Introduction

Alzheimer's disease (AD) is an age-related neuro-degenerative disease characterized by two types of lesions in the brain: neuritic plaques composed mainly of the amyloid -peptide (A) and neuro-fibrillary tangles (NFTs) composed mainly of hyperphosphorylated forms of the protein tau, and by a selective neuronal cell death.¹ A neurotoxicity is associated with morphological alterations in rat and in human neurons in culture—including neuronal shrinkage plus severe axonal and dendritic dystrophy—and similar dystrophic changes are seen in AD neurons that develop neurofibrillar pathology.² Remarkably, it has been observed that primary cultures of fetal rat hippocampal and human cortical neurons exposed to A induced the activation of glycogen synthase kinase-3 (GSK-3), the hyperphosphorylation of tau proteins, and the loss of the microtubular network.^3,4,5

GSK-3 is a key modulator of the Wnt signal transduction pathway.^6,7,8 According to the classical view of Wnt signaling,^9,10 in the presence of an extracellular Wnt ligand, membrane-anchored receptors of the Frizzled protein family transduce its signal to the intracellular space activating Dishevelled protein. Dishevelled, in turn, inactivates GSK-3 activity, through the formation of a multiprotein complex. As a result of GSK-3 inactivation, intracellular levels of -catenin increase, allowing its binding to components of the high mobility group family of transcription factors T-cell factor/lymphoid enhancer-binding factor (Tcf/LEF). Finally, -catenin–Tcf/LEF complexes enter the nucleus and activate the expression of Wnt-target genes. Alternatively, in the absence of a Wnt ligand, the activity of GSK-3 is switched on and thus it phosphorylates -catenin for ubiquitin-proteosome-mediated degradation.^11,12 As a net result, -catenin levels are diminished within the cytosol and therefore the expression of the Wnt-target genes is switched off.

Wnt signaling is essential in developmental and oncogenic processes.^13,14 More recently, it has been implicated in neurodegenerative disorders such as autism,^15,16 schizophrenia,^17,18 and AD.^19,20 Indeed, several studies have shown that the familial AD-linked presenilin-1 proteins form high molecular weight multiprotein complexes with the -catenin protein.^21,22,23 Further studies showed that -catenin levels were markedly reduced in AD patients carrying presenilin-1-inherited mutations,²⁴ and it has been suggested that similar mutations may disturb -catenin translocation to the nuclei,^25,26 likely affecting Wnt activity. Likewise, presenilin-1–-catenin complexes also contain GSK-3^27,28 and its substrate tau,²⁷ and it was suggested that presenilin-1-inherited mutations altered the affinity and thus the activity of GSK-3.^29,30

Considering that lithium mimics Wnt signaling by reversibly inactivating GSK-3,^31,32 here we examined whether lithium could act as a neuroprotective factor against A-dependent damage. Since it has been suggested that Wnt signaling inactivates GSK-3 via an intracellular pathway that involves the protein kinase C (PKC) enzyme,^7,8,33 we also studied the ability of PKC agonists/inhibitors to modulate A neurotoxicity. We report here that A fibrils induce the destabilization of endogenous levels of -catenin, and that activation of Wnt signaling by inhibiting GSK-3 activity either with lithium, the PKC activator phorbol 12-myristate 13-acetate (PMA) or with conditioned medium containing the Wnt-3a ligand protected post-mitotic neurons from A neurotoxicity. Furthermore, lithium reduced hippocampal degeneration, recovered -catenin levels, and substantially improved spatial memory deficits of rats injected with A fibrils. A preliminary account of this work has been presented elsewhere.³⁴

Materials and methods

A fibril formation

Human wild-type A_1–40 peptide (Bachem, CA, USA) was aggregated as described previously.³⁵ Preformed A fibrils were washed several times in either sterile phosphate-buffered saline (PBS) (pH 7.4), or sterile artificial cerebrospinal fluid (aCSF; 130 mM NaCl, 2.6 mM KCl, 4.3 mM MgCl₂, 1.8 mM CaCl₂), and then pelleted by centrifugation (30 min at 14 000 rpm in an Eppendorf microcentrifuge). A concentrations were determined from denaturing Tris-Tricine SDS-PAGE gels subjected to densitometric scanning and the data were processed using the GS365W program (Hoefer Scientific Instruments, CA, USA). The final pellet was resuspended at a concentration of 2.5 g/l.

Primary cultures of rat hippocampal neurons

Cultures were prepared from 18-day-old Sprague–Dawley rat fetuses.³⁶ The hippocampi were dissected under a stereomicroscope and individual cells were prepared by trypsinization (0.1%) in Hank's balanced salt solution. Cells were seeded for 2 h onto 24- to 96-well culture dishes or cover glasses coated with poly-D-lysine (50 g/ml) and adhesion medium: Dulbecco's modified Eagle's medium (DMEM, Gibco/BRL), 10% horse serum, 100 U/ml penicillin/streptomycin. The culture medium was then substituted with Neuro-basal media supplemented with B27, 100 g/ml streptomycin and 100 U/ml penicillin, and cells were incubated at 37°C and 8% CO₂. For experiments, 6 to 8-day-old hippocampal cell cultures were shifted to B27-free media.

Neuro 2A cells

Neuro 2A cells³⁷ were grown in DMEM supplemented with 5% fetal bovine serum (FBS), 100 g/ml streptomycin, 100 U/ml penicillin and 25 g/ml fungizone, and maintained at 37°C, in 5% CO₂ and saturated humidity. Cells were then induced to differentiate 48 h after plating by applying 5 mM dibutyryl cAMP.

