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Effect of estrogen receptor agonists treatment in MPTP mice: evidence of neuroprotection by an ERα agonist
Neuropharmacology 47 (2004) 1180–1188 www.elsevier.com/locate/neuropharm
Effect of estrogen receptor agonists treatment in MPTP mice: evidence of neuroprotection by an ERa agonist Myreille D’Astous, Marc Morissette, The´re`se Di Paolo Molecular Endocrinology and Oncology Research Center, Laval University Medical Center, CHUL, 2705 Laurier Boulevard, Quebec City, Que., G1V 4G2, Canada Faculty of Pharmacy, Laval University, Quebec City, Que., G1K 7P4, Canada Received 16 January 2004; received in revised form 27 July 2004; accepted 17 August 2004
Abstract Beneficial effects of 17b-estradiol (17b-E2) on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced striatal dopamine (DA) depletion are well documented but the mechanisms implicated are poorly understood. The present experiments investigated the effect of estrogen receptor (ER) agonists treatment in MPTP mice as compared to 17b-E2. The agonists specific for each subtype were 4,40 ,400 -(4-propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol (PPT) (ERa agonist), 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and D3-diol (5-androsten-3b, 17b-diol, also known as 5-androstenediol, androstenediol or hermaphrodiol) (ERb agonists). Biogenic amines were assayed by HPLC with electrochemical detection. 8 mg/kg of MPTP was administered to give a moderate depletion of striatal DA and its metabolite dihydroxyphenylacetic acid (DOPAC). Protection against MPTP-induced striatal DA and DOPAC depletion was obtained with PPT and 17b-E2 but not with DPN or D3-diol. The striatal dopamine transporter (DAT) was assayed by autoradiography with [125I]RTI-121-specific binding. A positive and significant correlation was observed between striatal DA concentrations and [125I]RTI-121-specific binding, suggesting that estrogenic treatment that prevented the MPTP-induced DA depletion also prevented loss of DAT. The effect of PPT suggests the implication of an ERa in the estrogenic neuroprotection against MPTP. Pointing out which ER is implicated in neuroprotection becomes helpful in designing more specific estrogenic drugs for protection of the aging brain. # 2004 Elsevier Ltd. All rights reserved. Keywords: MPTP; Estrogen; Agonists; Neuroprotection; Parkinson
1. Introduction Clinical studies have demonstrated a greater prevalence of Parkinson’s disease (PD) in men than in women (Baldereschi et al., 2000; Dluzen and McDermott, 2000b; Saunders-Pullman, 2003). Even though the cause of this disease is still unknown, the clinical evidence implies a role of sex hormones in the incidence of PD. Estrogens (Es) play an important role during brain development in neuronal differentiation, plasticity and Corresponding author. Molecular Endocrinology and Oncology Research Center, Laval University Medical Center, CHUL, 2705 Laurier Boulevard, Quebec City, Que., Canada G1V 4G2. Tel.: +1418-654-2296; fax: +1-418-654-2761. E-mail address: [email protected] (T. Di Paolo).
0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.08.020
cell survival (Beyer, 1999; Wise et al., 2001). In adult, Es act on many brain regions, including those implicated in reproduction and areas responsible for memory, cognition and mood (Wise et al., 2001). Many in vivo studies have demonstrated beneficial effects of Es (Garcia-Segura et al., 2001) against methamphetamine (Dluzen and McDermott, 2000a; Dluzen et al., 2002), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Callier et al., 2000, 2001; Ramirez et al., 2003), and ischemic injury in the brain (Simpkins et al., 1997; Dubal et al., 2001). However, the mechanisms by which Es protect against neuronal damages are still unknown. Es mediate their actions by two types of mechanisms, classified as genomic or nongenomic. The genomic mechanism implicates an interaction between Es and its nuclear receptors to activate genomic transcription (Pettersson and Gustafsson,
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2001). Other mechanisms, independents of estrogen receptors (ERs), are classified as non-genomic, such as the antioxidant capacity of Es (Moosmann and Behl, 1999), direct effect on the membrane (Ramirez and Zheng, 1996; Moss and Gu, 1999) or on its membrane receptor (Razandi et al., 1999). Using the MPTP mouse model, which reproduces the striatal dopamine (DA) loss seen in PD (Burns et al., 1983), we investigated the neuroprotective effects of 17b-estradiol (17b-E2) and the mechanisms implicated in this protection. We previously reported a neuroprotective role of 17b-E2 against MPTP toxicity (Callier et al., 2000; Grandbois et al., 2000; Callier et al., 2001; D’Astous et al., 2003). Evidence from these experiments suggests a genomic mechanism of action to explain the beneficial effects of estradiol on dopaminergic neurons. Indeed, the protection obtained with estradiol was only obtained with 17b-E2 (the active isomer of estradiol), while 17a-estradiol (17a-E2) did not protect (Callier et al., 2000). Other groups have also reported stereospecificity of neuroprotection by estradiol in this model (Ramirez et al., 2003). In this report, we investigated whether estradiol’s neuroprotective effects occur via one or both receptor subtypes, using ERa and ERb agonists. These molecules are described as ERa or ERb agonist based on their binding capacity to each receptor and on their transcriptional potency once bound to the receptor. 4,40 ,400 -(4-Propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol (PPT) binding affinity is 410 times greater for ERa than ERb (Stauffer et al., 2000). 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN) binding affinity is 72 times greater for ERb (Meyers et al., 2001), while D3diol (5-androsten-3b, 17b-diol) has a 2.8 times greater affinity for ERb (Kuiper et al., 1997). Binding affinity only demonstrates the capacity of a molecule to bind a specific receptor, whereas transcription assays measure its capacity to induce genomic transcription after binding to a receptor. PPT is reported to induce transcription only via ERa, at any concentration (Stauffer et al., 2000; Harrington et al., 2003). DPN can act as an agonist on both subtypes, with relative transcription potency 78 times greater for ERb at lower doses (Meyers et al., 2001). However, at high doses (108 M), DPN can also activate ERa in transcription assays (Harrington et al., 2003). No studies on relative transcription potency for each ER subtype are available for D3-diol. Herein, using chronic treatments with ERa and ERb agonists, we assessed the implication of ERs in neuroprotection against MPTP striatal DA toxicity in mice.
