An Estrogen Replacement Therapy Containing Nine Synthetic Plant-Based Conjugated Estrogens Promotes Neuronal Survival

Abstract

Epidemiological data from retrospective and case–control studies have indicated that estrogen replacement therapy can decrease the risk of developing Alzheimer’s disease. In addition, estrogen replacement therapy has been found to promote neuronal survival both in vivo and in vitro. We have shown that conjugated equine estrogens (CEE), containing 238 different molecules composed of estrogens, progestins, and androgens, exerted neurotrophic and neuroprotective effects in cultured neurons. In the current study, we sought to determine whether a steroidal formulation of nine synthetic conjugated estrogens (SCE) chemically derived from soybean and yam extracts is as effective as the complex multisteroidal formulation of CEE. Analyses of the neuroprotective efficacy indicate that SCE exhibited significant neuroprotection against beta amyloid, hydrogen peroxide, and glutamate-induced toxicity in cultured hippocampal neurons. Indices of neuroprotection included an increase in neuronal survival, a decrease in neurotoxin-induced lactate dehydrogenase release, and a reduction in neurotoxin-induced apoptotic cell death. Furthermore, SCE was found to attenuate excitotoxic glutamate-induced [Ca²⁺]_i rise. Quantitative analyses indicate that the neuroprotective efficacy of SCE was comparable to that of the multisteroidal CEE formulation. Data derived from these investigations predict that SCE could exert neuroprotective effects comparable to CEE in vivo and therefore could reduce the risk of Alzheimer’s disease in postmenopausal women.

Increasing evidence suggests that a loss in estrogen can increase a women’s risk of developing Alzheimer’s disease (AD) (1, 2), a neurodegenerative condition characterized by a progressive loss of cognitive capacity manifested as a deterioration of memory function throughout the course of the disease and the development of psychotic symptoms later in the disease process (3, 4). Epidemiological data from retrospective and case–control studies have indicated that postmenopausal women receiving estrogen replacement therapy (ERT) exhibit a decreased risk of developing AD (1, 3–8). Because women have a greater life span than men (mean age of 79.5 and 74.1, respectively, National Vital Statistics Reports, 2000) and because age is the greatest risk factor for AD (9), it would be anticipated that women have a greater incidence of this degenerative disease (3, 4). Although this is indeed true, age is not the only contributing variable. There appears to be an impact of gender on risk of AD. In age-matched analyses, women were still two to three times more likely to develop AD than their age-matched male counterparts (9). In vitro analyses have shown that 17β-estradiol and select forms of ERT can protect neurons against damage induced by beta amyloid (Aβ), hydrogen peroxide (H₂O₂), and glutamate (10–13).

Data from a number of clinical studies indicate that a loss of estrogen either through surgical or natural menopause results in a decline in memory function and that ERT can reverse menopause-associated memory deficits (9, 14–21). In vitro analyses from our laboratory and others have shown that 17β-estradiol can also significantly increase the outgrowth of neuronal projections and the formation of neural circuits in brain regions critical for memory function (12, 13, 22–27).

A formulation of conjugated equine estrogens (CEE) containing 238 different molecules composed of estrogens, progestins, and androgens (marketed under the trade name Premarin™), is the most frequently prescribed ERT in the United States (12). CEE is the ERT of the estrogen only arm of the Women’s Health Initiative studies of the National Institutes of Health, a large-scale and long-term clinical trial. Our previous research demonstrated that the CEE formulation of ERT exerts significant neuroprotective effects against toxic insults associated with AD in cultured neurons derived from brain regions affected by the disease (12). Furthermore, we found that CEE significantly enhances neuronal process outgrowth, a marker of memory formation (12).

In the present study, we sought to determine whether a select combination of estrogenic steroids derived from plant (soybean and yam) sources would exert neuroprotective effects comparable to those found for a multisteroidal formulation of ERT, CEE. To address this issue, we used the relatively newly developed ERT marketed under the trade name of Cenestin™, which is composed of nine estrogens chemically synthesized from a starting material derived from soybean and yam extracts. These nine estrogens within synthetic conjugated estrogens (SCEs) are also contained within CEE and include sodium estrone and sodium equilin which account for 84.8% of the formulation and seven other estrogenic compounds (17α-dihydroequilin, 17β-dihydroequilin, 17α-estradiol, 17β-estradiol, equilenin, 17α-dihydroequilenin, and 17β-dihydroequilenin), which account for the remaining 15.2% of the formulation (28). This formulation of ERT is FDA approved for the treatment of hot flashes, night sweats and other moderate-to-severe vasomotor symptoms associated with menopause (28).

Although this formulation of ERT has been proven effective in the prevention of vasomotor spasms, it is not yet known whether it will exert neuroprotection against toxic insults associated with neurodegenerative diseases. In the present study, we sought to determine whether SCE would exert neuroprotection against Aβ, H₂O₂, and glutamate-induced toxicity comparable with that observed with CEE. Four experimental strategies were used in the assessment of neuroprotective effects of SCE: neuron survival, release of lactate dehydrogenase (LDH), apoptotic neuron death, and attenuation of rise in [Ca²⁺]_i after exposure to excitotoxic concentration of glutamate.

Materials and Methods

Neuronal Culture.

