APP-dependent alteration of GSK3β activity impairs neurogenesis in the Ts65Dn mouse model of Down syndrome

Neurobiology of Disease 67 (2014) 24–36

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APP-dependent alteration of GSK3β activity impairs neurogenesis in the Ts65Dn 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

a r t i c l e

i n f o

Article history: Received 3 December 2013 Accepted 2 March 2014 Available online 15 March 2014 Keywords: Down syndrome Neurogenesis impairment GSK3β APP AICD 5-HT1A receptor Fluoxetine Lithium

a b s t r a c t Intellectual disability in Down syndrome (DS) appears to be related to severe neurogenesis impairment during brain development. The molecular mechanisms underlying this defect are still largely unknown. Accumulating evidence has highlighted the importance of GSK3β signaling for neuronal precursor proliferation/differentiation. In neural precursor cells (NPCs) from Ts65Dn mice and human fetuses with DS, we found reduced GSK3β phosphorylation 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 trisomic gene APP that directly bound to GSK3β. We restored GSK3β phosphorylation in trisomic aNPCs using either lithium, a well-known GSK3β inhibitor, or using a 5-HT receptor agonist or fluoxetine, which activated the serotonin receptor 5-HT1A. Importantly, this effect was accompanied by restoration of proliferation, cell fate specification and neuronal maturation. In agreement with results obtained in vitro, we found that early treatment with fluoxetine, which was previously shown to rescue neurogenesis and behavior in Ts65Dn mice, restored GSK3β phosphorylation. These results provide a link between GSK3β activity alteration, APP triplication and the defective neuronal production that characterizes the DS brain. Knowledge of the molecular mechanisms underlying neurogenesis alterations in DS may help to devise therapeutic strategies, potentially usable in humans. Results suggest that drugs that increase GSK3β phosphorylation, such as lithium or fluoxetine, may represent useful tools 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 decreased brain size of individuals with DS. Accumulating evidence in individuals with DS and DS mouse models shows that brain hypotrophy is due to proliferation impairment (Chakrabarti et al., 2007; Contestabile et al., 2007; Guidi et al., 2008; Guidi et al., 2011; Haydar et al., 2000; Lorenzi and Reeves, 2006; Roper et al., 2006). Proliferation impairment is worsened by altered cell fate specification with a reduction in neurogenesis and an increase in astrogliogenesis (Contestabile et al., 2007; Guidi et al., 2008; Guidi et al., 2011). Dendritic pathology is also a consistent feature and a possible substrate for cognitive impairment Abbreviations: AICD, amyloid precursor protein intracellular domain; APP, amyloid precursor protein; DS, Down syndrome; GSK3β, glycogen synthase kinase 3β; 5-HT, serotonin; aNPC, adult neural precursor cell. ⁎ Corresponding author at: Department of Biomedical and Neuromotor Sciences, Piazza di Porta San Donato 2, 40126 Bologna, Italy. Fax: +39 051 2091737. E-mail address: [email protected] (E. Ciani). Available online on ScienceDirect (www.sciencedirect.com). 1 Authors labeled with an asterisk contributed equally to the work.

http://dx.doi.org/10.1016/j.nbd.2014.03.003 0969-9961/© 2014 Elsevier Inc. All rights reserved.

in DS. In children and adults with DS, there is a marked reduction in dendritic branching and spine density (Becker, 1991; Prinz et al., 1997; Schulz and Scholz, 1992; Takashima et al., 1981; Takashima et al., 1989). This evidence suggests that proliferation impairment, cell fate specification and neuronal maturation defects may be key determinants of intellectual disability in individuals with DS. Trisomy 21 results in the triplication of over 400 genes (Sturgeon and Gardiner, 2011), which makes elucidation of the contribution of different genes in cognitive impairment a challenge. Recent evidence shows that the amyloid precursor protein (APP) gene is involved in important aspects of brain development, such as cell migration and cell cycle progression (Nalivaeva and Turner, 2013), suggesting the possible involvement of the triplicated gene APP in the neurodevelopmental alterations 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 the Ts65Dn mouse model of DS (Trazzi et al., 2011, 2013). It should be noted that APP is an extremely complex molecule that may be functionally important not only in its full-length configuration, but also as the source of numerous fragments with various effects on neural function (Zhou et al., 2011). For example, one fragment, the secreted APPsα, is neuroprotective, neurotrophic and regulates cell

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

excitability and synaptic plasticity, while the β-amyloid (Aβ) fragment appears to exert opposite effects. Less is known about the neural functions of the fragment called APP intracellular domain (AICD), but there is a growing interest in understanding AICD functions. For instance, this fragment appears to contribute to neurodegenerative disorders, such as Alzheimer's disease (Chang et al., 2006; Konietzko, 2012), by modulating neural cell survival (Muller et al., 2008). Moreover, AICD overexpression in transgenic mice impairs neurogenesis (Ghosal et al., 2010). By looking at the mechanisms whereby APP impairs neurogenesis in trisomic neuronal precursor cells, we found that AICD fragment was critically involved in the APP-dependent impairment of precursor cell proliferation, cell fate specification and neuronal maturation (Trazzi et al., 2011, 2013). Concerning the mechanism of action of AICD, it has been shown that AICD enters the nucleus and regulates gene expression by associating with 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 recently found that AICD is involved in the transcriptional regulation of Ptch1 in trisomic neural precursor cells (Trazzi et al., 2011). Excessive levels of AICD lead to overexpression of Ptch1, causing derangement of the Sonic Hedgehog (Shh) pathway (Trazzi et al., 2011). AICD may also exert actions independently from direct regulation of gene expression. In vitro studies showed that AICD can alter cell signaling (Leissring et al., 2002; Zhou et al., 2012) by interacting with proteins and probably regulating their stability or function. For instance, AICD directly interacts with glycogen synthase kinase 3β (GSK3β) promoting its kinase activity, with no effect on its transcription (Ryan and Pimplikar, 2005; Zhou et al., 2012). In the framework of the cellular mechanisms underlying brain alterations in DS, the direct link of AICD with GSKβ seems of relevance because GSK3β is a key component of a surprisingly large number of cellular processes and several diseases (Jope and Johnson, 2004). In particular, emerging evidence points at GSK3β as a key negative regulator in multiple neurodevelopmental processes, including neurogenesis, neuronal differentiation, neuronal migration and axon growth and guidance (Hur and Zhou, 2010). Moreover, changes in GSK3β activity have been associated with many psychiatric and neurodegenerative diseases (Tilleman et al., 2002). The broad spectrum of action of GSK3β in the normal and diseased brain suggests that deregulation of GSK3β activity dependent by the APP/AICD system may play a role in the brain phenotype of DS. We show here that increased levels of AICD alter GSK3β activity in neural precursor cells from the Ts65Dn mouse model of DS and that GSK3β deregulation negatively affects cell proliferation, cell fate specification and neuronal maturation.

