Experimental Neurology 253 (2014) 126–137

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Experimental Neurology

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The PSD protein ProSAP2/Shank3 displays synapto-nuclear shuttlingwhich is deregulated in a schizophrenia-associated mutation

Stefanie Grabrucker a,b, Christian Proepper b, Katharina Mangus a, Matti Eckert a, Resham Chhabra a,Michael J. Schmeisser b, Tobias M. Boeckers b, Andreas M. Grabrucker a,b,⁎a WGMolecular Analysis of Synaptopathies, Neurology Dept., Neurocenter of Ulm University, Albert-Einstein Allee 11, 89081 Ulm, Germanyb Institute for Anatomy and Cell Biology, Ulm University, Albert-Einstein Allee 11, 89081 Ulm, Germany

⁎ Corresponding author at: Ulm University, Anatomy aAnalysis of Synaptopathies, Albert-Einstein Allee 11, D-8731 500 1223214.

E-mail address: andreas.grabrucker@uni-ulm.de (A.M

0014-4886/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.expneurol.2013.12.015

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 July 2013Revised 12 December 2013Accepted 20 December 2013Available online 29 December 2013

Keywords:SCZSynapseShank3hnRNPAutismPSDNuclear shuttlingLRRTM1Synaptotagmin 1

Recently, mutations in ProSAP2/Shank3 have been discovered as one of the genetic factors for schizophrenia(SCZ). Here, we show that the postsynaptic density protein ProSAP2/Shank3 undergoes activity dependentsynapse-to-nucleus shuttling in hippocampal neurons. Our study shows that the de novo mutation (R1117X)in ProSAP2/Shank3 that was identified in a patient with SCZ leads to an accumulation of mutated ProSAP2/Shank3 within the nucleus independent of synaptic activity. Furthermore, we identified novel nuclearProSAP2/Shank3 interaction partners. Nuclear localization of mutated ProSAP2/Shank3 alters transcription ofseveral genes, among them already identified genetic risk factors for SCZ such as Synaptotagmin 1 andLRRTM1. Comparing the SCZ mutation of ProSAP2/Shank3 to the knockdown of ProSAP2/Shank3 we foundsome shared features such as reduced synaptic density in neuronal cultures. However, hippocampal neuronsexpressing the ProSAP2/Shank3 SCZ mutation furthermore show altered E/I ratio and reduced dendriticbranching. Thus, we conclude that the uncoupling of ProSAP2/Shank3 nuclear shuttling from synaptic activitymay represent a molecular mechanism that contributes to the pathology of SCZ in patients with mutations inProSAP2/Shank3.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Schizophrenia (SCZ) is a mental disorder characterized by theoccurrence of disordered thought processes and disturbed emotionalresponsiveness (Picchioni and Murray, 2007; Tandon et al., 2009; vanOs and Kapur, 2009). SCZ is associated with auditory hallucinations,paranoid and confused delusions as well as disorganized speech andthinking. The symptoms of SCZ usually manifest in young adulthood,with an overall lifetime prevalence of about 0.3 to 0.7% (van Os andKapur, 2009). Although thediagnosis is based onbehavioral observationand experience reports from the patients thus far, some genes werefound to be associated with SCZ (Greenwood et al., 2012; Sun et al.,2008; Zhao et al., 2013). Many of them have a central role in synapticfunction, including Dysbindin (DTNBP1) (Straub et al., 2002),Neuregulin 1 (NRG1) (Stefansson et al., 2002), Shank1 (Lennertz et al.,2012), ProSAP2/Shank3 (Gauthier et al., 2010), proteins of the Synapto-tagmin family and Leucine-rich repeat transmembrane neuronalprotein 1 (LRRTM1) (Inoue et al., 2007; Ludwig et al., 2009; Takashimaet al., 2011). Intriguingly, multiple studies have shown a linkage of

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. Grabrucker).

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SCZ and the 22q11–13 locus, which includes proline dehydrogenase(PRODH) and ProSAP2/Shank3 (Condra et al., 2007; DeLisi et al., 2002;Gill et al., 1996; Jorgensen et al., 2002; Karayiorgou et al., 1995;Mowry et al., 2004; Vallada et al., 1995).

ProSAP/Shank proteins are a family of scaffoldingmolecules found atexcitatory glutamatergic synapses. At the postsynaptic density (PSD),these proteins are capable of interacting with structural elements aswell as ligand gated ion channels, cell adhesion molecules such asneuroligins, and proteins regulating the dynamic assembly of F-actin(Kreienkamp, 2008). Two mutations in ProSAP2/Shank3 (R1117X andR536W) were identified in two families and were both reported to beassociated with SCZ (Gauthier et al., 2010). The ProSAP2/Shank3R1117X mutation results in a truncated protein, lacking the Homer-and Cortactin-binding sites and the sterile α motif (SAM) domainnecessary for synaptic localization of the protein (Boeckers et al.,2005; Grabrucker et al., 2011a). Here, we show that this mutation inProSAP2/Shank3, which leads to a translational STOP after the prolinerich region of the protein (R1117X human or R1119X rat sequence)(Gauthier et al., 2010), results in an accumulation of ProSAP2/Shank3within the nucleus. Moreover, we show that wild-type ProSAP2/Shank3is able to translocate from synapse to nucleus in an activity dependentmanner. However, the mutation leads to a ProSAP2/Shank3 proteinthat constitutively localizes within the nucleus, independent of synapticactivity.

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Gene expression can be altered upon synaptic activity and recentstudies revealed a number of synapto-nuclear protein messengers thathave been shown to translocate to the nucleus (Dieterich et al., 2008;Jordan and Kreutz, 2009; Kindler et al., 2009; Lai et al., 2008; Meffertet al., 2003; Mikenberg et al., 2007; Proepper et al., 2007; Schmeisseret al., 2009). Thus, it has been speculated that shuttling of synapticand dendritic proteins to the nucleus might be involved in plasticity-related gene expression and thereby contribute to learning andmemoryformation (Alberini, 2009; Flavell and Greenberg, 2008; Jordan andKreutz, 2009). We identified several nuclear interaction partnersof ProSAP2/Shank3R1119X, among them are multiple heterogeneousnuclear ribonucleoproteins (hnRNPs). Localization of ProSAP2/Shank3R1119X in the nucleus induced gene transcription of severalgenes, among them are already identified SCZ associated factors suchas Synaptotagmin 1 (Syt1) and Leucine-rich repeat transmembraneneuronal protein 1 (LRRTM1). Both genes were also shown to be up-regulated after induction of synaptic activity accompanied by nuclearenrichment of wt ProSAP2/Shank3. Moreover, neurons expressingProSAP2/Shank3R1119X show reduced synaptic density, altered E/Iratio and reduced dendritic branching, the later two features beingdistinct from a knockdown of ProSAP2/Shank3.

