Microglia, the immunocompetent cells of the central nervous system, can react to virtually any homeostatic modification by changing their morphology, motility, gene expression, and proinflammatory cytokine production and release (Hanisch and Kettenmann, 2007). In the inflamed brain, microglia can exert dual actions: besides being the cellular source of proinflammatory cytokines, these cells are indispensable for the phagocytic removal of infectious agents and cellular debris (Hanisch and Kettenmann, 2007; Rivest, 2009). Diverse signaling molecules—including growth factors, neurotransmitters, and morphogens—may instruct microglia functions (Hanisch, 2002; Pocock and Kettenmann, 2007), thus emphasizing that the temporal and spatial dynamics of communication between microglia and neurons, astrocytes or oligodendrocytes determine microglia fate.
WNTs bind Frizzled (FZD) family receptors (Schulte, in press; Schulte and Bryja, 2007) whose cooperation with particular co-receptors, such as LDL receptor-related protein 5/6 (LRP5/6), receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2), or related to receptor tyrosine kinase (RYK), defines downstream signal specificity (Hendrickx and Leyns, 2008). A main WNT signaling branch is the WNT/β-catenin pathway (Fig. S1) progressing through disheveled (DVL), glycogen synthase-3 (GSK-3), and the transcriptional regulator β-catenin (MacDonald et al., 2009). WNT signaling inhibits constitutive β-catenin phosphorylation by GSK-3 and its proteasomal degradation upon FZD-LRP5/6 activation allowing for β-catenin accumulation, nuclear import, and the regulation of gene transcription.
The pathophysiological significance of WNT/β-catenin signaling in neuronal differentiation, growth as well as dysfunction, is broadly accepted (Inestrosa and Arenas, 2010). Particularly, microglia activation is implicated in various neurodegenerative conditions (Morales et al., 2010; Morganti-Kossmann et al., 2007; Perry et al., 2002; Tai et al., 2007; Tansey and Goldberg, 2010; Weinstein et al., 2010). It is broadly accepted that microglia activity exacerbate the underlying, molecularly diverse disease pathologies by releasing proinflammatory cytokines to compromise neuronal survival and function (Hanisch and Kettenmann, 2007). Notably, progressive neuronal dysfunction in Alzheimer's disease (AD) and its transgenic models is associated with decreased WNT/β-catenin signaling (De Ferrari and Moon, 2006; Dinamarca et al., 2008; Inestrosa and Arenas, 2010). Accordingly, GSK3 blockade in a mouse model of AD improves memory performance suggesting that restoring WNT/β-catenin signaling may alleviate the underlying neuronal deficits (Toledo and Inestrosa, 2009).
Although microglia reside and act in an environment where multiple members of the WNT family of lipoglycoproteins are expressed (Inestrosa and Arenas, 2010), it is not known whether WNTs influence microglia functions, particularly, their proinflammatory transformation. Here, we report that β-catenin levels in active, mobile, and macrophage-like microglia are elevated in neurodegenerative conditions associated with chronic neuroinflammation and in the corresponding transgenic model. We show that microglia express appropriate combinations of WNT receptors to activate β-catenin signaling. By using WNT-3A, we show LRP6 and DVL phosphorylation, β-catenin stabilization, and nuclear import in cultured microglia, a specific proinflammatory gene expression pattern, and proinflammatory cytokine release. Therefore, we propose that WNT/β-catenin signaling in microglia is a potent proinflammatory signaling cascade.