25

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 cultures were obtained as previously reported (Trazzi et al., 2011). Cells were cultured in suspension in DMEM/F12 (1:1) containing B27 supplements (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 subculturing protocol consisted of neurosphere passaging every 7 days with whole culture media change (with freshly added FGF-2 and EGF). All experiments were done using neurospheres obtained after 2–3 passages from the initially prepared cultures. Most (98%) of the cells in neurospheres were positive for nestin, an established marker for neural and glial precursors. Cell cultures were kept in a 5% CO2 humidified atmosphere at 37 °C. Treatments during in vitro NPC differentiation were performed as follows:

In vitro differentiation Neurospheres were dissociated into a single cell suspension and plated onto poly-L-ornithine-coated 24-well chamber slides at a density of 3 × 104 cells per well. Cells were cultured for 2 days in DMEM/F12 medium containing EGF (20 ng/mL), FGF (20 ng/mL) and 2% fetal bovine serum (FBS) and then transferred to differentiation medium (EGF and FGF free plus 1% FBS) for 6 or 12 days. Every 2 days half of the medium was replaced with fresh differentiation medium.

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

Drugs The following drugs were administrated on alternate days: 1 μM fluoxetine, 100 nM (±)-8-Hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT), 2 mM Lithium chloride and 1 nM WAY-100635 maleate salt. All chemicals were purchased from Sigma-Aldrich.

Materials and methods Ts65Dn mice colony

Double-immunocytochemistry and analysis of neurite length in differentiated aNPCs

Female Ts65Dn mice carrying a segmental trisomy of chromosome 16 (Reeves et al., 1995) were obtained from Jackson Laboratories (Bar Harbour, ME, USA) and maintained on the original genetic background by mating them to C57BL/6JEi × C3SnHeSnJ (B6EiC3) F1 males. Animals were karyotyped by real-time quantitative PCR (qPCR) as previously described (Liu et al., 2003), and by PCR with primers spanning the translocation breakpoint of extra chromosome 1716 (Reinholdt et al., 2011). The animals had access to water and food ad libitum and were kept in a room with a 12:12 h dark/light cycle. Experiments were performed in accordance with the Italian and European Community law for the use of experimental animals and were approved by Bologna University Bioethical Committee. In this study all efforts were made to minimize animal suffering and to keep the number of animals used to a minimum.

For immunofluorescent staining, differentiated aNPC cultures were paraformaldehyde fixed and stained with antibodies against: glial fibrillary acidic protein (1:400; GFAP mouse monoclonal, Sigma) and ß tubulin III (1:100; rabbit polyclonal, Sigma) as primary antibodies, and with mouse FITC-conjugated (1:200; Sigma), and rabbit Cy3conjugated (1:200; Jackson Laboratories), as secondary antibodies. Samples were counterstained with Hoechst-33258. Ten random fields from each coverslip were photographed and counted. The number of positive cells for each marker was referred to the total number of Hoechst-stained nuclei. Evaluation of neurite length was performed by using the image analysis system Image Pro Plus (Media Cybernetics, Silver Spring, MD 20910, USA). The average neurite length per cell was calculated by dividing the total neurite length by the number of cells counted in the areas.

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

Immunocytochemistry and determination of the labeling index in cell cultures

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

Neurospheres were harvested on microscope slides by cytospin centrifugation (212 × g, 5 min, Shandon, Thermo, Dreieich, Germany). Specimens were fixed in 4% paraformaldehyde for 30 min. Blocking was done in 5% goat serum for 30 min followed by incubation with the following primary antibodies: anti-β-catenin (1:100, BD Transduction Laboratories), anti-phospho-GSK3β Ser9 (1:1000) (Cell Signaling Technology) and anti-GSK3β (1:1000) (Cell Signaling Technology). Detection was done with Cy3-conjugated anti-mouse or anti-rabbit antibodies (1:200, Jackson ImmunoResearch Laboratories). For proliferation analysis, neurospheres were treated with 10 μM BrdU as previously described (Trazzi et al., 2011), incubated with an anti-5-bromo-2-deoxyuridine (BrdU) monoclonal antibody (1:100; Roche Applied Science) and a Cy3-conjugated anti-mouse secondary antibody (1:200; Sigma). Samples were counterstained with Hoechst33258. Fluorescence images, taken from random microscopic fields (10–12 for each coverslip), were superimposed and used to determine the labeling index (LI), defined as percentage of cells labeled with BrdU over total cell number in three independent experiments in duplicate. Digital images were captured using NIS-Elements AR software (Nikon).

Ts65Dn mouse

Serotonin immunocytochemistry Differentiated aNPC cultures were paraformaldehyde fixed and stained with an anti-serotonin antibody (1:400; mouse monoclonal, Abcam) and with a mouse Cy3-conjugated (1:200; Jackson Laboratories), as secondary antibody. Samples were counterstained with Hoechst-33258.

