Estradiol inhibits GSK3 and regulates interaction of estrogen receptors, GSK3, and beta-catenin in the hippocampus

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 25 (2004) 363 – 373

Estradiol inhibits GSK3 and regulates interaction of estrogen receptors, GSK3, and beta-catenin in the hippocampus P. Cardona-Gomez, a,b M. Perez, c J. Avila, c L.M. Garcia-Segura, a and F. Wandosell c,* a

Laboratory of Cellular and Molecular Neuroendocrinology, Instituto Cajal, CSIC, Madrid 28002, Spain Neuroscience Group, School of Medicine University of Antioquia, PO Box 1226 Medellin, Colombia c Centro de Biologı´a Molecular "Severo Ochoa" CSIC-Universidad Auto´noma de Madrid, Madrid 28049, Spain b

Received 30 January 2003; revised 24 September 2003; accepted 7 October 2003

Estrogens regulate a wide set of neuronal functions such as gene expression, survival and differentiation in a manner not very different from that exerted by neurotrophins or by growth factors. The beststudied hormonal action is the transcriptional activation mediated by estrogen receptors. However, the direct effects of estrogen on growth factor signaling have not been well clarified. The present data show that estradiol, in vivo, induces a transient activation of GSK3 in the adult female rat hippocampus, followed by a more sustained inhibition, as inferred from phosphorylation levels of Tau. Similar data was obtained from cultured hippocampal neurons when treated with the hormone. The transient activation was confirmed by direct measure of GSK3 kinase activity. In addition, our results show a novel complex of estrogen receptor A, GSK3, and B-catenin. The presence of the hormone removes Bcatenin from this complex. There is a second complex, also affected by estradiol, in which Tau is associated with GSK3, B-catenin, and elements of the PI3 kinase complex. Considering the role of GSK3 in neurodegeneration, our data suggest that part of the neuroprotective effects of estrogen may be due to the control of GSK3. D 2004 Elsevier Inc. All rights reserved.

Introduction Considerable evidence from animal studies indicates that estradiol is neuroprotective (Garcia-Segura et al., 2001; Green and Simpkins, 2000; Lee and McEwen, 2001; Wise et al., 2001). At least part of the neuroprotective actions of estradiol are mediated by estrogen receptors, which are expressed in different brain areas, primarily, but not exclusively, in neurons (Shughrue et al., 1997; Simerly, 1993). In addition, estradiol has rapid effects in neurons that may be associated with new membrane forms of estrogen receptors or with transient association of classical estrogen receptors with specific membrane compartments (Toran-Allerand et al., 1999, 2002). Rapid effects of estradiol in the brain include the activation of signaling pathways coupled to tyrosine kinase receptors (Bi et al.,

* Corresponding author. Fax: +34-91-3974799. E-mail address: [email protected] (F. Wandosell). Available online on ScienceDirect (www.sciencedirect.com.) 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2003.10.008

2000; Cardona-Gomez et al., 2002; Honda et al., 2000; Ivanova et al., 2002a; Kuroki et al., 2001; Singh, 2001; Singh et al., 1999; Zhang et al., 2001). Furthermore, one of such receptors, the insulinlike growth factor-I (IGF-I) receptor, is involved in many different actions of estradiol in the brain, including the regulation of neuronal differentiation (Duenas et al., 1996; Ma et al., 1994; Toran-Allerand et al., 1988), synaptic plasticity (Cardona-Gomez et al., 2000; Fernandez-Galaz et al., 1999), neuronal survival after injury (Azcoitia et al., 1999; Cardona-Gomez et al., 2001), gonadotrophin secretion, and reproductive behavior (Quesada and Etgen, 2001, 2002). The mechanisms involved in the interaction of estradiol and IGF-I receptor are not well understood. Recent studies have shown that estrogen receptor a, but not h, interacts with IGF-I receptor and with the p85 subunit of the PI3K in the brain (Mendez et al., in press). In addition, systemic administration of estradiol to adult ovariectomized female rats results in a transient increase in tyrosine phosphorylation of the IGF-I receptor, in a transient interaction of the IGF-I receptor with the estrogen receptor a and in an enhanced interaction of estrogen receptor a with p85 (Mendez et al., in press). This suggests that estrogen receptor a may affect IGF-I actions in the brain by a direct interaction with some of the components of IGF-I receptor signaling. Such interactions may explain why IGF-I and estradiol synergistically activate in the brain the kinase Akt (Cardona-Gomez et al., 2002), a component of the IGF-I/insulin signaling pathway (Ivanova et al., 2002b; Singh, 2001; Zhang et al., 2001). Downstream of Akt, there is another protein that may have a key role in IGF-I/insulin signaling: glycogen synthase kinase 3 (GSK3) (Cross et al., 1995; Pap and Cooper, 1998). GSK3 has two highly conserved isoforms, a and h; for review, see Frame and Cohen (2001) and Grimes and Jope (2001). GSK3 is involved not only in the signaling pathways of insulin/IGF-I, but also in the pathways of Wnt/Wingless (Cook et al., 1996; Welsh and Proud, 1993), FGF (Hashimoto et al., 2002), or LPA (Sayas et al., 1999). GSK3 is highly expressed in the central nervous system (Takahashi et al., 1994) where it may participate in a broad range of biological processes. In particular, its inhibition is associated with the activation of survival pathways in neurons (Cross et al., 1995). One of the substrates of GSK3 is h-catenin, a protein that is both a cell adhesion molecule as well as a transcription factor (Cadigan and Nusse, 1997; Miller and Moon, 1996). Other sub-