HEK-293 cell line and conditioned medium containing the Wnt-3a ligand

HEK-293 cells were grown essentially as described.³⁸ Cells were transiently transfected with the expression vector containing HA-tagged mouse Wnt-3a under the control of a CMV promoter or with its control plasmid (Upstate Biotechnology, NY, USA), by using Lipofectamine-plus (Gibco/BRL) according to instructions from the manufacturer. For conditioned media, cells were grown to 95% confluence, washed with PBS, and maintained in serum-free DMEM over-night. The conditioned media were filtered through 0.22 M filter units, aliquoted, and stored at –80°C until use.

In vitro cytotoxicity of A fibrils

Hippocampal neurons or Neuro 2A cells (310³/100 l per well) were assayed for the cytotoxic effects of A (0.1–10 M) by measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a color compound.³⁵ The cytotoxic effects of A were assayed in the presence of increasing concentrations of LiCl (1 M–10 mM), PMA (50 nM–2.5 M), POC-16 (1–100 M) and AM-44 (1–100 M)³⁹ or with 0.2 ml/cm² conditioned media containing Wnt-3a.³⁸ MTT reduction was determined in a Labsystem Uniskam I spectrophotometer (Finland) at 540 and 650 nm. MTT values (in triplicate) correspond to 4–6 separate experiments and are expressed as the percentage of control (untreated) cells.

Immunocytochemistry and cell fractionation of hippocampal neurons

Hippocampal neurons grown on cover slides exposed to different treatments were washed once with PBS at 37°C, fixed with 4% paraformaldehyde/PBS for 4 h, extracted using 0.1%. Triton X-100/PBS, and immunostained using primary monoclonal antibodies (diluted 1:300 in PBS) for -catenin (Santa Cruz Biotech., CA, USA). The secondary antibody was a fluorescein-conjugated polyclonal antibody. Cells were viewed by optic fluorescence using a ZEISS LSM-400 confocal microscope (63oil immersion objectives) and recorded at 520520 pixels/image. Cellular morphology was evaluated using phase-contrast microscopy (Optiphot-NIKON, Japan). Cytosolic fractions of treated cells (1.210⁵ cells in 24-well culture dishes) were harvested by scraping and homogenized in buffer A (10 mM HEPES, 1.5 mM MgCl, 10 mM KCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, pH 7.5) containing a cocktail of protease inhibitors (2 mM phenylmethylfluorosulfonate, 10 mg/ml aprotinin, 10 g/ml leupeptin, and 10 g/ml trypsin inhibitor). The resulting pellet was resuspended in buffer B (20 mM HEPES, 1.5 mM MgCl, 0.4 mM NaCl, 0.5 mM DTT, 0.4 mM EDTA, 25% glycerol, pH 7.9) plus protease inhibitors, and centrifuged at 13 000 g for 30 min (4°C). Supernatants were separated and kept as cytosol. Fractions were loaded onto 10% SDS-PAGE gels and electrophoretically transferred onto nitrocellulose membranes at 1.5 mA (4°C for 2 h), in 25 mM Tris-HCl, 192 mM glycine (pH 8.2) and 20% methanol. The membranes were blocked with TBS/non-fat dried milk (5%) for 2 h and incubated overnight (4°C) with the anti--catenin (Santa Cruz Biotech., CA, USA), the anti-Phospho-GSK-3 (Ser9) (Upstate Biotechnology, NY, USA), or the anti-GSK-3 (Santa Cruz Biotech., CA, USA) antibodies diluted 1:1000 in TBS/non-fat dried milk 5%. The secondary antibody was labeled with alkaline phosphatase and detected using NBT and BCIP. The anti-tubulin antibody (Santa Cruz Biotech., CA, USA) was used as a load control.

Intrahippocampal injection with preformed A fibrils

Male Sprague–Dawley rats (200 g) were randomly divided into two groups: the saline-treated group (control) and the chronic LiCl-treated group. LiCl was dissolved in physiological saline (0.9% NaCl) and was administered intraperitoneally once a day with a load dose of 3 meq/kg for 2 weeks prior to surgery. A maintenance dose of 2 meq/kg was given during the following 2 weeks. Control rats were injected with physiological saline alone. Plasma LiCl concentrations were measured in a separate group of rats treated with chronic lithium doses, at the end of the pre- and post-treatment period (data not shown). After 2 weeks of lithium treatment, rats were anesthetized using Equitesin (2.5 ml/kg i.p.) and injected bilaterally into the dorsal hippocampus (-3.5 mm AP, 2.0 mm ML and -2.7 DV, according to Bregma) stereotaxically with a 10 l Hamilton syringe. In total, 3.0 l of A fibrils (7.5 g/l), ibotenic acid (3.0 g/3 l), or aCSF were administered at a rate of 0.5 l/min. On the second day after surgery, the animals were post-treated with LiCl or saline as control for 2 weeks before the Morris water maze task⁴⁰ was applied.

