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Keywords:

  • β-Catenin;
  • Neurogenesis;
  • Cell proliferation;
  • Subventricular zone;
  • Olfactory bulb;
  • Mash1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

The subventricular zone (SVZ) is the largest germinal zone in the mature rodent brain, and it continuously produces young neurons that migrate to the olfactory bulb. Neural stem cells in this region generate migratory neuroblasts via highly proliferative transit-amplifying cells. The Wnt/β-catenin signaling pathway partially regulates the proliferation and neuronal differentiation of neural progenitor cells in the embryonic brain. Here, we studied the role of β-catenin signaling in the adult mouse SVZ. β-Catenin-dependent expression of a destabilized form of green fluorescent protein was detected in progenitor cells in the adult SVZ of Axin2-d2EGFP reporter mice. Retrovirus-mediated expression of a stabilized β-catenin promoted the proliferation of Mash1+ cells and inhibited their differentiation into neuroblasts. Conversely, the expression of Dkk1, an inhibitor of Wnt signaling, reduced the proliferation of Mash1+ cells. In addition, an inhibitor of GSK3β promoted the proliferation of Mash1+ cells and increased the number of new neurons in the olfactory bulb 14 days later. These results suggest that β-catenin signaling plays a role in the proliferation of progenitor cells in the SVZ of the adult mouse brain.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

The largest germinal zone of the adult mammalian forebrain is the subventricular zone (SVZ) of the lateral ventricles, where new neurons are continuously generated [1, 2]. This region is composed of four cell types [3]: slowly dividing SVZ astrocytes (type B cells), rapidly dividing transit-amplifying cells (type C cells), migrating neuroblasts (type A cells), and ependymal cells (type E cells). Each of these cell types regulates its cell cycle by a different mechanism [4, [5], [6]7]. However, little is known about the signaling mechanisms that control cell proliferation in the adult SVZ.

The canonical Wnt pathway is an important regulator of mammalian neural development [8], and β-catenin is a critical downstream component of this pathway. Depending on its phosphorylation state, β-catenin can be found in the membrane, cytoplasm, or nucleus. In the absence of a Wnt signal, β-catenin is phosphorylated by casein kinase I and GSK3β and degraded by the ubiquitin-proteasome system. In the presence of a Wnt signal, GSK3β activity is inhibited, and unphosphorylated β-catenin accumulates in the cytoplasm and translocates into the nucleus where it promotes the transactivation of a variety of developmentally important genes [9].

Both in vitro and in vivo studies demonstrate that the Wnt/β-catenin pathway regulates the proliferation and differentiation of neural progenitor cells [10]. Several studies have demonstrated that the context-dependent role of this pathway in neuronal differentiation. For example, neuronal differentiation is induced in the pluripotent P19 cell line by overexpression of β-catenin [11] or pharmacological inhibition of GSK3β [12]. Neuronal differentiation is also induced in vitro by Wnt3a [13] or Wnt7a or by expression of a stabilized form of β-catenin [14].

In contrast, proliferation of neural progenitor cells is induced by activation of the β-catenin signaling pathway, and a GSK3β inhibitor can inhibit differentiation and maintain the self-renewal capacity of human and mouse embryonic stem cells [15]. Likewise, Wnt7a and Wnt7b stimulate the proliferation of neurogenic progenitors and primary neurospheres [16], and the growth of the hippocampus is inhibited in mice lacking Wnt3a [17] or LEF1 [18]. Stabilized β-catenin also causes an overgrowth of neural progenitor cells, resulting in a grossly enlarged brain [19]. A recent study has identified Wnt signaling as a regulatory pathway in adult hippocampal neurogenesis involved in the control of neuronal differentiation and the proliferation of progenitor cells [20]. Wnt3a and Wnt5a both increase the proliferation of cultured progenitor cells isolated from postnatal and adult mouse SVZ and promote their neuronal differentiation [21]. Thus, the Wnt/β-catenin signal can regulate the proliferation and/or differentiation of neural progenitor cells during development in a stage- and tissue-dependent manner. Here, we used pharmacological inhibitors and retroviral overexpression to study the in vivo effect of β-catenin signaling on neurogenesis in the adult SVZ. We found that β-catenin signaling regulates cell proliferation in the SVZ of mice in vivo.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Animals

Adult male C57/BL6 mice (9 weeks old) and Axin2-d2EGFP transgenic mice [22] were maintained on a 12-hour light/dark cycle with unlimited access to food and water. All animal-related procedures were approved by the Laboratory Animal Care and Use Committee of Keio University and the University of California San Francisco and were conducted in accordance with the guidelines of the NIH.