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strates of GSK3 are microtubule-associated proteins, such as Tau (Ishiguro et al., 1993). Phosphorylation of microtubule-associated proteins by GSK3h may be involved in the regulation of microtubule dynamics, neuritis growth, synaptogenesis, and synaptic plasticity. (Hall et al., 2000) Furthermore, GSK3h may be responsible for the hyperphosphorylation of Tau in Alzheimer’s disease (Lovestone et al., 1994). Since estradiol activates Akt in the brain (Cardona-Gomez et al., 2002; Ivanova et al., 2002b; Singh, 2001; Zhang et al., 2001), GSK3 may be involved in the neuroprotective effects of the hormone. As a first step to test this hypothesis, we have assessed in this study the effect of estradiol on the activity of GSK3 and on the activity of molecules located downstream of GSK3 signaling, such as Tau, in the hippocampus. The data presented here indicate that estradiol regulates the activity of GSK3 and the phosphorylation of Tau. Furthermore, we show that h-catenin is associated with estrogen receptor a and with GSK3h and that this association is regulated by estradiol. Finally, we have found that estradiol increases the association of Tau with h-catenin and the p85 subunit of the phosphatidylinositol 3-kinase and increases the association of Tau with phosphorylated GSK3h. These findings indicate that

GSK3 is involved in estradiol signaling in the brain, suggesting that GSK3 may mediate some neuroprotective and plastic effects of the hormone.

Results Estradiol transiently regulates the phosphorylation of GSK3 and Tau in the rat hippocampus The effect of estradiol on the phosphorylation of GSK3 and Tau was assessed in the hippocampus of adult ovariectomized rats in vivo. One hour after hormonal administration, there was a notable increase in phosphorylation of GSK3 in serine (Fig. 1A). By 6 h, the phosphorylation of serine had almost reached the control level. In parallel, the phosphorylation on tyrosine 216 increased. One hour after estradiol treatment, GSK3h-PTyr move up to 150%, then 6 h after treatment, the phosphorylation level decreased to75% of the control level (Fig. 1A). To demonstrate whether changes in the phosphorylation of GSK3 were associated to changes in the activity of the molecule,

Fig. 1. Estradiol modulates the phosphorylation of GSK3 in the hippocampus and its kinase activity. (A) Estradiol was administered to ovariectomized rats and after different time periods, the hippocampus was dissected and soluble cell extracts obtained. Western blots from soluble hippocampal extracts, obtained 30 min, 1 or 6 h (0.5, 1 and 6 h) after estradiol injection were incubated with anti-phospho Ser9/21-GSK3, or with anti-phospho Tyr216-GSK3, C represents the cell extract from control ovariectomized rats treated with vehicle. Data from GSK3-Pser and total GSK3 h were normalized with respect to neuron-specific hIIItubulin (loading control), present in each cell extract. Data from controls were considered as 1 or 100 relative units (RU). Data are means F SEM. Asterisk (*) indicates statistical significance ( P < 0.05) versus control value. (B) GSK3h was immunoprecipitated from soluble hippocampal extracts, obtained 1 or 6 h (1 h and 6 h) after hormonal treatment, or from control ovariectomized rats treated with vehicle (C). 40 Ag of each sample was immunoprecipitated (IP) with antiserum against GSK3. The final immunoprecipitate was resuspended in kinase buffer and the specific activity was determined using the GS1 peptide. The kinase activity was normalized with respect the total amount of GSK3 presented in each sample. Data from three different immunoprecipitates are represented.

