Ankyrin-G regulates neurogenesis and Wnt signaling by altering the subcellular localization of β-catenin

ORIGINAL ARTICLE

Ankyrin-G regulates neurogenesis and Wnt signaling byaltering the subcellular localization of β-cateninO Durak1,6, FC de Anda1,6,7, KK Singh1,8, MP Leussis2,3,4,9, TL Petryshen2,3,4, P Sklar5 and L-H Tsai1

Ankyrin-G is a scaffolding protein required for the formation of the axon initial segment in neurons. Recent genome-wideassociation studies and whole-exome sequencing have identified ANK3, the gene coding for ankyrin-G, to be a risk gene formultiple neuropsychiatric disorders, such as bipolar disorder, schizophrenia and autism spectrum disorder. Here, we describe anovel role for ankyrin-G in neural progenitor proliferation in the developing cortex. We found that ankyrin-G regulates canonicalWnt signaling by altering the subcellular localization and availability of β-catenin in proliferating cells. Ankyrin-G loss-of-functionincreases β-catenin levels in the nucleus, thereby promoting neural progenitor proliferation. Importantly, abnormalities inproliferation can be rescued by reducing Wnt pathway signaling. Taken together, these results suggest that ankyrin-G is requiredfor proper brain development.

Molecular Psychiatry advance online publication, 13 May 2014; doi:10.1038/mp.2014.42

INTRODUCTIONGenome-wide association studies have identified many genes thatare associated with a risk for mental disorders, such as bipolardisorder, schizophrenia and autism spectrum disorder (ASD).1–7

The ANK3 gene is among the most consistently replicated andstatistically significant bipolar risk gene identified in thesestudies.6–8 A number of single-nucleotide polymorphisms inANK3 have been associated with bipolar disorder, and several ofthese variants have been associated with altered white matterintegrity and changes in cortical–striatal–thalamic circuits, as wellas with poorer performance on tasks of sustained attention.9,10

A 2012 study described decreased levels of ANK3 mRNA expres-sion in schizophrenia patients.11 Moreover, whole-exome and-genome sequencing studies identified putative causative muta-tions in coding and noncoding regions of ANK3 in patientswith ASD and intellectual disabilities.12–14 However, it remainsunknown how alterations in ANK3 may contribute to thepathology of mental illness.ANK3 encodes a scaffolding protein, ankyrin-G, which, in mature

neurons, localizes to the axon initial segment and the nodes ofRanvier.15,16 Ankyrin-G is required for the assembly and main-tenance of the axon initial segment, which is established throughits interaction with scaffolding and transmembrane proteins, andvoltage-dependent sodium and potassium channels.16,17 Recently,several studies have suggested that ankyrin-G is required forestablishing and maintaining neuronal polarity, suggesting a centralrole for this protein in establishing intact neural circuitry.17,18

Additionally, ankyrin-G has been shown to be a binding partner of

E-cadherin in epithelial cells, and is required, along with β2-spectrin,for the localization of E-cadherin to cell adhesion sites, where itassembles a complex that includes the important Wnt pathwaycomponent β-catenin.19

Canonical Wnt signaling has an important role in neuralprogenitor proliferation in the developing central nervous system,and is also involved in dendrite development, synaptogenesis andthe establishment of axons.20–23 Recent studies using comparativegenome sequencing of human patients, as well as the examina-tion of neuronal development in rodents, have demonstrated theimportance of canonical Wnt signaling in neuropsychiatricdisorders, such as schizophrenia, bipolar disorder and ASD.24,25

For instance, the product of the gene disrupted in schizophrenia 1 hasbeen shown to regulate several aspects of cortical developmentsuch as neural progenitor proliferation and dendritogenesis.26–29

Additionally, the autosomal recessive primary microcephaly riskgene ASPM was shown to regulate Wnt signaling and progenitorproliferation during cortical development.30 These studies high-light the importance of the molecular pathways that underlie earlycortical development to the etiology of complex psychiatric andneurodevelopmental disorders.β-catenin is one of the key components of the Wnt signaling

pathway. Upon activation of canonical Wnt signaling, via theinteraction of the Wnt peptide with its membrane receptors LDL-receptor-related protein and Frizzled, β-catenin is stabilized in thecytoplasm through the inhibition of glycogen synthase kinase 3(GSK3β), which normally promotes the proteolytic degradationof β-catenin. Once stabilized, β-catenin then enters the nucleus,

1Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA; 2Psychiatric andNeurodevelopmental Genetics Unit, Center for Human Genetic Research and Department of Psychiatry, Massachusetts General Hospital, Boston, MA, USA; 3Department ofPsychiatry, Harvard Medical School, Boston, MA, USA; 4Stanley Center for Psychiatric Research, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge,MA, USA and 5Division of Psychiatric Genomics, Departments of Psychiatry, Neuroscience and Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY,USA. Correspondence: Dr L-H Tsai, Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 77 MassAvenue, 46-4235, Cambridge, MA 02139, USA.E-mail: [email protected] authors contributed equally to this work.7Current Address: Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany.8Current Address: Departments of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.9Current Address: Department of Psychology, Emmanuel College, Boston, MA, USA.Received 18 June 2013; revised 17 March 2014; accepted 20 March 2014

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where it binds to T-cell factor/lymphoid-enhancing factor(TCF/LEF) family transcription factors to activate the expressionof Wnt target genes.20 In addition to its central role in Wntsignaling, β-catenin also binds to type I cadherins at the cellmembrane, linking them to the actin cytoskeleton and therebyhaving a role in structural organization.31 Several lines of evidencesuggest that altering the levels of β-catenin localized to thecatenin–cadherin complex can affect the availability of β-cateninfor participation in Wnt signaling.31–34 This includes the observa-tion that the sequestration of β-catenin at the cell membrane viaoverexpression of the cytoplasmic domain of cadherins and therepression of E-cadherin have opposite effects on Wnt signaling.Importantly, it has previously been shown that overexpression ofβ-catenin can perturb the development of mammalian cortex.20

