Neurobiology of Disease 67 (2014) 24–36

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Neurobiology of Disease

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APP-dependent alteration of GSK3β activity impairs neurogenesis in theTs65Dn mouse model of Down syndrome

Stefania Trazzi 1, Claudia Fuchs 1, Marianna De Franceschi, Valentina Maria Mitrugno,Renata Bartesaghi, Elisabetta Ciani ⁎Department of Biomedical and Neuromotor Sciences, University of Bologna, 40126 Bologna, Italy

Abbreviations: AICD, amyloid precursor protein intraprecursor protein; DS, Down syndrome; GSK3β, glycogentonin; aNPC, adult neural precursor cell.⁎ Corresponding author at: Department of Biomedical a

di Porta San Donato 2, 40126 Bologna, Italy. Fax: +39 051E-mail address: elisabetta.ciani@unibo.it (E. Ciani).Available online on ScienceDirect (www.sciencedir

1 Authors labeled with an asterisk contributed equally t

http://dx.doi.org/10.1016/j.nbd.2014.03.0030969-9961/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 December 2013Accepted 2 March 2014Available online 15 March 2014

Keywords:Down syndromeNeurogenesis impairmentGSK3βAPPAICD5-HT1A receptorFluoxetineLithium

Intellectual disability in Down syndrome (DS) appears to be related to severe neurogenesis impairment duringbrain development. The molecular mechanisms underlying this defect are still largely unknown. Accumulatingevidence has highlighted the importance of GSK3β signaling for neuronal precursor proliferation/differentiation.In neural precursor cells (NPCs) from Ts65Dnmice and human fetuses with DS, we found reduced GSK3β phos-phorylation and, hence, increased GSK3β activity. In cultures of trisomic subventricular-zone-derived adult NPCs(aNPCs) we found that deregulation of GSK3β activity was due to higher levels of the AICD fragment of the triso-mic gene APP that directly bound to GSK3β. We restored GSK3β phosphorylation in trisomic aNPCs using eitherlithium, a well-known GSK3β inhibitor, or using a 5-HT receptor agonist or fluoxetine, which activated the sero-tonin receptor 5-HT1A. Importantly, this effect was accompanied by restoration of proliferation, cell fate specifi-cation and neuronal maturation. In agreement with results obtained in vitro, we found that early treatment withfluoxetine, which was previously shown to rescue neurogenesis and behavior in Ts65Dn mice, restored GSK3βphosphorylation. These results provide a link between GSK3β activity alteration, APP triplication and the defec-tive neuronal production that characterizes the DS brain. Knowledge of the molecular mechanisms underlyingneurogenesis alterations in DS may help to devise therapeutic strategies, potentially usable in humans. Resultssuggest that drugs that increase GSK3β phosphorylation, such as lithium or fluoxetine, may represent usefultools for the improvement of neurogenesis in DS.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Down syndrome (DS), caused by trisomy of chromosome 21(HSA21), is the most frequent genetic cause of cognitive impairment.Cognitive impairment has been attributed to the characteristically de-creased brain size of individuals with DS. Accumulating evidence in in-dividualswith DS and DSmousemodels shows that brain hypotrophy isdue to proliferation impairment (Chakrabarti et al., 2007; Contestabileet al., 2007; Guidi et al., 2008; Guidi et al., 2011; Haydar et al., 2000;Lorenzi and Reeves, 2006; Roper et al., 2006). Proliferation impairmentis worsened by altered cell fate specification with a reduction inneurogenesis and an increase in astrogliogenesis (Contestabile et al.,2007; Guidi et al., 2008; Guidi et al., 2011). Dendritic pathology is alsoa consistent feature and a possible substrate for cognitive impairment

cellular domain; APP, amyloidsynthase kinase 3β; 5-HT, sero-

nd Neuromotor Sciences, Piazza2091737.

ect.com).o the work.

in DS. In children and adults with DS, there is a marked reduction indendritic branching and spine density (Becker, 1991; Prinz et al.,1997; Schulz and Scholz, 1992; Takashima et al., 1981; Takashimaet al., 1989). This evidence suggests that proliferation impairment, cellfate specification and neuronal maturation defects may be key determi-nants of intellectual disability in individuals with DS.

Trisomy 21 results in the triplication of over 400 genes (SturgeonandGardiner, 2011),whichmakes elucidation of the contribution of dif-ferent genes in cognitive impairment a challenge. Recent evidenceshows that the amyloid precursor protein (APP) gene is involved in im-portant aspects of brain development, such as cell migration and cellcycle progression (Nalivaeva and Turner, 2013), suggesting the possibleinvolvement of the triplicated gene APP in the neurodevelopmentalalterations that characterize DS. In agreement with this hypothesis,we recently demonstrated that triplication of APP impairs proliferation,differentiation and maturation of neuronal precursor cells from theTs65Dn mouse model of DS (Trazzi et al., 2011, 2013).

It should be noted that APP is an extremely complex molecule thatmay be functionally important not only in its full-length configuration,but also as the source of numerous fragments with various effects onneural function (Zhou et al., 2011). For example, one fragment, the se-creted APPsα, is neuroprotective, neurotrophic and regulates cell

25S. Trazzi et al. / Neurobiology of Disease 67 (2014) 24–36

excitability and synaptic plasticity, while the β-amyloid (Aβ) fragmentappears to exert opposite effects. Less is known about the neuralfunctions of the fragment called APP intracellular domain (AICD), butthere is a growing interest in understanding AICD functions. Forinstance, this fragment appears to contribute to neurodegenerative dis-orders, such as Alzheimer's disease (Chang et al., 2006; Konietzko,2012), by modulating neural cell survival (Muller et al., 2008). More-over, AICD overexpression in transgenic mice impairs neurogenesis(Ghosal et al., 2010). By looking at the mechanisms whereby APP im-pairs neurogenesis in trisomic neuronal precursor cells, we found thatAICD fragment was critically involved in the APP-dependent impair-ment of precursor cell proliferation, cell fate specification and neuronalmaturation (Trazzi et al., 2011, 2013).

Concerning themechanism of action of AICD, it has been shown thatAICD enters the nucleus and regulates gene expression by associatingwith Fe65 protein and the histone acetyltransferase Tip60 (Baek et al.,2002; Cao and Sudhof, 2001; Gao and Pimplikar, 2001; Trazzi et al.,2011). Consistently with the transcriptional role of AICD, we have re-cently found that AICD is involved in the transcriptional regulation ofPtch1 in trisomic neural precursor cells (Trazzi et al., 2011). Excessivelevels of AICD lead to overexpression of Ptch1, causing derangementof the Sonic Hedgehog (Shh) pathway (Trazzi et al., 2011). AICD mayalso exert actions independently from direct regulation of gene expres-sion. In vitro studies showed that AICD can alter cell signaling (Leissringet al., 2002; Zhou et al., 2012) by interactingwith proteins and probablyregulating their stability or function. For instance, AICD directly inter-actswith glycogen synthase kinase 3β (GSK3β) promoting its kinase ac-tivity, with no effect on its transcription (Ryan and Pimplikar, 2005;Zhou et al., 2012).