MATERIALS AND METHODS
Human brains (for human tissue handling see Supp. Info.) were blocked, embedded in paraffin, and sectioned at a thickness of 7 μm. Sections were dewaxed in xylene, washed in ethanol, and endogenous peroxidase activity was blocked by 1% H2O2 in 100% methanol (30 min). Antigen retrieval was carried out by incubating sections in 5% urea in 0.1 M Tris–HCl (pH 9.5) and microwaving (800 W, 8 min) followed by incubation in ice-cold NaHBO4 [0.5%, in phosphate buffer (PB) for 5–10 min]. Chromogenic detection of β-catenin was performed by using rabbit anti-β-catenin IgG (1:1,000; Abcam, Cambridge, UK). Sections were blocked in 0.1% Triton X-100, 5% normal donkey serum (NDS), and 2% BSA in 0.1 M PB (pH 7.4) for 1 h at 22–24°C. Subsequently, sections were exposed to the primary antibody diluted in PB, 0.1% Triton X-100, 1% NDS, and 0.1% BSA (pH 7.4) overnight at 4°C. After repeated rinses in PB, sections were subjected to biotinylated donkey anti-rabbit IgG as secondary antibody [1:600; Jackson ImmunoResearch, Newmarket, UK; 2 h at 22–24°C in PB (pH 7.4)] followed by exposure to streptavidin-conjugated horseradish peroxidase [1:300; Jackson ImmunoResearch; 1 h at 22–24°C in PB (pH 7.4)] and development with 3,3′-diaminobenzidine tetrahydrochloride (20 mg/100 mL) and H2O2 (0.05%). Sections were coverslipped with Entellan (in toluene, Merck, Darmstadt, Germany).
Immunofluorescence histochemistry on human specimens after antigen retrieval and free-floating mouse brain (for information on mouse tissue preparation see Supp. Info.) sections was performed according to published protocols (Harkany et al., 2003). Sections were rinsed in PB and preincubated with 5% NDS (Jackson ImmunoResearch), 2% BSA, and 0.3% Triton X-100 in PB for 1 h (22–24°C). Sections were then exposed to combinations of the following primary antibodies: rabbit anti-β-catenin (1:1,000; Abcam) goat anti-ionized Ca2+-binding adaptor molecule 1 (IBA-1; 1:200; Abcam), mouse anti-glial fibrillary acidic protein (GFAP, 1:1,000, Millipore, Billerica, MA), mouse antihyperphosphorylated tau AT8 (1:1,000; Pierce/Thermo Scientific; Rockford, IL), and rabbit anti-CB2 cannabinoid receptor [CB2R; 1:500 (Van Sickle et al., 2005)], diluted in PB, 0.1% Triton X-100, 0.1% BSA, and 1% NDS and incubated overnight at 4°C. Subsequently, sections were rinsed in PB and incubated with a mixture of carbocyanine (Cy)-conjugated secondary antibodies raised in donkey (IgGH+L; 1:200; Jackson) diluted in PB/2% BSA (2 h at 22–24°C). Specimens were counterstained with Hoechst 33,342 (Sigma-Aldrich, St. Louis, USA). Finally, mouse sections were rinsed in PB, dipped in distilled water, mounted on fluorescence-free glass slides, air-dried, and coverslipped with Entellan (in toluene, Merck). For the human sections lipofuscin autofluorescence was blocked using 1% Sudan Black B (70% ethanol), and sections were mounted using aquamount (DAKO, Glostrup, Denmark).
Standard control experiments were performed by omission of primary antibodies and yielded the lack of any cellular labeling. We have minimized the likelihood of staining artifacts such as lipofuscin autofluorescence by using Sudan Black B (Schnell et al., 1999) in combination with spectral dye unmixing (Fig. S2).
Images were captured on a Zeiss 710LSM laser-scanning microscope using multi-track consecutive channel capture configuration and spectral unmixing. Image overviews (see Fig. 1) were captured using the tile-scanning function and differential interference contrast microscopy. Emission wavelengths for maximum separation of immunofluorescence signals were limited to 407–484 nm (Hoechst 33,342), 510–530 nm (Cy2), 560–610 nm (Cy3), and 650–720 nm (Cy5). Reconstruction of β-catenin+ microglia was carried out by capturing consecutive images at 63× primary magnification. Three-dimensional rendering and maximum intensity projections were prepared using the ZEN2009 software package (Zeiss). Images were processed and figures were assembled in CorelDraw X5 (Corel Corp., Ottawa, ON, Canada).
N13 microglia-like cells (Ferrari et al., 1996) were cultured as described (Hammarberg et al., 2003). For biochemical analysis or immunocytochemistry, cells were seeded either directly or on collagen-coated coverslips in 24 well plates (density: 5 × 104 cells/well). Serum-starved cells were stimulated with vehicle (0.1% BSA in PBS) with or without recombinant mouse WNT-3A (R&D Systems, Wiesbaden, Germany) as specified. In proliferation assays, N13 cell numbers were determined after stimulation in serum-free medium, trypsination, and cell-counting in a Bürker chamber (in triplicate, n = 6). For stimulation with Aβ, lyophilized Aβ(1–42) [from Dr. Botond Penke; University of Szeged, Hungary; Minkeviciene et al., 2009)] was dissolved directly in growth medium to yield fibrillar Aβ and added to N13 cells as a 10× solution.