Given that knockout mouse models for ProSAP2/Shank3 werereported to show autistic-like behavior (Jiang and Ehlers, 2013) andthat similar mutations leading to a C-terminally truncated ProSAP2/Shank3 protein were also found in autistic patients (Grabrucker et al.,2011b), these shared featuresmight underline the relationship betweenautism and SCZ (de Lacy and King, 2013).

Material and methods

Materials

Primary antibodies were purchased from Synaptic Systems(Gephyrin, Synaptotagmin 1), Stressgen (Bassoon), Sigma (β-actin,anti-MYC), Abcam (PRP8, PSD-95), Novus Biologicals (MED23,GAPDH), Santa Cruz (TRBP2), Lifespan Biosciences (ROA3, RALY),MyBiosource (ROA2, LRRTM1), Cell Signaling (ROA0, Histone H3),Clontech (GFP) and Chemicon (MAP2). ProSAP1/Shank2 and ProSAP2/Shank3 antibodies have been described previously (Grabrucker et al.,2011a; Schmeisser et al., 2012). Secondary Alexa Fluor conjugatedantibodieswere purchased from Invitrogen. Unless otherwise indicated,all other chemicals were obtained from Sigma-Aldrich.

Expression constructs

The pEGFP (C1-3), MYC (C1-3) (Clontech, Palo Alto, CA, USA) andFUGW vector systems (gift from Craig Garner) were used for theProSAP/Shank and RanBP9 expression constructs. Full-length ProSAP2/Shank3 cDNAs were cloned into these vectors. RNAi Oligonucleo-tides were purchased from Eurofins and cloned into a pSUPER andpFUGW H1 via a pZOff vector. ProSAP2/Shank3 target sequences di-rected against exon 21 were used as described previously (Verpelliet al., 2011): 5′-GGAAGTCACCAGAGGACAAGA-3′ (targeting the ratand mouse ProSAP2/Shank3 gene) (GenBank™ accession numberNM_021676 and NM_021423.3). The ProSAP2/Shank3R1119X andInsG mutant construct was generated using a PCR-based approach(Phusion DNA Polymerase from NEB) and site directed mutagenesis(Stratagene QuikChange II XL).

Cell culture, immunocytochemistry and treatments

Hippocampal cultures were prepared from rat (embryonic day 18)as described before (Grabrucker et al., 2009). All animal experimentswere performed in compliance with the guidelines for the welfare ofexperimental animals issued by the Federal Government of Germanyand by the local ethics committee (Ulm University) ID Number: O.103.

For immunofluorescence, the primary cultures were fixed with 4%paraformaldehyde (PFA)/4% sucrose/PBS at 4 °C for 20 min andprocessed for immunocytochemistry. After washing 3 × 5 min with1× PBS containing 0.2% Triton X-100 at RT, blocking was performedwith 10% FBS/1× PBS for 1 h at RT, followed by the incubation withprimary antibody at RT 2 h or 4 °C overnight. After a 3 × 5 minwashing-step with 1× PBS, incubation with the secondary antibodycoupled to Alexa488, Alexa568 or Alexa647 followed for 1 h. Cell nucleiwere counterstained with DAPI and coverslips subsequently mountedusing Vecta Mount (Vector Laboratories). For stimulations, growthmedia were supplemented with 50 mM KCl (HiK+), 50 μM NMDA or30 μM Glutamate for 1 min. Leptomycin B treatment was performedusing 20 nM. Cells were fixed and analyzed after 2 and 4 h. Treatmentwith 5 μM Colchicine or 1 μM Cytochalasin D was either performedalone or with stimulation for 1 min with HiK+ after 10 min pre-treatment with the toxins. Cells were fixed after 60 min. Vinblastinetreatment was performed with either 1 μg/ml vinblastine or vehicle(dimethylsulfoxide (DMSO)) in the culture medium at 37 °C for 3 h.After 2 h of incubation, cells were stimulated.

Cos7 and NIH cells were maintained in DMEM at 37 °C in 5% CO2.Cell lines were transfected using Polyfect (Invitrogen) following themanufacturer's protocol. Primary hippocampal neurons in 24-wellplates were transfected with OptiFect (Invitrogen) following themanufacturer's protocol.

Lentivirus production

Lentivirus was produced by transfecting a three-plasmid vectorsystem comprising a shuttle plasmid (FUGW) and two packagingplasmids (pSpax2 and pHCMV VSVg) into HEK293T cells (grownin DMEM + 10% fetal bovine serum and penicillin/streptomycin). Inbrief, transfections were conducted on confluent (90–100%) cellcultures with PolyFect (Qiagen) using 22.5 μg of total DNA per 10 cmPlate. 2 d after transfection, the virus-containingmediumwas collected,passed through a 0.45 μm filter to remove cell debris, and frozen at−80 °C. The viral titer was determined by fluorescence analysis ofinfected HEK293T cells (infective units).

Immunohistochemistry

Cryosections from wt C57BL/6JRj mice were thawed for 20 min andfixed in PFA for 20–30 min. After fixation, sections were washed3 × 10 min with PBS. Sections were permeabilized with 0.2% Triton inPBS for 2 h and washed again 3 × 10 min with PBS containing 0.05%Triton. Sections were then placed in 10% FCS in PBS for 2 h. Afterblocking, sections were incubated with the primary antibody at 37 °Cfor 2 h followed by 3 × 10 min washing with PBS containing 0.05%Triton. Sections were incubated with the secondary antibody at 37 °Cfor 1.5 h. After washing 3 × 15 min with PBS containing 0.05% Triton,sections were washed 2 × 5 min with PBS containing DAPI, rinsedwith H2O dest., and mounted with Vecta Mount (Vector Laboratories).

RT-PCR

RNA was prepared using the RNeasy Kit from Qiagen. QuantitativeRT-PCR analysis of total RNA was performed using the TranscriptorOne-Step RT-PCR Kit (Roche) and gene-specific primers. The β-actingene was used as a reference.