In the current study, we show that ankyrin-G is highly enrichedin the ventricular zone (VZ) of the embryonic brain and is requiredfor proper neural progenitor proliferation. Ankyrin-G loss-of-function leads to increased neural progenitor proliferation andincreased canonical Wnt signaling. This is accompanied by adisruption of the β-catenin/cadherin interaction and increase inthe nuclear pool of β-catenin. These results suggest that ankyrin-Gcan regulate canonical Wnt signaling via the fine-tuning ofavailable levels of β-catenin, thereby ensuring proper braindevelopment. As the importance of canonical Wnt signaling inneuropsychiatric disorders has become increasingly evident, ourresults shed light on the molecular events that, when influencedby human disease genes, may contribute to the etiology ofneuropsychiatric disorders.

MATERIALS AND METHODSDNA constructsShort hairpin RNA (shRNA) constructs used in this study were cloned intopLKO.2 PU6-shRNA/PUbiC-eGFP vector available from Broad Institute(Cambridge, MA, USA). Control non-targeting shRNA (Sigma-Aldrich,St Louis, MO, USA; SHC002) and shRNA2 targeting Ank3 were obtainedfrom the Petryshen lab at Massachusetts General Hospital (Boston, MA,USA). shRNA1 targeting Ank3 is obtained from Broad Institute. Thesequences can be found in Supplementary Table S1. Full-length HA-GSK3β(a kind gift from Dr Yingwei Mao at Penn State University, State College, PA,USA) were previously used in Mao et al.26 Super8XTOPFLASH, a gift from DrR Moon (University of Washington, Seattle, WA, USA) and a Renilla-Luc-TKreporter (pRL-TK; Promega, Madison, WI, USA) were used for testing TCFtranscriptional activity. pCAGIG-Venus was provided by Dr Zhigang Xie(Boston University, Boston, MA, USA).

AnimalsSwiss Webster pregnant female mice were purchased from Taconic(Hudson, NY, USA) for in utero electroporation. Ank3 +/− mice were kindgifts from Bennett Lab (Duke University, Durham, NC, USA) and have beendescribed elsewhere.35,36

Cell cultureP19 embryonic carcinoma cell line was cultured in Dulbecco’s modifiedEagle's medium GlutaMAX (10566-016; Invitrogen, Life Technologies,Grand Island, NY, USA) containing 10% fetal bovine serum and penicillin/streptomycin.

Antibodies and reagentsThe following primary antibodies were used in this study: rabbit anti-ankyrin-G antibody (H-215; Santa Cruz Biotechnology, Dallas, TX, USA),rabbit anti-β-catenin antibody (H-102; Santa Cruz Biotechnology), mouseanti-β-catenin antibody (610153; BD Transduction Laboratories, San Jose,CA, USA), mouse anti-ZO-1 antibody (33-9100; Invitrogen), chicken anti-green fluorescent protein (GFP) antibody (GFP-1020; Aves Labs, Tigard, OR,USA), mouse anti-5-bromo-2'-deoxyuridine (BrdU) antibody (M0744; CloneBu20a; DakoCytomation, Glostrup, Denmark), rabbit anti-Ki67 antibody(Clone SP6; Lab Vision/Thermo Scientific, Waltham, MA, USA), rabbit anti-phospho-histone H3 (Ser10) antibody (06-570; Millipore, Billerica, MA, USA),

mouse anti-GSK3β antibody (610202; BD Transduction), mouse anti-pY216GSK3β antibody (ab75745; Abcam, Cambridge, MA, USA), anti-actin(Sigma-Aldrich; A5316; clone AC-74) and mouse anti-E-cadherin antibody(610181; BD Transduction). Alexa-conjugated secondary antibodies (Jack-son ImmunoResearch, West Grove, PA, USA) were used for immunohis-tochemistry and immunocytochemistry. Recombinant human Wnt3a waspurchased from R&D Systems (catalog number: 5036-WN, Minneapolis,MN, USA). BrdU was purchased from Sigma-Aldrich (B5002-5G).

ImmunohistochemistryBrain sections. Embryonic cortical brains were drop-fixed overnight in 4%formaldehyde and then transferred to 30% sucrose/phosphate-bufferedsaline (PBS) solution at 4 °C. Brains were embedded in OCT compound(Electron Microscopy Sciences, Hatfield, PA, USA) and sliced into 14–20 μmsections using cryostat. Cryosections were rehydrated with 1× PBS andblocked for 1–2 h with blocking solution (1× PBS+10% donkey normalserum+0.3% Triton-X). Following blocking, the cryosections were incubatedwith primary antibodies overnight at 4 °C. Incubation with secondaryantibodies were performed for 1 h at room temperature. Finally, cryosec-tions were mounted using ProLong Gold Antifade Reagent (Invitrogen).

Cell cultures. Cell cultures were plated onto coverslips in 24-well plates.Following transfection with Lipofectamine 2000, cells were fixed with 4%formaldehyde at room temperature for 10min and then washed three timeswith 1×PBS. Following 30min blocking, they were incubated with primaryantibodies for 45–60min, washed again with 1×PBS, incubated with secon-dary antibodies for 30min and finally washed and mounted for imaging.