Synthesis and Characterization of Ro3303544

The indole maleimide Ro3303544 (supplemental online Fig. 1) is one of a series of highly selective GSK3β inhibitors (inhibitory concentration50 of 0.6 nM) that were prepared according to methods described in a published patent [23]. In brief, Ro3303544 was synthesized by the conversion of the corresponding furan-2,5-dione with ammonia in reflux in dimethylformamide. The furan-2,5-diones were synthesized from N-methyl-indol-3-yl-oxo-acetyl chloride and substituted with phenylacetic acid in dichloromethane in the presence of triethylamine.

For the assay of GSK3β enzyme activity, rabbit GSK3β cDNA was expressed as a GST fusion in BL21DE3 cells from the pGEX-3X vector [24]. To produce constitutively active GSK3β enzyme, 10 NH2-terminal amino acids were deleted [25]. Transformed BL21 DE3 cells were grown at 37°C to mid log-phase, and protein production was induced with isopropyl-beta-(d)-thiogalactopyranoside (0.4 mM) at 30°C for 2 hours. After the cells were homogenized, the extract was loaded on a glutathione-Sepharose 4B column, and GSK3β was eluted with glutathione buffer (50 mM Tris, pH 8, and 10 mM reduced glutathione). The eluate was collected in 3-minute fractions and assayed for GSK3β content on a 10% SDS-polyacrylamide gel electrophoresis. Fractions above 20% peak height were pooled and used in the assay. The GSK3β assay was performed in 50-μl reactions in a 96-well polypropylene plate; each reaction contained 20 mM magnesium chloride, 40 μM ATP, 2 mM dithiothreitol, 88.5 μM biotinylated and phosphorylated CREB-peptide substrate (biotin-KRREILSRRPS (p) YR), [γ-33P]ATP (1 μCi), and 2 μl of inhibitors in dimethyl sulfoxide (DMSO). Fifteen microliters of the pooled GSK3β elute was added, and the reaction mixture was incubated at 30°C for 1 hour. The reaction was stopped by transferring 25 μl of the reaction mixture to a phosphocellulose plate containing 130 μl of 1.85% phosphoric acid. The free radionucleotides in the membrane were washed off under vacuum with 1.85% phosphoric acid (five times). After the last wash, the plate was transferred to an adapter plate, 50 μl of scintillation cocktail (Microscint-20; catalog no. 20-133; PerkinElmer Life and Analytical Sciences, Waltham, MA, http://las.perkinelmer.com) was added to each well, and the amount of radioactivity was counted in a Top Count scintillation counter. Protein kinase C assays were performed using recombinant protein kinase C-α (catalog no. 14-232; Upstate, Charlottesville, VA, http://www.upstate.com) and substrate (catalog no. 17-139) according to the manufacturer's instructions. To assay the inhibitor's activity on other kinases, Ro3303544 was sent to Upstate Cell Signaling Solutions (Charlottesville, VA, http://www.upstate.com) for kinase panel testing.

β-Catenin Immunoblotting

7F2 mouse osteoblasts (ATCC CRL-12557) were cultured in growth medium in a six-well plate. Upon reaching confluence, the growth medium was replaced with differentiation medium (10 mM β-glycerophosphate and 50 μg/ml ascorbic acid) with 100, 300, or 500 nM Ro3303544 or vehicle control (DMSO). Twenty-four hours later, the cells were harvested, and nuclear and cytoplasmic extracts were prepared using a NE-PER kit (Pierce, Rockford, IL, http://www.piercenet.com), according to the manufacturer's instructions. Lysates (20 μg each) were subjected to immunoblotting with a monoclonal antibody against β-catenin (Transduction Laboratories, Lexington, KY, http://www.bdbiosciences.com/pharmingen). As a loading control, the blots were reprobed with either anti-β-actin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) or anti-Lamin A/C (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) monoclonal antibodies.