Experimental protocol of in vivo mice treatment Euploid (n = 4) and Ts65Dn (n = 4) mice received a daily subcutaneous 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/kg from P8 to P15). Age-matched euploid (n = 4) and Ts65Dn (n = 4) mice were injected with the vehicle. Each treatment group had approximately the same composition of males and females. Histological procedures and immunohistochemistry P2 mice were decapitated, brains removed and fixed by immersion in Glyo-Fix (Thermo Electron Corp., Waltham, MA, USA) for 48 h and embedded in paraffin. The forebrain was coronally sectioned in 8 μm thick sections that were attached to poly-lysine-coated slides. One out of 12 sections, in P2 animals were stained using anti-phospho-GSK3β Ser9 (1:100) (Cell Signaling Technology), anti-GSK3β (1:100) (Cell Signaling Technology), anti-phospho-CRMP2 Thr514 (1:100) (Cell Signaling 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 and processed as previously described (Contestabile et al., 2007). Sections were incubated with Cy3-conjugated anti-rabbit (1:200; Jackson Laboratories) secondary antibody. Human fetuses

Western blotting

Subjects

Total proteins from the hippocampus and neurosphere cultures of euploid 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% sodium deoxycholate, 0.1% SDS) supplemented with 1 mM PMSF and 1% protease and phosphatase inhibitor cocktail (Sigma). Protein concentration was determined by the Lowry method (Lowry et al., 1951). Equivalent amounts (50 μg) of protein were subjected to electrophoresis on a 4– 12% Mini-PROTEAN® TGX™ Gel (Bio-Rad) and transferred to a Hybond ECL nitrocellulose membrane (Amersham Life Science). The following primary antibodies were used: anti-GAPDH rabbit polyclonal (1:5000; Sigma) anti-Alzheimer precursor protein A4 clone 22C11 mouse monoclonal (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 Signaling Technology), 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 detection the nitrocellulose membrane was processed for antigen-retrieval as previously described (Ryan and Pimplikar, 2005). The blot was incubated with the anti-C-terminal APP rabbit primary antibody (1:8000; A8717 Sigma-Aldrich). Densitometric analysis of digitized images was performed with Scion Image software (Scion Corporation, Frederick, MD, USA).

Human fetal brains (17–21 weeks of gestation) were obtained after prior informed consent from the parents and according to the procedures approved by the Ethical Committee of the St. Orsola-Malpighi Hospital, Bologna, Italy. Regulations of the Italian Ministry of Health and the policy of Declaration of Helsinki were followed. All fetuses were derived from legal abortions and were collected with an average post-mortem delay of approximately 2 h. Three control fetuses with no obvious developmental or neuropathological abnormalities and three DS fetuses were used. Trisomy was karyotypically proved from the results of genetic amniocentesis procedures. Autopsies were performed at the Institute of Pathology of the St. Orsola-Malpighi Hospital. The gestational age of each fetus was estimated by menstrual history and crown-rump length.

AICD/GSK3β coimmunoprecipitation 6 × 107 euploid or Ts65Dn aNPCs were lysated in the buffer (50 mM Tris–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 inhibitor cocktail) and cleared by centrifugation (10,000 ×g, 30 min). Anti-GSK3β (1:100) (Cell Signaling Technology) was added to equal amounts (2 mg) of protein lysate for 4 h at 4 °C and afterward incubated

Histological procedures Brains were fixed by subdural perfusion with Metacarnoy fixative (methyl alcohol:chloroform:acetic acid 6:1:1) injected through the anterior and posterior fontanelles. After 24 h, brains were removed, and the hippocampal region of each hemisphere was coronally sectioned to obtain 3 blocks with a thickness of 2–3 mm. The first block roughly corresponded to the rostral third of the hippocampal formation. The blocks were post-fixed in formalin (4% buffered formaldehyde) for 5 days, embedded in paraffin, according to standard procedures and sectioned in 4–5 μm-thick coronal sections. GSK3β immunohistochemistry Serial sections from the first block of the right hemisphere were stained using anti-phospho-GSK3β Ser9 (1:100) (Cell Signaling Technology) and anti-GSK3β (1:100) (Cell Signaling Technology) antibodies. Antigens were retrieved with citrate buffer, pH 6.0 at 98 °C for 40 min.

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

After cooling at room temperature for 20 min, sections were washed with distilled water, buffered for 10 min in PBS 0.1 M (pH 7.2–7.4) and then incubated overnight with the primary antibody. Sections were incubated with Cy3-conjugated anti-rabbit (1:200; Jackson Laboratories) secondary antibody. Immune serum was omitted in negative controls. For each antibody, all sections were immunostained in a single batch to avoid possible immunostaining differences. Adjacent sections were used for phospho-GSK3β Ser9 and GSK3β immunohistochemistry, respectively. Five-six sections 200 μm apart were used for each antibody. Immunofluorescent image acquisition and quantification of human and mouse brain samples Fluorescence-labeled signals were acquired using an Eclipse TE 2000-S microscope (Nikon) equipped with an AxioCam MRm (Zeiss) digital camera. Image processing and analysis were carried out using the NIS Elements AR software (Nikon). All parameters used in the acquisition step were standardized (detector gain and exposure time) to maintain high reproducibility. Immunofluorescence was quantified within the regions of interest (the SVZ and granular cell layer of the hippocampus) by measuring the total fluorescence units in a box of 900 μm2 for the SVZ and 1600 μm2 for the granule cell layer, randomly placed at six different sites. The total fluorescence intensity in the SVZ and granule cell layer was normalized to the background fluorescence of the corpus callosum and fimbria, respectively, because these fiber tracts exhibit low immunoreactivity. Intensity values in each region of interest are expressed as percentage of the values in euploid samples. Real-time RT-qPCR Total RNA was extracted from the hippocampus and neurosphere cultures of euploid and Ts65Dn mice with TriReagent (Sigma-Aldrich) according to the manufacturer's instructions. cDNA synthesis was achieved with 1.0 μg of total RNA using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. The used primer sequences 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′-ACAGGGCG GTGGGGACTC-3′, and reverse, 5′-CAAGCAGGCGGGGACATAGG-3′ ; 5hydroxytryptamine (serotonin) receptor 2A (Htr2A; NM_172812.2), forward, 5′-GCCTACAAGTCTAGTCAGCTCCAG-3′, and reverse, 5′-ACATCTCT TCCGAGTGTTGGTTCC-3′; 5-hydroxytryptamine (serotonin) receptor 2C (Htr2C; NM_008312.4), forward, 5′-GGGTTGCTGCCACTGCTTTG-3′, and reverse 5′-ACACTACTAATCCTCTCGCTGACC-3′, solute carrier family 6 [neurotransmitter transporter serotonin (SERT; NM_010484.2), forward, 5′-GATCCCTGCTCACACTGACATC-3′ and reverse, 5′-CCATAGAACCAAGA CACGACGAC-3′ ; glyceraldehyde-3-phosphate dehydrogenase (GAPDH; NM_008084.2), forward, 5′-GAACATCATCCCTGCATCCA-3′, and reverse, 5′-CCAGTGAGCTTCCCGTTCA-3′. Real-time PCR was performed using a SYBR Premix Ex Taq kit (Takara) according to the manufacturer's instructions in an iQ5 real-time PCR detection system (Bio-Rad). Fluorescence was determined at the last step of every cycle. Real-time PCR assay was done under the following universal conditions: 2 min at 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 quantification was performed using the ΔΔ Ct method. Statistical analysis Results are presented as the mean ± standard error (SE) of the mean. Statistical significance was assessed by two-way analysis of variance (ANOVA), followed by Bonferroni's post hoc test or by the two-tailed Student's t-test. A probability level of P b 0.05 was considered to be statistically significant.

