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(The FASEB Journal. 2000;14:1202-1214.)
© 2000 FASEB

Mechanisms of antiapoptotic effects of estrogens in nigral dopaminergic neurons

HIDEYUKI SAWADA, MASAKAZU IBI*, TAKESHI KIHARA, MAKOTO URUSHITANI, KAZUHIRO HONDA, MIKI NAKANISHI*, AKINORI AKAIKE* and SHUN SHIMOHAMA1

Department of Neurology, Graduate School of Medicine, Kyoto University, Sakyoku, Kyoto 606-8507, Japan; and
* Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyoku, Kyoto 606-8501, Japan

1Correspondence: Department of Neurology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyoku, Kyoto 606-8507, Japan. E-mail: i53367{at}sakura.kudpc.kyoto-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Parkinson’s disease is characterized by the mesencephalic dopaminergic neuronal loss, possibly by apoptosis, and the prevalence is higher in males than in females. The estrogen receptor (ER) subtype in the mesencephalon is exclusively ER ß, a recently cloned novel subtype. Bound with estradiol, it enhances gene transcription through the estrogen response element (ERE) or inhibits it through the activator protein-1 (AP-1) site. We demonstrated that 17ß-estradiol provided protection against nigral neuronal apoptosis caused by exposure to either bleomycin sulfate (BLM) or buthionine sulfoximine (BSO). BLM and BSO-induced nigral apoptosis was blocked by inhibitors for caspase-3 or c-Jun/AP-1. The antiapoptotic effect by estradiol was blocked by ICI 182,780, an antagonist for ER, but not by a synthesized peptide that inhibits binding of the ER to the ERE. Estradiol had no effects on caspase-3 activation and c-Jun NH2-terminal kinase (JNK), which were activated by BLM. It also suppressed apoptosis by serum deprivation, which was independent of caspase-3 activation. Therefore, the antiapoptotic neuroprotection by estradiol is mediated by transcription through AP-1 site downstream from JNK and caspase-3 activation. Furthermore, 17{alpha}-estradiol, a stereoisomer without female hormone activity, also provided an antiapoptotic effect. Therefore, the antiapoptotic effect is independent of female hormone activity.—Sawada, H., Ibi, M., Kihara, T., Urushitani, M., Honda, K., Nakanishi, M., Akaike, A., Shimohama, S. Mechanisms of antiapoptotic effects of estrogens in nigral dopaminergic neurons.


Key Words: estrogen receptor ß • AP-1 element • Parkinson’s disease • neuroprotection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PARKINSON’S DISEASE IS a chronic, progressive degenerative disorder characterized by selective loss of mesencephalic dopaminergic neurons. In the development of the disease, exaggerated metabolic dopamine turnover, deposition of free iron, and decrease in glutathione content (1) cause oxidative stress against mesencephalic dopaminergic neurons. Oxidative stress can cause neuronal apoptosis (2 , 3) , and it has been revealed that radical induced-apoptosis is involved in the degenerative process of dopaminergic neurons (4 , 5) .

Epidemiological studies have indicated that the incidence and prevalence of Parkinson’s disease is higher in men than in women (6) . The ratio of the prevalence in females vs. males is 1:1.36 (7) ~1:3.7 (8) . Estrogens, which are female sex steroid hormones, are modulators of apoptosis in the uterine epithelium (9) and in breast carcinoma cells (10) . It has been discovered recently that estrogens provide neuroprotection against neuronal degeneration. There have been several reports that the decline of estrogen is related to the risk of Alzheimer’s disease in postmenopausal women and that replacement of estrogen reduces the morbidity of the disease (11 12 13 14) . Also, estradiol provides neuroprotection against glutamate- or radical-induced neurotoxicity in vitro (15 16 17 18 19) , including in nigral dopaminergic neurons (20) . Recent investigations reported that the neuroprotection by estradiol is at least partially mediated by conjugation of estradiol with glutathione (19) and not mediated by the estrogen receptor (21) . Furthermore, others have reported that activation of extracellular signal-regulated protein kinase (ERK), an isoform of mitogen-activated kinase, by estradiol mediates the neuroprotection (22 , 23) ; however, it is controversial whether or not the ERK activation by estradiol is mediated by estrogen receptor (22 23 24 25) .

Recently, a novel subtype (named ER ß) of the estrogen receptor (ER) was cloned and the classical subtype was renamed ER {alpha} (26) . Estradiol regulates gene transcription in two ways: through binding to the estrogen response element (ERE) and through the activator protein-1 (AP-1) enhance element of DNA. Estradiol enhances gene transcription through the ERE when bound to either ER {alpha} or ß. In contrast, it enhances transcription through the AP-1 site when bound to ER {alpha}, but suppresses it when bound to ER ß (27) . In the central nervous system, the dominant ER subtype is ER ß, especially in the cerebral cortex, cerebellum, and brainstem. In the substantia nigra of the mesencephalon, the subtype is exclusively ER ß (28 , 29) . In the process of apoptosis, the activator proteins c-Fos and c-Jun play a pivotal role and may regulate gene transcription through heteromer formation with the ER (30 , 31) . Therefore, estradiol may inhibit gene transcription through the AP-1 site, inhibiting apoptosis in nigral dopaminergic neurons.