β-Catenin Enzyme Immunoassay

After 24 hours of incubation in six-well plates containing GSK3β inhibitors, Jurkat T cells (5 × 105 cells per milliliter) were washed in phosphate-buffered saline (PBS), harvested, and lysed in 0.3 ml of radioimmunoprecipitation assay buffer (catalog no. 1 920 693; Boehringer Mannheim, Mannheim, Germany, http://www.boehringer.com) according to the manufacturer's instructions. β-Catenin protein in the lysate was analyzed by enzyme immunoassay (EIA). The EIA was performed in 96-microwell plates; capture antibody (mouse monoclonal anti-β-catenin; Zymed, Carlsbad, CA, http://www.invitrogen.com)-coated wells were washed three times (PBS, 0.05% Tween 20), blocked with assay diluent (blocked with assay diluent; BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml), and then incubated at room temperature for at least 72 hours. The wells were washed, 100 μl of cell extract or various concentrations of purified β-catenin protein were added, and the plates were incubated for 2 hours at room temperature. The wells were washed again, 100 μl of anti-β-catenin antibody (β-catenin H-102, sc-7199, rabbit IgG; Santa Cruz Biotechnology) in assay diluent (1:1,250) was added, and the plates were incubated at room temperature for 2 hours. Antibody binding was measured using 100 μl of a 1:2,000 dilution of biotinylated mouse monoclonal anti-rabbit IgG antibody (B5283; Sigma-Aldrich) and 3,3′,5,5′-tetramethylbenzidine (catalog no. 2642KK; BD Pharmingen) for color development, according to the manufacturer's instructions.

GSK3β Inhibitor and Bromodeoxyuridine Administrations

Mice received two intraperitoneal injections per day of vehicle (saline) or Ro3303544 GSK3β inhibitor (500 μM) for 5 consecutive days. Bromodeoxyuridine (BrdU) (Boehringer Mannheim) (50 mg/kg) was given intraperitoneally to mice at 1, 3, or 14 days before sacrifice (n = 5 each). For intracerebroventricular administration, the GSK3β inhibitor (100 μM) or vehicle alone was infused into the right lateral ventricle of the brain for 7 days with a mini-osmotic pump (model 1007D; flow rate, 0.5 μl/hour; Alzet Osmotic Pumps, Cupertino, CA, http://www.alzet.com). The cannula was implanted stereotaxically at the following coordinates: anterior, 0 mm; lateral, 1.1 mm; depth, 2.3 mm (relative to the bregma and the surface of the brain). After 6 days of infusion, BrdU was injected into the mice (50 mg/kg i.p.). Twenty-four hours later, the pump was removed, and the mice were sacrificed for analyses.

Retroviral Injections into the SVZ

PMX-IRES-GFP, a replication-incompetent retroviral vector containing an internal ribosome entry site (IRES) sequence followed by the coding sequence for enhanced green fluorescent protein (GFP) [26], was used for the production of recombinant retroviruses. pMX-ΔN90β-catenin-IRES-GFP and pMX-Dkk1-IRES-GFP were described previously [14]. Ecotropic virus-packaging (PLAT-E) cells were transfected with each plasmid using FuGENE 6 (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) and then cultured for 3 days at 37°C. Retroviral particles were collected from the culture supernatant by centrifugation at 6,000g for 16 hours at 4°C and were resuspended in serum-free Dulbecco's modified Eagle's medium-Ham's F-12 medium (1:1) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The titers of the retroviral stocks used in this study were 7.0–7.3 × 105 colony-forming units per milliliter. Two hundred nanoliters of a viral suspension was injected into the right SVZ at the following coordinates: anterior, 1 mm; lateral, 1 mm; and depth, 3.4–1.8 mm.

