Neuroscience Letters 373 (2005) 179–183
Estradiol and raloxifene protect cultured SN4741 neurons against oxidative stress Eric Biewenga, Leigh Cabell, Teresa Audesirk∗ Biology Department, University of Colorado at Denver, P.O. Box 173364, Denver, CO 80217-3364, USA Received 19 July 2004; received in revised form 24 September 2004; accepted 28 September 2004
Abstract A large body of research has documented neuroprotective effects of estrogen against oxidative stress. Some neurodegenerative diseases such as Parkinson’s disease, in which oxidative stress has been implicated as a contributing factor, affect more males than females, suggesting a possible protective effect of estrogen. We used the clonal substantia nigra cell line SN4741 to compare the neuroprotective properties of estrogen and raloxifene against oxidative stress, and to determine whether raloxifene acted as an estrogen agonist or antagonist in this system. We pretreated SN4741 cultures with ␣-estradiol, ␤-estradiol, and raloxifene, and exposed them to hydrogen peroxide. Low nanomolar levels of raloxifene, ␤-estradiol, and ␣-estradiol all significantly reduced cell death caused by oxidative stress. The estrogen receptor (ER) antagonist ICI 182,780 failed to reverse the neuroprotection by ␤-estradiol, suggesting that the effect is not mediated by a classical ER. Western blotting using an antibody to the C-terminus region of ER-␣ revealed two bands, one at approximately 67 kDa (corresponding to ER-␣) and a more prominent band at approximately 55–56 kDa. These results suggest that, in this cell line, both raloxifene and estrogen may be acting via a non-classical estrogen receptor. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Estrogen; Raloxifene; Estrogen receptor; Oxidative stress; Substantia nigra; SN4741; Parkinson’s disease; Hydrogen peroxide; ER-X
Parkinson’s disease (PD), characterized by degeneration of neurons of the substantia nigra (SN), is at least partially a result of oxidative stress [1,5,8,29]. Estrogen has been shown to be important in the survival, maturation, and functioning of dopaminergic cells of the mammalian midbrain. Both nuclear estrogen receptors and unidentified membrane receptors have been implicated in these effects (reviewed in ). Several lines of evidence support the hypothesis that estrogen may confer benefits against Parkinson’s disease. In most (but not all) epidemiological studies, PD has been found to affect more males than females. In some clinical trials, estrogen has been shown to improve motor disability in post-menopausal women suffering from PD (reviewed in [6,7]). In male mice, estrogen significantly reduced striatal dopamine depletion caused by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) ∗
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[4,9,19]. Estrogen also has well-documented antioxidant effects, which may contribute to its ability to reduce PD-related pathology [10,13,20–22]. Raloxifene is a selective estrogen receptor modulator (SERM) with tissue-specific, mixed agonist/antagonist properties at estrogen receptors (ER) [14,15] prescribed to post-menopausal women to help maintain bone density. Raloxifene has estrogenic effects on bone and lipids, but antagonizes the effects of estrogen in breast and endometrial tissue. Recent studies have shown that raloxifene exerts a variety of effects on the CNS. These studies (reviewed in ) provide evidence that raloxifene crosses the blood brain barrier in both humans and rats, and that it exerts brain region-specific agonistic or antagonistic effects on estrogen receptors. The effects of raloxifene on neurons at the cellular level remain largely unexplored. Because ␤-estradiol confers neuroprotection through a variety of mechanisms, it is important to determine whether drugs administered to aging women are likely to mimic or block the effects of estradiol, particularly
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on neurons that are important in Parkinson’s disease, such as those of the substantia nigra. The clonal cell line SN4741 was developed by Son et al.  to provide an in vitro model system to allow investigation of dopaminergic neurons derived from the substantia nigra. In the present study, we investigated the neuroprotective effects of ␤-estradiol and raloxifene on SN4741 cells subjected to oxidative stress, and evaluated the likelihood that the observed effects were mediated by classical ER. Cultured SN4741 neurons, derived from transgenic embryonic mouse substantia nigra neurons , were used to evaluate the effects of low level (10 nM) ␤-estradiol alone, and raloxifene (alone and in combination with estradiol) on cell death induced by exposure to hydrogen peroxide. To determine whether these effects were ER-mediated, we used the ER antagonist ICI 182,780 (which blocks both ER-␣ and ER-␤) and ␣-estradiol, a stereoisomer, which does not significantly activate classical ER. Western immunoblotting was used to determine the presence of ER. Neurons of the SN4741 cell line were cryopreserved in Eagle’s minimum essential medium (MEM) buffered with 10 mM NaHCO3 and 25 mM HEPES, supplemented with 2 mM l-glutamine, 2% glucose, 1 mM sodium pyruvate, 10% fetal bovine serum, 8% dimethyl sulfoxide (DMSO), and 15 mM KCl. For culturing, SN4741 neurons were thawed rapidly in a 37 ◦ C water bath. The cells were plated on untreated 35 mm plastic culture dishes containing 2 ml of culture medium (MEM supplemented with 10% fetal bovine serum and 3.5% glucose) and incubated at 37 ◦ C in a humidified, 5% CO2 atmosphere for 3 h to allow attachment. The medium was then replaced to remove residual DMSO. After 24 h, at which time the cells had multiplied to near confluence, the medium was replaced with culture medium supplemented with 0.5% fetal bovine serum. The cells were then incubated at 40 ◦ C in a humidified, 5% CO2 environment to promote differentiation. All experiments were initiated after 4 days incubation at 40 ◦ C, at which time approximately 90% of the cells had differentiated. Cultures were incubated at 40 ◦ C in ␤-estradiol (E2; 10 nM), ␣-estradiol (10 nM), raloxifene (10 nM), or ICI 182,780 (100 nM) dissolved in DMSO. Control cultures received an equal amount of DMSO. After 24 h of incubation in the additives listed above, H2 O2 was added (producing a final concentration of 50 M) and allowed to remain on the cultures for 24 h. Cell death was assayed using the Vybrant® Apoptosis Assay (Molecular Probes). The cells were visually analyzed under an inverted light microscope using both visible light and 490 nm UV light. In this assay, apoptotic cells fluoresce green under UV light, while necrotic cells fluoresce red. Healthy cells exclude the dyes and can be counted under visible light. A minimum of 50 cells were counted per dish. For Western immunoblotting, cells were cultured as described above, and then harvested on ice in 50 L of SDS sample buffer (62.5 mM Tris chloride, 2% SDS, 10% glycerol, 0.1 M dithiothreitol, 0.01% bromophenol blue, pH 6.8).
Fig. 1. Hydrogen peroxide significantly increased cell death over control levels (*). E2 alone did not significantly alter survival compared to controls, but significantly reduced H2 O2 -induced cell death (**) (N = 14, P < 0.05).
Cell extracts were immediately heated for 5 min in a boiling water bath, cooled on ice, and frozen at −20 ◦ C until use, usually within 48 h. Proteins were separated by SDS–polyacrylamide gel electrophoresis using a 12% gel. After transferring the proteins to nitrocellulose, the blot was blocked overnight with blocking buffer (I-Block; Tropix) at 4 ◦ C. The blots were incubated with antibodies against both ER-␣ (MC 20; Santa Cruz Biotechnology, Inc.) and ER-␤ (Zymed Laboratories). Blots were exposed to antibodies in blocking buffer for 1.5 h at room temperature, and rinsed three times for 5 min in blocking buffer. The blots were then incubated for 1.5 h in HRP-conjugated secondary antibodies (goat for ER-␤; Pierce Biotechnology, and rabbit for ER-␣; Stressgen Biotechnologies), rinsed 2× in blocking buffer and 3× in Tris-buffered saline for 5 min. Blots were then incubated with chemiluminescent stain (PicoWest; Pierce Biotechnology) for 5 min and photographed with a cooled CCD camera. Statistical significance was determined using the one-way ANOVA followed by the Student–Newman–Keuls test. Control cultures exhibited low levels of cell death, which was unaltered by 10 nM E2, but was significantly increased by H2 O2 exposure. Under these conditions and at this experimental time point, cell death was almost entirely necrotic as determined by the Vybrant assay. Pre-treatment with 10 nM E2 for 24 h significantly reduced cell death upon exposure to H2 O2 (Fig. 1). To determine if the neuroprotection by E2 was mediated by classical ER, the ER antagonist ICI 182,780 was added to cultures alone, in combination with E2, and with both E2 and H2 O2 . ICI 182,780 alone did not alter cell death over control levels, and ICI 182,780 did not significantly reduce the protective effects of E2 against H2 O2 . The isomer ␣-estradiol (␣-E2, which exhibits only weak estrogenic activity at classical ER) alone did not alter survival compared to controls, but ␣-E2 significantly reduced cell death induced by H2 O2 exposure (Fig. 2). Cultures were then treated with raloxifene alone, raloxifene in combination with H2 O2 , and raloxifene combined
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Fig. 4. Western immunoblotting revealed two bands of protein stained by the antibody against ER-␣, one migrating at about 67 kDa (corresponding to ER-␣) and the second, darker band at about 55–56 kDa. Multiple replicates of control cultures are shown.