In the framework of the cellular mechanisms underlying brainalterations in DS, the direct link of AICD with GSKβ seems of relevancebecause GSK3β is a key component of a surprisingly large number ofcellular processes and several diseases (Jope and Johnson, 2004).In particular, emerging evidence points at GSK3β as a key negativeregulator in multiple neurodevelopmental processes, includingneurogenesis, neuronal differentiation, neuronal migration andaxon growth and guidance (Hur and Zhou, 2010). Moreover, changesin GSK3β activity have been associated with many psychiatric andneurodegenerative diseases (Tilleman et al., 2002). The broad spectrumof action of GSK3β in the normal and diseased brain suggests thatderegulation of GSK3β activity dependent by the APP/AICD systemmay play a role in the brain phenotype of DS. We show here that in-creased levels of AICD alter GSK3β activity in neural precursor cellsfrom the Ts65Dnmousemodel of DS and that GSK3β deregulation neg-atively affects cell proliferation, cell fate specification and neuronalmaturation.

Materials and methods

Ts65Dn mice colony

Female Ts65Dn mice carrying a segmental trisomy of chromosome16 (Reeves et al., 1995) were obtained from Jackson Laboratories(Bar Harbour, ME, USA) and maintained on the original genetic back-ground by mating them to C57BL/6JEi × C3SnHeSnJ (B6EiC3) F1males. Animals were karyotyped by real-time quantitative PCR (qPCR)as previously described (Liu et al., 2003), and by PCR with primersspanning the translocation breakpoint of extra chromosome 1716(Reinholdt et al., 2011). The animals had access to water and food adlibitum and were kept in a roomwith a 12:12 h dark/light cycle. Exper-iments were performed in accordance with the Italian and EuropeanCommunity law for the use of experimental animals andwere approvedby Bologna University Bioethical Committee. In this study all effortswere made to minimize animal suffering and to keep the number ofanimals used to a minimum.

Subventricular-zone-derived adult NPC (aNPC) cultures and treatments

Cells were isolated from the SVZ of newborn (postnatal day 2)euploid (n= 15) and Ts65Dn (n= 15) mice and neurosphere cultureswere obtained as previously reported (Trazzi et al., 2011). Cellswere cultured in suspension inDMEM/F12 (1:1) containingB27 supple-ments (2%), FGF-2 (20 ng/mL), EGF (20 ng/mL), heparin (5 μg/mL)and antibiotics (penicillin: 100 units/mL; streptomycin: 100 μg/mL).Primary neurospheres were dissociated at day 8–10 using Accutase(PAA, Pasching, Austria) to derive secondary neurospheres. The sub-culturing protocol consisted of neurosphere passaging every 7 dayswith whole culture media change (with freshly added FGF-2 andEGF). All experiments were done using neurospheres obtained after2–3 passages from the initially prepared cultures. Most (98%) of thecells in neurospheres were positive for nestin, an established markerfor neural and glial precursors. Cell cultures were kept in a 5% CO2 hu-midified atmosphere at 37 °C. Treatments during in vitro NPC differen-tiation were performed as follows:

In vitro differentiation

Neurospheres were dissociated into a single cell suspension andplated onto poly-L-ornithine-coated 24-well chamber slides at a densityof 3 × 104 cells per well. Cells were cultured for 2 days in DMEM/F12medium containing EGF (20 ng/mL), FGF (20 ng/mL) and 2% fetalbovine serum (FBS) and then transferred to differentiation medium(EGF and FGF free plus 1% FBS) for 6 or 12 days. Every 2 days half ofthe medium was replaced with fresh differentiation medium.

Viral particle transduction

aNPCs were infected, at day 1 post-plating, with mouse APP shRNAlentiviral particles (MOI: 2.5; Santa Cruz Biotechnology), APP adenovi-rus particles (MOI: 25; Vector BioLabs) and AICD lentiviral particles(MOI: 5) (Trazzi et al., 2013). Twenty four hours later, the mediumwas replaced with a differentiation medium.

Drugs

The following drugs were administrated on alternate days: 1 μMfluoxetine, 100 nM (±)-8-Hydroxy-2-(dipropylamino)tetralinhydrobromide (8-OH-DPAT), 2 mM Lithium chloride and 1 nMWAY-100635 maleate salt. All chemicals were purchased fromSigma-Aldrich.

Double-immunocytochemistry and analysis of neurite length in differentiatedaNPCs

For immunofluorescent staining, differentiated aNPC cultureswere paraformaldehyde fixed and stained with antibodies against:glial fibrillary acidic protein (1:400; GFAP mouse monoclonal, Sigma)andß tubulin III (1:100; rabbit polyclonal, Sigma) as primary antibodies,and with mouse FITC-conjugated (1:200; Sigma), and rabbit Cy3-conjugated (1:200; Jackson Laboratories), as secondary antibodies.Samples were counterstained with Hoechst-33258. Ten random fieldsfrom each coverslip were photographed and counted. The number ofpositive cells for each marker was referred to the total number ofHoechst-stained nuclei. Evaluation of neurite length was performed byusing the image analysis system Image Pro Plus (Media Cybernetics,Silver Spring, MD 20910, USA). The average neurite length per cellwas calculated by dividing the total neurite length by the number ofcells counted in the areas.

26 S. Trazzi et al. / Neurobiology of Disease 67 (2014) 24–36

Immunocytochemistry and determination of the labeling index in cellcultures

Neurosphereswere harvested onmicroscope slides by cytospin cen-trifugation (212 × g, 5 min, Shandon, Thermo, Dreieich, Germany).Specimens were fixed in 4% paraformaldehyde for 30 min. Blockingwas done in 5% goat serum for 30 min followed by incubation withthe following primary antibodies: anti-β-catenin (1:100, BD Transduc-tion Laboratories), anti-phospho-GSK3β Ser9 (1:1000) (Cell SignalingTechnology) and anti-GSK3β (1:1000) (Cell Signaling Technology).Detection was done with Cy3-conjugated anti-mouse or anti-rabbitantibodies (1:200, Jackson ImmunoResearch Laboratories).

For proliferation analysis, neurospheres were treated with 10 μMBrdU as previously described (Trazzi et al., 2011), incubated with ananti-5-bromo-2-deoxyuridine (BrdU) monoclonal antibody (1:100;Roche Applied Science) and a Cy3-conjugated anti-mouse secondaryantibody (1:200; Sigma). Samples were counterstained with Hoechst-33258. Fluorescence images, taken from random microscopic fields(10–12 for each coverslip), were superimposed and used to determinethe labeling index (LI), defined as percentage of cells labeled withBrdU over total cell number in three independent experiments in dupli-cate. Digital images were captured using NIS-Elements AR software(Nikon).

Serotonin immunocytochemistry

Differentiated aNPC cultures were paraformaldehyde fixed andstained with an anti-serotonin antibody (1:400; mouse monoclonal,Abcam) and with a mouse Cy3-conjugated (1:200; Jackson Laborato-ries), as secondary antibody. Samples were counterstained withHoechst-33258.