L-buthionine-[S,R]-sulfoximine (BSO) is an irreversible specific inhibitor of {gamma}-glutamylcysteine synthetase (32) and causes deprivation of glutathione (33) . BSO-induced neurotoxicity can be used as a model of toxicity induced by oxidative stress in Parkinson’s disease because glutathione is depleted in the nigral dopaminergic neurons in the disease (34 35 36) . Bleomycin sulfate (BLM) is an anti-cancer drug with two molecular domains: one binds to nuclear DNA and the other acts as a radical generator (37) . BSO and BLM have been reported to induce apoptosis (38 39 40 41) and can be used as models of oxidative stress. In the present study, we examined the hypothesis that estrogens provide neuroprotection against apoptosis induced by experimental oxidative stress, achieved by exposure to BSO or BLM, in nigral dopaminergic neurons, and that the antiapoptotic effects are independent of female hormone activity and mediated by gene transcriptional regulation through the AP-1 site.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Eagle’s minimum essential medium (EMEM) was purchased from Nissui Pharmaceutical Co. (Tokyo, Japan). BLM was obtained from Calbiochem-Novabiochem AG (Läufelfingen, Switzerland). 1,7-bis(4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-dione (curcumin), BSO, and tamoxifen citrate were purchased from Sigma Chemical Co. (St. Louis, Mo.). ICI 182,780 was obtained from Tocris Cookson Incorporation (Ballwin, Mo.). Antiestrogen peptide (Yp537; H-Cys-Asn-Val-Val-Pro-Leu-Tyr (PO3H2)-Asp-Leu-Leu-Leu-Glu-OH) was purchased from Bachem Bioscience Inc. (Bubendorf, Switzerland). Specific inhibitors for caspase-1 (Ac-Trp-Glu-His-Asp-H; Ac-WEHD-CHO) (42) and caspase-3 (Ac-Asp-Met-Gln-Asp-H; Ac-DMQD-CHO) (43) were obtained from Peptide Institute Inc. (Osaka, Japan). Monoclonal anti-tyrosine hydroxylase (TH) antibody was obtained from Eugene Tech (Ridgefield Park, N.J..), and monoclonal anti-microtubule associated protein 2 (MAP 2) antibody for immunocytochemical studies was obtained from Sigma. Anti-c-Jun NH2-terminal kinase 1 (JNK1), anti-c-Jun-NH2-terminal kinase 2/3 (JNK 2/3), and anti-c-Jun antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Cell culture
Cultures of the rat mesencephalon were performed according to methods described previously (20 , 44 45 46) . The ventral two-thirds of the mesencephalon was dissected from rat embryos on the 16th day of gestation. The dissected regions included dopaminergic neurons in the substantia nigra and the ventral tegmental area, but not noradrenergic neurons in the locus ceruleus. Neurons were incubated with 0.1% trypsin at 37°C for 20 min and incubated with EMEM containing 40% fetal calf serum (FCS). They were gently triturated and plated out onto 0.1% polyethyleneimine-coated plastic coverslips at a density of 1.3 x 105 cells/cm2. The culture medium consisted of EMEM containing 10% FCS for the first 1 to 4 days in culture and horse serum (HS) from the 5th day onward. The animals were treated in accordance with guidelines published in the NIH Guide for the Care and Use of Laboratory Animals (47) .

Treatment of the cultures
In a pilot study, 24 h incubation was required to induce apoptotic morphological changes which were detected by TUNEL and the nuclear dye Hoechst 33258. Therefore, to investigate BSO- or BLM-induced neurotoxicity, cultured neurons were exposed to BSO or BLM for 24 h on the 8th day of culture, incubated in EMEM containing 10% HS for an additional 72 h, and fixed on the 12th day. To determine the effects of preincubation with estrogens on BSO- or BLM-induced neurotoxicity, the cultures were preincubated with 10 nM 17{alpha}- or 17ß-estradiol on the 7th day in culture for 24 h prior to BSO or BLM exposure. On the 8th day in culture, cells were exposed to EMEM containing BSO or BLM with 17{alpha}- or 17ß-estradiol for 24 h. Cells were incubated in medium without drugs for an additional 72 h and fixed on the 12th day. Control experiments were similar to treatment, using EMEM containing no drugs. Incubation with less than 0.1% dimethyl sulfoxide (DMSO) or 0.1% ethanol for 24 h was found to have no effect on neuronal survival rates, and so 17{alpha}- and 17ß-estradiol, ICI 182,780, and curcumin were dissolved in DMSO. Tamoxifen, Ac-WEHD-CHO, Ac-DMQD-CHO, and Yp537 were dissolved in ethanol. BSO and BLM were dissolved in water.

In a pilot study, concentrations of tamoxifen greater than 1 µM, Yp537 greater than 10 µM, Ac-WEHD-CHO greater than 100 µM and Ac-DMQD-CHO greater than 100 µM were toxic to cultured neurons (data not shown). Therefore, the drug concentrations used were 10–100 nM for tamoxifen, 1 µM for Yp537, and 1–10 µM for Ac-WEHD-CHO or Ac-DMQD-CHO.

Evaluation of neurotoxicity
The number of surviving neurons was determined using immunostaining as described in our previous studies (20 , 44 45 46) . Briefly, cultured cells were fixed and incubated with anti-TH (diluted 1:1000) or anti-MAP2 (diluted 1:400) antibodies for 24 h, with the secondary biotinylated antibody for 1 h, and with avidin–biotin complex solution (Vectastain) for 1 h. Finally, the cultures were reacted with diaminobenzidine solution for 6 min. The number of cells stained with anti-TH antibody in 10 to 20 randomly selected fields (x200, total magnification) was taken as the numbers of surviving dopaminergic neurons and those stained with anti-MAP2 antibody in 15 randomly selected fields (x400) as that of total neurons. Counts were made blind to the experimental treatment. In control cultures, 100 to 200 TH-positive neurons or 100 to 200 MAP2-positive neurons were counted for dopaminergic and total neurons, respectively. Neurotoxicity was evaluated by the reduction in neuronal survival rate in each experiment.

