− Mice

Accepted Manuscript Early correction of NMDAR function improves autistic-like social behaviors in adult –/– Shank2 mice Changuk Chung, Seungmin Ha, Hyojin Kang, Jiseok Lee, Seung Min Um, Haidun Yan, Ye-Eun Yoo, Taesun Yoo, Hwajin Jung, Dongwon Lee, Eunee Lee, Seungjoon Lee, Jihye Kim, Ryunhee Kim, Yonghan Kwon, Woohyun Kim, Hyosang Kim, Lara Duffney, Doyoun Kim, Won Mah, Hyejung Won, Seojung Mo, Jin Yong Kim, ChaeSeok Lim, Bong-Kiun Kaang, Tobias M. Boeckers, Yeonseung Chung, Hyun Kim, Yong-Hui Jiang, Eunjoon Kim PII:

S0006-3223(18)31896-1

DOI:

10.1016/j.biopsych.2018.09.025

Reference:

BPS 13655

To appear in:

Biological Psychiatry

Received Date: 26 July 2018 Revised Date:

20 September 2018

Accepted Date: 26 September 2018

Please cite this article as: Chung C., Ha S., Kang H., Lee J., Um S.M., Yan H., Yoo Y.-E., Yoo T., Jung H., Lee D., Lee E., Lee S., Kim J., Kim R., Kwon Y., Kim W., Kim H., Duffney L., Kim D., Mah W., Won H., Mo S., Kim J.Y., Lim C.-S., Kaang B.-K., Boeckers T.M., Chung Y., Kim H., Jiang Y.-H. & Kim E., –/– Early correction of NMDAR function improves autistic-like social behaviors in adult Shank2 mice, Biological Psychiatry (2018), doi: https://doi.org/10.1016/j.biopsych.2018.09.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Early correction of NMDAR function improves autistic-like social behaviors in adult Shank2–/– mice

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Changuk Chung,1,2,* Seungmin Ha,1,* Hyojin Kang,3 Jiseok Lee,2 Seung Min Um,1 Haidun Yan,4 Ye-Eun Yoo,1 Taesun Yoo,1 Hwajin Jung,2 Dongwon Lee,2 Eunee Lee,2 Seungjoon Lee,1 Jihye Kim,2 Ryunhee Kim,1 Yonghan Kwon,1 Woohyun Kim,1 Hyosang Kim,1 Lara Duffney,4 Doyoun Kim,2 Won Mah,5 Hyejung Won,6 Seojung Mo,7 Jin Yong Kim,7 Chae-Seok Lim,8 Bong-Kiun Kaang,8 Tobias M. Boeckers,9 Yeonseung Chung,10 Hyun Kim,7 Yong-Hui Jiang,4, 11,12,13,14 and Eunjoon Kim1,3,# 1

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Department of Biological Sciences, Korea Advanced Institute for Science and Technology (KAIST), Daejeon 305-701, Korea; 2Center for Synaptic Brain Dysfunctions, Institute for Basic Science (IBS), Daejeon 305-701, Korea; 3Department of Convergence Technology Research, KISTI, Daejeon 34141, Korea; 4Department of Pediatrics, Duke University, Durham, North Carolina, USA; 5Department of Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, Daegu, Korea; 6Department of Neurology, Center for Autism Research and Treatment, Semel Institute, David Geffen School of Medicine, University of California Los Angeles, California 90095, USA; 7 Department of Anatomy and Division of Brain Korea 21, Biomedical Science, College of Medicine, Korea University, Seoul 136-705, Korea; 8School of Biological Sciences, Seoul National University, Seoul, South Korea; 9Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany; 10Department of Mathematical Sciences, Korea Advanced Institute for Science and Technology (KAIST), Daejeon 305-701, Korea; 11Department of Neurobiology, Duke University, Durham, North Carolina, USA; 12Cell and Molecular Biology Program, Duke University, Durham, North Carolina, USA; 13Duke Institute of Brain Science, Duke University, Durham, North Carolina, USA; 14Genomics and Genetics Program, Duke University, Durham, North Carolina, USA; *These authors contributed equally to the work; #Corresponding author.