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Results Increased GSK3β activity in neuronal precursor cells from the Ts65Dn mouse Cultures of subventricular-zone-derived adult neuronal precursor cells (aNPCs) of Ts65Dn mice are a suitable model to study the mechanisms underlying neurogenesis impairment in DS because they exhibit reduced proliferation rate, impaired acquisition of a neuronal phenotype and neuronal maturation similarly to the in vivo condition (Trazzi et al., 2011, 2013). We first sought to establish whether GSK3β activity is altered in trisomic vs. euploid aNPCs. Since GSK3β kinase activity is inhibited through phosphorylation of serine 9, we examined GSK3β activity by using an anti-GSK3β phospho-specific antibody (Ser9). We found decreased GSK3β phosphorylation, evaluated both by immunocytochemistry (Figs. 1A,B) and Western blotting (Fig. 1C), in trisomic vs. euploid cultures, indicating an increased GSK3β activity in trisomic aNPCs. In contrast, trisomic and euploid aNPCs had similar levels of mRNA transcripts (data not shown) and total protein levels of GSK3β (Fig. 1B). GSK3β exerts its functions by modulating the activity of a wide range of substrates involved in gene transcription, including β-catenin (Ikeda et al., 1998). Active GSK3β controls the amount of β-catenin, by reducing β-catenin protein stability (Nemoto et al., 2009). We found that β-catenin expression was considerably lower in trisomic aNPCs compared to euploid aNPCs (Figs. 1A,B,C), which is in agreement with the increased activity of GSK3β. Phosphorylated Akt has been shown to play a central role in the inhibition of GSK3β (by increasing its phosphorylation) in response to insulin and insulin growth factors (Cross et al., 1995). We found no difference in the phosphorylation levels of Akt (Fig. 1D), indicating that Akt signaling is not involved in GSK3β activation in trisomic aNPCs. APP/AICD-dependent GSK3β activation in neuronal precursor cells from the Ts65Dn mouse We previously found that trisomic aNPCs had increased levels of AICD due to trisomic APP expression and that increased levels of AICD impair precursor cell proliferation, cell fate specification and neuronal maturation of trisomic aNPCs (Trazzi et al., 2011, 2013). Since AICD has been reported to directly interact with GSK3β reducing its phosphorylation at Ser9, thereby increasing its activity (Ryan and Pimplikar, 2005; Zhou et al., 2012), and that AICD overexpressing mice showed an activation of GSK3β (Ryan and Pimplikar, 2005), it may be hypothesized that increased levels of the APP/AICD system may underlie the observed GSK3β activation in trisomic aNPCs. We reduced APP expression by using lentiviral-mediated RNA interference in trisomic aNPCs and found an increase in the phosphorylation levels of GSK3β (Fig. 2A). We next examined the effect of increased levels of APP on GSK3β activity in euploid aNPCs. Infection with recombinant adenoviruses containing the APP sequence caused a decrease in GSK3β phosphorylation (Fig. 2A). This evidence suggests that the reduced phosphorylation of GSK3β (hence, increased activity) in trisomic aNPCs is APP-dependent. Next, we over-expressed AICD in euploid aNPCs. We found that AICD overexpression significantly decreased the phosphorylation levels of GSK3β (Fig. 2A), suggesting that the APPdependent phosphorylation reduction of GSK3β found in trisomic aNPCs was due to AICD accumulation. In order to establish whether alteration of GSK3β phosphorylation was retained after differentiation, we evaluated the phosphorylation levels of GSK3β in differentiated trisomic aNPC cultures. We found that differentiated trisomic cultures exhibited reduced phosphorylation levels of GSK3β (Fig. 2B). In agreement with the results obtained in proliferating aNPCs, APP interference significantly increased the phosphorylation levels of GSK3β in differentiated trisomic cultures (Fig. 2B) and APP or AICD overexpression decreased GSK3β phosphorylation levels in euploid cultures (Fig. 2B).

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

Ts65Dn

Euploid

Ts65Dn

Euploid

β-catenin/Hoechst

P-GSK3β/Hoechst

Euploid

B

50

Ts65Dn

125

β-catenin (% of Euploid)

75

GSK3β (% of Euploid)

100

125

**

P-GSK3β (% of Euploid)

125

100 75 50

100 75 50

25

25

25

0

0

0

125

125

75 50 25

100 75 50 25 0

0

125

*

β-catenin/GAPDH (% of Euploid)

100

***

D P-GSK3β/GSK3β (% of Euploid)

C

P-AKT/AKT (% of Euploid)

A

*

28

100 75 50 25 0

P-GSK3β

β-catenin

P-AKT

GSK3 β

GAPDH

AKT

Fig. 1. Altered GSK3β phosphorylation and β-catenin expression in aNPCs from the Ts65Dn mouse. A: Immunofluorescence of Ts65Dn and euploid neurospheres showing phospho-GSK3β Ser9, 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 euploid mice. 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 in trisomic 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).

