for 1 min before exposure. Changes in [Ca2+]i are presented as mean ± SEM from four independent experiments with at least 30 cells per experiment. Equal dye loading was determined as previously described (10).

Statistical analysis
Data are presented as mean ± SEM and analyzed by one-way ANOVA, followed by Student Newman-Keuls post hoc comparisons. Statistically significant differences between groups were considered at a P value less than 0.05. All experiments consisted of n = 6–8 wells per experimental group. Results are presented as the percent of vehicle-treated control cultures, which is 100%, and expressed as mean ± SEM. Data are derived from a minimum of three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neuroprotective effects of different temporal patterns and dosages of E2
The objective of this study was to determine whether the neuroprotective efficacy of E2 was dependent upon temporal patterns (acute, continuous, or intermittent) or treatment doses (10 or 200 ng/ml). Pretreatment with low concentration of E2 (10 ng/ml) in each of the three temporal patterns (Fig. 1AGo) significantly attenuated ßA-induced neurodegeneration. Treatments of Aß1–42 1.5 µM alone decreased the calcein fluorescence to 61 ± 5% of control (Fig. 1CGo; P < 0.01 compared with vehicle control), and increased ethidium homodimer fluorescence to 132 ± 5% of control (Fig. 1DGo; P < 0.01 compared with vehicle control). Pretreatment with low concentration of E2 (10 ng/ml) for 2 or 7 d continuously or intermittently three times during a 7-d period, increased survival rate after Aß1–42 exposure to 83 ± 5% after E2 for 2 d, 85 ± 4% after continuous E2 exposure for 7 d, and 81 ± 3% after three intermittent E2 exposures over the course of 7 d (Fig. 1CGo; P < 0.01 compared with Aß1–42 alone).

For acute, continuous, and intermittent interventions, the magnitude of therapeutic neuroprotective efficacy was 56, 61, and 51% respectively, compared with Aß1–42 alone. Correspondingly, 10 ng/ml E2 protected against neuron death after Aß1–42 exposure to 108 ± 4% after E2 for 2 d, 107 ± 3% after continuous E2 exposure for 7 d, and 121 ± 5% after three intermittent E2 exposures over the course of 7 d (Fig. 1CGo; P < 0.01 or P < 0.05 compared with Aß1–42 alone). This represents a significant therapeutic neuroprotective efficacy against neuronal death of 40 and 45% for acute and continuous 10 ng/ml E2 exposure, respectively, and a therapeutic neuroprotective efficacy of 34% for intermittent 10 ng/ml E2 exposure, compared with ßA1–42 alone.

In contrast, exposure to a high concentration of E2 (200 ng/ml) was ineffective in preventing neurodegeneration regardless of temporal mode of exposure (Fig. 1Go, C and D). Pretreatment of neurons with high dose of E2 (200 ng/ml) for 2 or 7 d continuously or intermittently three times during a 7-d period resulted in neuron survival after Aß1–42 exposure of 47 ± 5, 55 ± 5, and 49 ± 5% of control live cells, respectively, which was lower than 61 ± 5% of Aß1–42 1.5 µM alone (Fig. 1CGo; P < 0.05 compared with control cultures). Similarly, exposure to high-dose E2 resulted in neuronal death after ßA1–42 exposure of 141 ± 9, 125 ± 5, and 139 ± 7% of control, respectively (Fig. 1DGo). Although high-dose E2 was not neuroprotective and exacerbated ßA-induced neuronal death, exposure to 200 ng/ml of E2 did not alter neuronal survival nor death in the absence of Aß1–42 (data not shown). These results demonstrate that, in contrast to low-dose E2, high-dose E2 does not prevent Aß1–42-induced neurodegeneration and can exacerbate the neurodegenerative process.