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Key words: autism, treatment, synapse, Shank2, NMDA receptor, memantine

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Number of words: Abstract (248 words), Article body (excluding abstract, acknowledgments, financial disclosure, legends, and references) (3567 words). Number of figures: 5 main figures Supplement: 7 files (1 PDF, 6 Excel files) Running title: Early NMDAR alteration and correction in Shank2–/– mice

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Abstract Background: Autism spectrum disorders (ASD) involve neurodevelopmental

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dysregulations that lead to visible symptoms at early stages of life. Many ASD-related mechanisms suggested by animal studies are supported by demonstrated improvement in autistic-like phenotypes in adult animals following experimental reversal of

stages to cause autistic-like phenotypes is unclear.

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dysregulated mechanisms. However, whether such mechanisms also act at earlier

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Methods: We used Shank2–/– mice carrying a mutation identified in human ASD (exons 6 and 7 deletion) and combined electrophysiological and behavioral analyses to see if early pathophysiology at pup stages is different from late pathophysiology at juvenile and adult stages, and correcting early pathophysiology can normalize late

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pathophysiology and abnormal behaviors in juvenile and adult mice. Results: Early correction of a dysregulated mechanism in young mice prevents

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manifestation of autistic-like social behaviors in adult mice. Shank2–/– mice, known to display N-methyl-D-aspartate receptor (NMDAR) hypofunction and autistic-like

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behaviors at post-weaning stages after postnatal day 21 (P21), show the opposite synaptic phenotype—NMDAR hyperfunction—at an earlier pre-weaning stage (~P14). Moreover, this NMDAR hyperfunction at P14 rapidly shifts to NMDAR hypofunction after weaning (~P24). Chronic suppression of the early NMDAR hyperfunction by the NMDAR antagonist memantine (P7–21) prevents NMDAR hypofunction and autistic-like social behaviors from manifesting at later stages (~P28 and P56).

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Conclusions: Early NMDAR hyperfunction leads to late NMDAR hypofunction and autistic-like social behaviors in Shank2–/– mice, and early correction of NMDAR

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dysfunction has the long-lasting effect of preventing autistic-like social behaviors from developing at later stages. Introduction

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Autism spectrum disorders (ASD), characterized by social deficits and repetitive

behaviors, exhibit a strong genetic influence. While the number of candidate ASD-risk

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genes is rapidly increasing (1), our understanding of the underlying mechanisms has lagged, partly because of the intrinsic heterogeneity and complexity of causal mechanisms in different brain regions at different developmental times. Many ASD-related mechanisms suggested by animal studies are supported by

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demonstrated improvement in autistic-like phenotypes in adult animals following experimental reversal of dysregulated mechanisms (2-11). However, whether such

13).

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mechanisms also act at earlier stages to cause autistic-like phenotypes is unclear (12,

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Shank is a family of postsynaptic scaffolding proteins known to regulate

excitatory synapse development and function (14, 15) and involved in various psychiatric disorders, including ASD and Phelan McDermid Syndrome (4, 7, 16, 17). Shank2, the second member of the family also known as ProSAP1, has also been implicated in ASD and other disorders, including intellectual disability, developmental delay, and schizophrenia (18-31).

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More recently, studies on mice lacking Shank2 in the whole brain or specific cell types have further enhanced our understanding of the normal functions of Shank2 as

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well as ASD-related pathophysiology (32-45). For example, mice lacking exons 6–7 and exon 7of Shank2 display autistic-like behavioral abnormalities that are associated with altered N-methyl-D-aspartate receptor (NMDAR) function (37, 38).

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More specifically, Shank2–/– mice with exons 6–7 deletion show decreased

NMDAR-mediated synaptic currents and NMDAR-dependent synaptic plasticity (38).

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Importantly, the treatment of adult Shank2–/– mice with D-cycloserine (NMDAR agonist) rapidly normalizes social interaction, suggesting that suppressed NMDAR function may underlie the social deficits. However, it remains unclear whether the NMDAR hypofunction observed in adult Shank2–/– mice (exons 6–7) also acts at early stages to

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play important roles in the development of social deficits.