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2. Methods 2.1. Chemicals MPTP and 17b-E2 were purchased from Sigma Chemical (St. Louis, MO, USA), DPN and PPT from Tocris (Ellisville, MO, USA), while D3-diol was purchased from Steraloids Inc (Newport, RI, USA). 2.2. Animals and treatments C57Bl/6 male mice (10–12 weeks, 25 2 g) were purchased from Charles River Laboratories (Canada). Mice were distributed in groups of six to nine animals and each group received a pre-treatment of hormones or vehicle 5 days prior to the MPTP injections. The pre-treatment consisted of two daily injections (subcutaneous) of 17b-E2, PPT, DPN or D3-diol, while control mice received injections of vehicle (0.9% saline with 0.3% gelatin/0.2 ml). Estrogenic compounds concentrations were 2 lg/day (~80 lg/kg) for 17b-E2 and PPT, 3 lg/day (~120 lg/kg) for D3-diol and 6 lg/day (~240 lg/kg) for DPN. Doses were chosen based on their affinity for ERs (Kuiper et al., 1997; Stauffer et al., 2000; Meyers et al., 2001) and their transcriptional capacity (Stauffer et al., 2000; Meyers et al., 2001; Harrington et al., 2003), since no in vivo studies on the brain were available for DPN and D3-diol. On day 5, mice received four injections of MPTP (8 mg/kg, intraperitoneal) at 2 h interval, while the control group received saline solution. The treatments (estrogenic compounds or vehicle) were continued until day 10. On day 11, mice were decapitated, brains quickly removed v and frozen in isopentane (40 C). In another complementary experiment, intact C57Bl/6 male mice (without lesion) received estrogenic drug treatments. Mice were also treated for 10 days with the same concentrations of the drugs as described above, while one group received only the vehicle and served as control in this experiment. The Laval University Animal Care Committee approved all the animal studies. All efforts were made to minimize animal suffering and to reduce the number of mice used. 2.3. Striatal biogenic amines determination The left anterior striata were dissected, homogenized v in 250 ll of 0.1 M HClO4 at 4 C and then centrifuged v at 10 000 g for 10 min (4 C) to precipitate proteins as previously reported (D’Astous et al., 2003). The supernatants were used to determine the biogenic amine concentrations while the pellets were dissolved in NaOH 1 N to assay protein content. The concentrations of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxyphenylethylamine (3-MT), and homovanillic acid
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(HVA) as well as serotonin (5-HT) and its metabolites 5-hydroxyindoleacetic acid (5-HIAA) were measured by high performance liquid chromatography (HPLC) with electrochemical detection. Supernatants of striatal tissue were directly injected into the chromatograph consisting of a Waters 717 plus autosampler automatic injector, a Waters 515 pump equipped with a Beckman C-18 column (Waters Nova-Pak C18, 3 lm, 3:9 mm 150 cm), a BAS LC-4C electrochemical detector and a glassy carbon electrode. The mobile phase consisted of 0.025 M citric acid, 1.7 mM 1-heptane-sulfonic acid, and 10% methanol, in filtered distilled water, delivered at a flow rate of 1 ml/min. The final pH of 4.1 was obtained by addition of NaOH. The electrochemical potential was set at 0.8 V with respect to an Ag/AgCl reference electrode. The sensitivity of this assay was 13 pg for DOPAC, 7 pg for DA, 40 pg for HVA, 27 pg for 3-MT, 33 pg for 5HIAA and 40 pg for 5-HT. 2.4. DA transporter (DAT) autoradiography acid 3b-(4-[125I]Iodophenyl)tropane-2b-carboxylic isopropyl ester ([125I]-RTI-121) specific binding autoradiography was carried out as previously described (Carli et al., 1997; Callier et al., 2001). The binding was performed on slices containing the striatum at coordinates bregma 1.54 to 0.94 mm, according to the atlas of Franklin and Paxinos (1997). Three brain slices per animal were used in this protocol. Specific binding to DAT was measured using 20 pmol of [125I]-RTI-121 (2200 Ci/mmol), in the presence of 2 nM of mazindol to evaluate the non-specific binding. Slices were apposed to Kodak film for 40 h. Autoradiograms were analyzed using the software NIH 1.68. 2.5. In situ hybridization of preproenkephalin (PPE) mRNA In situ hybridization of preproenkephalin (PPE) mRNA was performed as previously described (Calon et al., 2002) on brain slices containing the striatum at coordinates bregma 1.54 to 0.94 mm (Franklin and Paxinos, 1997). Two brain slices per animal were used in this protocol. An oligonucleotide complementary to bases 304–350 of the mice PPE sequence (GenBank accession number M13227) was designed to determine specific binding while non-specific binding was assayed using a sens oligonucleotide probe. Slices were apposed to Kodak film for 5 days and analyzed using the software NIH 1.68. Relative optical densities for specific binding were obtained by subtracting non-specific from total binding.
2.6. Binding affinity assay Specific binding to the DAT was investigated in mice striatal homogenates under conditions previously described (Boja et al., 1995) with slight modifications. Mice striata were dissected, pooled, and weighted. The striata were then homogenized in a glass–teflon homogenizer in 100 volumes (w/v) of phosphate buffer (10.1 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2 mM KCl, pH 7.5) and centrifuged at 20 000g for 15 min. Washing procedures were repeated twice and the final pellet resuspended in 100 volumes (w/v) of the same ice-cold phosphate buffer. Mazindol, 17b-E2, PPT, DPN and D3-diol (concentrations of 106 and 107 M) were added directly into the incubation buffer in the presence of a fixed concentration of [125I]-RTI121 (20 pM) in a final volume of 2 ml. The binding reaction was initiated by the addition of 0.2 mg of striatal tissue. Samples were incubated at room temperature for 1 h. The reaction was stopped by rapid filtration through Whatman GF/C glass fiber filters under vacuum, followed by three rapid rinses (5 ml). Radioactivity on the filters was measured with a gamma counter. Percent binding for each compound was determined by dividing bound [125I]-RTI-121 in presence of the competing drugs on the value obtained for [125I]-RTI-121 total binding multiplied by 100. 2.7. Statistical analysis Statistical comparisons of biogenic amine concentrations, DAT-specific binding and PPE mRNA levels were performed using a one-way analysis of variance (ANOVA) followed by post hoc analysis with Fisher probability of least significance difference test. Coefficient of correlations and significance of the degree of linear relationship between DA and DOPAC concentrations versus DAT-specific binding were determined using a simple regression model.
3. Results In the present protocol, MPTP was administered in order to give a moderate striatal DA depletion, as measured by DA and its metabolites concentrations (Fig. 1). Indeed, the toxin decreased the DA content in the striatum of MPTP-treated mice to 59% of intact (F 5;36 ¼ 4:118, P ¼ 0:0046) (Fig. 1A). The metabolite DOPAC was decreased by 24% (76% of intact) (F 5;36 ¼ 3:850, P ¼ 0:0068) (Fig. 1B), while 3-MT and HVA remained unchanged (105 and 89% of intact) (F 5;36 ¼ 0:969, P ¼ 0:4495 for 3-MT; F 5;36 ¼ 1:222, P ¼ 0:3189 for HVA) (Fig. 1C and D). MPTP mice receiving PPT and 17b-E2 had higher striatal DA concentrations than the vehicle-treated
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Fig. 1. Effects of 17b-E2, PPT, DPN and D3-diol on striatal catecholamine concentrations in C57Bl/6 male mice treated with MPTP as compared to vehicle-treated intact animals. Mice were treated with estrogenic compounds or vehicle for 10 days and MPTP-treated mice received four injections of MPTP (8 mg/kg) on day 5. P < 0:05, P < 0:005 and P < 0:0005 versus intact þ vehicle; y P < 0:05 and yy P < 0:005 versus MPTP þ vehicle; u P < 0:05 versus MPTP þ 17b-E2 ; h P < 0:05 and hh P < 0:005 versus MPTP þ PPT. Values are the mean ðng=mg of proteinÞ SEM of six to nine mice per group.