Western blotting

Total proteins from the hippocampus and neurosphere cultures ofeuploid and Ts65Dn mice were homogenized in ice-cold RIPA buffer(50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton-X100, 0.5% sodiumdeoxycholate, 0.1% SDS) supplemented with 1 mM PMSF and 1% prote-ase and phosphatase inhibitor cocktail (Sigma). Protein concentrationwas determined by the Lowry method (Lowry et al., 1951). Equivalentamounts (50 μg) of protein were subjected to electrophoresis on a 4–12%Mini-PROTEAN® TGX™ Gel (Bio-Rad) and transferred to a HybondECL nitrocellulose membrane (Amersham Life Science). The followingprimary antibodies were used: anti-GAPDH rabbit polyclonal (1:5000;Sigma) anti-Alzheimer precursor protein A4 clone 22C11mousemono-clonal (cat: MAB348 1:500; Millipore) anti-phospho-AKT Ser473(1:1000), anti-AKT (1:1000), and anti-phospho-GSK3β Ser9 (1:1000)(Cell Signaling Technology), anti-GSK3β (1:1000) (Cell SignalingTechnology), anti-β-catenin (1:1000, BD Transduction Laboratories,)anti-phospho-CRMP2 Thr514 (1:1000) (Cell Signaling Technology),anti-CRMP2 (1:1000) (Cell Signaling Technology). For AICD detectionthe nitrocellulose membrane was processed for antigen-retrieval aspreviously described (Ryan and Pimplikar, 2005). The blot was incubat-ed with the anti-C-terminal APP rabbit primary antibody (1:8000;A8717 Sigma-Aldrich). Densitometric analysis of digitized images wasperformed with Scion Image software (Scion Corporation, Frederick,MD, USA).

AICD/GSK3β coimmunoprecipitation

6 × 107 euploid or Ts65Dn aNPCs were lysated in the buffer (50mMTris–HCl at pH 7.4, 150 mM NaCl, 0.1% NP40, 1 mM dithiothreitol DTT,supplemented with 1 mM PMSF and 1% protease and phosphatase in-hibitor cocktail) and cleared by centrifugation (10,000 ×g, 30 min).Anti-GSK3β (1:100) (Cell Signaling Technology) was added to equalamounts (2mg) of protein lysate for 4 h at 4 °C and afterward incubated

overnight with protein A-trisacryl (Pierce). For AICD detection the im-munoprecipitates were subjected to Western blot as described above.

Ts65Dn mouse

Experimental protocol of in vivo mice treatment

Euploid (n= 4) and Ts65Dn (n= 4) mice received a daily subcuta-neous injection (at 9–10 A.M.) of fluoxetine (Sigma-Aldrich) in 0.9%NaCl solution from P3 to P15 (dose: 5 mg/kg from P3 to P7; 10 mg/kgfrom P8 to P15). Age-matched euploid (n = 4) and Ts65Dn (n = 4)mice were injected with the vehicle. Each treatment group had approx-imately the same composition of males and females.

Histological procedures and immunohistochemistry

P2 mice were decapitated, brains removed and fixed by immersionin Glyo-Fix (Thermo Electron Corp., Waltham, MA, USA) for 48 h andembedded in paraffin. The forebrain was coronally sectioned in 8 μmthick sections that were attached to poly-lysine-coated slides. One outof 12 sections, in P2 animals were stained using anti-phospho-GSK3βSer9 (1:100) (Cell Signaling Technology), anti-GSK3β (1:100) (CellSignaling Technology), anti-phospho-CRMP2 Thr514 (1:100) (Cell Sig-naling Technology) or anti-CRMP2 (1:100) (Cell Signaling Technology)rabbit polyclonal antibodies. Sections were retrieved with citrate buffer(pH 6.0) at 98 °C for 40 min before incubation with the antibody andprocessed as previously described (Contestabile et al., 2007). Sectionswere incubated with Cy3-conjugated anti-rabbit (1:200; JacksonLaboratories) secondary antibody.

Human fetuses

Subjects

Human fetal brains (17–21 weeks of gestation) were obtained afterprior informed consent from the parents and according to the proce-dures approved by the Ethical Committee of the St. Orsola-MalpighiHospital, Bologna, Italy. Regulations of the Italian Ministry of Healthand the policy of Declaration of Helsinki were followed. All fetuseswere derived from legal abortions and were collected with an averagepost-mortem delay of approximately 2 h. Three control fetuses withno obvious developmental or neuropathological abnormalities andthree DS fetuses were used. Trisomy was karyotypically proved fromthe results of genetic amniocentesis procedures. Autopsies were per-formed at the Institute of Pathology of the St. Orsola-Malpighi Hospital.The gestational age of each fetus was estimated by menstrual historyand crown-rump length.

Histological procedures

Brains were fixed by subdural perfusion with Metacarnoy fixative(methyl alcohol:chloroform:acetic acid 6:1:1) injected through the an-terior and posterior fontanelles. After 24 h, brains were removed, andthe hippocampal region of each hemisphere was coronally sectionedto obtain 3 blocks with a thickness of 2–3 mm. The first block roughlycorresponded to the rostral third of the hippocampal formation. Theblocks were post-fixed in formalin (4% buffered formaldehyde) for5 days, embedded in paraffin, according to standard procedures andsectioned in 4–5 μm-thick coronal sections.

GSK3β immunohistochemistry

Serial sections from the first block of the right hemisphere werestained using anti-phospho-GSK3β Ser9 (1:100) (Cell Signaling Tech-nology) and anti-GSK3β (1:100) (Cell Signaling Technology) antibodies.Antigens were retrieved with citrate buffer, pH 6.0 at 98 °C for 40 min.

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After cooling at room temperature for 20 min, sections were washedwith distilled water, buffered for 10 min in PBS 0.1 M (pH 7.2–7.4)and then incubated overnight with the primary antibody. Sectionswere incubated with Cy3-conjugated anti-rabbit (1:200; JacksonLaboratories) secondary antibody. Immune serum was omitted in neg-ative controls. For each antibody, all sections were immunostained ina single batch to avoid possible immunostaining differences. Adjacentsections were used for phospho-GSK3β Ser9 and GSK3β immunohisto-chemistry, respectively. Five-six sections 200 μm apart were used foreach antibody.

Immunofluorescent image acquisition and quantification of human andmouse brain samples

Fluorescence-labeled signals were acquired using an Eclipse TE2000-S microscope (Nikon) equipped with an AxioCam MRm (Zeiss)digital camera. Image processing and analysis were carried out usingtheNIS Elements AR software (Nikon). All parameters used in the acqui-sition step were standardized (detector gain and exposure time) tomaintain high reproducibility. Immunofluorescence was quantifiedwithin the regions of interest (the SVZ and granular cell layer of thehippocampus) by measuring the total fluorescence units in a box of900 μm2 for the SVZ and 1600 μm2 for the granule cell layer, randomlyplaced at six different sites. The total fluorescence intensity in the SVZand granule cell layer was normalized to the background fluorescenceof the corpus callosum and fimbria, respectively, because these fibertracts exhibit low immunoreactivity. Intensity values in each region ofinterest are expressed as percentage of the values in euploid samples.

