Recent evidence suggests that an impaired activity of the Wingless-Int (Wnt) signaling pathway contributes to the pathophysiology of neuronal degeneration in Alzheimer's disease (AD) (De Ferrari and Inestrosa 2000; Garrido et al. 2002; Inestrosa et al. 2002). Wnt glycoproteins interact with different membrane receptors, including several members of the Frizzled family of seven transmembrane (7-TM) receptors and the low density lipoprotein receptor-related proteins, LRP5 and LRP6 (Dale 1998; Tamai et al. 2000). In the canonical Wnt signaling pathway, interaction of Wnts with Frizzled and LRP5/6 triggers a cascade of intracellular reactions leading to the inhibition of glycogen synthase kinase-3β (GSK3β) and an increased stability of cytosolic β-catenin, which migrates to the nucleus and activates gene expression by interacting with transcription factors of the TCF (T-cell factor)/LEF (lymphoid enhancer factor) family (Dale 1998; Willert and Nusse 1998; Novak and Dedhar 1999; Huelsken and Behrens 2002; Miller 2002). Interestingly, GSK3β has been implicated in the pathophysiology of neurofibrillary tangles (one of the major histopathological hallmarks of AD), owing to its ability to phosphorylate tau protein (Wagner et al. 1996; Utton et al. 1997). The canonical Wnt pathway is negatively modulated by the secreted protein Dickkopf-1 (Dkk-1) (Zorn 2001). Dkk-1 interacts with LRP5/6 and the transmembrane protein Kremen-2, thus inducing the rapid internalization of the Wnt coreceptor LRP6 (Mao et al. 2002). We have recently demonstrated that β-amyloid peptide induces Dkk-1 expression in cultured cortical neurons, and that Dkk-1 expression is causally related to tau hyperphosphorylation in β-amyloid peptide-treated neurons (Caricasole et al. 2004). Interestingly, LRP5, the Dkk-1 receptor, can also bind apolipoprotein E (ApoE) (Kim et al. 1998; Magoori et al. 2003), and LRP1, the ApoE receptor, can negatively modulate the Wnt pathway by interacting with Frizzled receptors (Zilberberg et al. 2004). Whether any of the three human isoforms can modulate the Wnt signaling pathway is presently unknown. This question is highly relevant because the isoform ApoE4 is the major risk factor for AD (Strittmatter and Roses 1995; Cedazo-Minguez and Cowburn 2001; Laws et al. 2003; Raber et al. 2004). Using PC12 cells transfected with a Wnt-responsive reporter gene, we now show that ApoE4 negatively modulates the canonical Wnt signaling pathway, and that, under all circumstances, ApoE4 inhibits the Wnt pathway to a greater extent than ApoE2 or ApoE3.
We examined the effect of the three human isoforms of apolipoprotein E (ApoE2, ApoE3, and ApoE4) on the canonical Wnt signaling pathway in undifferentiated PC12 cells. Addition of recombinant ApoE4 reduced Wingless-Int7a-stimulated gene expression at concentrations of 80 and 500 nm. Recombinant ApoE2 and ApoE3 were virtually inactive. Recombinant ApoE4 also inhibited Wnt signaling when combined with very low density lipoproteins (VLDLs) or in cells over-expressing the low density lipoprotein receptor-related protein, LRP6. In contrast, the enforced expression of LRP5 unmasked an inhibition by ApoE2 and ApoE3, which, however, were less effective than ApoE4 in inhibiting Wnt signaling. We also transfected PC12 cells with constructs encoding for the three human ApoE isoforms to examine whether endogenously expressed ApoE isoforms could modulate the Wnt pathway. Under these conditions, all three ApoE isoforms were able to inhibit Wnt signaling, although ApoE4 showed the greatest efficacy. Only the conditioned medium collected from cultures transfected with ApoE4 induced a significant inhibition of Wnt7a-stimulated gene expression, confirming that ApoE4 has an extracellular action that is not shared by the other ApoE isoforms. We conclude that ApoE4 behaves as an inhibitor of the canonical Wnt pathway in a context-independent manner.
glycogen synthase kinase-3β
low density lipoprotein receptor-related protein
very low density lipoprotein
Materials and methods
Recombinant human ApoE2, ApoE3, and ApoE4 were purchased from Panvera Invitrogen Corporation (Carlsbad, CA, USA) and dialyzed overnight in 0.1 m ammonium bicarbonate. The human very low density lipoprotein (VLDL) was purchased from Academy Biomedical Co. (Houston, TX, USA).