Use of animals has been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Southern California (Protocol No. 9052). Primary cultures of hippocampal neurons were generated as described in Brinton (12). Briefly, hippocampi were dissected from the brains of embryonic day 18 (E18 d) Sprague–Dawley rat fetuses, treated with 0.02% trypsin in Hank’s balanced salt solution (137 mM NaCl, 5.4 mM KCL, 0.4 mM KH₂PO₄, 0.34 mM Na₂HPO₄•7H₂0, 10 mM glucose, 10 mM HEPES) for 5 min at 37°C and dissociated by repeated passage through a series of fire polished constricted Pasteur pipettes. For morphological analyses between 20,000 and 40,000 cells were seeded onto poly-D-lysine-coated (10 μg/ml) 22-mm round coverslips, 23-mm gridded coverslips, or 4-well chamber slides, whereas cultures used for LDH analysis were plated onto poly-D-lysine-coated 24-well culture plates at a density of 10⁵ neurons/ml. Nerve cells were grown in Neurobasal medium (NBM; Invitrogen Corp., Carlsbad, CA) supplemented with B27, 5 U/ml penicillin, 5 μg/ml streptomycin, 0.5 mM glutamine, and 25 μM glutamate, at 37°C in 10% CO₂. Cultures grown in serum-free neurobasal medium (NBM) yields approximately 99.5% neurons and 0.5% glia. Microscopically, glial cells were not apparent in hippocampal cultures at the times these cultures were used for experimental analyses. The culture media were exchanged with glutamate-free NBM 3 days after the day of cell culture and the hippocampal neurons were fed with glutamate-free NBM twice weekly.

Neuronal Survival Analysis.

For analyses of neuronal survival, between 20,000 and 40,000 hippocampal neurons were seeded onto poly-D-lysine-coated 23-mm coverslips with a grid size of approximately 600 μm² composed of 520 alphanumeric grids. Neurons were treated with 10 ng/ml SCE or CEE beginning at 3 days of age and exposed for a total of 4 days before exposure to beta amyloid peptide_25–35 (Aβ_25–35). Neurons were counted at 7 days of age and grids with viable neurons were selected for analysis over the entire period of the experiment. One hundred neurons per coverslip were selected for study and there were three coverslips per condition for a total of 300 analyzed neurons per condition per experiment. After the time 0 neuron count, cultures were exposed to 8 μg/ml Aβ_25–35 in culture media with SCE or CEE for 48 hr. Viable neurons in the same grid were counted again after Aβ_25–35 exposure by morphological criteria: smooth, round neuronal soma with extensive neuritic arborizations. Neuronal viability was determined by three morphological criteria, phase brightness, possession of at least one or more neurites longer than the diameter of the cell body, and granulation-free neurites as described by Mattson (29). Statistically significant differences were determined by a one-way analysis of variance followed by a Newman-Keuls post-hoc analysis for individual comparisons.

Efflux Assay of LDH.

Neurotoxins used included 0.1 mM glutamate, 8 μg/ml Aβ_25–35, and 20 μM H₂O₂. Neuronal cultures grown in 24-well plates were pretreated with varying concentrations of SCE or 10 ng/ml CEE for 4 days prior to exposure to glutamate at room temperature for 5–10 min or to H₂O₂ at 37°C for 15 min in HEPES-buffered saline solution (HBSS) containing (in mM) 100 NaCl, 2.0 KCl, 2.5 CaCl₂, 1.0 MgSO₄, 1.0 NaH₂PO₄, 4.2 NaHCO₃, 12.5 HEPES and 10.0 glucose, or to Aβ_25–35 for 48 hr in NBM media. After exposure to glutamate and H₂O₂, cultures were washed twice with HBSS and replaced with fresh NBM media. Cultures were then returned to the incubator and assessment of LDH release in the media conducted 24 hr after exposure to the neurotoxins. LDH release into the culture media was measured using a Cytotoxicity Detection Kit from Boehringer Mannheim Biochemicals and absorption was read at 490 nm. Statistically significant differences between conditions were determined by a one-way analysis of variance followed by a Newman–Keuls post-hoc analysis for individual comparisons.

Glutamate Exposure.

One-week-old hippocampal neuronal cultures pretreated with varying concentrations of SCE or 10 ng/ml CEE for 4 days were exposed to 100 μM glutamate for 5–10 min at room temperature in HBSS. Immediately following glutamate exposure, cultures were washed twice with HBSS and replaced with fresh NBM. Cultures were returned to the culture incubator and allowed to incubate for 24 before LDH measurement.

Aβ_25–35 Exposure.

Aβ_25–35 was dissolved in sterile distilled water at a concentration of 1μg/ml as a stock solution. This stock was aliquoted and stored at −20°C. One-week-old hippocampal neuron cultures pretreated with varying concentrations of SCE or 10 ng/ml CEE for 4 days in NBM were exposed to 8 μg/ml Aβ_25–35 in the presence or absence of SCE or CEE, following the procedure described by Behl et al. (30). Briefly, NBM containing Aβ_25–35 alone or Aβ_25–35 plus varying concentrations of SCE or 10 ng/ml CEE were added to the neuronal cultures and the cultures were incubated in test substances for 48 hr at 37°C followed by LDH measurement.

H₂O₂ Exposure.

Dilution of H₂O₂ was made fresh from a 30% stock solution into HBSS just before each experiment. One-week-old hippocampal neuronal cultures pretreated with varying concentrations of SCE or 10 ng/ml CEE for 4 days were exposed to 20 μm H₂O₂ in HBSS for 15 min at 37°C. During exposure, SCE or CEE was added concurrently with H₂O₂. After 15 min the cultures were rinsed twice with HBSS, and fresh medium with SCE or CEE was added to the culture. Cultures were returned to the culture incubator and allowed to incubate for 24 h before LDH measurements on the next day.

TdT-Mediated dUTP-X Nick End Labeling (TUNEL) Analysis.

