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Endocrinology, doi:10.1210/en.2006-1210
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Endocrinology Vol. 148, No. 2 627-637
Copyright © 2007 by The Endocrine Society

17ß-Estradiol Protects Cortical Neurons Against Oxidative Stress-Induced Cell Death through Reduction in the Activity of Mitogen-Activated Protein Kinase and in the Accumulation of Intracellular Calcium

Yumiko Numakawa, Tomoya Matsumoto, Daisaku Yokomaku, Takahisa Taguchi, Etsuo Niki, Hiroshi Hatanaka1, Hiroshi Kunugi and Tadahiro Numakawa

Human Stress Signal Research Center (Y.N., E.N.), Neuronics Research Group, Special Division for Human Life Technology (T.M., D.Y., T.T., T.N.), National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka 563-8577, Japan; Division of Protein Biosynthesis (Y.N., T.M., D.Y., H.H., T.N.), Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan; and Department of Mental Disorder Research (H.K., T.N.), National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan

Address all correspondence and requests for reprints to: Tomoya Matsumoto, Division of Pharmacology/Neurobiology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland. E-mail: Tomoya.Matsumoto{at}unibas.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although many studies have suggested that estrogen acts as a neuroprotective agent in oxidative stress, the underlying mechanism has not been fully elucidated. In the present study, we examined the effect of 17ß-estradiol (17ß-E2) on H2O2-induced death signaling in cultured cortical neurons. Exposure of the cortical neurons to H2O2 triggered a series of events, including overactivation of p44/42 MAPK and intracellular Ca2+ accumulation via voltage-gated Ca2+ channels and ionotropic glutamate receptors, resulting in apoptotic-like cell death. The MAPK pathway might work as death signaling in our system, because the MAPK pathway inhibitor, U0126, blocked H2O2-induced MAPK activation, Ca2+ overload, and cell death. Interestingly, a similar inhibitory effect on H2O2-triggered MAPK activation, Ca2+ accumulation, and cell death was observed in cultures incubated with 17ß-E2 for 24 h before exposure to H2O2, suggesting that the protective effect of 17ß-E2 is induced via attenuating overactivation of the MAPK pathway. Furthermore, we found that ionotropic glutamate receptor subunits, including NR2A and GluR2/3, but not NR2B and GluR1, were down-regulated in the 17ß-E2-treated cultures. The down-regulation of these glutamate receptor subunits was also observed after chronic treatment with U0126. Therefore, it is possible that 17ß-E2 down-regulates the expression of the ionotropic glutamate receptors by reducing activity of the MAPK pathway, which might be important for the protective effect of 17ß-E2 against oxidative stress-induced toxicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GROWING ACCUMULATION of evidence suggests gender difference in the incidence of various neurodegenerative disorders such as cerebral stroke, Parkinson’s disease, and Alzheimer’s disease (1). For example, women have a lower risk of developing stroke than men, whereas this protection is lost in the postmenopausal years (2). In addition, postmenopausal estrogen replacement therapy might reduce the risk of these diseases (2, 3, 4), suggesting that certain levels of estrogens are essential for protection against these diseases.

In addition to contributing to sexual differentiation in the brain (5), estrogens play a role in numerous neuronal events in the central nervous system (CNS). For example, the addition of 17ß-estradiol (17ß-E2), one of the major estrogens, increases cell differentiation, survival, and the viability of various neurons, including cultured hypothalamic (1 pM of 17ß-E2) (6), amygdala (36 nM) (7), and neocortical neurons (1 nM) (8). Furthermore, estrogens could be involved in synaptic plasticity (9, 10). We have also reported that pretreatment of cultured hippocampal neurons with 17ß-E2 (1–10 nM) for 24 h enhances activity-dependent glutamate release (11). Many reports have shown that estrogens rescue neurons from oxidative stress, which is a hallmark of neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases (12, 13). Preapplication of 17ß-E2 (10–50 nM) for 24 h protected cultured cortical neurons against glutamate (0.1 mM)-induced toxicity (14, 15). Behl et al. have shown that pretreatment of cultured cortical neurons with 17ß-E2 (10 µM, 20 h) decreases cell death triggered by exposure to hydrogen peroxide (H2O2, 30 µM) (16). Furthermore, preincubation with 17ß-E2 (1–30 nM) for 48 h protects cultured hippocampal neurons against the toxicity of ß-amyloid (25 µM) whose accumulation in the brain is a key biochemical abnormality in Alzheimer’s disease (17). These in vitro studies support the role of 17ß-E2 as a neuroprotective agent.

One of the mechanisms underlying the protective effects of 17ß-E2 is via classical estrogen receptors, nuclear transcription factors known as ER{alpha} and ERß, although ER-independent protection was also reported when a high concentration of 17ß-E2 (10 µM) was applied (18). Activation of ERs induces the expression of neuroprotective genes. Pretreatment of NT2 neurons with 17ß-E2 (10 nM) for 24 h prevents H2O2 (100 µM)-induced toxicity (19). This survival effect of 17ß-E2 requires the ER-mediated up-regulation of Bcl-2, an antiapoptotic molecule. In addition, ER-dependent increase in Bcl-xL, another antiapoptotic protein, is involved in the neuroprotective action of 17ß-E2 in ß-amyloid-induced apoptosis (17). Interestingly, 17ß-E2 activates intracellular survival signaling via ERs. 17ß-E2 rapidly activates the MAPK pathway through ERs, resulting in the prevention of glutamate-induced toxicity in cultured cortical neurons (15). Treatment with 17ß-E2 (50–100 nM) for 3 d blocked ß-amyloid (10 µM)-induced neuronal cell death via activation of the ER/phosphatidylinositol 3-kinase (PI3-K) pathway in cultured hippocampal neurons (20). Therefore, physiological levels of 17ß-E2 could exert neuroprotective effects through ER-mediated intracellular signaling.

On the other hand, 17ß-E2 is reported to affect toxicity-induced intracellular Ca2+ accumulation (Ca2+ overload), which is a common event in neuronal cell death (21). Acute application of 17ß-E2 at 10 µM to immortalized hypothalamic neurons (GT1–7 cells) decreased the marked rise in Ca2+ induced by ß-amyloid (5–20 µM) (22). Similarly, acute exposure of cultured hippocampal neurons to 17ß-E2 (50 µM) attenuates glutamate (0.3 mM)-induced Ca2+ overload (23). These effects could be exerted in an ER-independent (22) or -dependent (23) manner. However, the effectiveness of 17ß-E2 at physiological levels on Ca2+ overload remains unclear. Furthermore, the mechanism underlying the reduction in Ca2+ accumulation resulting in neuroprotection by 17ß-E2, if any, is largely unknown.