Real-time RT-qPCR

Total RNAwas extracted from the hippocampus and neurosphere cul-tures of euploid and Ts65Dn mice with TriReagent (Sigma-Aldrich) ac-cording to the manufacturer's instructions. cDNA synthesis wasachieved with 1.0 μg of total RNA using the iScript cDNA synthesis kit(Bio-Rad) according to the manufacturer's instructions. The used primersequences are as follows: tryptophan hydroxylase 1 (TPH1; NM_009414.3, NM_001136084.2) forward, 5′-AGTTGCGGTATGACCTTGAT-3′,and reverse, 5′-AGGCGAGAGACATTGCTAA-3′; 5-hydroxytryptamine(serotonin) receptor 1A (Htr1A; NM_008308), forward, 5′-ACAGGGCGGTGGGGACTC-3′, and reverse, 5′-CAAGCAGGCGGGGACATAGG-3′ ; 5-hydroxytryptamine (serotonin) receptor 2A (Htr2A; NM_172812.2), for-ward, 5′-GCCTACAAGTCTAGTCAGCTCCAG-3′, and reverse, 5′-ACATCTCTTCCGAGTGTTGGTTCC-3′; 5-hydroxytryptamine (serotonin) receptor 2C(Htr2C; NM_008312.4), forward, 5′-GGGTTGCTGCCACTGCTTTG-3′, andreverse 5′-ACACTACTAATCCTCTCGCTGACC-3′, solute carrier family 6[neurotransmitter transporter serotonin (SERT; NM_010484.2), forward,5′-GATCCCTGCTCACACTGACATC-3′ and reverse, 5′-CCATAGAACCAAGACACGACGAC-3′ ; glyceraldehyde-3-phosphate dehydrogenase (GAPDH;NM_008084.2), forward, 5′-GAACATCATCCCTGCATCCA-3′, and reverse,5′-CCAGTGAGCTTCCCGTTCA-3′. Real-time PCR was performed using aSYBR Premix Ex Taq kit (Takara) according to the manufacturer'sinstructions in an iQ5 real-time PCR detection system (Bio-Rad).Fluorescence was determined at the last step of every cycle. Real-timePCR assay was done under the following universal conditions: 2 minat 50 °C, 10 min at 95 °C, 50 cycles of denaturation at 95 °C for 15 s,and annealing/extension at 60 °C for 1 min. Relative quantificationwas performed using the ΔΔ Ct method.

Statistical analysis

Results are presented as the mean ± standard error (SE) of themean. Statistical significance was assessed by two-way analysis ofvariance (ANOVA), followed by Bonferroni's post hoc test or by thetwo-tailed Student's t-test. A probability level of P b 0.05was consideredto be statistically significant.

Results

Increased GSK3β activity in neuronal precursor cells from the Ts65Dnmouse

Cultures of subventricular-zone-derived adult neuronal precursorcells (aNPCs) of Ts65Dn mice are a suitable model to study the mecha-nisms underlying neurogenesis impairment in DS because they exhibitreduced proliferation rate, impaired acquisition of a neuronal pheno-type and neuronal maturation similarly to the in vivo condition(Trazzi et al., 2011, 2013). We first sought to establish whether GSK3βactivity is altered in trisomic vs. euploid aNPCs. Since GSK3β kinase ac-tivity is inhibited through phosphorylation of serine 9, we examinedGSK3β activity by using an anti-GSK3β phospho-specific antibody(Ser9). We found decreased GSK3β phosphorylation, evaluated bothby immunocytochemistry (Figs. 1A,B) and Western blotting (Fig. 1C),in trisomic vs. euploid cultures, indicating an increased GSK3β activityin trisomic aNPCs. In contrast, trisomic and euploid aNPCs had similarlevels of mRNA transcripts (data not shown) and total protein levelsof GSK3β (Fig. 1B). GSK3β exerts its functions by modulating the activ-ity of a wide range of substrates involved in gene transcription, includ-ing β-catenin (Ikeda et al., 1998). Active GSK3β controls the amount ofβ-catenin, by reducing β-catenin protein stability (Nemoto et al., 2009).We found that β-catenin expression was considerably lower in trisomicaNPCs compared to euploid aNPCs (Figs. 1A,B,C), which is in agreementwith the increased activity of GSK3β. Phosphorylated Akt has beenshown to play a central role in the inhibition of GSK3β (by increasingits phosphorylation) in response to insulin and insulin growth factors(Cross et al., 1995). We found no difference in the phosphorylationlevels of Akt (Fig. 1D), indicating that Akt signaling is not involved inGSK3β activation in trisomic aNPCs.

APP/AICD-dependent GSK3β activation in neuronal precursor cells from theTs65Dn mouse

We previously found that trisomic aNPCs had increased levels ofAICD due to trisomic APP expression and that increased levels of AICDimpair precursor cell proliferation, cell fate specification and neuronalmaturation of trisomic aNPCs (Trazzi et al., 2011, 2013). Since AICDhas been reported to directly interact with GSK3β reducing its phos-phorylation at Ser9, thereby increasing its activity (Ryan andPimplikar, 2005; Zhou et al., 2012), and that AICD overexpressingmice showed an activation of GSK3β (Ryan and Pimplikar, 2005), itmay be hypothesized that increased levels of the APP/AICD systemmay underlie the observed GSK3β activation in trisomic aNPCs.

We reduced APP expression by using lentiviral-mediated RNA inter-ference in trisomic aNPCs and found an increase in the phosphorylationlevels of GSK3β (Fig. 2A). We next examined the effect of increasedlevels of APP on GSK3β activity in euploid aNPCs. Infection with recom-binant adenoviruses containing the APP sequence caused a decrease inGSK3β phosphorylation (Fig. 2A). This evidence suggests that the re-duced phosphorylation of GSK3β (hence, increased activity) in trisomicaNPCs is APP-dependent. Next, we over-expressed AICD in euploidaNPCs. We found that AICD overexpression significantly decreased thephosphorylation levels of GSK3β (Fig. 2A), suggesting that the APP-dependent phosphorylation reduction of GSK3β found in trisomicaNPCs was due to AICD accumulation.

In order to establish whether alteration of GSK3β phosphorylationwas retained after differentiation, we evaluated the phosphorylationlevels of GSK3β in differentiated trisomic aNPC cultures. We foundthat differentiated trisomic cultures exhibited reduced phosphorylationlevels of GSK3β (Fig. 2B). In agreementwith the results obtained in pro-liferating aNPCs, APP interference significantly increased the phosphor-ylation levels of GSK3β in differentiated trisomic cultures (Fig. 2B) andAPP or AICD overexpression decreased GSK3β phosphorylation levelsin euploid cultures (Fig. 2B).

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Fig. 1.AlteredGSK3β phosphorylation andβ-catenin expression in aNPCs from the Ts65Dnmouse. A: Immunofluorescence of Ts65Dn and euploid neurospheres showing phospho-GSK3βSer9, GSK3β and β-catenin expression. Scale bar: 20 μm. B: Quantification of phospho-GSK3β Ser9, GSK3β and β-catenin immunofluorescence in neurospheres from Ts65Dn and euploidmice. C, D: Western blot quantification of phospho-GSK3β Ser9, β-catenin phospho-AKT Ser473 (normalized to total GSK3β, GAPDH and total AKT content, respectively) expression intrisomic and euploid neurospheres. Lower panels show representative examples of Western blots. Data (in B–D), given as percentage of the euploid condition, are expressed as mean ±SE (4 euploid and 4 Ts65Dn mice). *P b 0.05, **P b 0.01 ***P b 0.001 (two-tailed Student's t-test).