Effect of E2 treatment either before or after exposure to ßA insult on neuroprotection
To determine the window of opportunity for estrogen-induced neuroprotection against ßA1–42 we compared the neuroprotective efficacy of E2 in either a prevention or a treatment exposure paradigm. For the prevention model, hippocampal neurons were pretreated with E2 (10 ng/ml) 2 d prior and during Aß1–42 exposure (Fig. 2AGo). For the treatment paradigm, E2 was administered simultaneously with Aß1–42 exposure, or 1 or 2 d after Aß1–42 exposure (Fig. 2AGo). As above, 3 d of Aß1–42 exposure reduced neuron survival to 48 ± 8% of control (Fig. 2BGo; P < 0.01 compared with control) and increased neuronal death to 125 ± 6% of control (Fig. 2CGo; P < 0.01 compared with control). In the prevention mode of exposure, neurons treated with 10 ng/ml E2 before and during Aß1–42 exposure exhibited a significantly greater survival of 72 ± 7% of control relative to Aß1–42 alone (Fig. 2BGo; P < 0.01 compared with ßA1–42 alone) and essentially no evidence of neuronal death (102 ± 5% relative to control; Fig. 2CGo; P < 0.01 compared with ßA1–42 alone). Simultaneous exposure of neurons with 10 ng/ml E2 and Aß1–42 increased neuron survival to 60 ± 6% of control (Fig. 2BGo) and reduced neuronal death to 108 ± 4% of control (Fig. 2CGo; P < 0.05 compared with Aß1–42 alone). Exposure of hippocampal neurons to 10 ng/ml E2 at 1 or 2 d after Aß1–42 exposure was ineffective at promoting neuron survival, resulting in neuron survival rates of 53 ± 7 and 39 ± 7% (Fig. 2BGo; P > 0.05 compared with control) and neuronal death rates of 118 ± 3 and 128 ± 5% of control respectively (Fig. 2CGo; P < 0.05 compared with control). These results indicate that low-dose estrogen in a prevention mode of exposure is a significant and efficacious neuroprotectant against Aß1–42-induced neurodegeneration.

To determine whether E2 promoted recovery from ßA-induced neurodegeneration, hippocampal neurons were exposed to Aß1–42 (1.5 µM) for 3 d followed by 0 or 2 d of ßA1–42 withdrawal or for 5 d of continuous Aß1–42 exposure (Fig. 3AGo). E2 (10 ng/ml) was present for either the last 2 d of the recovery from Aß1–42 or for the last 2 d of continuous Aß1–42 exposure (Fig. 3AGo). As above, a 3-d exposure to ßA1–42 resulted in a decrease in cell survival to 48 ± 8% of control (Fig. 3BGo; P < 0.01 compared with control) and increased the rate of membrane damage to 125 ± 6% of control (Fig. 3CGo; P < 0.01 compared with control). Removal of Aß1–42 for 2 d after a 3-d exposure resulted in a survival rate of 65 ± 5% relative to control cultures (Fig. 3BGo; P < 0.01 compared with control cultures; P < 0.05 compared with Aß1–42 3-d with no recovery) and neuronal death at 128 ± 4% of control (Fig. 3CGo; P < 0.01 compared with control) indicating a partial recovery of enzymatic activity, but not membrane integrity, upon removal of ßA1–42 toxin. Exposure to 10 ng/ml E2 during the 2-d washout period prevented the recovery resulting in a survival rate, similar to that of the 3-d Aß1–42 exposure without washout, of 58 ± 6% of control (Fig. 3BGo; P < 0.01 compared with control) and a rate of neuronal death of 123 ± 6% of control (Fig. 3CGo; P < 0.01 compared with control). Five days of continuous Aß1–42 exposure resulted in a survival rate of 44 ± 5% relative to control cultures (Fig. 3BGo; P < 0.01 compared with control culture) and neuronal death of 142 ± 4% of control (Fig. 3CGo; P < 0.01 compared with control). The neurodegenerative effect of Aß1–42 was potentiated by exposure to E2 (10 ng/ml) during the final 2 d of the 5-d Aß1–42 exposure resulting in a reduced neuron survival rate of 36 ± 5% of control (Fig. 3BGo; P < 0.01 compared with control) and a neuronal death rate of 146 ± 6% of control (Fig. 3CGo; P < 0.01 compared with control).