In the present study, we used primary cortical neurons as a model system to address the mechanism underlying the protective effect of 17ß-E2 on oxidative stress-induced toxicity. We found that pretreatment with 17ß-E2 at 10 nM for 24 h clearly protected neurons against H2O2-induced cell death by blocking the series of events triggered by exposure to H2O2, including overactivation of the MAPK pathway and Ca2+ overload (via voltage-gated Ca2+ channels and ionotropic glutamate receptors) in a classical ER-dependent manner. Interestingly, the down-regulation of ionotropic glutamate receptors was observed in the 17ß-E2-treated cultures. The MAPK pathway inhibitor, U0126, mimicked the effects of 17ß-E2. These results indicate a novel mechanism underlying the neuroprotective effect of 17ß-E2 in oxidative stress.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
H2O2 was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 17ß-E2 and 17{alpha}-E2 (Sigma, St. Louis, MO) were dissolved in ethanol. 17ß-E2 conjugated to bovine serum albumin (Sigma) was dissolved in PBS. U0126 was purchased from Promega (Madison, WI). ICI182,780 and tamoxifen were obtained from Tocris Cookson Inc. (St. Louis, MO). Nifedipine was purchased from Research Biochemicals International (Boston, MA). Other reagents were obtained from Sigma.

Cell culture
Primary dissociated cortical cultures were prepared from the cerebral cortex of 2-d-old rats (SLC, Shizuoka, Japan) as previously reported (24, 25, 26). Briefly, the cells were gently dissociated with a plastic pipette after digestion with papain (90 U/ml; Worthington Biochemical, Lakewood, NJ) at 37 C, and the cells were then plated at a final density of 5 x 105 cells/cm2 on polyethylenimine-coated 48-well plates or 3.5-cm dishes (Corning, Corning, NY), except for a Ca2+ imaging experiment (see Ca2+ imaging). The culture medium consisted of 5% precolostrum newborn calf serum, 5% heated-inactivated horse serum, and 90% DF medium, a 1:1 mixture of DMEM and Ham’s F-12 medium containing 15 mM HEPES buffer (pH 7.4), 30 nM Na2SeO3, and 1.9 mg/ml NaHCO3. A total of 0.5 or 2.0 ml of the culture medium was added to the 48-well plates or 3.5-cm dishes to maintain the cultured cells. After maintenance for 4–6 d, analysis was performed. Under these conditions, most of the cultured cells (approximately 80% of the total cells) were microtubule-associated protein 2 (MAP2, a neuronal marker)-positive at 5 d in vitro, whereas glial fibrillary acidic protein-positive glial cells were also observed as a minor population (25, 26). Dissociated hippocampal (from 2-d-old rats) (11), cerebellar (from 5-d-old rats) (27), and amygdala (2-d-old rats) neurons were also prepared. The culture maintenance before the analysis was carried out in the same way as that of the cortical culture.

Drug application
After maintaining the cultured neurons for 4–6 d, 17ß-E2 was added to the cultures by bath application. Except for analyzing the dose dependency of 17ß-E2, we used 17ß-E2 at a final concentration of 10 nM. Twenty-four hours after 17ß-E2 incubation, H2O2 dissolved in the DF medium was bath-applied to the cultured cells to trigger cell death and then the cultures were maintained in the presence of H2O2. Except for analyzing the dose dependency of H2O2, the final concentration of H2O2 was 50 µM. Twelve hours after H2O2 application, we performed immunostaining with anti-MAP2 antibody, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, or 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay to estimate the number of surviving or dying cells (see below). We confirmed that pretreatment with ethanol (the vehicle for 17ß-E2) did not have any effect on cell survival compared with no treatment (data not shown). For time-course analysis, the cortical cultures were exposed to H2O2 for 1, 3, 6, 12, and 24 h. Inhibitors including cycloheximide (1 µM), actinomycin D (0.1 µM), U0126 (5, 10, or 20 µM), 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM; 0.1 or 1 µM), nifedipine (0.1 or 1 µM), d-(-)-2-amino-5-phosphonopentanoic acid (AP5; 1 or 10 µM), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 or 10 µM) were applied 30 min before adding H2O2. ICI182,780 or tamoxifen (1 or 10 µM, respectively) was coapplied with 17ß-E2, and the cultures were incubated for 24 h before H2O2 stimulation. To examine the effect of 17ß-E2 or of U0126 on the expression of ionotropic glutamate receptors, these chemicals were added to the cultures, and the cultures were then lysed 24 h after drug incubation without H2O2 stimulation followed by Western blotting (see below). In our system, before H2O2 exposure, the medium was replaced with fresh culture medium containing 17ß-E2 and/or inhibitors.

Immunostaining
Immunostaining with anti-MAP2 (Sigma) antibody was performed as follows. First, the cultured cells were fixed in 4% paraformaldehyde containing 0.05% Triton X-100 at room temperature (25 C) for 20 min. After three washes with PBS, the cells were incubated overnight with anti-MAP2 antibody at 4 C (1:1000). Secondary antibody was applied at room temperature for 1 h. To visualize the staining, a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) together with 0.02% (w/v) 3,3'-diaminobenzidine 4 HCl dissolved in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% (vol/vol) H2O2 and 0.1% (w/v) (NH4)Ni(SO4)2 was used. The number of MAP2-positive cells was then counted. Representative data from a sister culture are shown in the figures. N indicates the number of randomly selected fields in the wells for each experimental condition in a culture plate. All experiments for this analysis were performed using three separate cultures to confirm reproducibility.

TUNEL assay
To determine cell death (double-strand breaks in DNA), a TUNEL assay was performed using the DeadEnd Colorimetric Apoptosis Detection kit (Promega). Briefly, cultured cells were fixed with 4% paraformaldehyde containing 0.05% Triton X-100 at room temperature for 40 min. After washing three times with PBS, the cells were incubated with biotinylated nucleotide mixture together with terminal deoxynucleotidyl transferase enzyme. TUNEL-positive cells were detected using hydrogen peroxide and 3,3'-diaminobenzidine 4 HCl and then counted. Representative data from a sister culture are shown in the figures. N indicates the number of randomly selected fields in the wells for each experimental condition in a culture plate. All experiments were performed using three separate cultures to confirm reproducibility.

MTT assay
To determine the viability of the cultured cells, we carried out an MTT assay. The metabolic activity of mitochondria was estimated by measuring the mitochondrial-dependent conversion of the tetrazolium salt, MTT. MTT was applied to the cultures at a final concentration of 0.5 mg/ml for 1.5 h at 37 C. The medium was then aspirated, and acidified isopropyl alcohol was added to solubilize the colored formazan product. Absorbance was determined at 550 nm on a scanning multiwell plate reader (Bio-Rad Laboratories Inc., Hercules, CA) after agitating the plates. All experiments were performed using 3–5 separate cultures to confirm reproducibility. Representative data from a sister culture are shown in the figures. N indicates the number of wells of a plate for each experimental condition.