28 S. Trazzi et al. / Neurobiology of Disease 67 (2014) 24–36

In order to establish whether the decreased phosphorylation levelsof GSK3β in trisomic aNPCs were mediated by a direct AICD–GSK3β in-teraction,we used an immunoprecipitation assay. In anti-GSK3β immu-noprecipitates of trisomic aNPCs we found immunoreactivity for AICD(Fig. 2C), indicating that AICD directly binds to GSK3β. AICD bindingwas also found in euploid aNPCs (Fig. 2C), though AICD immunoreactiv-ity was lower, in agreement with the lower levels of AICD in euploidaNPCs.

Normalization of GSK3β activity in neuronal precursor cells from theTs65Dn mouse restores cell proliferation

To determine whether excessive activation of the GSK3β underliesimpaired cell proliferation in trisomic aNPCs, we treated aNPCs withtwo inhibitors of GSK3β activity. We used lithium, a well-known inhib-itor of GSK3β activity that acts both indirectly, by increasing the inhibi-tory phosphorylation of GSK3β, and directly, by antagonizing its kinaseactivity (Jope, 2003). Since recent evidence shows that the activationof the serotonin receptor 5-HT1A by serotonin or the selective agonist

8-OH-DPAT inhibits GSK3β activity by increasing GSK3β phosphoryla-tion (Polter et al., 2012), we additionally used 8-OH-DPAT. We foundthat in trisomic aNPCs both lithium and 8-OH-DPAT completely re-storedGSK3βphosphorylation levels (Fig. 3A). This effectwas accompa-nied by restoration of the proliferation impairment that characterizestrisomic aNPCs (Figs. 3B,C). In euploid aNPCs, lithium increased GSK3βphosphorylation, while 8-OH-DPAT did not affect GSK3β phosphoryla-tion (Supplementary Fig. 1A). In euploid aNPCs the lithium-induced in-crease in GSK3β phosphorylation was accompanied by an increase incell proliferation (Supplementary Fig. 1B).

Normalization of GSK3β activity in neuronal precursor cells from theTs65Dn mouse restores cell fate specification and neuronal maturation

Since decreased GSK3β phosphorylation characterized also differen-tiated trisomic aNPCs (Fig. 2B), we sought to establish whether the im-paired neuronal fate acquisition and maturation that characterizetrisomic aNPCs (Trazzi et al., 2011, 2013) are due to increased GSK3β

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Fig. 2.APP/AICD dependent GSK3β phosphorylation in aNPCs from the Ts65Dnmouse. A:Western blot quantification of phospho-GSK3β Ser9 expression, normalized to total GSK3β con-tent, in trisomic and euploid neurospheres. Trisomic aNPCswere infected at day 1 post-plating with APP shRNA lentiviral particles at amultiplicity of infection (MOI) of 2.5. Euploid aNPCswere infected with APP adenovirus particles (MOI: 25) or AICD lentiviral particles (MOI: 5). Cells were harvested 24 h after infection. Right panel: Example of Western blot analysis ofphospho-GSK3β Ser9 and GSK3β expression in aNPCs from euploid (EU) and Ts65Dn (TS) mice. B: Western blot quantification of phospho-GSK3β Ser9 expression, normalized to totalGSK3β content, in 6-day differentiated aNPCs. aNPCswere infected at day 1 post-plating as indicated above. Twenty-four hours later, themediumwas replacedwith a differentiationme-dium. Right panel: Example ofWestern blot analysis of phospho-GSK3β Ser9 and GSK3β expression in differentiated aNPCs from euploid (EU) and Ts65Dn (TS)mice. Data (in A,B), givenas percentage of the untreated euploid condition, are expressed as mean ± SE (5 euploid and 4 Ts65Dn mice). *P b 0.05, **P b 0.01 as compared to the euploid condition; #P b 0.05 ascompared to the untreated trisomic condition (Bonferroni test after ANOVA). C: Interactions between GSK3β and AICD in vivo. Ts65Dn and euploid cell lysates of aNPCs wereimmunoprecipitated with anti-GSK3β antibodies (IP α-GSK3β). The amount of immunoprecipitated GSK3β was determined by immuno-blot analyses using anti-GSK3β antibodies(IB α-GSK3β). GSK3β-associated AICD was revealed by anti-C-terminal APP immunoblotting (IB α-AICD). The AICD expression was confirmed with total cell lysates (input).

29S. Trazzi et al. / Neurobiology of Disease 67 (2014) 24–36

activity and whether these defects can be restored by treatments thatnormalize GSK3β phosphorylation.

Treatmentwith lithium and 8-OH-DPAT completely restored GSK3βphosphorylation levels in trisomic differentiated cultures (Fig. 4A), aneffect that is similar to that observed in undifferentiated aNPCs(Fig. 3A).We found that differentiated aNPCs expressed tryptophan hy-droxylase 1 (TPH1) (Supplementary Fig. 2A), the rate-limiting enzyme

in serotonin synthesis, and were immunoreactive for serotonin (Sup-plementary Fig. 2B), suggesting the presence of serotonergic neuronsin the population of aNPCs. However, it must be noted that serotoninwas present in the differentiating culture medium, due to serum addi-tion (necessary for culture differentiation). Therefore, we cannot ruleout that serotonin immunoreactivity may be due to the uptake of exog-enous serotonin. In view of the presence of serotonin, we treated aNPCs

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Fig. 3. Effect of GSK3β phosphorylation on cell proliferation of aNPCs from the Ts65Dn mouse. A: Western blot quantification of phospho-GSK3β Ser9 expression, normalized to totalGSK3β content, in trisomic (n = 4) and euploid (n = 4) neurospheres. aNPCs were treated with Lithium chloride (2 mM) or (±)-8-Hydroxy-2-(dipropylamino)tetralin hydrobromide(DPAT; 100 nM) for 72 h starting from day 1 post-plating. B: Labeling index (LI), defined as percentage of BrdU-positive cells over total cell number, was determined for neurospheresfrom euploid (n = 4) and Ts65Dn (n = 3) mice. Neurospheres were treated as indicated in A. BrdU (10 μM) was added for the last 6 h and thereafter cells were processed for BrdUimmunocytochemistry. C: Images of BrdU positive cells (red) in euploid (EU) and Ts65Dn (TS) neurospheres. Cell nuclei were stained using Hoechst dye (blue). Scale bar: 20 μm. Data(in A,B), given as percentage of the untreated euploid condition, are expressed as mean ± SE. **P b 0.01, ***P b 0.001, as compared to the euploid condition; ##P b 0.01 as compared tothe untreated trisomic condition (Bonferroni test after ANOVA).

30 S. Trazzi et al. / Neurobiology of Disease 67 (2014) 24–36

with fluoxetine, a selective serotonin reuptake inhibitor that increasesserotonin availability. We found that fluoxetine caused an effect onGSK3β phosphorylation similar to that obtained by the activation ofthe 5-HT1A receptor through the agonist 8-OH-DPAT (Fig. 4A).