Primary microglia were isolated from newborn mouse brains (Prinz et al., 1999). Cultures typically contained >95% microglia, as validated by cytochemistry using Griffonia simplicifolia isolectin B4 (Sigma-Aldrich). Cultures were challenged 24 h after plating.
β-Catenin Localization In Vitro
Cells were fixed in 4% paraformaldehyde (10 min) in PB. β-Catenin was detected by using mouse anti-β-catenin antibody (1:1,000; BD Transduction Laboratories). A Cy3-coupled donkey-anti-mouse antibody (1:500; Jackson) was used as secondary antibody in combination with SYTOX Green or DAPI nuclear stains (Molecular Probes/Invitrogen, Eugene, OR). Images were captured on an LSM710 (Zeiss) laser-scanning microscope.
Immunoblotting of lysates from cultured cells (Bryja et al., 2007) and mouse cortices (Martín-Ibañez et al., 2006) was performed as described (Table S3). Signals were visualized by the enhanced chemiluminescence method (GE Healthcare, Pollards Wood, UK). Densitometry was done with the Scion Image software.
Primary mouse microglia were plated in 96 well plates (density: 105 cells/well) and stimulated with WNT-3A (300 ng/mL; R&D Systems) for 24 h. Supernatants were analyzed for IL-6, IL-12 (including the IL-12 isoforms p70, p40, and p402), and tumor necrosis factor α (TNFα) using mouse-specific antibody pairs and mouse protein standards designed for ELISA applications (R&D Systems, MN). Colorimetric reactions were quantified on a microplate reader (Victor, 1420 Multilabel counter, Perkin Wallac, Waltham, MA).
Reverse Transcriptase-PCR and Quantitative Real-Time PCR
RNA was isolated from N13 cells and primary microglia using the RNeasy Mini kit and the RNeasy Micro kit (Qiagen, Hilden, Germany), respectively. cDNA was transcribed using the high-capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). RT-PCR was performed according to a standard protocol: 94°C for 5 min followed by 30 cycles of 94°C for 30 s, Tanneal (Table S4) for 45 s, 72°C for 1 min, followed by 72°C for 10 min (2720 Thermo Cycler, Applied Biosystems). Genomic mouse-tail DNA served as positive control, while nontranscribed (minus reverse transcriptase/-RT) RNA was used as negative control. PCR products were analyzed by 2% agarose/ethidium bromide gel electrophoresis. Quantitative real-time PCR was carried out with the TaqMan gene expression assay (Applied Biosystems) according to the manufacturer's instructions. Mm00443260_g1 (TNFα), Mm99999064_m (IL6), and Mm01288991_g1 (IL12) primer pairs were from Applied Biosystems. Reactions were performed in triplicates on an ABI Prism 7000 Sequence detector (Applied Biosystems). Results were presented as the difference in the number of cycles to reach the detection threshold (Ct, cycle at threshold), using 18S rRNA (Applied Biosystems/Ambion) as internal reference standard (ΔCt = Ctcytokine − Ct18S). ΔCt was presented as relative fold change in gene expression (mean ± SEM).
Affimetrix Expression Analysis
Three independent harvests of primary microglia were seeded in six well plates and stimulated with 0.1% BSA in PBS (vehicle control) or 300 ng/mL recombinant WNT-3A for 6 h after serum-deprivation overnight. RNA was prepared as described earlier and analyzed on an Affimetrix Mouse Gene 1.0 ST Array (Bioinformatics and Expression Analysis Core Facility, Department of Biosciences and Nutrition, Karolinska Institutet; www.bea.ki.se). RNA was quality controlled on an Agilent BioAnalyzer (Agilent RNA 6000 Nano assay). Functional grouping was done according to the gene ontology terminology (www.geneontology.org; GO). Data were selected by their P value being <0.05 and their fold change (FC) ≤−2 or ≥2 from the functional grouping GO:0006955 (immune response). Overlap with the following groups is indicated: GO:0006954 (inflammatory response), GO:0035556 (intracellular signal transduction), and GO:0001817 (regulation of cytokine production).
