This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mize, A. L. Articles by Dorsa, D. M. Search for Related Content PubMed PubMed Citation Articles by Mize, A. L. Articles by Dorsa, D. M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ESTRADIOL GLUTAMIC ACID HYDROCHLORIDE

Estrogen Receptor-Mediated Neuroprotection from Oxidative Stress Requires Activation of the Mitogen-Activated Protein Kinase Pathway

Departments of Pharmacology (A.L.M.) and Psychiatry and Behavioral Sciences (R.A.S., D.M.D.), University of Washington, Seattle, Washington 98195; and Department of Physiology and Pharmacology (R.A.S., D.M.D.), Oregon Health Science University, Portland, Oregon 97201

Address all correspondence and requests for reprints to: Robert A. Shapiro, Ph.D., Oregon Health Science University, Department of Physiology and Pharmacology, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. E-mail: shapiror{at}ohsu.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is well documented that estrogen mediates responses by both genomic and nongenomic mechanisms, both of which are important for cell survival. Because direct evidence showing that the estrogen receptors (ERs) and/or ß can activate rapid signaling that may mediate neuroprotection is lacking, the hippocampal-derived cell line, HT22, was stably transfected with ER (HTER), ERß (HTERß), or a mutated form of ER (HTERHE27), which lacks the ability to mediate ER element-mediated transcription. Treatment of HT22, HTER, HTERß, and HTERHE27 cells with glutamate (5 mM) resulted in a significant decrease in cell viability. Pretreatment for 15 min with 10 nM 17ß-estradiol resulted in a 50% increase in the number of living cells in HTER and HTERß cells but not in HT22 cells. The ER antagonist ICI 182,780 and the MEK inhibitor PD98059 prevented 17ß-estradiol-mediated protection. In HTERHE27 cells, 17ß-estradiol rapidly phosphorylated ERK2 (within 15 min), in the absence of estrogen response element-mediated transcription. Treatment of HTERHE27 cells with 10 nM 17ß-estradiol partially reversed the cell death produced by glutamate treatment. This study demonstrates that activation of either ER or ERß can result in neuroprotection and that activation of the MAPK pathway is an important part of the neuroprotective mechanism.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE GONADAL SEX steroid hormone estradiol serves many important functions. In addition to its classical reproductive role, numerous studies demonstrate that it plays an important trophic and protective role in the brain. Epidemiological evidence suggests that estrogen replacement therapy for postmenopausal women is associated with an improvement of some measures of cognitive performance, protection against cognitive deterioration, and decreased incidence of Alzheimer’s disease (1, 2, 3, 4, 5). In addition, beneficial effects of estrogen on the mortality and morbidity associated with cerebral stroke have also been demonstrated (6, 7, 8).

At the cellular level, estrogen is known to exert neuroprotective effects in various model systems. In vitro studies have shown that 17ß-estradiol reduces neuronal damage caused by serum deprivation (9, 10), ß-amyloid treatment (11, 12), and exposure to glutamate (12, 13). Although activation of estrogen receptors has been suggested in mediating neuroprotection, the complex mechanisms by which estrogen protects neurons against injury are not completely understood.

Although estrogen is generally thought to mediate its effects by activation of transcription via nuclear receptors, increasing evidence suggest that estrogen may also cause rapid activation of signal transduction pathways. As examples, estrogen is known to produce mobilization of intracellular calcium (14) production of cAMP (15, 16), activation of Akt (17) as well as MAPK, ERK1, and ERK2 (11, 18, 19, 20). Several lines of evidence suggest estrogen neuroprotection may be mediated by rapid intracellular signaling events rather than estrogen response element (ERE)-mediated gene transcription. For example, activation of protein kinase A, protein kinase C, and MAPK have been linked to neuroprotection in various cellular model systems (19, 21, 22). In addition, recent reports from our laboratory have shown that estrogen receptor (ER) activation of MAPK is important to protect neuronal cells from ß-amyloid toxicity (11). However, it is not clear whether nuclear estrogen receptor activation of rapid signaling pathways is sufficient to elicit neuroprotection in the absence of ERE-mediated transcription.

Our study was conducted to examine whether expression of ER and ERß are required for low doses of estrogen to elicit neuroprotection against glutamate toxicity in a neuronal cell line. In addition, we have examined the relative role of MAPK activation vs. classical ERE-mediated transcription in eliciting estrogen-mediated neuroprotection.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemicals
17ß-Estradiol, glutamate, and PD98059 were purchased from Sigma (St. Louis, MO). ICI 182,780 was purchased from Tocris Cookson (Ballwin, MO).