Evaluation of the number of cells with either a neuronal (β-tubulinIII-positive cells) or an astrocytic (GFAP-positive cells) phenotypeshowed that lithium, 8-OH-DPAT and fluoxetine treatments increasedthe percentage of new neurons (Figs. 4B,C) and decreased the percent-age of new astrocytes (Figs. 4B,C) in trisomic cultures. Importantly, thepercentage of cells of each phenotype became similar to that of untreat-ed euploid cultures (Fig. 4C), indicating that treatments that are able torestore GSK3β phosphorylation levels also restore the process of cellfate specification. Co-exposure of cells to WAY-100635 (WAY), a selec-tive antagonist of the serotonin receptor 5-HT1A, and either 8-OH-DPATor fluoxetine prevented restoration of fate acquisition, indicating thatthe effect of 8-OH-DPAT and fluoxetinewasmediated by the 5-HT1A re-ceptor (Fig. 4C). Inhibition of the 5-HT1A receptor byWAY had no effecton cell fate specification (Fig. 4C). In euploid aNPCs, while lithium in-creased the acquisition of a neuronal phenotype and reduced the acqui-sition of an astrocytic phenotype (Supplementary Fig. 1C), no effect wasinduced by 8-OH-DPAT or fluoxetine treatment (SupplementaryFig. 1C).

In view of the role of the GSK3β in neuronal maturation (Kim et al.,2011), we sought to determine whether increased GSK3β activity alsounderlies reduced neurite length of trisomic aNPCs. Evaluation ofneurite length of the new neurons after treatment with lithium, 8-OH-DPAT and fluoxetine showed that in trisomic aNPC cultures neuritelength underwent a large increase and became similar to that of un-treated euploid cultures (Fig. 4D). Co-treatment with WAY preventedthe neurite length increase induced by 8-OH-DPAT and fluoxetine treat-ments (Fig. 4D). Unlike in trisomic aNPC cultures, in euploid culturestreatments had no effect on neurite length (Supplementary Fig. 1D).

In order to establish themechanismwhereby 8-OH-DPAT and fluox-etine were effective in trisomic but not in euploid cultures, we exam-ined the expression of serotonin receptors involved in neurogenesis/differentiation (5-HT1A, 5-HT2A, 5-HT2C) and of the serotonin transport-er (SERT) by quantitative real time PCR (RT-qPCR). No differenceswere found between trisomic and euploid cultures in the expressionof any of these genes (Supplementary Fig. 2C), indicating that thedifferent response to treatments was not due to the differences inserotonin receptor/transporter expression. Taken together, results sug-gest an impairment of the serotonin pathway downstream to themem-brane receptors in trisomic aNPCs that is overcome by increasingactivation of the receptors, with consequent increase in GSK3βphosphorylation.

Increased GSK3β activity in the SVZ and hippocampus of Ts65Dn mice

Basedon thehighGSK3β activity in trisomic aNPC cultures (Figs. 1A,B),we next sought to establish whether a similar up-regulation occurredalso in the in vivo condition. We analyzed GSK3β phosphorylationlevels in the two major sites of adult neurogenesis in the brain, thesubventricular zone (SVZ) of the lateral ventricle and the subgranularzone (SGZ) of the dentate gyrus (DG) of newborn Ts65Dn and euploidmice. We found in both analyzed regions a decrease in the phosphory-lation levels of GSK3β, evaluated by immunohistochemistry (Figs. 5A,B). This effect was confirmed in hippocampal homogenates byWesternblot analysis (Fig. 5C). Similarly to evidence obtained in cultures of tri-somic aNPCs, we found an increased expression of APP and accumula-tion of AICD in the hippocampus of Ts65Dn mice (Fig. 5D), whichmost likely accounts for alteration in the phosphorylation levels ofGSK3β.

In order to establish whether changes in the phosphorylationstatus of GSK3β translated into activity changes, we examined the

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Fig. 4. Effect of GSK3β phosphorylation on cell fate specification and reduced neurite length of aNPCs from the Ts65Dnmouse. A:Western blot quantification of phospho-GSK3β Ser9 ex-pression, normalized to total GSK3β content, in trisomic (n = 3) and euploid (n = 3) aNPCs. Trisomic aNPCs were treated with Lithium chloride (2 mM), (±)-8-Hydroxy-2-(dipropylamino)tetralin hydrobromide (DPAT; 100 nM) or fluoxetine (1 μM). Drugs were administrated on alternate days throughout the entire differentiation period (6-days) startingfrom day 1 post-plating. Right panel: Example of Western blot analysis of phospho-GSK3β Ser9 and GSK3β expression in differentiated aNPCs treated as specified in A. B: Representativedouble-fluorescence images of 6-day differentiated aNPCs immunopositive forβ tubulin III (red) andGFAP (green). Scale bar: 60 μm. C: Percentages ofβ tubulin III- andGFAP-positive cellsin 6-day differentiated aNPC cultures from euploid (EU; n = 8) and Ts65Dn (TS; n = 8) mice. aNPCs were treated with Lithium chloride (2 mM), (±)-8-Hydroxy-2-(dipropylamino)tetralin hydrobromide (DPAT; 100 nM), fluoxetine (Fluo; 1 μM),WAY-100635 maleate salt (WAY; 1 nM), DPAT plusWAY or Fluo plusWAY throughout the entire differentiation period.Values represent mean ± SE. *P b 0.05; **P b 0.01; ***P b 0.001, as compared to the euploid condition; ##P b 0.01 as compared to the untreated trisomic condition (Bonferroni test afterANOVA). D:Quantification of neurite outgrowth ofβ tubulin III-positive cells fromdifferentiated aNPC cultures from euploid (n=6) and Ts65Dn (n=5)mice. aNPC cultureswere treatedas specified in A. Values represent mean ± SE. ***P b 0.001 as compared to euploid condition; ##P b 0.01 as compared to untreated trisomic samples (Bonferroni test after ANOVA).

31S. Trazzi et al. / Neurobiology of Disease 67 (2014) 24–36

phosphorylation levels of the collapsin response mediator protein-2(CRMP-2), a well-known target of GSK3β. CRMP-2, a critical proteinfor specifying axon/dendrite fate, binds and stimulatesmicrotubule sta-bility but fails to bind microtubules upon phosphorylation, causing im-paired neurite outgrowth (Fukata et al., 2002; Jope and Johnson, 2004).We examined the phosphorylation status of CRMP2 in the dentate gyrusof Ts65Dn mice, and found that it was considerably higher in compari-son with euploid mice (Fig. 5E). The increased phosphorylation of

CRMP2 in Ts65Dn mice was confirmed byWestern blot analysis in hip-pocampal homogenates (Fig. 5F).