GraphPad Prism 5.0 (GraphPad Software, San Diego, USA) was used to perform nonlinear regression analysis, bar graphs, and group comparisons. Data were analyzed by either one-way ANOVA followed by Bonferroni's multiple comparison post hoc test or Student's t-test (independent group design) as appropriate. *P < 0.05, **P < 0.01, and ***P < 0.001.
β-Catenin Accumulates in Microglia in Alzheimer's Disease
We chose to study β-catenin expression by microglia in AD, because this neurodegenerative condition presents significant chronic neuroinflammation and microglia activation (Querfurth and LaFerla, 2010). Immunohistochemistry to detect β-catenin was performed on clinicopathologically verified postmortem material from AD patients and aged-matched controls. We observed two types of β-catenin immunoreactivity in controls and Braak stage II–V cases: (a) low-to-moderate staining of perikarya, morphologically reminiscent of pyramidal cells (Fig. 1A,A1) and (b) intensely labeled nuclei (Fig. 1A1,B1) with quasi-random distribution across all cortical laminae in the temporooccipital region (Fig. 1). In contrast, β-catenin immunoreactivity redistributes in cortices from Braak VI subjects: besides being scantly dispersed in large (neuronal) somata, a meshwork of fine-caliber processes of multipolar cells with (peri-)nuclear β-catenin immunoreactivity (Fig. 1C), likely representing micro- or astroglia (Fig. 1C1) emerges. We find closely associated β-catenin and IBA-1 immunoreactivities, the latter being a microglia marker (Fig. 1D) in Braak VI cases. We have verified that IBA-1+ cells around AT8+ dystrophic neurons were activated microglia by their co-expression of CB2Rs (Fig. 1E) (Walter et al., 2003). Thus, our data from a human AD cohort suggest a shift from neuronal toward (micro-)glial β-catenin expression in patients manifesting significant AD-related neuroinflammation.
Surveying microglia undergo phasic transformation to reach a macrophage-like stage with phagocytic capacity (Fig. 2A). Therefore, we have determined whether β-catenin is co-expressed at any particular morphological state(s) of IBA-1+ microglia (Fig 2A1). β-Catenin immunoreactivity is not detectable in either surveying/resting microglia with ramified morphology (Fig. 2A1/a2) or chronically active microglia (Fig. 2A1/a1). In contrast, activated, mobile microglia (Fig. 2A1/a3) progressing toward a macrophagelike state are β-catenin+ (2A1/a4). Whilst we acknowledge the resolution limits of light microscopy in rounded-down β-catenin+/IBA-1+ microphage-like cells, our data suggest β-catenin localized in the cytosol, submembraneously, or in the nucleus of such cells (Fig. 2A1/a4). Colocalization experiments of β-catenin and GFAP show that β-catenin levels are generally low in astrocytes, irrespective of the AD stage analyzed (Fig. 2A1/a2–a4).
We have performed regression analysis to assess whether β-catenin levels could correlate with CB2R or GFAP in microglia and astroglia, respectively, in the AD brain (Fig. 2B,B1). We find significantly increased CB2R levels in severe AD when compared with age-matched controls (P < 0.05) or moderate AD subjects (P < 0.01). We find a quasi-random relationship between β-catenin and CB2R levels in control and moderate AD subjects. However, our data support a close positive association between β-catenin and CB2R protein expression in severe AD. Although a similar relationship between β-catenin and GFAP contents appears in both moderate and severe AD cohorts (Fig. 2B,C), the considerable population spread of GFAP expression negates any association. Analysis of SNAP-25 protein levels, a marker of glutamatergic synapses in human brain (Garbelli et al., 2008; Matteoli et al., 2009), revealed significantly reduced protein concentrations in moderate but not severe AD (Fig. S3D,D1) and a lack of expressional relationship with β-catenin. Collectively, our neuroanatomical and biochemical analysis suggest that β-catenin expression is significantly increased in microglia in AD.
β-Catenin Accumulation in Microglia Is Age-Dependent But Unrelated to Acute β-Amyloid Toxicity
We confirmed our above data in APdE9 mice (Jankowsky et al., 2004; Minkeviciene et al., 2009), exhibiting considerable microgliosis and astrogliosis coinciding progressive β-amyloid production. We provide added support for chronic neuroinflammation in APdE9 mice by showing increased TNFα mRNA expression (see Fig. 3) in brains of 7 and 12 months old APdE9 mice [P < 0.05 vs. control (7 months); P < 0.005 vs. control (12 months)].