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MPTP mice. The ERb agonists DPN and D3-diol treated MPTP mice had striatal DA concentrations similar to the MPTP vehicle-treated group. Striatal DA content for the PPT-treated mice was significantly greater than the D3-diol-treated group (Fig. 1A). For the metabolite DOPAC, a similar pattern of protection as for the DA content was observed, although the decrease induced by MPTP on this metabolite was less important. The MPTP-induced decrease in DOPAC was statistically significant for the DPN and D3-diol, but not for the 17b-E2 treated groups, with regards to the intact group (Fig. 1B). PPT administration to MPTP-mice prevented this decrease, since DOPAC values were significantly different and higher than those of the MPTP-vehicle group. In addition, the striatal DOPAC content for the PPT group was significantly greater than DPN and D3-dioltreated groups (Fig. 1B). The two other DA metabolites, HVA and 3-MT, remained unchanged after MPTP alone or in combination with the agonists treatments (Fig. 1C and D). MPTP treatment led to an increase in striatal DA turnover, as assessed by the HVA/DA ratio (F 5;36 ¼ 3:396, P ¼ 0:0129) (Fig. 1G). PPT and 17b-E2 prevented this increase, but not D3-diol or DPN. The ratios DOPAC/DA (Fig. 1E) and 3-MT/DA (Fig. 1F) showed no statistical differences between the groups (F 5;36 ¼ 1:784, P ¼ 0:1409 for DOPAC/DA; F 5;36 ¼ 2:235, P¼ 0:0718 for 3-MT/DA), although they displayed a similar pattern of changes between the treatment groups as for the HVA/DA ratio, but to a lesser extent. To verify the specificity of the lesion or treatments, striatal indolamine concentrations were measured. There were no variation of striatal 5-HT or its metabolite 5-HIAA after MPTP alone or in combination with the estrogenic drug treatments (F 5;36 ¼ 0:388, P ¼ 0:8539 for 5-HT; F 5;35 ¼ 0:942, P ¼ 0:4665 for 5HIAA) (Table 1). For comparison, catecholamine concentrations were measured in non-lesioned animals treated with these agonists. There were no change of striatal DA or its metabolites concentrations after a treatment with any of the estrogenic drug tested (F 4;52 ¼ 0:132, P ¼ 0:9698
Table 1 Effects of 17b-E2, PPT, DPN and D3-diol on striatal indolamine concentrations in C57Bl/6 male mice treated with MPTP as compared to vehicle-treated intact animals. Values are the mean ðng=mg of proteinÞ SEM of six to nine mice per group Groups
5-HT (ng/mg of protein)
5-HIAA (ng/mg of protein)
Intact þ vehicle MPTP þ vehicle MPTP þ 17b-E2 MPTP þ PPT MPTP þ DPN MPTP þ D3-diol
16:6 1:2 17:6 1:4 17:9 0:8 18:6 0:5 17:1 1:3 17:7 1:1
6:3 0:7 5:8 0:6 6:5 0:5 6:4 0:5 5:2 0:4 6:4 0:3
for DA; F 4;50 ¼ 2:371, P ¼ 0:0649 for DOPAC; F 4;52 ¼ 1:894, P ¼ 0:1254 for HVA; F 4;52 ¼ 2:094, P ¼ 0:0949 for 3-MT) (Table 2). As another marker of striatal DA neurons, we measured the effect of the lesion and treatments on [125I]RTI-121-specific binding to the DAT by autoradiography (Fig. 2). Following the MPTP lesion, DAT-specific binding was significantly decreased by 42% (58% of intact) (F 5;34 ¼ 4:397, P ¼ 0:0034) (Fig. 2A). MPTP mice that received DPN or D3-diol showed decreased levels of DAT-binding, similar to the MPTP group. However, mice pre-treated with PPT or 17b-E2 showed greater striatal DAT-binding than the vehicle-treated MPTP mice. Striatal DAT-specific binding for PPT- and 17b-E2-treated mice were significantly greater than the D3-diol group, while only PPT-treated mice were statistically higher than the DPN-treated group. A positive and significant correlation was observed between striatal DAT-specific binding and DA concentrations for individual (F 1;38 ¼ 36:967, P ¼ 0:0001) and group values (F 5;1 ¼ 41:250, P ¼ 0:0030), and also between DAT-binding and DOPAC (F 5;1 ¼ 19:929, P ¼ 0:0111) (Fig. 2B and C). We also measured the effect of the treatments on PPE mRNA levels by in situ hybridization as a striatal non-dopaminergic marker. In MPTP mice, 17b-E2 treatment decreased the levels of PPE mRNA, although not significantly (0.115 versus 0.123 for control; values expressed in relative optical densities). On
Table 2 Effects of treatment for 10 days of intact C57Bl/6 male mice with 17b-E2, PPT, DPN and D3-diol on striatal catecholamine concentrations. Values are the mean ðng=mg of proteinÞ SEM of six to nine mice per group Groups
DA (ng/mg of protein)
DOPAC (ng/mg of protein)
HVA (ng/mg of protein)
3-MT (ng/mg of protein)
Vehicle 17b-E2 PPT DPN D3-diol
109:5 6:1 114:3 4:8 113:2 5:2 114:5 7:7 116:1 9:8
6:7 0:5 6:0 0:2 6:3 0:4 7:4 0:6 8:0 0:9
8:3 0:7 9:1 1:0 8:4 0:6 10:0 1:2 11:5 1:2
10:1 1:5 7:4 0:8 6:9 0:6 7:2 0:7 9:3 1:0
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Fig. 2. (A) Effects of 17b-E2, PPT, DPN and D3-diol on the dopamine transporter (DAT)-specific binding in the striatum of C57Bl/6 male mice treated with MPTP as compared to vehicle-treated intact animals. P < 0:05 and P < 0:005 versus intact þ vehicle; y P < 0:05 and yy P < 0:01 versus MPTP þ vehicle; u P < 0:05 versus MPTP þ 17b-E2 ; h P < 0:05 and hh P < 0:005 versus MPTP þ PPT. Values are the mean ðpercentage of intact þ vehicle; where 100% equals 1:516 fmol=mg of tissueÞ SEM of six to nine mice per group. (B) Correlation between striatal DA concentrations and [125I]RTI-121-specific binding to the DAT in C57Bl/6 MPTP male mice treated with agonists or vehicle as compared to vehicle-treated intact animals. Correlations and statistical analyses were performed on individual values for DA (B) and for group values for DA as well as DOPAC concentrations (C).
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the other hand, PPT-, DPN- and D3-diol-treated mice showed increased levels of PPE mRNA in the striatum of MPTP mice (0.151, 0.182 and 0.194, respectively), levels which were significantly greater than vehicletreated intact mice (F 5;36 ¼ 22:519, P ¼ 0:0001). Competition for [125I]RTI-121 binding was used to determine if the estrogenic compounds investigated in this experiment had affinity for the DAT. At 106 M, the maximal concentration injected sub-cutaneously to the animals, we observed that only mazindol competed completely with the ligand [125I]RTI-121 for DAT binding (100% for mazindol), while all the estrogenic compounds showed little or no binding to the transporter in this assay (17b-E2, 10%; PPT, 23%; DPN, 7% and D3-diol, 6% competition of [125I]RTI-121 binding to the DAT). Since PPT showed weak binding to the transporter at 106 M concentration, we measured its binding at 107 M, which was 0%. 4. Discussion Using ER agonists, we observed that neuroprotection against MPTP toxicity depends on the selectivity of the agonist. Indeed, PPT, a specific ERa agonist, provided complete protection against MPTP-induced DA depletion in the striatum of C57Bl/6 mice. 17b-E2, which is the active isomer of estradiol, also protected against MPTP. These results could be explained by the fact that 17b-E2 binds equally ERa and ERb (Kuiper et al., 1997) and PPT binds preferentially ERa (Stauffer et al., 2000). Also, PPT only activates gene transcription through ERa (Stauffer et al., 2000). These results suggest that ERa mediates the neuroprotective potential of estradiol in this model. Other evidence came from the ERb agonists, DPN and D3-diol. These two estrogenic compounds preferentially bind and activate ERb (Kuiper et al., 1997; Meyers et al., 2001) and failed to prevent the depletion in DA induced by the toxin. The same pattern of protection by PPT was observed for the metabolite DOPAC. Results from DAT-specific binding also suggest that the neuroprotective effect of estradiol is mediated by ERa, since PPT efficiently protected against the decrease of binding to the transporter. The two ERb agonists used in this protocol, DPN and D3-diol, failed to protect against the decrease in DAT-specific binding induced by MPTP. The positive and significant correlation between DA and DAT binding shows that PPT protects two markers of dopaminergic neurons, namely the DA content and also the transporter. Moreover, the significant correlation between DAT binding and DOPAC suggests that neuroprotection occurs pre-synaptically, since both of these markers are located on pre-synaptic dopaminergic neurons and therefore implies that protection is not the