Ca2+ imaging
Imaging analysis of intracellular Ca2+ was performed as previously reported (24, 28). Briefly, dissociated cells were cultured on polyethyleneimine-coated glasses (Matsunami, Osaka, Japan) attached to flexiperm (IN VITRO, Kalkberg, Germany). After being washed, the cells were incubated for 1 h at 37 C with 10 µM Fluo-3 AM (Molecular Probes, Eugene, OR) diluted in HEPES-buffered Krebs Ringer buffer, a modified HEPES-buffered Krebs Ringer solution containing 130 mM NaCl, 5 mM KCl, 1.2 mM NaH2PO4, 1.8 mM CaCl2, 10 mM glucose, and 25 mM HEPES (pH 7.4). The dye intensity was monitored using a confocal microscope (TMD-300 controlled by RCM 8000, Nikon, Tokyo, Japan) or a fluorescent microscope (Axiovert 200 controlled by the Slide BookTM 3.0, Zeiss, Tokyo, Japan). The obtained image data were analyzed with RCM 8000 or Slide BookTM 3.0. The changes in intracellular Ca2+ were analyzed by quantifying the fluorescence intensity at the cell body. The ratio value, F/F0:F0 or F refers to the fluorescence intensity before or after H2O2 stimulation, respectively. The dye fluorescence intensities were relatively stable with minimum bleach for more than 30 min in the vehicle solution. To confirm reproducibility, imaging experiments were performed at least four times with independent cultures. Representative data are shown in the figures.

Western blotting
Western blotting was performed as previously described (29). Briefly, cells were lysed in sodium dodecyl sulfate lysis buffer containing 1% sodium dodecyl sulfate, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA (pH 8.0), 10 mM NaF, 2 mM Na3VO4, 0.5 mM phenylarsine oxide, and 1 mM phenylmethylsulfonyl fluoride. To estimate the activation of MAPK/ERK, anti-phospho MAPK (1:1000; Cell Signaling Technology Inc., Beverly, MA) and anti-MAPK (ERK) (1:1000; Cell Signaling) antibodies were used. Furthermore, immunoblotting with anti-GluR1 (1:1000; Chemicon International Inc., Temecula, CA), anti-GluR2/3 (1:1000; Chemicon International Inc.), anti-NR2A (1:1000; Sigma), anti-NR2B (1:1000; Sigma), and anti-class III ß-tubulin (TUJ1) (1:4000, Berkeley Antibody Co., Richmond, CA) antibodies were carried out. Immunoblotting was performed at least three times with independent cultures. Representative blot images from a sister culture are shown.

Statistical analysis
The data shown in this study are expressed as means ± SD. Statistical significance was evaluated with one-way analysis of variance followed by Newman-Keuls test for multiple groups, and probability values of less than 5% were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
H2O2 induces cell death in cultured cortical neurons
To investigate the protective effect of 17ß-E2 against oxidative stress in CNS neurons, we used an H2O2-induced cell death system in cultured cortical neurons. We have recently shown that exposure of the cultures to H2O2 causes neuronal cell death (26); thus, summarized data are shown in the present study to characterize the cell death system. First, the dose dependency of H2O2-induced cell death was examined. The results of the MTT survival assay are shown in Fig. 1AGo. At a final concentration of 1 or 10 µM, exposure to H2O2 for 12 h did not trigger cell death. In contrast, H2O2 at 50 or 100 µM caused significant neuronal cell death. Next, the time course of cell death induced by H2O2 (50 µM) was examined. Approximately 20% loss of neurons was observed 1–6 h after H2O2 exposure and, thereafter, cell death proceeded in a time-dependent manner (Fig. 1BGo). Thirty-six hours after H2O2 application, almost all neurons died (data not shown). In the following experiments, cultured neurons were exposed to H2O2 at 50 µM for 12 h. To determine whether cell death was apoptotic-like, the effects of cycloheximide (a protein synthesis inhibitor) and actinomycin D (an RNA synthesis inhibitor) were tested. The number of the MAP2 (a neuronal marker)-positive surviving neurons was reduced after H2O2 exposure compared with the control, and the reduction of MAP2-positive cells was rescued by cycloheximide or actinomycin D (Fig. 1CGo). This phenomenon was confirmed by MTT assay (Fig. 1DGo).


Figure 1
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  		FIG. 1. H2O2 causes cell death in cultured cortical neurons. A, H2O2 induced neuronal cell death in a dose-dependent manner. H2O2 was applied to the cortical cultures at 1, 10, 50, or 100 µM (final concentration). After 12 h, cell survival was determined by MTT assay. The data represent mean ± SD (n = 4, n indicates the number of wells of a plate for each experimental condition). ***, P < 0.001 vs. 0 µM of H2O2. B, H2O2 induced cell death in a time-dependent manner. The cortical neurons were exposed to H2O2 (final 50 µM) for 0, 1, 3, 6, 12, or 24 h followed by undergoing MTT assay. The data represent mean ± SD (n = 8, n indicates the number of wells of a plate for each experimental condition). ***, P < 0.001 vs. 0 h. C and D, Cycloheximide (CHX) or actinomycin D (Act D) (a protein or an RNA synthesis inhibitor, respectively) blocked H2O2-induced cell death. CHX (1 µM) or Act D (0.1 µM) was applied 30 min before exposure to H2O2 (final 50 µM). Twelve hours after adding H2O2, cell survival was determined by immunostaining with anti-MAP2 (a neuronal marker) antibody (C) or by MTT assay (D). C, scale bar, 100 µm. D, the data represent mean ± SD (n = 8, n indicates the number of wells of a plate for each experimental condition). ***, P < 0.001; **, P < 0.01 vs. –H2O2. ###, P < 0.001 vs. +H2O2.

 
17ß-E2 prevents CNS neurons from H2O2-induced cell death
Using this cell death system, we first examined the dose-dependent effect of 17ß-E2 on H2O2-induced cell death. Pretreatment with 17ß-E2 at 0.1 nM or more significantly reduced cell death induced by H2O2, and the effects plateaued at 100 nM (Fig. 2AGo). Protection by 17ß-E2 was confirmed by immunostaining with anti-MAP2 antibody and TUNEL assay. The number of MAP2-positive surviving neurons was reduced in H2O2-applied cultures compared with the control, and 17ß-E2 increased the number of surviving neurons in H2O2-induced oxidative stress (Fig. 2BGo, upper). Furthermore, the TUNEL-positive dying cells were increased by exposure to H2O2, and the increase was blocked by 17ß-E2 pretreatment (Fig. 2BGo, lower), suggesting that 17ß-E2 blocked apoptosis-like cell death. In addition to cultured cortical neurons, the effects of 17ß-E2 in cultured amygdala, cerebellar, and hippocampal neurons were investigated. As shown, the marked H2O2-induced cell death occurred in these cultured neurons and 17ß-E2 protected them (Fig. 2CGo). These results suggest that 17ß-E2 protects CNS neurons against oxidative stress-induced toxicity.


Figure 2
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  		FIG. 2. Pretreatment with 17ß-E2 protects cultured cortical neurons against H2O2-induced cell death. A, 17ß-E2 exerted a protective effect in a dose-dependent manner. 17ß-E2 was applied to the cortical neurons at 0.1, 1, 10, 100, or 1000 nM (final concentration). After 24 h, the cultures were then incubated with H2O2 (final 50 µM) for 12 h in the presence of 17ß-E2 followed by MTT assay. The data represent mean ± SD (n = 4, n indicates the number of wells of a plate for each experimental condition). ***, P < 0.001; **, P < 0.01 vs. –H2O2. ###, P < 0.001 vs. +H2O2. B, The protective effect of 17ß-E2 was determined by MAP2 staining and TUNEL assay. The cultured neurons were preincubated with 17ß-E2 (10 nM) for 24 h followed by the addition of H2O2 (final 50 µM) in the presence of 17ß-E2. After 12 h, MAP2 staining or TUNEL assay was performed. The upper portion shows MAP2-positive cells. The lower portion shows the TUNEL-positive dying cells. Scale bar, 100 µm. C, 17ß-E2 exerted a protective effect on cultured amygdala, cerebellar granule, and hippocampal neurons. These cultures were pretreated with 17ß-E2 (10 nM) for 24 h and then incubated with H2O2 (final 50 µM) in the presence of 17ß-E2 for 12 h. Cell survival was determined by MTT assay. The data represent mean ± SD (n = 6, n indicates the number of wells of a plate for each experimental condition). ***, P < 0.001; **, P < 0.01 vs. –H2O2. ###, P < 0.001 vs. +H2O2.