Western blot analysisTransfected cells were lysed and run on 8% sodium dodecylsulfate-polyacrylamide gels at 60–120 constant voltage to separate, and trans-ferred onto Immobilon-P PVDF membranes (Millipore) at a constantcurrent. Membranes were blocked using 5% bovine serum albuminprepared in TBS-T (50 mM Tris-HCl (pH 7.4), 150mM NaCl, 0.1% Tween-20)for 30min at room temperature. Membranes were incubated with theprimary antibodies overnight at 4 °C. Next, they were washed three timeswith TBS-T, followed by incubation with horseradish peroxidase-con-jugated secondary antibodies (GE Healthcare, Pittsburgh, PA, USA) for 1 hat room temperature. Following washing with TBS-T, immunoreactivitysignals were detected by enhanced chemiluminescence (Perkin-Elmer,Waltham, MA, USA).

qPCRTotal RNA was collected using the RNeasy Plus Kit (Qiagen, Valencia, CA,USA) 48 h after transfection. Reverse transcription of the mRNA transcriptsto produce cDNA for quantitative polymerase chain reaction (qPCR) wasachieved using the SuperScript III Reverse Transcriptase (Invitrogen). qPCRwas performed using SsoFast Evagreen Supermix (Bio-Rad, Hercules, CA,USA) on CFX96 Real-Time PCR Detection System (Bio-Rad). The reactionswere run in triplicates and average of these triplicates were used forstatistical analysis. β-Actin was used as internal control. Primer sequencesused for qPCR can be found in Supplementary Table 1.

ImmunoprecipitationAt 48 h after transfection, transfected cells were lysed in 1 × lysis buffer(150mM NaCl, 0.1% NP-40, 50 mM Tris (pH 7.5), 5 mM EDTA) with proteaseinhibitors. Whole-tissue lysates of Ank3 +/− mice brains were prepared byhomogenization in 500 μl solution, including 50mM Tris, 120mM NaCl and0.5% NP-40 with protease inhibitors. Protein (0.5 mg) from each conditionwas added to protein A sepharose beads (GE Healthcare) conjugated withβ-catenin antibody, and incubated overnight at 4 °C. The beads were thenwashed with RIPA and lysis buffers before boiling in Laemmli samplebuffer. Following sodium dodecylsulfate-polyacrylamide gel electrophor-esis to separate the proteins, blots were probed with E-cadherin antibody.

In utero electroporationThe Institutional Animal Care and Use Committee of MassachusettsInstitute of Technology approved all experiments. In utero electroporationwas performed as described elsewhere.37 Briefly, pregnant Swiss Webstermice were anesthetized by intraperitoneal injections of ketamine 1%/xylazine2mgml− 1, the uterine horns were exposed, and the plasmids mixed with

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Fast Green (Sigma-Aldrich) were microinjected into the lateral ventricles ofembryos. Five pulses of current (50ms on/950ms off) were deliveredacross the head of the embryos. The following voltages were used fordifferent ages: 28–30 V for E13 and 32–35 V for E15. In the DNA mixture,the shRNA plasmid concentration was two- to threefold higher than that ofpCAGIG-Venus. For rescue experiments with GSK3β, the ratio between theankyrin-G shRNA and GSK3β was 3:1 in coexpression DNA mixture.

Luciferase assayLuciferase assays were performed as described elsewhere.26 P19 cells at1 × 105 cell per well density were plated into 24-well plates withoutantibiotics. Cells were transfected with 0.8 μg of shRNA plasmid along with50 ng of Super8XTOPFLASH and 10 ng of pRL-TK. The media were replacedwith one containing antibiotics 2 h after transfection. Either 24 or 36 h aftertransfection, cells were stimulated with recombinant human Wnt3a foreither 16 or 12 h, respectively, in Wnt-stimulated condition. TCF/LEFreporter activity was measured using the Dual-Luciferase Reporter AssaySystem (Promega). For the rescue experiments, 0.2 μg of HA-GSK3β wasco-transfected with 0.6 μg of ankyrin-G shRNA. Firefly luciferase activitywas normalized to Renilla luciferase activity in all conditions.

RESULTSAnkyrin-G is necessary for proper embryonic neural progenitorproliferationTo understand whether ankyrin-G has a role in embryonic cerebralcortical development, we first examined the expression pattern ofankyrin-G in the mouse embryonic day 15 (E15) brain, at whichperiod the proliferation and differentiation of cortical neurons areboth at high levels.38 We found that ankyrin-G is highly expressedin the ventricular surface of developing cortex (Figure 1a).Previous studies have shown that endfeet of radial glial cellsat the ventricular surface contact neighboring cells throughcell junctions, which is composed of two distinct domains:ZO1/mammalian Par3 (mPar3) (apical) and β-catenin/N-cadherin(basal).39 A more careful examination of ankyrin-G expression atthe ventricular surface revealed that ankyrin-G is expressed inboth the domains colocalizing with both β-catenin (Figure 1a, toprow of images) and ZO1 (Figure 1a, bottom row of images).β-Catenin is well known to regulate multiple steps of neurogen-esis, including proliferation, differentiation and radial migra-tion.21,31 The similarity in the expression patterns of β-cateninand ankyrin-G encouraged us to ask whether ankyrin-G regulatesneural progenitor proliferation at early stages of corticogenesis. Toaddress this question, we selected two small hairpin RNAs(shRNA1 and shRNA2) based on their ability to downregulateendogenous ankyrin-G in the P19 embryonic carcinoma cell line,as assayed by western blot and qPCR (Supplementary Figures S1a and b). We used these two shRNAs to examine the effect ofankyrin-G knockdown on embryonic neural proliferation. Weperformed in utero electroporation at E13, using a combination ofeither a non-targeting scrambled shRNA (Control) or ankyrin-GshRNA expression constructs, in addition to a GFP expressionconstruct to label electroporated cells. This was followed by pulselabeling either at E15 or E16 with BrdU, which incorporates intothe newly synthesized DNA of replicating cells during the S phaseof the cell cycle. The brains were harvested either 2 or 24 hfollowing the BrdU injection and immunolabeled with antibodiesagainst BrdU, the proliferative marker Ki67 (which is only absentfrom cells in G0 phase30), and GFP. We found that ankyrin-Gknockdown resulted in a significant increase in BrdU incorporationin the GFP-positive (GFP+) cell population compared with controlsafter both 2 h (Figures 1b and c) and 24 h (SupplementaryFigures S2a and b) of BrdU labeling, indicating an overall increasein cell proliferation during the BrdU labeling period. Additionally,the number of Ki67-negative (Ki67−) cells within the GFP+ BrdU+