Cell culture
HT22 cells were given as a kind gift from Dr. Pamela Maher (The Scripps Research Institute, La Jolla, CA). These cells were grown on 100-mm tissue culture dishes and maintained in DMEM (Sigma) media supplemented with 5% fetal bovine serum and 1% Pen-Strep (Gem Cell, Woodland, CA) at 37 C in a 5% CO₂ atmosphere. Cell density was maintained 70% or less confluency as described previously (23).

Plasmids and oligonucleotide mutagenesis
The zinc finger mutation (C202H;C205H) corresponding to HE27, described previously (23), was introduced into ERHEGO/pcDNA3.1 hygromycin by a sequence overlap extension procedure. Overlapping PCR fragments were first generated in separate reactions and then reannealed and extended in a second PCR. In the first step, two oligonucleotides (complement and reverse complement) containing the desired nucleotides were synthesized for each mutation. Two fragments were then synthesized by PCR using a T7 primer and the reverse complement (5'-GAATACTTCTCTTGAAGAAGGCCTTGTGGCCCTCATGACACCAGACTCCATAATGG-3') to generate the 5' fragment and with the complement (5'-CCATTATGGAGTCTGGTCTCATGAGGGCCACAAGGCCTTCTTCAAGAGA-AGTATTC3') and a 3' oligo (5'TGTACACTCCAGAATTAAGC-3') to generate the 3' fragments. Altered nucleotides are indicated in bold type. A novel BspH1 site is underlined. After gel purification, 10 ng of each fragment were mixed and used as a template in a second PCR with the two outer oligonucleotides. The resulting 1400-bp fragment was digested with HindIII and a 1020-bp fragment used to replace the corresponding fragment in ERHEGO/pcDNA3.1 hygromycin. All nucleotide changes were confirmed by DNA sequencing.

Generation of HT22 stable transfectants
Stable transfectants were generated as described previously (11). Briefly, HT22 cells were grown to approximately 60% confluency before being transfected with the lipid transfection reagent, TransFast (Promega Corp., Madison, WI). The pCDNA3.1 hygromycin (7.3 µg) containing either the full-length human ER cDNA (24), a gift from Dr. Pierre Chambon (Strasbourg, France), or rat ERß cDNA (25), a gift from George Kuiper (Karolinska Institute, Sweden), was added at a 1:1 ratio with Transfast per 100-mm plate. Medium was changed 24 h later, and hygromycin (125 µg/ml) was added 72 h after transfection for selection of ER-expressing clones. Single colonies were isolated after the 10th day of growth in selective conditioned media and tested for receptor expression by immunoblotting.

Activation of ERK2
ER-positive cells were grown to 70–80% confluency on 100-mm plates. Eighteen hours before treatment, the medium was replaced with phenol red-free DMEM supplemented with 1% charcoal-stripped serum. Cells were treated with ethanol vehicle (0.1% final concentration) or 17ß-estradiol (10 nM) for the indicated times. The medium was removed, and the cells were washed in ice-cold PBS. Cells were rinsed with ice-cold PBS buffer, scraped into immunoprecipitation buffer (1 M HEPES, 0.1 M EGTA, 0.5 M EDTA, 0.5 M Na⁺ pyrophosphate, 1 M NaF, 1 mM NaVO₄, and 9 mM NaCl) and incubated on ice for at least 5 min. The samples were then sonicated for 2 min followed by centrifugation at 15,000 rpm for 10 min. The supernatant was transferred to a new tube and protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce Chemical Co., Rockford, IL). The proteins samples were diluted in Laemmeli sodium dodecyl sulfate sample buffer and equal volumes of cell lysate were loaded and resolved by electrophoresis on 4–12% Bis-Tris precast gels (Invitrogen, Carlsbad, CA) in running buffer [50 mM 2-(N-morpholine)ethane sulfonic acid, 50 mM Tris base, 0.1% sodium dodecyl sulfate, and 1 mM EDTA] as described by the manufacturer. The proteins were transferred to polyvinylidene diflouride membranes and blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.2% Tween 20 for 1 h at room temperature. ERK2 phosphorylation was detected using mouse antiphospho-p44/42 MAPK (1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) that recognizes phospho-THR202 and THR204 forms of ERK1 and 2. Phosphorylation at these sites has been correlated with increased activity (26, 27, 28). Total ERK2 was detected using rabbit anti-ERK2 (1:10,000, Santa Cruz Biotechnology, Inc.).