MicroArray

MicroArray (mouse, whole Genome, Affymetrix Chips) analysis wasperformed at imaGenes GmbH (Berlin). Raw data was analyzed andadjusted to quality standards, normalized and finally evaluated bystatistical methods like t-Test, principal-component-analysis (PCA)and hierarchical clustering.

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Protein biochemistry

For immunoprecipitation experiments, whole-cell lysates ormembrane-associated fractions from rat brains (P2) were extractedwith RIPA buffer at 4 °C for 2–3 h by overhead shaking before beingcentrifuged for 15 min at 13,000 ×g. The resulting supernatant wasthen used for immunoprecipitation using the μMACS magnetic beadseparation system (Miltenyi Biotec). Subcellular fractions were isolatedas described previously with minor modifications (Schmeisser et al.,2012). Proteins were separated by SDS-PAGE and blotted onto nitrocel-lulose membranes. Immunoreactivity was visualized using HRP-conjugated secondary antibodies and the SuperSignal detection system(Pierce, Upland, USA).

Nuclear and cytoplasmic extracts from hippocampal Neurons (DIV14)were prepared using theNE-PER Nuclear and Cytoplasmatic Extrac-tion Kit (Thermo Scientific) following the manufacturer's protocol witha protease inhibitor from Roche (Complete Mini).

Animals

ProSAP2/Shank3 mutants were generated by Genoway (Lyon,France) and raised on a C57BL/6 background. ProSAP2/Shank3 mutantshave been characterized before (Schmeisser et al., 2012).

Mass-spectrometry

Mass spectrometrywas performed at the Core FacilityMass Spectro-metric Proteomics of the University Clinics Hamburg-Eppendorf (UKE).Cells were transfected with GFP-ProSAP2/Shank3R1119X and proteincomplexes purified using the μMACS magnetic GFP-microbead system(Miltenyi). The eluate was loaded on a SDS gel and Coomassie stainingwas performed. Detected bands were cut out and analyzed using massspectrometry. Results were corrected for known false positives.

Statistics

ImmunocytochemistryAcquisition and evaluation of all images were performed under

“blinded” conditions. 10 cells of each condition were imaged. All signalswithin the optic fields were quantified in the following way: Fluores-cence images were obtained with an upright Axioscope microscopeequipped with a Zeiss CCD camera (16 bits; 1280 × 1024 ppi) usingAxiovision software (Zeiss) and the number of signals per 10 μmdendrite length as well as dendritic branching (number of primary,secondary, tertiary and quarterly dendrites) analyzed and quantifiedusing ImageJ 1.46p. Statistical analysis was performed using MicrosoftExcel and SPSS and tested for significance using t-tests followed byANOVA (p b 0.05*; b0.01**; b0.001***).

Fig. 1. ProSAP2/Shank3 is able to shuttle from synapse to nucleus. A) Hippocampal neuronsimmunocytochemistry labeling ProSAP2/Shank3, Abi-1 and nuclei (DAPI). Compared to non-nucleus is visible in cells treated with NMDA. B) Cell lysate from hippocampal neurons was pr1 h and compared to un-stimulated control cells. Cell fractionation was performed and the puand Histone H3 (17 kDa, nuclear) byWestern blot analysis (upper panel). A clear separation bwere analyzed byWestern blot. Using similar protein concentrations per lane, an enrichment ofbe seen (lower panels). The graph displays a semi-quantitative analysis of theWestern blot resnormalized against Histone H3 under control conditions and after stimulation with NMDA. A sstimulation. C) Application of the nuclear export inhibitor leptomycin B (20 nM) results in an entagged ProSAP2/Shank3 in the nucleus after 4 h of treatment while hardly any nuclear signal isleptomycinB results in an accumulation of nuclear ProSAP2/Shank3 after 4 h. Themean gray valmean nuclear and dendritic signal intensity from 10 cells is shown. The increase in nuclear staAdditional stimulation of neuronswithHiK+ (50 mMKCl) for 1 min increases nuclear localizatinot treated with leptomycin B but stimulated return to baseline (control) conditions 4 h aftedisturbance of cytoskeletal components by cytochalasin D, but not cholchicine. Hippocampal ncells and cells stimulated for 1 min with HiK+ (with and without presence of cytochalasin D anand themean ratio between nuclear and somatic signal intensities of 10 cells per condition is shosection of a wt mouse hippocampus (area CA1) with labeling of ProSAP2/Shank3 and nuclei (shows control staining without primary ProSAP2/Shank antibody).

Western blot and RT-PCR quantificationEvaluation of bands was performed using ImageJ. Three indepen-

dent experiments were performed and images scanned (600 dpi). Theindividual bands were selected and the integrated density wasmeasured. All bandswere normalized toβ-actin and the ratios averagedand tested for significance with α level of significance set at 0.05(p b 0.05*; b0.01**; b0.001***).

Results

ProSAP2/Shank3 shuttles into the nuclear compartment upon synapticactivation

Recently, a number of PSD proteins functioning as synapto-nuclearprotein messengers have been characterized (Jordan and Kreutz,2009). Among them a number of ProSAP2/Shank3 interacting proteinssuch as Abi-1 and Lapser1 (Proepper et al., 2007; Schmeisser et al.,2009) have been previously reported to translocate from synapse-to-nucleus in an activity dependentmanner. However, it might be possiblethat a whole complex of proteins is transported from the PSD to the nu-cleus. Therefore, we stimulated hippocampal neurons with HiK+,NMDA (Fig. 1A) and Glutamate (Fig. S1A) and investigated ProSAP2/Shank3 localization after 1 h. The previously published translocationof Abi-1 was used as positive control (Proepper et al., 2007) (Fig. 1A)and PSD-95 as negative control (Fig. S1B). Interestingly, we detectedendogenous ProSAP2/Shank3 within the nucleus (Figs. 1A, S1A) 1 hafter 1 min stimulation. This localizationwas detected using several dif-ferent polyclonal serum antibodies against the N-terminal part ofProSAP2/Shank3 and to a lesser extent using a polyclonal serum anti-body against the C-terminal part of ProSAP2/Shank3. Additionally,using cytoplasmic and nuclear fractions, we could detect ProSAP2/Shank3 in the nuclear fraction 1 h after 1 min stimulation with NMDAand HiK+ (Fig. 1B). Apart from the three isoforms ProSAP2/Shank3α,β and γ, a band at 130 kDa showed an increase after stimulation (notused for analysis of total nuclear ProSAP2/Shank3 levels (Fig. 1Bgraph)).