Apoptosis induced by neurotoxic insults was determined using TUNEL. Neurotoxins used included beta amyloid_1–42 (Aβ_1–42) and H₂O₂. Aβ_1–42 was dissolved in 10 mM HCl at a concentration of 1 mM as a stock solution. This stock was stored at −20°C and was aggregated in 0.1 M phosphate buffer (PBS) for 3 days before use. Neuronal cultures grown on four-well chamber slides were pretreated with 10 ng/ml SCE or CEE for 4 days before exposure to Aβ_1–42 or H₂O₂. Pretreated hippocampal neurons were incubated with either pre-aggregated Aβ_1–42 for 3 days or exposed to 20 μM H₂O₂ for 15 min followed by a 24-hr incubation. The treated cells were rinsed with PBS and fixed with 95% methanol for 5 min at 4C°. Subsequently, neurons were incubated with the TUNEL reaction mixture (In Situ Cell Death Detection Kit [Fluorescein], Boehringer Mannheim Biochemicals, Indianapolis, IN) for 60 min at 37°C. After washing with PBS for three times, neurons were mounted with Mounting Medium containing DAPI (Vector Laboratories, Inc., Burlingame, CA) to stain the nucleus. Apoptosis was qualified by fluorescence microscopy. The percentage of apoptotic cells was calculated by counting TUNEL-positive and DAPI-stained (total) cells individually in three high-power (400×) fields per treatment.

Fura-2 Intracellular Ca²⁺ Imaging.

The [Ca²⁺]i in hippocampal neurons was measured by ratiometric Ca²⁺ imaging with the Ca²⁺-sensitive fluorescence dye fura-2. Before imaging, neurons were loaded with 2 μM fura-2 acetoxymethyl ester for 30–45 min at 37°C in HEPES-Buffered Solution (HBS), containing (in mM): 100 NaCl, 2.0 KCl, 1.0 CaCl₂, 1.0 MgCl₂, 1.0 NaH₂PO₄, 4.2 NaHCO₃, 12.5 HEPES, and 10.0 glucose. Excess fura-2 dye was removed by washing with HBS buffer and then the neurons were incubated in HBS buffer for 30 min at 37°C to equilibrate. The coverslip with fura-2 AM-loaded neurons was removed and attached to the perfusion cell chamber for imaging. Coverslips were then placed onto coverslip clamp chamber MS-502S (ALA Scientific Instruments, Westbury, NY) for the Ca²⁺ imaging analysis. Fluorescence measurements of [Ca²⁺]_i were performed using the InCyt2^TM fluorescence imaging system (Intracellular Imaging, Inc., Cincinnati, OH). Neurons were placed on the stage of an inverted microscope (MT-2, Olympus) equipped with epifluorescence optics (20× , Nikon). The perfusion solution is HBS and the perfusion system connected to the perfusion chamber was balanced using two variable speed pumps. Imaging was performed at room temperature. Fluorescence was excited at wavelengths of 340 and 380 nm alternatively using a rotating 4-wheel filter changer. The Ca²⁺ concentration curve was recorded during the experiment or images were saved for analysis after the experiment. To minimize the background noise of the fura-2 signal, successive values (16 sample images, 8 background images) were averaged (13 images / min). After 30 s of basal image acquisition, 100 μM glutamate was perfused into the chamber throughout the remaining observation time. To exclude any effect of mechanical stimulation, control HBS buffer was perfused without glutamate and the Ca²⁺ response observed for 5 min, and no [Ca²⁺]_i rise was observed following the perfusion of HBS buffer.

Chemicals.

Aβ_25–35 was obtained from Bachem, CA. Aβ_1–42 was purchased from U.S. Peptide, INC. SCE was acquired in liquid concentrate form from Duramed Pharmaceuticals Inc., at a concentration of 125 mg/ml of the estrogenic formulation, stored at 2–8°C, diluted to final concentration in culture media just before use. CEE was acquired in lyophilized form from the University of Southern California Health Science Campus Pharmacy in 5-ml ampoules of the therapeutic formulation Premarin^™ containing 25 mg of the estrogenic formulation, 200 mg lactose, 12.5 mg sodium citrate, and 0.2 mg simethicone, stored at room temperature, dissolved in sterile H₂O before use and diluted to final concentration in culture media. Inert components, including 200 mg of lactose, 12.5 mg sodium citrate, and 0.2 mg simethicone, were obtained from Wyeth-Ayerst laboratories as vehicle for control groups. Fura-2 AM was purchased from Molecular Probes. Cytotoxicity Detection Kit (for LDH analysis) and the In Situ Cell Death Detection Kit (for TUNEL analysis) were purchased from Boehringer Mannheim Biochemicals.

Results

Neuroprotective Efficacy of SCE against Aβ.

The first series of neuroprotection experiments investigated the impact of SCE on hippocampal neuron morphological integrity and survival before and after a 48-hr exposure to the toxic peptide Aβ_25–35. Videomicroscopy was performed, and upon microscopic inspection, neurons prior to exposure to Aβ_25–35 under different conditions exhibited markers of viable neurons with smooth dark nerve cell bodies, phase bright halos and extensive neuronal extensions. After 48 hr exposure to 8 μg/ml Aβ_25–35, degeneration of cultured hippocampal neuron processes and toxicity were apparent (Fig. 1). Neurons pretreated for 4 days with 10 ng/ml of SCE exhibited no or less Aβ_25–35-induced degeneration of hippocampal neuronal processes (Fig. 1 ).

In an attempt to determine whether SCE impacted hippocampal neuron survival comparable to that induced by CEE, quantitative analysis of neuronal survival was conducted (Fig. 2 ). After exposure to 8 μg/ml Aβ_25–35 for 48 hr, the percentage of viable neurons significantly decreased compared to the control condition (95.33 ± 1.76% for control group and 62.11 ± 2.39% for Aβ_25–35 group, ⁺ P < 0.01). Neurons pretreated with 1, 10, or 100 ng/ml SCE for 4 days significantly increased the number of viable neurons compared to Aβ_25–35 alone group (92.43 ± 1.59%, 83.11 ± 1.76%, and 91.24 ± 1.23% respectively, **P < 0.01). The SCE-induced magnitude of survival was comparable to 10 ng/ml CEE-induced neuronal survival (87.06 ± 1.95%, **P < 0.01). Together, results of these experiments indicate that SCE exerted significant protective effect against Aβ_25–35-induced cell death comparable with that of CEE.