Statistical analysis
Statistical analysis was performed by one-way ANOVA and post hoc multiple comparison by Newman-Keuls’ method when variance in the data was statistically equivalent. Uniformity of variance was analyzed statistically by Bartlett’s test. Statistical significance was defined as P<0.05.

LD50 of the neurotoxicity induced by BSO and BLM exposure
The dose response curve for BSO toxicity was fitted to the curve using the following Michaelis Menten’s equation.

where [Survival (%)] is survival rate of the cultured neurons, [BSO] is the BSO concentration (µM), LD50 is the 50% lethal dose of BSO toxicity. The value of LD50 and the 95% confidential intervals were determined by the computer software (GraphPad Prism Version 3.0, GraphPad Software Inc.) and convergence was reached when three consecutive iterations changed the sum-of-squares by less than 0.001%. Curve fitting for BLM toxicity was performed similarly.

Evaluation of apoptotic features in cultured cells
To reveal the nuclear morphological changes in cultured neurons, cells were stained with the nuclear dye Hoechst 33258. On the 8th day, cells were exposed to BLM and BSO, then fixed with neutral formaldehyde on the 9th day. They were incubated with 1 mM Hoechst 33258 at room temperature (RT) for 30 min and washed three times with phosphate-buffered saline (PBS). Cells were observed under ultraviolet illumination using fluorescent microscopy.

In situ visualization of DNA fragmentation at the single cell level was performed by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method (48) using the Apoptosis Detection kit (Wako, Tokyo, Japan). To avoid detachment of fragmented DNA, cells were fixed with 4% neutral formaldehyde saline immediately after experiments on the 8th or 9th day in culture and washed twice with PBS, then exposed to buffer containing sodium citrate (0.1%) and Triton X-100 (0.1%) on ice for 2 min. Cells were washed for 20 s, incubated with TdT buffer at 37°C for 10 min, and washed twice with PBS. Endogenous peroxidase was inactivated with 3% H2O2 for 5 min at RT. Then the cells were washed for 10 min and incubated with buffer containing peroxidase-conjugated antibody at 37°C for 10 min. After washing with PBS for 10 min, cells were reacted with diaminobenzidine at RT for 5 min and counterstained with methylgreen solution.

Caspase-3 activity
Caspase-3 activity was measured according to methods described previously (49) . Briefly, the caspase-3 activity was detected by the cleavage of the colorimetric caspase-3 substrate, acetyl-Asp-Glu-Val-Asp-p-nitroanilidine (DEVD-pNA), using an assay kit, ApoAlert CPP32 (Clontech, Palo Alto, Calif.). Cultured neurons were harvested using a cell scraper and resuspended in 10 mM PBS. Cells were collected and incubated with the cell lysis buffer included in the kit for 10 min on ice and centrifuged at 12,000 rpm for 3 min at 4°C. The supernatant was added to the reaction buffer with dithiothreitol and the DEVD-p-NA, and incubated for 1 h at 37°C. Relative caspase-3 activity was measured as optical density at 405 nm. The relative activity was standardized by a protein concentration that was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, Calif.).

Western blot analysis
Cells were fixed on the 9th day in culture to assay level of Bcl-2, JNK 1, JNK 2/3 by Western blotting. Cells were washed twice with cold TBS, harvested using a cell scraper, and lysed in the buffer containing Tris (40 µM), ß-glycerophosphate (50 mM), EGTA (0.8 mM), Triton X-100 (2%), phenylmethylsulfonylfluoride(1 mM), aprotinine (1%), dithiothreitol(2 mM), and vanadate (1 mM) on ice. Lysates were centrifuged at 150,000 r.p.m. at 4°C for 30 min. The protein was denatured by boiling at 100°C for 4 min. An aliquot (15 µg as protein) of the supernatant was loaded onto a sodium dodecyl sulfate polyacrylamide gel, separated electrophoretically, and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Lab., Inc.). After the PVDF membrane was incubated with 10 mM TBS with 1.0% Tween 20 and 5% dehydrated skim milk (Difco Lab., W. Molesley, Surrey, U.K.) to block nonspecific protein binding, the membrane was incubated with primary antibody [anti-Bcl-2 (1:1000), anti-JNK 1 (1:500), anti-JNK 2/3 (1:500)] or secondary antibody (horseradish peroxidase-linked antibody, diluted 1:1000). Subsequently, membrane-bound horseradish peroxidase-labeled antibodies were detected by an enhanced chemiluminescence detection system (ECL-plus, Amersham) and exposed to Fuji X-ray film.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Neuronal death by BLM and BSO
Exposure to BSO or BLM for 24 h caused significant toxicity to both dopaminergic and nondopaminergic neurons. The 50% lethal dose (LD50) of BSO toxicity was 10.7 µM (6.61–14.7 µM; 95% confidential interval) and 4.37 µM (2.64–6.11 µM) for dopaminergic and nondopaminergic neurons, respectively. Comparison by a two-tailed t test showed that the LD50 in dopaminergic neurons was significantly higher than that in nondopaminergic neurons (P=0.0049) The LD50 of BLM toxicity was 42.8 µM (30.8–54.8 µM) and 33.2 µM (20.1–46.3 µM) for dopaminergic and nondopaminergic neurons, respectively, so that the difference of LD50 was not statistically significant (P=0.279, two-tailed t test) (Fig. 1 ). After exposure to BSO or BLM or after serum deprivation, cultured cells were labeled with TUNEL. No TUNEL-positive cells were seen in control experiment (Fig. 2a ,b ,c ,d ). Nuclear staining with Hoechst 33258 demonstrated that nuclei were homogeneously stained in the control experiment. After exposure to BSO or BLM or serum deprivation treatment, nuclear chromatin was aggregated or condensed (Fig. 2e ,f ,g ,h ).