In the present study, we found an increase in NMDAR function at an earlier stage (~postnatal day or P14), which sharply contrasts with the decreased NMDAR function at

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~P21. Importantly, suppression of the early NMDAR hyperfunction by the treatment of Shank2–/– mice with memantine (NMDAR antagonist) during P7–21 normalized NMDAR

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function and social interaction at juvenile (~P28) as well as adult (~P56) stages. These results suggest that early NMDAR hyperfunction induces NMDAR hypofunction and social deficits at later stages, and that early correction of NMDAR function has longlasting effects. Methods and materials Animals 4

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Shank2–/– mice lacking the exons 6–7 of the Shank2 gene have been previously reported (38). Shank2–/– mice lacking exon 7 (37) and exon 24 (33) have also been

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previously reported. All mutant and their WT littermates of Shank2–/– mice lacking exons 6–7 were generated on and backcrossed to a C57BL/6N background for more than 20 generations. Shank2–/– mice lacking exons 6–7 under a different background (C57BL/6J)

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were generated by backcrossing the original mice in the genetic background of

C57BL/6N with C57BL/6J for more than six generations. Shank2–/– mice lacking exon 7

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have been previously published (37), and backcrossed to and maintained in a C57BL/6J background for more than 5 generations.

Further information on methods is available in Supplementary Information. Results

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Distinct transcriptomic profiles in Shank2–/– brains at P14 and P25 Mice lacking Shank2/ProSAP1 (Shank2–/– mice; exons 6–7; global deletion) display

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autistic-like social deficits that are rapidly improved by acute D-cycloserine (NMDAR agonist) treatment at juvenile and adult stages (38). However, excitatory

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synaptogenesis in mice is most active during the first three postnatal weeks (46), and expression levels of Shank2 protein in the whole brain peak at ~P14 (Figure 1A), although Shank2 mRNA levels did not show a similar pattern (Figure 1B). We thus sought to identify phenotypes that are most prominent at P14 through comparative RNA-Seq transcriptomic analysis of wild-type (WT) and Shank2–/– brains at P21, a stage around weaning where active synaptogenesis is largely completed (Table

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S1). We found 16 differentially expressed genes (DEGs; FDR < 0.05, fold change > 2.0) in naïve six pairs of WT versus Shank2–/– mice at P14, and 35 DEGs at P25 (Table S2).

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However, because the small number of the identified DEGs did not yield significant gene ontology terms or pathways, we next performed gene set enrichment analysis (GSEA; software.broadinstitute.org/gsea/) (Figure 1C), which aims to achieve unbiased

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results by testing the enrichment of a list of ranked, whole genes, rather than a subset above an arbitrary cutoff (fold-change or significance), in pre-curated gene sets (47, 48).

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The two lists of genes from Shank2–/– versus WT (KO/WT) mice at P14 and P25, ranked by differential expression, were distinctly enriched for a large number of target gene sets in the C2 category (curated gene sets) (Table S3). Importantly, the top 10 enriched target gene sets for P14 and P25 were largely distinct; and in the case of the

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four overlapping gene sets associated with ribosome/translational terms, the directions of enrichments were opposite (Figure S1).

Additional GSEA using target gene sets in the C5 category (gene ontology)

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revealed that the KO/WT-P14, but not KO/WT-P25, gene list was enriched for gene sets associated with excitatory postsynaptic compartments, including excitatory synapse,

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postsynaptic membrane, and postsynaptic density (Figure 1D; Figure S2; Table S4). Because Shank2–/– mice show autistic-like behaviors accompanied by reduced

NMDAR function (38), we examined NMDAR-related enrichments. The KO/WT-P14 but not KO/WT-P25 gene list was enriched for gene sets associated with NMDAR activation and NMDAR-dependent synaptic plasticity (long-term potentiation and long-term

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depression) (Figure 1E; Figure S2). Collectively, these results suggest that Shank2 deletion in mice leads to distinct transcriptomic changes at P14 and P25.