Perfusion and fixation of rat brains

Animals were anesthetized using Equitesin (2.5 ml/kg i.p.) and injected with heparin (4 USP/kg i.p.) before perfusion. Rats were perfused through the heart with buffer containing 0.1% sodium nitrite, followed by fixation with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 30 min. Brains were removed from their skulls and post-fixed in the same fixative solution for 3 h at room temperature, followed by 10% sucrose in PB overnight at 4°C. After fixation, brains were coded to ensure unbiased processing and analysis. Finally, the brains were cut into 40 m coronal sections with a cryostat (Leitz 1900) at -20°C. Sections from the same brain were divided into groups for analysis by Nissl staining (0.3% cresyl violet), as previously described,⁴¹ and for immunohistochemical staining (see below).

Immunohistochemical staining of hippocampal sections

All sections were mounted on gelatin-coated slides, air dried, dehydrated by serial rinses in graded ethanol solutions, cleared with xylene and cover-slipped with Canada (Merck, Damstadt, Germany). Free floating immunohistochemical procedures were performed as described.⁴¹ Throughout experiments, washing and dilution of immunoreagents was carried out using 0.01 M PBS and 0.2% Triton X-100 (PBS+T); two PBS+T washes were performed following each antibody incubation. Immunodetection of A was performed using rabbit anti-A (1:500) polyclonal antibody (Sigma Co, St Louis) incubated overnight at 4°C. GSK-3 was detected with a goat polyclonal antibody (Santa Cruz Biotech., CA, USA) diluted 1:300 in PBS+T. A horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was used as a second antibody (1:600) and incubated for 1 h at room temperature. After two washes, staining was generated by incubating in 0.6% DAB for 15 min, adding H₂O₂ at a final concentration of 0.01% and incubating for 4 min. Hippocampal sections were pre-treated with 0.5% H₂O₂ for 30 min to reduce endogenous peroxidase activity, followed by treatment with 5% normal goat serum (DAKO) at room temperature for 1 h to avoid non-specific binding. -catenin was immunodetected with the anti -catenin antibody (1:200, Santa Cruz Biotech., CA, USA) and a secondary antibody conjugated to FITC (1:300). Sections were observed under a fluorescent microscope. Finally, amyloid deposits were also visualized by Thioflavin-S (Th-S) staining, as described previously.³⁵ To quantify -catenin-positive cells, fluorescence microscopy images were placed under a transparency sheet divided into nine equal squares. Eight different persons, without knowing the corresponding treatments, counted -catenin-positive cells in each picture by this method. Then, the values obtained were graphed as a vertical point scatter plot. The mean of the eight observations is shown in each case.

TUNEL assay

In order to detect the onset of apoptotic processes, we used the In Situ Cell Death Detection Kit, TMR red TUNEL assay (Roche Molecular Biochemicals, Mannheim, Germany). Rats injected either with A fibrils or with ibotenic acid were fixed by perfusion 24 h after injection and the brains were cut into 30 m sections. To reduce the fluorescent background induced by paraformaldehyde fixation, sections mounted in gelatin-coated slides were treated with 0.15 M glycine pH 7.4 at RT for 15 min following incubation with NaBH₄ for 15 min at RT. The sections were permeabilized with 0.1% TX-100 in 0.1% sodium citrate at 4°C. Then, brain sections were incubated with the TUNEL reaction mixture for 60 min at 37°C in a humidified atmosphere in the dark. After brain sections were washed twice with blocking buffer (PBS, 0.1% TX-100, 0.5% BSA), coverslips were mounted with fluorescent medium and directly analysed under a fluorescent microscope and by confocal microscopy. As a negative control, brain sections were incubated with Label Solution (without terminal transferase) instead of TUNEL reaction mixture. As a positive control, brain sections were incubated with DNase I to induce DNA strand breaks. Negative and positive controls are not shown.

Behavioral tests

All animals were trained in a circular water maze⁴⁰ (1.6 m diameter, 75 cm deep and painted black) using a two trial per day regime. Rats were trained for 5 consecutive days, followed by 2 days off, and then trained for 4 additional days. Each trial began when rats were allowed to swim, always starting at the same eastern point (see below) and ended when the animal resided on a platform for 5 s. Rats were then removed from the maze, dried and returned to their cages. For descriptive data collection, imaginary lines sub-divided the pool into four equal quadrants. These lines intersected the edge of the pool at the arbitrary cardinal start locations called north, south, east and west. The platform (9 cm in diameter) was located in the center of the northwest quadrant (hidden quadrant) and the pool was divided into three equidistant concentric annuli. Trial data were gathered using a water maze video tracking system (HVS Imagem, Hampton, UK). Water depth and temperature were maintained at 50 cm and 19–21°C.

Results

Lithium prevents the metabolic impairment induced by A in differentiated neuronal cells

We have previously observed the ability of A fibrils and A-acetylcholinesterase complexes to induce neuronal cell death to the rat pheochromocytoma PC12 cell line and to primary cultures of chick retina cells.^34,35 Here, differentiated Neuro 2A cells exposed to 10 M of A fibrils for 24 h diminished their metabolic activity in ca 50% as indicated by MTT reduction (Figure 1a). Acute treatment with an optimal dose of 100 M lithium (Figure 1b) protected differentiated Neuro 2A cells from the neurotoxic effects of A and determined a significant increase in cellular activity for those cells (>80% MTT reduction). Primary cultures of hippocampal neurons, which represent a population of mature excitatory neurons maintained throughout development and adulthood, showed a significantly impaired cellular metabolism when exposed to A fibrils (ca 45% MTT reduction), although this was not so when co-incubated with 100 M lithium (Figure 1a). Under light microscopy, differentiated Neuro 2A (Figures 1c–f), as well as hippocampal cells (Figures 1g–j), showed clear morphological alterations upon exposure to 10 M A fibrils. In particular, differentiated Neuro 2A and hippocampal neurons, which under control conditions formed an extensive network of well-spread shapes and a profusion of long neurites fibrils showed somatic shrinkage plus severe axonal and dendritic dystrophy (Figures 1e and i). A total of 100 M lithium protected post-mitotic neurons by way of preserving their normal shape, axonal processes, and neurites (Figures 1f and j). Finally, neurons incubated only with 100 M lithium did not display altered morphologies (Figures 1d and h).