In order to establish whether the decreased phosphorylation levels of GSK3β in trisomic aNPCs were mediated by a direct AICD–GSK3β interaction, we used an immunoprecipitation assay. In anti-GSK3β immunoprecipitates of trisomic aNPCs we found immunoreactivity for AICD (Fig. 2C), indicating that AICD directly binds to GSK3β. AICD binding was also found in euploid aNPCs (Fig. 2C), though AICD immunoreactivity was lower, in agreement with the lower levels of AICD in euploid aNPCs. Normalization of GSK3β activity in neuronal precursor cells from the Ts65Dn mouse restores cell proliferation To determine whether excessive activation of the GSK3β underlies impaired cell proliferation in trisomic aNPCs, we treated aNPCs with two inhibitors of GSK3β activity. We used lithium, a well-known inhibitor of GSK3β activity that acts both indirectly, by increasing the inhibitory phosphorylation of GSK3β, and directly, by antagonizing its kinase activity (Jope, 2003). Since recent evidence shows that the activation of the serotonin receptor 5-HT1A by serotonin or the selective agonist

8-OH-DPAT inhibits GSK3β activity by increasing GSK3β phosphorylation (Polter et al., 2012), we additionally used 8-OH-DPAT. We found that in trisomic aNPCs both lithium and 8-OH-DPAT completely restored GSK3β phosphorylation levels (Fig. 3A). This effect was accompanied by restoration of the proliferation impairment that characterizes trisomic aNPCs (Figs. 3B,C). In euploid aNPCs, lithium increased GSK3β phosphorylation, while 8-OH-DPAT did not affect GSK3β phosphorylation (Supplementary Fig. 1A). In euploid aNPCs the lithium-induced increase in GSK3β phosphorylation was accompanied by an increase in cell proliferation (Supplementary Fig. 1B).

Normalization of GSK3β activity in neuronal precursor cells from the Ts65Dn mouse restores cell fate specification and neuronal maturation Since decreased GSK3β phosphorylation characterized also differentiated trisomic aNPCs (Fig. 2B), we sought to establish whether the impaired neuronal fate acquisition and maturation that characterize trisomic aNPCs (Trazzi et al., 2011, 2013) are due to increased GSK3β

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

A

29

Proliferation

** **

#

100

**

P-GSK3β/GSK3β (% of Euploid)

125

75

EU

50

P-GSK3β β

25

GSK3β β shRNA

0

shRNA APP AICD

- + - - - + - - - +

-

B

APP AICD

TS

-

-

EU

+ -

- - - + - - +

Differentiation EU

100

* **

#

**

P-GSK3β/GSK3β (% of Euploid

125

75

EU

P-GSK3β β GSK3β β

50

-

- + - - - + - - - +

Euploid

Ts65Dn

shRNA

25

APP 0

- + - - - + - - - +

EU

TS

APP AICD

Input

IP

Input

-

shRNA

C

TS

AICD

IP

10kDa

AICD

6kDa

IB: α−AICD 50kDa

GSK3β β 37kDa

IB: α−GSK3β β Fig. 2. APP/AICD dependent GSK3β phosphorylation in aNPCs from the Ts65Dn mouse. A: Western blot quantification of phospho-GSK3β Ser9 expression, normalized to total GSK3β content, in trisomic and euploid neurospheres. Trisomic aNPCs were infected at day 1 post-plating with APP shRNA lentiviral particles at a multiplicity of infection (MOI) of 2.5. Euploid aNPCs were 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 of phospho-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 total GSK3β content, in 6-day differentiated aNPCs. aNPCs were infected at day 1 post-plating as indicated above. Twenty-four hours later, the medium was replaced with a differentiation medium. Right panel: Example of Western blot analysis of phospho-GSK3β Ser9 and GSK3β expression in differentiated aNPCs from euploid (EU) and Ts65Dn (TS) mice. Data (in A,B), given as 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 as compared to the untreated trisomic condition (Bonferroni test after ANOVA). C: Interactions between GSK3β and AICD in vivo. Ts65Dn and euploid cell lysates of aNPCs were immunoprecipitated 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).

activity and whether these defects can be restored by treatments that normalize GSK3β phosphorylation. Treatment with lithium and 8-OH-DPAT completely restored GSK3β phosphorylation levels in trisomic differentiated cultures (Fig. 4A), an effect that is similar to that observed in undifferentiated aNPCs (Fig. 3A). We found that differentiated aNPCs expressed tryptophan hydroxylase 1 (TPH1) (Supplementary Fig. 2A), the rate-limiting enzyme

in serotonin synthesis, and were immunoreactive for serotonin (Supplementary Fig. 2B), suggesting the presence of serotonergic neurons in the population of aNPCs. However, it must be noted that serotonin was present in the differentiating culture medium, due to serum addition (necessary for culture differentiation). Therefore, we cannot rule out that serotonin immunoreactivity may be due to the uptake of exogenous serotonin. In view of the presence of serotonin, we treated aNPCs

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BrdU/Hoechst 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 total GSK3β 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 neurospheres from 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 BrdU immunocytochemistry. 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 to the untreated trisomic condition (Bonferroni test after ANOVA).

with fluoxetine, a selective serotonin reuptake inhibitor that increases serotonin availability. We found that fluoxetine caused an effect on GSK3β phosphorylation similar to that obtained by the activation of the 5-HT1A receptor through the agonist 8-OH-DPAT (Fig. 4A). Evaluation of the number of cells with either a neuronal (β-tubulin III-positive cells) or an astrocytic (GFAP-positive cells) phenotype showed that lithium, 8-OH-DPAT and fluoxetine treatments increased the percentage of new neurons (Figs. 4B,C) and decreased the percentage of new astrocytes (Figs. 4B,C) in trisomic cultures. Importantly, the percentage of cells of each phenotype became similar to that of untreated euploid cultures (Fig. 4C), indicating that treatments that are able to restore GSK3β phosphorylation levels also restore the process of cell fate specification. Co-exposure of cells to WAY-100635 (WAY), a selective antagonist of the serotonin receptor 5-HT1A, and either 8-OH-DPAT or fluoxetine prevented restoration of fate acquisition, indicating that the effect of 8-OH-DPAT and fluoxetine was mediated by the 5-HT1A receptor (Fig. 4C). Inhibition of the 5-HT1A receptor by WAY had no effect on cell fate specification (Fig. 4C). In euploid aNPCs, while lithium increased the acquisition of a neuronal phenotype and reduced the acquisition of an astrocytic phenotype (Supplementary Fig. 1C), no effect was induced by 8-OH-DPAT or fluoxetine treatment (Supplementary Fig. 1C). In view of the role of the GSK3β in neuronal maturation (Kim et al., 2011), we sought to determine whether increased GSK3β activity also underlies reduced neurite length of trisomic aNPCs. Evaluation of neurite length of the new neurons after treatment with lithium, 8-OHDPAT and fluoxetine showed that in trisomic aNPC cultures neurite length underwent a large increase and became similar to that of untreated euploid cultures (Fig. 4D). Co-treatment with WAY prevented the neurite length increase induced by 8-OH-DPAT and fluoxetine treatments (Fig. 4D). Unlike in trisomic aNPC cultures, in euploid cultures treatments had no effect on neurite length (Supplementary Fig. 1D).