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Reversal of NMDAR function in Shank2–/– mice during P14 and P25

Based on these results, we next measured excitatory synaptic transmission mediated by NMDARs and AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

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receptors) in the hippocampus, a brain region implicated in ASD (49-51), in Shank2–/– mice at P14 and P25. We found an increase in the ratio of NMDAR/AMPAR-mediated

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synaptic transmission at Schaffer collateral-CA1 pyramidal (SC-CA1) synapses at ~P14, as compared with that at WT synapses (Figure 2A; all electrophysiology data summarized in Table S5).

In contrast, basal synaptic transmission, mainly mediated by AMPARs, and

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paired-pulse ratio (a measure of presynaptic release probability) were normal at Shank2–/– SC-CA1 synapses (Figure S3). In addition, there were no changes in miniature excitatory postsynaptic currents (mEPSCs), mediated by AMPARs, and

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miniature inhibitory postsynaptic currents (mIPSCs) in Shank2–/– CA1 pyramidal

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neurons (Figure S4). These results suggest that NMDAR-, but not AMPAR-, mediated synaptic transmission is increased at P14 Shank2–/– SC-CA1 synapses. At ~P24 (P21–27), however, the NMDA/AMPA ratio at Shank2–/– SC-CA1

synapses was decreased, as compared with that at WT synapses (Figure 2B), consistent with the results from the previous study (38). This study also demonstrated normal basal transmission at Shank2–/– SC-CA1 synapses and normal mEPSCs in Shank2–/– CA1 pyramidal neurons. These results suggest that NMDAR-, but not 7

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AMPAR-, mediated synaptic transmission is reduced at P25 Shank2–/– SC-CA1 synapses.

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This age-dependent shift in NMDAR function was similar in males and females (Figure S5). In addition, the increased NMDAR function at P14 Shank2–/– synapses involved mainly the GluN2B subunit of NMDARs, as supported by the slower decay

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kinetics of the Shank2–/– NMDAR currents at P14, but not at ~P24 (Figure 2C-D), and the higher sensitivity of the currents to the GluN2B-specific inhibitor ifenprodil, as

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compared with those in WT mice (Figure 2E).

In addition to the hippocampus, layer II/III pyramidal neurons in the medial prefrontal cortex (mPFC), another ASD-related brain region (52), showed a similar switch of NMDAR function: an increased NMDA/AMPA ratio at Shank2–/– synapses at

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P14, but a decreased ratio at ~P24 (Figure S6).

When mEPSCs were measured in these mPFC neurons, the amplitude of

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mEPSCs in P14 Shank2–/– neurons was normal (Figure S7A), suggesting, together with the increased NMDA/AMPA ratio, that NMDAR-mediated transmission is selectively

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increased. The mEPSC frequency, however, was decreased in these Shank2–/– mPFC neurons, which, together with the normal paired pulse ratio (Figure S7B), suggests that excitatory synapse number is decreased in P14 Shank2–/– mPFC neurons. These results collectively suggest that Shank2 deletion induces a rapid temporal

switch of NMDAR function from hyper- to hypofunction across the period of P14 to P24 in the hippocampus and mPFC.

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To explore the mechanisms underlying the distinct NMDAR function at P14 and P25 Shank2–/– synapses, we examined total and phosphorylation levels of NMDAR

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subunits in Shank2–/– brains. Phosphorylation, but not total, levels of NMDAR subunits (GluN2B-Ser1303 and GluN1-Ser896), which have been shown to regulate surface and synaptic trafficking of GluN2B (53), were increased at P14 but decreased at P21

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(Figure S8A-B).

Other glutamate receptor (GluA and mGluR) subunits were largely normal in total

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levels in both P14 and P21 Shank2–/– brains. In addition, signaling molecules were also largely normal, with moderate changes in the phosphorylation of ERK1, CREB, and p38 (Figure S8C-D).

Early memantine normalizes NMDAR function in juvenile and adult Shank2–/– mice

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We next tested whether the early NMDAR hyperfunction at P14 is causally associated with NMDAR hypofunction at later stages by chronically suppressing NMDAR hyperfunction at ~P14 (P7–21) and examining NMDAR phenotypes in juvenile (~P28)

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and adult (~P56) Shank2–/– mice.