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we directly measured the kinase activity from hippocampal soluble extracts from female ovariectomized rats treated with estradiol. GSK3h was immunoprecipitated and the specific activity was determined, as indicated in Experimental methods. One hour after estradiol administration, GSK3 activity showed almost two-fold increase, compared to control levels. Kinase activity decreased to basal levels, 6 h after the administration of the hormone (Fig. 1B). To determine whether the differences on kinase activity were relevant for some GSK3 substrates, we focused our analysis on Tau. Tau phosphorylation was also modulated in the hippocampus after estradiol administration to ovariectomized rats (Fig. 2). The phosphorylation of PHF-1 epitope transiently increased 30 min after treatment. The increase was small and did not reach significance. Then, phosphorylation decreased 1 h after hormonal treatment. The level of PHF1-Tau phosphorylation returned to control conditions 6 h after hormone administration. In addition, a significant increase in Tau-1 immunoreactivity was detected 1 h after the administration of estradiol. Six hours after hormone treatment, Tau-1 immunoreactivity returned to control levels. The changes detected in Tau appeared to be specific, since estradiol did not affect the phosphorylation of another GSK3 substrate, MAP2C, as assessed by an antiserum that recognizes phospho-epitope 305, an epitope sensitive to GSK3 (data not shown). Estradiol transiently regulates the phosphorylation of GSK3 and Tau in primary hippocampal neurons Considering that GSK3 is present in both neurons and glia, it is important to evaluate whether the changes detected in GSK3 phosphorylation are due to the direct action of estrogen on hippocampal neurons or may be a glia-mediated action. Thus, we decided to analyze the effect of estrogen on cultured hippocampal neurons.

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Estradiol treatment increased the level of phosphorylation in serine of GSK3-a and -h. The increase of phosphorylation was dose-dependent, being observed at concentrations of the hormone ranging from 50 to 200 nM (Fig. 3A). These changes were also evaluated analyzing the phosphorylation level of tau. Our data showed a clear increase in the phosphorylation of the PHF-1 epitope, 1 h after hormonal treatment, at concentrations of estradiol ranging from 100 to 500 nM (Fig. 3A). The phosphorylation of the PHF-1 epitope was transient, reaching a maximum 30 min after estradiol addition to the cultures (Fig. 3B). The analysis of Tau-1 phospho-epitope demonstrated the lowest level of dephosphorylation 1 h after hormone addition (Fig. 3B). b-catenin is modified by estradiol treatment The data obtained support the hypothesis that GSK3 activity is modified and that its substrate Tau is differentially phosphorylated after estradiol treatment. In a consecutive experiment, we assessed whether the modification in GSK3 activity affected hcatenin, since this molecule is a specific substrate of GSK3h (Cadigan and Nusse, 1997; Kim and Kimmel, 2000). As shown in Fig. 4, estradiol treatment caused a modest increase in the total amount of h-catenin. This change was detected between 30 min and 1 h after the administration of the hormone. Levels of hcatenin reached control values 6 h after the hormonal treatment (Fig. 4A). To evaluate whether the change on h-catenin was detectable in a specific subcellular compartment, cellular fractions were prepared from the hippocampus, as indicated in Experimental methods. Soluble, membrane, and nuclear fractions were analyzed by Western blot using anti h-catenin antiserum. Our data showed a moderate increase of h-catenin content in the soluble fractions, 1 h after hormone treatment. In addition, h-catenin showed a slight increase in the membrane fraction, 6 h after hormone treatment.

Fig. 2. Estradiol modifies the phosphorylation of Tau in the hippocampus. (A) Samples from the hippocampus of estradiol-treated rats, killed 30 min, 1 h or 6 h after hormonal administration (0.5, 1 and 6 h), were incubated with antibodies against Tau. C represents the cell extract from ovariectomized controls. Antibodies used were phosphorylation-dependent (PHF-1 or Tau 1) or phosphorylation-independent (against total Tau). The data from each time-point were normalized with respect to the neuron-specific hIII-tubulin (loading control) present in each cell extract. Diagrams show the mean normalized densitometry values and the corresponding SEMs, for each phosphorylation-dependent antibodies. In addition, the ratio of total Tau/tubulin is represented. The value from control rats was taken as 1 RU or 100 RU, in the case of Tau versus Tubulin. Asterisk (*) indicates statistical significance ( P < 0.05) versus control value.

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As shown in Fig. 5, GSK3 co-immunoprecipitated with ERa. However, the interaction of GSK3 and ERa was not significantly affected by estradiol treatment (Fig. 5). Nevertheless, an increased phosphorylation, in serine 9, of GSK3 molecules associated with ERa, was observed 1 h after estradiol administration (Fig. 5). This effect was preceded by decreased phosphorylation in tyrosine 216, 30 min after the administration of the hormone. In addition, an interaction between ERa and h-catenin was also detected (Fig. 5). h-catenin was dissociated from ERa 30 min after estradiol treatment (Fig. 5). The interaction of Tau with GSK3 is modified by estradiol