PC12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with glutamine, 2 mm, and 10% fetal bovine serum and maintained at 37°C in a 5% CO2 humidified atmosphere.
Western blot analysis of β-catenin levels in the nuclear fraction
Western blot analysis was performed as described previously (Iacovelli et al. 2002). Briefly, 24-h post-transfection PC12 cells were lysed in Triton X lysis buffer [10 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mm sodium orthovanadate, 50 mm sodium fluoride, and 10 mmβ-glycerophosphate] for 15 min. The cell lysates were clarified by centrifugation (10 000 g for 10 min). Nuclear fractions were isolated by differential centrifugations using a commercially available kit (Pierce, Rockford, IL, USA) (Calderone et al. 2003). The purity of the fractions was verified by western blotting using an antibody against the nuclear protein poly(ADP-ribose) polymerase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein cell lysates (80 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and probed using a polyclonal anti-β-catenin antibody (Cell Signaling Technology, Beverly, MA, USA) at 1 µg/mL dilution and polyclonal anti-apolipoprotein E at 1 : 1000 dilution (Chemicon, Temecula, CA, USA). The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences, Milan, Italy), using horseradish peroxidase-linked secondary antibodies.
Construction of plasmids was as follows. For the Wnt7a expression construct, the Wnt7a complementary DNA (cDNA) was amplified from human adult brain cDNA by using primers carrying EcoRV and XbaI restriction sites at the flanking ends. The amplified cDNA was sequenced and subcloned into the EcoRV and XbaI restriction enzyme sites of the eucaryotic expression pCIN4 vector (Rees et al. 1996; Caricasole et al. 2002). The expression plasmid for Wnt1 was provided by Marc van de Westering, Hubrecht Laboratory, Utrecht, the Netherlands. The expression plasmids for human LRP5 and LRP6 and for mouse Dkk-1 were kind gifts from Drs C. Niehers and M. Semenov, and construction details were reported previously (Tamai et al. 2000; Wu et al. 2000; Mao et al. 2001). The expression plasmids for human ApoE2, ApoE3, and ApoE4 were kind gifts from Dr J. S. Owen from the University of London, and the construction details were reported previously (Stannard et al. 2001).
Transient transfection assays for reporter studies
Transfection and reporter assays were carried out essentially as described (Caricasole et al. 2002). Transient transfections of PC12 cells were carried out in triplicate, employing LipofectAMINE 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. PC12 cells (1.2 × 105) were plated 1 day before transfection in 96-well culture plates. A total of 0.65 µg of DNA was transfected into each well, including luciferase reporter plasmid (100 ng), expression constructs (200 ng), and carrier plasmid DNA (pGEM4Z, to 320 ng; Promega, Madison, WI, USA) as appropriate, in 100 µL of OPTIMEM Medium (Gibco, Rockville, MD, USA) lacking fetal calf serum. Four hours later, cells were added with an additional 100 µL of the plating medium containing 20% fetal calf serum. In some experiments, the transfection medium was removed after 4 h and substituted with the conditioned medium collected by cultures transfected with constructs encoding for ApoE2, ApoE3, or ApoE4. The luciferase reporter plasmid is the p4TCF, comprising four copies of a TCF-responsive element upstream of a TATA element luciferase coding sequence transcriptional unit (Bettini et al. 2002). In some experiments, cells were cotransfected with the Renilla luciferase CMV-driven internal reporter (20 ng; Promega) for normalization. However, because ApoE treatments affected the constitutive expression of Renilla luciferase, data were normalized by µg of proteins. Human recombinant ApoE2, ApoE3, and ApoE4 (3.9-13-39-80-500 nm) were added to cultures 4 h after transfection. Control cultures were added with 0.1 m ammonium bicarbonate. Luciferase activity was measured after 24 h using the Promega luciferase assay reagent and read using a Berthold LUMAT LB3907 tube luminometer.