In order to establish the mechanism whereby 8-OH-DPAT and fluoxetine were effective in trisomic but not in euploid cultures, we examined the expression of serotonin receptors involved in neurogenesis/ differentiation (5-HT1A, 5-HT2A, 5-HT2C) and of the serotonin transporter (SERT) by quantitative real time PCR (RT-qPCR). No differences were found between trisomic and euploid cultures in the expression of any of these genes (Supplementary Fig. 2C), indicating that the different response to treatments was not due to the differences in serotonin receptor/transporter expression. Taken together, results suggest an impairment of the serotonin pathway downstream to the membrane receptors in trisomic aNPCs that is overcome by increasing activation of the receptors, with consequent increase in GSK3β phosphorylation. Increased GSK3β activity in the SVZ and hippocampus of Ts65Dn mice Based on the high GSK3β activity in trisomic aNPC cultures (Figs. 1A,B), we next sought to establish whether a similar up-regulation occurred also in the in vivo condition. We analyzed GSK3β phosphorylation levels in the two major sites of adult neurogenesis in the brain, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) of newborn Ts65Dn and euploid mice. We found in both analyzed regions a decrease in the phosphorylation levels of GSK3β, evaluated by immunohistochemistry (Figs. 5A, B). This effect was confirmed in hippocampal homogenates by Western blot analysis (Fig. 5C). Similarly to evidence obtained in cultures of trisomic aNPCs, we found an increased expression of APP and accumulation of AICD in the hippocampus of Ts65Dn mice (Fig. 5D), which most likely accounts for alteration in the phosphorylation levels of GSK3β. In order to establish whether changes in the phosphorylation status 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 Ts65Dn mouse. A: Western blot quantification of phospho-GSK3β Ser9 expression, 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) starting from 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: Representative double-fluorescence images of 6-day differentiated aNPCs immunopositive for β tubulin III (red) and GFAP (green). Scale bar: 60 μm. C: Percentages of β tubulin III- and GFAP-positive cells in 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 plus WAY or Fluo plus WAY 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 after ANOVA). D: Quantification of neurite outgrowth of β tubulin III-positive cells from differentiated aNPC cultures from euploid (n = 6) and Ts65Dn (n = 5) mice. aNPC cultures were treated as 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).

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

CRMP2 in Ts65Dn mice was confirmed by Western blot analysis in hippocampal homogenates (Fig. 5F). Fluoxetine treatment restores GSK3β pathway activity in the hippocampus of Ts65Dn mice Recently, we found that neonate Ts65Dn mice treated (P2–P15) with fluoxetine exhibited full recovery of hippocampal neurogenesis,

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

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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 lateral ventricle (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 expression 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: Western blot quantification of phospho-GSK3β Ser9 expression in the hippocampus of P2 euploid (n = 4) and Ts65Dn (n = 4) mice. Lower panels: Representative examples of Western 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 of Western 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. Immunofluorescence 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 of phospho-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, subventricular zone.

granule cell dendritic maturation and hippocampus-dependent behavior (Bianchi et al., 2010b; Guidi et al., 2013). The mechanism underlying the regulation of hippocampal neurogenesis by fluoxetine in Ts65Dn mice is still unknown. Quantification of GSK3β phosphorylation in the hippocampal region of P15 untreated mice showed that also at this age Ts65Dn mice had lower GSK3β phosphorylation in comparison with euploid mice (Fig. 6A). After treatment with fluoxetine, the levels of 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 comparison 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). This data suggests that modulation of GSK3β activity by fluoxetine may be involved in the positive impact of fluoxetine on brain development in Ts65Dn mice.

Increased GSK3β activity in the trisomic fetal brain Recently, we found that human fetuses with DS had remarkably fewer proliferating cells in all germinal zones of the hippocampal region (Contestabile et al., 2007; Guidi et al., 2008). To establish whether, similarly to the mouse model, fetuses with DS exhibit increased GSK3β activity, we examined the phosphorylation levels of GSK3β in the granular layer of the DG (Fig. 7A) and in the ventricular zone of the hippocampus (Fig. 7B). At the investigated gestational age (17–21 weeks of gestation), both regions exhibit intense mitotic activity (Contestabile et al., 2007; Guidi et al., 2008). Immunohistochemical analysis showed that the phosphorylation levels of GSK3β were lower in both the granular layer of the DG (−35%) and in the ventricular zone of the hippocampus (−25%) of fetuses with DS (Figs. 7A,B), indicating that a defect in GSK3β activity is present also in neuronal precursor cells of human subjects with DS.

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

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+Fluo Fig. 6. Effect of fluoxetine on GSK3β and CRMP2 phosphorylation in the hippocampus of euploid and Ts65Dn mice. A,B: Western blot quantification of phospho-GSK3β Ser9 expression and phospho-CRMP2 Thr514 expression in the hippocampus of P15 untreated euploid (n = 4) and Ts65Dn (n = 4) mice and euploid (n = 4) and Ts65Dn (n = 3) mice treated with fluoxetine (Fluo). Protein levels were normalized respectively to total GSK3β and CRMP2 content and expressed as percent of untreated condition (100%). Representative examples of Western blots on 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 condition; #P b 0.05 as compared to untreated trisomic samples (Bonferroni test after ANOVA).