Secondary goat antimouse antibodies (1:5000, Santa Cruz Biotechnology, Inc.) or goat antirabbit antibodies (1:5000) conjugated to horseradish peroxidase were used for detection by enhanced chemiluminescence (NEN Life Science Products, Boston, MA) on film. The resulting film samples were scanned and analyzed with an image analysis program (NIH Image, Scion Corp., Frederick, MD). Data are presented as a ratio of phospho-ERK2/total ERK2 in the sample, normalized to vehicle-treated samples.

Transient transfections
HT22 cells (5 x 10⁵ cells/well) were plated into 24-well plates and transfected with 1.0 µg/well tk-ERE-luciferase (kindly provided by Dr. Peter Burbach, Rudolf Magnus Institute, University of Utrecht) using the TransFast protocol. One microgram pCH110, a ß-galactosidase reporter (Amersham Pharmacia, Uppsala, Sweden) was added to each well to normalize for transfection efficiency. After 24 h in DMEM, cells were transfected at 37 C in Transfast reagent in media supplemented with 10% charcoal-stripped calf serum for 1 h. Media was then replaced, and cells were allowed to recover for 24 h before pharmacological manipulation.

Cell treatments
Cells were grown to 70–80% confluency in 12-well plates. Twenty-four hours before treatment the medium was replaced with phenol red free DMEM supplemented with charcoal-stripped fetal bovine serum (1%). Glutamate was diluted to a final concentration of 5 mM in culture medium and cells were exposed for 24 h. 17ß-Estradiol was initially dissolved in 95% ethanol at a concentration of 1 mM and diluted to the appropriate concentration (10 nM) in culture medium. Exposure to 17ß-estradiol was initiated 15 min before glutamate addition. Ethanol was used at a final concentration of 0.1% as a vehicle control. This concentration of ethanol had no effect on cell viability or glutamate toxicity. ICI 182,780 and PD98059 were made as 1000x stocks in 100% dimethylsulfoxide and were added to cells 15 min before 17ß-estradiol exposure.

Dimethylthiazoldiphenyltetra-zoliumbromide (MTT) assay
The MTT assay measures cellular viability by assessing mitochrondrial activity in the cells. After completion of incubation, 200 µl of a 5 mg/ml MTT stock in PBS was added to each well, and the incubation continued for 4 h. After removal of the MTT solution, 1 ml solubilization solution containing 50% dimethylformamide, 20% sodium dodecyl sulfate (pH 4.8) was added. Absorption values at 570 nM were read on a Packard Spectra spectrophotometer.

Calcein acetomethyl ester (AM)
Following incubation of cells in glutamate, cells were rinsed once with PBS and incubated with 1 µM calcein AM dye (Molecular Probes, Inc., Eugene, OR) at 37 C for 15 min, washed twice with PBS, and coverslips applied. Green fluorescent cells are the product of mitochondrial cleavage of calcein AM and were observed using a Optiphot 2 microscope (Nikon) with the EF-D fluorescence attachment and G-1B and DM510 filters and counted as living cells.

Statistical analysis
The significance of differences among groups was determined by one-way ANOVA followed by a Tukey’s multiple comparison test. P < 0.05 was considered significant, and each group consisted of 6–12 wells or plates. All values are expressed as mean ± SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Figure 1 demonstrates that HT22, HTER, and HTERß cells are sensitive to glutamate exposure, in which greater than 50% of cells were killed after a 24-h treatment. To further characterize the steps leading to cell death, various compounds were tested to determine their ability to block glutamate-induced cell death. As suggested by previous reports, both N-methyl-D-aspartate and kainate glutamate receptor antagonists failed to prevent glutamate toxicity in HTER and HTERß cells (Table 1). However, cystine was able to completely reverse the cellular death.

View larger version (19K): [in this window] [in a new window]   Figure 1. Glutamate significantly decreases cell survival in HT22, HTER, and HTERß cells. HT22, HTER, and HTERß cells were treated with glutamate (5 mM) for 24 h. Cellular viability was assessed using the MTT assay. Data are expressed as the percentage of the maximal number of living cells in a vehicle-treated control. All results represent the mean ± SEM from three to four separate platings. *, Statistically different from vehicle-treated cells, P < 0.05.

View larger version (52K): [in this window] [in a new window]   Figure 2. Fifteen-minute pretreatment with low-dose 17ß-estradiol protects HTER and HTERß cells from glutamate toxicity. HT22, HTER, and HTERß cells were treated with 17ß-estradiol (1 µM or 10 nM) for 15 min, followed by glutamate (5 mM) for 24 h. Cellular viability was assessed by calcein AM staining. The number of living cells was assessed by fluorescence with calcein AM. A, Numbers of living cells are expressed as percent of vehicle-treated controls. All results represent the mean ± SEM from three to four separate platings. *, Statistically different from glutamate-treated cells, P < 0.05. B, Representative micrograph showing the presence of living cells, compared with vehicle-treated controls.