Immunocytochemistry

Brains were perfusion-fixed with 4% paraformaldehyde and postfixed in the same fixative overnight, and 50 μm sections were cut on a Vibratome sectioning system (VT1000S; Leica, Heidelberg, Germany, http://www.leica.com). After three rinses in PBS, some sections were incubated either in acetone for 20 seconds on ice (for staining β-catenin) or in 2 N HCl for 30 minutes (for staining BrdU, proliferating cell nuclear antigen [PCNA], and Mash1). The sections were then incubated for 1 hour in TNB blocking solution (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), overnight with primary antibodies, followed by a 60-minute incubation at room temperature with biotinylated secondary antibodies (1:200) or Alexa Fluor-conjugated secondary antibodies (1:200; Molecular Probes Inc., Eugene, OR, http://www.probes.invitrogen.com), unless otherwise noted. Biotinylated antibodies were visualized using the ABC Elite kit (Vector Laboratories) and TSA (PerkinElmer Life and Analytical Sciences). The primary antibodies (final dilution and source) used in this study were as follows: mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:100) (catalog number G3893; Sigma-Aldrich), rabbit polyclonal anti-GFAP (1:1,000) (catalog number Z0334; Dako, Glostrup, Denmark, http://www.dako.com), mouse monoclonal anti-PSA-NCAM IgM (1:250, a gift from Tatsunori Seki) [27], mouse monoclonal anti-Mash1 (1:100) (catalog number 556604; BD Pharmingen), rat monoclonal anti-BrdU (1:100) (catalog number ab6326; Abcam, Cambridge, U.K., http://www.abcam.com), rabbit anti-GFP (1:200) (catalog number 598; MBL International Corp., Woburn, MA, http://www.mblintl.com), goat anti-doublecortin (anti-DCX) (1:200) (catalog number sc-8066; Santa Cruz Biotechnology), anti-phosphohistone H3 (1:200) (catalog number 06-570; Upstate), anti-β-catenin (1:200) (catalog number 610154; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), anti-PCNA (1:200) (catalog number NAO3T; Siemens Medical Solutions Diagnostics, Oncogene Science Biomarker Group, Cambridge, MA, http://www.oncogene.com), anti-NeuN (1:200) (catalog number MAB377; Chemicon, Temecula, CA, http://www.chemicon.com), and mouse monoclonal anti-cleaved Caspase-3 (1:200) (catalog number 9661; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com).

These antibodies have been shown to be specific in the following ways. The anti-GFP and anti-BrdU antibodies used in this study did not show any staining in brain sections of normal, untreated mice. The antibodies against anti-GFAP [28], PSA-NCAM [28], Mash1 [29], DCX [30], phosphohistone H3 [31], PCNA [32], NeuN [33], and cleaved Caspase-3 [34] have been widely used as specific markers for the immunohistochemistry of brain sections. The labeling patterns we obtained with these antibodies were consistent with previous reports. The anti-β-catenin antibody used in this study was purchased from Becton Dickinson and stains a single band of 92 kDa on Western blots (according to the manufacturer's technical information). This antibody also produced a staining pattern similar to that previously reported [35]. The number of SVZ cells containing nuclear β-catenin, which were detected using this antibody, was increased by GSK3β inhibitor injection, as expected for the specific labeling of β-catenin (Fig. 3). Finally, we further confirmed the specificity of this antibody by staining brain sections of mice that had received injections of pMX-IRES-GFP or pMX-ΔN90β-catenin-IRES-GFP retrovirus into the SVZ. The β-catenin signal was clearly colocalized with GFP in the SVZ treated with pMX-ΔN90β-catenin-IRES-GFP but not that treated with pMX-IRES-GFP (supplemental online Fig. 1), indicating that the signal produced by this antibody represents specific immunoreactivity with the β-catenin protein.

Quantitative Analysis of Immunohistochemistry

Using a Vibratome sectioning system, we first prepared a complete set of serial sections of the forebrain. We then selected coronal sections from two brains at the same level of the SVZ or olfactory bulb sections from five brains, all taken at the same level, and used them to count the number of cells positive for each marker. Quantitative analyses of marker expression were performed using a Zeiss LSM 510 Laser Scanning Confocal Microscope. For SVZ analyses, we chose two coronal sections from each brain from two anterior SVZ regions: one contained the anterior half of the anterior horn (between 1.18 mm anterior and 0.58 mm anterior to the bregma) and the other contained the posterior half (between 0.58 mm anterior and 0.02 mm posterior to the bregma). We counted all the detectable cells labeled with each marker on both sides of the SVZ, which was identified by its higher cell density. For counting nuclear β-catenin-containing cells, we used three-dimensional image stacks.

Statistical Analysis

All the quantified data are expressed as the mean ± SD. Retroviral results were evaluated for statistical significance by analysis of variance with Dunnett's test. Differences between two means were analyzed by the unpaired Student's t-test and were considered statistically significant when the p value was less than 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Axin2-d2EGFP Reporter Is Expressed in Type B and C Cells in SVZ