 
The protective effect of 17ß-E2 is via classical ERs
Because previous studies have suggested that 17ß-E2 has the ability to scavenge free radicals (1), the effects of acute 17ß-E2 preapplication or of coapplication with H2O2 were examined. In both cases, 17ß-E2 did not block H2O2-induced cell death (Table 1Go, A and B), suggesting that 17ß-E2 is unlikely to play a role as a radical scavenger in our system. Furthermore, 17{alpha}-E2, a stereoisomer of 17ß-E2, failed to prevent H2O2-induced death (Table 1CGo), indicating the specificity of 17ß-E2. We next examined whether ERs are involved in the protective effect of 17ß-E2. Both ICI182,780 and tamoxifen (each an estrogen receptor antagonist) canceled the protective effect of 17ß-E2 (Table 2Go, A and B). In addition, H2O2 still killed the neurons after pretreatment with membrane-impermeable 17ß-E2 conjugated to bovine serum albumin (Table 3Go), suggesting that membrane ER is not involved. These results suggest that 17ß-E2 exerts its protective effect through activation of classical cytosolic/nuclear ERs.


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  		TABLE 1. Acute preapplication (A) or coapplication of 17ß-E2 (B) or long-term preapplication of 17{alpha}-E2 (C) failed to protect cultured cortical neurons against H2O2-induced cell death

 

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  		TABLE 2. ERs antagonists, ICI182,780 (A) and tamoxifen (B), blocked the neuroprotective effects of 17ß-E2 against H2O2-induced cell death

 

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  		TABLE 3. 17ß-E2-BSA failed to protect cultured cortical neurons against H2O2-induced cell death

 
17ß-E2 exerts its effect by reducing H2O2-induced activation of MAPK
To address the mechanism involved in 17ß-E2-mediated protection, we first focused on intracellular signaling. Because the p44/42 MAPK (ERK1/2) pathway has been suggested to be important for neuronal survival (26) or death (30), the effect of U0126 (a specific MEK inhibitor, an upstream molecule of MAPK) on H2O2-induced cell death was tested. As shown in Fig. 3AGo, U0126 inhibited cell death in a dose-dependent manner (MTT assay). Another MEK inhibitor, PD98059, was also effective (data not shown). Similarly, the number of MAP2-positive or TUNEL-positive cells under oxidative stress was increased or reduced by U0126 in a dose-dependent manner, respectively (Fig. 3BGo, a and b). No additional or synergistic effect of coapplication of U0126 and 17ß-E2 compared with treatment with 17ß-E2 solely was observed (Fig. 3BGo, a and b). We then performed Western blotting to determine H2O2-induced activation (phosphorylation) of p44/42 MAPK (ERK1/2). As shown, H2O2 significantly activated p44/42 MAPK and U0126 blocked the activation (Fig. 3CaGo), suggesting involvement of the MAPK pathway in H2O2-induced cell death. The expression of TUJ1 (class III ß-tubulin, a neuronal marker) was shown as a negative control (Fig. 3CaGo). Next, we determined p44/42 MAPK activation stimulated by H2O2 with or without 17ß-E2 preincubation. As expected, H2O2-induced p44/42 MAPK activation was strongly reduced after 17ß-E2 treatment (Fig. 3CbGo). No significant change in TUJ1 level was observed (Fig. 3CbGo). It is of note that some level of MAPK activation remains in cultures exposed to H2O2 in the presence of U0126 (Fig. 3CaGo) or of 17ß-E2 (Fig. 3CbGo).


Figure 3
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  		FIG. 3. The MAPK pathway is involved in H2O2-induced cell death, and reduction in H2O2-stimulated activation of MAPK by 17ß-E2 is important for its protective effect. A, U0126 (a specific inhibitor of MEK, an upstream molecule of MAPK) exerted a protective effect in a dose-dependent manner. U0126 (5, 10, or 20 µM) was applied 30 min before exposure to H2O2 (50 µM). Twelve hours after adding H2O2, MTT assay was performed. The data represent mean ± SD (n = 6, n indicates the number of wells of a plate for each experimental condition). ***, P < 0.001 vs. –H2O2. ###, P < 0.001; ##, P < 0.01 vs. +H2O2. B, The protective effect of U0126 was confirmed by MAP2 staining (a) and by TUNEL assay (b). Note that U0126 did not exert any additional or synergistic effects compared with treatment with 17ß-E2 solely. U0126 (5 or 10 µM) was applied to the cortical cultures 30 min before exposure to H2O2 (50 µM) with or without 17ß-E2 pretreatment (10 nM, 24 h). After 12 h, MAP2 staining or TUNEL assay was performed and the positive cells were counted, respectively. The data represent mean ± SD (n = 20, n indicates the number of randomly selected fields from six wells for each experimental condition in a culture plate). The MAP2- or TUNEL-positive number was normalized to –H2O2 or +H2O2, respectively. ***, P < 0.001 vs. –H2O2. ###, P < 0.001 vs. +H2O2. C (a) U0126 or (b) 17ß-E2 reduced activation of MAPK stimulated by H2O2, although some levels of activated MAPK still remain in the U0126- or 17ß-E2-treated cultures, respectively. The levels of total p44/42 MAPK (ERK1/2), phosphorylated p44/42 MAPK (pERK1/2), and TUJ1 are shown, respectively (Western blotting). (a) U0126 (10 µM) was applied to the cortical cultures 30 min before exposure to H2O2 (50 µM). After 6 h, the cell lysates were collected. (b) 17ß-E2 was preapplied to the cultures for 24 h, and H2O2 (50 µM) was then added. The cell lysates were collected 0, 1, 3, and 6 h after H2O2 addition.

 
17ß-E2 blocks MAPK-mediated Ca2+ accumulation stimulated by H2O2
To further characterize the protective effects of 17ß-E2, we next focused on Ca2+ overload triggered by H2O2, because excess Ca2+ influx is essential for the induction of cell death (21). First, we monitored intracellular Ca2+ accumulation after H2O2 exposure and found that H2O2 induced Ca2+ increase gradually (Fig. 4AGo and Bb), whereas the vehicle for H2O2 (water) did not have any effect on intracellular Ca2+ mobilization (Fig. 4BaGo). Interestingly, Ca2+ accumulation stimulated by H2O2 was significantly reduced by U0126 or 17ß-E2 treatment (Fig. 4BGo, c and d, respectively). Quantitative data on intracellular Ca2+ for each experimental condition were obtained by analyzing the fluorescent intensity at the cell body. The number of cells with intracellular Ca2+ changes after H2O2 exposure was counted. As a result, H2O2-induced Ca2+ accumulation was U0126- and 17ß-E2-sensitive (Fig. 4CGo, a–d). These results suggest that decrease in the Ca2+ overload by 17ß-E2 treatment is due to reduction in H2O2-induced activation of the MAPK pathway.