Analysis of LDH release was conducted to investigate the effect of SCE on neuronal membrane integrity following exposure to Aβ_25–35 (Fig. 3 ). Hippocampal neurons were pretreated with SCE at five different concentrations (0.01, 0.1, 1, 10, and 100 ng/ml) for 4 days prior to exposure to 8 μg/ml Aβ_25–35 peptide for 48 hr. Within the same experiment, 10 ng/ml CEE was used as positive control. Relative to Aβ_25–35 alone, SCE induced significant reduction in LDH release at 0.1, 1, 10, and 100 ng/ml (64.67 ± 10.19%, 58.56 ± 7.68%, 68.67 ± 5.92%, and 70.56 ± 6.67%, respectively, **P < 0.01). The reduction in LDH release was comparable to that induced by 10ng/ml CEE (55.22 ± 6.23%, **P < 0.01).

TUNEL staining was used to detect DNA damage in neurons undergoing apoptotic cell death. The impact of SCE and CEE pretreatment on Aβ_1–42-induced apoptotic cell death was investigated in cultured hippocampal neurons. In addition, DAPI was used to label neuronal nuclei to quantitate the total number of neurons in the field (Fig. 4A ). Fluorescent images of TUNEL staining show that Aβ_1–42 induced a marked increase in the number of neurons undergoing apoptosis. SCE alone was indistinguishable from the control culture. SCE at 10 ng/ml greatly reduced the number of TUNEL-positive cells induced by Aβ_1–42, comparable to that induced by 10 ng/ml CEE (Fig. 4A ). To quantify the neuroprotective effect of SCE and CEE against Aβ_1–42-induced apoptosis, TUNEL-positive and DAPI-stained neurons were counted and the percentage of TUNEL-positive neurons calculated (Fig. 4B ). Statistical analysis of Mean ± SEM from three separate experiments indicates that Aβ_1–42 significantly increased the percentage of apoptotic cells compared to control (3.87 ± 0.56% for control group and 39.21 ± 8.29% for Aβ_1–42-treated group, ⁺ P < 0.01). SCE induced a near 50% decrease in the percentage of neurons undergoing apoptosis compared to Aβ_1–42 alone (20.02 ± 4.98% for SCE-treated group, 39.21 ± 8.29% for Aβ_1–42-treated group, **P < 0.01), which was comparable to CEE-mediated decrease in Aβ_1–42-induced apoptosis (13.93 ± 3.93%, **P < 0.01).

Neuroprotective Efficacy of SCE Against H₂O₂.

H₂O₂ acts as a precursor for the hydroxyl free radical, HO⁻, and is well documented to be a free radical generator (31). H₂O₂-induced damage was selected for investigation as oxidative damage in brain significantly increases with age and is hypothesized to contribute to the degenerative profile of AD (32–34).

Biochemical analysis of the impact of SCE on LDH release following H₂O₂ exposure was investigated (Fig. 5). Hippocampal neurons were pretreated with SCE at five different concentrations (0.01, 0.1, 1, 10, and 100 ng/ml) for 4 days prior to exposure to 20 μM H₂O₂ for 15 min. Within the same experiment, 10 ng/ml CEE was used as positive control. Relative to H₂O₂ alone, SCE induced significant reduction in LDH release at 1, 10, and 100 ng/ml (82.27 ± 3.82%, 73.27 ± 6.69%, and 76.67 ± 6.34%, respectively, *P < 0.05, **P < 0.01). CEE at 10 ng/ml induced a greater reduction in LDH release (56.00 ± 3.82% compared with H₂O₂ alone, **P < 0.01), suggesting that CEE might exert greater efficacy as a free radical scavenger than SCE.

TUNEL analysis was conducted to determine H₂O₂-induced apoptotic cell death and the impact of SCE and CEE. TUNEL-positive neurons were labeled with fluorescein while DAPI staining was used to label neuronal nuclei to quantitate the total number of neurons in the field (Fig. 6A and B ). H₂O₂ induced a marked increase in the percentage of neurons undergoing apoptosis (1.94 ± 0.90% for control group and 44.36 ± 3.26% for H₂O₂-treated group, ⁺ P < 0.01). The percentage of TUNEL-positive hippocampal neurons in cultures treated with SCE in the absence of H₂O₂ was indistinguishable from that of control neurons. A significant reduction in TUNEL-positive neurons occurred in cultures pretreated with SCE prior to exposure to H₂O₂ (25.30 ± 5.48%, **P < 0.01). The reduction of TUNEL-positive neurons in the SCE treated cultures was lower than that observed in cultures pretreated with 10 ng/ml CEE (12.46 ± 1.66%, **P < 0.01), suggesting again that CEE might exert greater efficacy as a free radical scavenger than SCE.

Neuroprotective Efficacy of SCE Against Excitotoxic Glutamate.