Pretreatment requirement for E2 neuroprotection
Because E2 administered after Aß1–42 exposure did not prevent Aß1–42-mediated neuronal death, we sought to determine the optimal E2 exposure paradigm for maximal neuroprotection. Hippocampal neurons were treated with E2 (10 ng/ml) for 2 d either 1 or 2 d before or at the same time of Aß1–42 exposure (Fig 4AGo). As above, 3 d of Aß1–42 exposure significantly reduced neuronal survival to 46 ± 8% of control (Fig. 4BGo; P < 0.01 compared with control) and increased neuronal death to 142 ± 3% of control (Fig. 4CGo; P < 0.01 compared with control). Our standard E2 prevention paradigm of 2 d E2 (10 ng/ml) pretreatment and continued presence during 3-d Aß1–42 exposure significantly increased neuronal survival to 80 ± 7% of control (Fig. 4CGo; P < 0.01 compared with Aß1–42 alone) and reduced neuronal death to 120 ± 3% of control (Fig. 4CGo; P < 0.01 compared with Aß1–42 alone). Both of the other pretreatment paradigms (pretreatment only and the 1-d/1-d), but not the simultaneous treatment, induced significant neuroprotection with neuronal survival rates of 73 ± 6, 74 ± 8, and 52 ± 5% of control (Fig. 4BGo) and neuronal death rates of 123 ± 3, 137 ± 4, and 139 ± 2% of control (Fig. 4CGo). The ineffectiveness of simultaneous E2 exposure on ßA1–42-induced neurotoxicity indicates the requirement for pretreatment with E2.

Persistence of E2-induced neuroprotection
To determine the duration of E2-induced neuroprotection, neurons were pretreated with 10 ng/ml E2 for 2 d followed by E2 withdrawal for 0, 1, or 2 d before Aß1–42 (1.5 µM) exposure for 3 d (Fig. 5AGo). As above, the Aß1–42-induced neuron death was significantly attenuated by the standard E2 pretreatment paradigm (10 ng/ml; 2 d prior and continued presence). For Aß1–42 neuronal survival and death were 78 ± 6 and 146 ± 4% of control, respectively (Fig. 5Go, B and C; P < 0.01 compared with control). For E2, pretreatment rates of neuronal survival and death were 78 ± 6 and 120 ± 3% of control, respectively (Fig. 5Go, B and C; P < 0.01 compared with ßA1–42 alone). The 2-d E2 pretreatment protective effect persisted as evidenced by the improved neuronal survival in the 0, 1-, and 2-d withdrawal groups with survival rates of 73 ± 7% (P < 0.01 compared with Aß1–42 alone), 64 ± 5% (P < 0.05 compared with Aß1–42 alone), and 63 ± 6% (P < 0.05 compared with Aß1–42 alone) of control, respectively (Fig. 5BGo). The rate of neuronal death for the 1- and 2-d withdrawal groups were 145 ± 5 and 137 ± 4% of control (Fig. 5CGo), whereas the protection against neuronal death was only evident in the 0-d withdrawal group with 123 ± 4% of control (Fig. 5CGo; P < 0.01 compared with ßA1–42 alone).

E2 regulation of calcium homeostasis
To address the potential mechanism underlying the disparity in outcome induced by low- vs. high-dose E2 exposure, we investigated the impact of low vs. high doses of E2 on calcium homeostasis. Resting steady-state intracellular calcium levels were unchanged in response to low 10 ng/ml E2, whereas it was significantly increased by high 200 ng/ml E2 by 18.7% (Fig. 6Go; P < 0.05 compared with vehicle control). Exposure to Aß1–42 for 24 h resulted in a significant increase (27.7% increase over vehicle control) in resting intracellular calcium level (Fig. 6Go; P < 0.05 compared with vehicle control). The Aß1–42-induced increase in resting calcium levels was significantly decreased by pretreatment with 10 ng/ml E2 (Fig. 6Go; P < 0.05 compared with Aß1–42 alone), but not by pretreatment to 200 ng/ml dose (Fig. 6Go).


