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Originally published In Press as doi:10.1074/jbc.M411936200 on March 1, 2005 Originally published In Press as doi:10.1074/jbc.M411936200 on January 19, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11615-11625, March 25, 2005
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Trolox and 17{beta}-Estradiol Protect against Amyloid {beta}-Peptide Neurotoxicity by a Mechanism That Involves Modulation of the Wnt Signaling Pathway*

Rodrigo A. Quintanilla{ddagger}§, Francisco J. Muñoz§, Maria J. Metcalfe{ddagger}, Maureen Hitschfeld{ddagger}, Gonzalo Olivares{ddagger}, Juan A. Godoy{ddagger}, and Nibaldo C. Inestrosa{ddagger}||

From the {ddagger}Centro de Regulación Celular y Patología "Joaquín V. Luco," Millennium Institute of Fundamental and Applied Biology, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile and Unitat de Senyalització Cellular, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, 08003 Barcelona, Spain

Received for publication, October 20, 2004 , and in revised form, January 18, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stress is a key mechanism in amyloid {beta}-peptide (A{beta})-mediated neurotoxicity; therefore, the protective roles of 17{beta}-estradiol (E2) and antioxidants (Trolox and vitamin C) were assayed on hippocampal neurons. Our results show the following: 1) E2 and Trolox attenuated the neurotoxicity mediated by A{beta} and H2O2 as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assays, quantification of apoptotic cells, and morphological studies of the integrity of the neurite network. 2) Vitamin C failed to protect neurons from A{beta} toxicity. 3) A{beta}-mediated endoperoxide production, reported to induce cell damage, was decreased in the presence of E2 and Trolox. 4) Two key Wnt signaling components were affected by E2 and Trolox; in fact, the enzyme glycogen synthase kinase 3{beta} was inhibited by both E2 and Trolox, and both compounds were able to stabilize cytoplasmic {beta}-catenin. 5) E2 activated the expression of the Wnt-5a and Wnt-7a ligands, and at the same time, E2, through the {alpha}-estrogen receptor, was able to prevent the excitotoxic A{beta}-induced rise in bulk-free Ca2+ as an alternative pathway to increase cell viability. 6) Finally, the Wnt-7a ligand protected against cytoplasmic calcium disturbances induced by A{beta} treatment. Our results suggest that control of oxidative stress, regulation of cytoplasmic calcium, and activation of Wnt signaling may prevent A{beta} neurotoxicity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer disease (AD)1 is a neurodegenerative disease characterized by neuronal cell death, dystrophic neurites, neurofibrillary tangles, and senile plaques (1). Senile plaques are composed by the amyloid {beta}-peptide (A{beta}), a 40–42-amino acid peptide that originates from the proteolytic cleavage of the amyloid precursor protein (2). There is also evidence relating the etiopathology of AD with oxidative stress induced by A{beta} in the brain of AD patients (3–6). A{beta} increases the production of intraneuronal reactive oxygen species (ROS) and stimulates hydrogen peroxide (H2O2) levels through metal ion reduction (7, 8). Free radicals peroxidize membrane lipids (9) and oxidize proteins (10). In vitro experiments also support the observation that the neurotoxic effect of A{beta} is mediated by free radical mechanisms (5, 11, 12) and alteration of Ca2+ homeostasis (13). Furthermore, several studies have reported neuroprotection by antioxidants against A{beta}-mediated cytotoxicity (14–16). Also, 17{beta}-estradiol (E2; estrogen) treatment apparently has beneficial effects on AD (17, 18). In addition, E2 prevents A{beta}-induced cell death by activation of the {alpha}-ER (19) and preserves neuronal viability and function in cortical neurons exposed to glutamate toxicity (20). Also, there is evidence that E2 prevents morphological neurodegenerative changes in hippocampal neurons caused by A{beta} deposits (21).

On the other hand, neurofibrillary tangles are intracellular aggregates of paired helical filaments produced by hyperphosphorylation of the microtubule-associated protein tau (23). It has been proposed that glycogen synthase kinase-3{beta} (GSK-3{beta}) is a key enzyme in the hyperphosphorylation of tau protein (24). Increased paired helical filaments were observed in transgenic mice overexpressing GSK-3{beta} (25). In addition, GSK-3{beta} accumulates in the cytoplasm of pre-tangle neurons following the pathological progression of the disease (26). In cortical and hippocampal neurons challenged with A{beta}, there is an activation of GSK-3{beta} (24, 27) and tau hyperphosphorylation (23, 28) correlating with cell death (24, 28). Besides, GSK-3{beta} is involved in the signaling of insulin and insulin-like growth factor I (29) and also in wingless/Wnt signaling (30). Wnt signaling is essential for development, synaptogenesis, and oncogenic processes (31–35), and it has even been proposed to play a role in neurodegenerative disorders such as schizophrenia (36, 37) and AD (38, 39). Wnt proteins inhibit GSK-3{beta}, disrupting a multiprotein complex comprising GSK-3{beta} and its substrates in the Wnt signaling pathway, which, unlike other growth factors, do not require a priming phosphate (29). Wnt signaling also inactivates GSK-3{beta} via activation of the protein kinase C (PKC) enzyme (40, 41), which is decreased in the cortex of AD patients (42); besides, PKC activation is an important step in the estrogen protection mechanism against A{beta} neurotoxicity (43). In the absence of a Wnt ligand, GSK-3{beta} phosphorylates {beta}-catenin for ubiquitin-proteasome mediated degradation (44). Decreased {beta}-catenin-mediated transcription has been proposed to increase apoptosis in neurons challenged with A{beta} (45). Therefore, a loss of Wnt signaling function might play a role during {beta}-amyloid neurotoxicity (39, 41, 46, 47), and key Wnt signaling components are affected in AD (35).

In the present work, we have studied the neuroprotective properties of vitamin C, E2, and Trolox (a hydrosoluble analogue of vitamin E) on rat hippocampal neurons challenged with A{beta} and H2O2. Incubation with E2 and Trolox reduces the neuronal cell death induced by A{beta} treatment, as evaluated by the MTT assay and the TUNEL assay for apoptosis. Both Trolox and E2 decreased intraneuronal ROS levels. We also found that E2 and Trolox can modulate the Wnt signaling pathway. In fact, both treatments decreased the level of the GSK-3{beta} activity in neurons exposed to A{beta}. Moreover, such treatment increased {beta}-catenin levels and regulated the expression of Wnt ligand genes. Moreover, treatment of hippocampal neurons with E2 prevented the A{beta}-induced rise in the cytoplasmic calcium levels. Similar results were observed in the presence of conditioned medium enriched with the Wnt-7a ligand. Taken together, these results established that E2 and Trolox protect neurons exposed to A{beta}. The mechanism involved is apparently related to modulation of the Wnt signaling pathway and regulation of the cytoplasmic calcium content of neuronal cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Synthetic A{beta}1–40 peptide corresponding to the human A{beta} wild-type sequence was obtained from Chiron Corp. Inc. (Emeryville, CA) and Calbiochem. Chemicals (E2, Trolox, and vitamin C), culture media, and sera were obtained from Sigma, Roche Applied Science, Merck, and Invitrogen, and Fluo-3 AM was obtained from Molecular Probes (Leiden, The Netherlands). Propyl pyrazole triol (PPT) ({alpha}-agonist of ER) and diarylpropionitrile (DPN) ({beta}-agonist of ER) were purchased from Tocris Cookson (Ellisville, MO), and ICI 182780 (antagonist of ER) was donated by Elger Entech (Jena, Germany).