β-Catenin levels were found increased with age in IBA-1+ microglia in the polymorph layer of the dentate gyrus (Fig. 4A) in wild-type mice (Fig. 4A1,A2). The density of both β-catenin+ microglia and β-catenin− astroglia in the dentate gyrus of aged APdE9 mice (Fig. 4A3) as well as total β-catenin levels in hippocampus (129.2 ± 13.1%, n = 3/group; P < 0.05; Fig. 4C,C1) exceeded that of age-matched littermate controls (Fig. 4A2). High-resolution microscopy suggests perinuclear β-catenin in microglia (Fig. 4B,B1) but not astrocytes. Although our analysis focused on the hippocampus, the brain region primarily affected by β-amyloid (Aβ) overexpression in APdE9 mice, β-catenin+ microglia were also found in the cerebral cortex, striatum, and basal forebrain (data not shown). Cumulatively, our findings in postmortem AD brains and APdE9 mouse hippocampi reveal progressive and significant β-catenin stabilization in proinflammatory microglia.
Considering that Aβ can bind FZDs, such as, for example, FZD5 (Magdesian et al., 2008), we hypothesized that Aβ may be critical to stabilize β-catenin in microglia. Therefore, we exposed N13 microglia-like cells to Aβ(1–42) for 6 h (data not shown) and 24 h (Fig. 4D) and monitored β-catenin levels. We find that Aβ elevates inducible nitric oxide synthase (iNOS) expression, a marker of microglia activation (Hanisch and Kettenmann, 2007). However, β-catenin levels remained unchanged suggesting that Aβ per se may not regulate β-catenin levels in microglia.
WNT-3A Increases Microglial β-Catenin Levels
We have performed a ligand array coupled to Western analysis of β-catenin and LRP6 phosphorylation (P-LRP6) to identify factor(s) inducing β-catenin stabilization and signaling in microglia. We included ligands (Table S2) that have previously been reported to affect β-catenin stabilization or are otherwise important for microglia function (Jin et al., 2008; Pocock and Kettenmann, 2007). As Figure 5A shows, WNT-3A and LiCl, the latter used as a positive control given its inhibitory effect on GSK-3β, but not lipopolysaccaride, thrombin, interferon α/β, TNFα, insulin, glutamate, ATP/UTP, N-ethylcarboxiamidoadenosine, isoproterenol, or apomorphine-induced β-catenin accumulation in N13 microglialike cells by 60 min. Only WNT-3A exposure led to LRP6 phosphorylation.
WNTs are expressed in the adult brain (Malaterre et al., 2007). Here, we confirmed WNT-1, WNT-2, WNT-3, and WNT-7A expression, all of which can activate WNT/β-catenin signaling (Hwang et al., 2004; Shimizu et al., 1997), in adult mouse brain (Fig. 5B). Therefore, we hypothesize that WNTs may represent a novel class of signaling molecules modulating microglia functions.
Microglia Express WNT Receptors
A prerequisite of functional WNT signaling is the expression of WNT receptors. By RT–PCR, we identify that both N13 cells and primary mouse microglia express a variety of WNT receptors invariably dominated by FZD4, FZD5, FZD7, FZD8, LRP5, and LRP6 (Fig. 5C). Neither cell type expressed ROR1, ROR2, or RYK, atypical receptor tyrosine kinases that can cooperate with FZDs or act as cell-autonomous WNT receptors. Although minor differences in WNT receptor expression exist between N13 and primary microglia (Fig. 5C), our data validate the use and responsiveness to WNT-3A of both models in functional assays in vitro.
WNT/β-Catenin Signaling in Microglia
Cytosolic, perinuclear, and nuclear β-catenin localization in microglia in vivo suggests that WNTs could signal through β-catenin in this cell type. To test this possibility, we explored the potential of purified, recombinant WNT-3A to stimulate β-catenin signaling in microglia. We emphasize that WNT-3A was used as a model ligand to establish signaling mechanisms in proof-of-principle studies. Yet, the molecular identity of WNTs activating microglia in vivo may vary and might be specific to certain brain areas or particular disease conditions.