Moreover, application of the nuclear export inhibitor leptomycin Bto GFP-ProSAP2/Shank3 and MYC-ProSAP2/Shank3 transfected NIHcells (Fig. 1C) and staining for endogenous ProSAP2/Shank3 in NIHand hippocampal neurons (Figs. 1C,D) revealed that under this condi-tion, GFP/MYC-ProSAP2/Shank3 and endogenous ProSAP2/Shank3 aremainly seen within the nuclear compartment. Intriguingly, provokingsynaptic activity by HiK+ stimulation facilitates the reversible accumu-lation of ProSAP2/Shank3 in hippocampal neurons (Figs. 1D, S1C).Treatment of hippocampal neurons with cytochalasin D but not colchi-cine prevented nuclear translocation, suggesting that ProSAP2/Shank3nuclear transport is directly or indirectly dependent on a functionalactin cytoskeleton rather than microtubular system (Figs. 1E,F).

were stimulated for 1 min with 50 μM NMDA at DIV 14 and fixed after 1 h followed bystimulated control neurons, a clear enrichment of ProSAP2/Shank3 and Abi-1 within theepared at DIV 14. Cells were stimulated for 1 min with HiK+ or NMDA and incubated forrity of the fraction is controlled using the marker proteins GAPDH (37 kDa, cytoplasmic)etween cytoplasmic and nuclear fraction can be seen. Additionally, ProSAP2/Shank3 levelsProSAP2/Shank3 isoforms in the nuclear fraction after stimulationwith HiK+ orNMDAcanults showing the mean nuclear protein expression levels from three different experimentsignificant increase of total ProSAP2/Shank3 levels in the nuclear fraction can be seen afterrichment of endogenous ProSAP2/Shank3 aswell as overexpressedMYC-tagged and GFP-detectable under control condition. D) Similarly, treatment of hippocampal neurons withue of dendritic andnuclear ProSAP2/Shank3 stainingswasmeasured and the ratio betweenining under leptomycin B treatment is significant after 4 h and visible as a trend after 2 h.on of ProSAP2/Shank3 and a significant enrichment is visible already after 2 h. Cells that arer stimulation. E,F) The nuclear translocation of ProSAP2/Shank3 can be prevented by theeurons DIV 14 were treated with cytochalasin D and colchicine and compared to controld colchicine). The nuclear and somatic signal intensity of ProSAP2/Shank3 was measured,wn. CytochalasinD, but not colchicine significantly reduced nuclear translocation. G) BrainDAPI) (right panel). Also in vivo, some nuclei are positive for ProSAP2/Shank3 (left panel

129S. Grabrucker et al. / Experimental Neurology 253 (2014) 126–137

However, treatment with vinblastine that inhibits microtubule assem-bly also affected nuclear localization of ProSAP2/Shank3 in stimulatedneurons (data not shown).

Using hippocampal brain sections of wildtypemice and immunohis-tochemistry labeling ProSAP2/Shank3, we were also able to detectProSAP2/Shank3 within the nucleus of several neurons (Fig. 1G).

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An SZ associated mutation in ProSAP2/Shank3 shows constitutive nuclearlocalization

Interestingly, the C-terminal deletion mutation of ProSAP2/Shank3associated with SCZ in human patients (ProSAP2/Shank3R1119X)(Fig. 2A) not only shows no localization to the PSD due to the deletionof the SAM domain, but is constitutively found within the nucleus ofneurons and NIH cells (Fig. 2B). In contrast, the C-terminal part that isdeleted in the SCZ associated mutant localizes to postsynapses due tothe presence of the SAM domain (Fig. 2C).

Importin is known to transport proteins into the nucleus by bindingto a specific nuclear localization signal (NLS). Previous work has shownthat neuronal importins are present in dendrites and synapses(Thompson et al., 2004) and importin-α and -β shuttle from synapsesto the nucleus in an NMDA receptor (NMDAR)-dependent manner(Thompson et al., 2004). We thus investigated, if importin-α can befound in a complexwith ProSAP2/Shank3 and ProSAP2/Shank3R1119X.Immunoprecipitation assays reveal that both, wt ProSAP2/Shank3 aswell as the SCZ associated ProSAP2/Shank3R1119X can be found associ-ated with importin-α (Fig. 2D). Moreover, overexpression of Ran-binding protein 9 (RANBP9), a key protein involved in the translocationof proteins through the nuclear pore complex increases nuclear accu-mulation of ProSAP2/Shank3 (Fig S1D). Durand et al. (2007) identifieda mutation in ProSAP2/Shank3 termed “InsG” in a patient with autism.The insertion of a guanine (InsG) creates a frame-shift at nucleotide3680, thereby leading to a modification of the C-terminal sequenceand truncation of the protein. Similarly to ProSAP2/Shank3R1119X,this mutated ProSAP2/Shank3 is found in the nucleus (Fig. S1E).

Intriguingly, sequence analysis using NLStradamus software (NguyenBa et al., 2009) reveals two putative NLS sequences within the ProSAP2/Shank3 protein (Fig. S1F). These putative NLS sequences are still presentin ProSAP2/Shank3R1119X. The two clusters of amino acids might bepart of a bipartite NLS. However, one of the NLS might also function asmonopartite NLS with the consensus sequence K-K/R-X-K/R. Thus, toinvestigate, if these putative NLS sequences are indeed functional andmediate the nuclear transport of ProSAP2/Shank3, we cloned andexpressed a ProSAP2/Shank3 with larger C-terminal truncation nowexcluding the predicted NLS sites. However, our results show that thisGFP-ProSAP2ΔC-term protein still localizes to the nucleus (Fig. 2E).