View larger version (40K): [in this window] [in a new window]   Figure 3. ICI 182,780 blocks 17ß-estradiol-mediated neuroprotection against glutamate-induced toxicity in HTER and HTERß cells. HTER and HTERß cells were treated with 17ß-estradiol (10 nM) for 15 min in the presence and absence of ICI 182,780 (1 µM), followed by glutamate (5 mM) for 24 h. Cellular viability was assessed as described and presented in Fig. 2.

View larger version (43K): [in this window] [in a new window]   Figure 4. PD98059 blocks 17ß-estradiol-mediated neuroprotection against glutamate induced toxicity in HTER and HTERß cells. HTER and HTERß cells were treated with 17ß-estradiol (10 nM) for 15 min in the presence and absence of PD98059 (50 µM), followed by glutamate (5 mM) for 24 h. Cellular viability was assessed as described in Fig. 2.

View larger version (22K): [in this window] [in a new window]   Figure 5. 17ß-Estradiol treatment increases ERK2 phosphorylation in HTER HE27 cells but does not activate an ERE reporter gene construct. A, Lysates from HTER HE27 cells treated with 10 nM 17ß-estradiol for 5, 15, or 30 min were analyzed by immunoblotting for changes in phosphorylation of ERK2. Lysates were probed with an antiphospho-specific p42/44 MAPK (ERK1/ERK2) antibody. Total ERK2 was detected using an anti-ERK2 antibody that recognizes both phosphorylated and unphosphorylated ERK2. Relative amounts of phosphorylated ERK2 were determined from densitometric scanning of ECL-exposed film. Data are represented as fold increase in relative phosphorylated ERK2, compared with vehicle treatment ± SEM. All experiments were performed three times in triplicate, and asterisks (*) indicate a P value of 0.05 or less, as determine by one-way ANOVA. B, Transiently transfected HTER HE27 cells were treated with vehicle or 50 nM 17ß-estradiol for 24 h and assayed for luciferase and ß-galactosidase activity. Data are represented as the ratio of luciferase/ß-galactosidase activity. All experiments were performed three times in triplicate, and asterisks (*) indicate a P value of 0.05 or less, as determined by one-way ANOVA.

View larger version (49K): [in this window] [in a new window]   Figure 6. Ffiteen-minute pretreatment with low-dose 17ß-estradiol partially protects HTER HE27 cells from glutamate toxicity. HTER HE27 and HTER cells were treated with 17ß-estradiol (10 nM) for 15 min, followed by glutamate (5 mM) for 24 h. Cellular viability was assessed as described in Fig. 2. *, Significantly different from vehicle-treated controls (P 0.05). **, Significantly different from cells treated with vehicle or glutamate (P 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Glutamate is thought to be a major cause of neuronal cell death in a number of different neurodegenerative diseases (30, 31, 32). Two pathways for glutamate toxicity have been described; excitotoxicity (33), which occurs through the activation of glutamatergic receptors (34, 35), and oxidative glutamate toxicity, which is mediated via a series of disturbances to the redox homeostasis of the cell (36). These pathways are incompletely characterized, but both result in the production of free radicals (31, 36). Our results are in accordance with others (23), showing that HT22 cells die via the oxidative pathway from glutamate toxicity, as glutamatergic receptor antagonists fail to block glutamate induced toxicity. It has been reported that exposure of HT22 cells to glutamate results in an inhibition of cystine transport into the cell (36), leading to an inability to maintain intracellular glutathione levels. The low level of intracellular glutathione reduces the cell’s ability to counteract oxidative reactions within the cell and, ultimately, cell death. Our results support this notion because a high dose of cystine is able to protect the cells from glutamate toxicity.

There is significant debate over whether estrogen’s neuroprotective actions are mediated by one of the known ERs or nonspecific antioxidant mechanisms. The theory that at supraphysiological doses estrogen acts through antioxidant mechanism to prevent cell death has been clearly established. In doses greater than 10 µM 17ß-estradiol can reduce phospholipid oxidation in a cell-free system (37). More recently it has been shown that micromolar doses of estrogen are able to protect estrogen receptor-negative neuronal cell lines form cytotoxicity (12, 38). There is, however, also a body of evidence showing that exposure to lower, more physiological doses of estrogen is possible to achieve neuroprotection in cells containing ERs. For example, Gollapudi and Oblinger (39) found that estradiol exposure attenuates serum deprivation toxicity in PC12 cells transfected with ER but not those transfected with a control plasmid. In addition, in an in vivo model of ischemia, ER was found to be the critical link in mediating the protective effects of physiological concentrations of estradiol in brain injury (40). Furthermore, we have previously reported that a short pretreatment with 17ß-estradiol protects cells from ß-amyloid toxicity, and this protection is dependent on ER expression (11).