We first analyzed activation of β-catenin-induced gene expression in each cell type in the SVZ using Ax2-d2EGFP transgenic mice. In these mice, the expression of a green fluorescent protein with reduced stability (d2EGFP) is driven by the promoter and first intron of the Axin2 gene, a direct target of the Wnt pathway whose activation is mediated by Tcf/LEF factors [22]. The 5.6-kilobase Axin2 genomic sequence, including the promoter and first intron, which contains eight Tcf/LEF consensus-binding sites, is sufficient to direct the tissue-specific expression of d2EGFP in transgenic embryos. Therefore, the Ax2-d2EGFP reporter is a sensitive and reliable reporter for monitoring the activation of β-catenin signaling in vivo. Coronal sections of the Ax2-d2EGFP reporter mouse brains showed d2EGFP expression in the SVZ within the lateral wall of the lateral ventricles (Fig. 1A). To identify each cell type in the SVZ, we used the following markers: GFAP, which is expressed in type B cells [3]; Mash1, which is expressed in type C cells [29, 36] and in a subpopulation of type B cells (Z.M. and A.A.-B., unpublished data); and DCX, which is expressed in type A cells [3] (Fig. 1B–1M). GFP was detected in 42.5% ± 5.4% of Mash1+ cells. GFP was also detectable in a subpopulation of GFAP+ type B cells, although the fibrous and fragmentary pattern of GFAP staining prevented quantitative analysis. GFP was not detected in DCX+ type A cells. These results suggest that the β-catenin signaling is activated in type B and C cells in the adult SVZ.

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Figure Figure 1.. β-Catenin-responsive cells in the adult SVZ. (A): Coronal section of Ax2dEGFP reporter mouse shows GFP expression in cells of the SVZ, on the lateral wall of the lateral ventricle. (B–M): At higher magnification, GFP-expressing cells were found coexpressing GFAP (B–E) or Mash1 (F–I), but not Dcx (J–M). Abbreviations: Ax2dEGFP, Axin2–d2EGFP; DAPI, 4,6-diamidino-2-phenylindole; Dcx, doublecortin; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein.

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β-Catenin Signaling Increases the Number of Mash1+ Cells

We next tested the effects of β-catenin signaling on SVZ cells. To activate or inactivate β-catenin signaling specifically in mitotic SVZ cells, we injected the following retroviral vectors [14]: pMX-IRES-GFP (as a control retrovirus), pMX-ΔN90β-catenin-IRES-GFP (to express a constitutively active form of β-catenin) [37], and pMX-mDKK-IRES-GFP (to express DKK-1, which antagonizes frizzled receptors and inhibits β-catenin signaling) [38, [39]40]. Two days after the injections, we quantified the percentage of infected cells expressing GFP that were also positive for Mash1 (for type C cells and a subset of B cells) or DCX (type A cells) [41]. The percentage of Mash1+ cells was significantly increased by ΔN90β-catenin and decreased by mDKK-1 (Fig. 2A–2K; *, p < .05; **, p < .01; n = 5). On the other hand, the percentage of DCX+ type A cells was decreased after ΔN90β-catenin expression and was increased after mDKK-1 expression (Fig. 2L–2U; **, p < .01; n = 5). Taken together, these results indicate that β-catenin signaling increases the percentage of Mash1-positive cells in the SVZ.

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Figure Figure 2.. The β-catenin signal increases the number of type C cells in the subventricular zone. (A–K): Effects of a retrovirally introduced stabilized form of β-catenin or Dkk1 on the maintenance of type C cells. (A): Timeline of the experiment. (B–J): Sections of subventricular zone harvested 2 days after the stereotaxic injection of pMX-IRES-GFP (B–D), pMX-ΔN90β-catenin-IRES-GFP (E–G), or pMX-mDKK-IRES-GFP (H–J), stained for Mash1 (red). Infected cells and their progenies expressed GFP (green). (K): The percentage of Mash1-positive cells in the GFP-positive cell population was significantly decreased by mDKK-1 (*, p < .05) and increased by ΔN90β-catenin (**, p < .01). (L–U): Effects of a retrovirally introduced stabilized form of β-catenin or Dkk1 on the differentiation of type C cells into type A cells. (L–T): Sections of subventricular zone harvested 2 days after the stereotaxic injection of pMX-IRES-GFP (L–N), pMX-ΔN90β-catenin-IRES-GFP (O–Q), or pMX-mDKK-IRES-GFP (R–T), stained for Dcx (red). Infected cells and their progenies expressed GFP (green). (U): The percentage of Dcx-positive cells in the GFP-positive cell population was significantly increased by DKK1 (**, p < .05) and decreased by ΔN90β-catenin (**, p < .05). Scale bar = 50 μm. Abbreviations: Dcx, doublecortin; GFP, green fluorescent protein; SVZ, subventricular zone.