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Fig. 7. Altered GSK3β phosphorylation in human fetuses with DS. A,B: Examples of phospho-GSK3β Ser9 fluorescence immunohistochemistry at the level of the hippocampal dentate gyrus (A) and the ventricular zone of the hippocampus (B) of a control (gestational week GW 19; n = 3) and a DS (GW 19; n = 3) fetus. The dashed line square indicates the region where the immunofluorescence 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 hippocampal 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, are expressed 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.

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Discussion This study shows that GSK3β signaling is altered in the DS brain and that increased GSK3β activity is related to increased levels of the APP intracellular fragment (AICD). Disruption of GSK3β signaling adversely affects 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 useful tools 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 associated with deficits in GSK3β signaling (Lei et al., 2011; Salcedo-Tello et al., 2011), we thought it was important to establish whether GSK3β activity is altered in DS at early phases of development. Indeed, we found an alteration in GSK3β activity in aNPCs of Ts65Dn mice and in neuronal precursors of human fetuses with DS. Importantly, an increase in GSK3β activity was observed not only in proliferating but also in differentiated aNPCs (Figs. 2B and 5C). These results strongly suggest that the increased GSK3β activity that characterizes trisomic cells may be a determinant of the reduced neurogenesis and dendritic atrophy that characterize the trisomic brain starting from early phases of development. GSK3β is also expressed in the adult brain and is particularly enriched in the hippocampus, neocortex, and cerebellum (Yao et al., 2002), suggesting a role of this kinase also in adulthood. Overexpression of GSK3β in the hippocampus results in tau-dependent neurodegeneration of this region (Gomez de Barreda et al., 2010) and the neurodegeneration observed in Alzheimer's disease (AD) is also thought to result from tau hyperphosphorylation. A unique characteristic of DS is that subjects have a high risk of developing AD from 35 years of age onwards. The observed increase in GSK3β activity in the trisomic brain may underlie the development of AD-like pathology due to tau hyperphosphorylation. GSK3β regulates a wide variety of developmental events by influencing a broad range of substrates involved in gene transcription, for instance β-catenin (Ikeda et al., 1998), or regulating cytoskeletal dynamics, for instance CRMP2 (Yoshimura et al., 2005). GSK3β plays a key role in controlling the amount of β-catenin, by negatively regulating β-catenin protein stability (Wada, 2009). The current finding in trisomic aNPCs of decreased levels of β-catenin is consistent with the increased GSK3β activity. Recent evidence shows that β-catenin, besides being involved in a variety of functions (Moon et al., 2004; Toledo et al., 2008), plays an important role in regulating proliferation of neural stem cells. In these cells, βcatenin acts downstream from the canonical Wnt-signaling pathway (Chenn and Walsh, 2002; Chenn and Walsh, 2003) and appears to increase proliferation by decreasing cell cycle exit (Chenn and Walsh, 2002). Our results suggest that deregulation of the GSK3β/β-catenin system may concur to impair proliferation of trisomic aNPCs. Inhibition of GSK3β promotes dendritic growth in sympathetic, cortical and hippocampal neurons (Lim and Walikonis, 2008; Naska et al., 2006). Conversely, abnormally increased GSK3β activity contributes to dendrite degeneration under pathological conditions (Lin et al., 2010). A recent study shows that GSK3β mediates phosphorylation of CRMP2 and demonstrates that the GSK3β/CRMP2 pathway is an important mediator of cerebellar granule neuron dendritogenesis (Tan et al., 2013). Our findings suggest a correlation between altered GSK3β activity and increased CRMP2 phosphorylation in the trisomic brain, which may provide new insights into the understanding of the mechanisms underlying dendritic hypotrophy in DS. Overexpression of the APP/AICD system underlies deregulation of GSK3β activity in trisomic aNPCs We have previously reported that increased levels of AICD impair the 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 Ptch1 promoter nucleosomes (Trazzi et al., 2011). Here we provide evidence that the APP/AICD system contributes to regulate the GSK3β activity in trisomic aNPCs. This is in agreement with a study that demonstrated the activation of GSK3β and phosphorylation of CRMP2 in transgenic mice expressing AICD (Ryan and Pimplikar, 2005). Since the levels of mRNA transcripts and total protein levels of GSK3β were not changed in trisomic mice, the AICD-dependent modulation of GSK3β activation must be mediated by a non-transcriptional mechanism. This is consistent with evidence that AICD, in addition to modulate transcription, brings about its effects without being present in the nucleus. Interestingly, recent evidence shows that AICD associates with GSK3β and enhances its kinase activity, as indicated by decreased Ser9 phosphorylation (Zhou et al., 2012). Current findings provide evidence that in trisomic aNPCs AICD directly associates with GSK3β and increases its activity by phosphorylating Ser9. It is well known that several signaling pathways induce the inhibition of GSK3β by phosphorylating the same residue (Fang et al., 2002). Hence, we cannot exclude that other pathways may contribute, in addition to the APP/AICD system, to the impairment of GSK3β activity that characterizes trisomic aNPCs. The role of triplicated genes in the neurological phenotype of DS is still poorly understood. Our data suggest that the trisomic gene APP may be a key determinant of impaired neurogenesis in DS, through AICD-dependent mechanisms. In addition to modulating the Shh pathway (Trazzi et al., 2011), AICD appears to play an important role in the modulation of GSK3β activity and signaling. Interestingly, recent evidence shows that AICD transgenic mice exhibit impaired neurogenesis (Ghosal et al., 2010) and deficits in working memory, that are blocked by lithium treatment (Ghosal et al., 2010). The demonstration of a causal link between overexpression of the APP/AICD system and deregulation of GSK3β activity in DS may be a starting point for studies that can shed light on the mechanisms that underlie the DS neurological phenotype. GSK3 inhibitors as pharmacotherapy for Down syndrome Changes in GSK3β activity have been associated with many psychiatric and neurodegenerative diseases (Tilleman et al., 2002) and it has become increasingly apparent that GSK3β might be a common therapeutic 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 GSK3β in psychiatric disorders has revealed that GSK3β can be regulated by antidepressants acting on serotonergic (5-HT) neurotransmission (Li et al., 2004). Importantly, activation of the 5-HT1A receptor leads to phosphorylation of GSK3β (Li et al., 2004; Polter et al., 2012) and there is evidence that administration of fluoxetine results in enhanced GSK3β 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 activity in trisomic aNPCs and that this effects is accompanied by restoration of neurogenesis (cell proliferation, fate specification and neurite outgrowth). These data suggest that normalization of GSK3β activity may play a central role in restoration of neurogenesis. However, in view of the indirect action of lithium on other cellular signaling pathways (Kang et al., 2003; Pardo et al., 2003; Sasaki et al., 2006) and the multiple signal transduction pathways affected by 5-HT receptors, we cannot exclude the contribution of additional mechanisms in neurogenesis restoration. Early treatment with fluoxetine has been previously shown to correct neurogenesis, dendritic hypotrophy and behavior in Ts65Dn mice (Bianchi et al., 2010b; Guidi et al., 2013). We show here that this treatment restores GSK3β activity, suggesting that this effect may be involved in the positive impact of treatment in the trisomic brain. Consistently with current findings that lithium restores GSK3β activity and