A nuclear protein complex of ProSAP2/Shank3

Many interaction partners of ProSAP2/Shank3 have been describedso far, such as Abi-1, ProSAPiP1, α-Fodrin, Sharpin, GKAP/SAPAP, CRIL,SSTR2, Dynamin-2, Homer, and Cortactin (Boeckers et al., 2002). How-ever, the focus so far was on synaptic proteins. The results shownabove indicate that a second protein complex linked to ProSAP2/Shank3 might exist, distinct from this PSD bound complex, within thenucleus. We thus further identified proteins that might be part of thenuclear ProSAP2/Shank3 complex. To do this, we expressed GFP-ProSAP2/Shank3R1119X to ensure nuclear localization of ProSAP2/Shank3 and purified the protein via its GFP tag. To identify putativeinteraction partners, we separated the proteins in complex with thepurified GFP-ProSAP2/Shank3R1119X on SDS-Page and performedmass spectrometry to characterize the visible proteins after Coomassiestaining (Fig. S2A). Interestingly, among others (PRP8, MYO5A,STRUM, ATS10, GRD2I, PABP1, IF4A3, TRBP2), we could detect sever-al proteins that are part of a RNA polymerase II (MED14, MED23) andhnRNP complex (RALY, ROA3, ROA2, ROA0) (Fig. S2B). To furtherverify these putative interaction partners, we performed co-immunoprecipitation and co-localization experiments. The resultsshow that MED23, the hnRNPs ROA0, ROA2, ROA3 as well as RALY canbe found in a complex with precipitated GFP-ProSAP2/Shank3R1119X(Fig. 3). Moreover, transfection of GFP-ProSAP2/Shank3 and GFP-ProSAP2/Shank3R1119X and subsequent labeling of MED23, TRBP2and RALY, ROA3, ROA2, ROA0 reveal a clear co-localization of ROA3,

ROA2, ROA0 with ProSAP2/Shank3R1119X within the nucleus(Fig. S2C). However, the interactionwith TRBP2 could not be confirmedin co-immunoprecipitation experiments and co-localization experi-ments revealed a predominantly somatic distribution of RALY andTRBP2. Based on these results, it is likely, that nuclear ProSAP2/Shank3 influences gene transcription via assembly or modification ofan hnRNP complex within the nucleus.

Nuclear ProSAP2/Shank3R1119X leads to altered gene and protein expression

To investigate the role of nuclear ProSAP2/Shank3 in transcriptionalregulation, we expressed GFP-ProSAP2/Shank3R1119X in NIH cells andcompared gene expression to GFP control and shRNA ProSAP2/Shank3(Fig. S3A) transfected cells via whole genome microarray chip-analysis(Figs. 4A,B). We found the expression of 57 genes to be significantlyaltered in ProSAP2/Shank3 knockdown cells (among them Neurogenicdifferentiation 4 (Neurod4), chimerin 2 (Chn2), stargazin (Cacng2),DEP domain containing 2 (Depdc2) and adenomatosis polyposis coli 2(Apc2)) (Fig. S3B), and the expression of 43 genes to be significantlyaltered in ProSAP2/Shank3R1119X expressing cells. Among them gapjunction membrane channel protein alpha 9 (Gja9), cyclic AMP-regulatedphosphoprotein 21 (Arpp21), VPS10 domain receptor protein SORCS 1(Sorcs1), insulin receptor substrate 4 (Irs4), RAB27A, cystic fibrosistransmembrane conductance regulator homolog (Cftr), leucine richrepeat transmembrane neuronal 1 (Lrrtm1), synaptotagmin I (Syt1) andcholinergic receptor, nicotinic, alpha polypeptide 5 (Chrna5) (Fig. 4A).Intriguingly, besides these candidate genes, we found the expressionof 7 genes to be similarly regulated in GFP-ProSAP2/Shank3R1119Xand shRNA ProSAP2/Shank3 transfected NIH cells leading to thehypothesis that ProSAP2/Shank3R1119X might function in many wayssimilar to a loss of ProSAP2/Shank3 (Fig. S3C). However, the expressionof both Syt1 and Lrrtm1was not altered under ProSAP2/Shank3 knock-down conditions and ProSAP2/Shank3 transgenic mice (Schmeisseret al., 2012) show no difference in Syt1 protein levels (Fig. S3D). Thus,to measure if the observed changes in gene expression lead to alter-ations in protein levels in NIH cells, we quantified protein concentra-tions of Syt1 and Lrrtm1 in NIH cell lysate. The results show asignificant increase in Syt1 protein and a trend towards an increase inLrrtm1 in NIH cells transfected with GFP-ProSAP2/Shank3R1119X(Fig. 4B). Based on these data, a role of wt ProSAP2/Shank3 in thenucleus can be hypothesized. Since ProSAP2/Shank3 translocated intothe nucleus in an activity dependent manner (Fig. 1A), we thereforestimulated hippocampal neurons DIV 14 with HiK+ and quantifiedRNA expression levels of the genes observed to be altered before(Fig. 4A) using RT-PCR. The results show that indeed, the significantup-regulation of Arpp21, Rab27a, as well as Lrrtm1 and Syt1 can bereproduced in hippocampal neurons 60 min after the stimulation. How-ever, RNA levels returned to baseline levels after 90 min, with theexception of Sorcs1 that showed a significant increase (Fig. 4C).

We subsequently verified the observed changes in gene expressionin neurons by infection of GFP-ProSAP2/Shank3R1119X and qt RT-PCR(Figs. 4D, S4A). Similarly to the experiments described before, wefound that compared to control (GFP infected) neurons, the expressionlevels of Cftr, LRRTM1 and Syt1 are significantly higher in ProSAP2/Shank3R1119X expressing neurons.

Expression of ProSAP2/Shank3R1119X leads to altered synaptic density anddendritic branching

Given that synapse-to-nucleus messaging may be an importantfactor for synaptic plasticity, we analyzed, if the expression of theProSAP2/Shank3R1119X mutation leads to changes in dendriticbranching, synapse density or the ratio of excitatory to inhibitory synap-ses (E/I) and also included the knockdown of ProSAP2/Shank3 forcomparison. While dendritic arborization at DIV 14 is generallynot affected in hippocampal neurons expressing control-DNA (GFP or

Fig. 2.An SZ associatedmutation in ProSAP2/Shank3 shows impaired activity dependent synapto-nuclear signaling. A) The SCZ associatedmutation in ProSAP2/Shank3 leads to aC-terminally truncated protein. B) Expression of this mutated ProSAP2/Shank3 (R1119X) results in permanent nuclear localization of the protein in NIH cells as well as hippocampalneurons, which is independent of the Myc or GFP tag. The localization of wt ProSAP2/Shank3 is shown for comparison. C) Hippocampal neurons (DIV 14) transfected with GFP-ProSAP2/Shank3C-term and Homer1 and DAPI staining. The C-terminal SAM domain (including Homer binding site) missing in ProSAP2/Shank3R1119X shows synaptic localizationand no enrichment in the nucleus. D) Both, the mutated and wt ProSAP2/Shank3 proteins can be found in complex with importin α as shown by immunoprecipitation experimentsusing cell lysate from NIH cells overexpressing wt or mutant GFP-ProSAP2/Shank3. A ProSAP2/Shank3 antibody (against the N-terminus) was used to precipitate the protein complexfrom cell lysate. In the Western blot, an antibody directed against the N-terminus of ProSAP2/Shank3 confirmed the binding to the column (left panel, Eluate). An antibody againstimportin α could readily detect its antigen in the precipitate (Eluate). As positive control, cell lysate was used (Input lane) and a negative control was performed with unspecific IgG(Ctrl IgG). E) ProSAP2/Shank3 with larger C-terminal truncation excluding the predicted NLS sites still shows localization of GFP-ProSAP2/Shank3ΔC-term in the nucleus.