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  		Figure 1. Neurotoxicity induced by exposure to buthionine sulfoximine (BSO) and bleomycin sulfate (BLM). On the 8th day in culture, cultured cells were exposed to 1–100 µM BSO (a) or 1–100 µM BLM (b) for 24 h. The cells were then incubated for an additional 72 h without drugs, then fixed on the 12th day. They were stained immunocytochemically with anti-tyrosine hydroxylase (TH) and anti-microtubule-associated protein 2 (MAP2) antibodies for evaluation of the survival of dopaminergic and total neurons, respectively. Neurotoxicity was evaluated by a decrease in dopaminergic and nondopaminergic neuronal survival, which was presented as relative ratio (%) to control experiment. BSO toxicity was dose dependent and the 50% lethal dose (LD50) of BSO was 10.7 µM for dopaminergic neurons and 4.4 µM for nondopaminergic neurons (a). The LD50 of BLM was 42.8 µM and 33.2 µM for dopaminergic and nondopaminergic neurons, respectively (b).



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  		Figure 2. TUNEL staining and nuclear staining with Hoechst 33258 in the control experiment (a/e), BSO exposure (b/f), BLM exposure (c/g), and after serum deprivation (d/h). TUNEL staining. To detect DNA cleavage and avoid detachment of fragmented DNA, cultured cells were labeled with TUNEL immediately after exposure to 10 µM BSO, 100 µM BLM, and after serum deprivation for 48 h. In the control experiment, cultured cells were stained light green with the nonspecific nuclear dye methylgreen, but there were no TUNEL-positive cells (a). There were TUNEL-positive cells (dark brown) after exposure to 10 µM BSO (b) and to 100 µM BLM (c), and after serum deprivation for 48 h (d). Bar = 20 µm. Nuclear staining. To detect nuclear morphological changes, cells were stained with Hoechst 33258. In the control experiment, nuclei were stained homogeneous (e). After exposure to BSO (f) or BLM (g) or after serum deprivation (h), nuclear chromatin was aggregated (arrows). Bar = 10 µm

Apoptotic neurotoxicity induced by BLM was blocked by Ac-WEHD-CHO (caspase-1 inhibitor) and Ac-DMQD-CHO (caspase-3 inhibitor); that induced by BSO was blocked by Ac-DMQD-CHO but not by Ac-WEHD-CHO (Fig. 3a, b ). Furthermore, BSO- and BLM-induced neurotoxicity was blocked by coadministration of curcumin, an inhibitor for c-Jun/AP-1 (Fig. 3c, d ). Caspase-3 activity was elevated by BLM treatment. The elevated caspase activity by BLM was not affected by curcumin (Fig. 4 ).



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  		Figure 3. Neurotoxicity induced by BSO and BLM was dependent on caspase-3 (a, b) and c-Jun/AP-1 (c, d) in dopaminergic ({blacksquare}) and nondopaminergic ({square}) neurons. a) Neurotoxicity induced by 3 µM BSO was not blocked by the caspase-1 inhibitor Ac-WEHD-CHO, but was significantly blocked by an inhibitor of caspase-3, Ac-DMQD-CHO. *P<0.001 compared to control, {xi}P<0.001 compared to BSO. n = 3 coverslips/experiment. Error bars represent SEM. b) Neurotoxicity induced by exposure to 30 µM BLM was significantly blocked by preincubation with either Ac-WEHD-CHO (a caspase-1 inhibitor) or Ac-DMQD-CHO (a caspase-3 inhibitor) in both dopaminergic and nondopaminergic neurons. *P<0.001 compared to control, {xi}P<0.001 compared to BLM. n = 3 coverslips/experiment. Error bars represent SEM. c, d) Neurotoxicity induced by BSO was blocked by curcumin, an inhibitor for c-Jun/AP-1 proteins in both dopaminergic and nondopaminergic neurons. Cultured cells were exposured to 3 µM BSO or 30 µM BLM for 24 h with or without curcumin. Curcumin was administered 1 h prior to BSO or BLM exposure and simultaneously coadministered with BSO or BLM. *P<0.001 compared to control, {xi}P<0.001 compared to BSO or BLM.



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  		Figure 4. Caspase-3 activity was not blocked by a c-Jun/AP-1 inhibitor. Caspase-3 was elevated about twofold by BLM. The elevated caspase-3 activity was not suppressed by a c-Jun/AP-1 inhibitor. Cultured cells were treated with 30 µM BLM for 6 h with or without administration of curcumin. Curcumin was administered 30 min prior to BLM exposure and was administered simultaneously with BLM treatment.

Protection by estradiol against BLM- and BSO-induced neuronal death
Preincubation with 1 or 10 nM 17ß-estradiol significantly protected both types of neurons against toxicity induced by BSO. 17ß-Estradiol preincubation exerted significant neuroprotection against BLM neurotoxicity in both dopaminergic and nondopaminergic neurons dose dependently (Fig. 5a, b ). In contrast, 100 nM tamoxifen, an antiestrogen agent, exaggerated BLM neurotoxicity, and the surviving neuronal number was decreased from 650 ± 60 to 311 ± 15 cells/cm2 for dopaminergic neurons and from 10,966 ± 537 to 7,841 ± 256 cells/cm2 for nondopaminergic neurons. It also significantly exaggerated the BSO neurotoxicity to dopaminergic neurons (from 544±54 to 314±21 cells/cm2) and nondopaminergic neurons (from 8,507±652 to 723±224 cells/cm2) (Fig. 5c ). Coadministration with 1 µM ICI 182,780, a pure antagonist for ERß, antagonized the neuroprotection provided by 17ß-estradiol, but an inhibitor for ERß homodimerization, Yp537 (1 µM), did not block the neuroprotection. ICI 182,780 (1 µM) or Yp537 (1 µM) alone did not exert any detectable effects on neuronal survival (Fig. 6a ). The neuroprotection provided by 17ß-estradiol was significant when administered for longer than 2 h; preincubation for 4 h or longer did not provide additional neuroprotection (Fig. 6b ).