Primary Rat Embryo Hippocampal Neuronal Cultures—Hippocampal neurons were isolated from 18-day-old Sprague-Dawley rat embryos. All experiments were carried out in accordance with the directives of the Council of the European Communities No. 86/609/CEE. Pregnant rats were killed by CO2 inhalation. Then, embryos were rendered hypothermic and decapitated. Hippocampi were aseptically dissected and trypsinized for 20 min. After centrifugation for 1 min, cells were seeded in phenol red-free Dulbecco's modified Eagle's medium plus 10% horse serum into 1% poly-L-lysine-coated plates. After 120 min, medium was removed, and Neurobasal medium was added containing 1% B27 supplement from Invitrogen, plus streptomycin and penicillin. On day 3 of culture, hippocampal cells were treated with 2 µM 1-{beta}-D-arabinofuranosylcytosine for 24 h to reduce the number of proliferating non-neuronal cells. Cultured hippocampal neurons were used for the experiments on day 6.

Aggregation of Amyloid {beta}-Peptide—A{beta} fibrils were obtained by dissolving freeze-dried aliquots of A{beta}1–40 in Me2SO. Peptide stock aliquots were diluted in 0.1 M Tris-HCl (pH 7.4) to a final concentration of 100 µM A{beta}. Solutions were stirred continuously (1300 rpm) at room temperature for 48 h. The quality of amyloid was verified by different criteria including turbidometry, Congo red binding, and thioflavine T spectrofluorometry (48, 49). Aliquots of the homogenate were transferred to a denaturing buffer and resolved by Tris-glycine SDS-PAGE to quantify the concentration of A{beta} contained in the fibrils. This was achieved by densitometric scanning using different A{beta} concentrations as standard.

Cell Viability Assay (MTT Reduction)—MTT assays were performed as described previously (50, 51). Hippocampal neurons (2 x 104 cells/100 µl/well) were assayed in B27- and phenol red-free medium. Neurons were pre-incubated for 2 h with 100 µM vitamin C, 10 µM E2, 100 µM Trolox, or medium (control). Then, 5 µM A{beta} or 100 µM H2O2 was added to the wells. Cells were incubated for 24 h at 37 °C, after which cell viability was measured by the MTT method. MTT reduction was determined in a Lab Systems Uniskam I spectrophotometer at 540 and 650 nm.

Immunofluorescence Studies—Hippocampal neurons were seeded onto poly-L-lysine-coated coverslips (20,000 neurons/cover) in 24-well culture plates. After 6 days in Neurobasal medium plus B27, neurons were washed and exposed to A{beta} and antioxidants. Then, neurons were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Immunostaining was done with polyclonal anti-NF-200 antibody (1:300; Sigma). Antibody detection was made using rhodamine-conjugated anti-rabbit IgG antibody (Sigma). Coverslips were mounted and analyzed under a Zeiss confocal microscope. Neuronal morphology was analyzed by confocal microscopy, and the number and length of neurites were evaluated using Image-Pro Plus software (Media Cybernetic, Silver Spring, MD).

Neuronal Apoptosis by TUNEL Assay—Programmed cell death was evaluated by the TUNEL method using the In Situ POD Cell Death Detection Kit (Roche Applied Science). Hippocampal neurons were plated on poly-L-lysine-coated coverslips (50,000 neurons/cover). After 5 days in Neurobasal medium plus B27, hippocampal neurons were washed, starved for 2 h, and treated for 6 h. Then, neurons were fixed in fresh paraformaldehyde (4%) and permeabilized with 0.1% Triton X-100, and staining was performed as indicated by the manufacturer. Samples were analyzed under a light microscope (Olympus BX51TF), and the percentage of apoptotic cells (brown staining) on different fields was determined using the Image-Pro Plus program (Media Cybernetics), the data were subdivided into 10 intervals (from 0% to 100% apoptosis), and the frequency of each interval was calculated.

Measurement of Intracellular Free Radicals—Hippocampal neurons were incubated with the fluorochrome 2,7-dichlorofluorescein diacetate at 5 µM for 2 h. Then, wells were washed three times with Hank's solution, and neurons were pretreated for 2 h with the corresponding antioxidants in phenol red-free Dulbecco's modified Eagle's medium. A{beta} fibrils (5 µM) were added to each well and incubated for 4 h. Cells were trypsinized, and fluorescence was monitored (excitation, 495 nm; emission, 520 nm) using a Shimadzu spectrofluorometer. Results are expressed as percentages relative to control neurons arbitrarily set at 100%.

GSK-3{beta} Activity—After 5 days in Neurobasal medium plus B27, hippocampal neurons were treated for 6 h. Then, neurons were washed twice with cold Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 7.4) and lysed (25 mM HEPES, 125 mM NaCl, 25 mM NaF, 30 µM Na2P2O7·10H2O, 1 mM EDTA, 1% Nonidet P-40, 1 mM NaVO3, 100 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 1 µg/ml pepstatin, and 2 µM leupeptin). To immunoprecipitate GSK-3{beta}, samples were incubated overnight with 10 µl of rabbit polyclonal IgG anti-GSK-3{beta} (Santa Cruz Biotechnology), and then 25 µl of protein A-agarose (Santa Cruz Biotechnology) were added for 4 h. Immune complexes were centrifuged (425 x g, 4 °C, 1 min) and washed six times with cold immunoprecipitation buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM NaF, 30 µM Na2P2O7·10H2O, 1 mM NaVO3, 100 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 1 µg/ml pepstatin, and 2 µM leupeptin). Samples were resolved by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Then, membranes were incubated with goat anti-phosphotyrosine polyclonal IgG p-GSK-3{beta} (Tyr-216) or goat polyclonal anti-phosphoserine p-GSK-3{beta} (Ser-9) (Santa Cruz Biotechnology). Bands were revealed by the enhanced chemiluminescence method (Santa Cruz Biotechnology) using an anti-goat horseradish peroxidase-conjugated secondary antibody. Films were scanned, and densitometry was carried out with MATRIX software (Quantavision).