Fig. 3. Estradiol modulates the phosphorylation of GSK3 and modifies the phosphorylation of Tau, in primary hippocampal neurons. (A) Hippocampal neurons were cultured in defined medium for 48 h. Then, estradiol was supplemented to the culture medium at different concentrations. One hour after the addition of the hormone, cell extracts were obtained and Western blots were analyzed using different antibodies against GSK3, anti PHF1Tau or against the neuron-specific hIII-tubulin (loading control). The figure shows that 200 nM estradiol induced the highest level of phosphorylation of GSK3 in serine. The highest levels of phosphorylated PHF1-Tau were obtained with hormonal concentrations ranging from 200 to 500 nM. (B) Hippocampal neurons were cultured in defined medium for 48 h. Then, estradiol (200 nM) was supplemented to the culture medium. At different times (30 min, 1 or 6 h) after the addition of the hormone, cell extracts were obtained and the Western blots analyzed using different antibodies against Tau-1 or anti PHF1-Tau or against the neuron-specific hIII-tubulin (loading control). The figure shows that booth phosphoepitopes have a transient modification on phosphorylation levels after estradiol treatment.

However, the amount of h-catenin associated with the nuclear fraction was unaffected by estradiol (Fig. 4B). Estrogen receptor a interacts with GSK3 and b-catenin in the brain The rapid change in the phosphorylation pattern of GSK3 may support the hypothesis that ER and GSK3 may be part of a multimolecular complex. To assess whether estrogen receptor a (ERa) interacts with GSK3 in vivo, ovariectomized rats were treated with estradiol or vehicle. ERa was then immunoprecipitated from hippocampal homogenates. Western blots from these immunoprecipitates were further analyzed using antibodies against GSK3 PSer9, GSK3 Ptyr216/279, h-catenin, and ER as a control.

It has been recently described that the amino-terminal region of Tau may bind GSK3 (Sun et al., 2002). To determine whether the modification of GSK3 activity is associated to a modification in the population of Tau bound to GSK3, immunoprecipitation experiments were performed using an antiserum that recognizes Tau independently of its state of phosphorylation. Hippocampal extracts from ovariectomized rats treated with estradiol or with vehicle were immunoprecipitated and the immunocomplexes analyzed by Western blot with antibodies against h-catenin, PI3K (p85), GSK 3h, and GSK3h P-Ser, as well as Tau as a control. In control rats, Tau was associated with a fraction of GSK3, hcatenin, as well as with the regulatory subunit of PI3K (p85). Estradiol treatment increased the amount of h-catenin and P85 associated to Tau (Fig. 6). Whereas total GSK3h diminished in the complex; the hormone induced an increase in the association of Tau with the fraction of GSK3-h phosphorylated on serine (Fig. 6).

Discussion Our findings indicate that estradiol regulates GSK3 activity in the adult rat hippocampus in vivo. GSK3 activity is negatively regulated by phosphorylation of serines and positively regulated by phosphorylation of tyrosines (Bhat et al., 2000; Cohen and Frame, 2001; Cross et al., 1995). Estradiol administration to ovariectomized rats resulted in a transient increase in the phosphorylation of GSK3, in serine 9 in the alpha isoform and in serine 21 in the beta isoform, and a transient decrease in the phosphorylation in tyrosine 276/216 (a/h) in the hippocampus. We demonstrated that this transient activation may be reproduced by direct action of estradiol on cultured hippocampal neurons, suggesting that glial cells are not necessary for this hormonal effect. The direct measure of the GSK3 kinase activity demonstrated an initial increase, in clear correlation with the phospho-tyrosine isoforms, followed by a decrease below the basal level, 6 h after the treatment. Similar transient activation has been reported after IGF-1 treatment (Lesort et al., 1999).This activation may serve to rearrange the cytoskeleton or interaction between cytoskeleton and interaction (Maas et al., 2000), mainly increasing MT dynamics, which would be essential for growth of neurites, or for neuronal plasticity (Cardona-Gomez et al., 2000). Different pathways may control GSK3 activity. Of these, the Insulin/IGF-I pathway has been deeply studied. Estradiol and IGF-I interact in the brain to regulate many neural functions (Cardona-Gomez et al., 2002; Duenas et al., 1996; Garcia-Segura

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Fig. 4. Estradiol modifies the amount of h-catenin in the hippocampus. (A) Western blots from soluble hippocampal extracts from ovariectomized rats killed 30 min, 1 h or 6 h after the administration of estradiol. Extracts were incubated with antibodies against total h-catenin or against the neuron-specific hIII-tubulin. Each time-point was normalized with respect to the neuron-specific tubulin (loading control) present in each cell extract, and the control taken as 100 relative units (RU). Data shown are the mean normalized densitometry values and the corresponding SEMs. Asterisk (*) indicates statistical significance ( P < 0.05) versus control value. (B) Soluble (Sol), membrane (Mn.) and nuclear fractions (Nucl.) were obtained from hippocampus from ovariectomized rats killed 1 or 6 h after the administration of estradiol, or untreated (C). Identical amounts of protein were analyzed by Western blots from each fraction, using an antiserum against h-catenin. The data show an evident increase of h-catenin immunoreactivity, 1 h after hormone administration in the soluble fraction (Sol.), whereas no change was detected in the nuclear fraction (Nucl.).