The role of ERß in mediating neuroprotection is more controversial. For example, in studies by Dubal et al. (40), ERß was not sufficient for protection against ischemia in an in vivo model. Kim et al. (41) did not observe neuroprotection against ß-amyloid peptide toxicity in HT22 cells stably transfected with ERß. However, we observed pronounced neuroprotection in HTERß cells. In support of our findings, studies by Sawada and Shimohama (42) demonstrated estrogen to be neuroprotective in mesencephalon dopaminergic neurons, which exclusively express ERß. In addition, Wang et al. (43) demonstrated the importance of ERß in the survival of neurons throughout the brain by measuring a neuronal deficit in ERß knockout mice. In their studies, the neuronal loss was increased with age, suggesting a role for ERß in the prevention of neurodegenerative diseases.

The time frame of MAPK activation in HTER and HTERß cells (11) correlates to the pretreatment time required to see neuroprotection. Indeed, PD98059 blocked neuroprotection, suggesting that transient phosphorylation of MAPK results in estrogen’s ability to be neuroprotective. It is possible that estrogen is eventually activating transcription of genes important for neuroprotection. This has been shown to occur in SK-N-BE2C cells transfected with ER. In these cells, the membrane-impermeable E2-BSA acted through nongenomic mechanisms at the cell membrane to facilitate the ability of estrogen to activate an ERE-luciferase reporter. The events were proven to act through the classical ER at the cell membrane because ICI 182,780 blocked the transcriptional effects (44). Alternatively, genes responding to activation of the MAPK pathway, independent of ERE activity, may also be induced. For example, BCL2 is induced by estrogen in primary neuron cultures, and it has been recently reported that cAMP response element activation in the BCL promoter may be critical for this effect (45). Therefore, it is possible that a 15-min pretreatment with estrogen is neuroprotective because of the subsequent transcription of multiple genes (i.e. a nonclassical pathway for estrogen activation of transcription).

The generation of a cell line expressing a mutated form of ER allowed us to demonstrate more clearly that rapid signaling was indeed important for neuroprotection. This mutated receptor contains two point mutations in the zinc finger domain, rendering the receptor unable to bind the palindromic ERE on DNA to activate ERE-mediated transcription (29). We clearly showed that HTERHE27 is incapable of inducing luciferase activity from an ERE, verifying the critical role of these cystine residues in interacting with this element. Although the receptor was unable to activate an ERE reporter gene, HTERHE27 was fully capable of rapidly activating MAPK in a time frame similar to what was seen in cells expressing ER or ERß (11). In addition, 10 nM 17ß-estradiol phosphorylated MAPK approximately 5-fold, which is the same fold activation observed in HT22 cells expressing the wild-type ER (11). When examining the neuroprotective capabilities of HTERHE27, significant neuroprotection was seen (approximately two thirds of neuroprotection observed in wild-type ER cells). This further suggests that estrogen’s ability to activate the MAPK pathway is important for neuroprotection against glutamate toxicity. These results are consistent with our earlier reports, demonstrating that estrogen-mediated neuroprotection against glutamate toxicity required activation of the MAPK pathway in primary cortical neurons (21). The involvement of the MAPK pathway in the protection of neurons from excitotoxicity has been further confirmed by the ability of PD98059 to block estrogen’s effects on protection from N-methyl-D-aspartate and kainate induced toxicity in hippocampal slice cultures (46). In addition, estrogen activation of MAPK through the ERs provides neuroprotection in primary neuronal cultures against a variety of toxic insults including quinolinic acid toxicity (47) and glutamate excitotoxicity (21).

Although clear and significant neuroprotection was found in the HTERHE27 cell line, estrogen was not able to block 100% of the cell death brought about by glutamate exposure. This suggests that multiple pathways may be involved in the complex mechanism of neuroprotection. It is possible that acutely, estrogen activates MAPK to establish one pathway of neuroprotection, although longer treatments with the hormone work through the classical ERE-mediated transcriptional pathway to up-regulate protective genes within the cell. Further studies need to be conducted to delineate more clearly how the different pathways work together to mediate the neuroprotection pathway.