Fig. 2. Cell death was not changed by ␣-E2, E2, and ICI 182,780 alone, but was significantly increased by H2 O2 (*). ␣E2, E2, and the combination of ICI 182,780 and E2 all showed significant neuroprotection against H2 O2 induced cell death (**) (N = 10–14, P < 0.05).
with both E2 and H2 O2 . Both raloxifene alone and E2 alone provided significant protection against H2 O2 -induced cell death; the degree of protection was unchanged when raloxifene and E2 were combined, suggesting a similar mechanism of action (Fig. 3). Western blots were used to determine the presence of ER-␣ and ER-␤ in the SN4741 cultures. Antibodies to the C-terminus region of ER-␣ revealed a band at the approximate molecular weight of 67 kDa, corresponding to ER-␣, and a second, much darker band at approximately 55–56 kDa (Fig. 4). No ER-␤ was detected. In the current study, both ␣ and ␤ isomers of estradiol as well as the SERM raloxifene conferred protection against death caused by oxidative stress induced by H2 O2 in SN4741 cells. The protection conferred by ␤-estradiol was not re-
Fig. 3. Raloxifene alone did not alter cell death compared to controls. H2 O2 significantly increased cell death (*). Raloxifene, E2 and a combination of raloxifene and E2 all conferred comparable levels of neuroprotection against H2 O2 -induced cell death (**) (N = 10, P < 0.05).
versed by the ER antagonist ICI 182,780, suggesting that it is not mediated by classical ER. Numerous studies utilizing diverse experimental systems, including primary neuronal cultures, transformed cell lines, explants, and living animals have implicated a variety of mechanisms underlying neuroprotection by ␤-estradiol against oxidative stress. Consistent with our results, some of these have reported antioxidant effects of low nanomolar concentrations of estrogen which were not mediated by classical ER in cell cultures. For example, the non-ER-activating enantiomer of ␤-E2 (Ent-E2) was as potent as ␤-E2 in conferring protection in SK-N-SH and HT-22 cell lines against H2 O2 neurotoxicity . Neuroprotection against oxidative stress induced by the HIV coat protein gp120 was demonstrated in primary rat hippocampal cultures. The protection was conferred equally by both ␣-E2 and ␤-E2, and was not reversed by the ER antagonist tamoxifen . In contrast to our findings in SN4741 cells, other studies have documented neuroprotection by ␤-E2 that appears to be mediated by classical ERs. For example, ischemic injury in neonatal rat cortical explant cultures was significantly reduced by pretreatment with low nanomolar levels of ␤estradiol. The effect was blocked by ICI 182,780, and was not duplicated by ␣-estradiol, consistent with mediation by a classical ER . In vivo studies using male mice demonstrated that both ␤-estradiol and raloxifene reduced MPTPinduced loss of striatal dopamine (released from terminals of substantia nigra neurons), while ␣-estradiol was ineffective [4,9], also suggesting that a classical ER was involved. A similar study  supported the hypothesis that a classical ER mediated E2 protection against MPTP toxicity, but did not observe neuroprotection by raloxifene. A classical ER (ER-␣) has also been implicated in protection of the murine cholinergic cell line SN56 against amyloid-␤ neurotoxicity . Extremely high (M) concentrations of estradiol have antioxidant properties that have been attributed to the structure of its phenol ring [2,18,28] rather than to ER activation. However, it is unlikely that estrogen in the low nanomolar concentrations used in our study is acting in this manner. Therefore we hypothesize that the neuroprotection reported here is mediated by an ER, but with different properties from either ER-␣ or ER-␤.
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Recent research by Toran-Allerand et al. supports the existence of a previously undescribed plasma membrane ER, designated ER-X, found in mouse, rat and baboon [24–26]. This 62–63 kDa ER binds antibodies to the C-terminus region of ER␣, is activated by picomolar concentrations of both ␣-E2 and ␤-E2, and its activity is not antagonized by ICI 182,780. In the study described here, the putative ER responsible for neuroprotection is also activated by both ␣-E2 and ␤-E2, and is insensitive to ICI 182,780. Western blots utilizing an antibody to the C-terminus region of ER-␣, revealed two protein bands. A faint band at approximately 67 kDa corresponds to the molecular weight of ER-␣. A much darker band was visible at a molecular weight of about 55–56 kDa (Fig. 4), based on comparison with protein standards. Although we cannot rule out that we are observing a breakdown product of ER-␣, our careful handling of harvested cell extracts generally precludes this. Attempts to determine the exact molecular weight of a specific protein and compare it to weights reported by other laboratories can be somewhat problematic because proteins used as commercial standards may run at slightly different locations. Thus, the receptor that we hypothesize to mediate neuroprotection in this system shares some of the properties of ER-X, but may have a lower molecular weight. It may also be another addition to the large and increasing number of ER subtypes which are being described . Raloxifene may be acting through the same non-classical ER, since it also conferred neuroprotection, and its effects were not additive when combined with E2. Further characterization and subcellular localization of this hypothesized non-classical ER in the SN4741 cell line, and assessment of its similarity to ER-X, awaits further study. The use of a substantia-nigra derived cell line suggests lines of inquiry into possible mechanisms by which estradiol may delay or ameliorate symptoms of Parkinson’s disease. It would be of interest to compare this transformed substantia nigra cell line to primary cultures of substantia nigra neurons with respect to the neuroprotective properties of estradiol and raloxifene, as well as the presence of non-classical ER.
Acknowledgments We thank Dr. Gerald Audesirk for consultation and critical reading of the manuscript. SN4741 neurons were kindly provided by Jin Son of Cornell University. This work was supported by the NIH Grant AG19648 from the National Institute of Aging.
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