The GSK-3{beta} kinase activity assay was performed in immunoprecipitation buffer with 10 µCi of [{gamma}-32P]ATP and 62.5 µM phospho-glycogen synthase peptide-2 (GS-2; Upstate Biotechnology) as substrate. After 30 min at 30 °C, the reaction was analyzed by two different methods. 1) Samples were adsorbed to p81 phosphocellulose paper (Whatman, Pleasanton, CA) and washed three times for 15 min with 0.75% orthophosphoric acid, and then radioactivity was counted in a scintillation counter. 2) The reaction was stopped with protein charge buffer, and samples were resolved by Tris-glycine 16% SDS-PAGE; the gel was dried, and GSK-3{beta} phosphorylation peptide substrate was assayed using autoradiography. Films were scanned, and densitometry was carried out with MATRIX software.

Cytoplasmic {beta}-Catenin Stabilization—After 5 days in culture, hippocampal neurons were treated for 6 h in Neurobasal medium alone. Then, neurons were washed twice with cold phosphate-buffered saline and lysed (10 µM HEPES, 1.5 µM MgCl2, 10 µM KCl, 1 µM EDTA, 1 µM dithiothreitol, 1 µM phenylmethylsulfonyl fluoride, 1 µg/µl pepstatin, 1 µg/ml aprotinin, and 10% glycerol) for 10 min at 4 °C. Samples were centrifuged (4000 x g, 4 °C, 15 min), and supernatants were kept at 4 °C to evaluate {beta}-catenin. Samples were resolved by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Then, membranes were incubated with a mouse anti-{beta}-catenin antibody (Santa Cruz Biotechnology). Bands were revealed by the enhanced chemiluminescence method (Santa Cruz Biotechnology) using an anti-goat horseradish peroxidase-conjugated secondary antibody. Films were scanned, and densitometry was carried out as indicated above.

RNA Extraction and RT-PCR—After 6 days in Neurobasal medium plus B27, hippocampal neurons were kept in Neurobasal medium for 4 h. Then, cells were lysed with TRIzol reagent according to the manufacturer's instructions. RNA concentration was calculated by the absorbance measurement at 260 and 280 nm (UV-150–02 Shimadzu spectrophotometer). In the RT-PCR assay, Moloney murine leukemia virus reverse transcriptase was used for the RT reaction. The PCR was performed with 2.5 µl of RT reaction mixture (1 µl of reaction buffer, 0.5 µl of 10 mM deoxynucleotide triphosphate, 1.5 µl of 25 mM MgCl2, 0.125 µl of primers at 100 µM, 1 µl of Taq polymerase, and 16.75 µl of diethyl pyrocarbonate-treated water). PCR conditions were as follows: for Wnt-5a, 1 min at 94 °C, 1 min at 58 °C, and 1 min at 72 °C for 30 cycles; and for Wnt-7a, 1 min at 94 °C, 1 min at 54 °C, and 1 min at 72 °C for 30 cycles. {beta}-Actin was measured as a housekeeping gene, and its PCR amplification conditions were the same as those of the gene measured. RT-PCR primers for Wnt-5a, Wnt-7a, and {beta}-actin were as follows: Wnt-5a, 5'-TGAACCTNCACAACAAYGAGGCGGG-3' (forward) and 5'-GAAGCGGCTGTTGACCTGTAC-3' (reverse); Wnt7a, 5'-TGAACCTNCACAACAAYGAGGCGGG-3' (forward) and 5'-GCTTCTTGATCTTCAGGAAGCGGCTGTTGACCTGTAC-3' (reverse); and {beta}-actin, 5'-TCTACAATGAGCTGCGTGTG-3' (forward) and 5'-TACATGGCTGGGGTGTTGAA-3' (reverse). PCR products were analyzed by electrophoresis on a 1% agarose gel poured in Tris acetic EDTA buffer. The band intensity for Wnt-5a and Wnt-7a was measured with MATRIX software, and normalized with {beta}-actin band intensity.

Calcium Imaging and Fluorescence Measurements—Hippocampal neurons grown on glass coverslips were loaded for 30 min (37 °C) with 5 µM Fluo-3 in its AM form (Fluo-3 AM) in Krebs Ringer HEPES-glucose containing 0.02% pluronic acid. Coverslips were washed three times and left in Krebs Ringer HEPES-glucose for 10 min until cell fluorescence reached a plateau. Fluorescence was imaged with a LSM Pascal Zeiss confocal microscope as described previously (52). Background was measured in parts of the field devoid of neurons and found to be not significantly different from the signal recorded in neurons depleted of dye with 100 µM digitonin. This value was subtracted from cell measurements. The fluorescence intensity variation was recorded from 20–25 neurons on average per experiment. Estimation of fluorescence intensity of Fluo-3 AM was presented as the pseudoratio ({Delta}F/F) indicated by the following equation: {Delta}F/F = (F - Fbase)/(Fbase - B), where F is the measured fluorescence intensity of the Ca2+ indicator, Fbase is the fluorescence intensity before the stimulation, and B is the background signal determined from the average of areas adjacent to the neurons (52, 53). Besides, from dye saturation, {Delta}F/F is thought to approximately reflect [Ca2+] if there is no change in dye concentration, intracellular environment, or path length (53). Data are presented as the means ± S.E. from four independent experiments with at least 20–25 neurons per experiment.

Quantification of Copper—The Cu concentration was determined by means of a graphite furnace atomic adsorption spectrophotometer (SIMMA 6100; PerkinElmer Life Sciences). Calibration was performed against a standard curve prepared using dilutions of Cu standards, and the sample values were normalized to the total protein content (54).

Statistical Analysis—Data were expressed as the means ± S.E. of the values from the number of experiments indicated in the corresponding figures. Data were evaluated statistically by using Student's t test, with p < 0.05 considered significant. Analysis of variance was used to compare apoptosis distribution between different populations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Trolox and E2 Protect against A{beta}-mediated Toxicity in Rat Hippocampal Neurons
We evaluated the neuroprotective properties of three different antioxidant molecules: Trolox (a derivative of vitamin E), E2, and vitamin C. Hippocampal neurons challenged with A{beta} were pretreated with Trolox, E2, or vitamin C (Fig. 1). After 24 h, cell viability was measured by the MTT reduction assay (Fig. 1A). As we expected, A{beta} decreased cell viability (40%). This effect was partially prevented by the presence of Trolox and E2 (p < 0.05). However, vitamin C was unable to protect hippocampal neurons. This latter result is in contrast with reports that implicated vitamin C in the protection of neurons exposed to A{beta} (55). When neurons were exposed to H2O2, results similar to those observed with A{beta} were obtained (Fig. 1B). These results are in agreement with reports that implicated ROS generation in A{beta}-induced neuronal death (4, 7, 8). In order to characterize the mechanism by which E2 and Trolox protect against neuronal cell death, we evaluated apoptosis using the TUNEL assay. Fig. 2A illustrates a plot of apoptotic neurons (percentage of decrease) treated with A{beta} in the presence of Trolox, E2, and vitamin C. Results indicate that the co-incubation of A{beta} with E2 or Trolox prevented the appearance of the apoptotic neuronal population; however, vitamin C was not able to prevent apoptosis. The effect was strong for Trolox (70%), but it was not as strong for E2 (25%). These observations are consistent with the cell survival studies presented above. On the other hand, increased oxidative stress has been shown to contribute to the neurodegenerative process in AD (4, 5). Indeed, A{beta} has been shown to induce oxidative stress in many in vivo and in vitro models of AD (56). The formation of intracellular free radicals in response to the action of A{beta} was evaluated on hippocampal neurons (Fig. 2B). Neurons treated with A{beta} showed a high production of endogenous free radicals, and this effect was inhibited when cells were pretreated with Trolox (p < 0.005) or E2 (p < 0.05). No effect was observed in neurons pretreated with 100 µM vitamin C compared with those treated with only 5 µM A{beta}. In this context, Shea and Ortiz (57) reported that E2 reduces ROS levels in A{beta}-treated neuronal cultures. Our results suggests an antioxidant effect for Trolox and E2 in A{beta}-treated hippocampal neurons.