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  		Figure 5. Neurotoxicity by BSO and BLM was blocked by 17ß-estradiol (a, b) but was exaggerated by an antiestrogen, tamoxifen citrate (c) in both dopaminergic ({blacksquare}) and nondopaminergic ({square}) neurons. a) On the 7th day in culture, cultured cells were preincubated with or without 10 nM 17ß-estradiol for 24 h and then exposed to 10 µM BSO. 17ß-Estradiol was administered for 24 h prior to BSO exposure and coadministered with BSO for 24 h. The cells were incubated for an additional 72 h without drugs and fixed on the 12th day. Exposure to BSO caused significant neuronal death in both dopaminergic and nondopaminergic neurons (*). Preincubation with 17ß-estradiol provided significant neuroprotection against neurotoxicity induced by BSO in both dopaminergic and nondopaminergic neurons ({xi}). *P<0.001 compared to control, {xi}P<0.001 compared to 10 µM BSO. n = 4 coverslips/experiment. Error bars represent SEM. b) Cultured cells were preincubated with or without 17ß-estradiol (0.1–10 nM) and then exposed to 30 µM BLM for 24 h. Exposure to BLM caused significant neurotoxicity in both dopaminergic and nondopaminergic neurons (*). Preincubation with 17ß-estradiol provided neuroprotection in a dose-dependent manner, having a significant effect at doses greater than 1.0 nM in dopaminergic and at doses greater than 10 nM in nondopaminergic neurons ({xi}). *P<0.001 compared to control. {xi}P<0.001 compared to 30 µM BLM. n = 4 coverslips/experiment. Error bars represent SEM. c) Tamoxifen (Tam) enhanced BLM- and BSO-induced neurotoxicity. Cultured neurons were pretreated with or without tamoxifen (100 nM) and then exposed to 30 µM BLM or 3 µM BSO. The neurotoxicity induced by BLM or BSO was significantly exaggerated by pretreatment with tamoxifen (*, {xi}). Incubation with 100 nM tamoxifen for 48 h had no effect on neuronal survival. *P<0.001 compared to 30 µM BLM. {xi}P<0.001 compared to 3 µM BSO. n = 4 coverslips/experiment. Error bars represent SEM.



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  		Figure 6. Neuroprotection by 17ß-estradiol was blocked by an estrogen receptor antagonist (a) and was dependent on preincubation time (b) in both dopaminergic ({blacksquare}) and nondopaminergic ({square}) neurons. a) Antiapoptotic protection by 17ß-estradiol (ßE2) was blocked by ICI 182,780 but not by Yp537. Administration of 1 µM ICI 182,780 with 17ß-estradiol (10 nM) significantly blocked the antiapoptotic effects of 17ß-estradiol (*). In contrast to 1 µM ICI 182,780, 1 µM Yp537 did not block the antiapoptotic effects. ICI 182,780 was administered 30 min prior to 17ß-estradiol treatment and simultaneously administered to estradiol. NS: not significant. *P<0.001 compared to ßE2/BSO. n = 4 coverslips/experiment. Error bars represent SEM. b) Time dependency of neuroprotection provided by 17ß-estradiol. Cultured neurons were preincubated with 17ß-estradiol for 2, 4, 8, 16, and 24 h prior to 3 µM BSO exposure. Preincubation for longer than 2 h provided significant antiapoptotic protection in both dopaminergic and nondopaminergic neurons. There was no significant difference between neuronal survival after a 2 h preincubation and that after a 4 h or longer preincubation. *P<0.001 compared to 3 µM BSO. n = 4 coverslips/experiment. Error bars represent SEM.

The activity of caspas-3, which was elevated by BLM, was not suppressed by preincubation with 10 nM 17ß-estradiol (Fig. 7 ). Immunoblotting assay of Bcl-2 revealed that 17ß-estradiol increased the amount of Bcl-2, but the up-regulation of Bcl-2 required preincubation for 8 h or longer (Fig. 8 ). JNK 1 and JNK 2/3 were elevated by BSO or BLM treatment. The elevated activity of JNK 1 was suppressed neither by 17ß-estradiol nor by Ac-DMQD-CHO. In contrast to JNK 1, JNK 2/3 activity was slightly suppressed by estradiol but was almost completely suppressed by Ac-DMQD-CHO (Fig. 9 ).



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  		Figure 7. Caspase-3 was not affected by 17ß-estradiol pretreatment. Caspase activity, which was elevated about twofold by BLM, was not suppressed by 17ß-estradiol. Cultured cells were treated with 30 µM BLM for 6 h with or without pretreatment with 10 nM 17ß-estradiol. Estradiol was administered 6 h prior to BLM treatment and simultaneously administered with BLM.



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  		Figure 8. Western blotting analysis for Bcl-2. Cultured cells were incubated with 10 nM 17ß-estradiol for 2, 4, 8, 24 h on the 8th day in culture and analyzed for Bcl-2 by Western blotting. Bcl-2 was elevated by estradiol incubation, but the elevation required 4 h or longer incubation.