et al., 2000; Quesada and Etgen, 2001, 2002) and to promote neuroprotection (Azcoitia et al., 1999). Indeed, it has been shown that ER may bind in the brain to PI3K, a well-known effector of IGF-I receptor (Mendez et al., in press). In addition, in neural cultures and in the brain in vivo, estrogen activates Akt (CardonaGomez et al., 2002; Ivanova et al., 2002a,b; Zhang et al., 2001), a kinase downstream of PI3K. In turn, Akt controls GSK3 activity (Alessi and Cohen, 1998; Alessi et al., 1996; Rylatt et al., 1980) by phosphorylation on serines 9 and 21 (Cohen and Frame, 2001; Scheid and Woodgett, 2001). We may therefore postulate that the Akt pathway may be responsible for the GSK3 phosphorylation on serine 9 by estradiol. However, the kinase responsible for the tyrosine phosphorylation remains to be determined (Lesort et al., 1999). To explore the implications of the regulation of GSK3 by estradiol, we focused our analysis on some downstream elements that have been reported to be associated with GSK3 activity. Downstream of GSK3 are microtubule-associated proteins, such

as Tau, MAP2, and MAP1B (Goold et al., 1999; Sanchez et al., 2000). Indeed, GSK3 is also known as Tau protein kinase (Ishiguro et al., 1993) and is one of the best candidates for generating the hyperphosphorylated Tau that is characteristic of PHFs in Alzheimer’s disease (Lovestone et al., 1994). Administration of estradiol resulted in a decreased phosphorylation of Tau, as revealed by the increase of Tau 1 phospho-epitope and the final decrease of PHF-1 phospho-epitope. Similar results were obtained in vivo and in vitro where the final amount of Tau-1 phosphoepitope was higher than the control level. The time course of the hormonal induction of Tau dephosphorylation is compatible with an effect mediated via GSK3 plus an additional, still unidentified, phosphatase activity. Another interesting observation is that some potential GSK3 phospho-epitopes are not affected. For instance, the phosphoepitope 305-MAP2c (Sanchez et al., 1996) was not affected in our experiments. This may be explained taking into account that two different GSK3 substrates have been identified: primed,

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Fig. 5. The association between GSK3, h-catenin and ERa was modified by estradiol. Soluble hippocampal extracts from ovariectomized rats, killed 30 min or 1 h after estradiol administration, were immunoprecipitated with an antiserum against estrogen receptor a (ERa). Western blots from the immunoprecipitates were analyzed with antibodies against h-catenin, GSK3 h, GSK3h-PSer, GSK3-P Tyr, or ERa as an immunoprecipitation internal control. The histograms show the densitometric values (mean F SEM) for h-catenin, total GSK3-h and GSK3-Pser, normalized to the ERa value of each sample. In all cases, the control was taken as 100 relative units. Asterisk (*) indicates statistical significance ( P < 0.05) versus control value.

previously phosphorylated by another kinase at a site that is four amino acids C-terminal to the target residue, and unprimed (Cho and Johnson, 2003). The ability of GSK3 to phosphorylate several substrates in Tau, together with a differential accessibility to phosphatases, may result in the generation of different patterns of Tau phosphorylation that may have distinct functional implications. Sustained activation of GSK3 may result in neurodegenerative damage (Bhat et al., 2000; Pap and Cooper, 1998) while the dephosphorylation of Tau, the transient phosphorylation of GSK3 (Lesort et al., 1999) and the long-term decrease of GSK3 activity, all of them observed in our study, have been associated with the promotion of neuronal survival by several signaling pathways (Welsh and Proud, 1993). Therefore, the observed effects of estradiol on GSK3 may be part of the neuroprotective mechanisms of the hormone. In addition, by regulating the phosphorylation of GSK3 and cytoskeletal proteins, such as Tau, estradiol may affect the morphology and plasticity of neuronal processes and synaptic contacts (Garcia-Segura et al., 1994; McEwen, 2002; Woolley and McEwen, 1992). In addition to being involved in the phosphorylation of microtubule-associated proteins, GSK3h kinase activity controls the stability of h-catenin, a key protein in the Wnt/wingless signaling pathway (Cadigan and Nusse, 1997; Dominguez and Green, 2001; Miller and Moon, 1996). The inhibition of GSK3h activity by the Wnt ligand, permits the nuclear translocation and transcriptional activity of h-catenin, whereas the activation of GSK3 may redirect h-catenin to the proteasome pathway (Barth et al., 1997). Previous studies have suggested that h-catenin may