Detection of apolipoprotein E levels in the extracellular medium
ApoE levels in the extracellular medium of PC12 cells transfected with constructs encoding for ApoE2, ApoE3, or ApoE4 were detected with the ApoE4/Pan-ApoE ELISA kit (MBL, Naka-ku, Nagoya, Japan) using the pan-ApoE conjugated solution according to the manufacturer's instructions.
We examined the modulation of the ‘canonical’ Wnt signaling pathway by the three different isotypes of ApoE (ApoE2, ApoE3, and ApoE4) using undifferentiated PC12 cells transfected with a TCF-luciferase reporter construct responsive to Wnt. These cells constitutively express the Wnt coreceptors, Frizzled-2, -5, and -7, and LRP6, but not LRP5 (Caricasole et al. 2003). Cells were cotransfected with a plasmid encoding for Wnt7a, an established activator of the canonical Wnt signaling pathway (Caricasole et al. 2003; Eickholt et al. 2002; Hall et al. 2002). This led to a substantial expression of the reporter gene, which we routinely detected 18–24 h after transfection. We studied the influence of ApoE isoforms on the Wnt pathway either by adding human recombinant ApoE2, ApoE3, and ApoE4, or cotransfecting cells with expression plasmids encoding for the three human ApoE isotypes. Addition of recombinant ApoE2, ApoE3, and ApoE4 did not induce changes in TCF/luciferase expression by themselves (i.e. in the absence of Wnt7a) at concentrations ranging from 4 to 500 nm. Concentrations of 80 and 500 nm of recombinant ApoE4 substantially reduced Wnt signaling in Wnt7a-expressing cells, whereas recombinant ApoE2 was inactive (up to 500 nm). Recombinant ApoE3 reduced Wnt signaling by approximately 20% at concentrations of 500 nm only (Fig. 1). We also examined the effect of recombinant ApoE isoforms in cells cotransfected with vectors encoding for LRP5, LRP6, or Dkk-1. Cultured PC12 cells constitutively express LRP6, but not LRP5 mRNA (Caricasole et al. 2003). Transfection of PC12 cells with LRP5- or LRP6-encoding vectors amplifies Wnt7a signaling to a variable extent (see Caricasole et al. 2003). In this particular experiment, we observed a slight amplification of Wnt7a-stimulated luciferase expression (about 15–25%) in cells transfected with LRP5 or LRP6 (Fig. 2a). Interestingly, however, transfection with LRP5 or LRP6 differentially affected the effect of recombinant ApoE isoforms on the Wnt pathway. In cells transfected with LRP6, only recombinant ApoE4 reduced Wnt signaling, similarly to what was observed in control cells. In contrast, all recombinant ApoE isoforms reduced Wnt signaling in cells expressing LRP5, although ApoE4 showed the greatest efficacy (Fig. 2a). In cells expressing Dkk-1, a negative modulator of the canonical Wnt pathway (Zorn 2001), the response to Wnt7a was reduced by about 45%. Recombinant ApoE4 could still reduce Wnt7a-stimulated gene expression in the presence of Dkk-1, whereas equimolar concentrations of recombinant ApoE3 (80 nm) were inactive (Fig. 2a). We have also tested recombinant ApoE isoforms (80 nm) in the presence of equimolar concentrations of recombinant VLDL. VLDL alone induced a slight reduction in Wnt signaling, which was not further amplified by recombinant ApoE2 or ApoE3. In contrast, recombinant ApoE4 co-applied with VLDL significantly reduced Wnt7a-stimulated luciferase expression (Fig. 2b). Taken collectively, these data show that recombinant ApoE4 showed a greater ability than recombinant ApoE2 and ApoE3 in inhibiting Wnt7a-stimulated luciferase expression in PC12 cells. To further explore the modulation of the canonical Wnt signaling pathway by ApoE isoforms, we examined the nuclear levels of β-catenin in cells expressing Wnt7a and treated with recombinant ApoE2, ApoE3, and ApoE4 (all at 80 nm). Western blot analysis performed on extracts from nuclear proteins showed a substantial increase in the levels of β-catenin in cultures expressing Wnt7a (Fig. 3). This was expected because activation of the canonical Wnt signaling pathway increases the levels of β-catenin available for nuclear translocation (Willert and Nusse 1998; Novak and Dedhar 1999; Huelsken and Behrens 