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β-Catenin Signaling Promotes Cell Proliferation in the SVZ

We next examined the effect of a novel highly potent and selective inhibitor of GSK3β enzyme activity (Ro3303544; supplemental online Fig. 2) on cellular proliferation in the adult SVZ. Since pharmacological inhibition of GSK3β enzyme activity results in activation of β-catenin signaling, this enabled us to examine the functional importance of this pathway in cells within the SVZ. Ro3303544 inhibits the GSK3β kinase activity at nanomolar concentrations but does not inhibit other kinases tested (supplemental online Table 1). In vitro treatment of two different cell types with this inhibitor produced a dose-dependent increase in cytoplasmic and nuclear β-catenin (supplemental online Fig. 3A, 3B), indicating activation of the canonical Wnt pathway.

Administration of Ro3303544 over a 5-day period resulted in a marked increase in the number of cells with nuclear β-catenin relative to those that received vehicle control (Fig. 3A–3G; **, p < .01; n = 5). The number of Mash1+ cells with detectable nuclear β-catenin was increased after Ro3303544 administration (Fig. 3H; *, p < .05; n = 5). These results indicate that administration of Ro3303544 activated β-catenin signaling in Mash1+ cells in the adult SVZ.

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Figure Figure 3.. Administration of GSK3β inhibitor enhances the nuclear localization of β-catenin. SVZ sections stained for β-catenin (green) and Hoechst (blue) from mice that received two intraperitoneal injections per day of vehicle (A–C) or Ro3303544 (D–F) for 5 consecutive days. A strong nuclear β-catenin signal could be observed in many cells in the Ro3303544-injected animals (white arrows). (G) Ro3303544-injected mice had a significantly increased number of nuclear β-catenin-containing cells (**, p < .01 vs. control). (H) Ro3303544-injected mice had a significantly increased number of nuclear β-catenin-containing Mash1-positive type C cells (*, p < .05). Scale bar = 10 μm. Abbreviation: SVZ, subventricular zone.

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To study the effect of this drug on cell proliferation, mice were treated with Ro3303544 by intraperitoneal infusion for 5 days (Fig. 4A–4D, 4F) or by intracerebroventricular administration for 7 days (Fig. 4E), followed by BrdU injections to label proliferating cells and sacrificed 1 day later. The number of SVZ cells that were labeled with BrdU or phosphohistone H3, which is a M phase cell cycle marker [42], was assessed in drug- and vehicle-treated mice. There was a significant increase in the number of labeled cells in the SVZ of Ro3303544-treated mice relative to the vehicle-infused control group (Fig. 4A–4F; *, p < .05; **, p < .01; n = 5 mice each). There was no significant difference in the number of cleaved-Caspase3-positive cells in the SVZ between the animals treated with Ro3303544 and vehicle alone (data not shown; p = .17; n = 5). This suggests that the drug-induced increase in the number of BrdU+ cells was caused by increased proliferation rather than increased cell survival [43, 44].

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Figure Figure 4.. β-Catenin signaling promotes cell proliferation in the SVZ. (A–F): Effect of GSK3β inhibitor on the number of proliferating cells in the SVZ. Mice received intraperitoneal injections of vehicle or Ro3303544 for 5 consecutive days. After the injection period, the mice received an intraperitoneal injection of BrdU and were perfused 24 hours later (A). (B, C): BrdU-labeled proliferating cells in the SVZ of mice given control vehicle (B) or Ro3303544 (C). (D, E): Significantly increased numbers of BrdU-labeled cells were observed in the SVZ of mice that received the Ro3303544 injections i.p. (D) or icv (E), compared with the saline-injected controls. (F): Significantly increased numbers of phosphohistone H3-positive cells were observed in the SVZ of mice that received Ro3303544 injections intraperitoneally compared with the saline-injected control mice. GSK3β inhibitor increases the type C cell population in the SVZ. (G–I): Mash1-positive type C cells (red) in the SVZ of mice that received two intraperitoneal injections per day for 5 consecutive days of vehicle (G) or Ro3303544 (H). (I): Significantly increased numbers of Mash1-positive cells were observed in the SVZ of mice injected with Ro3303544 compared with the control mice. (J–Q): Mice were treated with vehicle or Ro3303544 for 5 days. BrdU was injected on the third day in the treatment, and the mice were sacrificed 3 days later (J). (K–P): Sections of the SVZ of mice that received saline (K–M) or Ro3303544 (N–P) injections, stained for BrdU (green) and Mash1 (red). (Q): The percentage of Mash1-positive cells in the BrdU-labeled cell population was significantly increased in the Ro3303544-treated mice compared with the controls (*, p < .05). Scale bars = 20 μm (K–P). Abbreviations: BrdU, bromodeoxyuridine; icv, intracerebroventricularly; SVZ, subventricular zone.