Our studies clearly show that ER and ERß expression is necessary for estrogen-mediated neuroprotection against glutamate toxicity. In HTER and HTERß cells, 17ß-estradiol provided neuroprotection following a 15-min pretreatment. Neuroprotection was blocked by ICI 182,780 and PD98059, demonstrating that estrogen activation of MAPK via the ER is necessary for 17ß-estradiol to be neuroprotective. In addition, in cells expressing the mutated form of ER, HE27, moderate neuroprotection was still observed. These data suggest that either ER or ERß can mediate estrogen neuroprotection in a neuronal cell model and that rapid activation of the MAPK pathway is indeed important in the mechanism of neuroprotection. Therefore, a further understanding of estrogen rapid signaling may be important to increase neuroprotection and prevent neurodegenerative diseases.

	Footnotes

 
This work was supported by NIH Grant T32-AG-00057 (to A.L.M.), a project in the University of Washington Alzheimer’s Center AG 05136-18, and NIH Grant NS-20311 (to D.M.D.).

Abbreviations: AM, Acetomethyl ester; ER, estrogen receptor; ERE, estrogen response element; MTT, dimethylthiazoldiphenyltetra-zoliumbromide.

Received July 9, 2002.

Accepted for publication September 23, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Paganinihill A, Henderson VW 1994 Estrogen deficiency and risk of Alzheimer’s disease in women. Am J Epidemiol 140:256–261[Abstract/Free Full Text]
2. Robinson D, Friedman L, Marcus R, Tinklenberg J, Yesavage J 1994 Estrogen replacement therapy and memory in older women. J Am Geriatr Soc 42:919–922[Medline]
3. Sherwin BB 1994 Estrogenic effects on memory in women. Ann N Y Acad Sci 743:213–230[Abstract]
4. Birge SJ 1996 Is there a role for estrogen replacement therapy in the prevention and treatment of dementia? J Am Geriatr Soc 44:865–870[Medline]
5. Costa MM, Reus VI, Wolkowitz OM, Manfredi F, Lieberman M 1999 Estrogen replacement therapy and cognitive decline in memory-impaired post-menopausal women. Biol Psychiatry 46:182–188[CrossRef][Medline]
6. Lafferty FW, Fiske ME 1994 Postmenopausal estrogen replacement: a long-term cohort study. Am J Med 97:66–77[CrossRef][Medline]
7. Grodstein F, Stampfer MJ, Manson JE, Colditz GA, Willett WC, Rosner B, Speizer FE, Hennekens CH 1996 Postmenopausal estrogen and progestin use and the risk of cardiovascular disease. N Engl J Med 335:453–461[Abstract/Free Full Text]
8. Hurn PD, Macrae IM 2000 Estrogen as a neuroprotectant in stroke. J Cereb Blood Flow Metab 20:631–652[CrossRef][Medline]
9. Bishop J, Simpkins JW 1994 Estradiol treatment increases viability of glioma and neuroblastoma cells in vitro. Mol Cell Neurosci 5:303–308[CrossRef][Medline]
10. Green PS, Bishop J, Simpkins JW 1997 17-Estradiol exerts neuroprotective effects on SK-N-SH cells. J Neurosci 17:511–515[Abstract/Free Full Text]
11. Fitzpatrick JL, Mize AL, Wade CB, Harris JA, Shapiro RA, Dorsa DM 2002 Estrogen-mediated neuroprotection against ß-amyloid toxicity requires expression of estrogen receptor or ß and activation of the MAPK pathway. J Neurochem 82:674–682[CrossRef][Medline]
12. 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]
13. 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]
14. Morley P, Whitfield JF, Vanderhyden BC, Tsang BK, Schwartz JL 1992 A new, nongenomic estrogen action: the rapid release of intracellular calcium. Endocrinology 131:1305–1312[Abstract]
15. Razandi M, Pedram A, Greene GL, Levin ER 1999 Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ER and ERß expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319[Abstract/Free Full Text]
16. Aronica SM, Kraus WL, Katzenellenbogen BS 1994 Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription. Proc Natl Acad Sci USA 91:8517–8521[Abstract/Free Full Text]
17. Ahmad S, Singh N, Glazer RI 1999 Role of AKT1 in 17ß-estra. Biochem Pharmacol 58:425–430[CrossRef][Medline]
18. Migliaccio A, Di DM, Castoria G, de FA, Bontempo P, Nola E, Auricchio F 1996 Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J 15:1292–1300[Medline]
19. Watters JJ, Campbell JS, Cunningham MJ, Krebs EG, Dorsa DM 1997 Rapid membrane effects of steroids in neuroblastoma cells: effects of estrogen on mitogen activated protein kinase signaling cascade and c-fos immediate early gene transcription. Endocrinology 138:4030–4033[Abstract/Free Full Text]
20. Singh M, Setalo G, Guan X, Warren M, Toran AC 1999 Estrogen-induced activation of mitogen-activated protein kinase in cerebral cortical explants: convergence of estrogen and neurotrophin signaling pathways. J Neurosci 19:1179–1188[Abstract/Free Full Text]