2002; Miller 2002). Addition of ApoE2 and ApoE3 (both at 80 nm) did not affect nuclear levels of β-catenin both in the absence or presence of Wnt7a. In contrast, addition of equimolar concentrations of ApoE4 reduced the increase in β-catenin levels induced by Wnt7a without affecting the basal levels of the protein (Fig. 3). Finally, we attempted to confirm the negative modulation of the Wnt pathway by ApoE4 by cotransfecting PC12 cells with expression vectors encoding for human ApoE2, ApoE3, and ApoE4, and using the expression of TCF/luciferase in response to Wnt7a as a measure of the Wnt signaling pathway. Empty vectors containing the CMV promoter were used as controls in these experiments. Tranfection with vectors containing ApoE2, ApoE3, or ApoE4 induced a large expression of the corresponding proteins, as shown by western blot analysis (Fig. 4a). As opposed to data obtained with recombinant ApoE isoforms, all transfected ApoE isoforms reduced TCF/luciferase expression under basal conditions, in cells expressing Wnt7a, and in cells expressing Wnt7a + Dkk-1. However, the reduction was significantly higher in cells expressing ApoE4 than in cells expressing ApoE2 or ApoE3 (Fig. 4b). We also examined the effect of transfected ApoE isoforms on luciferase expression stimulated by transfected Wnt1. Transfected ApoE2 and ApoE3 induced only a trend to a reduction in Wnt1 signaling, whereas transfected ApoE4 significantly reduced Wnt1 signaling in PC12 cells (Fig. 4b). To examine whether the effect of transfected ApoE isoforms was intracellular or extracellular, we carried out experiments in which the conditioned medium collected from cultures transfected with individual ApoE isoforms for 24 h was transferred to recipient cultures 4 h after transfection with Wnt7a. ApoE levels in the conditioned medium (means ± SEM of three determinations) were below detection in cells transfected with the empty vector, 21.4 ± 3.5 µg/mL (0.63 ± 0.10 µm) in cells transfected with the ApoE2-encoding construct, 17.4 ± 0.9 µg/mL (0.51 ± 0.03 µm) in cells transfected with the ApoE3-encoding construct, and 23.8 ± 1.75 µg/mL (0.70 ± 0.05 µm) in cells transfected with the ApoE4-encoding construct. The conditioned medium collected from cultures transfected with ApoE4 reduced Wnt signaling in recipient cells, whereas the medium collected from cultures transfected with ApoE2 or ApoE3 had no effect on Wnt7a-stimulated luciferase expression (Fig. 4c).
ApoE isoforms transport lipids from the blood to recipient cells expressing one or more members of the LDL receptor family (Herz and Beffert 2000). This family includes the LDL receptors, the VLDL receptor, the ApoE receptor-2, the multiple epidermal growth factor repeat containing protein, MEGF7, LRP1, LRP1b, and megalin (LRP2). More distant family members are LRP5 and LRP6, which also behave as Wnt coreceptors (Schneider and Nimpf 2003; Herz and Bock 2002). LRP5 binds ApoE in vitro (Kim et al. 1998), and mice lacking LRP5 and ApoE show severe hypercholesterolemia, impaired fat tolerance, and advanced atherosclerosis (Magoori et al. 2003). Evidence for an interaction between LRP6 and ApoE is still lacking. Binding of ApoE to its receptors promotes intracellular events that go beyond receptor internalization and cholesterol import (Strittmatter and Bova Hill 2002; Han 2004). For example, ApoE negatively modulates the action of Reelin, a secreted protein that is critically involved in brain development and interacts with VLDL receptor and ApoE receptor-2 (D'Arcangelo et al. 1999; Koch et al. 2002; Tissir and Goffinet 2003). In cultured cortical neurons, ApoE activates the mitogen-activated protein kinase (MAPK) pathway, inhibits JUN kinase, and regulates GSK3β activity (Cedazo-Minguez et al. 2003; Hoe et al. 2005). Data presented here provide direct evidence that ApoE modulates the Wnt signaling pathway. Modulation was influenced by LRP5 expression, depended on the source of ApoE isoforms (exogenous vs. endogenous), and, more importantly, varied in relation to the isotype of ApoE. Mid/high nanomolar concentrations of ApoE4 negatively