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  		FIG. 1.
Protective effect of Trolox and E2 against A{beta}-mediated neurotoxicity. Hippocampal neurons were pretreated with 100 µM Trolox, 10 µM E2, and 100 µM vitamin C and treated with 5 µM A{beta}. Cell viability was evaluated by MTT reduction assay (A) after 24 h of incubation with A{beta}. Data are the means ± S.E. of 9–11 experiments performed in triplicate. Cell viability was also assayed after 24 h of incubation with 100 µM H2O2 (B). Data are the means ± S.E. of 6 experiments performed in triplicate. *, p < 0.05; **, p < 0.005 by non-paired Student's t test.

 



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  		FIG. 2.
Trolox and E2 prevent the apoptotic process and decrease the intracellular ROS levels in A{beta}-treated neurons. The apoptotic process and ROS levels were observed in hippocampal neurons treated with 5 µM A{beta} in the presence of Trolox and E2. A, apoptotic cells stained by TUNEL were counted after 6 h of treatment, and the apoptosis level is represented as the percentage of decrease (100% apoptosis is represented by A{beta}-treated neurons). Data are the means of two representative experiments. B, production of endoperoxides was evaluated on hippocampal neurons treated with 5 µM A{beta} in the absence or presence of 100 µM vitamin C, 100 µM Trolox, or 10 µM E2. Data are the means ± S.E. of 6 experiments performed in triplicate. E2: *, p < 0.05; **, p < 0.005 by non-paired Student's t test.

 
Trolox and E2 Maintain the Structural Integrity of Hippocampal Neurons Exposed to A{beta}
The loss of the neuronal connection and the neurite network is considered a marker of serious neuronal damage. A{beta} triggers an extensive loss of neurites in different neuronal cells, and this effect can be prevented by treatment with lithium salts (39) and Wnt-3a conditioned medium (47). Therefore, we evaluated the integrity of the neurite network in cells challenged with A{beta} in the presence of Trolox, E2, and vitamin C by labeling the neurons with an anti-neurofilament antibody (Fig. 3). Fig. 3A shows a graph that quantified the number of neurites/cell under different treatments. A{beta} generates an extensive loss of neurites, preventing the normal connection between cells (Fig. 3C), in relation to the control neurons (Fig. 3B). This morphological pattern of neurodegeneration is correlated with the pattern of cell death obtained by both the MTT assay (Fig. 1A) and the TUNEL assay (Fig. 2A). No major protective effect on neurites was observed when neurons were pretreated with vitamin C (Fig. 3D). However, neurons pretreated with E2 (Fig. 3E) or Trolox (Fig. 3F) and challenged with A{beta} showed an almost normal neurite network allowing interconnection among neurons. Pretreatments with Trolox and E2 maintained the neurite network similar to that observed in the control neurons (data not shown). These results indicate that Trolox and E2 prevent the neurodegenerative changes induced by A{beta} treatment. In addition, in other models, E2-induced neuroprotection was correlated with the formation of a more dynamic microtubular system, an event that increases microtubule stability and prevents the neurite loss induced by A{beta} (21). Those results are consistent with the effects on A{beta}-treated hippocampal neurons observed here.



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  		FIG. 3.
Trolox and E2 maintain the structural integrity of hippocampal neurons challenged with A{beta}. Hippocampal neurons were treated for 8 h. A shows the number of neurites from hippocampal neurons per cell; the number of neurites decreased to 50% when neurons were treated with A{beta}, pretreatment with vitamin C results in the 80% neurites in cultures, but pretreatment of neurons with E2 and Trolox protects the network from A{beta}-induced toxicity. Neurons were stained with anti-neurofilament antibody searching for the integrity of the neurite network (B-F). The treatments were as follows: controls, B; 5 µM A{beta}, C; pretreatment with 100 µM vitamin C + 5 µM A{beta}, D; pretreatment with 10 µM E2 + A{beta}, E; and Trolox + A{beta}, F. Pictures were taken from representative experiments (n = 3) performed in duplicate.

 
Trolox and E2 Modulate Activation of the Neuronal Wnt Signaling Pathway
GSK-3{beta} Activity—A loss of function of the Wnt signaling pathway has been found to play a role during {beta}-amyloid neurotoxicity (35, 39, 47), and key components of this pathway are affected in AD, i.e. {beta}-catenin is reduced, and GSK-3{beta} is activated in pre-neurofibrillary lesions (26, 45). We decided to evaluate the changes observed in GSK-3{beta} activity by following the phosphorylation of a GS-2 peptide substrate in vitro. The radioactive labeling on GS-2 peptide is considerably augmented in response to A{beta} (Fig. 4A, a). This correlates with the GSK-3{beta} activity levels, immunodetected with an antibody that specifically indicated phosphorylation of the Tyr-216 residue, which represents the activation of GSK-3{beta} (Fig. 4A, b). This observation is followed with a decrease in the phosphorylation of the P-Ser-9 residue, detected by Western blot (Fig. 4A, c). It is well established that Ser-9 phosphorylation down-regulates GSK-3{beta} (22). In fact, when neurons were pretreated with E2 or Trolox, a significant decrease (p < 0.005) in the activity of GSK-3{beta} was obtained (Fig. 4B), and the incorporation of 32P on GS-2 peptide decreases, as also occurs with P-Tyr-216 on GSK-3{beta} (Fig. 4A, a and b). However, the phosphorylation on P-Ser-9 increases (Fig. 4A, c). These results established that the increase in GSK-3{beta} activity induced by A{beta} is completely prevented by coincubation with either Trolox or E2, indicating that both compounds interfere with the neurotoxic cascades triggered by A{beta} on hippocampal neurons.