Figure 4
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  		FIG. 4. H2O2 induces MAPK-mediated Ca2+ overload, which is attenuated by 17ß-E2. A, Intracellular Ca2+ was accumulated after H2O2 (50 µM) exposure. Ca2+ imaging in cultured cortical neurons was performed with the Ca2+ indicator, fluo-3AM (10 µM). Left and middle, Representative gray scale and pseudocolor images shown were acquired 1 min before H2O2 addition. Right was acquired 15 min after exposure to H2O2 (50 µM). Scale bar, 20 µm. B, Temporal changes in the levels of intracellular Ca2+ in six cortical neurons randomly selected are shown. (a) The vehicle (water) had no effect. (b) Ca2+ accumulation progressively occurred after H2O2 exposure, which was blocked by U0126 (c) and 17ß-E2 (d). The percentage change in Ca2+ is shown as a [(F-F0)/F0] x 100. F0, basal fluorescent intensity at the cell body before exposure to H2O2. C, Frequency histogram of the percentage change in intracellular Ca2+ concentration. The number of cells with intracellular Ca2+ changes of more than 10% was significant in the H2O2-exposed cultures without drug pretreatment (b) compared with the control (a), U0126- (c), or 17ß-E2-pretreated cultures (d). The percentage is [(F-F0)/F0] x 100. F0 and F, the fluorescent intensity at the cell body 1 min before and 15 min after H2O2 addition, respectively. The number of cells that responded to H2O2 or the vehicle with intracellular Ca2+ changes of less than 0, 0–10, 10–20, 20–30, or more than 30% was counted, respectively. A total of 27–29 neurons were randomly selected from three coverslips for each experimental condition. B and C, The cultures were exposed to (a) the vehicle (water) or (b) H2O2 (50 µM). B (c), C (c), U0126 (10 µM) was applied 30 min before exposure to H2O2 (50 µM). B (d), C (c), Twenty-four hours after pretreatment with 17ß-E2 (10 nM), H2O2 (50 µM) was applied.

 
Ca2+ influx via voltage-gated Ca2+ channels and ionotropic glutamate receptors is involved in H2O2-induced cell death
We next examined the involvement of Ca2+ influx in cell death in our system because 17ß-E2 influenced Ca2+ accumulation induced by H2O2 (Fig. 4Go). In the presence of BAPTA-AM, an intracellular Ca2+ chelator, cell death triggered by H2O2 was inhibited (Fig. 5AGo), suggesting that Ca2+ accumulation is a critical step for cell death induced by H2O2. No additional or synergistic effect of BAPTA-AM and 17ß-E2 coapplication compared with sole 17ß-E2 addition was observed (Fig. 5AGo). Similar results as those with the application of BAPTA-AM were obtained in the case of nifedipine (an L-type Ca2+ channel inhibitor), CNQX [an {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-sensitive glutamate receptor antagonist], or AP5 [an N-methyl-D-aspartate (NMDA)-sensitive glutamate receptor antagonist] application, respectively (Fig. 5Go, B and C). These results suggest that Ca2+ influx via not only voltage-gated Ca2+ channels, but also ionotropic glutamate receptors is an essential process for H2O2-dependent toxicity and that 17ß-E2 influences Ca2+ signaling.


Figure 5
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  		FIG. 5. Ca2+ influx via voltage-gated Ca2+ channels and ionotropic glutamate receptors is involved in H2O2-induced cell death. A, BAPTA-AM (a membrane-permeable Ca2+ chelator); B, nifedipine (a voltage-gated Ca2+ channel inhibitor); and C, CNQX and AP5 (AMPA and NMDA glutamate receptor antagonists, respectively) blocked H2O2-induced death. None of these drugs exerted any additional or synergistic effects compared with treatment with 17ß-E2 solely. BAPTA-AM (0.1 or 1 µM), nifedipine (0.1 or 1 µM), CNQX (1 or 10 µM), and AP5 (1 or 10 µM) were applied to the cultured neurons 30 min before exposure to H2O2 (50 µM) with or without 17ß-E2 pretreatment (10 nM, 24 h). Twelve hours after adding H2O2, MTT assay was carried out. The data represent mean ± SD (n = 8, the number of wells of a plate for each experimental condition). ***, P < 0.001; **, P < 0.01 vs. –H2O2. ###, P < 0.001 vs. +H2O2.

 
The levels of ionotropic glutamate receptors are down-regulated after chronic treatment with 17ß-E2 and the MAPK pathway inhibitor
How does the decrease in H2O2-induced Ca2+ accumulation by 17ß-E2 exposure occur? We examined the possibility that a change in the levels of ionotropic glutamate receptors such as NMDA and AMPA receptors is involved, because their antagonists significantly inhibited cell death in our system (Fig. 5CGo). Indeed, the expression of ionotropic glutamate receptor subunits, including NR2A and GluR2/3, was clearly decreased in the 17ß-E2-treated cultures compared with the control, although no significant change in the levels of NR2B and GluR1 was observed (Fig. 6Go). Interestingly, long-term application of U0126 for 24 h also reduced the levels of these glutamate receptor subunits (Fig. 6Go). TUJ1 expression was shown as a negative control. Therefore, 17ß-E2 might down-regulate ionotropic glutamate receptors via inactivating the MAPK pathway.


Figure 6
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  		FIG. 6. Long-term treatment with 17ß-E2 or U0126 down-regulates the levels of ionotropic glutamate receptor subunits NR2A and GluR2/3, but not of NR2B and GluR1. Cultured cortical neurons were incubated with 17ß-E2 (10 nM) or U0126 (10 µM) for 24 h before the cell lysates were collected without H2O2 exposure. The levels of NMDA receptor subunits (NR2A and NR2B), AMPA receptor subunits (GluR1 and GluR2/3), and TUJ1 were determined (Western blotting).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, 17ß-E2 protected cortical neurons against oxidative stress by reducing H2O2-induced activation of the MAPK pathway. After exposure to H2O2, a Ca2+ overload via voltage-gated Ca2+ channels and ionotropic glutamate receptors occurred in a MAPK pathway-dependent manner, resulting in neuronal cell death, which was prevented by 17ß-E2 treatment. Furthermore, 17ß-E2 down-regulated the expression of ionotropic glutamate receptors, which might be due to inactivation of the MAPK pathway.