modulated Wnt signaling independently of the context, whereas ApoE2 and ApoE3 acted as Wnt inhibitors in the presence of LRP5 only or when endogenously expressed in transfected cells. Under the latter conditions, however, ApoE4 was always more effective than ApoE2 and ApoE3. ApoE4 might inhibit the canonical Wnt pathway acting at different levels of signal propagation. In the absence of LRP5, ApoE4 might inhibit Wnt signaling by interacting with LRP1, which is expressed by PC12 cells (Bu et al. 1998; Chiabrando et al. 2002). LRP1 is known to interact with Frizzled receptors and to down-regulate the canonical Wnt pathway in HEK 293 cells (Zilberberg et al. 2004). However, whether or not LRP1 also negatively regulates the Wnt pathway in PC12 cells is currently unknown. A role for LRP6 in the effect we have seen is unlikely because ApoE4 inhibited the Wnt pathway to the same extent in cells overexpressing LRP6 and in cells expressing Dkk-1, which is known to bind LRP6 (Mao et al. 2001). The enforced expression of LRP5 unmasked the action of ApoE2 and ApoE3 and amplified the action of ApoE4, suggesting that interaction of ApoE isoforms with LRP5 perturbs the coactivation of LRP5 and Frizzled by Wnt. All ApoE isoforms were more active when endogenously expressed after cell transfection than when exogenously applied to the culture medium. Possible explanations are that endogenous ApoE isoforms are post-translationally modified (and therefore more stable) or interact intracellularly with adaptor proteins or enzymes that lie along the Wnt signaling pathway. We assessed whether part of the action of transfected ApoE isoforms was extracellular by transferring the conditioned medium to recipient cells challenged with Wnt7a. The concentrations of ApoE isoforms detected in the conditioned media did not differ substantially among cultures transfected with ApoE2, ApoE3, or ApoE4 encoding vectors (0.6, 0.5, and 0.7 µm, respectively), and were close to the maximal concentrations of recombinant ApoE isoforms used in our experiments (see Fig. 1). The medium of cultures transfected with ApoE4 was able to significantly reduce Wnt signaling, whereas the media of cultures transfected with ApoE2 or ApoE3 had no effect on Wnt signaling. We can speculate that all ApoE isoforms may act intracellularly to inhibit Wnt signaling, whereas only ApoE4 has an additional extracellular effect. The latter possibility is consistent with data obtained using recombinant ApoE isoforms.
If demonstrated in neurons, inhibition of the canonical Wnt signaling pathway by ApoE4 may be relevant for the pathophysiology of AD. The presence of the ε4 allele encoding for ApoE4 is a major risk factor for sporadic AD, and reduces the age of onset for familial AD (Corder et al. 1998). The underlying mechanisms are still debated. ApoE4 may influence the extracellular deposition and/or clearance of β-amyloid peptide (Strittmatter et al. 1993; LaDu et al. 1994; Ma et al. 1994; Wisniewski et al. 1994; Bales et al. 1999; Holtzman et al. 2000; Irizarry et al. 2000) or may be toxic to neurons by promoting oxidative stress (Miyata and Smith 1996), alterations of cytoskeleton dynamics (Nathan et al. 1994, 1995), tau hyperphosphorylation, and formation of neurofibrillary tangles (Strittmatter et al. 1994;. Tesseur et al. 2000; Huang et al. 2001; Ljungberg et al. 2002). Inhibition of Wnt signaling by ApoE4 might contribute to the pathological cascade leading to neuronal degeneration in AD because an impairment of Wnt signaling has been associated with this disorder (De Ferrari and Inestrosa 2000; Inestrosa et al. 2002). Expression of Dkk-1 is causally related to tau protein hyperphosphorylation in cultured neurons challenged with β-amyloid, and Dkk-1 colocalizes with neurofibrillary tangles in degenerating neurons of the AD brain (Caricasole et al. 2004). An additive inhibition of Wnt by ApoE4 and Dkk-1 (as shown by our data in PC12 cells) might severely impair the canonical Wnt signaling pathway with ensuing GSK3β activation, β-catenin degradation, tau protein hyperphosphorylation, and neuronal death.