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  		FIG. 4.
Effect of E2 and Trolox on GSK-3{beta} activity in hippocampal neurons. The activity of GSK-3{beta} was evaluated on hippocampal neurons treated by 32P incorporation into GS-2 substrate and phosphorylation on Ser-9 or Tyr-219 by Western blot. The neurons were treated with 5 µM A{beta} in the absence or presence of 100 µM vitamin C, 100 µM Trolox, or 10 µM E2 (A, a). The radioactivity of the controls was assumed as 100%. Data are the means ± S.E. of 3 experiments performed in duplicate (B). Representative autoradiography in a SDS-PAGE for GSK-3{beta} activity is shown, with the corresponding control of total GSK-3{beta} (A, c). Representative Western blots against phosphorylated Tyr-216-GSK-3{beta} (A, b) and phosphorylated Ser-9-GSK-3{beta} (A, c) are shown with the corresponding controls (bottom of the figures). Each lane corresponds to control cells treated with 5 µM A{beta}, A{beta} + 100 µM vitamin C, A{beta} + 10 µM E2, and A{beta} + 100 µM Trolox. **, p < 0.005 by non-paired Student's t test.

 
{beta}-Catenin Stabilization—Regulation of {beta}-catenin stability is a crucial control point in Wnt signaling pathways (40). GSK-3{beta} induces the phosphorylation of {beta}-catenin and triggers its degradation through the ubiquitin-proteasome pathway (44, 58). Therefore, we investigated whether the protective effect of Trolox and E2 in A{beta}-induced neurodegeneration was related to {beta}-catenin stabilization. We found that A{beta} decreases the levels of cytoplasmic {beta}-catenin as described previously (39, 47), and this change can be prevented by E2, Trolox, and vitamin C (Fig. 5). Taken together, these results suggest that Trolox and E2 can regulate the neuronal Wnt signaling pathway, modulating GSK-3{beta} activity and stabilizing the cytoplasmic {beta}-catenin levels. These parameters are the most representative hallmarks involved in regulation of the Wnt signaling pathway (35, 59, 60).



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  		FIG. 5.
Trolox and E2 stabilize intraneuronal {beta}-catenin levels in A{beta}-treated neurons. Cytoplasmic {beta}-catenin stabilization was evaluated on hippocampal cells (bar graph). The hippocampal neurons were treated with 5 µM A{beta} in the absence or presence of 100 µM vitamin C, 10 µM E2, or 100 µM Trolox. Data are the means ± S.E. of 3 separate experiments performed in duplicate. A representative Western blot for {beta}-catenin is shown at the top, with the corresponding expression of tubulin.

 
Trolox and E2 Modulate Expression of the Wnt Genes in Hippocampal Neurons
Because GSK-3{beta} and {beta}-catenin are involved in the Wnt signaling pathway and Trolox and E2 modulated their activation, we decided to study the expression of Wnt ligand genes in response to these antioxidants (Fig. 6, A and B). In RT-PCR experiments, we found that E2 increases the expression of canonical and non-canonical Wnt ligands (35), Wnt-7a and Wnt-5a (Fig. 6A, a and b), as well as the Wnt target gene engrailed-1 (en-1) (Fig. 6A, c) mRNA levels. These increases in mRNA level are independent of the variations in the total amount of mRNA, as observed in Fig. 6B for the intraneuronal actin mRNA levels. Trolox treatment induces a modest increase in the Wnt-7a and Wnt-5A mRNA levels (Fig. 6B, a and b) in comparison with the effect of E2. This is an interesting observation because the typical neuroprotective action of Trolox is via an antioxidant mechanism (61). Moreover, expression of en-1 (a target gene of the Wnt pathway) is required to prevent apoptosis in mesencephalic dopaminergic neurons, an event that supports the role of the Wnt pathway in neuroprotection (62). These observations could be related to an alternative protective mechanism for neurons, in which both E2 and Trolox can modulate Wnt ligand genes and Wnt target genes in hippocampal neurons exposed to A{beta}.



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  		FIG. 6.
Effect of Trolox and E2 on the Wnt pathway genes in hippocampal neurons. Trolox and E2 increased the mRNA levels in hippocampal neuron extracts. The Wnt-7a (A, a), Wnt-5a (A, b), and en-1 (A, c) mRNA expression was evaluated in hippocampal cells treated with 10 µM E2 or 100 µM Trolox. Representative images from a RT-PCR for Wnt-5a, Wnt-7a, and en-1 are shown. The mRNA levels were expressed in relation to the {beta}-actin RNA levels (B). Data are the means ± S.E. (bars) of 3 separate experiments performed in triplicate. *, p < 0.05 by non-paired Student's t test.

 
E2 Reverts the Cytoplasmic Calcium Increase Produced by A{beta} in Primary Hippocampal Neurons
It has been documented that E2 mediates responses by genomic and non-genomic mechanisms, both of which are important for neuronal survival (63, 64). There is evidence that at supraphysiological doses, E2 acts through an antioxidant mechanism to prevent neuronal cell death (63). More recently, in neurotoxicity studies, it has been shown that E2 is able to protect ER-negative neuronal cell lines (64, 65). However, there is also evidence showing E2 neuroprotection in cells that express ERs. For instance, E2 exposure attenuates toxicity in PC12 cells transfected with {alpha}-ER exposed to serum deprivation (66). On the other hand, E2 is neuroprotective against N-methyl-D-aspartate (NMDA)- and kainate-mediated neurotoxicity, an effect mediated by mitogen-activated protein kinase activation and stimulation of NMDA receptor phosphorylation (67). This effect is concomitant with a severe decline in the cytoplasmic calcium levels induced by glutamate in cortical neurons (68). We studied the cytoplasmic calcium changes in hippocampal neurons loaded with Fluo-3 AM. The intensity of the fluorescence changes is representative of the variations in the cytoplasmic calcium levels in hippocampal neurons (13, 53). A{beta} treatment produces an increased entry of calcium for 30 min, following regulation of calcium entry for 1 h (Fig. 7B, b), as compared with control neurons (Fig. 7B, a). The cytosolic calcium changes are illustrated in Fig. 7A, which shows the neuronal calcium levels in each condition, and representative confocal photographs of neurons labeled with Fluo-3 AM are presented. When we used E2 at physiological concentrations (10 nM) in experiments on calcium homeostasis, E2 treatment decreased calcium influx in A{beta}-treated neurons (Fig. 7A, c). The specific ER blockade with 10 nM of antagonist ICI 182780 induced a similar A{beta} bulk calcium cytoplasmic rise in neurons exposed to A{beta} in the presence of E2 (Fig. 7A, d). This result suggests a role for ERs in the modulation of intracellular calcium perturbed by A{beta}. The activation of ERs with specific agonists for {alpha}-ER or {beta}-ER shows different responses. PPT, an {alpha}-ER agonist, prevents the bulk rise in calcium produced by A{beta} (Fig. 7A, e). On the other hand, DPN, a {beta}-ER agonist, does not protect against the rise in the cytoplasmic level of calcium in hippocampal neurons exposed to A{beta} (Fig. 7A, f). A summary plot in Fig. 7C shows the cytoplasmic calcium behavior observed in each condition and represents quantification of four independent experiments. Taken together, these observations support the role of {alpha}-ER in regulation of cytoplasmic calcium in A{beta}-treated neurons. Previous studies have demonstrated that E2 treatment of hippocampal neurons attenuates the excytotoxic glutamate-induced rise in bulk-free cytoplasmic Ca2+, despite potentiation of the influx of Ca2+ induced by glutamate (68). Besides, E2 can regulate the ATP-induced Ca2+ transients, an event that may be involved in nociceptive signaling in the peripheral nervous system (69).