population was significantly decreased following ankyrin-G knock-down (Figures 1b and d and Supplementary Figures S2a and c),indicating that ankyrin-G knockdown reduces the number of cells

exiting the cell cycle. To further demonstrate that ankyrin-G isimportant for neural progenitor proliferation, a complementaryexperiment was conducted using embryonic Ank3 +/− mice inwhich the brain-specific isoform of the ankyrin-G is deleted.A previously published study demonstrated that Ank3 +/− miceexhibit bipolar disorder-like behavioral alterations such as reducedanxiety and increased motivation for reward.35 E15 Ank3 +/−brains were pulse-labeled with BrdU, harvested and immunola-beled with antibodies against BrdU and Ki67. Images covering thesame area were taken from five to seven consecutive brain slicesper animal. Consistent with our in utero electroporation assay, weobserved a significant increase in the number of BrdU-positivecells in Ank3 +/− mice compared with wild-type littermates(Figures 1e and f). The increase in BrdU-labeled cells wasaccompanied by a significant decrease in cell-cycle exit (Figures1e and g). These results further confirm that ankyrin-G is a positiveregulator of neural progenitor proliferation.Consistent with the observation of increased proliferation, we

also observed a significant increase in mitotic activity in thein utero transfected GFP+ population in ankyrin-G knockdownanimals based on immunoreactivity for phosphohistone H3,30

which labels mitotic cells (Figures 1h and i). In addition, weobserved a reduced percentage of GFP+ cells in the cortical plate(CP) following ankyrin-G knockdown compared with controls(Figure 1j). Reduction in GFP+ cells in the CP could be due to eithera delay in neuronal differentiation or an early neuronal migrationdefect. To rule out the latter possibility, we analyzed the distribu-tion of GFP+ cells exclusively in the intermediate zone/CPpopulation and found no significant difference between controland ankyrin-G-knockdown conditions (data not shown). The factthat the increase in GFP+ cells in the VZ correlates with thereduction in GFP+ cells in the CP is suggestive of a neuronaldifferentiation phenotype. To test this possibility, we examinedimmunoreactivity for Tuj1, a marker of differentiated neurons.30

Our results showed that ankyrin-G knockdown reduced thepercentage of GFP+ cells overlapping with the Tuj1+ cells inthe cortex, which are mostly localized to the CP and interme-diate zone (Figures 1k and l). Overall, these results suggest thatankyrin-G knockdown increases progenitor proliferation throughreducing cell-cycle exit and neuronal differentiation in thedeveloping brain.

Ankyrin-G regulates Wnt reporter activityThe observation of abundant ankyrin-G expression in the VZ in apattern similar to β-catenin expression and the upregulation ofneural progenitor proliferation following ankyrin-G knockdown,combined with the well-known role of Wnt signaling in prolife-ration, led us to ask whether ankyrin-G has a role in canonical Wntsignaling, of which β-catenin is a core component. Additionally,the Wnt pathway has recently been implicated in several psychia-tric disorders via its regulatory function in cortical neurogene-sis.26,30 To measure Wnt-mediated transcriptional activity, we useda luciferase reporter construct containing seven copies of the TCF/LEF-binding site (8XSuperTOPFLASH), which can be activated byβ-catenin downstream of canonical Wnt signaling.40,41 Murine P19cells were transfected with either control or ankyrin-G-targetingshRNA constructs together with the luciferase reporter construct.We found that, following knockdown of ankyrin-G, TCF/LEFreporter activity was increased in the absence of Wnt3a stimula-tion compared with controls (Figure 2a, left). Wnt3a treatmentincreased the TCF/LEF reporter activity by ~ 2-fold, and AnkGknockdown further upregulated the luciferase activity by ~1.5-fold(Figure 2a, right). These results suggest that ankyrin-G negativelyregulates the canonical Wnt signaling pathway. To furtherdecipher this mechanism, we examined the protein expressionlevels of several canonical Wnt pathway components followingankyrin-G knockdown in P19 cells. We observed that neither the


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protein (Figures 2b and c) nor the mRNA (Supplementary FigureS1c) levels of β-catenin were altered following ankyrin-G knock-down compared with control conditions in P19 cells. Moreover,we did not detect differences in GSK3β or phospho-GSK3β (pY216

GSK3β) abundance at the whole-cell level following ankyrin-Gknockdown (Figures 2b and c), suggesting that ankyrin-G does notdirectly regulate the abundance of these canonical Wnt pathwaycomponents.