First, we verified that recombinant WNT-3A induces β-catenin stabilization and nuclear import in N13 cells as well as primary microglia (Fig. 6A). We find increased β-catenin immunoreactivity in microglia when stimulated with recombinant WNT-3A (Fig. 5A). Importantly, β-catenin underwent nuclear translocation as revealed by β-catenin, SYTOX green/DAPI colocalization (Fig. 6A).
Next, we determined WNT/β-catenin-signaling kinetics by detecting P-LRP6 and β-catenin upon WNT-3A stimulation for up to 6 h (Fig. 6B). In agreement with previous studies in other cell types (Bryja et al., 2007), WNT-3A induced β-catenin stabilization and P-LRP6 accummulation. WNT-3A-induced changes in P-LRP6 and β-catenin levels occurred simultaneously with increases detected from 15-min poststimulation and reaching saturation by 120 min. Therefore, we used 2-h stimulation to establish dose-response relationships upon WNT-3A stimulation in N13 cells (Fig. 6C,C′). Nonlinear regression analysis of WNT-3A-induced P-LRP6 or β-catenin stabilization estimated EC50 values at 205 ng/mL (115–367 ng/mL) and 147 ng/mL (84–256 ng/mL), respectively.
An upstream regulator of β-catenin is the phosphoprotein (DVL), which becomes phosphorylated and shifted (PS-DVL) upon WNT stimulation (Bryja et al., 2007; Cong et al., 2004). WNT-3A at concentrations significantly stabilizing β-catenin progressively induced PS-DVL3 formation (from 30-min poststimulation; Fig. 6D). All three mammalian DVL isoforms responded to WNT-3A stimuli (2 h) with PS-DVL formation (Fig. 6D′).
We have excluded the possibility that the above WNT-3A-induced signaling events were specific to N13 cells by stimulating primary mouse microglia with WNT-3A. WNT-3A increased both P-LRP6 and β-catenin levels at 30 and 100 ng/mL (Fig. 6E), concentrations previously shown to be effective in N13 cells. Similarly, the temporal induction of P-LRP6 and β-catenin was similar to those seen in N13 cells (Fig. 6E). Collectively, these data suggest that WNT-3A signaling through P-LRP6 activates DVL and results in β-catenin stabilization and nuclear translocation in microglia.
WNT-3A Modulates Microglia Activity
Our findings show that microglia express a repertoire of receptors suited to mediate WNT-3A responses. However, the cellular consequence(s) and physiological response(s) WNT-3A imposes on microglia, including cell proliferation and proinflammatory cytokine (IL-6, IL-12, and TNFα) expression, and release remain to be determined.
Because enhanced proliferation is a hallmark of generalized microglia activation (Hanisch and Kettenmann, 2007), and β-catenin-dependent signaling pathways can exert mitogenic effects in select cellular systems (Boland et al., 2004; Castelo-Branco et al., 2003; Willert et al., 2003), we studied whether WNT-3A affects N13 cell growth. WNT-3A (300 ng/mL) stimulation did not affect the rate of N13 cell proliferation when compared with serum-starved control cells (Fig. 7A). Treatment with fetal calf serum (10%), used as positive control (Kloss et al., 1997), provided the expected increase in N13 cell proliferation (P < 0.001, n = 6 in triplicate).
Proinflammatory cytokine release is a hallmark of activated microglia and contributes to chronic neuroinflammation (Farfara et al., 2008). Therefore, we tested whether exposure of primary mouse microglia to WNT-3A affects the expression and release of the proinflammatory cytokines IL-6, IL-12, or TNFα. WNT-3A (300 ng/mL, 24 h) increased mRNA levels and facilitated release of corresponding cytokines in the medium (Fig. 7B,B′). Thus, WNT-3A can potently exacerbate neuroinflammatory responses.