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  		FIG. 7.
Effect of E2, PPT, and DPN on the cytoplasmic calcium increase produced by A{beta} in primary hippocampal neurons. The cytoplasmic calcium level was evaluated in hippocampal neurons loaded with Fluo-3 AM. A shows representative confocal photographs of neurons loaded with Fluo 3AM, which represent the cytoplasmic calcium levels for each condition, and neurons were treated as follows: a, control; b; A{beta}; c, A{beta} + E2; d, A{beta} + 10 nM antagonist ICI 182780; e, A{beta} + E2 + PPT (1 nM); and f, A{beta} + E2 DPN (1 nM). B, immediately before treatment with A{beta}, hippocampal neurons were treated with the following: a, vehicle; b, A{beta}; c, A{beta} + E2 (10 nM); d, A{beta} + ER antagonist ICI 182780; e, A{beta} + PPT (1 nM); and f, A{beta} + DPN (1 nM). E2 treatment induced a significant decrease in calcium when applied to hippocampal neurons treated with A{beta}. Incubation with PPT (a specific agonist of {alpha}-ER) prevents the cytoplasmic increase in A{beta}-treated neurons. C, a summary of the results described above, expressed in fluorescence units representative for each condition. Data are the means ± S.E. (bars) of 4 separate experiments performed in duplicate.

 
Effect of the Wnt Signaling Pathway on the Cytoplasmic Calcium Increase Induced by A{beta}
Previous studies from our laboratory suggest a significant role of the Wnt signaling pathway in protection from the neuronal cell death elicited by A{beta} (39, 47). Here, we evaluated whether the Wnt neuronal pathway is involved in modulation of the cytoplasmic calcium perturbations induced by A{beta} (Fig. 8). Therefore, we studied the cytosolic calcium changes in hippocampal neurons exposed to A{beta} in the absence (Fig. 8A, b) or presence of the Wnt-7a ligand (Fig. 8A, d). Results indicated that Wnt-7a, a ligand of the canonical Wnt pathway (35), was very successful in protecting hippocampal neurons challenged with A{beta} (Fig. 8A, d). This observation is presented with the corresponding graph for each condition (Fig. 8A) and the fluorescence photographs (Fig. 8B, b and d) that represent the intracellular calcium levels in each condition. The incubation with Wnt-7a ligand does not induce any disturbance in the cytoplasmic calcium level in relation with control neurons (Fig. 8A, a and c). No effect was observed when Wnt-5a, a ligand of the non-canonical Wnt pathway, was studied (data not shown). This is surprising because some non-canonical pathways result in changes in cytoplasmic Ca2+ levels (35). These results suggest a novel role of the Wnt signaling pathway in the regulation of cytosolic calcium disturbances induced by A{beta} treatment. Cytoplasmic calcium regulation may be involved in both neuronal protection against cellular death and the morphological neurite changes induced by A{beta}. These effects have been reported for both the Wnt neuronal pathway (39, 47) and the E2 neuroprotection effect observed here. Therefore, our studies support the role of Wnt signaling and E2 in the protection induced in A{beta}-treated neurons.



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  		FIG. 8.
Effect of the neuronal Wnt-7a ligand on the cytoplasmic calcium increase induced by A{beta}. Co-incubation with Wnt-7a conditioned medium prevents the A{beta}-induced cytosolic calcium increase in hippocampal neurons. The neurons were loaded with Fluo-3 AM, and the fluorescence changes were registered in a confocal microscope. A shows representative fluorescence plots of control neurons (a), A{beta}-treated neurons (b), Wnt-7a-treated neurons (c), and neurons treated with A{beta} + Wnt-7a medium (d). Incubation with Wnt-7a ligand induced a significant decrease in the cytoplasmic calcium levels in hippocampal neurons treated with A{beta}. Data are representative of 3 separate experiments. B shows representative confocal photographs of control neurons (a'), A{beta}-treated neurons (b'), neurons in Wnt-7a medium (c'), and A{beta}-treated neurons in the presence of Wnt-7a conditioned medium (d') that represent the cytoplasmic calcium levels for each condition. Data are representative of 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Oxidative stress has been implicated in the characteristic neurodegeneration found in AD (4–6). In the present work, we studied the neuroprotective effect of several molecules with antioxidant properties on the modulation of the Wnt signaling pathway and the cytosolic calcium increase induced by A{beta}. Nonetheless, the protective role of antioxidants shows some controversial reports. There are studies reporting no protection with vitamin E and Trolox against 25 µM A{beta}1–42 and A{beta}25–35 in rat hippocampal cells (56, 70) and 50 µM A{beta}1–42 in PC12 cells (71). The lack of protection in those studies may be due to the extremely high concentration of A{beta} used. Contrary to this evidence, neuroprotection with vitamin E against the challenge to A{beta}1–40,A{beta}1–42, and A{beta}25-35 has been reported for PC12 cells (72), neocortical synaptosomes (12), and vascular cells (51). These controversial results suggest that antioxidant protection involves several effects, which are not necessarily linked to a decrease in ROS levels. Our results show that E2 and Trolox significantly attenuated the cell death induced by A{beta} in hippocampal neurons. E2 and Trolox protect against the neurodegenerative changes induced by A{beta}, including the neurite loss network and the pathological cytoplasmic calcium influx. Besides, Trolox and E2 increase the expression of Wnt-7a and Wnt-5a ligand genes and the Wnt target gene en-1, indicating a novel role in modulation of the Wnt signaling pathway in hippocampal neurons. In fact, Trolox decreases the caspase-3 activity deregulated in A{beta}-treated neurons (data not shown). This observation indicates a role of antioxidants in several death effectors. However, an effect of Trolox on the cytoplasmic calcium dyshomeostasis induced by glutamate and H2O2 was discarded in several reports (61). Nevertheless, pretreatment with LiCl or Wnt-3a conditioned medium completely inhibited H2O2-induced mitochondrial collapse and DNA fragmentation in HEK-293 cells (74). These results suggest that the Wnt signaling pathway can rescue neurons from H2O2-induced toxicity and that the protective action of Trolox in our system could be mediated by modulation of the ROS species and the Wnt signaling pathway. Antioxidants could act not only by eliminating the membrane damage elicited by free radicals (75) but also by inhibiting the intracellular damage triggered by A{beta}. Endoperoxides have been proposed to mediate A{beta} toxicity in neuronal (11, 72) and vascular cells (51). In relation to AD, the interaction among copper, amyloid precursor protein, and A{beta} can result in ROS generation and subsequent oxidative stress (7, 8, 60). Toxicity of A{beta} in neuronal cultures was consistent with catalytic H2O2 production increased by the presence of copper (8). In fact, we actually checked the copper concentration in our hippocampal neuron culture (see Table I). We found concentrations of copper in the range of 0.2–3 µM in the different mediums used in our studies and the hippocampal culture process. Thus, it is probable that the neurodegenerative effects of A{beta} may be mediated by the complexing of A{beta} with copper. Besides, recent studies from Huang et al. (76) indicate that nucleation-driven aggregation of A{beta} is dependent upon metal ions, such as copper and zinc, present at trace concentrations and that metal chelation attenuated the formation of soluble A{beta} oligomers from a cell-free culture medium, an event that is involved in the toxicity mechanism of A{beta} against hippocampal neurons. Moreover, evidence for apoptotic cell death induced by oxidative damage has been reported in AD brains (4). Also, the severe decline in ROS levels in A{beta}-treated neurons observed with E2 and Trolox treatment is in agreement with the results of previous studies by Shea and Ortiz (57).