Glutamate-induced excitotoxicity is a multifaceted process that includes excessive influx of Na⁺ and Ca²⁺ into neurons through glutamate activated cation channels (35). The influx of Ca²⁺ can lead to a lethal rise in intracellular Ca²⁺ and the generation of free radicals (36). Although free radicals can induce neuronal damage directly, they also induce secondary and tertiary events that further decrease the viability and survival of neurons. Studies by Mattson and his co-workers have shown that generation of reactive oxygen species impairs ion-motive ATPase activities, which can lead to a disruption of Ca²⁺ homeostasis and cell death (37). This series of catastrophic events has been postulated to be a precursor to the toxicity induced by glutamate. Moreover, glutamate-induced excitotoxicity has been proposed to be one of the contributing factors to the neurodegenerative characteristic of AD and other neuropathological states (38, 39), which led us to investigate the impact of SCE on glutamate-induced toxicity.

Videomicroscopy was performed to investigate the impact of SCE on hippocampal neuron morphological integrity and survival prior to and following exposure to 100 μM glutamate. Upon microscopic inspection, neurons prior to glutamate exposure exhibited markers of viability with smooth dark cell bodies, phase bright halos and extensive neuronal extensions. After exposure to excitotoxic glutamate, degeneration of cultured hippocampal neuron processes was apparent (Fig. 7). Those neurons pretreated for 4 days with 10 ng/ml of SCE exhibited features of viability comparable to control neurons with smooth dark cell bodies, phase bright halos and extensive neuronal extensions.

Quantitative biochemical assessment of plasma membrane damage demonstrated that SCE significantly reduced glutamate-induced LDH release (Fig. 8 ). Hippocampal neurons were pretreated with SCE at five different concentrations (0.01, 0.1, 1, 10, and 100 ng/ml) for 4 days prior to exposure to 100 μM glutamate for 5 min. Within the same experiment, 10 ng/ml CEE was used as positive control. Relative to 100 μM glutamate alone, SCE induced a significant reduction in LDH release at 0.1, 1, 10, and 100 ng/ml (83.93 ± 3.41%, 79.93 ± 4.82%, 69.02 ± 6.91%, and 77.27 ± 8.03%, respectively, *P < 0.05, **P < 0.01), which was comparable to 10 ng/ml CEE-induced reduction in LDH release (58.27 ± 5.31%, **P < 0.01).

Attenuation of Excitotoxic Glutamate-Induced [Ca²⁺]_i by SCE.

Ca²⁺ influx plays an important role in neuronal damage and death induced by glutamate. To test the hypothesis that SCE protects neurons against glutamate-induced excitotoxicity by attenuating glutamate-induced rise in [Ca²⁺]_i, intracellular Ca²⁺ imaging was performed using the Ca²⁺-sensitive fluorophore fura-2. Primary hippocampal neurons were pretreated with 10 ng/ml SCE for 2 days prior to monitoring [Ca²⁺]_i in response to 100 μM glutamate. Glutamate induced a rapid increase in [Ca²⁺]_i that was significantly attenuated by pretreatment of 10 ng/ml SCE (Fig. 9A ). Quantitative analysis of [Ca²⁺]_i indicated that 100 μM excitotoxic glutamate induced a rise of [Ca²⁺]_i to approximately 500 nM in control neurons, while in neurons pretreated with 10ng/ml of SCE for 2 days, the [Ca²⁺]_i rise induced by glutamate was significantly lower (approximately 340 nM; Fig. 9B ). Note that SCE treatment diminished the magnitude of [Ca²⁺]_i rise without altering the onset time of the Ca²⁺ response.

Attenuation of excitotoxic glutamate-induced [Ca²⁺]_i rise by SCE was compared to that induced by CEE (Fig. 10A ). The magnitude of SCE-induced attenuation to 100 μM glutamate-induced [Ca²⁺]_i was comparable to that observed with CEE. Furthermore, statistical analyses of two experiments with at least 25 neurons per condition per experiment indicate that neurons treated with SCE or CEE for 2 days exhibited a comparable reduction of glutamate-elevated [Ca²⁺]_i relative to control neurons (69.52 ±11.34% and 71.12 ± 6.82% of glutamate alone respectively, *P < 0.05; Fig. 10B ).

Discussion

The purpose of this study was to determine whether SCE, an estrogenic formulation containing nine synthetic plant-based conjugated estrogens, could exert a neuroprotective effect in cultured hippocampal neurons against three different neurotoxic agents, Aβ (including Aβ_25–35 and Aβ_1–42), H₂O₂ and excitotoxic glutamate. Each of these neurotoxic agents was selected because of their role in degeneration associated with AD (40, 41). Three experimental strategies, neuronal survival, LDH release and neuronal apoptosis, were conducted to determine the neuroprotective action of SCE. To assess neuronal survival, neurons were monitored and individually analyzed for survival following the criteria described by Mattson et al. (29). As a biochemical marker, LDH was used to assess plasma membrane damage. LDH is a stable cytoplasmic enzyme present in all cells including neurons and is rapidly released into media when the plasma membrane is damaged. To assess early stage neuroprotective effect, apoptotic cell death in response to Aβ_1–42 and H₂O₂ was determined. Furthermore, because excitotoxic glutamate-induced cell death is due to an increased level of [Ca²⁺]_i (35, 42), intracellular Ca²⁺ imaging was conducted to determine the mechanism of SCE-mediated neuroprotection against glutamate.