result of inhibition of degradation enzymes, such as monoamine oxidase. Moreover, with binding assays, we showed that the estrogenic compounds used in this protocol had weak affinity for the DAT at 106 M. This concentration is the dose injected to the animals in this protocol and does not take into account the pharmacokinetic, such as distribution and elimination of the compounds. The quantity of the estrogenic compounds that actually reaches the brain is therefore far less than the concentration tested here in vitro. Since PPT competed a little at this concentration, we also measured its affinity at 107 M and measured no competition for binding to the transporter at this concentration. Accordingly, the positive effects of 17b-E2 and PPT cannot be attributed to an inhibition of the toxic effect of MPTP by their blockade of the transporter. To further support the idea of neuroprotection, nonlesioned animals were treated with the same agonists and revealed no changes in DA and metabolites content. These results indicate that the positive effects of PPT on these parameters following a MPTP lesion are a reflection of neuroprotection rather than neurostimulation of the remaining neurons. We also verified if the lack of effect of ERb agonists was due to differential pharmacokinetic or brain penetration. Since no in vivo studies were available for DPN and knowing that it can activate both subtype of ER when administered in a larger dose (Meyers et al., 2001), we kept the dose as low as possible to avoid cross activation of ERa. For this reason, with the same animals, we verified DPN and D3-diol capacities to induce transcription in the striatum. Both ERb agonists were effective to increase PPE mRNA in these mice after a MPTP lesion. Therefore, we can conclude that these agonists are effective in inducing transcription in the striatum and that an absence of positive effect on DA content and DAT binding for the ERb agonists cannot be attributed to differential brain penetration. Other models of neuronal toxicity have also suggested that neuroprotection by Es involves ERa activation. With knockout mice for each subtype of ERs, Dubal et al. (2001) demonstrated that neuroprotection by Es against stroke was mediated by ERa. Recently, systemic administration of 17b-E2 was shown to prevent the activation of microglia and this anti-inflammatory effect of estradiol against LPS toxicity was exclusively linked to ERa (Vegeto et al., 2003). Evidence suggests independent roles of each type of ERs in various areas of the brain. Even though the two types of receptors coexist in different brain region, ERa and ERb mRNA (Couse et al., 1997; Kuiper et al., 1997) and protein (Mitra et al., 2003) are also found independently in other regions. Although some groups failed to detect ER in the striatum or substantia nigra (SN) of mice (Struble et al., 2003), others have demonstrated the presence of mRNA (Kuiper et al., 1997) or
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protein (Mitra et al., 2003) in these regions. However, there seems to be a variation in the intensity of expression of the mRNA or protein depending on the subtype of ER, brain areas and/or gender studied. In addition to differential distribution, the two subtypes respond differently to lesion. In a stroke model, Dubal et al. (1999) demonstrated that ERs were differently modulated after an injury. Indeed, while ERa was upregulated following a stroke, ERb was down-regulated (Dubal et al., 1999). Furthermore, a mechanical injury of the brain also up-regulated ERa in astrocytes in a primate model (Blurton-Jones and Tuszynski, 2001). Another evidence for a different role for each ER comes from their differences in transcriptional activity. Even though estradiol binds equally ERa and ERb, the transcriptional capacity of each receptor is not the same, as reported in various in vitro models (Mosselman et al., 1996; Pettersson et al., 1997; Watanabe et al., 1997). These studies report that ERa is more efficient than ERb to induce genomic transcription. Furthermore, ERb is only 30% as efficient as ERa to induce transcriptional activity (Pettersson and Gustafsson, 2001). Lastly, although the two types of receptors have a strong sequence homology, especially in the DNA binding-domain, lower similarities are present in the Cterminal ligand binding-domain and in the N-terminal transactivation domain (Mosselman et al., 1996; Kuiper et al., 1997). Recently, this difference in ligand binding-domain sequence has been highlighted by Mendez (Mendez et al., 2003), as they demonstrated that only ERa interacts and binds directly the insulinlike growth factor I (IGF-I) to activate the Akt/PI3K signaling pathway. Since activation of this signaling pathway by estradiol is known to provide neuroprotection (Azcoitia et al., 1999), dissimilarities in ERs could account for their difference in neuroprotective ability. Different binding or transcriptional capacity, presence or absence of coactivators for each ER could explain this difference in neuroprotection. We believe that like IGF-I, many other intracellular components could specifically bind ERa over ERb, and activate specific signaling pathways. This could also lead to the activation of different transcription factors and also, to the transcription of different genes linked to the survival of dopaminergic neurons. Although evidence from our group and others suggest that estrogens provide neuroprotection through genomic mechanisms (Callier et al., 2000; Ramirez et al., 2003; Dubal et al., 2001; Vegeto et al., 2003), neuroprotection with estrogens have been obtained via non-genomic mechanism as well (Behl et al., 1997; Green et al., 1997, 2001; Moosmann and Behl, 1999). The antioxidant activity has been linked to the presence of a phenolic ring on the estrogen molecule (Moosmann and Behl, 1999). Since 17b-E2, PPT and DPN all possess one or more phenolic rings, we should
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see similar protection between these compounds if the antioxidant effect of Es was implicated. Even though DPN contains two phenolic rings, it did not protect against MPTP, while 17b-E2 has only one and did protect. This suggests that antioxidant activity of estrogenic compounds cannot explain their neuroprotection effect in MPTP mice. Other non-genomic mechanisms could be implicated in the protection against MPTP toxicity, many of which we could not confirm or infirm with this protocol. However, results from MPTP experiments suggest a genomic mechanism of action for E. The stereospecificity for estradiol protection (Callier et al., 2000; Ramirez et al., 2003) and the results presented herein with ER agonists proposed a genomic mechanism for Es protection. Since Es can act on the brain through many mechanisms, which type of mechanism (genomic or non-genomic) could depend on the type of lesion and/or treatment and brain areas.
Acknowledgements This research was supported by a grant from the Canadian Institutes of Health Research (CIHR) to T.D.P. M.D. holds a CIHR studentship.