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Figure 1. Ankyrin-G regulates progenitor cell proliferation in developing cortex. (a) Ankyrin-G (red) is highly expressed in the VZ of developingcortex. (b) Images of E16 mouse cortices electroporated at E13 with non-targeting (left panel, Control) and ankyrin-G-directed small hairpin(right panel, AnkG shRNA) and GFP expression plasmid. Single-pulse BrdU was injected 2 h before brain dissection. Images were stained forGFP (green), BrdU (red) and Ki67 (white). Arrows indicate BrdU and GFP double-positive cells, and arrowheads indicate Ki67, BrdU and GFPtriple-positive cells. (c) Ankyrin-G knockdown resulted in increased BrdU incorporation (Control, n= 3; shRNA1 and shRNA2, n= 4). (d) Ankyrin-Gknockdown decreased cell-cycle exit (Control, n= 3; shRNA1 and shRNA2, n= 4). (e) Images of E15 Ank3 +/− brains stained for BrdU (green)and Ki67 (white). Images were taken by keeping the width of the images constant, and covering all the cortical layers (VZ, SVZ, intermediatezone (IZ) and CP). (f) Number of BrdU-positive cells per brain slice is significantly increased in Ank3 +/− animals compared with wild-typelittermates (WT, n= 2; Ank3 +/− , n= 3). (g) Cell-cycle exit in the BrdU-positive population is decreased in Ank3 +/− animals compared withwild-type littermates (WT, n= 2; Ank3 +/− , n= 3). (h) Images of E16 mouse cortices electroporated at E13. Images were stained for GFP (middleimages in each set), phosphohistone H3 (PHH3) (left images in each set) and DAPI. Arrows indicate GFP and PHH3 double-positive cells. (i)Ankyrin-G knockdown increased mitotic index as measure by PHH3 staining (Control, n= 4; shRNA1 and shRNA2, n= 3). (j) Distribution ofGFP+ cells in different cortical zones 72 h after transfection at E16. Consistent with proliferative effect of ankyrin-G, the percentage of GFP+

cells increased in the VZ/SVZ after ankyrin-G knockdown (Control, n= 7; shRNA1, n= 5 and shRNA2, n= 6). (k) Images of E16 mouse corticeselectroporated at E13. Images were stained for GFP (middle images in each set), Tuj1 (left images in each set) and DAPI. (l) Fraction of GFP+

cells overlapping with Tuj1 staining is reduced after ankyrin-G knockdown (Control, n= 7; shRNA1, n= 5 and shRNA2, n= 8). All analyses, one-way analysis of variance (one-way ANOVA) followed by Dunnett’s multiple comparison test, except panel (f) and (g), where unpaired t-test isused; *Po0.05; **Po0.01; ***Po0.001. Scale bar: 10 μm (a), 50 μm (b and e) and 100 μm (h and k).

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Figure 2. Ankyrin-G regulates Wnt signaling. (a) Ankyrin-G negatively regulates canonical Wnt signaling. Ankyrin-G knockdown resulted insignificant increase in luciferase activity with (right) or without (left) Wnt3A stimulation (no Wnt stimulation, n=4; Wnt stimulation, n=8).(b) Ankyrin-G knockdown does not alter the expression of canonical Wnt signaling proteins. Sample western blots shown for several componentsof canonical Wnt signaling. (c) Quantification of protein expression levels does not show significant difference after ankyrin-G knockdowncompared with control, except ankyrin-G levels (please see Supplementary Figure 1 for ankyrin-G quantification) (n⩾3, in all cases). (d) Ankyrin-Gknockdown reduces interaction between E-cadherin and β-catenin in P19 cells. Immunoprecipitation (IP) with β-catenin antibody followed byimmunoblotting (IB) E-cadherin antibody. Input, 5% of the total protein used for immunoprecipitation. (e) Fold change of E-cadherin levelsnormalized to loading immunoprecipitated β-catenin (Control and shRNA2, n=5; shRNA1, n=3). (f) Ankyrin-G knockdown reduces interactionbetween E-cadherin and β-catenin in E14 Ank3 +/− mice brain lysates. IP with β-catenin antibody followed by IB E-cadherin antibody. Input, 5% ofthe total protein used for immunoprecipitation. (g) Fold change of E-cadherin levels normalized to immunoprecipitated β-catenin (WT, n=7;Ank3 +/− , n=6). All analyses, one-way analysis of variance (one-way ANOVA) followed by Dunnett’s multiple comparison test, except for (a) whereTukey’s multiple comparison test is used, and (g) where unpaired t-test is used; *Po0.05; **Po0.01; ***Po0.001.


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Ankyrin-G knockdown alters the subcellular localization ofβ-cateninIn addition to its central role in Wnt-mediated TCF/LEF transcrip-tion, β-catenin is a core member of a cell–cell adhesion complexcomprised of cadherins and catenins (the cadherin–catenincomplex).31 Although it was previously thought that the cadherin-bound pool of β-catenin cannot be made available for Wntsignaling, recent studies have suggested instead the existence ofan interplay between Wnt signaling and this cell adhesioncomplex.31–34 Specifically, studies show that sequestration ofβ-catenin at cell–cell adhesion sites via E-/N-cadherin overexpres-sion downregulates Wnt signaling and that the absence ofE-cadherin in embryonic stem cells results in the accumulation offree β-catenin in the nucleus.42 Importantly, ankyrin-G has beenidentified to be a molecular partner of E-cadherin in epithelial cellsand is required for the proper accumulation of E-cadherin tocell–cell contact sites.19 Therefore, we set out to evaluate howankyrin-G knockdown affects the integrity of the adherens junc-tions, as well as to determine changes in the subcellular localiza-tion and levels of β-catenin following ankyrin-G knockdown. First,following ankyrin-G knockdown in P19 cells, we examined theassociation of β-catenin and E-cadherin by immunoprecipitationfollowed by western blot analysis. We found that the amount ofE-cadherin co-immunoprecipitated with β-catenin was markedlydecreased in ankyrin-G knockdown conditions compared withcontrols (Figures 2d and e). We then repeated this experimentusing brain lysates from E14 Ank3 +/− mice. Consistent with theshRNA-knockdown assay, the interaction between β-catenin andE-cadherin is significantly reduced in Ank3 +/− mice comparedwith wild-type littermates (Figures 2f and g).We next used immunohistochemistry to determine whether the