Consistent with previous studies (14, 15, 17, 19, 20, 31), chronic (24-h) treatment with physiological levels of 17ß-E2 was effective for neuroprotection against oxidative stress-induced toxicity (Fig. 2Go), which implies the involvement of intracellular survival pathways rather than the ability of 17ß-E2 as a radical scavenger (1). Chronic preincubation with {alpha}-tocopherol (10 nM) or brain-derived neurotrophic factor [(BDNF), 100 ng/ml] protected cultured cortical or hippocampal neurons against H2O2 (50 µM)- or glutamate (125 µM)-induced toxicity in a MAPK pathway- and PI3-K pathway-dependent manner, respectively (26, 32). The potency of 17ß-E2 was also reported. Preapplication of 17ß-E2 (10 nM, 24 h) protected cultured cortical neurons against glutamate (0.1 or 0.3 mM)-induced toxicity through the MAPK or PI3-K pathway (15, 31). Previously, we showed that 17ß-E2 (10 nM) caused activation of both MAPK and Akt (downstream of the PI3-K pathway) in cultured hippocampal neurons, whereas the peaks of MAPK and Akt activations were within 1 h (11). However, this acute action of 17ß-E2 might not play a role in neuroprotection in the present study, because short-term treatment with 17ß-E2 failed to prevent H2O2-stimulated toxicity (Table 1Go, A and B). Thus, in the present study, we focused on H2O2-induced MAPK activation after chronic treatment with 17ß-E2.

Although the MAPK pathway plays a role in neuronal survival, this pathway could also serve as death signaling. Sustained activation of MAPK contributes to glutamate (5 mM)-induced cortical cell death (33). Consistently, in our system, H2O2-induced MAPK activation was required for cell death (Fig. 3Go). A previous study also showed MAPK activation in cultured cortical neurons exposed to H2O2 (300 µM); however, U0126 did not rescue but slightly enhanced cell death (34). This discrepancy may be the result of differences in the level of the basal activity of MAPK. Basal levels of MAPK activation in our cultures seem to be higher than those in their system. Furthermore, U0126 inhibited H2O2-induced (not basal) activation of MAPK (Fig. 3CaGo), whereas basal MAPK activation in their system was abolished (34). Similarly, 17ß-E2 attenuated the H2O2-induced activation of MAPK but did not abolish basal activation (Fig. 3CbGo). It is possible that basal activity of the MAPK pathway under a certain threshold might be necessary for survival, whereas its activation over the threshold is toxic.

Perturbation of intracellular Ca2+ homeostasis is a common feature of apoptotic cell death in neurodegenerative disorders (21). Intracellular Ca2+ gradually increases in cultured cortical neurons exposed to glutamate (0.5 mM), resulting in cell death (35). Similar progressive Ca2+ accumulation occurred after adding H2O2 (Fig. 4Go), which was essential for cell death (Fig. 5AGo). Glutamate (0.5 mM)-induced Ca2+ accumulation via ionotropic glutamate receptors, leading to cortical cell death, was reported (36). Nifedipine prevented AMPA (10 µM)- or kinate (30 µM)-induced cortical cell death (37). In our system, NMDA and AMPA receptor antagonists and nifedipine blocked H2O2-induced cell death (Fig. 5Go, B and C), suggesting the involvement of ionotropic glutamate receptors and voltage-gated Ca2+ channels. We next examined if the MAPK pathway is involved in Ca2+ accumulation, because both H2O2-stimulated MAPK activation and Ca2+ overload were necessary for cell death (Figs. 3–5GoGoGo). We found for the first time that H2O2-stimulated Ca2+ overload occurred in a MAPK pathway-dependent manner (Fig. 4Go). How does the MAPK pathway contribute to H2O2-induced Ca2+ overload? Some studies including our previous study demonstrated the MAPK-dependent release of glutamate (28, 38). Thus, it is possible that overactivation of MAPK may cause excessive release of glutamate. Furthermore, another study indicated that extracellular glutamate increased after exposing cultured cerebellar or cortical neurons to xanthine (100 µM) and xanthine oxidase (45 mU/ml) or to H2O2 (200 µM) (39, 40). Taking these findings together, H2O2-stimulated activation of MAPK might trigger abnormal release of glutamate, resulting in Ca2+ overload via ionotropic glutamate receptors and voltage-gated Ca2+ channels.

Acute preincubation of cultured hippocampal neurons with 17ß-E2 at 50 µM attenuated glutamate (0.3 mM)- or NMDA (30 µM)-induced Ca2+ overload (23, 41). The reduction in Ca2+ overload by brief application of 17ß-E2 at such a high concentration could be due to the modulation of membrane properties (22) and interaction with NMDA receptors (41). In contrast, the effect of physiological levels of 17ß-E2 has been recently reported. Treatment of cultured hippocampal neurons with 17ß-E2 for several days at 1 nM decreased kinate (10 µM)-induced Ca2+ accumulation (42). In the present study, a reduction in H2O2-stimulated Ca2+ overload was observed after chronic 17ß-E2 treatment at 10 nM (Fig. 4Go), implying that the treatment time required for attenuating Ca2+ overload depends on the 17ß-E2 concentration. We then focused on the effect of 17ß-E2 on the expression of ionotropic glutamate receptors, because their antagonists blocked cell death (Fig. 5CGo). We found for the first time that NMDA- and AMPA-receptor subunits (NR2A and GluR2/3) were clearly down-regulated in the 17ß-E2-treated cultures (Fig. 6Go). Interestingly, similar down-regulation was observed when MAPK activation was chronically inhibited (Fig. 6Go), suggesting that 17ß-E2 decreases glutamate receptor expression via reducing the activity of the MAPK pathway. As described previously, the MAPK pathway is involved in glutamate release (28, 38). The neuronal activity positively regulates the expressions of NMDA and AMPA receptors (43, 44). Thus, inactivation of the MAPK pathway by 17ß-E2 treatment might suppress neuronal activity, resulting in the down-regulation of glutamate receptors.

Cytosolic/nuclear ER-mediated neuroprotection has been reported (15, 17, 20), whereas the involvement of membrane-associated ERs has been suggested (23). In our system, ER antagonists blocked the protective effects of 17ß-E2 (Table 2Go). Furthermore, 17ß-E2-bovine serum albumin (a membrane-impermeable form) failed to prevent H2O2-induced cell death (Table 3Go). 17{alpha}-E2 did not rescue the neurons (Table 1CGo), although 17{alpha}-E2 could induce MAPK activation via membrane-associated ERs (45). Taken together, 17ß-E2 might exert neuroprotective effects through cytosolic/nuclear ERs, although the possibility of extranuclear roles of ERs could not be excluded.

We addressed the possibility that neurotropins mediate neuroprotection by 17ß-E2, because ERs could regulate the transcription of BDNF and nerve growth factor (NGF) (46, 47). In addition, preapplication of NGF (10 ng/ml), BDNF (100 ng/ml), or basic fibroblast growth factor (10 ng/ml) for 48 h protected the cultured hippocampal neurons against glutamate (10–100 µM)-induced toxicity (48). Thus, it is possible that growth factors play a role as a mediator in neuroprotection by 17ß-E2. However, application of BDNF, NGF, basic fibroblast growth factor, or insulin-like growth factor-1 (100 ng/ml, respectively) did not inhibit H2O2-induced cell death in our system (data not shown). TrkB-IgG, an endogenous BDNF scavenger, did not have any effect on neuroprotection by 17ß-E2 (data not shown). Thus, the involvement of growth factors in our system is unlikely.