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  		TABLE I
Copper concentration in hippocampal culture medium Determinations are representative of three different samples used for quantification. Calibration was against a standard curve prepared using dilutions of Cu standards (54).

 
Vitamin C (100 µM) failed to inhibit A{beta}-mediated cytotoxicity. Similar results were obtained with A{beta}25–35 on rat hippocampal cells (70). However, another study has reported neuroprotection by vitamin C, although at concentrations higher than those used in the present study (55). The neuroprotective role of vitamin C is controversial due to its putative physiological role related to restoration of the antioxidant properties of vitamin E (77). Besides, other authors have attributed pro-oxidant properties to vitamin C at high concentrations (78), which are in agreement with our results using higher concentrations of vitamin C (data not shown). Additionally, vitamin C, 3,4-dihydroxyphenylalanine, and dopamine were excellent substrates for A{beta}-copper-mediated production of H2O2 (8). Vitamin C can modify its structure and becomes a powerful pro-oxidant molecule, inducing a neurotoxic effect in hippocampal neurons (8, 78). Additionally, GSK-3{beta} activity increased in HT22 neuronal cells exposed to glutamate and H2O2, suggesting that GSK-3{beta} is involved in control of oxidative stress resistance in these cells (79). However, in our studies, vitamin C was able to stabilize cytoplasmic {beta}-catenin levels, an event involved in Wnt signaling activation. This effect can be explained by the activation of PKC mediated by vitamin C (80). Modification of the regulatory domain of PKC by pro-oxidant agents stimulates cellular PKC activity (80). Additionally, we reported that activation of PKC by phorbol 12-myristate 13-acetate drives the accumulation of cytoplasmic {beta}-catenin and activates the transcriptional activation via {beta}-catenin/T-cell factor/lymphoid enhancer factor-1 of Wnt target genes (41). Besides, vitamin C can increase the expression of developmental genes in neuronal cells and does not induce any changes in neuronal cell death protection genes (81). Additionally, Western blot and immunohistochemistry for {beta}-catenin in brain cortex, brain stem, and cerebellum revealed differential accumulation of this protein in different types of brain cells including neurons, astrocytes, and oligodendrocytes at different developmental stages (82).

E2 has been reported as a neuroprotective agent against A{beta}1–40, A{beta}1–42, and A{beta}25-35 in assays at 4–48 h on HT22 cells (11), hippocampal cells (14), and PC12 and Neuro-2a cells (15, 83). The antioxidant property of E2 (63) is independent of its classical mechanisms of action (17). In addition to its antioxidant activity, other biological actions such as a decrease in A{beta} release (84, 85), an enhancement of amyloid aggregate uptake by microglial cells (86), an increase in the synthesis of cholineacetyltransferase (87), and the promotion of neuronal growth (88) have been described, making estrogens useful tools for the design of future therapeutic strategies in AD. Additionally, there is evidence showing E2 neuroprotection in cells that express estrogen receptors. In addition, in an in vivo model of ischemia, {alpha}-ER was found to be critical to protect against brain injury (89). The role of {beta}-ER in mediating neuroprotection is more controversial. {beta}-ER was not sufficient to protect against ischemia (89). Besides, E2 does not induce protection against {beta}-amyloid toxicity in HT22 cells stably transfected with {beta}-ER. The effects of E2 on neuronal morphology and cytoplasmic calcium regulation have been reported previously (21, 57). These studies demonstrated that E2 treatment of hippocampal neurons attenuates the excitotoxic glutamate-induced rise in bulk-free cytoplasmic Ca2+ (68). Additionally, E2 preserved both neuronal viability and function in an in vitro glutamate toxicity model (20). In addition, E2 can regulate activation of the NMDA receptors by the mitogen-activated protein kinase pathway, an event that is involved in the long terminal potentials neuronal response (67). In our system, E2 was able to prevent the cytosolic calcium induced by A{beta}. This effect seems to be regulated by activation of {alpha}-ERs, without participation of {beta}-ERs. These results are in agreement with above-mentioned reports that relate the E2 neuroprotection mechanism with the activation of {alpha}-ERs in neurons exposed to A{beta} and glutamate. However, because E2 can regulate NMDA receptor activation, we cannot exclude the possibility that E2 can decrease the rise in cytosolic calcium induced by A{beta} by this pathway. Thus, the role of {alpha}-ERs in the regulation of cytosolic neuronal calcium is a novel contribution of this study and can explain the different actions of E2 in the neuroprotection mechanism.

A recent report from Cardona-Gómez et al. (90) showed that in vivo, E2 induces a transient activation of GSK-3{beta} in the adult female rat hippocampus, followed by a more sustained inhibition, as inferred from the tau phosphorylation levels. In addition, these results showed a novel complex that includes {alpha}-ER, GSK-3{beta}, and {beta}-catenin. Considering the role of GSK-3{beta} in neurodegeneration, our data suggest that part of the neuroprotective effects of E2 may be due to the control of GSK-3{beta} and to the formation of the {alpha}-ER, GSK-3{beta}, and {beta}-catenin complex in A{beta}-treated neurons. These observations are consistent with a potential role of E2 in modulation of the Wnt signaling pathway in neurons challenged with A{beta}. A scheme showing the main effects promoted by A{beta} and the contribution of Trolox and E2 for the neuronal viability is depicted in Fig. 9.