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scrambled RNAi with GFP), GFP-ProSAP2/Shank3 or GFP-ProSAP2/Shank3 shRNA, the expression of GFP-ProSAP2/Shank3R1119X leads toa significant reduction in primary and secondary dendrites (Fig. 5A).Moreover, synapse density is significantly reduced in neurons expressingGFP-ProSAP2/Shank3R1119X (Fig. 5B) similar to neurons with knock-down of ProSAP2/Shank3 (Grabrucker et al., 2011a; Roussignol et al.,2005). Given that the overexpression of wt ProSAP2/Shank3 leads to anincrease in the number of synapses per unit dendrite length, whileProSAP2/Shank3 knockdown results in a decrease due to impairedsynapse maturation (Grabrucker et al., 2011a; Roussignol et al., 2005),the ProSAP2/Shank3R1119X mutation behaves comparable to the latterphenotype. Since, similar to the patient situation, where endogenous wtProSAP2/Shank3 is still present, ProSAP2/Shank3R1119X expression

was performed in a wildtype ProSAP2/Shank3 expression background,we next evaluated, if the presence of ProSAP2/Shank3R1119X affectsthe localization and levels of wt ProSAP2/Shank3 at the synapse. There-fore, we quantified the signal intensity of ProSAP2/Shank3 opposite to aBassoon signal in ProSAP2/Shank3R1119X expressing neurons with anantibody directed against the c-term of ProSAP2/Shank3 that is unableto bind to the truncated version and compared the intensity values tountransfected cells in the same field of view (Figs. 5C, S4B). The resultsshow a significant reduction of endogenous levels of ProSAP2/Shank3 inProSAP2/Shank3R1119Xexpressing cells compared to control transfectedcells (Fig. 5C).

Finally, we investigated if the observed changes lead to alterationsof the ratio of excitatory to inhibitory synapses (E/I). We measured

Fig. 3. Identification of nuclear ProSAP2/Shank3 interaction partners. ThemutatedGFP-ProSAP2/Shank3 (R1119X)was expressed inNIH cells and immunoprecipitation experimentswereperformed using cell lysate. GFP-ProSAP2/Shank3R1119X (153 kDa) was precipitated using anti-GFP magnetic beads and an anti-GFP antibody confirmed the binding to the column(Input, Eluate). In theWestern blot, an antibody against PRP8 (238 kDa),MED23 (130 kDa), TRBP2 (45 kDa), ROA3 (34 kDa), RALY (31 kDa), ROA2 (37 kDa) andROA0 (32 kDa) detectedits antigen in the precipitate (Eluate). As positive control, cell lysate was used (Input lane) and a negative control was performed with unspecific IgG (Ctrl IgG). While TRBP2 was notprecipitated, all other proteins (PRP8, MED23, ROA3 (hnRNPA3), RALY (hnRNP-associated with lethal yellow), ROA2 (hnRNPA2/B1), ROA0 (hnRNPA0)) showed clear enrichment inthe eluate compared to the negative control supporting the data obtained from mass spectrometry.

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synaptic density of inhibitory synapses in neurons expressing GFP, GFP-ProSAP2/Shank3R1119X or GFP-ProSAP2/Shank3 shRNA (Fig. 5D).Overexpression of wt ProSAP2/Shank3 does not affect inhibitory synapsedensitymeasured byGephyrin signal density. Similarly,we could observeno differences in the number of inhibitory synapses upon GFP-ProSAP2/Shank3R1119X or GFP-ProSAP2/Shank3 shRNA expression, whichin turn leads to a change in the (E/I) ratio of neurons expressing theGFP-ProSAP2/Shank3R1119X mutation (Fig. 5E) since the number ofexcitatory synapses decreases.

Discussion

Attention deficits and impaired memory are core features of schizo-phrenia (SCZ). These impairments are either due to a global corticaldysfunction or problems of specific components e.g. the hippocampus,where a dysfunction during episodic memory retrieval in SCZ wasreported (Heckers et al., 1998). Cytoarchitectural changes in the hippo-campal formation including alterations in presynaptic and dendritic

Fig. 4. Nuclear ProSAP2/Shank3R1119X leads to altered gene and protein expression. A) WhoShank3R1119X expressing NIH cells and compared to GFP expressing control cells. We found thcells, among them the neuronal expressed gene gap junction membrane channel protein alpprotein SORCS 1 (Sorcs1), insulin receptor substrate 4 (Irs4), RAB27A, cystic fibrosis transmem1 (Lrrtm1), synaptotagmin 1 (Syt1) and cholinergic receptor, nicotinic, alpha polypeptide 5 (CNIH cells. Western blot analysis of protein concentrations of ProSAP2/Shank3, Syt1 and Lrrtmupregulation of Syt1 and a trend (p = 0.0805#) towards an upregulation of Lrrtm1 can be seeDIV 14 for 1 min with HiK+ show regulation of RNA expression levels of the genes observedArpp21, Rab27a, as well as Lrrtm1 and Syt1 can be seen. 90 min after stimulation RNA levels rinfected with GFP-ProSAP2/Shank3R1119X compared to control neurons (infected with GFPstandard. Left panel: Quantification ofmRNA expression levels of thegenes observed to be altereneurons (infectedDIV 0). Themean of three ratios between the gene and corresponding actin sia trend is visible for Gja9 and Arpp21. Exemplary expression-bands are shown in the right pan

markers are prominent among the various neuropathological abnor-malities found in SCZ (Harrison and Eastwood, 2001). Intriguingly,many of the genes implicated in SCZ can be found at glutamatergicexcitatory synapses and thus, the glutamate system recently came intofocus as underlying pathophysiology of SCZ. In particular, a potentialrole of dysfunction of subtypes of glutamatergic receptors, as well as alink between SCZ and glutamate receptor genes was suggested(Kantrowitz and Javitt, 2012; Moghaddam and Javitt, 2012). Moreover,evidence for a link between SCZ and the NMDA system is mounting(Gilmour et al., 2012; Kantrowitz and Javitt, 2010). Therefore, it is notsurprising that it was shown that SCZ involves molecular changes inthe glutamatergic pathways mediating excitatory communicationbetween multiple brain regions (Kristiansen et al., 2010) and thatpostmortem studies revealed alterations in glutamate receptors andtheir modulators in SCZ (Tsai and Coyle, 2002).