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We first tested whether early memantine treatment rescues NMDAR function in

adult Shank2–/– mice. Young Shank2–/– mice orally administered the NMDAR antagonist memantine (20 mg/kg, twice daily) for 2 weeks before weaning (P7–21) showed normalized NMDA/AMPA ratio at Shank2–/– hippocampal SC-CA1 synapses at P25–31 (Figure 3A), without affecting mEPSCs (Figure S9).

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In addition, early memantine rescued NMDAR-dependent long-term potentiation (LTP) induced by high frequency stimulation at Shank2–/– SC-CA1 synapses, both

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immediately after memantine treatment (P25–31) and at an adult stage (>P56) (Figure 3B-C), with no difference between males and females (Figure S10). In contrast, early memantine treatment had no effect on LTP at WT SC-CA1 synapses at either stage.

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Early memantine treatment for a shorter period (P7–14) could rescue the

NMDA/AMPA ratio even at P14, without affecting mEPSCs (Figure 3D; Figure S11),

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indicative of a strong and immediate effect of early memantine treatment on the normalization of NMDAR function. These results collectively suggest that early memantine treatment induces immediate and long-lasting normalization of NMDAR function.

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Early memantine improves social interaction in juvenile and adult Shank2–/– mice We next tested whether early memantine treatment can rescue abnormal behaviors in

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Shank2–/– mice at later (juvenile and adult) stages. Early memantine treatment (20 mg/kg, twice daily; P7–21) significantly improved social interaction of juvenile (P28–35)

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Shank2–/– mice in the direct interaction test (Figure 4A-B). Social isolation or weaning of mice before direct social-interaction tests had no effect on the results (Figure S12). In contrast, early memantine treatment did not affect social interaction in WT mice (Figure 4B). Memantine also improved social interaction of adult (~P56) Shank2–/– mice in the three-chamber test (54) (Figure 4C–E; Figure S13), indicative of a long-lasting effect.

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When we tested olfactory function and social hierarchy, known to significantly affect social interaction in mice (55-60), olfactory function was similar between

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genotypes, as reported previously (38), whereas Shank2–/– mice were submissive to WT mice in both tube and urine tests (Figure S14A–C), suggesting that Shank2 deletion affects social hierarchy in mice. This submissiveness, however, did not correlate with

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social interaction at the individual mouse level (Figure S14D–G). In addition, this

submissiveness was not improved by early memantine treatment (Figure S14B-C).

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Early memantine treatment modestly improved hyperactivity in adult Shank2–/– mice in the open-field test (Figure S15A–C). However, increasing habituation to a new environment eliminated the drug effect (Figure S15 D–G). In addition, the hyperactivity of Shank2–/– mice in a familiar environment was not normalized by early memantine

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treatment (Figure S16). Early memantine treatment did not rescue other behavioral abnormalities of Shank2–/– mice, including reduced ultrasonic vocalizations, enhanced repetitive behaviors, and anxiolytic-like behavior (Figure S17).

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Shank2–/– mice

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Early D-cycloserine or late memantine fails to normalize social interaction in

Importantly, treating Shank2–/– mice with D-cycloserine instead of memantine from P7 to P21 did not improve social interaction or hyperactivity, similar to the results from WT mice (Figure 5A–C; Figure S18). Intriguingly, early memantine-treated WT mice showed decreased NMDAR function at ~P21–25, as shown by the decreased NMDA/AMPA ratio and normal mEPSC amplitude (Figure S19); the mEPSC frequency was also reduced, likely through compensatory mechanisms. These results suggest that 11

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artificially elevated NMDAR function in WT mice during an early stage (P7–21) is sufficient to induce NMDAR hypofunction in juvenile mice (~P21–25) but not abnormal

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behaviors in adult mice. Treating Shank2–/– mice with memantine at a late stage (P25–39) failed to improve social interaction or hyperactivity (Figure 5D–F; Figure S20). Acute

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memantine treatment at P56 also failed to correct social interaction or hyperactivity (Figure S21). These results collectively suggest that early NMDAR hyperfunction in

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Shank2–/– mice should be corrected early and in the right direction.