Results of these analyses indicate that SCE induced significant neuroprotection against each of the neurotoxins investigated. SCE-induced neuroprotection was manifested by an increase in viable neurons, a decrease in LDH release and a decrease in the number of neurons undergoing apoptotic cell death. Furthermore, the neuroprotective efficacy of SCE was comparable to that of CEE, a much more multisteroidal formulation. These data provide the basis for the prediction that SCE could decrease the risk of developing AD in postmenopausal women. In addition, SCE was found to attenuate excitotoxic glutamate-induced [Ca²⁺]_i rise. One of the mechanisms for glutamate-induced neurotoxicity is elevated intracellular Ca²⁺. Reports in the literature of the magnitude of [Ca²⁺]_i induced by 100 μM excitotoxic glutamate vary between 200 nM to over 1 μM, with the majority falling around 500 nM (43–46). In our system, excitotoxic level of glutamate (100 μM) induces an intracellular Ca²⁺ rise to approximately 500 nM, which is comparable to that most consistently reported in the literature. In neurons pretreated with SCE the excitotoxic glutamate-induced rise in intracellular Ca²⁺ concentration is decreased to approximately 340 nM, which falls close to a nontoxic [Ca²⁺]_i level. It is well known that the high [Ca²⁺]_i induced by high concentrations of glutamate is a key component of glutamate-induced cytotoxicity (35, 36). The attenuation of the excitotoxic glutamate-induced rise in [Ca²⁺]_i to near nontoxic range provides a probable mechanism by which SCE reduces the glutamate cytotoxicity and promotes neuronal survival. Furthermore, a strong correlation exists between neuroprotective molecules and attenuation of glutamate-induced intracellular Ca²⁺ rise in literature. For example, neuroprotective molecules conjugated equine estrogens, 17β-estradiol, progesterone and 19-norprogesterone attenuate excitotoxic glutamate-induced intracellular Ca²⁺ rise, whereas nonneuroprotective molecules such as medroxyprogesterone acetate do not (12, 47, 48).

A small degree of variance has been observed in the data sets. For example, most of the results for SCE (Figs. 2, 3 , and 5 ) do not display a doses-dependent response, whereas the results in Figure 8 do. Several possible explanations can account for this variability. First of all, three different neurotoxic agents, β-amyloid, H₂O₂, and glutamate, were used in this study, and the molecular mechanisms by which these molecules are toxic are different. For example, H₂O₂ directly causes oxidative stress in the neurons whereas glutamate toxicity initiates toxicity by exceeding calcium buffering capacity of the neuron and mitochondria, which leads to accumulation of free radicals within mitochondria. Second, different analyses (survival, LDH release and TUNEL) were used to determine neuroprotection, and these different metrics measure different cellular responses. For example, LDH determines the integrity of neuronal membrane whereas TUNEL staining is an indicator of strand breaks in DNA, which is a prelude to apoptosis. Finally, the mechanisms by which SCE exerts neuroprotection against these three neurotoxins may also differ as described below.

Potential Mechanisms of SCE-Induced Neuroprotection

Mounting evidence indicates that nuclear estrogen receptors are involved in estrogen-induced neuroprotection. The ERα subtype has been suggested to be a critical mechanistic link in mediating the protective effects of physiological levels of 17β-estradiol as deletion of ERα completely abolishes the protective actions of estradiol in all regions of the brain; whereas the ability of 17β-estradiol to protect against brain injury is totally preserved in the absence of ERβ (49). Furthermore, in vitro evidence suggests that transfection of HT22 cells with human ERα, but not ERβ, restores the protective effect of estrogen against Aβ (50). However, other studies have demonstrated otherwise, suggesting that ERβ is involved in estrogen-mediated effect in the brain. Gustafsson’s group reported that the brains of ERβ knockout mice show several morphological abnormalities (51) and disruption of ERβ gene impairs spatial learning in female mice (52), suggesting that ERβ could influence the development of degenerative diseases of the central nervous system, such as AD. Based on these data, a reasonable hypothesis is that both ER receptor subtypes are required for the full estrogen effect in the brain. We are currently pursuing the estrogen receptor subtype(s) necessary for SCE neuroprotection against toxic insults.

Several other mechanisms may underlie estrogen-inducible neuronal protection against toxic insults such as Aβ, H₂O₂ and glutamate toxicity. One of the common mechanisms by which these neurotoxic agents affect neurons is their ability to trigger downstream oxidative processes by the generation and accumulation of reactive oxygen and nitrogen species, leading to excessive oxidation of cellular lipids, proteins and DNA (53). Oxidative stress is a well-described hallmark of neurodegenerative diseases and is also a by-product of the inflammatory processes in the brain (32). As the brain is particularly vulnerable to changes in the oxidative environment (32), the beneficial action of estrogens might be related to its capacity to interfere with oxidative neurotoxic insults. 17β-estradiol is a monophenolic compound that is similar to the lipophilic free-radical scavenger α-tocopherol (vitamin E). Phenolic structures are well-described inhibitors of lipid peroxidation in biochemical cell-free assays (54, 55). In addition, because SCE is an estrogenic formulation containing nine conjugated estrogens, an obvious question is which of these estrogens exert an antioxidant effect. Data from the Behl and Simpkins research groups would predict that hydroxylated estrogens at the three position of the A phenolic ring structure would exert a neuroprotective effect (11, 56). We are currently testing this hypothesis by investigating the neuroprotective effect of individual estrogenic components of the SCE formulation. These neuroprotective data on each estrogen will further shed light on the mechanisms of estrogen-mediated neuroprotection.

Although the full spectrum of the mechanisms required for estrogen-inducible neuroprotection remains to be elucidated, the Src/mitogen-activated protein kinase (MAPK) signaling cascade appears to be pivotal. Studies from our laboratory and others have demonstrated that Src kinase is activated rapidly after 17β-estradiol treatment (57, 58). Singh and Toran-Allerand reported that 17β-estradiol activated the MAPK signaling pathway in brain (59). Singer et al. followed up this observation and demonstrated that blockade of this pathway abolished 17β-estradiol-induced neuroprotection against glutamate-induced toxicity (60). Src/MAPK pathway can mediate anti-apoptotic signaling pathway through increased expression of the anti-apoptotic protein Bcl-2 (61, 62). The antiapoptotic protein Bcl-2 is localized to the mitochondrial membrane and its expression significantly enhances mitochondrial Ca²⁺ sequestration (63), which can account for SCE-induced attenuation of excitotoxic glutamate-induced [Ca²⁺]_i rise. In addition to increasing the magnitude of Ca²⁺ sequestration by mitochondria, Bcl-2 enhances the tolerability of mitochondria for increased levels of mitochondrial Ca²⁺ that otherwise result in dissipation of mitochondrial membrane potential and cell death (64). Another 17β-estradiol -activated pathway that potentially interacts with the estrogen-inducible MAPK signaling and confers neuroprotection is the Akt/protein kinase B pathway. Recently, Singh reported estradiol activation of the Akt/Protein kinase B, which can mediate anti-apoptotic signaling through increased expression of Bcl-2 (62).