Figure 1.

Lithium protects post-mitotic neurons against the metabolic impairment induced by A fibrils. (a) Cell metabolic activity (MTT assay) of differentiated Neuro 2A cells (black bars), and primary cultures of hippocampal neurons (white bars) treated for 24 h with 10 M A fibrils in the presence of increasing LiCl concentrations. (b) Dose–response effects of LiCl on metabolic activity of differentiated Neuro 2A cells () and hippocampal neurons (). Values represent the meanSEM of MTT reduction assays in relation to control cells, for four independent experiments carried out in triplicate. Asterisks indicate statistical significance at P<0.005 (Student's t-test). (c–j) Representative contrast-phase microscopy of differentiated Neuro 2A cells (c–f) and 8-day-old hippocampal neurons (g–j) maintained under control conditions (c,g, respectively), incubated with 100 M lithium alone (d,h), treated with 10 M A fibrils (e,i), or treated with 10 M A fibrils and co-incubated with 100 M lithium (f,j). Bar 10 m.

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A fibrils induce the destabilization of endogenous levels of -catenin and this effect is prevented by pre-treating neurons with lithium

It has been previously observed that transfection of a dominant-negative construct of -catenin—which inhibits -catenin signaling through the Tcf/LEF family of transcription factors—augmented apoptosis in primary cultures of rat hippocampal cells exposed to 40 M A fibrils.²⁴ Therefore, here we examined the levels and distribution of endogenous -catenin in hippocampal neurons exposed to A fibrils. As can be seen in Figure 2, quantitative Western blot analysis of hippocampal neurons, incubated with increasing concentrations of A fibrils (2.5–10 M) for 4 h, revealed a significant dose-dependent decrease in the levels of cytosolic -catenin up to ca. 35%, compared to control hippocampal neurons (Figure 2a). We next hypothesized that the neuroprotective effect of lithium was achieved by way of -catenin stabilization. Indeed, this effect was reversed in a dose-dependent manner when cells were co-incubated with A fibrils (10 M) and increasing concentrations of lithium (10 M–1 mM) (Figure 2b). Examined under the confocal microscope, -catenin displayed predominantly a cytoplasmic localization in control hippocampal neurons (Figure 2c), and this distribution was enhanced to some extent when cells were incubated with low doses of lithium (100 M) (Figure 2d). -catenin was also seen associated to plasma and nuclear membranes and low levels of the protein were also observed in patchy distributions within cell nuclei (Figures 2c and d), a fact that is consistent with its normal role in cell adhesion and nuclear/transcriptional events. Conversely, -catenin almost disappeared from the cytoplasm of hippocampal cells when exposed to 10 M A fibrils (Figure 2e). Remarkably, neurons similarly treated with A fibrils, but this time in the presence of 100 M lithium, retained the distribution of -catenin in the cytosol (Figure 2f), as it was observed for control cells.

Figure 2.

A-neurotoxicity induces the destabilization of endogenous -catenin. (a, b) Quantitative Western blot analysis of cytosolic -catenin levels upon exposure to A fibrils. (a) Cytosolic fractions of -catenin obtained from hippocampal neurons treated with increasing concentrations of A fibrils for 4 h. (b) Similar experiments, but this time hippocampal neurons were treated for 4 h with 10 M A fibrils alone or with increasing concentrations of LiCl. Values represent the meanSEM of -catenin normalized against tubulin (load control) in three independent experiments. (c–f) Representative immunofluorescence of endogenous -catenin in 8-day-old cultured hippocampal neurons, examined by laser-confocal microscopy. Under control conditions, a predominantly cytosolic location (arrowheads) was observed for -catenin (c). This effect was enhanced in the presence of 100 M LiCl (d). In cells treated for 4 h with 10 M A fibrils, -catenin was found associated to the plasma and nuclear membranes and was generally absent from the cytoplasm (arrowheads) (e). Hippocampal neurons treated with 10 M A fibrils but in the presence of 100 M LiCl retained cytoplasmic levels of -catenin (arrowheads) (f). Bar 10 m.

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PKC isoenzymes modulate neuronal death and -catenin destabilization induced by A fibrils