21. 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]
22. Rydel RE, Greene LA 1988 cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. Proc Natl Acad Sci USA 85:1257–1261[Abstract/Free Full Text]
23. Maher P, Davis JB 1996 The role of monoamine metabolism in oxidative glutamate toxicity. J Neurosci 16:6394–6401[Abstract/Free Full Text]
24. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[CrossRef][Medline]
25. Kuiper GG, Enmark E, Pelto HM, Nilsson S, Gustafsson J-Å 1996 Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
26. Robbins DJ, Zhen E, Owaki H, Vanderbilt CA, Ebert D, Geppert TD, Cobb MH 1993 Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. J Biol Chem 268:5097–5106[Abstract/Free Full Text]
27. Robbins DJ, Cobb MH 1992 Extracellular signal-regulated kinases 2 autophosphorylates on a subset of peptides phosphorylated in intact cells in response to insulin and nerve growth factor: analysis by peptide mapping. Mol Biol Cell 3:299–308[Abstract]
28. Payne DM, Rossomando AJ, Martino P, Erickson AK, Her JH, Shabanowitz J, Hunt DF, Weber MJ, Sturgill TW 1991 Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J 10:885–892[Medline]
29. Kumar V, Green S, Staub A, Chambon P 1986 Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J 5:2231–2236[Medline]
30. Greenamyre JT, Penney JB, Young AB, D’Amato CJ, Hicks SP, Shoulson I 1985 Alterations in L-glutamate binding in Alzheimer’s and Huntington’s diseases. Science 227:1496–1499[Abstract/Free Full Text]
31. Choi DW 1992 Excitotoxic cell death. J Neurobiol 23:1261–1276[CrossRef][Medline]
32. Lees GJ 1993 Contributory mechanisms in the causation of neurodegenerative disorders. Neuroscience 54:287–322[CrossRef][Medline]
33. Olney JW 1969 Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science 164:719–721[Abstract/Free Full Text]
34. Choi DW 1988 Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:623–634[CrossRef][Medline]
35. Michaels RL, Rothman SM 1990 Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations. J Neurosci 10:283–292[Abstract]
36. Murphy TH, Miyamoto M, Sastre A, Schnaar RL, Coyle JT 1989 Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron 2:1547–1558[CrossRef][Medline]
37. Sugioka K, Shimosegawa Y, Nakano M 1987 Estrogens as natural antioxidants of membrane phospholipid peroxidation. FEBS Lett 210:37–39[CrossRef][Medline]
38. Green PS, Gridley KE, Simpkins JW 1998 Nuclear estrogen receptor-independent neuroprotection by estratrienes: a novel interaction with glutathione. Neuroscience 84:7–10[CrossRef][Medline]
39. Gollapudi L, Oblinger MM 1999 Stable transfection of PC12 cells with estrogen receptor (ER): protective effects of estrogen on cell survival after serum deprivation. J Neurosci Res 56:99–108[CrossRef][Medline]
40. Dubal DB, Zhu H, Yu J, Rau SW, Shughrue PJ, Merchenthaler I, Kindy MS, Wise PM 2001 Estrogen receptor , not ß, is a critical link in estradiol-mediated protection against brain injury. Proc Natl Acad Sci USA 98:1952–1957[Abstract/Free Full Text]
41. Kim H, Bang OY, Jung MW, Ha SD, Hong HS, Huh K, Kim SU, Mook-Jung I 2001 Neuroprotective effects of estrogen against ß-amyloid toxicity are mediated by estrogen receptors in cultured neuronal cells. Neurosci Lett 302:58–62[CrossRef][Medline]
42. Sawada H, Shimohama S 2000 Neuroprotective effects of estradiol in mesencephalic dopaminergic neurons. Neurosci Biobehav Rev 24:143–147[CrossRef][Medline]
43. Wang L, Andersson S, Warner M, Gustafsson JA 2001 Morphological abnormalities in the brains of estrogen receptor beta knockout mice. Proc Natl Acad Sci USA 98:2792–2796[Abstract/Free Full Text]
44. Vasudevan N, Kow LM, Pfaff DW 2001 Early membrane estrogenic effects required for full expression of slower genomic actions in a nerve cell line. Proc Natl Acad Sci USA 98:12267–12271[Abstract/Free Full Text]
45. Linford NJ, Yang Y, Cook DG, Dorsa DM 2001 Neuronal apoptosis resulting from high doses of the isoflavone genistein: role for calcium and p42/44 mitogen-activated protein kinase. J Pharmacol Exp Ther 299:67–75[Abstract/Free Full Text]
46. Bi R, Broutman G, Foy MR, Thompson RF, Baudry M 2000 The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus. Proc Natl Acad Sci USA 97:3602–3607[Abstract/Free Full Text]
47. Kuroki Y, Fukushima K, Kanda Y, Mizuno K, Watanabe Y 2001 Neuroprotection by estrogen via extracellular signal-regulated kinase against quinolinic acid-induced cell death in the rat hippocampus. Eur J Neurosci 13:472–476[CrossRef][Medline]