be involved in transcriptional regulation by steroid hormone receptors. h-catenin interacts with the androgen receptor in prostate cancer cells (Truica et al., 2000; Yang et al., 2002) and in a gonadotropin-releasing hormone neuronal cell line (Pawlowski et al., 2002). In the neuronal cell line, activated androgen receptor shuttles h-catenin to the nucleus (Pawlowski et al., 2002) and in both prostate cells and neurons, the interaction of h-catenin with androgen receptor modulates transcriptional activity (Pawlowski et al., 2002; Truica et al., 2000; Yang et al., 2002). In pituitary GH3 cells, estradiol decreases, while the ER antagonist ICI 182,780 increases h-catenin expression (Heinrich et al., 1999; Smith et al., 1999). Our data indicates that, in the hippocampus, estradiol affects the association of h-catenin to soluble and membrane fractions and does not affect the association to the nuclear fraction. This finding suggests that estradiol does not have an effect on transcriptional regulation by h-catenin in the hippocampus at the times analyzed. Our findings indicate that h-catenin is associated, in a multimolecular complex, with ERa and GSK3h in the brain. Estradiol does not affect the association of ERa´ and GSK3h. However, the amount of GSK3h phosphorylated in serine is increased by the hormone. Nevertheless, estradiol decreases the association of ERa´ with h-catenin. This suggests that the hormonal regulation of GSK3h kinase activity results in the dissociation of h-catenin from the molecular complex with GSK3h, allowing its interaction with other partners or its relocation to the proteasome pathway (Barth et al., 1997; Miller and Moon, 1996). However, this latter alternative may be excluded, considering that the total amount of h-catenin is increased within 1 h after estradiol treatment (see Fig. 7).

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Fig. 6. The association between Tau, GSK3 and h-catenin is modified by estradiol. Hippocampal extracts from ovariectomized rats, killed 30 min or 1 h after estradiol administration, were immunoprecipitated with an antiserum against total Tau. Western blots from the immunoprecipitates were analyzed with antibodies against h-catenin, GSK3 h, GSK3h-PSer, PI3K-regulatory subunit (p85), or total Tau as an immunoprecipitation internal control. The histograms show the mean normalized densitometric values and the corresponding SEMs for h-catenin, total GSK3-h, GSK3-Pser, and p85 with respect to the total Tau value in each sample. In all cases the control was taken as 100 relative units. Asterisk (*) indicates statistical significance ( P < 0.05) versus control value.

A second important observation is that Tau forms a multimolecular complex with GSK3h in the brain in vivo. In addition, the regulatory subunit of the PI3K, p85, and h-catenin also form part of this complex. It is important to note that estradiol treatment increased the association of Tau with aˆ-catenin, p85, and GSK3aˆPSer. All these findings suggest that estradiol regulates Tau phosphorylation and this, in turn, modulates the interaction of p85, GSK3h, and h-catenin with Tau (see Fig. 7). We have to remember that the interaction of GSK3 with Tau depends on Tau phosphorylation level (Sun et al., 2002) and that the interaction of Tau with membranes follow similar rules (Maas et al., 2000). Thus, we may speculate that this second pool of GSK3-Tau-h-catenin (Sun et al., 2002), that is increased by estrogen treatment, may be in the proximity of cellular membranes or membrane receptors, such as the IGF-I receptor. Indeed, the increase of total h-catenin, 1 h after the hormonal treatment, correlates with the increase of this protein in the soluble fraction. The estrogen-regulated interaction of ERa with GSK3h and hcatenin represents a new and unexplored signaling mechanism of ERs in the brain. Acting through GSK3h, h-catenin, and Tau, ERa may be involved in the regulation of neuronal cytoskeleton, neuritic growth, synapse formation, and the promotion of neuronal survival. Therefore, the interaction of estradiol signaling with GSK3h and h-catenin may explain the well-characterized neuroprotective effects of estradiol, as well as the ability of the hormone to induce neuritic growth and axonal regeneration, and to prevent or delay the development of Alzheimer’s disease (Garcia-Segura et al., 2001; Lee and McEwen, 2001; McEwen, 2002; Wise et al., 2001).

In conclusion, neuroprotective mechanisms of estrogen may include short-term effects, associated to the regulation of kinases such as GSK3, which may modify the cytoskeleton and membraneassociated cytoskeleton, and long-term effects, associated to modifications in the transcription of neuroprotective genes.