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Meyers, M.J., Sun, J., Carlson, K.E., Marriner, G.A., Katzenellenbogen, B.S., Katzenellenbogen, J.A., 2001. Estrogen receptor-beta potency-selective ligands: structure-activity relationship studies of diarylpropionitriles and their acetylene and polar analogues. J. Med. Chem. 44 (24), 4230–4251. Mitra, S.W., Hoskin, E., Yudkovitz, J., Pear, L., Wilkinson, H.A., Hayashi, S., Pfaff, D.W., Ogawa, S., Rohrer, S.P., Schaeffer, J.M., McEwen, B.S., Alves, S.E., 2003. Immunolocalization of estrogen receptor beta in the mouse brain: comparison with estrogen receptor alpha. Endocrinology 144 (5), 2055–2067. Moosmann, B., Behl, C., 1999. The antioxidant neuroprotective effects of estrogens and phenolic compounds are independent from their estrogenic properties. Proc. Natl. Acad. Sci. USA 96 (16), 8867–8872. Moss, R.L., Gu, Q., 1999. Estrogen: mechanisms for a rapid action in CA1 hippocampal neurons. Steroids 64 (1–2), 14–21. Mosselman, S., Polman, J., Dijkema, R., 1996. ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett. 392 (1), 49–53. Pettersson, K., Gustafsson, J.A., 2001. Role of estrogen receptor beta in estrogen action. Annu. Rev. Physiol. 63, 165–192. Pettersson, K., Grandien, K., Kuiper, G.G., Gustafsson, J.A., 1997. Mouse estrogen receptor beta forms estrogen response elementbinding heterodimers with estrogen receptor alpha. Mol. Endocrinol. 11 (10), 1486–1496. Ramirez, V.D., Zheng, J., 1996. Membrane sex-steroid receptors in the brain. Front. Neuroendocrinol. 17 (4), 402–439. Ramirez, A.D., Liu, X., Menniti, F.S., 2003. Repeated estradiol treatment prevents MPTP-induced dopamine depletion in male mice. Neuroendocrinology 77 (4), 223–231. Razandi, M., Pedram, A., Greene, G.L., Levin, E.R., 1999. Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells. Mol. Endocrinol. 13 (2), 307–319. Saunders-Pullman, R., 2003. Estrogens and Parkinson disease: neuroprotective, symptomatic, neither, or both? Endocrine 21 (1), 81– 87. Simpkins, J.W., Rajakumar, G., Zhang, Y.Q., Simpkins, C.E., Greenwald, D., Yu, C.J., Bodor, N., Day, A.L., 1997. Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat. J. Neurosurg. 87 (5), 724– 730. Stauffer, S.R., Coletta, C.J., Tedesco, R., Nishiguchi, G., Carlson, K., Sun, J., Katzenellenbogen, B.S., Katzenellenbogen, J.A., 2000. Pyrazole ligands: structure–affinity/activity relationships and estrogen receptor-alpha-selective agonists. J. Med. Chem. 43 (26), 4934–4947. Struble, R.G., Rosario, E.R., Kircher, M.L., Ludwig, S.M., McAdamis, P.J., Watabe, K., McAsey, M.E., Cady, C., Nathan, B.P., 2003. Regionally specific modulation of brain apolipoprotein E in the mouse during the estrous cycle and by exogenous 17beta estradiol. Exp. Neurol. 183 (2), 638–644. Vegeto, E., Belcredito, S., Etteri, S., Ghisletti, S., Brusadelli, A., Meda, C., Krust, A., Dupont, S., Ciana, P., Chambon, P., Maggi, A., 2003. Estrogen receptor-alpha mediates the brain antiinflammatory activity of estradiol. Proc. Natl. Acad. Sci. USA 100 (16), 9614–9619. Watanabe, T., Inoue, S., Ogawa, S., Ishii, Y., Hiroi, H., Ikeda, K., Orimo, A., Muramatsu, M., 1997. Agonistic effect of tamoxifen is dependent on cell type, ERE-promoter context, and estrogen receptor subtype: functional difference between estrogen receptors alpha and beta. Biochem. Biophys. Res. Commun. 236 (1), 140– 145. Wise, P.M., Dubal, D.B., Wilson, M.E., Rau, S.W., Liu, Y., 2001. Estrogens: trophic and protective factors in the adult brain. Front. Neuroendocrinol. 22 (1), 33–66.
Effect of estrogen receptor agonists treatment in MPTP mice: evidence of neuroprotection by an ERa agonist Myreille D’Astous, Marc Morissette, The´re`se Di Paolo Molecular Endocrinology and Oncology Research Center, Laval University Medical Center, CHUL, 2705 Laurier Boulevard, Quebec City, Que., G1V 4G2, Canada Faculty of Pharmacy, Laval University, Quebec City, Que., G1K 7P4, Canada Received 16 January 2004; received in revised form 27 July 2004; accepted 17 August 2004
Abstract Beneficial effects of 17b-estradiol (17b-E2) on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced striatal dopamine (DA) depletion are well documented but the mechanisms implicated are poorly understood. The present experiments investigated the effect of estrogen receptor (ER) agonists treatment in MPTP mice as compared to 17b-E2. The agonists specific for each subtype were 4,40 ,400 -(4-propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol (PPT) (ERa agonist), 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and D3-diol (5-androsten-3b, 17b-diol, also known as 5-androstenediol, androstenediol or hermaphrodiol) (ERb agonists). Biogenic amines were assayed by HPLC with electrochemical detection. 8 mg/kg of MPTP was administered to give a moderate depletion of striatal DA and its metabolite dihydroxyphenylacetic acid (DOPAC). Protection against MPTP-induced striatal DA and DOPAC depletion was obtained with PPT and 17b-E2 but not with DPN or D3-diol. The striatal dopamine transporter (DAT) was assayed by autoradiography with [125I]RTI-121-specific binding. A positive and significant correlation was observed between striatal DA concentrations and [125I]RTI-121-specific binding, suggesting that estrogenic treatment that prevented the MPTP-induced DA depletion also prevented loss of DAT. The effect of PPT suggests the implication of an ERa in the estrogenic neuroprotection against MPTP. Pointing out which ER is implicated in neuroprotection becomes helpful in designing more specific estrogenic drugs for protection of the aging brain. # 2004 Elsevier Ltd. All rights reserved. Keywords: MPTP; Estrogen; Agonists; Neuroprotection; Parkinson
1. Introduction Clinical studies have demonstrated a greater prevalence of Parkinson’s disease (PD) in men than in women (Baldereschi et al., 2000; Dluzen and McDermott, 2000b; Saunders-Pullman, 2003). Even though the cause of this disease is still unknown, the clinical evidence implies a role of sex hormones in the incidence of PD. Estrogens (Es) play an important role during brain development in neuronal differentiation, plasticity and Corresponding author. Molecular Endocrinology and Oncology Research Center, Laval University Medical Center, CHUL, 2705 Laurier Boulevard, Quebec City, Que., Canada G1V 4G2. Tel.: +1418-654-2296; fax: +1-418-654-2761. E-mail address: [email protected] (T. Di Paolo).
0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.08.020
cell survival (Beyer, 1999; Wise et al., 2001). In adult, Es act on many brain regions, including those implicated in reproduction and areas responsible for memory, cognition and mood (Wise et al., 2001). Many in vivo studies have demonstrated beneficial effects of Es (Garcia-Segura et al., 2001) against methamphetamine (Dluzen and McDermott, 2000a; Dluzen et al., 2002), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Callier et al., 2000, 2001; Ramirez et al., 2003), and ischemic injury in the brain (Simpkins et al., 1997; Dubal et al., 2001). However, the mechanisms by which Es protect against neuronal damages are still unknown. Es mediate their actions by two types of mechanisms, classified as genomic or nongenomic. The genomic mechanism implicates an interaction between Es and its nuclear receptors to activate genomic transcription (Pettersson and Gustafsson,
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2001). Other mechanisms, independents of estrogen receptors (ERs), are classified as non-genomic, such as the antioxidant capacity of Es (Moosmann and Behl, 1999), direct effect on the membrane (Ramirez and Zheng, 1996; Moss and Gu, 1999) or on its membrane receptor (Razandi et al., 1999). Using the MPTP mouse model, which reproduces the striatal dopamine (DA) loss seen in PD (Burns et al., 1983), we investigated the neuroprotective effects of 17b-estradiol (17b-E2) and the mechanisms implicated in this protection. We previously reported a neuroprotective role of 17b-E2 against MPTP toxicity (Callier et al., 2000; Grandbois et al., 2000; Callier et al., 2001; D’Astous et al., 2003). Evidence from these experiments suggests a genomic mechanism of action to explain the beneficial effects of estradiol on dopaminergic neurons. Indeed, the protection obtained with estradiol was only obtained with 17b-E2 (the active isomer of estradiol), while 17a-estradiol (17a-E2) did not protect (Callier et al., 2000). Other groups have also reported stereospecificity of neuroprotection by estradiol in this model (Ramirez et al., 2003). In this report, we investigated whether estradiol’s neuroprotective effects occur via one or both receptor subtypes, using ERa and ERb agonists. These molecules are described as ERa or ERb agonist based on their binding capacity to each receptor and on their transcriptional potency once bound to the receptor. 4,40 ,400 -(4-Propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol (PPT) binding affinity is 410 times greater for ERa than ERb (Stauffer et al., 2000). 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN) binding affinity is 72 times greater for ERb (Meyers et al., 2001), while D3diol (5-androsten-3b, 17b-diol) has a 2.8 times greater affinity for ERb (Kuiper et al., 1997). Binding affinity only demonstrates the capacity of a molecule to bind a specific receptor, whereas transcription assays measure its capacity to induce genomic transcription after binding to a receptor. PPT is reported to induce transcription only via ERa, at any concentration (Stauffer et al., 2000; Harrington et al., 2003). DPN can act as an agonist on both subtypes, with relative transcription potency 78 times greater for ERb at lower doses (Meyers et al., 2001). However, at high doses (108 M), DPN can also activate ERa in transcription assays (Harrington et al., 2003). No studies on relative transcription potency for each ER subtype are available for D3-diol. Herein, using chronic treatments with ERa and ERb agonists, we assessed the implication of ERs in neuroprotection against MPTP striatal DA toxicity in mice.