reduced interaction between E-cadherin and β-catenin might leadto alterations in the subcellular localization of β-catenin. P19 cellswere transfected with either nuclear GFP-expressing scrambledshRNA (Control) or ankyrin-G shRNA constructs for 48 h, and thenfixed and immunolabeled with antibodies against ankyrin-G andeither β-catenin or E-cadherin. In control cells, as observed pre-viously,19 E-cadherin is localized to the cell membrane, assuggested by the immunoreactivity surrounding the cell. How-ever, ankyrin-G knockdown disrupted the localization of E-cad-herin to the cell periphery, resulting instead in a distribution that ismore diffuse throughout the cell (Figures 3a and b). This disruptedE-cadherin phenotype was observed in over 85% of ankyrin-G-knockdown cells, compared with 20% of control cells (Figure 3a).Consistent with previous observations, β-catenin is mostlylocalized to the plasma membrane in control shRNA-transfectedcells (Figure 3c). In ankyrin-G-knockdown cells, however, β-cateninlocalization to the cell periphery was less conspicuous (Figures 3cand d).

The β-catenin on the cell membrane is presumably in a complexwith E-cadherin.31 Therefore, since the overall levels of β-catenindo not change in the absence of ankyrin-G (Figures 2b and c andSupplementary Figure S1c), we hypothesized that reduced levelsof E-cadherin/β-catenin at the plasma membrane followingankyrin-G knockdown might increase the levels of free, non-membrane-bound β-catenin that would then be available for Wntsignaling. We measured nuclear β-catenin levels in transfectedcells expressing control shRNA or ankyrin-G shRNAs, as well asnuclear GFP, and observed a marked increase in the amount of β-catenin immunoreactivity localized to the nucleus (Figures 3c–e).Finally, to confirm that this phenomenon is observed in the brainin vivo, we examined the localization of intracellular β-catenin incortical progenitor cells in sections from in utero transfected E16embryos as well as from E15 Ank3 +/− brains. These sectionswere immunolabeled with antibodies against β-catenin, Ki67 (tolabel cycling cells) and 4',6-diamidino-2-phenylindole (DAPI).In both in utero ankyrin-G knockdown and Ank3 +/− animals,a greater level of β-catenin immunoreactivity was observedthroughout the cytoplasm and the nucleus of Ki67+ cells comparedwith those from control animals (Figures 3f and h, respectively).Additionally, consistent with the in vitro assay, increased levels ofnuclear β-catenin are observed in proliferating cortical neuralprogenitor cells (Figures 3f–i). These data suggest that ankyrin-Gknockdown reduces the levels of E-cadherin localized to cadherin-–catenin complexes at the plasma membrane, which in turnresults in an increased abundance of β-catenin in the nucleus,which is then available for the activation of Wnt signaling.

Increased GSK3β levels ameliorated heightened Wnt signalingcaused by ankyrin-G loss-of-functionOur findings suggest that ankyrin-G is a negative regulator of theWnt signaling pathway, as reduced levels of ankyrin-G lead toupregulated TCF/LEF reporter activity and nuclear β-cateninaccumulation. To test this hypothesis directly, we examined theeffect of the overexpression of GSK3β, which phosphorylatesβ-catenin and targets it for ubiquitin-dependent proteasomaldegradation,43,44 on the ankyrin-G loss-of-function phenotype. InP19 cells, increased TCF/LEF luciferase reporter activity, observedas the result of ankyrin-G shRNA knockdown, was completelyreversed by the overexpression of GSK3β (Figure 4a). These resultsindicate that ankyrin-G functionally interacts with other Wntsignaling proteins such as GSK3β and that the effects of ankyrin-Gknockdown on Wnt signaling activity can be normalized byreducing β-catenin abundance. Taken together, these resultsfurther suggest that ankyrin-G is a negative regulator of Wntsignaling.

Figure 3. Ankyrin-G knockdown increased nuclear β-catenin levels. (a) Top panels are control cells transfected with non-targeting shRNA(Control) and lower panels are cells transfected with ankyrin-G targeting shRNA (AnkG shRNA). Ankyrin-G knockdown disrupts E-cadherinlocalization to cell membrane. P19 cells are stained for ankyrin-G (white), E-cadherin (red), GFP (green) and DAPI (blue). (b) Orthogonal imagesof single cells showing that ankyrin-G knockdown disrupt E-cadherin localization to cell membrane. Arrows point to E-cadherin expression onthe cell membrane. (c) Ankyrin-G knockdown disrupts β-catenin localization to cell membrane, and increases nuclear β-catenin levels. P19 cellsare stained for ankyrin-G (white), β-catenin (red), GFP (green) and DAPI (blue). (d) Orthogonal images of single cells showing that nuclearβ-catenin levels are increased after ankyrin-G knockdown in P19 cells. Cell nuclei are circled in each image. Arrows point to β-cateninexpression on the cell membrane. (e) Quantification of nuclear β-catenin levels showing increased levels of β-catenin after ankyrin-Gknockdown (Control, n= 127; shRNA1, n= 165; shRNA2, n= 130; three different cultures in all cases). (f) Ankyrin-G knockdown increasesnuclear β-catenin levels in proliferating neural progenitors in vivo. Images of E16 mouse cortices electroporated at E13 with non-targeting (toppanel, Control) and ankyrin-G-directed small hairpin (bottom panel, AnkG shRNA) and GFP expression plasmid. Images were stained for GFP(green), Ki67, cell-cycle marker (white), β-catenin (red) and DAPI (blue). Arrows indicate Ki67 and GFP double-positive cells. (g) Quantificationof nuclear β-catenin levels showing increased levels upon ankyrin-G knockdown compared with control (Control, n= 16; shRNA1 and shRNA2,n= 13; two different animals per condition). (f) Loss of ankyrin-G also results in increased nuclear β-catenin levels in proliferatingneuroprogenitors in vivo. E15 brain slices from wild-type (top panels) and Ank3 +/− animals are stained for Ki67 (red), β-catenin (green) andDAPI (blue). (g) Quantification of nuclear β-catenin levels showing increased levels in Ank3 +/− animals compared with wild-type littermates(WT, n= 16; Ank3 +/− , n= 22; two different animals per condition). All analyses, one-way analysis of variance (one-way ANOVA) followed byDunnett’s multiple comparison test, except for (i) where unpaired t-test is used; *Po0.05; **Po0.01; ***Po0.001. Scale bar: 10 μm.