Several studies have suggested that Wnt signaling inactivates GSK-3 via an intracellular pathway that involves the protein kinase C (PKC) enzyme.^7,8,33 Therefore, we decided to evaluate the ability to prevent the metabolic impairments induced by A fibrils with PKC agonists/inhibitors. Figure 4a summarizes the effect of PKC agonists/inhibitors on cell viability, as measured by the MTT assay, of hippocampal neurons exposed to A fibrils for 24 h. While inhibitors of classical Ca²⁺-dependent PKC isoenzymes POC-16 and AM-44³⁹ enhanced the cytotoxic effect of 5 M A fibrils, the phorbol ester PMA, which is a potent activator of a broad range of PKC isoenzymes, protected cells against the neurotoxic effects of the same concentration of A fibrils (Figure 3a). Such an effect on cell viability seemed to be partially dependent on the increased resistance/vulnerability induced by treatment of neurons solely with PMA or with the PKC inhibitors POC-16 and AM-44 (Figure 3a). It has been previously observed in the HEK-293 cell line that PKC modulated the accumulation of -catenin.⁸ Therefore, we next examined the effect of PMA and POC-16 on the destabilization of -catenin levels induced by A fibrils. As seen in Figure 3b, while PMA recovered in a dose-dependent manner the destabilization of -catenin levels induced by 10 M A fibrils, 10 M POC-16 had the opposite effect by further reducing neuronal levels of -catenin. Given that PKC has been suggested to phosphorylate and inactivate GSK-3 in vitro,⁴² and considering that inactivation of GSK-3 has been proposed to occur through specific phosphorylation of its N-terminal residue Ser9,⁴³ we finally evaluated whether the neuroprotective effect of PMA was achieved through PKC-dependent phosphorylation of GSK-3. Figure 3c shows the temporal course of GSK-3 inhibition as a function of PMA-dependent activation of PKC. As can be observed, hippocampal neurons exposed to 500 nM PMA are induced to phosphorylate transiently the residue Ser9 of GSK-3 (as recognized by a phospho-specific antibody against phospho-Ser9), which suggests that inhibition of GSK-3 is an essential requisite for protection against the cytotoxic effects of A fibrils. Indeed, hippocampal neurons exposed to 5 M A fibrils for 24 h were induced to decrease in ca. 30% the phosphorylation of residue Ser9 of GSK-3, with respect to control levels (Figure 3c). According to what was expected, this cytotoxic effect induced by A fibrils was reversed when neurons were treated simultaneously with 2.5 M PMA, but not when cells were co-incubated with 10 M POC-16 (Figure 3c).

Figure 4.

Wnt-3a protects hippocampal neurons from A neurotoxicity. (a) Immunodetection of HA-tagged mouse Wnt-3a in conditioned media from HEK-293 cells. (b) Cell metabolic activity (MTT) of hippocampal neurons exposed to increasing concentrations of A fibrils in the presence of HEK-293-conditioned medium containing a transfection vector without (black bars) or with the Wnt-3a insert (white bars).

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Figure 3.

PKC agonists/inhibitors modulate A neurotoxicity. (a) Metabolic activity of hippocampal neurons exposed to A fibrils in the absence or presence of different agonists/inhibitors of PKC activity. [A]=5 M; [POC-16]=50 M; [AM-44]=50 M; [PMA]=1 M. (b) -Catenin destabilization induced by A fibrils is recovered by PMA but not by POC-16. Quantitative Western blot analysis of cytosolic fractions of -catenin obtained from hippocampal neurons treated for 4 h with 10 M A fibrils alone or in the presence of 1–2.5 M PMA and 10 M POC-16. (c) Left panel: PKC-dependent transient phosphorylation of GSK-3 at Ser9 induced by 500 nM PMA. Right panel: Quantitative Western blot analysis of residue Ser9 of GSK-3 levels upon exposure to 5 M A fibrils for 4 h in the absence or presence of 1 M PMA and 10 M POC-16.

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Conditioned medium containing the Wnt-3a ligand prevents A-dependent neurotoxicity

Several Wnt proteins regulate early patterning events in the developing nervous system.⁴⁴ In particular, the gene products for Wnt-3, Wnt-3a, Wnt-7b, and Wnt-8b participate as intercellular signaling molecules in the developing forebrain,^44,45,46 a region that subsequently gives rise to the hippocampus, which in turn is affected in AD brains. Therefore, we next examined whether activation of Wnt signaling by a Wnt ligand itself could prevent the metabolic impairments induced by A fibrils. In order to do so, we prepared conditioned medium by transfecting the HEK-293 cell line³⁸ with an expression vector containing HA-tagged mouse Wnt-3a, which is under the control of a CMV promoter (see Materials and methods). Figure 4a shows that Wnt-3a can be efficiently recovered from the conditioned medium of HEK-293 cells. Remarkably, hippocampal cells exposed to 5–10 M A fibrils were protected from its cytotoxic effects only when co-incubated in the presence of HEK-293-conditioned medium containing the Wnt-3a protein, but not when co-incubated with conditioned medium containing the expression vector without the insert coding for the ligand (Figure 4b).

Neuronal degeneration of the dorsal hippocampal region in rats injected with A fibrils is reversed with chronic lithium treatment