Although we reveal select changes upon WNT-3A challenge, proinflammatory microglia transformation are likely to trigger widespread changes in complex cytokine-signaling networks. Therefore, we determined genome-wide expressional changes upon WNT-3A exposure (300 ng/mL, 6 h) in primary mouse microglia. Affimetrix gene-expression profiling shows (see Fig. 8) WNT-3A-induced upregulation of immune response genes, particularly, IL-6 and TNFα. IL-12 mRNA also increased by 6 h; however, sample variability rendered the changes statistically nonsignificant (+7.73-fold increase, P = 0.32). Because quantitative PCR confirmed our expression analysis, we used the gene-expression microarray approach for in-depth analysis of WNT-3A's inflammatory profile. We grouped WNT-3A-regulated genes according to GO: “immune response” (see Fig. 8). Soluble proinflammatory factors such as IL-6, IL-1α, IL-15, oncostatin M, chemokines (CXCL11, CCL7, CXCL2, 10, CCL2, CCL4, and 5) and complement factor 3 were upregulated. Members of the TNF-superfamily (TNFSF9, 10, 15, and TNF) and factors involved in immune signaling or function (MX1 and 2, TLR3, ICAM1, SP110, NFKB2, IRGM1, MYD88, and IRAK3) were also induced (see Fig. 8). Furthermore, we find that WNT-3A robustly induces iNOS/NOS2, CD69, matrix metalloproteases (MMP13 and 14), prostaglandin-endoperoxide synthase 2, or cyclooxygenase 2 (PTGS2/COX-2; Table S5). Thus, our microarray analysis supports our hypothesis that WNT-3A can trigger proinflammatory microglia transformation.
Microglia serve as a crucial point of convergence for innate immune responses in the brain (Rivest, 2009). Our results suggest that β-catenin signaling is active in microglia in the inflamed brain and implicate enhanced cytosolic β-catenin stabilization and nuclear import in proinflammatory microglia transformation under conditions of chronic neuroinflammation. Previous studies have shown that peripheral macrophages can use WNT-based communication to modulate inflammatory responses (Pereira et al., 2009), suggesting that brain microglia could also process WNT signals. A prerequisite of relaying WNT signals intracellularly is the expression of WNT receptors. We show that both N13 and primary microglia express such receptors, including various FZDs and LRP5/6. However, we did not explore the identity of specific WNT receptor complexes in microglia. Instead, we have focused on identifying cellular response patterns to WNT-3A, thus providing primary data on the cellular functions of WNT/β-catenin signaling in this particular cell type. WNT-3A is the prototypic WNT that activates WNT/β-catenin signaling. In vitro, WNT-3A induces WNT/β-catenin signaling (LRP6 phosphorylation, PS-DVL formation, β-catenin stabilization/nuclear translocation) in both N13 cells and primary microglia with expected kinetics, and dose-response relationships (Bryja et al., 2007; MacDonald et al., 2009; Willert et al., 2003)].
Neuroinflammation in AD
AD is accompanied by a severe inflammatory reaction with microglia implicated to act in both detrimental and beneficial fashions (Querfurth and LaFerla, 2010). Our data show increased β-catenin levels in microglia transiting from a surveying to an active macrophage-like stage in postmortem AD brains. This notion suggests that enhanced β-catenin signaling is an intricate part of the inflammatory transformation cascade in microglia and participates in exacerbating AD-related neuroinflammation. Furthermore, β-catenin stabilization with disease progression from moderate to severe AD correlates with an increased expression of CB2Rs, expressed in activated (IBA-1+) microglia rather than with astrocytic or neuronal markers, emphasizing the selective elevation of β-catenin levels in microglia.
APdE9 Mice and Microglial β-Catenin
Compatible with the human AD pathology, APdE9 mice show important features of chronic inflammation, such as microgliosis and astrogliosis, increased production of inflammatory cytokines, and upregulation of the complement system (Akiyama et al., 2000). Important differences exist between various models of chronic neuroinflammation, regarding, for example, the extent of tissue damage, gliosis, or blood–brain-barrier permeability. Interestingly, aging is also associated with a progressive inflammatory reaction and a transformation in microglia morphology and function (Miller and Streit, 2007). In aged wild-type mice, we also observed an increased density of IBA-1+ microglia accumulating β-catenin. Excess density of β-catenin+/IBA-1+ microglia in the APdE9 phenotype prompts us to speculate that β-catenin and also WNT/β-catenin communication could serve as a fundamental means of microglia communication in neuroinflammation.