Developmental switch of NMDAR function is not observed in other Shank2–/– mutant mice

We next tested if the temporal shift in NMDAR function in our Shank2–/– mice also occur

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in other Shank2-mutant mouse lines (33, 37). Shank2–/– mice lacking exon 24 (Shank2dEx24) showed reduced NMDAR function at P21, similar to the reported NMDAR hypofunction at ~2–4 months (33), but normal NMDAR function at P14 (Figure S22). In

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addition, Shank2–/– mice lacking exon 7 (Shank2-dEx7) showed enhanced NMDAR

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function at P21, similar to the reported NMDAR hyperfunction at ~P24 (37), but normal NMDAR function at P14 (Figure S23). Moreover, a switch in the background of our Shank2–/– mice lacking exons 6–7

from C57BL/6N to C57BL/6J had no effect on the reduced NMDAR function at ~P24 (Figure S24). Therefore, temporal patterns of NMDAR functions in different Shank2–/– mouse lines seem to be distinct.

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Lastly, we sought to identify additional molecular differences between Shank2–/– mice lacking exons 6–7 and those lacking exon 7, which show opposite NMDAR

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functions at ~P21 (37, 38) and minimally overlapping transcriptomic profile (34). Intriguingly, Shank2–/– mice lacking exons 6–7 had undetectable levels of the shorter Shank2a splice variant, whereas Shank2–/– mice lacking exon 7 had markedly increased

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(~6-fold) levels of the Shank2a variant, while both mouse had similarly decreased levels of the longer Shank2b splice variant (Figures S25 and S26). Whether this differential

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splice variant expression leads to differences in NMDAR function remains to be determined. Our molecular modeling, however, predicted that the short peptide (57 aalong) produced from the Shank2b splice variant in Shank2-dEx7 mice by protein truncation may form a structure that partly mimics the N-terminal region of the Shank2 PDZ domain and compete with the normal Shank2 PDZ domain for binding to the C-

(Figure S27).

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Discussion

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terminal PDZ-binding domains of GKAPs/SAPAPs in a dominant-negative manner

Our study provides early NMDAR hyperfunction at ~P14 as a causal mechanism for the

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NMDAR hypofunction and social deficits in Shank2–/– mice at juvenile (~P28) and adult (~P56) stages. In support of this, Shank2–/– mice display a rapid reversal of NMDAR function during P14 and P21 in the hippocampus (Figure 2; Figures S3–5) as well as in the mPFC (Figures S6 and S7). Importantly, early memantine treatment (P7–21) normalizes NMDAR function and social interaction without affecting other behaviors at juvenile and adult stages (Figures 3 and 4; Figures S9–17). In addition,

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pharmacological correction of NMDAR function in a wrong direction (early Dcycloserine), or during a wrong time window (late memantine), does not normalize

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social interaction in Shank2–/– mice (Figure 5; Figures S18 and S20). These results strongly suggest that early NMDAR hyperfunction induces late NMDAR hypofunction and social deficits in Shank2–/– mice, and early correction of NMDAR function has long-

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lasting effects on NMDAR function and social interaction.

Regarding how early memantine treatment induces long-lasting influences on

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electrophysiology and behavior in Shank2–/– mice, it is possible that once the NMDAR function is normalized by early chronic memantine treatment at the juvenile stage, the continuing lack of Shank2 expression after the end of early memantine treatment period (P7–21) may not induce a further change in NMDAR function. This hypothesis could be

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tested by inducing a Shank2 KO by a genetic method that starts from ~P21 after the completion of normal brain development during embryonic and early postnatal stages. Our results are reminiscent of a recent report that showed early correction of serotonin

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levels in infants through mother milk in a mouse model of autism carrying the human 15q11-13 duplication has long-lasting effects, rescuing serotonin levels as well as social

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behaviors in adults (61), which, together with our results, strongly suggest that early corrections are important. Early memantine treatment of WT mice does not affect NMDAR function

(Figures 3 and 4). This might be attributable to that the daily and total doses of memantine are in the range that can normalize NMDAR function in Shank2–/– mice but that do not suppress the normal NMDAR function in WT mice. Intriguingly, early D-

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cycloserine treatment of WT mice induces a decrease in NMDAR function at the juvenile stage (Figures S19), although it does not affect behaviors at the adult stage

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(Figure 5A–C). This could be attributable to that the doses of D-cycloserine may be just enough to induce a decrease in NMDAR activity but not to impair behaviors in WT mice, which may require a continuing deficit such as Shank2 KO. Alternatively, D-cycloserine

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may affect all NMDAR-expressing neurons, whereas Shank2 KO may mainly affect Shank2-expressing neurons.