Figure 6
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  		FIG. 6. E2 regulation of calcium homeostasis. Neurons were pretreated with 10 or 200 ng/ml E2 for 48 and 24 h during ßA1–42 (1.5 µm) exposure. After Aß exposure, calcium concentration was measured using fura 2-AM ratiometric imaging. A, Representative psuedocolor images of primary hippocampal neurons. B, Bar graphs representing mean ratios (calcium concentration) taken from at least 10 individual neurons per coverslip and at least three coverslips per condition. *, P < 0.05 compared with control; +, P < 0.05 compared with Aß1–42 alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The largely positive findings of gonadal hormone action in brain derived from basic science are in contrast to the adverse outcomes of the WHIMS hormone therapy clinical trial in women of advanced age. These profound disparities have led to a growing reassessment of gonadal hormone action in the central nervous system including a reexamination of the basic science model systems. To address these issues, we investigated the impact of low vs. high E2 on neuroprotective efficacy against ßA1–42-induced neurodegeneration. We further investigated the impact of prevention vs. treatment modes of E2 exposure. Results of these analyses indicate that E2 exposure at low concentration (10 ng/ml) exerted neuroprotection in each of the three temporal patterns of perimenopausal estrogen fluctuation. In contrast, high E2 concentration (200 ng/ml) was ineffective at inducing neuroprotection. Although high E2 (200 ng/ml) alone was not toxic, neurons exposed to high E2 exhibited greater Aß1–42-induced neurodegeneration. In the prevention and treatment models, E2 was most effective when present before and during Aß1–42 insult, whereas E2 was ineffective at reversing Aß1–42-induced degeneration and exacerbated Aß1–42-induced neuronal death when administered after Aß1–42 exposure. To determine the persistence of E2-induced neuroprotection, we designed an E2 withdrawal paradigm. Results of these analyses indicated that E2-induced neuroprotection persisted for 3 d after a 48 h E2 exposure. These in vitro data are remarkably consistent with epidemiological analyses indicating that women who receive ET at the time of the menopause, in a prevention mode well before age-associated degeneration is rampant, have a lower risk of developing Alzheimer’s disease than women who have never received ET or HT (3, 11, 12, 13, 14). Results from our in vitro"treatment" paradigm, are also remarkably consistent with the clinical data, indicating that women treated with HT in their 60s or 70s, when age-associated insults have already occurred in some women, estrogen and hormone therapy can exacerbate neurodegeneration in a subpopulation of those women (3, 4, 13, 15).

Points of convergence and divergence between basic and clinical science
Overall, basic science analyses using both in vitro and in vivo model systems indicated that estrogen, typically E2 but also CEE, exerted a robust protection of neurons against insults associated with Alzheimer’s disease (1, 2, 3, 4, 5, 16, 17, 18, 19, 20, 21, 22, 23, 24). Moreover, these same estrogens in the same model systems activated biochemical, genomic, cellular, and behavioral mechanisms of memory (16, 17, 18, 19, 20, 21, 22, 23). Basic science findings were consistent with studies conducted in young to middle aged women undergoing hysterectomy/oophorectomy and who were immediately replaced with estrogen therapy (24, 25). These studies indicted that estrogen therapy reversed the decline in cognitive function associated with surgically induced menopause (26, 27, 28). Basic science findings also correlated with results of both retrospective and prospective observational studies indicating that estrogen or hormone therapy promoted neurological health and reduced the risk of Alzheimer’s disease (3, 14, 29, 30, 31, 32).

In contrast to the basic science and observational studies, results of the hormone therapy arm of WHIMS, the combination of CEE and MPA, indicated that women receiving hormone therapy had a 2-fold greater risk of developing Alzheimer’s disease than women in the placebo arm of the randomized double-blind clinical trial (4). Analyses of the estrogen-only therapy arm of the WHIMS trial indicated that women on CEE were not statistically different than women in the placebo arm of the trial but that there was a trend toward greater risk of Alzheimer’s disease and mild cognitive impairment, which became statistically significant when the data from the estrogen therapy trial were combined with the hormone therapy trial (15).