Within the CNS, proteins of the ProSAP/Shank family are so far foundexclusively at the PSD of excitatory synapses and two mutations inProSAP2/Shank3 were reported to be associated with SCZ (Gauthier

le genome microarray chip-analysis was performed using total RNA from GFP-ProSAP2/e expression of 43 genes to be significantly altered in ProSAP2/Shank3R1119X expressingha 9 (Gja9), cyclic AMP-regulated phosphoprotein 21 (Arpp21), VPS10 domain receptorbrane conductance regulator homolog (Cftr), leucine rich repeat transmembrane neuronalhrna5). B) The observed changes in gene expression lead to alterations in protein levels in1 from three independent experiments was performed using NIH cell lysate. A significantn in ProSAP2/Shank3R1119X overexpressing cells. C) Hippocampal neurons stimulated atto be altered before (A) about 60 min after the stimulation. A significant up-regulation ofeturned to baseline with the exception of Sorcs1. D) Gene expression is altered in neurons). Three independent experiments were performed and the actin signal used as internald before (A,C) at DIV 14 fromnormalized against actin expression levels using hippocampalgnal is shown. A significantly higher expression of Cftr, Lrrtm1 and Syt1was detected, whileel.

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et al., 2010). Intriguingly, one of these mutations in ProSAP2/Shank3associated with the disorder leads to a protein with truncation that isno longer able to cluster at the PSD. The overexpression of ProSAP2/Shank3R1119X leads to a reduction in dendritic branching and excitato-ry synapse density. However, the number of inhibitory synapses is notaffected, thus leading to a shift in the E/I ratio of hippocampal neurons.

Although the expression of ProSAP2/Shank3R1119X was performed inneurons in a wildtype background, we detected a significant down-regulation of endogenous ProSAP2/Shank3 in cells expressing the SCZassociated mutant of ProSAP2/Shank3. Additionally, expression ofProSAP2/Shank3 shRNA as well as ProSAP2/Shank3R1119X changesthe transcription levels of a subset of genes in a similar manner.

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Syt1 and Lrrtm1 mRNA levels are increased upon synaptic localiza-tion of ProSAP2/Shank3. Lrrtm1 is amaternally suppressed gene associ-ated with SCZ (Francks et al., 2007; Ludwig et al., 2009). It is expressedpredominantly in the nervous system by postmitotic neurons and theprotein, which is restricted to excitatory differentiations, showssynaptogenic activity (Francks et al., 2007). Syt1 has a regulatory rolein the membrane interactions during trafficking of vesicles at synapticsites. Syt1 mRNA levels have been reported to be increased in youngerschizophrenia patients (Sokolov et al., 2000) and post-mortem brainsof SCZ patients (Mudge et al., 2008).

Reduced synaptic clustering of wt ProSAP2/Shank3 by ProSAP2/Shank3R1119X would also imply that reduced clustering of ProSAP2/Shank3 due to zinc deficiency (Grabrucker et al., 2011a, 2013) shouldalso lead to SCZ-like phenotypes in some cases. Intriguingly, it wasalready shown that SCZ can be caused by gestational zinc deficiencyand it was speculated that the resulting psychosis is due to a combinationof dietary or otherwise induced zinc deficiency resulting in a lack of zincreleasing capacity in the hippocampus (Andrews, 1990). However,compared to the synaptic loss of ProSAP2/Shank3, overexpression ofProSAP2/Shank3R1119X, which leads to constitutive localization ofProSAP2/Shank3 within the nucleus, also shows some unique features.It is likely that autistic patients with related mutations leading toa truncation of the C-terminal domain such as “InsG” that similarlyshows localization to the nucleus might likewise share these features. In-triguingly, SCZ and autism seem to overlap at multiple levels. Bothdisorders share deficits in social behavior, disruption of emotionalprocessing and impairments in executive functions on a behavioral level(Cheung et al., 2010). Moreover, abnormalities affecting glutamatergicsynapses are present in both disorders. Finally, SCZ and autism shareseveral genetic factors such as mutations in ProSAP2/Shank3 andenvironmental risk factors, such as zinc deficiency and pre- and perinatalinflammation (Meyer et al., 2011).

Shuttling of synaptic proteins into the nucleus may be importantto convey additional specificity required for the integration of multiplesignaling pathways (Jordan and Kreutz, 2009; Karpova et al., 2012).Proteins like AIDA-1d, Jacob, Abi-1 and Lapser1 (Dieterich et al., 2008;Jordan et al., 2007; Karpova et al., 2013; Proepper et al., 2007;Schmeisser et al., 2009) undergo synapse-to-nucleus shuttling andsupport this model. Most of the identified PSD proteins found in thenucleus so far possess a nuclear localization signal (NLS) as part oftheir sequence. Intriguingly, sequence analysis using NLStradamussoftware (Nguyen Ba et al., 2009) revealed two putative NLSsequences within the ProSAP2/Shank3 protein. However, our resultsshow that they are not necessary for nuclear localization of ProSAP2/Shank3.

Upon nuclear localization, which we found to be dependent on anintact actin cytoskeleton, an increase in the transcription rate of severalgenes can be observed, which did not occur under control conditionsand ProSAP2/Shank3 shRNA expression. Regulation of the same set ofgenes could be shown following synaptic activation, although in a tran-sient manner. Given that ProSAP2/Shank3 has no capacity to directlybind DNA and act as transcription factor, it can be assumed that it inter-acts with specific regulatory protein complexes within the nucleus tomediate changes in gene transcription. We identified several nuclearinteracting proteins, including Med14 and Med23 (mediator ofpolymerase II transcription 14 and 23). Intriguingly, mutations inMed23 have been linked to intellectual disability (Hashimoto et al.,2011). Besides, Med14 and Med23, we furthermore found severalheterogeneous nuclear ribonucleoproteins (hnRNPs) in a complex withProSAP2/Shank3. The hnRNPs are predominantly nuclear RNA-bindingproteins that form complexes with RNA polymerase II transcripts. Theseproteins are involved in many cell functions, ranging from transcriptionand pre-mRNA processing in the nucleus to cytoplasmic transport, trans-lation and turnover of mRNA (Krecic and Swanson, 1999).