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We next studied the effects of pharmacologically activating β-catenin signaling on SVZ progenitor cells. Intraperitoneal administration of Ro3303544 for 5 days (two injections per day) resulted in a marked increase in the number of Mash1+ cells in the SVZ (Fig. 4G–4I; *, p < .05; **, p < .01; n = 5). Of note, the percentage of Mash1+ cells that were labeled with BrdU on day 4 was significantly increased after Ro3303544 administration (Fig. 4J–4Q; *, p < .05; n = 5). Similarly, retroviral injection studies indicated that β-catenin signaling is required and sufficient for promoting cell proliferation in the SVZ (supplemental online Fig. 4). Taken together, these results suggest that pharmacological or retroviral activation of β-catenin signaling increases the number of proliferating Mash1+ cells in the SVZ.

Activation of β-Catenin Signaling Increases the Number of Newborn Neurons in the Olfactory Bulb

We next tested whether increased β-catenin signaling-induced expansion of the progenitor pool within the SVZ results in increased neurogenesis in the olfactory bulb (OB). To assess this, we counted the number of newly formed neurons in mice appearing after administration of Ro3303544. Mice were treated with Ro3303544 or vehicle for 5 days, followed by BrdU injection, and then sacrificed 14 days after the last injection. BrdU/NeuN-double-positive cells in the OB were counted to indicate the number of newly formed neurons. Administration of Ro3303544 resulted in a marked increase in the number of BrdU-NeuN double-positive cells in the OB (Fig. 5A–5D; **, p < .01; n = 5). There was no significant difference in the percentage of BrdU+ cells that were NeuN+ between these two groups (control, 68.3% ± 6.8%; Ro3303544, 76.1% ± 2.6%), indicating that the Ro3303544 treatment did not affect the percentage of neurons formed. These data suggest that the β-catenin signaling-induced expansion of the progenitor population results in more new neurons in the OB.

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Figure Figure 5.. GSK3β inhibitor increases the number of newly generated neurons in the OBs. (A): Ro3303544 or vehicle was intraperitoneally administered for 5 days. The mice received an intraperitoneal injection of BrdU on the final day of the treatment and were perfused 14 days later. (B, C): Newborn neurons (white arrows) stained for BrdU (green) and NeuN (red) in the OB of mice treated with saline (B) or Ro3303544 (C). (D): Significantly increased numbers of BrdU/NeuN double-positive newborn neurons were observed in the Ro3303544-treated mice compared with the control (**, p < .01). Abbreviations: BrdU, bromodeoxyuridine; OB, olfactory bulb.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Although its functional effect on neural development has been extensively investigated, relatively little is known about the effect that β-catenin signaling has on neurogenesis in the adult brain. The lack of information results from the fact that modulation of this signaling pathway causes embryonic lethality. In the present study, we used a pharmacological inhibition and retroviral expression to activate or inhibit the pathway, to demonstrate that β-catenin signaling promotes proliferation of Mash1+ cells in the SVZ and increased number of new neurons in the OB of the adult mouse brain. A recent study showed that Wnt ligands (Wnt1, Wnt5a, and Wnt7a) and other components of the Wnt signaling pathway-including the Fz receptors (Fz3, Fz7, and Fz10), soluble Frizzled-like receptors (sFrp1 and sFrp2), and Tcf3 are expressed in the adult SVZ [45]. Furthermore, in vitro experiments using a reporter plasmid have demonstrated that the canonical Wnt pathway is active in neuronally committed progenitors in the adult hippocampus [20]. Analysis of mice expressing the Axin2-d2EGFP reporter transgene (Fig. 1) indicates that β-catenin signaling is activated in type B and C cells in the adult SVZ. Consistently, nuclear β-catenin was detectable in GFAP+ and Mash1+ cells but not in DCX+ cells (data not shown). In this study, we focused on the role of the β-catenin signaling on type C cells, the most actively proliferating cells in the adult SVZ.