Fluoxetine treatment restores GSK3β pathway activity in the hippocampusof Ts65Dn mice

Recently, we found that neonate Ts65Dn mice treated (P2–P15)with fluoxetine exhibited full recovery of hippocampal neurogenesis,

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Fig. 5. Altered GSK3β and CRMP2 phosphorylation in Ts65Dn mice. A: Examples of phospho-GSKβ Ser9 fluorescence immunohistochemistry at the level of the rostral part of the lateralventricle (left panels) and of the hippocampal dentate gyrus of a P2 euploid and a Ts65Dn mouse. Scale bar: 40 μm. B: Immunofluorescence quantification of phospho-GSK3β Ser9 ex-pression in the subventricular zone (SVZ) of the lateral ventricle (LV) and the subgranular zone (SGZ) of the dentate gyrus (DG) in euploid (n= 4) and Ts65Dn (n= 4)mice. C:Westernblot quantification of phospho-GSK3β Ser9 expression in the hippocampus of P2 euploid (n= 4) and Ts65Dn (n= 4)mice. Lower panels: Representative examples ofWestern blots. D:Western blot quantification of APP and AICD expression in the hippocampus of P2 euploid (n=4) and Ts65Dn (n= 4)mice. Lower panels: Representative examples ofWestern blots. E:Examples of phospho-CRMP2 Thr514 fluorescence immunohistochemistry at the level of the hippocampal dentate gyrus of a P2 euploid and a Ts65Dn mouse. Scale bar: 40 μm. Immu-nofluorescence quantification of phospho-CRMP2 expression in the granule cell layer (GCL) of the DG in euploid (n = 4) and Ts65Dn (n = 4) mice. F: Western blot quantification ofphospho-CRMP2 expression in the hippocampus of P2 euploid (n = 4) and Ts65Dn (n = 4) mice. Data (in B–F), given as percentage of the euploid condition, are expressed as mean ±SE. *P b 0.05, **P b 0.01 (two-tailed Student's t-test). Abbreviations: GRL, granule cell layer; H, hilus; LV, lateral ventricle; Mol, molecular layer; SGZ, subgranular zone; SVZ, subventricularzone.

32 S. Trazzi et al. / Neurobiology of Disease 67 (2014) 24–36

granule cell dendritic maturation and hippocampus-dependent be-havior (Bianchi et al., 2010b; Guidi et al., 2013). The mechanism un-derlying the regulation of hippocampal neurogenesis by fluoxetine inTs65Dnmice is still unknown. Quantification of GSK3β phosphorylationin the hippocampal region of P15 untreated mice showed that also atthis age Ts65Dnmice had lower GSK3β phosphorylation in comparisonwith euploid mice (Fig. 6A). After treatment with fluoxetine, the levelsof phosphorylated GSK3β in Ts65Dn mice were completely restored(Fig. 6A), which confirms the results obtained in vitro (Figs. 3A, 4A).Phosphorylation of CRMP2 was also higher in P15 Ts65Dn in compari-son with euploid mice (Fig. 6B). After treatment with fluoxetine,Ts65Dn mice exhibited a decrease in the phosphorylation of CRMP2,that became similar to that of untreated euploid mice (Fig. 6B). Thisdata suggests that modulation of GSK3β activity by fluoxetine may beinvolved in the positive impact of fluoxetine on brain development inTs65Dn mice.

Increased GSK3β activity in the trisomic fetal brain

Recently, we found that human fetuses with DS had remarkablyfewer proliferating cells in all germinal zones of the hippocampal region(Contestabile et al., 2007; Guidi et al., 2008). To establish whether, sim-ilarly to the mouse model, fetuses with DS exhibit increased GSK3β ac-tivity, we examined the phosphorylation levels of GSK3β in the granularlayer of the DG (Fig. 7A) and in the ventricular zone of the hippocampus(Fig. 7B). At the investigated gestational age (17–21 weeks of gesta-tion), both regions exhibit intense mitotic activity (Contestabile et al.,2007; Guidi et al., 2008). Immunohistochemical analysis showed thatthe phosphorylation levels of GSK3β were lower in both the granularlayer of the DG (−35%) and in the ventricular zone of the hippocampus(−25%) of fetuseswithDS (Figs. 7A,B), indicating that a defect in GSK3βactivity is present also in neuronal precursor cells of human subjectswith DS.

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Fig. 6. Effect offluoxetine onGSK3β andCRMP2 phosphorylation in the hippocampusof euploid and Ts65Dnmice. A,B:Western blot quantification of phospho-GSK3β Ser9 expression andphospho-CRMP2Thr514 expression in the hippocampus of P15 untreated euploid (n=4) and Ts65Dn (n=4)mice and euploid (n=4) and Ts65Dn (n=3)mice treatedwith fluoxetine(Fluo). Protein levelswere normalized respectively to total GSK3β and CRMP2 content and expressed as percent of untreated condition (100%). Representative examples ofWestern blotson the right. Data (in A and B), given as percentage of the euploid condition, are expressed as mean ± SE. *P b 0.05, **P b 0.01 (two-tailed Student's t-test) as compared to euploid con-dition; #P b 0.05 as compared to untreated trisomic samples (Bonferroni test after ANOVA).

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Fig. 7.AlteredGSK3βphosphorylation inhuman fetuseswithDS. A,B: Examples of phospho-GSK3β Ser9fluorescence immunohistochemistry at the level of thehippocampal dentate gyrus(A) and the ventricular zone of the hippocampus (B) of a control (gestational week GW19; n= 3) and a DS (GW19; n= 3) fetus. The dashed line square indicates the region where theimmunofluorescence was quantified. Scale bar: 40 μm. On the right: Quantification of phospho-GSK3β and total GSK3β fluorescence at the level of the granular layer (GCL) of the hippo-campal dentate gyrus (A) and of the ventricular zone of the hippocampus (B) in control and Down syndrome fetuses. Data (in A and B), given as percentage of the euploid condition, areexpressed as mean ± SE. **P b 0.01 (two-tailed Student's t-test). Abbreviations: GCL, granule cell layer; LV, temporal horn of the lateral ventricle.

33S. Trazzi et al. / Neurobiology of Disease 67 (2014) 24–36

34 S. Trazzi et al. / Neurobiology of Disease 67 (2014) 24–36

Discussion

This study shows that GSK3β signaling is altered in the DS brain andthat increased GSK3β activity is related to increased levels of the APP in-tracellular fragment (AICD). Disruption of GSK3β signaling adversely af-fects proliferation, cell fate specification and neuronal maturation.Pharmacological inhibition of GSK3β activity restores all these processes,suggesting that drugs that inhibit GSK3β activity may represent usefultools for the improvement of key processes of brain development in DS.

Impairment of GSK3β activity and signaling characterizes trisomic aNPCs

Since a number of neurological disorders are associatedwith deficitsin GSK3β signaling (Lei et al., 2011; Salcedo-Tello et al., 2011), wethought it was important to establish whether GSK3β activity is alteredin DS at early phases of development. Indeed, we found an alteration inGSK3β activity in aNPCs of Ts65Dn mice and in neuronal precursors ofhuman fetuses with DS. Importantly, an increase in GSK3β activitywas observed not only in proliferating but also in differentiated aNPCs(Figs. 2B and 5C). These results strongly suggest that the increasedGSK3β activity that characterizes trisomic cells may be a determinantof the reduced neurogenesis and dendritic atrophy that characterizethe trisomic brain starting from early phases of development.

GSK3β is also expressed in the adult brain and is particularly enrichedin the hippocampus, neocortex, and cerebellum (Yao et al., 2002), sug-gesting a role of this kinase also in adulthood. Overexpression ofGSK3β in the hippocampus results in tau-dependent neurodegenerationof this region (GomezdeBarreda et al., 2010) and the neurodegenerationobserved in Alzheimer's disease (AD) is also thought to result from tauhyperphosphorylation. A unique characteristic of DS is that subjectshave a high risk of developing AD from 35 years of age onwards. The ob-served increase in GSK3β activity in the trisomic brain may underlie thedevelopment of AD-like pathology due to tau hyperphosphorylation.