Experimental methods Animals and experimental treatments Wistar albino female rats from our in-house colony were kept in a 12:12 h light/dark cycle and received food and water ad libitum. Animals were handled following the European Union (86/609/EEC) guidelines and special care was taken to minimize animal suffering and to reduce the number of animals used to a minimum. One-month-old animals were bilaterally ovariectomized under tribromoethanol anesthesia. One month after ovariectomy, animals were subcutaneously injected with 17h-estradiol (300 Ag, Sigma, St. Louis, MO) or sesame oil vehicle, and killed 30 min, or 1, 6, 12, or 24 h later. This dose of estradiol results in high physiological proestrus concentration of E2 in plasma by 24 h, followed by a decrease to low levels by 48 h (Shull and Gorski, 1989). It was selected since previous studies have shown that this is the optimal dose in vivo for the protection of hippocampal neurons against neurotoxicity and for the activation of growth factor signaling cascades in the brain (Azcoitia et al., 1999; Cardona-Gomez et al., 2002; Picazo et al., in press).

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Neurobasal-B27 medium (GIBCO), supplemented with 1 mM glutamine plus antibiotics (Pen./Strept). After 30 h, 17h-estradiol (1  10 7 to 5  10 7 M) or vehicle was added to the cultures. Cell extracts were obtained at different times (30 min, 1 or 6 h) after the hormonal treatment. Western blotting and antibodies

Fig. 7. Possible signaling pathways triggered by estradiol, according to the findings of this study. Estrogen modifies the kinase activity of GSK-3 in the hippocampus. Among the main targets of GSK-3 in neurons are the microtubule-associated protein Tau and h-catenin. From the present data two different macromolecular complexes, regulated by estradiol, may be postulated. (A) In the first complex, Tau, GSK3, h-catenin and the PI3K regulatory subunit (p85) are associated. Estradiol increases the amount of Tau (dephosphorylated), h-catenin and the fraction of GSK3 phosphorylated in serine (inhibited GSK3). And a final localization near to cytoplasmic membrane may be suggested. (B) In the second complex, ERa is associated with h-catenin and GSK3. Estradiol increases the level of GSK3-PSer in the complex and induces the detachment of h-catenin, from this complex. Part of the physiological and neuroprotective actions of estrogens may be due to changes in Tau phosphorylation by GSK-3/ phosphatases, which may result in microtubule rearrangements (cytoskeletal and synaptic remodeling). A second set of actions may be due to the transcriptional activity (synthesis of pro-survival genes and or inhibition of pro-apoptotic genes).

Preparation of cellular fractions Hippocampus from ovariectomized rats, killed 1 or 6 h after estradiol or vehicle treatments, were homogenized in 100 mM Tris, pH 7.4, 3 mM MgCl2, 0.32 M sucrose, and 0.1% Triton X100 (Buffer A), containing 100 AM phenylmethylsulfonyl fluoride, 1 Ag/ml aprotinin and leupeptin (Sigma) and 100 AM vanadate. Soluble fraction was obtained after centrifugation at 2500 rpm for 15 min at 4jC. The pellet was resuspended in Buffer A and located on the top of a sucrose cushion, composed by two layers. The first one was Buffer A containing 1.9 M sucrose and the second, Buffer A containing 2 M sucrose. The sample was then centrifuged for 1 h at 10.000 rpm, at 4jC. Membrane fraction was in the top of the 2 M sucrose layer whereas the nuclear fraction was in the bottom. Primary hippocampal neurons Hippocampal neurons were obtained from E17-18 mouse embryos as previously described (Sanchez et al., 1996, 2000). Briefly, trypsin-dissociated cells were plated on poly-L-lysine (50 Ag/ml) plus laminin (10 Ag/ml) coated plastic dishes or slides (1  105 cells/P24 well or 2  106 cells/P60 dish) and cultured in