It is not yet determined whether the neuroprotection conferred by SCE is mediated by a genomic mechanism, a scavenging antioxidant mechanism or by activation of intracellular signaling cascades and subsequent kinase activation and gene expression. We are currently pursuing SCE activation of these and other signaling pathways in an effort to determine the mechanisms of SCE-induced neuroprotection against each of the neurotoxic agents investigated in the current study.

The current study demonstrated that SCE and CEE induced comparable neuroprotective effects against neurotoxic agents. Furthermore, the magnitude of SCE-exerted neuroprotection observed in the current study is comparable to the published CEE effect (12). Since the neuroprotective efficacy of SCE is provided by a formulation of nine estrogens while CEE’s neuroprotection is provided by a complex multisteroidal formulation of estrogens, progestins and androgens, the comparability of SCE and CEE-exerted neuroprotection suggests that additional steroid components within CEE are not required for the neuroprotective efficacy.

In conclusion, the present data indicate that SCE exerted significant neuroprotective efficacy against a broad range of neurotoxins. These in vitro data provide a plausible mechanism by which ERT could sustain neuronal integrity in the presence degenerative insults associated with AD. These data lead to the prediction that SCE could reduce the risk of developing AD by promoting neuronal survival in the face of neurotoxic insults. A definitive test of this prediction requires a randomized placebo-controlled double-blind clinical trial.

Figure 1.

Neuroprotective effect of SCE against Aβ_25–35-induced toxicity in cultured hippocampal neurons. Hippocampal neurons pretreated with 10 ng/ml SCE for 4 days were exposed to 8 μg/ml Aβ_25–35 for 48 hr. Photomicrographic images show fields of hippocampal neurons grown under the indicated conditions. Hippocampal neurons under control conditions exhibit indicators of viability, with dark cell bodies, phase bright halos, and abundant clearly defined neuronal processes. Hippocampal neurons exposed to 8 μg/ml Aβ_25–35 for 48 hr exhibit indicators of degeneration with shrunken cell bodies and fragmented neuronal processes. Hippocampal neurons grown in the presence of 10 ng/ml SCE and then exposed to Aβ_25–35 exhibit features of neuronal viability comparable to those of control neurons with dark cell bodies, phase bright halos and abundant clearly defined neuronal processes. Scale bar = 100 μm.

Figure 2.

Impact of SCE on survival of Aβ_25–35-treated hippocampal neurons. Hippocampal neurons were treated with indicated concentrations of SCE or 10 ng/ml CEE for 4 days before exposure to 8 μg/ml Aβ_25–35. Viable neurons on randomly selected grids were counted one hour prior to Aβ_25–35 exposure and following 48 hr of Aβ_25–35 exposure_. SCE significantly enhanced neuronal survival following exposure to Aβ_25–35 comparable to that induced by CEE. Data are presented as percent of control neuron survival (mean ± SEM from 3 separate experiments, 100 neurons were counted per neuronal culture, seven to eight cultures were analyzed/condition experiment). ⁺ P < 0.01 compared with control cultures, ** P < 0.01 compared with Aβ_25–35-treated cultures.

Figure 3.

Impact of SCE on Aβ_25–35-induced LDH release. Hippocampal neuronal cultures were treated with varying concentrations of SCE or 10 ng/ml CEE for 4 days followed by exposure to 8μg/ml Aβ_25–35 for 48 hr. The media was then harvested from all cultures and assayed for LDH content. SCE induced a significant decrease in LDH release after Aβ_25–35 exposure comparable to that induced by CEE. Results are expressed as mean ± SEM percent of Aβ_25–35-induced LDH release and were combined across three separate experiments, n = 12 per condition. ** P < 0.01 compared with Aβ_25–35-treated cultures.

Figure 4.

Impact of SCE on Aβ_1–42-induced apoptosis in cultured hippocampal neurons. Hippocampal neuronal cultures were treated with 10 ng/ml SCE or CEE for 4 days and subsequently exposed to 1.5 μM Aβ_1–42 for 3 days. A, Fluorescent microscopy of TUNEL staining shows no or few apoptotic neurons in control and SCE or CEE-treated cultures. After exposure to Aβ_1–42, the number of TUNEL-positive cells was markedly increased, whereas in SCE or CEE-pretreated groups, the number of apoptotic cells were greatly reduced compared to Aβ_1–42-treated cultures. B, Percent of TUNEL-positive cells was calculated by counting TUNEL-positive and DAPI-stained neurons in 3 high power (400x) fields per condition. Data are expressed as Mean ± SEM from three separate experiments, approximately 50 neurons were counted per neuronal culture, three cultures were analyzed / condition / experiment. ⁺ P < 0.01 compared with control cultures; ** P < 0.01 compared with Aβ_1–42-treated cultures. Scale bar = 100 μm.

Figure 5.