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  		Figure 9. Western blotting analysis for JNK. Cultured cells were exposed to 30 µM BLM or 3 µM BSO for 24 h, with or without preincubation with 10 nM 17ß-estradiol or 10 µM Ac-DMQD-CHO, and analyzed for JNK 1 and JNK 2/3. BLM caused the elevation of JNK1 or JNK2/3. Estradiol provided no or little effect on the elevation of JNK1 and JNK2/3. The elevation of JNK2/3 was suppressed by Ac-DMQD-CHO.

17{alpha}-Estradiol, a stereoisomer with little biological activity as a female sex hormone, also significantly protected dopaminergic and nondopaminergic neurons against apoptosis induced by BSO (Fig. 10 ). Preincubation with either 17{alpha}- or 17ß-estradiol significantly reduced the number of TUNEL-labeled cells after BSO exposure (the number of TUNEL-positive cells induced by BSO was decreased from 2,223±271 cells/cm2 to 185±46 cells/cm2 and 46±26 cells/cm2 by 17{alpha}- and 17ß-estradiol, respectively) (Fig. 10) . Table 1 presents the neuroprotection of 17{alpha}- and 17ß-estradiol against apoptosis induced by various agents. Preincubation with either 17{alpha}- or 17ß-estradiol provided significant neuroprotection against apoptosis induced by exposure to BLM and by serum deprivation, as well as that induced by BSO. In contrast to preincubation with estradiol, preincubation with other steroids, such as corticosterone, testosterone, and cholesterol, had no effect on neuronal apoptosis induced by BLM exposure (Table 2 ).



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  		Figure 10. Antiapoptotic protection by 17{alpha}- and 17ß-estradiol against BSO toxicity. a) Survival of dopaminergic ({blacksquare}) and nondopaminergic ({square}) neurons after BSO exposure preincubated with or without 17{alpha}- ({alpha}E2) or 17ß-estradiol (ßE2). Exposure to BSO caused significant neuronal death in both dopaminergic and nondopaminergic neurons in a dose-dependent manner (*). Preincubation with either 17{alpha}- or 17ß-estradiol provided significant neuroprotection ({xi}) in both types of neurons. *P<0.001 compared to control. {xi}P<0.001 compared to 3 µM BSO exposure. n = 4 coverslips/experiment. Error bars represent SEM. b) The number of TUNEL-positive cells after 10 µM BSO exposure preincubated with or without 17{alpha}- and 17ß-estradiol. To evaluate nuclear DNA cleavage, cultured cells were labeled using the TdT-mediated dUTP nick-end labeling (TUNEL) method after BSO exposure with or without estradiol pretreatment. In control experiments, fewer than 50 cells/cm2 were labeled. After BSO exposure, 2223 ± 271 cells/cm2 were labeled in the absence of estradiol preincubation. The number of positive labeled cells after BSO exposure was decreased to 185 ± 46 cells/cm2 and to 46 ± 26 cells/cm2 by preincubation with 17{alpha}- and 17ß-estradiol, respectively. *P<0.001 compared to control. {xi}P<0.001 compared to BSO exposure. n = 4 coverslips/experiment. Error bars represent SEM. c) Immunostaining with anti-tyrosine hydroxylase antibody to detect dopaminergic neurons (Anti-TH) and TUNEL to detect apoptotic cells (TUNEL) in the control experiment (control), after exposure to BSO and after BSO preincubated with 17{alpha}- or 17ß-estradiol ({alpha}E2/BSO, ßE2/BSO, respectively). Anti-TH: Compared to the control experiment, BSO exposure reduced both the number of dopaminergic neurons and the number and length of the neurites. Preincubation with 17{alpha}- or 17ß-estradiol preincubation resulted in the survival of more dopaminergic neurons with longer neurites. Bar = 100 µm. TUNEL: In the control experiment, cultured cells were stained light green with the nonspecific nuclear dye methylgreen, but no cells were labeled with TUNEL. After exposure to BSO, the number of cells decreased; most cells were labeled with TUNEL (stained dark brown), indicating nuclear DNA cleavage. After exposure to BSO with 17{alpha}-estradiol pretreatment, only one cell was labeled in this field (arrow). The number of surviving cells (stained with methylgreen) was greater after BSO exposure with 17ß-estradiol pretreatment than after BSO without pretreatment. None of the cells was labeled with the TUNEL method. Bar = 100 µm


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  		Table 1. Neuroprotection provided by estrogens against apoptosisa


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  		Table 2. Anti-apoptotic effects provided by steroidsa

As shown in Fig. 3a, b , apoptotic neurotoxicity induced by BLM was blocked by Ac-WEHD-CHO (caspase-1 inhibitor) and Ac-DMQD-CHO (caspase-3 inhibitor), and that induced by BSO was blocked by Ac-DMQD-CHO but not by Ac-WEHD-CHO. In contrast, neuronal death induced by serum deprivation was not blocked by either inhibitor (Fig. 11 ).