Intracerebral injection of A fibrils has been used successfully to produce animal models of AD.^47,48 Therefore, we injected stereotaxically male Sprague–Dawley rats in the dorsal hippocampus with preformed A fibrils, and the resulting morphological changes were evaluated in vivo in both lithium-treated and untreated animals (see Materials and methods) (Figure 5). In contrast to control animals (Figure 5a), sections of hippocampi injected with preformed A fibrils showed abundant neuronal loss in the upper leaf of the dentate gyrus (Figure 5b). Ibotenate (IBO), used as a control for neuronal damage,⁴⁹ also induced extensive death of hippocampal neurons (Figure 5c). Chronic lithium treatment (Figure 5d) prevented these outcomes in both A-treated and IBO-treated rat brains (Figures 5e and f, respectively). Whether neurodegeneration induced by injecting A fibrils or ibotenic acid was either due to apoptotic or necrotic processes was further examined by the TMR red TUNEL fluorescent assay (see Materials and methods) (Figure 6). As can be observed, while control rats treated with saline showed a few apoptotic nuclei around the injection site (Figure 6a), rats injected with vehicle and treated with lithium did not present either apoptotic neurons or tissue damage (Figure 6d). On the contrary, rats injected with A fibrils showed a dramatic increase in the density of apoptotic nuclei in the vicinity of the lesion (Figure 6b), which is consistent with previous findings that suggest that A-dependent degeneration was due to the onset of apoptotic processes. Interestingly, rats injected with A fibrils and treated with lithium did not display apoptotic nuclei around the lesion (Figure 6e), with respect to rats treated solely with saline. However, and despite the extensive tissue damage in rats injected with ibotenic acid, there were no apparent apoptotic nuclei (Figure 6c), indicating that the neuronal death induced by IBO occurred probably due to necrotic processes. Moreover, although rats injected with IBO and treated with lithium did not show apoptotic neurons, the tissue in the vicinity of the injection site was nevertheless protected (Figure 6f). Micrographs obtained at higher magnification by confocal microscopy (63, zoom 3) also revealed the presence of apoptotic nuclei in saline-treated rats injected with A fibrils in contrast with lithium-treated rats injected with A fibrils (Figures 6g and h, respectively).

Figure 5.

Lithium prevents neurodegeneration induced by injections of A fibrils or IBO into rat dorsal hippocampus. Representative hippocampal slices of rats subjected to cytotoxic insults were stained using Nissl staining to evaluate neuronal loss. (a) Vehicle injection: aCSF/saline. (b) Injection of 10 M A fibrils. (c) Injection of 3 g (6.3 mM) IBO. (d–f) Rats similarly injected as in (a–c), respectively, but receiving chronic lithium treatment (100 M LiCl). Number of animals in each treatment: (a) aCSF/saline: 5; (b) A/saline: 9; (c) IBO/saline: 14; (d) aCSF/LiCl: 7; (e) A/LiCl: 5; (f) IBO/LiCl: 9. Note that chronic LiCl treatment diminished neuronal loss in the dentate gyrus in rats injected with either A fibrils or ibotenate. Panels are shown at 4 magnification.

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Figure 6.

A fibrils injection induced apoptosis in rat hippocampus. Brain sections of treated animals were stained using TUNEL assay to detect apoptosis. Brain sections were analysed under a fluorescence microscope (a–f) or by confocal microscopy (g,h). (a) Vehicle injection: aCSF/saline. (b,g) Injection of 10 M A fibrils. (c) Injection of 3 g (6.3 mM) IBO. (d) aCSF/saline. (e,h) Injection of 10 M A fibrils, but receiving chronic lithium treatment (100 M LiCl). (f) Injection of 3 g (6.3 mM) IBO in the presence of lithium. Panels (a–f) are shown at 40 magnification; panels (g,h) are shown at 63 magnification, zoom 3.

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Lithium treatment rescues -catenin destabilization in vivo

The results presented so far suggested that loss of function of the Wnt pathway might indeed be involved in the onset of neurodegenerative processes in vivo. Therefore, we then examined by in situ immunodetection whether endogenous levels of -catenin were altered in brain sections of rats injected either with A fibrils or ibotenic acid, and whether the neuroprotective effects of lithium, as seen above, were achieved through -catenin stabilization (Figure 7). It can be observed that control rats receiving saline displayed abundant -catenin immunoreactivity around the injection site (Figure 7a). In agreement with what is expected from Wnt signaling, control rats injected with vehicle but receiving chronic lithium treatment showed an increase in density of cells positive for -catenin (Figure 7d), meaning that the protein has been stabilized by the effect of lithium, and thus allowed to enhance its levels. On the contrary, a decrease in the density of cells positive for -catenin was observed when rats were injected with A fibrils (Figure 7b). Chronic lithium treatment allowed the recovering of cells positive for -catenin to control levels (Figure 7e). Finally, we did observe a low number of -catenin positive cells around the lesion site in rats injected with ibotenic acid (Figure 7c), which was probably due to necrosis. However, a slight increase of -catenin-positive cells was detected in animals subjected to the same treatment but that had received chronic lithium treatment (Figure 7f). A quantification of these observations (see Materials and methods) showed that effective chronic lithium treatment increased the density of -catenin-positive cells in comparison with animals treated with saline (Figure 7g). Therefore, our results demonstrate that A-dependent neurotoxicity targets -catenin-responsive neurons for degeneration and that chronic lithium treatment protects these cells against such cytotoxic insults probably by direct inhibition of GSK-3. A fact consistent with such a possibility is that in the hippocampal region of A-treated rats, we observed a strong GSK-3 immunoreactivity (Figure 8d) around deposits of A fibrils, visualized either with the amyloid dye Thioflavine-S or with an anti-A antibody (Figures 8e and f, respectively). Control animals showed neither GSK-3 immunoreactivity nor A deposits (Figures 8a–c).

Figure 7.

-Catenin destabilization induced by A fibrils in vivo. Representative immunofluorescence microscopy of endogenous -catenin in hippocampal slices of rats injected either with A fibrils or ibotenate into the dorsal hippocampus. (a) aCSF/saline. (b) Injection of 10 M A fibrils. (c) Injection of 3 g (6.3 mM) IBO. (d–f) Rats similarly injected as in (a–c), respectively, but receiving chronic lithium treatment (100 M LiCl). Panels are shown at 40 magnification. (g) -catenin-positive cells were counted (see Materials and methods) and a vertical point scatter plot was generated. The horizontal line represents the average of the eight points of each treatment.