The impact of estrogen or hormone therapy formulation remains unresolved. The analyses of Sherwin (25, 26) used an estrogen therapy formulation and route of administration, im route of administration of 10 mg E2 valerate once every 4 wk, drastically different from the oral administration of CEE alone or in combination with MPA common to most users in observational studies and the clinical trials (3, 4, 14, 15, 25, 31, 32). In fact, the Sherwin model of treatment most closely parallels that of the basic science rodent and nonhuman primate models (33, 34, 35). Results of our in vitro analyses indicated that the complex formulation of CEE (Premarin) promoted morphogenesis and neuroprotection against neurodegenerative insults (5). Further analyses indicated that the neurotrophic and neuroprotective effects were mediated by some but not all estrogens contained within CEE (36). We found that the complex formulation of CEE and select estrogens within this mixture were as effective as E2 with a dose response profile that would predict a wider therapeutic range than E2 (5). These data were consistent with the epidemiological studies in which Premarin was the most frequently used estrogen replacement therapy (17). The profile of response changed dramatically when MPA (Depo-Provera; PremPro) was added. Our analyses indicated that MPA was the best antagonist to estrogen action in neurons that we had thus far identified. Unlike progesterone, which was neuroprotective alone and synergistic with E2, MPA was neither neuroprotective nor did it synergize with E2, quite the contrary. MPA antagonized E2-induced neuroprotection (37, 38). Moreover, MPA exacerbated glutamate excitotoxic-induced neuron death (39). Curiously, MPA did induce phosphorylated ERK without translocation to the nucleus but did block E2-induced ERK nuclear translocation (10). Results of the WHIMS trial in which the hormone therapy group (CEE plus MPA) had a 2-fold greater risk of developing Alzheimer’s disease strongly suggest that the addition of MPA in vivo or in vitro has deleterious outcomes for the brain (4).

Healthy cell bias of estrogen action hypothesis
Two common denominators transcend the analytic levels of observation from cellular in vitro paradigms to human clinical studies to observations of health outcomes of thousands of women using estrogen or hormone therapy. The first common denominator between the basic science and observational studies is that neurons/brains were healthy before exposure to estrogen or hormone therapy. Second, neurons/brains were exposed to estrogen or hormone therapy before exposure to neurological insults associated with Alzheimer’s disease. In contrast, in the WHIMS cohort of women, 65 yr of age or older, with no indicators of neurological disease but with variable health status, estrogen and hormone therapy for 5 yr increased the risk of developing Alzheimer’s disease. Thus, we posit a hypothesis of a healthy cell bias of estrogen action, which is manifested as a continuum. As neurological health progresses from healthy to unhealthy, so too do the benefits to adverse outcomes of estrogen or hormone therapy. If neurons are healthy at the time of estrogen exposure, their response to estrogen is beneficial for both neurological function and survival. In contrast, if neurological health is compromised, estrogen exposure over time exacerbates neurological demise. The healthy cell bias of estrogen action hypothesis provides a lens through which to assess the disparities in outcomes across the domains of scientific inquiry and to access future applications of estrogen and hormone therapeutic interventions.

Estrogen regulation of calcium signaling and the divergence point between benefit and harm
Our findings that Aß1–42-induced increase in resting calcium was prevented by low dose of E2 and not by the high dose of E2 is consistent with our findings on the impact of low vs. high dose of E2 on neuron survival. Moreover, these findings are consistent with a large body of evidence that indicate a pivotal role of calcium dysregulation during aging and in the neurodegenerative demise of neurons exposed to Aß1–42 (40, 41, 42). Calcium is a ubiquitous intracellular signal responsible for controlling numerous cellular processes (40). Exceeding the normal spatial and temporal boundaries for Ca2+ can result in cell death through both necrosis and apoptosis. Loss of [Ca2+]i homeostasis is implicated in several brain disorders, including stroke and severe epileptic seizures, and in the pathogenesis of Alzheimer’s disease (41, 42). Aging is associated with dysregulation of intracellular Ca2+ homeostasis, which is linked with or is causative for neurodegenerative disease (43, 44). The Ca2+ homeostasis dysregulation hypothesis of brain aging and neurodegeneration proposes that basal levels of [Ca2+]i increase in aging neurons and that restoration of calcium homeostasis is compromised (45, 46, 47). In earlier work, we discovered that in healthy neurons derived from aged rat hippocampi, E2 restored calcium homeostasis in aged neurons to that of neurons derived from middle aged rat hippocampi (48).