GSK3β regulates a wide variety of developmental events by influenc-ing a broad range of substrates involved in gene transcription, for instanceβ-catenin (Ikeda et al., 1998), or regulating cytoskeletal dynamics, for in-stance CRMP2 (Yoshimura et al., 2005). GSK3β plays a key role in control-ling the amount of β-catenin, by negatively regulating β-catenin proteinstability (Wada, 2009). The currentfinding in trisomic aNPCs of decreasedlevels of β-catenin is consistentwith the increased GSK3β activity. Recentevidence shows that β-catenin, besides being involved in a variety offunctions (Moon et al., 2004; Toledo et al., 2008), plays an importantrole in regulating proliferation of neural stem cells. In these cells, β-catenin acts downstream from the canonical Wnt-signaling pathway(Chenn and Walsh, 2002; Chenn and Walsh, 2003) and appears to in-crease proliferation by decreasing cell cycle exit (Chenn and Walsh,2002). Our results suggest that deregulation of the GSK3β/β-catenin sys-tem may concur to impair proliferation of trisomic aNPCs.

Inhibition of GSK3β promotes dendritic growth in sympathetic, cor-tical and hippocampal neurons (Lim and Walikonis, 2008; Naska et al.,2006). Conversely, abnormally increased GSK3β activity contributes todendrite degeneration under pathological conditions (Lin et al., 2010).A recent study shows that GSK3β mediates phosphorylation of CRMP2and demonstrates that the GSK3β/CRMP2 pathway is an importantme-diator of cerebellar granule neuron dendritogenesis (Tan et al., 2013).Our findings suggest a correlation between altered GSK3β activity andincreased CRMP2 phosphorylation in the trisomic brain, which mayprovide new insights into the understanding of themechanisms under-lying dendritic hypotrophy in DS.

Overexpression of the APP/AICD system underlies deregulation of GSK3βactivity in trisomic aNPCs

We have previously reported that increased levels of AICD impairthe Shh pathway through Ptch1 overexpression, its inhibitory regulator(Trazzi et al., 2011). We found that AICD, by binding to the Ptch1

promoter, induces Ptch1 overexpression through acetylation of Ptch1promoter nucleosomes (Trazzi et al., 2011). Here we provide evidencethat the APP/AICD system contributes to regulate the GSK3β activityin trisomic aNPCs. This is in agreement with a study that demonstratedthe activation of GSK3β and phosphorylation of CRMP2 in transgenicmice expressing AICD (Ryan and Pimplikar, 2005). Since the levels ofmRNA transcripts and total protein levels of GSK3β were not changedin trisomic mice, the AICD-dependent modulation of GSK3β activationmust be mediated by a non-transcriptional mechanism. This is consis-tent with evidence that AICD, in addition to modulate transcription,brings about its effects without being present in the nucleus. Interesting-ly, recent evidence shows that AICD associateswithGSK3β and enhancesits kinase activity, as indicated by decreased Ser9 phosphorylation (Zhouet al., 2012). Current findings provide evidence that in trisomic aNPCsAICD directly associates with GSK3β and increases its activity by phos-phorylating Ser9. It iswell known that several signaling pathways inducethe inhibition of GSK3β by phosphorylating the same residue (Fang et al.,2002). Hence,we cannot exclude that other pathwaysmay contribute, inaddition to the APP/AICD system, to the impairment of GSK3β activitythat characterizes trisomic aNPCs.

The role of triplicated genes in the neurological phenotype of DS isstill poorly understood. Our data suggest that the trisomic gene APPmay be a key determinant of impaired neurogenesis in DS, throughAICD-dependent mechanisms. In addition to modulating the Shh path-way (Trazzi et al., 2011), AICD appears to play an important role in themodulation of GSK3β activity and signaling. Interestingly, recent evi-dence shows that AICD transgenic mice exhibit impaired neurogenesis(Ghosal et al., 2010) and deficits in working memory, that are blockedby lithium treatment (Ghosal et al., 2010). The demonstration of a caus-al link between overexpression of the APP/AICD system and deregula-tion of GSK3β activity in DS may be a starting point for studies thatcan shed light on the mechanisms that underlie the DS neurologicalphenotype.

GSK3 inhibitors as pharmacotherapy for Down syndrome

Changes in GSK3β activity have been associated with many psychi-atric and neurodegenerative diseases (Tilleman et al., 2002) and it hasbecome increasingly apparent that GSK3β might be a common thera-peutic target for different classes of psychiatric drugs (Beaulieu, 2007).For instance, lithium, which is an inhibitor of GSK3 (Klein and Melton,1996), has been used in humans as a mood stabilizer for over 50 years(Martinez, 2008). Further investigation of the involvement of GSK3βin psychiatric disorders has revealed that GSK3β can be regulated by an-tidepressants acting on serotonergic (5-HT) neurotransmission (Li et al.,2004). Importantly, activation of the 5-HT1A receptor leads tophosphorylation of GSK3β (Li et al., 2004; Polter et al., 2012) andthere is evidence that administration of fluoxetine results in enhancedGSK3β phosphorylation in the frontal cortex of mice (Beaulieu, 2007).Current findings show that lithium and 5-HT1A receptor activation(through 8-OH-DPAT or fluoxetine) are able to restore GSK3β activityin trisomic aNPCs and that this effects is accompanied by restorationof neurogenesis (cell proliferation, fate specification and neurite out-growth). These data suggest that normalization of GSK3β activity mayplay a central role in restoration of neurogenesis. However, in view ofthe indirect action of lithium on other cellular signaling pathways(Kang et al., 2003; Pardo et al., 2003; Sasaki et al., 2006) and the multi-ple signal transduction pathways affected by 5-HT receptors, we cannotexclude the contribution of additional mechanisms in neurogenesisrestoration.

Early treatment with fluoxetine has been previously shown tocorrect neurogenesis, dendritic hypotrophy and behavior in Ts65Dnmice (Bianchi et al., 2010b; Guidi et al., 2013). We show here that thistreatment restores GSK3β activity, suggesting that this effectmay be in-volved in the positive impact of treatment in the trisomic brain. Consis-tently with current findings that lithium restores GSK3β activity and

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neurogenesis in trisomic aNPCs, treatmentwith lithium in adult Ts65Dnmice rescues neurogenesis, synaptic plasticity and memory (Bianchiet al., 2010a; Contestabile et al., 2013).

In view of the role of GSK3 in key neurodevelopmental processes,the current finding that GSK3β activity is elevated in human fetal brainswith DS suggests that deregulation of GSK3β activity may contribute tothe impairment of brain development. No effective therapies are avail-able at present for the rescue of neurogenesis and cognitive disabilityin individuals with DS. GSK3 inhibitors are currently in clinical trialsfor several neurological disorders (Eldar-Finkelman and Martinez,2011). We suggest that pharmacotherapy with GSK3β inhibitors maybe a potential tool for improving brain development in DS.

Acknowledgments

This work was supported by grants from the University of Bologna(RFO12BARTE) through funding for basic research given to E.C. and R. B.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nbd.2014.03.003.

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