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2. Methods 2.1. Chemicals MPTP and 17b-E2 were purchased from Sigma Chemical (St. Louis, MO, USA), DPN and PPT from Tocris (Ellisville, MO, USA), while D3-diol was purchased from Steraloids Inc (Newport, RI, USA). 2.2. Animals and treatments C57Bl/6 male mice (10–12 weeks, 25 2 g) were purchased from Charles River Laboratories (Canada). Mice were distributed in groups of six to nine animals and each group received a pre-treatment of hormones or vehicle 5 days prior to the MPTP injections. The pre-treatment consisted of two daily injections (subcutaneous) of 17b-E2, PPT, DPN or D3-diol, while control mice received injections of vehicle (0.9% saline with 0.3% gelatin/0.2 ml). Estrogenic compounds concentrations were 2 lg/day (~80 lg/kg) for 17b-E2 and PPT, 3 lg/day (~120 lg/kg) for D3-diol and 6 lg/day (~240 lg/kg) for DPN. Doses were chosen based on their affinity for ERs (Kuiper et al., 1997; Stauffer et al., 2000; Meyers et al., 2001) and their transcriptional capacity (Stauffer et al., 2000; Meyers et al., 2001; Harrington et al., 2003), since no in vivo studies on the brain were available for DPN and D3-diol. On day 5, mice received four injections of MPTP (8 mg/kg, intraperitoneal) at 2 h interval, while the control group received saline solution. The treatments (estrogenic compounds or vehicle) were continued until day 10. On day 11, mice were decapitated, brains quickly removed v and frozen in isopentane (40 C). In another complementary experiment, intact C57Bl/6 male mice (without lesion) received estrogenic drug treatments. Mice were also treated for 10 days with the same concentrations of the drugs as described above, while one group received only the vehicle and served as control in this experiment. The Laval University Animal Care Committee approved all the animal studies. All efforts were made to minimize animal suffering and to reduce the number of mice used. 2.3. Striatal biogenic amines determination The left anterior striata were dissected, homogenized v in 250 ll of 0.1 M HClO4 at 4 C and then centrifuged v at 10 000 g for 10 min (4 C) to precipitate proteins as previously reported (D’Astous et al., 2003). The supernatants were used to determine the biogenic amine concentrations while the pellets were dissolved in NaOH 1 N to assay protein content. The concentrations of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxyphenylethylamine (3-MT), and homovanillic acid
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(HVA) as well as serotonin (5-HT) and its metabolites 5-hydroxyindoleacetic acid (5-HIAA) were measured by high performance liquid chromatography (HPLC) with electrochemical detection. Supernatants of striatal tissue were directly injected into the chromatograph consisting of a Waters 717 plus autosampler automatic injector, a Waters 515 pump equipped with a Beckman C-18 column (Waters Nova-Pak C18, 3 lm, 3:9 mm 150 cm), a BAS LC-4C electrochemical detector and a glassy carbon electrode. The mobile phase consisted of 0.025 M citric acid, 1.7 mM 1-heptane-sulfonic acid, and 10% methanol, in filtered distilled water, delivered at a flow rate of 1 ml/min. The final pH of 4.1 was obtained by addition of NaOH. The electrochemical potential was set at 0.8 V with respect to an Ag/AgCl reference electrode. The sensitivity of this assay was 13 pg for DOPAC, 7 pg for DA, 40 pg for HVA, 27 pg for 3-MT, 33 pg for 5HIAA and 40 pg for 5-HT. 2.4. DA transporter (DAT) autoradiography acid 3b-(4-[125I]Iodophenyl)tropane-2b-carboxylic isopropyl ester ([125I]-RTI-121) specific binding autoradiography was carried out as previously described (Carli et al., 1997; Callier et al., 2001). The binding was performed on slices containing the striatum at coordinates bregma 1.54 to 0.94 mm, according to the atlas of Franklin and Paxinos (1997). Three brain slices per animal were used in this protocol. Specific binding to DAT was measured using 20 pmol of [125I]-RTI-121 (2200 Ci/mmol), in the presence of 2 nM of mazindol to evaluate the non-specific binding. Slices were apposed to Kodak film for 40 h. Autoradiograms were analyzed using the software NIH 1.68. 2.5. In situ hybridization of preproenkephalin (PPE) mRNA In situ hybridization of preproenkephalin (PPE) mRNA was performed as previously described (Calon et al., 2002) on brain slices containing the striatum at coordinates bregma 1.54 to 0.94 mm (Franklin and Paxinos, 1997). Two brain slices per animal were used in this protocol. An oligonucleotide complementary to bases 304–350 of the mice PPE sequence (GenBank accession number M13227) was designed to determine specific binding while non-specific binding was assayed using a sens oligonucleotide probe. Slices were apposed to Kodak film for 5 days and analyzed using the software NIH 1.68. Relative optical densities for specific binding were obtained by subtracting non-specific from total binding.
2.6. Binding affinity assay Specific binding to the DAT was investigated in mice striatal homogenates under conditions previously described (Boja et al., 1995) with slight modifications. Mice striata were dissected, pooled, and weighted. The striata were then homogenized in a glass–teflon homogenizer in 100 volumes (w/v) of phosphate buffer (10.1 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2 mM KCl, pH 7.5) and centrifuged at 20 000g for 15 min. Washing procedures were repeated twice and the final pellet resuspended in 100 volumes (w/v) of the same ice-cold phosphate buffer. Mazindol, 17b-E2, PPT, DPN and D3-diol (concentrations of 106 and 107 M) were added directly into the incubation buffer in the presence of a fixed concentration of [125I]-RTI121 (20 pM) in a final volume of 2 ml. The binding reaction was initiated by the addition of 0.2 mg of striatal tissue. Samples were incubated at room temperature for 1 h. The reaction was stopped by rapid filtration through Whatman GF/C glass fiber filters under vacuum, followed by three rapid rinses (5 ml). Radioactivity on the filters was measured with a gamma counter. Percent binding for each compound was determined by dividing bound [125I]-RTI-121 in presence of the competing drugs on the value obtained for [125I]-RTI-121 total binding multiplied by 100. 2.7. Statistical analysis Statistical comparisons of biogenic amine concentrations, DAT-specific binding and PPE mRNA levels were performed using a one-way analysis of variance (ANOVA) followed by post hoc analysis with Fisher probability of least significance difference test. Coefficient of correlations and significance of the degree of linear relationship between DA and DOPAC concentrations versus DAT-specific binding were determined using a simple regression model.
3. Results In the present protocol, MPTP was administered in order to give a moderate striatal DA depletion, as measured by DA and its metabolites concentrations (Fig. 1). Indeed, the toxin decreased the DA content in the striatum of MPTP-treated mice to 59% of intact (F 5;36 ¼ 4:118, P ¼ 0:0046) (Fig. 1A). The metabolite DOPAC was decreased by 24% (76% of intact) (F 5;36 ¼ 3:850, P ¼ 0:0068) (Fig. 1B), while 3-MT and HVA remained unchanged (105 and 89% of intact) (F 5;36 ¼ 0:969, P ¼ 0:4495 for 3-MT; F 5;36 ¼ 1:222, P ¼ 0:3189 for HVA) (Fig. 1C and D). MPTP mice receiving PPT and 17b-E2 had higher striatal DA concentrations than the vehicle-treated
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Fig. 1. Effects of 17b-E2, PPT, DPN and D3-diol on striatal catecholamine concentrations in C57Bl/6 male mice treated with MPTP as compared to vehicle-treated intact animals. Mice were treated with estrogenic compounds or vehicle for 10 days and MPTP-treated mice received four injections of MPTP (8 mg/kg) on day 5. P < 0:05, P < 0:005 and P < 0:0005 versus intact þ vehicle; y P < 0:05 and yy P < 0:005 versus MPTP þ vehicle; u P < 0:05 versus MPTP þ 17b-E2 ; h P < 0:05 and hh P < 0:005 versus MPTP þ PPT. Values are the mean ðng=mg of proteinÞ SEM of six to nine mice per group.