This article has been cited by other articles:

				M. M. McCARTHY Estradiol and the Developing Brain Physiol Rev, January 1, 2008; 88(1): 91 - 134. [Abstract] [Full Text] [PDF]

				T.-V. V. Nguyen, M. Yao, and C. J. Pike Flutamide and Cyproterone Acetate Exert Agonist Effects: Induction of Androgen Receptor-Dependent Neuroprotection Endocrinology, June 1, 2007; 148(6): 2936 - 2943. [Abstract] [Full Text] [PDF]

				T. Jover-Mengual, R. S. Zukin, and A. M. Etgen MAPK Signaling Is Critical to Estradiol Protection of CA1 Neurons in Global Ischemia Endocrinology, March 1, 2007; 148(3): 1131 - 1143. [Abstract] [Full Text] [PDF]

				K. Moriarty, K. H. Kim, and J. R. Bender Estrogen Receptor-Mediated Rapid Signaling Endocrinology, December 1, 2006; 147(12): 5557 - 5563. [Abstract] [Full Text] [PDF]

				S.-M. HO, Y.-K. LEUNG, and I. CHUNG Estrogens and Antiestrogens as Etiological Factors and Therapeutics for Prostate Cancer Ann. N.Y. Acad. Sci., November 1, 2006; 1089(1): 177 - 193. [Abstract] [Full Text] [PDF]

				Y. Xu, W. Zhang, J. Klaus, J. Young, I. Koerner, L. C. Sheldahl, P. D. Hurn, F. Martinez-Murillo, and N. J. Alkayed Role of cocaine- and amphetamine-regulated transcript in estradiol-mediated neuroprotection PNAS, September 26, 2006; 103(39): 14489 - 14494. [Abstract] [Full Text] [PDF]

				A. J. Mhyre, R. A. Shapiro, and D. M. Dorsa Estradiol Reduces Nonclassical Transcription at Cyclic Adenosine 3',5'-Monophosphate Response Elements in Glioma Cells Expressing Estrogen Receptor Alpha Endocrinology, April 1, 2006; 147(4): 1796 - 1804. [Abstract] [Full Text] [PDF]

				Y. Merot, F. Ferriere, E. Debroas, G. Flouriot, D. Duval, and C. Saligaut Estrogen receptor alpha mediates neuronal differentiation and neuroprotection in PC12 cells: critical role of the A/B domain of the receptor J. Mol. Endocrinol., October 1, 2005; 35(2): 257 - 267. [Abstract] [Full Text] [PDF]

				J. A. Arreguin-Arevalo and T. M. Nett A Nongenomic Action of 17{beta}-Estradiol as the Mechanism Underlying the Acute Suppression of Secretion of Luteinizing Hormone Biol Reprod, July 1, 2005; 73(1): 115 - 122. [Abstract] [Full Text] [PDF]

				J. M. Wang, P. B. Johnston, B. G. Ball, and R. D. Brinton The Neurosteroid Allopregnanolone Promotes Proliferation of Rodent and Human Neural Progenitor Cells and Regulates Cell-Cycle Gene and Protein Expression J. Neurosci., May 11, 2005; 25(19): 4706 - 4718. [Abstract] [Full Text] [PDF]

				X. Yu, R. V. S. Rajala, J. F. McGinnis, F. Li, R. E. Anderson, X. Yan, S. Li, R. V. Elias, R. R. Knapp, X. Zhou, et al. Involvement of Insulin/Phosphoinositide 3-Kinase/Akt Signal Pathway in 17{beta}-Estradiol-mediated Neuroprotection J. Biol. Chem., March 26, 2004; 279(13): 13086 - 13094. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mize, A. L. Articles by Dorsa, D. M. Search for Related Content PubMed PubMed Citation