6


In vivo defects in neurogenesis caused by ankyrin-G knockdowncan be rescued via depletion of β-catenin levels in the developingcortexGiven that the ankyrin-G-mediated increase in TCF/LEF activity canbe normalized via the expression of a core member (GSK3β) of the

Wnt signaling pathway, we hypothesized that the neurogenesisdefects caused by in vivo knockdown of ankyrin-G could berescued via similar manipulations. To examine whether theoverexpression of GSK3β can normalize the effects of ankyrin-Gknockdown, we performed in utero electroporation at E13 with

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control or ankyrin-G shRNA and coexpressed either control emptyvector or GSK3β overexpression construct. Pulse labeling withBrdU was performed at E16 and brains were harvested 2 h later. Asshown earlier, knockdown of ankyrin-G resulted in increased BrdUincorporation in the GFP+ cell population, indicating increased

proliferation (Figures 4b, c and e). Consistent with the TCF/LEFluciferase assay, the coexpression of GSK3β with ankyrin-G shRNAled to levels of BrdU incorporation that were indistinguishablefrom the control condition (Figures 4b, d and e). Additionally,overexpression of GSK3β was able to rescue the reduced cell-cycle

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exit phenotype associated with ankyrin-G downregulation(Figures 4b–d and f). Finally, we have observed normal GFP+ celldistribution upon coexpression of GSK3β with ankyrin-G shRNA(Figure 4g). These findings demonstrate that the neurogenesisdefects resulting from the in vivo knockdown of ankyrin-G can berescued via dampening Wnt signaling through modulation ofother core members of this signaling pathway. These in vivoneurogenesis data, in addition to our in vitro TCF/LEF reporterdata, suggest that ankyrin-G exerts its effect on neural progenitorproliferation via controlling the pool of β-catenin participating inWnt signaling (Figure 4h).

DISCUSSIONIn the current work, we describe a novel role for ankyrin-G in braindevelopment. Extensive work has addressed the role of ankyrin-Gas a scaffolding protein required for neuronal polarity and theformation of the axon initial segment.15,16 Our findings indicatethat ankyrin-G is also an important regulator of neural progenitorcells in the developing cortex. In the current literature, one of thehypotheses posits that early neurodevelopmental changes pre-dispose the individual to the development of schizophrenia inadulthood.45,46 Furthermore, ASDs are often associated withincreased brain size and malformation of multiple brain regionsincluding the cortex, suggesting alteration in aspects of neuro-genesis, such as the proliferation and differentiation of corticalneurons. Our study further supports these findings and alsosuggests that neurodevelopmental changes likely also underliebipolar disorder etiology.Recently, it was shown that ankyrin-G regulates the production

of olfactory bulb interneurons in the adult brain via itsupregulation in radial glia destined to become SVZ ependymalcells.47 In this environment, ankyrin-G is required for the SVZ nicheassembly through the lateral adhesion of neural progenitors.47

Consistent with this study, we show that ankyrin-G regulatesproliferation and new neuron production in the developing brainvia a mechanism that involves E-cadherin localization to the cellmembrane and alterations in subcellular β-catenin levels. Ourresults suggest, for the first time, that ankyrin-G modulatessubcellular compartmentalization of β-catenin, and therebycontrols the pool of cellular β-catenin available to participate inWnt signaling in cortical progenitor cells (Figure 4h).Our data suggest a model in which ankyrin-G regulates

β-catenin anchoring at the progenitor cell membrane via itsinteraction with E-cadherin. Thus, when ankyrin-G is down-regulated, the localization of E-cadherin to the cell membrane isreduced, and β-catenin becomes available to act at the nucleus,where it modulates gene transcription (Figure 4h). In supportof this model, it has previously been shown that ankyrin-Gdownregulation decreases N-cadherin attachment to the cellmembrane in postnatal radial glia progenitors during the SVZniche formation.47 Therefore, by regulating the levels of nuclear