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Our data also provide evidence suggesting that the GluN2B component contributes to the distinct NMDAR functions at P14 and P21 Shank2–/– synapses (Figure 2). Specifically, P14 Shank2–/– synapses show markedly increased GluN2B component, whereas P21 Shank2–/– synapses show normal levels of GluN2B

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component. Therefore, the increased NMDAR-mediated currents at P14 may be mainly mediated by GluN2B-containing NMDARs with slower decay kinetics, whereas the decreased NMDAR-mediated currents at P21, where both GluN2A and GluN2B are

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reduced, may involve both GluN2B-dependent and -independent mechanisms. Regarding further details, our immunoblot analyses suggest candidate

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mechanisms involving GluN1/GluN2B phosphorylation (Figure S8). It is known that GluN1-Ser896 phosphorylation promotes the ER-to-plasma membrane trafficking of GluN1 (62). In addition, CaMKII/PKC-dependent GluN2B-Ser1303 phosphorylation promotes synaptic retention of GluN2B through mechanisms involving the binding of GluN2B to MAGUKs (i.e. PSD-95) and AP2-dependent endocytosis of GluN2B (63-66). Therefore, the increased phosphorylation of GluN1-Ser896 and GluN2B-Ser1303 at

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P14 Shank2–/– synapses may promote surface and synaptic localization of NMDARs, whereas the decreased phosphorylation of GluN1-Ser896 and GluN2B-Ser1303 at P21

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Shank2–/– synapses may suppress synaptic retention of NMDARs. These mechanisms might contribute to the differential contributions of the GluN2B component to P14 and P21 Shank2–/– NMDAR currents. However, it should be pointed out that these

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biochemical changes could reflect those from many different types of cells with some limitations in the interpretation.

/–

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Developmental switches of NMDAR function were not observed in other Shank2– mouse mutant lines. Shank2-dEx7 mice show normal NMDAR function at ~P14 but

show increased NMDAR function at ~P21, as reported previously (67). In addition, Shank2-dEx24 mice showed reduced NMDAR function at ~P21, as reported previously

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(33), but normal NMDAR function at ~P14, partly similar to our mouse line. This discrepancy might be attributable to the differential expression of Shank2 splice variants in these mouse lines (Figures S25–26). For instance, we found a marked difference in

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the levels of the Shank2a transcript in Shank2-dEx6-7 and Shank2-dEx7 lines and, by molecular modeling (Figure S27), the possibility that a short truncated peptide derived

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from the Shank2a transcript in Shank2-dEx7 mice inhibits the normal interaction between Shank2 and SAPAPs, which may affect NMDAR activity or signaling, in a dominant-negative manner, although the presence of such short peptide could not be directly determined by immunoblot analyses. In addition, the exon 24 deleted in Shank2-dEx24 mice encodes the proline-rich region containing the Homer- and cortactin-binding sites (33), known to mediate the formation of a polymeric network structure and regulate the actin cytoskeleton in the postsynaptic density (68, 69). 16

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Although further details remain to be studied, these results are at the very least in line with that, in humans, different SHANK2 mutations, i.e. exons 6–7 and exon 7 deletions,

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are associated with distinct ASD symptoms (37). Similarly, two different mutations of SHANK3 (a SHANK2 relative) from ASD and schizophrenia patients, respectively, cause ASD- and schizophrenia-like phenotypes in mice (70).

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NMDAR dysfunction, which would suppress normal brain development and

function, has been suggested as an important candidate mechanism that may underlie

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ASD (71). Disturbances in NMDAR function, as a subset of excitatory synaptic function, would also induce the imbalance between synaptic and neuronal excitation and inhibition, another key mechanism implicated in ASD (72, 73). Given that NMDAR function is an important target for dynamic regulation and fine-tuning for normal brain

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development and function (53, 74), our results suggest that NMDAR dysfunctions observed in many other animal models of ASD (71, 75-90) may need to be further examined for their temporal changes. This might eventually help us develop more

dysfunctions.