The mechanism by which E2 promotes calcium homeostasis converges on mitochondrial calcium dynamics. Results of our analyses indicate that E2 increased mitochondrial sequestration of Ca2+ in response to excitotoxic glutamate, which resulted in a decrease in [Ca2+]i and a concomitant rise in intramitochondrial Ca2+ content (10, 49). E2-induced increase in mitochondrial Ca2+ sequestration is temporally correlated with an increase in Bcl-2 expression, which could protect against deleterious effects of increasing mitochondrial Ca2+ levels (50, 51). We proposed that, by increasing mitochondrial Ca2+ uptake capacity and the Bcl-2-induced resistance to Ca2+-induced respiratory inhibition, E2 prevents neurodegenerative insults due to a loss in Ca2+ homeostasis. Further studies demonstrated that pretreatment of cultured hippocampal neurons derived from aged rat brain prevented age-related loss of Ca2+ homeostasis and restored the Ca2+ homeostatic capacity of hippocampal neurons to that of neurons derived from middle aged rat hippocampi (48). These findings from previous analyses coupled with those of the current study, indicate that low-dose E2 in a prevention mode of administration activates mechanisms that protect neurons against neurodegenerative insults that induce a loss of Ca2+ homeostatic control. Because high levels of E2 resulted in elevated calcium to levels similar to those produced by Aß1–42 without concomitant neuronal death, elevated calcium cannot be the sole cause of Aß1–42-mediated neuronal death. However, the data indicate that factors which increase intracellular [Ca2+]i, such as high-dose E2 or Aß1–42, will exacerbate neurodegeneration due to loss of Ca2+ homeostatic control. These findings have obvious therapeutic implications for both the use of estrogen therapy and for control of Ca2+ homeostasis in aging or diseased neurons.

Conclusion
In summary, the profound disparities between the largely positive basic science findings of gonadal steroid action in brain and the adverse outcomes of recent hormone therapy clinical trials in women who are either aged postmenopausal or postmenopausal with Alzheimer’s disease, could be explained by a healthy cell bias of estrogen action. Model systems that simultaneously investigate the impact of gonadal hormones administered under either prevention or treatment conditions can be highly relevant to the target population, menopausal women considering the health benefits and risks of hormone therapy. The availability of both in vitro and in vivo model systems for Alzheimer’s disease are readily available to the scientific community and can be rapidly deployed to rigorously test the healthy cell bias of estrogen action hypothesis. Should the data support the hypothesis, these findings would provide the scientific foundation upon which clinical decisions regarding appropriate conditions for estrogen and hormone therapeutic interventions can be developed.


   Acknowledgments
 
This work was supported by the National Institutes of Aging (2PO1 AG14751-09; Project 2), the Kenneth T. and Eileen L. Norris Foundation, and the L. K. Whittier Foundation to R.D.B.


   Footnotes
 
Disclosure statement: The authors have nothing to disclose.

First Published Online August 17, 2006

Abbreviations: Aß1–42, ß-Amyloid 1–42; [Ca2+]i, intracellular Ca2+ concentration; CEE, conjugated equine estrogen; E2, 17ß-estradiol; MPA, medroxyprogesterone acetate; NBM, neurobasal medium; WHIMS, Women‘s Health Initiative Memory Study.

Received April 17, 2006.

Accepted for publication August 8, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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