In the in vitro system, ß-amyloid-increased H2O2 in cultured neurons was confirmed (49). Hence, our system may be useful not only for addressing the mechanisms underlying neuronal cell death in neurodegenerative disorders such as Alzheimer’s disease, but also for estimating candidate drugs that could block H2O2-induced toxicity. In our system, a series of events, including overactivation of the MAPK pathway, and Ca2+ overload, followed by neuronal cell death, occurred after exposure to H2O2. It is possible that chronic preincubation with physiological levels of 17ß-E2 attenuated the Ca2+ overload and cell death via down-regulation of ionotropic glutamate receptors through the attenuation of MAPK activation. These findings may provide important information concerning the effect of postmenopausal estrogen replacement therapy for neurodegenerative disorders.


   Footnotes
 
T.M.’s current address: Division of Pharmacology/Neurobiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

D.Y.’s current address: Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan.

T.T.’s current address: Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka 563-8577, Japan.

This work was supported by grants from the Japan Foundation for Aging and Health (to T.N.) and Ministry of Education, Culture, Sports, Science and Technology (to T.N.).

Author Disclosure Summary: All of the authors have nothing to disclose.

First Published Online November 2, 2006

1 H.H. died on May 25, 2001, and has been listed as an author because this study was originally started in his laboratory. Back

Abbreviations: AMPA, {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AP5, d-(-)-2-amino-5-phosphonopentanoic acid; BAPTA-AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-AM; BDNF, brain-derived neurotrophic factor; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CNS, central nervous system; 17ß-E2, 17ß-estradiol; ER, estrogen receptor; HEPES, N-2-hydroxyethylpiperadine-N'-2-ethane sulfonic acid; MAP2, microtubule-associated protein 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate; PI3-K, phosphatidylinositol 3-kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.

Received September 5, 2006.