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MPTP mice. The ERb agonists DPN and D3-diol treated MPTP mice had striatal DA concentrations similar to the MPTP vehicle-treated group. Striatal DA content for the PPT-treated mice was significantly greater than the D3-diol-treated group (Fig. 1A). For the metabolite DOPAC, a similar pattern of protection as for the DA content was observed, although the decrease induced by MPTP on this metabolite was less important. The MPTP-induced decrease in DOPAC was statistically significant for the DPN and D3-diol, but not for the 17b-E2 treated groups, with regards to the intact group (Fig. 1B). PPT administration to MPTP-mice prevented this decrease, since DOPAC values were significantly different and higher than those of the MPTP-vehicle group. In addition, the striatal DOPAC content for the PPT group was significantly greater than DPN and D3-dioltreated groups (Fig. 1B). The two other DA metabolites, HVA and 3-MT, remained unchanged after MPTP alone or in combination with the agonists treatments (Fig. 1C and D). MPTP treatment led to an increase in striatal DA turnover, as assessed by the HVA/DA ratio (F 5;36 ¼ 3:396, P ¼ 0:0129) (Fig. 1G). PPT and 17b-E2 prevented this increase, but not D3-diol or DPN. The ratios DOPAC/DA (Fig. 1E) and 3-MT/DA (Fig. 1F) showed no statistical differences between the groups (F 5;36 ¼ 1:784, P ¼ 0:1409 for DOPAC/DA; F 5;36 ¼ 2:235, P¼ 0:0718 for 3-MT/DA), although they displayed a similar pattern of changes between the treatment groups as for the HVA/DA ratio, but to a lesser extent. To verify the specificity of the lesion or treatments, striatal indolamine concentrations were measured. There were no variation of striatal 5-HT or its metabolite 5-HIAA after MPTP alone or in combination with the estrogenic drug treatments (F 5;36 ¼ 0:388, P ¼ 0:8539 for 5-HT; F 5;35 ¼ 0:942, P ¼ 0:4665 for 5HIAA) (Table 1). For comparison, catecholamine concentrations were measured in non-lesioned animals treated with these agonists. There were no change of striatal DA or its metabolites concentrations after a treatment with any of the estrogenic drug tested (F 4;52 ¼ 0:132, P ¼ 0:9698
Table 1 Effects of 17b-E2, PPT, DPN and D3-diol on striatal indolamine concentrations in C57Bl/6 male mice treated with MPTP as compared to vehicle-treated intact animals. Values are the mean ðng=mg of proteinÞ SEM of six to nine mice per group Groups
5-HT (ng/mg of protein)
5-HIAA (ng/mg of protein)
Intact þ vehicle MPTP þ vehicle MPTP þ 17b-E2 MPTP þ PPT MPTP þ DPN MPTP þ D3-diol
16:6 1:2 17:6 1:4 17:9 0:8 18:6 0:5 17:1 1:3 17:7 1:1
6:3 0:7 5:8 0:6 6:5 0:5 6:4 0:5 5:2 0:4 6:4 0:3
for DA; F 4;50 ¼ 2:371, P ¼ 0:0649 for DOPAC; F 4;52 ¼ 1:894, P ¼ 0:1254 for HVA; F 4;52 ¼ 2:094, P ¼ 0:0949 for 3-MT) (Table 2). As another marker of striatal DA neurons, we measured the effect of the lesion and treatments on [125I]RTI-121-specific binding to the DAT by autoradiography (Fig. 2). Following the MPTP lesion, DAT-specific binding was significantly decreased by 42% (58% of intact) (F 5;34 ¼ 4:397, P ¼ 0:0034) (Fig. 2A). MPTP mice that received DPN or D3-diol showed decreased levels of DAT-binding, similar to the MPTP group. However, mice pre-treated with PPT or 17b-E2 showed greater striatal DAT-binding than the vehicle-treated MPTP mice. Striatal DAT-specific binding for PPT- and 17b-E2-treated mice were significantly greater than the D3-diol group, while only PPT-treated mice were statistically higher than the DPN-treated group. A positive and significant correlation was observed between striatal DAT-specific binding and DA concentrations for individual (F 1;38 ¼ 36:967, P ¼ 0:0001) and group values (F 5;1 ¼ 41:250, P ¼ 0:0030), and also between DAT-binding and DOPAC (F 5;1 ¼ 19:929, P ¼ 0:0111) (Fig. 2B and C). We also measured the effect of the treatments on PPE mRNA levels by in situ hybridization as a striatal non-dopaminergic marker. In MPTP mice, 17b-E2 treatment decreased the levels of PPE mRNA, although not significantly (0.115 versus 0.123 for control; values expressed in relative optical densities). On
Table 2 Effects of treatment for 10 days of intact C57Bl/6 male mice with 17b-E2, PPT, DPN and D3-diol on striatal catecholamine concentrations. Values are the mean ðng=mg of proteinÞ SEM of six to nine mice per group Groups
DA (ng/mg of protein)
DOPAC (ng/mg of protein)
HVA (ng/mg of protein)
3-MT (ng/mg of protein)
Vehicle 17b-E2 PPT DPN D3-diol
109:5 6:1 114:3 4:8 113:2 5:2 114:5 7:7 116:1 9:8
6:7 0:5 6:0 0:2 6:3 0:4 7:4 0:6 8:0 0:9
8:3 0:7 9:1 1:0 8:4 0:6 10:0 1:2 11:5 1:2
10:1 1:5 7:4 0:8 6:9 0:6 7:2 0:7 9:3 1:0
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Fig. 2. (A) Effects of 17b-E2, PPT, DPN and D3-diol on the dopamine transporter (DAT)-specific binding in the striatum of C57Bl/6 male mice treated with MPTP as compared to vehicle-treated intact animals. P < 0:05 and P < 0:005 versus intact þ vehicle; y P < 0:05 and yy P < 0:01 versus MPTP þ vehicle; u P < 0:05 versus MPTP þ 17b-E2 ; h P < 0:05 and hh P < 0:005 versus MPTP þ PPT. Values are the mean ðpercentage of intact þ vehicle; where 100% equals 1:516 fmol=mg of tissueÞ SEM of six to nine mice per group. (B) Correlation between striatal DA concentrations and [125I]RTI-121-specific binding to the DAT in C57Bl/6 MPTP male mice treated with agonists or vehicle as compared to vehicle-treated intact animals. Correlations and statistical analyses were performed on individual values for DA (B) and for group values for DA as well as DOPAC concentrations (C).