Impact of SCE on H₂O₂-induced LDH release. Hippocampal neuronal cultures pretreated with varying concentrations of SCE or 10 ng/ml CEE for 4 days were exposed to 20μM H₂O₂ for 15 min followed by media exchange for H₂O₂-free NBM plus or minus SCE or CEE. The media was harvested from all cultures and assayed for LDH content 24 hr after H₂O₂ exposure. SCE significantly decreased H₂O₂-induced LDH release, comparable to CEE-exerted effect. Results are expressed as mean ± SEM percent of H₂O₂-induced LDH release and are combined across three separate experiments, n = 12 per condition. * P < 0.05, ** P < 0.01 compared with H₂O₂-treated cultures.

Figure 6.

Impact of SCE on H₂O₂-induced apoptosis in cultured hippocampal neurons. Hippocampal neuronal cultures were treated with 10 ng/ml SCE or CEE for 4 days and subsequently exposed to 20 μM H₂O₂ for 15 min, and TUNEL analysis was conducted 24 hr after H₂O₂ treatment. A, The fluorescent microscopy of TUNEL staining showed no or few apoptotic neurons in control and SCE or CEE-treated neurons. Exposure to H₂O₂ markedly increased the number of TUNEL-positive cells. In SCE or CEE-pretreated groups, the number of apoptotic cells was greatly reduced compared to H₂O₂-treated cultures. B, Percent of TUNEL-positive cells was calculated by counting TUNEL-positive and DAPI-stained neurons in 3 high power (400x) fields per condition. Data are expressed as Mean ± SEM from three separate experiments, approximately 50 neurons were counted per neuronal culture, 3 cultures were analyzed / condition / experiment. ⁺ P < 0.01 compared with control cultures; ** P < 0.01 compared with H₂O₂-treated cultures. Scale bar = 100 μm.

Figure 7.

Neuroprotective effect of SCE against glutamate-induced toxicity in cultured hippocampal neurons. Hippocampal neurons were treated with 10 ng/ml SCE for 4 days and subsequently exposed to 100 μM glutamate for 5 min followed by media exchange for glutamate-free NBM plus or minus SCE or CEE. Photomicrographic images were taken 24 hr following glutamate exposure. Hippocampal neurons under control conditions appear healthy with dark cell bodies, phase bright halos and abundant clearly defined neuronal processes. Hippocampal neurons exposed to 100 μM glutamate for 5 min and visualized 24 hr later display shrunken cell bodies and degeneration of neuronal processes. Neuronal cultures grown in the presence of 10 ng/ml SCE exhibit a marked reduction in features of degeneration and appear comparable to control neurons not exposed to excitotoxic glutamate. Scale bar = 100 μm.

Figure 8.

Impact of SCE on glutamate-induced LDH release. Hippocampal neuronal cultures were pretreated for 4 days with varying concentrations of SCE or 10 ng/ml CEE and subsequently exposed to 100μM glutamate for 5 min followed by media exchange for glutamate-free NBM plus or minus SCE or CEE. The media was harvested from all cultures and assayed for LDH content 24 hr after glutamate exposure. SCE induced significant decrease in LDH release following exposure to glutamate comparable to that induced by CEE. Results are expressed as mean ± SEM percent of glutamate-induced LDH release and are combined across three separate experiments, n = 12 per condition. * P < 0.05; ** P < 0.01 compared with glutamate-treated cultures.

Figure 9.

SCE-induced attenuation of excitotoxic glutamate-induced [Ca²⁺]_i rise in cultured hippocampal neurons. Hippocampal neurons grown in the presence or absence of 10 ng/ml SCE for 2 days were loaded with fura-2 and fluorescent Ca²⁺ imaging was conducted to determine the [Ca²⁺]_i. A, Color-coded images of Ca²⁺ fluorescence in neurons cultured in the absence (upper panels) and presence of SCE (lower panel) before and after exposure to glutamate. At rest (left panels), [Ca²⁺]_i was low in control and SCE-pretreated neurons (top and bottom panels). After exposure to 100 μM glutamate (right panels), SCE-pretreated neurons (bottom right panel) exhibited a lower rise in [Ca²⁺]_i than control neurons (top right panel). Color code bar indicates [Ca²⁺]_i in nM. B, Quantitative analysis of [Ca²⁺]_i in hippocampal neurons after exposure to 100 μM glutamate. Pretreatment with 10 ng/ml SCE attenuated the rise in [Ca²⁺]_i induced by excitotoxic glutamate. Data are expressed as the average of two experiments with at least 25 neurons per condition per experiment. Scale bar = 50 μm.

Figure 10.

Comparison between SCE and CEE-induced attenuation of glutamate-induced [Ca²⁺]_i rise in cultured hippocampal neurons. Hippocampal neurons grown in the presence or absence of 10 ng/ml SCE or CEE for 2 days were loaded with fura-2 and fluorescent Ca²⁺ imaging was conducted to determine the [Ca²⁺]_i. A, [Ca²⁺]_i in hippocampal neurons after exposure to 100 μM glutamate. Hippocampal neurons pretreated with 10 ng/ml SCE or CEE exhibited a lower Ca²⁺ response to excitotoxic glutamate than the control neurons, with SCE and SCE showing comparable attenuating effects. B, Statistical analysis of changes in [Ca²⁺]_i in response to excitotoxic glutamate. SCE and CEE showed comparable attenuating effects on 100 μM glutamate-induced [Ca²⁺]_i rise. Data are expressed as mean ± SEM percent of control glutamate-induced [Ca²⁺]_i and are combined across two separate experiments with at least 25 neurons per condition per experiment. * P < 0.05 compared with control cultures.

Footnotes

This work was supported by the National Institutes of Aging Grant PO1 AG1475: Project 2, Duramed Pharmaceuticals Inc. and the Norris Foundation.

1

To whom requests for reprints should be addressed at the Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089–9121. E-mail:

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