Accepted for publication October 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Behl C 2002 Oestrogen as a neuroprotective hormone. Nat Rev Neurosci 3:433–442[Medline]
  2. Hurn PD, Macrae IM 2000 Estrogen as a neuroprotectant in stroke. J Cereb Blood Flow Metab 20:631–652[CrossRef][Medline]
  3. Dluzen DE, McDermott JL 2000 Gender differences in neurotoxicity of the nigrostriatal dopaminergic system: implications for Parkinson’s disease. J Gend Specif Med 3:36–42[Medline]
  4. Henderson VW 2006 Estrogen-containing hormone therapy and Alzheimer’s disease risk: understanding discrepant inferences from observational and experimental research. Neuroscience 138:1031–1039[CrossRef][Medline]
  5. Simerly RB 2002 Wired for reproduction: organization and development of sexually dimorphic circuits in the mammalian forebrain. Annu Rev Neurosci 25:507–536[CrossRef][Medline]
  6. Chowen JA, Torres-Aleman I, Garcia-Segura LM 1992 Trophic effects of estradiol on fetal rat hypothalamic neurons. Neuroendocrinology 56:895–901[Medline]
  7. Arimatsu Y, Hatanaka H 1986 Estrogen treatment enhances survival of cultured fetal rat amygdala neurons in a defined medium. Brain Res 391:151–159[Medline]
  8. Brinton RD, Tran J, Proffitt P, Montoya M 1997 17ß-Estradiol enhances the outgrowth and survival of neocortical neurons in culture. Neurochem Res 22:1339–1351[CrossRef][Medline]
  9. Foy MR 2001 17ß-estradiol: effect on CA1 hippocampal synaptic plasticity. Neurobiol Learn Mem 76:239–252[CrossRef][Medline]
  10. Parducz A, Hajszan T, Maclusky NJ, Hoyk Z, Csakvari E, Kurunczi A, Prange-Kiel J, Leranth C 2006 Synaptic remodeling induced by gonadal hormones: neuronal plasticity as a mediator of neuroendocrine and behavioral responses to steroids. Neuroscience 138:977–985[CrossRef][Medline]
  11. Yokomaku D, Numakawa T, Numakawa Y, Suzuki S, Matsumoto T, Adachi N, Nishio C, Taguchi T, Hatanaka H 2003 Estrogen enhances depolarization-induced glutamate release through activation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase in cultured hippocampal neurons. Mol Endocrinol 17:831–844[Abstract/Free Full Text]
  12. Andersen JK 2004 Oxidative stress in neurodegeneration: cause or consequence? Nat Med 10(Suppl):S18–S25
  13. Bossy-Wetzel E, Schwarzenbacher R, Lipton SA 2004 Molecular pathways to neurodegeneration. Nat Med 10(Suppl):S2–S9
  14. Singer CA, Rogers KL, Strickland TM, Dorsa DM 1996 Estrogen protects primary cortical neurons from glutamate toxicity. Neurosci Lett 212:13–16[CrossRef][Medline]
  15. Singer CA, Figueroa-Masot XA, Batchelor RH, Dorsa DM 1999 The mitogen-activated protein kinase pathway mediates estrogen neuroprotection after glutamate toxicity in primary cortical neurons. J Neurosci 19:2455–2463[Abstract/Free Full Text]
  16. Behl C, Skutella T, Lezoualc’h F, Post A, Widmann M, Newton CJ, Holsboer F 1997 Neuroprotection against oxidative stress by estrogens: structure-activity relationship. Mol Pharmacol 51:535–541[Abstract/Free Full Text]
  17. Pike CJ 1999 Estrogen modulates neuronal Bcl-xL expression and ß-amyloid-induced apoptosis: relevance to Alzheimer’s disease. J Neurochem 72:1552–1563[CrossRef][Medline]
  18. Behl C, Widmann M, Trapp T, Holsboer F 1995 17-ß estradiol protects neurons from oxidative stress-induced cell death in vitro. Biochem Biophys Res Commun 216:473–482[CrossRef][Medline]
  19. Singer CA, Rogers KL, Dorsa DM 1998 Modulation of Bcl-2 expression: a potential component of estrogen protection in NT2 neurons. Neuroreport 9:2565–2568[Medline]
  20. Zhang L, Rubinow DR, Xaing G, Li BS, Chang YH, Maric D, Barker JL, Ma W 2001 Estrogen protects against ß-amyloid-induced neurotoxicity in rat hippocampal neurons by activation of Akt. Neuroreport 12:1919–1923[CrossRef][Medline]
  21. Mattson MP 2000 Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1:120–129[Medline]
  22. Kawahara M, Kuroda Y 2001 Intracellular calcium changes in neuronal cells induced by Alzheimer’s ß-amyloid protein are blocked by estradiol and cholesterol. Cell Mol Neurobiol 21:1–13[CrossRef][Medline]
  23. Huang Y, Huang YL, Zhang S, Zhu YC, Yao T 2004 Estradiol acutely attenuates glutamate-induced calcium overload in primarily cultured rat hippocampal neurons through a membrane receptor-dependent mechanism. Brain Res 1026:254–260[CrossRef][Medline]
  24. Numakawa T, Yamagishi S, Adachi N, Matsumoto T, Yokomaku D, Yamada M, Hatanaka H 2002 Brain-derived neurotrophic factor-induced potentiation of Ca2+ oscillations in developing cortical neurons. J Biol Chem 277:6520–6529[Abstract/Free Full Text]
  25. Matsumoto T, Numakawa T, Yokomaku D, Adachi N, Yamagishi S, Numakawa Y, Kunugi H, Taguchi T 2006 Brain-derived neurotrophic factor-induced potentiation of glutamate and GABA release: different dependency on signaling pathways and neuronal activity. Mol Cell Neurosci 31:70–84[CrossRef][Medline]
  26. Numakawa Y, Numakawa T, Matsumoto T, Yagasaki Y, Kumamaru E, Kunugi H, Taguchi T, Niki E 2006 Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. J Neurochem 97:1191–1202[CrossRef][Medline]
  27. Numakawa T, Matsumoto T, Adachi N, Yokomaku D, Kojima M, Takei N, Hatanaka H 2001 Brain-derived neurotrophic factor triggers a rapid glutamate release through increase of intracellular Ca2+ and Na+ in cultured cerebellar neurons. J Neurosci Res 66:96–108[CrossRef][Medline]
  28. Numakawa T, Yokomaku D, Kiyosue K, Adachi N, Matsumoto T, Numakawa Y, Taguchi T, Hatanaka H, Yamada M 2002 Basic fibroblast growth factor evokes a rapid glutamate release through activation of the MAPK pathway in cultured cortical neurons. J Biol Chem 277:28861–28869[Abstract/Free Full Text]
  29. Numakawa T, Ishimoto T, Suzuki S, Numakawa Y, Adachi N, Matsumoto T, Yokomaku D, Koshimizu H, Fujimori KE, Hashimoto R, Taguchi T, Kunugi H 2004 Neuronal roles of the integrin-associated protein (IAP/CD47) in developing cortical neurons. J Biol Chem 279:43245–43253[Abstract/Free Full Text]
  30. Yamagishi S, Matsumoto T, Numakawa T, Yokomaku D, Adachi N, Hatanaka H, Yamada M, Shimoke K, Ikeuchi T 2005 ERK1/2 are involved in low potassium-induced apoptotic signaling downstream of ASK1–p38 MAPK pathway in cultured cerebellar granule neurons. Brain Res 1038:223–230[CrossRef][Medline]
  31. Honda K, Sawada H, Kihara T, Urushitani M, Nakamizo T, Akaike A, Shimohama S 2000 Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons. J Neurosci Res 60:321–327[CrossRef][Medline]
  32. Almeida RD, Manadas BJ, Melo CV, Gomes JR, Mendes CS, Graos MM, Carvalho RF, Carvalho AP, Duarte CB 2005 Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways. Cell Death Differ 12:1329–1343[CrossRef][Medline]
  33. Stanciu M, Wang Y, Kentor R, Burke N, Watkins S, Kress G, Reynolds I, Klann E, Angiolieri MR, Johnson JW, DeFranco DB 2000 Persistent activation of ERK contributes to glutamate-induced oxidative toxicity in a neuronal cell line and primary cortical neuron cultures. J Biol Chem 275:12200–12206[Abstract/Free Full Text]
  34. Crossthwaite AJ, Hasan S, Williams RJ 2002 Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca2+ and PI3-kinase. J Neurochem 80:24–35[CrossRef][Medline]
  35. Matsumoto Y, Yamamoto S, Suzuki Y, Tsuboi T, Terakawa S, Ohashi N, Umemura K 2004 Na+/H+ exchanger inhibitor, SM-20220, is protective against excitotoxicity in cultured cortical neurons. Stroke 35:185–190[Abstract/Free Full Text]
  36. Hartley DM, Kurth MC, Bjerkness L, Weiss JH, Choi DW 1993 Glutamate receptor-induced 45Ca2+ accumulation in cortical cell culture correlates with subsequent neuronal degeneration. J Neurosci 13:1993–2000[Abstract]
  37. Weiss JH, Hartley DM, Koh J, Choi DW 1990 The calcium channel blocker nifedipine attenuates slow excitatory amino acid neurotoxicity. Science 247:1474–1477[Abstract/Free Full Text]
  38. Jovanovic JN, Czernik AJ, Fienberg AA, Greengard P, Sihra TS 2000 Synapsins as mediators of BDNF-enhanced neurotransmitter release. Nat Neurosci 3:323–329[CrossRef][Medline]
  39. Satoh T, Numakawa T, Abiru Y, Yamagata T, Ishikawa Y, Enokido Y, Hatanaka H 1998 Production of reactive oxygen species and release of L-glutamate during superoxide anion-induced cell death of cerebellar granule neurons. J Neurochem 70:316–324[Medline]
  40. Mailly F, Marin P, Israel M, Glowinski J, Premont J 1999 Increase in external glutamate and NMDA receptor activation contribute to H2O2-induced neuronal apoptosis. J Neurochem 73:1181–1188[CrossRef][Medline]
  41. Weaver Jr CE, Park-Chung M, Gibbs TT, Farb DH 1997 17ß-Estradiol protects against NMDA-induced excitotoxicity by direct inhibition of NMDA receptors. Brain Res 761:338–341[CrossRef][Medline]
  42. Hilton GD, Bambrick LL, Thompson SM, McCarthy MM 2006 Estradiol modulation of kainic acid-induced calcium elevation in neonatal hippocampal neurons. Endocrinology 147:1246–1255[Abstract/Free Full Text]
  43. Bai X, Wong-Riley MT 2003 Neuronal activity regulates protein and gene expressions of GluR2 in postnatal rat visual cortical neurons in culture. J Neurocytol 32:71–78[CrossRef][Medline]
  44. Zhang C, Wong-Riley M 1999 Expression and regulation of NMDA receptor subunit R1 and neuronal nitric oxide synthase in cortical neuronal cultures: correlation with cytochrome oxidase. J Neurocytol 28:525–539[CrossRef][Medline]
  45. Wade CB, Robinson S, Shapiro RA, Dorsa DM 2001 Estrogen receptor (ER){alpha} and ERß exhibit unique pharmacologic properties when coupled to activation of the mitogen-activated protein kinase pathway. Endocrinology 142:2336–2342[Abstract/Free Full Text]
  46. Simpkins JW, Green PS, Gridley KE, Singh M, de Fiebre NC, Rajakumar G 1997 Role of estrogen replacement therapy in memory enhancement and the prevention of neuronal loss associated with Alzheimer’s disease. Am J Med 103:19S–25S
  47. Sohrabji F, Miranda RCG, Toran-Allerand CD 1995 Identification of a putative estrogen response element in the gene encoding brain-derived neurotrophic factor. Proc Natl Acad Sci USA 92:11110–11114[Abstract/Free Full Text]
  48. Mattson MP, Lovell MA, Furukawa K, Markesbery WR 1995 Neurotrophic factors attenuate glutamate-induced accumulation of peroxides, elevation of intracellular Ca2+ concentration, and neurotoxicity and increase antioxidant enzyme activities in hippocampal neurons. J Neurochem 65:1740–1751[Medline]
  49. Behl C, Davis JB, Lesley R, Schubert D 1994 Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77:817–827[CrossRef][Medline]



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