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the other hand, PPT-, DPN- and D3-diol-treated mice showed increased levels of PPE mRNA in the striatum of MPTP mice (0.151, 0.182 and 0.194, respectively), levels which were significantly greater than vehicletreated intact mice (F 5;36 ¼ 22:519, P ¼ 0:0001). Competition for [125I]RTI-121 binding was used to determine if the estrogenic compounds investigated in this experiment had affinity for the DAT. At 106 M, the maximal concentration injected sub-cutaneously to the animals, we observed that only mazindol competed completely with the ligand [125I]RTI-121 for DAT binding (100% for mazindol), while all the estrogenic compounds showed little or no binding to the transporter in this assay (17b-E2, 10%; PPT, 23%; DPN, 7% and D3-diol, 6% competition of [125I]RTI-121 binding to the DAT). Since PPT showed weak binding to the transporter at 106 M concentration, we measured its binding at 107 M, which was 0%. 4. Discussion Using ER agonists, we observed that neuroprotection against MPTP toxicity depends on the selectivity of the agonist. Indeed, PPT, a specific ERa agonist, provided complete protection against MPTP-induced DA depletion in the striatum of C57Bl/6 mice. 17b-E2, which is the active isomer of estradiol, also protected against MPTP. These results could be explained by the fact that 17b-E2 binds equally ERa and ERb (Kuiper et al., 1997) and PPT binds preferentially ERa (Stauffer et al., 2000). Also, PPT only activates gene transcription through ERa (Stauffer et al., 2000). These results suggest that ERa mediates the neuroprotective potential of estradiol in this model. Other evidence came from the ERb agonists, DPN and D3-diol. These two estrogenic compounds preferentially bind and activate ERb (Kuiper et al., 1997; Meyers et al., 2001) and failed to prevent the depletion in DA induced by the toxin. The same pattern of protection by PPT was observed for the metabolite DOPAC. Results from DAT-specific binding also suggest that the neuroprotective effect of estradiol is mediated by ERa, since PPT efficiently protected against the decrease of binding to the transporter. The two ERb agonists used in this protocol, DPN and D3-diol, failed to protect against the decrease in DAT-specific binding induced by MPTP. The positive and significant correlation between DA and DAT binding shows that PPT protects two markers of dopaminergic neurons, namely the DA content and also the transporter. Moreover, the significant correlation between DAT binding and DOPAC suggests that neuroprotection occurs pre-synaptically, since both of these markers are located on pre-synaptic dopaminergic neurons and therefore implies that protection is not the
result of inhibition of degradation enzymes, such as monoamine oxidase. Moreover, with binding assays, we showed that the estrogenic compounds used in this protocol had weak affinity for the DAT at 106 M. This concentration is the dose injected to the animals in this protocol and does not take into account the pharmacokinetic, such as distribution and elimination of the compounds. The quantity of the estrogenic compounds that actually reaches the brain is therefore far less than the concentration tested here in vitro. Since PPT competed a little at this concentration, we also measured its affinity at 107 M and measured no competition for binding to the transporter at this concentration. Accordingly, the positive effects of 17b-E2 and PPT cannot be attributed to an inhibition of the toxic effect of MPTP by their blockade of the transporter. To further support the idea of neuroprotection, nonlesioned animals were treated with the same agonists and revealed no changes in DA and metabolites content. These results indicate that the positive effects of PPT on these parameters following a MPTP lesion are a reflection of neuroprotection rather than neurostimulation of the remaining neurons. We also verified if the lack of effect of ERb agonists was due to differential pharmacokinetic or brain penetration. Since no in vivo studies were available for DPN and knowing that it can activate both subtype of ER when administered in a larger dose (Meyers et al., 2001), we kept the dose as low as possible to avoid cross activation of ERa. For this reason, with the same animals, we verified DPN and D3-diol capacities to induce transcription in the striatum. Both ERb agonists were effective to increase PPE mRNA in these mice after a MPTP lesion. Therefore, we can conclude that these agonists are effective in inducing transcription in the striatum and that an absence of positive effect on DA content and DAT binding for the ERb agonists cannot be attributed to differential brain penetration. Other models of neuronal toxicity have also suggested that neuroprotection by Es involves ERa activation. With knockout mice for each subtype of ERs, Dubal et al. (2001) demonstrated that neuroprotection by Es against stroke was mediated by ERa. Recently, systemic administration of 17b-E2 was shown to prevent the activation of microglia and this anti-inflammatory effect of estradiol against LPS toxicity was exclusively linked to ERa (Vegeto et al., 2003). Evidence suggests independent roles of each type of ERs in various areas of the brain. Even though the two types of receptors coexist in different brain region, ERa and ERb mRNA (Couse et al., 1997; Kuiper et al., 1997) and protein (Mitra et al., 2003) are also found independently in other regions. Although some groups failed to detect ER in the striatum or substantia nigra (SN) of mice (Struble et al., 2003), others have demonstrated the presence of mRNA (Kuiper et al., 1997) or
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protein (Mitra et al., 2003) in these regions. However, there seems to be a variation in the intensity of expression of the mRNA or protein depending on the subtype of ER, brain areas and/or gender studied. In addition to differential distribution, the two subtypes respond differently to lesion. In a stroke model, Dubal et al. (1999) demonstrated that ERs were differently modulated after an injury. Indeed, while ERa was upregulated following a stroke, ERb was down-regulated (Dubal et al., 1999). Furthermore, a mechanical injury of the brain also up-regulated ERa in astrocytes in a primate model (Blurton-Jones and Tuszynski, 2001). Another evidence for a different role for each ER comes from their differences in transcriptional activity. Even though estradiol binds equally ERa and ERb, the transcriptional capacity of each receptor is not the same, as reported in various in vitro models (Mosselman et al., 1996; Pettersson et al., 1997; Watanabe et al., 1997). These studies report that ERa is more efficient than ERb to induce genomic transcription. Furthermore, ERb is only 30% as efficient as ERa to induce transcriptional activity (Pettersson and Gustafsson, 2001). Lastly, although the two types of receptors have a strong sequence homology, especially in the DNA binding-domain, lower similarities are present in the Cterminal ligand binding-domain and in the N-terminal transactivation domain (Mosselman et al., 1996; Kuiper et al., 1997). Recently, this difference in ligand binding-domain sequence has been highlighted by Mendez (Mendez et al., 2003), as they demonstrated that only ERa interacts and binds directly the insulinlike growth factor I (IGF-I) to activate the Akt/PI3K signaling pathway. Since activation of this signaling pathway by estradiol is known to provide neuroprotection (Azcoitia et al., 1999), dissimilarities in ERs could account for their difference in neuroprotective ability. Different binding or transcriptional capacity, presence or absence of coactivators for each ER could explain this difference in neuroprotection. We believe that like IGF-I, many other intracellular components could specifically bind ERa over ERb, and activate specific signaling pathways. This could also lead to the activation of different transcription factors and also, to the transcription of different genes linked to the survival of dopaminergic neurons. Although evidence from our group and others suggest that estrogens provide neuroprotection through genomic mechanisms (Callier et al., 2000; Ramirez et al., 2003; Dubal et al., 2001; Vegeto et al., 2003), neuroprotection with estrogens have been obtained via non-genomic mechanism as well (Behl et al., 1997; Green et al., 1997, 2001; Moosmann and Behl, 1999). The antioxidant activity has been linked to the presence of a phenolic ring on the estrogen molecule (Moosmann and Behl, 1999). Since 17b-E2, PPT and DPN all possess one or more phenolic rings, we should
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see similar protection between these compounds if the antioxidant effect of Es was implicated. Even though DPN contains two phenolic rings, it did not protect against MPTP, while 17b-E2 has only one and did protect. This suggests that antioxidant activity of estrogenic compounds cannot explain their neuroprotection effect in MPTP mice. Other non-genomic mechanisms could be implicated in the protection against MPTP toxicity, many of which we could not confirm or infirm with this protocol. However, results from MPTP experiments suggest a genomic mechanism of action for E. The stereospecificity for estradiol protection (Callier et al., 2000; Ramirez et al., 2003) and the results presented herein with ER agonists proposed a genomic mechanism for Es protection. Since Es can act on the brain through many mechanisms, which type of mechanism (genomic or non-genomic) could depend on the type of lesion and/or treatment and brain areas.
Acknowledgements This research was supported by a grant from the Canadian Institutes of Health Research (CIHR) to T.D.P. M.D. holds a CIHR studentship.
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