Genetic approaches are needed to directly and specifically study the effect of increased or decreased β-catenin activity on neuronal development. In adult transgenic mice that express the stabilized form of β-catenin, which lacks the GSK3β phosphorylation site, under the control of nestin second intronic enhancer, the SVZ is enlarged [46]. However, it is likely that the transgene expression in these mice affects neural development from embryonic stages, which makes it difficult to study its function in adult neurogenesis. Therefore, we injected retroviral vectors to activate or inhibit β-catenin signaling specifically in dividing cells in the adult mouse SVZ. Our results clearly demonstrate that β-catenin signaling is sufficient to increase the percentage of dividing Mash1+ cells (Fig. 4; supplemental online Fig. 4).

To study the role of β-catenin signaling in the adult brain, lithium has been used as an inhibitor for GSK3β. Administration of lithium increases neurogenesis in the DG of adult rodents [47, 48]. However, lithium's targets and actions are still unclear; it affects multiple signaling cascades other than GSK3β in the brain [49]. To overcome these technical limitations, we used a recently developed specific inhibitor for GSK3β. In our pharmacological study, administration of Ro3303544 over a 5-day period resulted in a marked increase in the number of Mash1+ cells with detectable nuclear β-catenin (Fig. 3H). Our data show that β-catenin signaling is sufficient for promoting cell proliferation in the SVZ and increasing the number of proliferating Mash1+ cells (Fig. 4). These data indicate that β-catenin signaling in the adult mouse SVZ plays a role in the proliferation or production of Mash1+ cells.

In the nucleus, β-catenin binds TCF/LEF1 and activates the expression of target genes that are involved in the G1-S transition, such as cyclin D1 [50, 51] and c-Myc [52]. The proliferation of progenitor cells in the adult mammalian SVZ seems to be regulated in part by cyclin D1 transcription [53] and c-myc [54]. Therefore, it is possible that β-catenin signaling could promote the proliferation of SVZ progenitor cells through these factors. In our retrovirus injection experiments (Fig. 2), activated β-catenin did not prevent the differentiation of type C cells into neurons, suggesting that other factors, such as Noggin [55] and brain-derived neurotrophic factor [56], can overcome the proliferation-stimulating effect of β-catenin and induce differentiation.

Neurogenesis can be controlled at multiple points, such as cell proliferation, differentiation, and migration. The present study demonstrates that the activated β-catenin signal expanded the population of proliferating SVZ neuronal precursors, which resulted in an increase in the number of newborn neurons in the OB (Fig. 5). Since type C cells proliferate more rapidly than type B cells [3], regulation of the proliferation of type C cells could be the key step controlled by β-catenin signaling. However, the precise stage at which Wnt signaling is acting in SVZ progenitor cells remains to be further investigated. Our results indicate that a sizable population of type B cells also responds to β-catenin activation, and this could result in the production of larger numbers of type C cells. Thus, our work demonstrates that β-catenin is involved in the control of proliferation of neural progenitor cells that give rise to new neurons in the SVZ. Clinically, strategies for increasing the number of new neurons in the adult brain may be useful for regenerating damage to the central nervous system [2], including cerebral infarction [33, 57], cerebral contusion [58], and Alzheimer disease [59]. The analysis of β-catenin function will add to our understanding of the mechanisms of adult neurogenesis, which may lead to the development of novel therapies for human brain damage.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

We are grateful to Dr. Tatsunori Seki for the PSA-NCAM antibody, Dr. Frank Costantini for Ax2-d2EGFP mouse, and members of our laboratories for valuable discussions. This work was supported by Bridgestone Corporation and grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan; The Ministry of Health, Labor and Welfare, Japan; Japan Science and Technology Agency (Core Research for Evolutional Science and Technology); the Mochida Memorial Foundation for Medical and Pharmaceutical Research; the Inamori Foundation; and the Takeda Foundation. Work in the Alvarez-Buylla laboratory was supported by NIH Grant HD32116 and a gift from Frances and John Bowes, and by NIH Grant P30 DK063720 to the Diabetes Center Microscopy Core.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
doc_Supplemental_fig2.pdf18KSupplemental Figure 2
doc_Supplemental_Figure_3.pdf57KSupplemental Figure 3
Adachi_Supplemental_Table.pdf67KSupplemental Table
doc_Supplement_Figure4.pdf1085KSupplemental Figure 4
doc_supplemental_fig1.tif2784KSupplemental Figure 1

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