β-catenin available for Wnt signaling, ankyrin-G acts to regulatenegatively progenitor cell proliferation. Accordingly, we were ableto counteract the effects of ankyrin-G knockdown upon corticalprogenitor proliferation by dampening Wnt signaling via theoverexpression of GSK3β, major component of this pathway.These findings support a hitherto unknown role for ankyrin-G

in canonical Wnt signaling. One of the most commonly usedmedications for the treatment of bipolar disorder, lithium, hasbeen shown to inhibit the function of GSK3β, and this has beensuggested to underlie some of lithium’s therapeutic effects.48 Thismechanism of lithium action further implicates the canonical Wntpathway in psychiatric disorders. Although the current study doesnot have a direct line of evidence suggesting the loss of functionof ankyrin-G manifests itself in behavioral changes, a recent studyusing the same Ank3 +/− mice line demonstrated that loss ofankyrin-G results in bipolar disorder-like behavioral alterationssuch as reduced anxiety, which can be considered as higher risktaking, and increased motivation for reward, both of which arefeatures of bipolar disorder.35 Additionally, they demonstratedthat behavioral changes caused by ankyrin-G knockdown can bereversed by lithium administration.35 However, little is knownabout the contribution of Wnt signaling to the etiology of mentalillnesses.We showed previously that deficits in neuronal proliferation via

modulation of GSK3β/β-catenin signaling underlie neurodevelop-mental phenotypes reminiscent of psychiatric disorders, such asschizophrenia.26 Furthermore, a recent analysis of genome-wideassociation study data examining pathways implicated in bipolardisorder found that the cadherin and Wnt signaling pathways areenriched in this disease and confirmed ANK3 as a bipolar riskgene.8 Our study makes the novel observation that ankyrin-G mayserve as a common regulator of both cadherin and Wnt signalingpathways by altering the availability of β-catenin. We demonstratethe impact of ankyrin-G loss-of-function on neurogenesis in theembryonic brain, supporting the importance of Wnt signaling inthe etiology of neurodevelopmental disorders. These results givenew insights into the study of psychiatric disease etiology, andsupport the idea that alterations in neuronal proliferation are animportant element underlying the pathology of mental illness.Finally, it is worth discussing that ankyrin-G is also highly

expressed at the apical domain of radial glial progenitor endfeet atthe ventricular surface. mPar3 is exclusively expressed at apicaldomain during interphase where β-catenin expression is mini-mal.39 Additionally, it was shown that mPar3 regulates asymmetriccell division of radial glial progenitors via Notch signaling in thedeveloping neocortex. Therefore, we cannot rule out thepossibility that ankyrin-G may also interact with mPar3 in radialglial progenitors, and through which it regulates neural progenitorproliferation. This regulation would be expected to be a β-catenin-independent pathway. Future studies will be needed to addresswhether ankyrin-G also interacts with mPar3 in neural progenitorsin the developing neocortex.

Figure 4. Dampening Wnt signaling rescues phenotypes associated with ankyrin-G knockdown. (a) GSK3β overexpression rescues increasedWnt-mediated luciferase activity (in all cases, n= 4; Tukey’s multiple comparison test). (b–d) Images of E16 mouse cortices electroporated atE13 with non-targeting (b, Control) or ankyrin-G-directed small hairpin along with either empty vector (c, AnkG shRNA) or GSK3βoverexpression construct (d, AnkG shRNA+GSK3β), and GFP expression plasmid. Images were stained for GFP (green), BrdU (red) and Ki67(blue). (e) Increased BrdU incorporation associated with ankyrin-G knockdown is reduced to control levels when ankyrin-G shRNA iscoexpressed with GSK3β (Control+Empty Vector, n= 4; AnkG shRNA+Empty Vector, n= 2; AnkG shRNA+ GSK3β, n= 4; Tukey’s multiplecomparison test). (f) Coexpression of GSK3β with ankyrin-G shRNA rescues the cell-cycle exit phenotype assayed using Ki67 proliferativemarker (Control+Empty Vector, n= 4; AnkG shRNA+Empty Vector, n= 2; AnkG shRNA+ GSK3β, n= 4; Tukey’s multiple comparison test).(g) Distribution of GFP+ cells in different cortical zones 72 h after transfection at E16. Coexpression of GSK3β with ankyrin-G shRNA reducedthe percentage of GFP+ cells in the VZ compared with ankyrin-G knockdown condition (Control+Empty Vector, n= 4; AnkG shRNA+EmptyVector, n= 2; AnkG shRNA+GSK3β, n= 4; Tukey’s multiple comparison test). (h) Working model for ankyrin-G function during corticaldevelopment. (Left) In control condition ankyrin-G localizes E-cadherin to plasma membrane where it participates in cadherin–catenincomplex. (Right) In ankyrin-G-knockdown condition E-cadherin localized to the plasma membrane is reduced, which in turn results inincreased free β-catenin available for Wnt signaling. Upregulation of Wnt signaling results in increased neural progenitor proliferation in thedeveloping cortex. *Po0.05; **Po0.01; ***Po0.001. Scale bar: 100 μm.


9


CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGMENTSWe thank Drs A Mungenast, Y Mao, A Bero and J Gräff for critical reading of themanuscript. We are thankful to Y Mao, D Rei, T Soda, P Giusti and J Gräff for technicalhelp and suggestions with the project. Super8XTOPFLASH luciferase reporterconstruct was a kind gift from Dr Randall Moon (University of Washington).We also thank ME Taylor and AS Gomes for their help with production of Ank3 mice.OD is a Henry Singleton (1940) Fellow (Brain and Cognitive Sciences, MassachusettsInstitute of Technology). FCdA was supported by a postdoctoral fellowship from theSimons Foundation (Simons Center for the Social Brain, Massachusetts Institute ofTechnology). This work was partially supported by an NIH RO1 Grant (MH091115) toL-HT, a grant from the Stanley Medical Research Institute to L-HT and TLP, and theHoward Hughes Medical Institute.

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Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp)


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Ankyrin-G regulates neurogenesis and Wnt signaling by altering the subcellular localization of β-catenin