Animals were killed by decapitation and their brains quickly removed. The hippocampus was dissected out and homogenized with lysis buffer containing 150 mM NaCl, 20 mM Tris, pH 7.4, 10% glycerol, 1 mM EDTA, 1% NP40, 100 AM phenylmethylsulfonyl fluoride, 1 Ag/ml aprotinin and leupeptin (Sigma) and 100 AM vanadate. Cultured hippocampal neurons were washed with PBS and resuspended in a buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 100 mM NaF, 1 mM sodium ortho-vanadate, 5 mM EDTA and protease inhibitors cocktail (COMPLETE TM), and homogenized with a Polytron. Soluble fraction was obtained by centrifugation at 13,000  g for 10 min at 4jC. Sodium dodecylsulphate – polyacrylamide gel electrophoresis (SDS-PAGE) (6, 10 or 12%) was carried out using a mini-protein system (Bio-Rad) with high- or low-range molecular weight standards (Bio-Rad). Proteins (30 – 100 Ag) were loaded in each lane with loading buffer containing 0.375 M Tris, pH 6.8, 50% glycerol, 10% SDS, 0.5 M DTT and 0.002% bromophenol blue. Samples were heated at 100jC for 3 min before gel loading. After electrophoresis, proteins were transferred to nitrocellulose membranes (Amersham) for 1 to 2 h, at 250 mA by using an electrophoretic transfer system (mini Trans-blot Electrophoretic Transfer Cell). The membranes were then washed for 30 min with TTBS (Tris – HCl 20 mM pH 7.5, NaCl 500 mM, 0.05% Tween-20, pH 7.4) and 5% bovine serum albumin. TTBS was used for all subsequent incubations. After washing in TTBS, the membranes were incubated overnight at 4jC with the primary antibodies. The following antibodies were used: anti-GSK3h (mouse monoclonal, Transduction Laboratories, diluted 1:2500, ref. no. G22320); rabbit polyclonal antibodies against GSK-3a/hPTyr(279216) (ref. no. 446004) and against GSK-3a/h-PSer(21/9) (ref. no. 44600) were both purchased from Biosource and used diluted 1:1000; anti-Tau-1 (mouse monoclonal, Chemicon International, 1:1000, ref. no. MAB2420), anti-h III-tubulin (mouse monoclonal, Promega, ref. no. G712A), and anti-PHF-1 (mouse monoclonal, kindly supplied by Dr. P. Davies, Albert Einstein College of Medicine, Bronx, NY, USA, diluted 1:20), anti-betacatenin (mouse monoclonal, Transduction Labs, diluted 1:800, ref. no. 61015 3), anti-estrogen receptor alpha (ERa) (rabbit polyclonal, Santa Cruz Biotechnology; diluted, 1:1000, ref. no. sc542), and rabbit polyclonal antisera 305 that recognizes an epitope dependent on phosphorylation of threonines 1620 and 1623 of MAP-2 (Sanchez et al., 2000). After washing in TTBS, the membranes were incubated for 1 h at room temperature with the secondary antibodies (anti-rabbit IgG peroxidase conjugated or anti-mouse IgG peroxidase conjugated; Jackson ImmunoResearch Laboratories Inc.; diluted 1:20,000). The membranes were then washed again with TTBS and developed with the chemiluminescence ELC Western blotting system (Amersham), followed by apposition of the membranes to autoradiographic films (Hyperfilm ECL, Amersham). Autoradiographic films were analyzed using a densitometer (Biorad, model 65800, software version 9.3.0). The results from each membrane

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were normalized to control values (rats treated with vehicles). To minimize inter-assay variation, samples from all experimental groups were processed in parallel. Immunoprecipitation experiments Samples from rat hippocampus were extracted in lysis buffer containing 150 mM NaCl, 20 mM Tris, pH 7.4, 10% glycerol, 1 mM EDTA, 1% NP40, 100 AM phenylmethylsulfonyl fluoride, 1 Ag/ml aprotinin and leupeptin (Sigma), and 100 AM vanadate. The extracts were cleared by centrifugation at 14,000 rpm for 2 min. Protein assay was carried out and 1 mg of extract was incubated overnight at 4jC in the presence of either an anti-estrogen receptor alpha (ERa) rabbit polyclonal antiserum (Santa Cruz Biotechnology; 1:250, ref. no. sc542) or a polyclonal antiserum against Tau (Santa Cruz Biotechnology; 1:250, ref. no. SC1995). Protein A-Sepharose beads were added and the incubations were prolonged for a further 2 h. The immune complexes were washed three times with immunoprecipitation lysis buffer before being analyzed by SDS-PAGE and immunoblotting. Proteins were separated using 10% SDS-PAGE, transferred to nitrocellulose membranes (Amersham), and probed with anti-GSK3a/h (Biosource, ref. no. 44610), anti-p Ser9/21GSK3 (Biosource, ref. no. 44600), anti-pTyr216/279GSK3 (Biosource, ref. no. 44604), anti-h-catenin, and anti-PI3K regulatory subunit (p85) (Trejo and Pons, 2001) antibodies. Antibodies against ER or Tau were used as an internal control, and lysates were used as positive controls. Statistical analysis Since the F test revealed significant homogeneous variance between groups, a one-way analysis of variance (ANOVA) test was appropriate for multiple statistical comparisons. Tukey’s post hoc test was used to determine significant differences between independent groups. A probability of P < 0.05 in a two-tailed test was adopted as the threshold of statistical significance. At least five rats were analyzed for each experimental group. The sample size, n, referred to in the statistical analysis was the number of animals. Data are expressed as mean F SEM, normalized with respect to a loading control.

Acknowledgments We thank Dr. J. Dı´az-Nido and Dr J.J. Lucas for helpful comments. This research was supported by grants from Spanish CICYT, MCYT (SAF 2002-00652) and the Commission of the European Communities, specific RTD program Quality of Life and Management of Living Resources, QLK6-CT-2000-00179 and by an institutional grant from Ramon Areces Foundation. Project 1115-05-11095 by COLCIENCIAS at University of Antioquia (to G.P.C-G.). G.P.C-G. is a postdoctoral fellow from Fundacio´n Carolina, AECI, Spain.

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