In particular, we identified a complex of hnRNPs associated withProSAP2/Shank3 that was reported to exist in RNA-transporting

granules (Kanai et al., 2004). In neurons, the local translation of selectedmRNAs at dendritic spine shafts—among them ProSAP/Shank mRNA(Böckers et al., 2004), plays an important role in long-term neuronalplasticity (LTP) (Kindler et al., 2005). Besides hnRNPA/B, hnRNPA0,hnRNPA1, hnRNP-D, hnRNP-U and FMR1, FXR1, FXR2, the eukaryoticinitiation factors eIF2α, eIF2β and eIF2γ were reported to be part ofRNA transporting granules (Kanai et al., 2004). Here, we could alsoidentify eIF4A3 as nuclear ProSAP2/Shank3 interacting protein. mRNAsthat are locally translated require dendritic targeting elements (DTEs).One of the key trans-acting factors that recognize DTEs in neurons ishnRNPA2/B2 (Muslimov et al., 2011). Former studies showed thathnRNPA2/B1 levels may be disturbed in Alzheimer's disease leading toaltered post-transcriptional regulation (Ishikawa et al., 2004; Mizukamiet al., 2005). Interestingly, CFTR levels have been reported to correlatewith hnRNPA1 expression levels and CFTR is among the up-regulatedgenes in ProSAP2/Shank3R1119X expressing cells (Nissim-Rafinia et al.,2000). Moreover, Abi-1, a synaptic interaction partner of ProSAP2/Shank3 that has been demonstrated to also undergo activity dependentsynapse to nucleus shuttling has been shown to bind to hnRNPK in thenucleus, thereby regulating the balance between filopodia formationand synaptic maturation in neurons (Proepper et al., 2011).

Conclusions

Taken together, we conclude that ProSAP2/Shank3 is a protein thatcan undergo synapse-to-nucleus shuttling to influence the transcriptionlevels of several target genes possibly via alteration of hnRNP complexes.While in a healthy background, this is a transient and activity dependentmechanism, in a pathological background for example caused by muta-tions in ProSAP2/Shank3, this might be a constitutively active mecha-nism. Among the up-regulated genes in NIH cells and hippocampalneurons,we found Synaptotagmin 1 and LRRTM1both already associatedwith SCZ. Thus, depending on the site of mutation, this previouslyundescribed function of nuclear ProSAP2/Shank3 may be disturbed andcontribute additionally to the phenotype seen by the loss of synapticPSD-bound ProSAP2/Shank3. However, the possible role of nuclearProSAP2/Shank3 and the up-regulation of target genes such as Lrrtm1and Syt1 in the development of SCZ needs to be investigated in moredetail in future research.

Author's contributions

SG, CP, KM andME carried out the in vitro studies andMJS providedmouse brain-lysate. AMG drafted the manuscript with RC and TMB. Allauthors read and approved the final manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.Supplementary data to this article can be found online at http://dx.

doi.org/10.1016/j.expneurol.2013.12.015.

Acknowledgments

The authors gratefully acknowledge the professional technicalassistance of U. Pika-Hartlaub. We would also like to acknowledge theassistance of the Core Facility Mass Spectrometric Proteomics of theUniversity Clinics Hamburg-Eppendorf (UKE) supported in part by theDFG and the BMBF. AMG and MJS are supported by Baustein 3.2(L.SBN.0081 to MJS, L.SBN.0083 to AMG). SG, RC and ME are membersof the International Graduate School in Molecular Medicine at UlmUniversity.

Fig. 5. Expression of ProSAP2/Shank3R1119X leads to impaired dendritic branching and reduced excitatory synapse density. A) Hippocampal neurons were transfected DIV 10–14 withcontrol-DNA (GFP), GFP-ProSAP2/Shank3, GFP-ProSAP2/Shank3R1119X or GFP-ProSAP2/Shank3 shRNA and stained against MAP2. The number of primary, secondary, tertiary andquaternary dendrites was counted from 10 cells and the mean number of dendrites is shown. A significant reduction in primary and secondary dendrites was visible in GFP-ProSAP2/Shank3R1119X expressing neurons. B) Hippocampal neurons were transfected DIV 10–14 with control-DNA (GFP or scrambled shRNA with GFP), GFP-ProSAP2/Shank3, GFP-ProSAP2/Shank3R1119X or GFP-ProSAP2/Shank3 shRNA and stained against a synaptic marker (Homer1). Synapse density is significantly reduced in neurons expressing GFP-ProSAP2/Shank3R1119X and neurons with knockdown of ProSAP2/Shank3. The overexpression of wt ProSAP2/Shank3 leads to an increase in the number of synapses per unit dendrite length.Five dendritic sections were analyzed for each cell and averaged. The mean synapse density from 10 cells per condition is shown. C) The presence of ProSAP2/Shank3R1119X affectsthe localization and levels of wt ProSAP2/Shank3 at the synapse. The signal intensity of ProSAP2/Shank3 opposite to a presynaptic Bassoon signal in Control (eGFP expressing) andProSAP2/Shank3R1119X expressing neurons with an antibody directed against the C-term of ProSAP2/Shank3 unable to bind to the truncated protein was measured. The mean ratioof the signal intensity of synapses from 10 cells (transfected/untransfected) for each group is shown. A significant decrease in ProSAP2/Shank3 levels can be seen in ProSAP2/Shank3R1119X expressing neurons. D) Hippocampal neurons were transfected DIV 10–14 with control-DNA (GFP), GFP-ProSAP2/Shank3R1119X or GFP-ProSAP2/Shank3 shRNA andstained against Gephyrin, a marker for inhibitory synapses. Five dendritic sections were analyzed for each cell and averaged. The mean synapse density from 10 cells per condition isshown. No significant change in inhibitory synapse density can be seen. E) Given that the number of inhibitory synapses is unchanged but a reduction of excitatory synapseswas observed,we calculated the E/I ratio of hippocampal neurons expressing control-DNA (GFP), GFP-ProSAP2/Shank3R1119X or GFP-ProSAP2/Shank3 shRNA. A significant reduction can be seen inneurons expressing the GFP-ProSAP2/Shank3R1119X mutation.

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