Inflammatory Fingerprint of WNT-3A on Microglia
Enhanced β-catenin signaling in microglia in the diseased brain could affect the immunocompetence of these cells by mediating either an anti-inflammatory response to compensate ongoing inflammation or a proinflammatory reaction exacerbating chronic inflammation. Our in vitro data show that WNT-3A-induced β-catenin in cultured microglia parallels a strong proinflammatory response manifesting in the increased expression of microglia “activity markers”: IL-6, IL-12, and TNFα expression and release (see Fig. 7). Gene expression analysis (see Fig. 8) corroborates these observations by identifying an immuno-response network provoked by WNT-3A, including cytokines, chemokines, and innate immune response factors. WNT-3A-evoked reprogramming may allow microglia cells to increase their activation status (e.g., CD40 and ICAM1), intercellular communication with other microglia, astrocytes, T-, and B-cells (CD40, CD28, chemokines, and cytokines), and phagocytic activity (TLR3 and MYD88) (Lynch, 2009). In fact, CCL2 is recognized as one of the driving forces of the acute inflammatory response in the brain (Lu et al., 1998).
Upregulation of IL-6, a destructive, proinflammatory cytokine, is particularly intriguing in an AD-context as IL-6 may link downstream neurotoxicity ubiquitously following microglia activation. In summary, the overall shift towards a proinflammatory phenotype upon WNT-3A stimulation suggests that WNTs can affect essential immune functions in the central nervous system and orchestrate the infiltration of peripheral immune cells.
β-Catenin and AD
Several molecular mechanisms regulate β-catenin levels (e.g., GSK-3), and many become impaired in AD (Hooper et al., 2008; Pei et al., 1999). Thus, it is of importance to characterize the signaling pathways as well as their cell type-specificities that impact β-catenin signaling in AD. Recent studies concentrated on establishing the molecular complexity of WNT/β-catenin signaling in neurons (Inestrosa and Arenas, 2010) and propose that WNT-induced inhibition of GSK-3 diminishes Aβ neurotoxicity by reducing tau hyperphosphorylation (Inestrosa et al., 2007). This concept is supported by findings demonstrating that WNT-3A engaging FZD1 decreases Aβ neurotoxicity (Alvarez et al., 2004; Chacón et al., 2008), inhibition of GSK-3 abrogates Aβ processing (Phiel et al., 2003), and LiCl treatment improves behavioral outcome in an AD mouse model (Toledo and Inestrosa, 2009). The association of Dickkopf-1, a WNT-inhibitor, with neuronal degeneration reinforces the beneficial effects of WNT signaling on neuronal survival (Caricasole et al., 2004). Thus, decreased neuronal β-catenin is detrimental for nerve cell survival. Compensation for the loss of neuronal β-catenin by enhanced WNT signaling or pharmacological inhibition of GSK3 therefore appears to have neuroprotective potential (Alvarez et al., 2004; Chacón et al., 2008; De Ferrari et al., 2003).
Our studies point to an increase in β-catenin levels in microglia, thus raising an important question: is enhanced β-catenin signaling in microglia in brain inflammation beneficial or detrimental for disease outcome? On one hand microglia activation is—particularly in the case of AD—associated with positive effects based on the reduction of the Aβ load by phagocytosing microglia. If this function turns out to be prominently affected by treatment with GSK3 inhibitors, there is the potential for beneficial therapeutic effects through support of proinflammatory microglia. On the other hand, WNT-3A inflicting a robust proinflammatory phenotype in cultured microglia suggests that this pathway might enhance the ongoing inflammation with a negative outcome. Therefore, we conclude that selective blockade of WNT signaling in microglia could be of therapeutic significance. Current drug development, however, promotes GSK3 inhibitors for the treatment of AD to counteract the decrease of neuronal β-catenin and the increase in GSK3-mediated tau phosphorylation (Hooper et al., 2008; Hu et al., 2008). However, treatment with a GSK3 inhibitor would inevitably enhance microglial WNT signaling and could thus exacerbate proinflammatory responses, possibly imposing enhanced neurotoxicity as long-term side effect. Thus, it is necessary to evaluate the net effect of GSK3 inhibition by weighing in both neuronal tau hyperphosphorylation and microglia-dependent inflammatory processes.
The authors declare no conflict of interest. Dr. Botond Penke (University of Szeged, Hungary) is acknowledged for providing β-amyloid(1–42) peptide. J.M. is recipient of a research fellowship from ART UK.