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  		Figure 11. Neurotoxicity induced by serum deprivation treatment was not blocked by inhibitors for caspases-1 and -3. Serum deprivation (SD) for 48 h caused significant neuronal death in both dopaminergic ({blacksquare}) and nondopaminergic ({square}) neurons. Neurotoxicity induced by SD was not affected by inhibitors for caspases-1 (Ac-WEHD-CHO) and -3 (Ac-DMQD-CHO). N.S., not significant. n = 3 coverslips/experiment. Error bars represent SEM.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
BSO- and BLM-induced neurotoxicity
Exposure to BSO or BLM caused significant neuronal death in cultured mesencephalic neurons. In the process of neuronal death by these agents, TUNEL revealed nuclear DNA cleavage, a characteristic feature of apoptosis (48) . However, DNA damage in necrotic cells may cause nonspecific staining by TUNEL (50 51 52) . In addition to DNA cleavage, nuclear staining using Hoechst 33258 showed the aggregation and condensation of nuclear chromatin. Furthermore, neurotoxicity by BLM and BSO was significantly blocked by a caspase-3 inhibitor. Taken together, the neuronal death induced by BSO and BLM is taken as apoptosis according to the morphological and biochemical criteria of apoptotic cell death (53) .

Although BSO was toxic to both dopaminergic and nondopaminergic neurons, the LD50 was higher for dopaminergic neurons than for nondopaminergic neurons, showing that dopaminergic neurons were relatively resistant to the toxicity. The reason why dopaminergic neurons were less vulnerable to BSO is unknown. However, dopaminergic neurons are resistant to another oxygen radical, nitric oxide (44 , 45) , and they may have a great ability to scavenge radicals (34) . Therefore, they may be relatively resistant in general to toxicity induced by reactive oxygen species.

BSO-induced apoptosis is thought to be mediated by an increase in intracellular H2O2 because it deprives the cells of glutathione and suppresses glutathione peroxidase. Apoptosis induced by reactive oxygen species such as H2O2 or O2- is mediated by caspase-1 (54 , 55) or caspase-3 (56) . In the present study, BSO-induced apoptosis was not blocked by Ac-WEHD-CHO, a caspase-1 inhibitor, but by Ac-DMQD-CHO, a caspase-3 inhibitor. Therefore, BSO-induced neurotoxicity was mediated by caspase-3 activation. BLM-induced apoptosis is mediated by the activation of caspases-1 and -3, because it was blocked by caspase-1 and caspase-3 inhibitors. Caspase-3 is thought to be activated downstream of caspase-1 (57) . Therefore, the pathway of BLM-induced neuronal apoptosis involves activation of caspase-1, followed by activation of caspase-3.

Apoptosis induced by anti-cancer drugs and oxygen radicals such as hydrogen peroxide requires the activation of AP-1 and transcriptional activation through the AP-1 element (30 , 31 , 58 59 60 61 62) . Also, in the present study apoptosis by BSO or BLM was blocked by curcumin, a pharmacological inhibitor for c-Jun/AP-1 proteins. c-Jun was activated by JNK, a family of mitogen activator protein kinases, and JNK 2/3 was suppressed by a caspase-3 inhibitor, as shown in Fig. 9 . Furthermore, activation of caspase-3 by BLM was not blocked by curcumin, as shown in Fig. 4 . Therefore, the c-Jun/AP-1 proteins were activated downstream of caspase-3. These data are consistent with a recent report by Srivastava (63) .Therefore, apoptosis by BSO or BLM is possibly mediated by the AP-1 activation downstream of caspase-3.

Neuroprotection by estrogens
17ß-Estradiol provides neuroprotection against the apoptosis induced by these agents. The antiapoptotic neuroprotection was not attributable to a general steroid structure because other steroids provided no protection against apoptosis. A possible mechanism is the antioxidant property of estradiol. Steroids with hydroxyl group in C3 position of the A ring provided an antioxidant property (17) . In our previous report, 17ß-estradiol suppressed intracellular oxygen radicals and provided neuroprotection against oxidative stress; however, antioxidant property requires a much higher concentration of estradiol (10–100 µM) (20) . Furthermore, antiapoptotic neuroprotection was blocked by ICI 182,780, which has hydroxyl group in C3. Therefore, it is unlikely that the antiapoptotic effect of estradiol is due to the antioxidant property of estradiol.

Recently, it has been reported that estradiol causes up-regulation of Bcl-2 (64 65 66) , which suppresses cytochrome c release and the ensuing caspase-3 activation. In our mesencephalic culture, estradiol caused up-regulation of Bcl-2, but it required estradiol treatment for longer than 8 h (Fig. 8) . As shown in Fig. 6 , preincubation for only 2 h provided significant antiapoptotic neuroprotection. It is unlikely that up-regulation of Bcl-2 plays a pivotal role in antiapoptotic protection because of different time dependency. Furthermore, we recently reported that glial cell line-derived neurotrophic factor (GDNF) causes up-regulation of Bcl-2 in the mesencephalic neurons and provides neuroprotection against BSO- and BLM-induced apoptosis, but GDNF does not protect neurons from apoptosis by serum deprivation (49) . In contrast to GDNF, estradiol provided neuroprotection from apoptosis induced by serum deprivation treatment. Therefore, antiapoptotic protection by estradiol in the present study includes other mechanisms besides Bcl-2 up-regulation.

The neuroprotection provided by 17ß-estradiol was thought to be mediated by the ER because it was antagonized by ICI 182,780, a pure antagonist for ER ß. 17ß-Estradiol has affinity for both subtypes of ER (29) , ER {alpha} and ER ß, but the neuroprotection of nigral dopaminergic neurons is probably mediated by ER ß, because it has been revealed that the ER subtype in the substantia nigra of the mesencephalon is exclusively ER ß (28 , 29) . Gene expression as a result of ligand-bound ER includes both primary and secondary responses, with the former directly regulated by the ER and the latter regulated by products of the primary response. The primary response to ligand-bound ER is mediated in two ways: through the ERE and through the AP-1 element. ER ß activated by 17ß-estradiol binds to DNA as a homodimer and enhances gene transcription through the ERE. However, it also suppresses gene transcription from the AP-1 element by coupling with he AP-1 proteins c-Fos and c-Jun (27) . Tamoxifen acts as a pure antagonist for ER ß (67) and, when bound to ER ß, regulates transcription through either the ERE or the AP-1 element in a reverse manner: it suppresses transcription through ERE and enhances it through AP-1 site (27) . In this study, it exaggerated the apoptotic toxicity induced by BSO and BLM. Therefore, the antiapoptotic effect of 17ß-estradiol could be mediated by the activation of antiapoptotic genes through the ERE or by the suppression of proapoptotic genes through the AP-1 enhancer element.