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  		FIG. 9.
Trolox and 17{beta}-estradiol protect against the A{beta} toxicity modulating the Wnt pathway, ROS, and calcium levels. The oxidative stress generated by A{beta} can be modulated with antioxidant molecules; the damage includes lipid peroxidation (1) or involves the A{beta}-mediated activation of receptor (2). The role of the Wnt neuronal pathway in neurodegeneration induced by A{beta} has been reported previously (39), and specific activation with ligands (Wnt-3a and Wnt-7a) can prevent the viability and extensive neurite network loss induced by amyloidogenic stress (3). The E2 neuronal protection mechanism involved control of the cytoplasmic calcium levels elevated by A{beta} (4). This effect can be mediated by inhibition of calcium-specific receptors or activation of {alpha}-ER (5).

 
An interesting observation of the present study relates to the finding that hippocampal cultures exposed to either A{beta} or H2O2 lose their neurite network, a hallmark of early neuronal damage. In fact, the initial steps of AD dementia are associated with the loss of neuronal connections and the appearance of dystrophic neurites (91). Our results show that Trolox and E2 significantly attenuated loss of the neurite network and its functional activity when hippocampal neurons were exposed to A{beta} and H2O2. Moreover, it has been reported that neurons challenged by A{beta} have an increase in the activity of GSK-3{beta} related with the hyperphosphorylation of tau protein and the loss of the microtubular network (24), and more recently, the inhibition of GSK-3{beta} by E2 has been demonstrated in rat hippocampus (90). In the present work, we found that E2 and Trolox significantly inhibit GSK-3{beta} activity in neurons exposed to A{beta}. The mechanisms mediating this effect could involve the classical intracellular pathways directed to neuroprotection such as activation of PKC, phosphatidylinositol 3-kinase/AKT, or extracellular signal-regulated kinase signaling, however, further work is needed to address the specific mechanism involved in the activation of GSK-3{beta} by A{beta} and the inhibition of the enzyme by E2 and Trolox. Additionally, it has been shown that GSK-3{beta} activation is involved in growing axons and cones and in the regulation of microtubule and actin filament stability (92). In this context, our studies complement the work of Shah et al. (21), which showed that E2-induced neuroprotection increases microtubule stability and prevents the neurite loss induced by the A{beta} peptide. In addition, the activation of axin (a scaffold protein involved in the Wnt pathway) through dishevelled stabilizes the neuronal microtubule network, resulting in local changes in the length of the neurites and in the increase of the axonal area (93). Therefore, we think there is strong evidence to support the involvement of Wnt signaling pathway activation in control of the microtubule network. In our studies, Trolox and E2 are capable of producing this modification, probably by modulating the Wnt signaling pathway. Trolox and E2 induce the expression of Wnt-5a and Wnt-7 ligand genes. These findings could be related to an alternative mechanism of protection from neuronal cell death by Trolox and E2. Additionally, there are other mechanisms involved in E2-induced neuroprotection in A{beta}-treated neurons. Previous findings in our laboratory indicate that the anti-apoptotic gene bcl-2 is regulated by Wnt signaling (60); in fact, lithium, a compound that mimics Wnt signaling activation (94), and Wnt-3a ligand cause a time-dependent increase in bcl-2 mRNA levels in primary rat hippocampal neurons (60). Also, the {beta}-catenin analogue plakoglobin induces bcl-2 expression (95). Previous findings have demonstrated that A{beta} decreases neuronal bcl-2 protein levels in vitro (96) and that bcl-2 immunoreactivity is reduced in tangle-bearing neurons of AD patients (97). Our results indicate that antioxidants increase the production of Wnt ligand mRNA, and by doing this, they could activate the Wnt/{beta}-catenin/bcl-2 cascade that induces neuroprotection because there is evidence that antioxidants also promote bcl-2 expression (68).

Preclinical trials and epidemiological studies suggest that several agents may help prevent the development of AD or slow down disease deterioration. The preclinical evidence supporting the use of antioxidants to prevent or slow down AD is interesting. Our results correlate with epidemiological studies reporting a delay in the onset of AD in patients who have been treated with E2 or antioxidants (17, 98–100), although there is still controversy about the epidemiological studies (101). Moreover, clinical and experimental data suggest that E2 may play a role in the genesis and perpetuation of colorectal cancer (102). The mechanism by which E2 exerts proliferative properties has been assumed to be exclusively mediated by genomic actions. Interestingly, E2 induced a rapid cellular alkalinization of crypts and cancer cells that was sensitive to Na/H exchanger blockade or PKC inhibition (103). Recent postmenopausal E2 therapy has been associated with improved cognitive functioning, a reduced risk of dementia in women with Parkinson disease, and decreased risk of AD (103). Additionally, in epithelial cells of the choroid plexus, AD patients had lower {alpha}-ER densities compared with non-AD patients (73, 103). In other studies, it was determined that a steroidal formulation of nine synthetic conjugated E2 derived from soybean and yam extracts exerted neurotrophic and neuroprotective effects in cultured neurons (73). Indices of neuroprotection included an increase in neuronal survival, a decrease in neurotoxin-induced lactate dehydrogenase release, a reduction in neurotoxin-induced apoptotic cell death, and an attenuated glutamate-induced [Ca2+]i rise.

The novel finding presented in this study provides alternative mechanisms for some of the actions of E2 and Trolox in neuroprotection. These represent effects on neuronal calcium and regulation of cytoskeletal and growth-associated genes related to the Wnt signaling pathway.


   FOOTNOTES
 
* This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico Grant 3980024 and Field-Initiated Studies Program Grant 01-1029 (to F. J. M.) and Fondo de Areas Prioritarias Biomedicine Grant 13980001 (to N. C. I.) and by the Millennium Institute of Fundamental and Applied Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

|| To whom correspondence should be addressed: Centro de Regulación Celular y Patología "Joaquín V. Luco"-Fondo de Areas Prioritarias Center, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile. Tel.: 56-2-6862724; Fax: 56-2-6862959; E-mail: ninestr{at}genes.bio.puc.cl.

1 The abbreviations used are: AD, Alzheimer disease; A{beta}, amyloid {beta}-peptide; E2, 17{beta}-estradiol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GSK-3{beta}, glycogen synthase kinase-3{beta}; ER, estrogen receptor; NMDA, N-methyl-D-aspartate; RT, reverse transcription; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; PKC, protein kinase C; PPT, propyl pyrazole triol; DPN, diarylpropionitrile; AM, acetoxymethyl ester. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Mauricio Gonzalez (Instituto de Nutrición y Tecnología de Alimentos, Chile) for help with the Cu measurements, Alexis Parada for the kind gift of estrogen receptor ligands, and Gonzalo Olivares for help during the early phases of this work.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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