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neurogenesis in trisomic aNPCs, treatment with lithium in adult Ts65Dn mice rescues neurogenesis, synaptic plasticity and memory (Bianchi et 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 brains with DS suggests that deregulation of GSK3β activity may contribute to the impairment of brain development. No effective therapies are available at present for the rescue of neurogenesis and cognitive disability in individuals with DS. GSK3 inhibitors are currently in clinical trials for several neurological disorders (Eldar-Finkelman and Martinez, 2011). We suggest that pharmacotherapy with GSK3β inhibitors may be 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. References Baek, S.H., Ohgi, K.A., Rose, D.W., Koo, E.H., Glass, C.K., Rosenfeld, M.G., 2002. Exchange of N-CoR corepressor and Tip60 coactivator complexes links gene expression by NF-kappaB and beta-amyloid precursor protein. Cell 110, 55–67. Beaulieu, J.M., 2007. Not only lithium: regulation of glycogen synthase kinase-3 by antipsychotics and serotonergic drugs. Int. J. Neuropsychopharmacol. 10, 3–6. Becker, L.E., 1991. Synaptic dysgenesis. Can. J. Neurol. Sci. 18, 170–180. Bianchi, P., Ciani, E., Contestabile, A., Guidi, S., Bartesaghi, R., 2010a. Lithium restores neurogenesis in the subventricular zone of the Ts65Dn mouse, a model for Down syndrome. Brain Pathol. 20, 106–118. Bianchi, P., Ciani, E., Guidi, S., Trazzi, S., Felice, D., Grossi, G., Fernandez, M., Giuliani, A., Calza, L., Bartesaghi, R., 2010b. Early pharmacotherapy restores neurogenesis and cognitive performance in the Ts65Dn mouse model for Down syndrome. J. Neurosci. 30, 8769–8779. Cao, X., Sudhof, T.C., 2001. A transcriptionally [correction of transcriptively] active complex of APP with Fe65 and histone acetyltransferase Tip60. Science 293, 115–120. Chakrabarti, L., Galdzicki, Z., Haydar, T.F., 2007. Defects in embryonic neurogenesis and initial synapse formation in the forebrain of the Ts65Dn mouse model of Down syndrome. J. Neurosci. 27, 11483–11495. Chang, K.A., Kim, H.S., Ha, T.Y., Ha, J.W., Shin, K.Y., Jeong, Y.H., Lee, J.P., Park, C.H., Kim, S., Baik, T.K., Suh, Y.H., 2006. Phosphorylation of amyloid precursor protein (APP) at Thr668 regulates the nuclear translocation of the APP intracellular domain and induces neurodegeneration. Mol. Cell. Biol. 26, 4327–4338. Chenn, A., Walsh, C.A., 2002. Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science 297, 365–369. Chenn, A., Walsh, C.A., 2003. Increased neuronal production, enlarged forebrains and cytoarchitectural distortions in beta-catenin overexpressing transgenic mice. Cereb. Cortex 13, 599–606. Contestabile, A., Fila, T., Ceccarelli, C., Bonasoni, P., Bonapace, L., Santini, D., Bartesaghi, R., Ciani, E., 2007. Cell cycle alteration and decreased cell proliferation in the hippocampal dentate gyrus and in the neocortical germinal matrix of fetuses with Down syndrome and in Ts65Dn mice. Hippocampus 17, 665–678. Contestabile, A., Greco, B., Ghezzi, D., Tucci, V., Benfenati, F., Gasparini, L., 2013. Lithium rescues synaptic plasticity and memory in Down syndrome mice. J. Clin. Invest. 123, 348–361. Cross, D.A., Alessi, D.R., Cohen, P., Andjelkovich, M., Hemmings, B.A., 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 378, 785–789. Eldar-Finkelman, H., Martinez, A., 2011. GSK-3 inhibitors: preclinical and clinical focus on CNS. Front. Mol. Neurosci. 4, 32. Fang, X., Yu, S., Tanyi, J.L., Lu, Y., Woodgett, J.R., Mills, G.B., 2002. Convergence of multiple signaling cascades at glycogen synthase kinase 3: Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase Cdependent intracellular pathway. Mol. Cell. Biol. 22, 2099–2110. Fukata, Y., Itoh, T.J., Kimura, T., Menager, C., Nishimura, T., Shiromizu, T., Watanabe, H., Inagaki, N., Iwamatsu, A., Hotani, H., Kaibuchi, K., 2002. CRMP-2 binds to tubulin heterodimers to promote microtubule assembly. Nat. Cell Biol. 4, 583–591. Gao, Y., Pimplikar, S.W., 2001. The gamma-secretase-cleaved C-terminal fragment of amyloid precursor protein mediates signaling to the nucleus. Proc. Natl. Acad. Sci. U. S. A. 98, 14979–14984. Ghosal, K., Stathopoulos, A., Pimplikar, S.W., 2010. APP intracellular domain impairs adult neurogenesis in transgenic mice by inducing neuroinflammation. PLoS One 5, e11866.

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