The neuroprotection provided by 17ß-estradiol in the present study was not antagonized by Yp537. Yp537 is a synthesized 12-amino acid phosphotyrosyl peptide containing a selected sequence surrounding tyrosine-537, a constitutively phosphorylated site, on the estrogen receptor (68) . It blocks the homodimer formation of the ER and effects of the pathway regulated by the ERE, but not the effects of the activation of the AP-1 site, because ligand-bound ER attaches to the AP-1 consensus element by making a heteromer with he AP-1 proteins Fos and Jun. These findings suggest that the 17ß-estradiol-ER ß complex inhibits the transcription of certain proapoptotic genes from the AP-1 element that suppress apoptosis. Therefore, the antiapoptotic effect by estradiol is thought to be mediated by suppression of proapoptotic genes through the AP-1, but not ERE.

Serum deprivation for 48 h caused significant neurotoxicity resulting in a large number of TUNEL-positive cells. Nuclear staining using Hoechst 33258 showed that there was nuclear aggregation after serum deprivation treatment. Therefore, neuronal death caused by serum deprivation was accompanied by the nuclear morphological features characteristic of apoptosis and by DNA cleavage, and was thought to be apoptosis. In contrast to apoptosis induced by BSO or BLM, that induced by serum deprivation was not blocked by either Ac-WEHD-CHO or Ac-DMQD-CHO. These data are consistent with a recent report that apoptosis induced by potassium deprivation does not require activation of caspase-3 (69) and indicate that serum deprivation caused apoptosis at least partially by a direct pathway to cascades downstream from the activation of caspase-3, such as activation of the AP-1 proteins or endonuclease in the apoptotic process (70) . Estradiol provided antiapoptotic protection against serum deprivation treatment. Furthermore, estradiol had no effect on caspase-3 or JNK activation. Therefore, the key proapoptotic gene products inhibited by estradiol are most likely downstream from the activation of caspase-3 and JNK (Fig. 12 ). In addition to 17ß-estradiol, 17{alpha}-estradiol also provided significant antiapoptotic neuroprotection, consistent with a recent study by Green et al. (71) using a neuroblastoma cell line. However, the estradiol-induced neuroprotection reported by Green et al. might be mediated by a different mechanism from that in nigral neurons because the neuroprotection in their report was not antagonized by tamoxifen, in contrast to our results. Although it is not known which subtype of ER mediates neuroprotection in the neuroblastoma cell line, the different response to tamoxifen may be related to the subtypes of ER mediating neuroprotection, since tamoxifen does not antagonize transcription regulation through AP-1 when mediated by ER {alpha} (27 , 67) .



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  		Figure 12. Proposed model for the antiapoptotic protection provided by estradiol to mesencephalic neurons. Buthionine sulfoximine (BSO), a {gamma}-glutamylcysteine synthetase inhibitor, causes depletion of glutathione (GSH), results in elevation of hydrogen peroxide, and causes apoptosis mediated by caspase-3 (Casp-3) activation. Bleomycin sulfate (BLM) causes apoptosis mediated by activation of caspases-1 (Casp-1) and -3. Neurotoxicity by BSO and by BLM is blocked by curcumin, an inhibitor for c-Jun/AP-1. BLM activates caspase-3 and JNK 2/3. Neuroprotection by estradiol was blocked by ICI 182,780 but not by Yp537. Furthermore, activation of caspase-3 or JNK 2/3 was not blocked by estradiol. Therefore, estradiol provided neuroprotection by suppression of proapoptotic gene transcription through the AP-1 site, which was downstream of caspase-3 activation and the ensuing JNK activation.

Compared to 17ß-estradiol, 17{alpha}-estradiol has few of the biological effects of female sex steroid hormones. A ligand binding study showed that 17{alpha}-estradiol has affinity for ER ß (relative binding affinity of 17{alpha}- and 17ß-estradiol is 0.11:1) (29) . Therefore, 17{alpha}-estradiol can activate ER ß and cause the same primary response as that of 17ß-estradiol, at least when an appropriate dose is applied. The antiapoptotic protection by estradiol is probably the results of a primary response initiated by estradiol because preincubation for only 2 h provided significant antiapoptotic protection in the present study. The negative biological estrogenic effects of 17{alpha}-estradiol may be a result not of the difference in affinity, but rather of the difference in secondary response induced by 17{alpha}- and 17ß-estradiol, since the 17-{alpha} isomer is bound to ER {alpha} and ER ß for a shorter duration than the 17ß-isomer (72) . 17{alpha}-Estradiol, or its analog, may be a candidate for antiapoptotic therapy in neurodegenerative diseases affecting neurons expressing ER ß, such as Parkinson’s disease, because it has few of the biological effects associated with female sex hormones.


   ACKNOWLEDGMENTS
 
This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, and by grants from the Ministry of Welfare of Japan, the Smoking Research Foundation, the Inamori Foundation, and the Yamanouchi Foundation for Research on Metabolic Disorders.


   FOOTNOTES
 
Received for publication April 7, 1999. Revised for publication January 10, 2000.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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