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Figure 8.

A deposition in the dorsal hippocampus induces GSK-3 immunoreactivity. Representative hippocampal slices of control animals (a–c) and rats injected with 10 M A fibrils (d–f). (a,d) Anti-GSK-3 antibody. (b,e) Amyloid staining with Thioflavine-S. (c,f) Anti-A antibody. Panels are shown at 10 magnification.

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Lithium averts the behavioral impairments induced by hippocampal injection of A fibrils

Severe behavioral deficits have been observed in animals exposed to A^47,50 and in transgenic mice overexpressing mutant forms of the hAPP.^51,52 Therefore, the possibility to ameliorate with chronic lithium treatment the spatial memory deficits induced by intrahippocampal administrations either of A fibrils or ibotenic acid was further investigated. Treated rats were trained for 2 weeks in the Morris water maze paradigm with the hidden platform and the learning score was recorded (see Materials and methods). Representative paths taken by control and treated animals are shown in Figure 9a. It can be observed that during the first week of maze trials and compared to control rats injected solely with vehicle (Figure 9b), animals injected with A fibrils or IBO but receiving chronic lithium treatment (Figures 9c and d, respectively) showed a faster learning response than rats lacking lithium. In the second week, all groups treated with lithium displayed similar and reduced escape latency values with respect to control animals. Therefore, our results suggest that chronic lithium treatment heightened spatial memory and thus prevented the behavioral impairment induced either by A fibrils or IBO, although in the latter case to a lesser extent.

Figure 9.

Lithium prevents spatial memory deficits after injection of A fibrils into rat dorsal hippocampi. (a) Paths taken from representative animals studied in each case at day 8 and 11 (top and bottom, respectively). (b–d) Graphs showing escape latency vs time after training in the Morris water maze for animals subjected to different treatments. Control animals (b). Animals injected with 100 M A fibrils (c) and IBO (d) presented high escape latency values, suggesting impaired spatial learning. In the presence of 100 M lithium, animals presented low escape latency values (c,d), similar to control animals treated with or without lithium, indicating that lithium prevented the memory loss induced by injected A or IBO, although in the latter case recovery was not complete at early stages. Values represent the meanSEM of three independent experiments. Number of animals in each treatment: aCSF/saline: 5; aCSF/LiCl: 7; A/saline: 9; A/LiCl: 5; IBO/saline: 14; IBO/LiCl: 9.

Full figure and legend (127K)

Discussion

In this study, we have found that A-dependent neurotoxicity alters the activity of key components of the Wnt signal transduction pathway and that activation of this signaling cascade is sufficient to prevent such cytotoxic effects. Previously, A neurotoxicity has been linked to the abnormal phosphorylation state of tau, as is observed in AD brains.^3,4,5 Given that hyperphosphorylation of tau occurs primarily at (Ser or Thr)-Pro motifs, it was suggested that proline-directed kinases were responsible for this activity. Among this group of kinases, it is presently accepted that GSK-3 plays a major role in the stability of MAPs such as tau,^4,53,54,55 and the responsive target for Wnt signaling MAP-1B.^34,56 These observations suggested that the activity of the GSK-3 enzyme should be kept tightly regulated in mature neurons. Consistent with such idea is the observation in AD brains in situ that neurons containing tangle-like inclusions that parallel the sequence of the development of neurofibrillary changes are positive for active, but not inactive, GSK-3.⁵⁷

Here we have observed that lithium, which inactivates GSK-3 and thus acts as a positive regulator in the Wnt signaling pathway,^31,32 prevents the cytotoxic effects of A fibrils to post-mitotic neurons —viz. differentiated Neuro 2A cells and primary cultures of hippocampal neurons—and recovers neurodegeneration and behavioral impairments in animals injected either with preformed A fibrils or IBO in the dorsal hippocampus in vivo. Our findings expand on previous studies in vitro, which showed that lithium protected rat cortical neurons, cerebellar granule cells and the PC12 cell line from A-induced cytotoxic stresses,^58,59 and with experiments in vivo, which suggested that chronic lithium prevented focal cerebral ischemia⁶⁰ and increased neurogenesis of hippocampal cells.⁶¹ Likewise, our findings are in agreement with a protective effect of lithium on the altered behavior of rats treated with IBO in the basal forebrain cholinergic system.⁴⁹

Previous experiments indicated that the neuroprotective effect of lithium was related to the specific dephosphorylation of tau proteins.^62,63,64 Additionally, lithium does not inhibit competitively both inositol-1-monophosphatase and inositol polyphosphate 1-phosphatase, inducing a down-regulation of phosphoinositide-coupled receptors.^65,66 Moreover, lithium has been reported to modulate the expression of proteins linked to the onset of apoptotic processes, such as bcl-2 and p53,⁶⁷ which have in turn been suggested to play a role in the development of AD. In the present study, the neuroprotective effect of lithium may be explained by its modulation of GSK-3 activity acting as Wnt signaling component. Indeed, our results confirmed previous observations, which suggested that A neurotoxicity altered the activity of the GSK-3 enzyme.^4,5 Furthermore, we have found that A neurotoxicity compromises the stability of endogenous -catenin in vitro and in vivo, likely affecting its translocation to the nucleus where it acts as a cofactor for the transcription of Wnt target genes, in a manner that can be prevented using low doses of lithium. Such observations are in general agreement with previous results showing that presenilin-1-inherited mutations, which lead to the most aggressive forms of AD, may affect the levels, trafficking or the phosporylation state of -catenin.^24,25,26

It has been observed that conditional transgenic mice overexpressing GSK-3 in the adult brain displayed tau hyperphosphorylation, decreased -catenin levels, and neurodegeneration.⁶⁸ Accordingly, our results indicate that deposition of A fibrils in the hippocampal formation led to the appearance of apoptotic processes in the vicinity of the lesion probably due to enhanced GSK-3 activity, which ultimately led to -catenin destabilization. On the other hand, and from a mechanistic perspective, the neurodegenerative effects induced by injecting toxic doses of ibotenic acid are consistent with the idea that a necrotic process is taking place. Nevertheless, although different in nature, the neuronal degeneration induced either by A fibrils or IBO exposure was prevented when animals received chronic lithium treatment. Moreover, animals exposed to cytotoxic insults, but receiving chronic lithium treatment, performed substantially better in the water maze task than rats similarly challenged but receiving saline treatment. Altogether these results indicate that components of the Wnt signaling cascade might have an active role in keeping cell-homeostatic processes in highly specialized regions of the adult brain, by acting as neuroprotective factors against a diverse range of cytotoxic insults.

Wnt signaling inactivates GSK-3 via activation of the PKC enzyme.^7,8 Regarding AD, analyses of afflicted brains have revealed PKC deficits in the frontal cortex,^69,70 and reduced PKC activity has also been reported for AD fibroblasts.^71,72 Although it was initially suggested that the activity of PKC was related to the abnormal processing of the APP, thus affecting A production,^73,74 recent studies indicated that PKC isoenzymes are differentially degraded upon exposure to A.⁷⁵ These observations suggested that in AD brains, the proposed regulatory role of GSK-3 activity on the stability of either MAPs or on the -catenin molecule may well relate to deficits in PKC activity acting as a Wnt signaling component. Indeed, here we have found that while POC-16 and AM-44, which are inhibitors of classical Ca²⁺-dependent PKC isoenzymes,³⁹ enhanced the metabolic impairments induced by A fibrils (namely cell viability, -catenin destabilization, and GSK-3 activation), the phorbol-ester PMA prevented such neurotoxic effects. Our results are in general agreement with previous observations in PC12 cells and dorsal root ganglion cultures,^76,77 which support the notion of a protective role for PKC upon A insults.

Wnt signaling is revealing itself to be increasingly complex and is only now beginning to be understood.^9,10 Although new modulators of this signaling cascade are being identified and their functions discerned at a rapid pace, there is a major need to establish whether the function of Wnt components is sustained throughout neurodegeneration of the adult human brain, as occurs in oncogenic processes.^13,14 In this respect, we have found that the metabolic impairments induced by A fibrils in hippocampal neurons were reversed selectively when these cells were co-incubated with conditioned medium containing Wnt-3a. Although current experiments are being undertaken in our lab to establish properly the role that Wnt ligands play in A neurotoxicity, or molecules that help to present its signal to its receptor, our results are suggestive of a major protective function of such molecules during the onset of neurodegenerative processes (Figure 10). Regarding AD, several genes coding for components of Wnt signaling are located in close proximity to genetic markers that account for putative locus for late-onset AD pedigrees (GV De Ferrari and NC Inestrosa, unpublished observations). Indeed, Wnt8B⁴⁵ and secreted Frizzled related protein-5 (SFRP-5)⁷⁸ are located around marker D10S1671 in the long arm of chromosome 10.⁷⁹ Likewise, Wnt1⁸⁰ and Wnt10B⁸¹ are located around markers D12S390 and D12S96 in the long arm of chromosome 12.^82,83 Finally, the gene coding for the low-density lipoprotein receptor-related protein-6 (LRP-6),⁸⁴ which functions as a co-receptor of Wnt signaling,⁸⁵ is located close to marker D12S1623 in the short arm of chromosome 12.⁸⁶

Figure 10.

A neurotoxicity induces loss of function of Wnt signaling components. Abnormal processing of APP leads to increased deposition/toxicity of the A peptide. Increased levels of A alter the normal activity of Ca²⁺-dependent PKC isoenzymes and enhance the activity of GSK-3. Activated GSK-3 targets -catenin for its degradation and tau proteins () for hyperphosphorylation. Degraded -catenin no longer can act as a cofactor for the transcription of Wnt target genes (eg C-jun); thus loss of function of Wnt signaling has occurred. Wnt signaling is tissue specific; therefore, survival of neurons that depend on its trophic support in selected regions of the brain is compromised (eg hippocampus), leading to cognitive decline as is observed in AD brains.

Full figure and legend (71K)

In conclusion, our results indicate that A-dependent neurotoxicity leads to a loss of function of Wnt signaling components,¹⁹ opening the possibility that lithium or compounds that mimic Wnt signaling may be used as putative candidates for therapeutic intervention in AD patients.

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Acknowledgements

We thank Dr FJ Muñoz and S Lienlaff for helping us in the early stages of this project. This work was supported by grants FONDAP Biomedicine No. 13980001 and Millenium Institute for Fundamental and Applied Biology (MIFAB) No. P 99-007-F. NCI and MB are recipients of a Presidential Chair in Science from the Chilean government. NCI is a John Simon Guggenheim Memorial Foundation Fellow.