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cautious treatment strategies for ASD patients with distinct temporal patterns of NMDAR

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In conclusion, our results suggest that Shank2 deletion in mice leads to NMDAR

hyperfunction at an early stage that is causally associated with NMDAR hypofunction and social deficits at later stages. In addition, our study suggests that early correction of NMDAR hyperfunction in Shank2–/– mice has long-lasting effects, preventing synaptic and social abnormalities from manifesting at later stages. Acknowledgments 17

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This work was supported by the National Research Foundation (NRF) grant (2013M3C7A1056732 to H.K.), NRF-2013-Fostering Core Leaders of the Future Basic

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Science Program (to C.C.), Global PhD Fellowship Program (NRF-2013H1A2A1032785 to S.H. and NRF-2015H1A2A1033937 to R.K.), the National Honor Scientist

Program (NRF-2012R1A3A1050385 to B.K.K.), the National Institute of Health grants

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(MH098114, MH104316, and HD087795 to Y.H.J.), Korea Institute of Science and

Technology Information (K-17-L03-C02-S01 to H.K.), and the Institute for Basic Science

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(IBS-R002-D1 to E.K.).

Financial disclosures

The authors report no biomedical financial interests or potential conflicts of interest.

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Figure legends

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Figure 1. Distinct transcriptomic profiles in Shank2–/– brains at P14 and P25. (A) Levels of Shank2 proteins in whole mouse brains increase during postnatal (P) days, reaching a plateau at ~P14. For quantification, Shank2 signals at each developmental

age groups. (n = 6 male mice for each stage).

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stages were normalized to those of α-tubulin and also to the average values of each

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(B) Levels of Shank2 mRNAs in the mouse brain during development, revealed by in situ hybridization of sagittal sections. The limited correlation between Shank2 protein and mRNA levels might be attributable to that the transcripts are differentially translated or that the in situ images do not cover the whole brain. For quantification, Shank2

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signals normalized to the brain area at each developmental stages were normalized to the average values of each age groups. (n = 3 male mice for each stage). Scale bar, 10 mm.

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(C) A schematic of gene set enrichment analysis (GSEA; software.broadinstitute.org/gsea/). The direction (positive/negative) and amplitude of

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enrichments reflect the extent to which the genes in a target gene set falls at the top (positive) or bottom (negative) of the ranked input gene list. (D) GSEA using KO/WT-P14 and KO/WT-P25 gene lists from Shank2–/– mice and target gene sets in the C5-CC category (gene ontology_cellular component). NES, normalized enrichment score accounting for the degree of overrepresentation at the top or bottom of a ranked list; FDR, false discovery rate. Positive and negative NES values are

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indicated in green and violet, respectively. (n = 6 mice for WT and KO at P14 and P25, ***FDR < 0.001; see Table S4 for details).

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(E) GSEA using KO/WT-P14 and KO/WT-P25 gene lists from Shank2–/– mice and NMDAR-related gene sets in the C2-all category. ‘Activation of NMDA receptor…’

indicates ‘Activation of NMDA receptor upon glutamate binding and postsynaptic event’.

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(n = 6 mice for WT and KO at P14 and P25, *FDR < 0.05, ***FDR < 0.001; see Table S3 for details).

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Figure 2. Reversal of NMDAR function in the Shank2–/– hippocampus during P14 and P25.

(A and B) NMDAR function is enhanced at Shank2–/– SC-CA1 synapses at ~P13–15, but reduced at ~P21–27, as shown by the NMDA/AMPA ratio. (n = 18 neurons from 11

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mice for WT and 16 (11) for KO (Shank2–/–) at P13–15, and 13 (8) for WT and 10 (6) for KO at P21–27, *P < 0.05, **P < 0.01, Student t-test; see Table S5 for the summary of

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electrophysiology results, and Table S6 for statistical details). (C and D) Reduced decay of NMDAR currents at Shank2–/– SC-CA1 synapses at ~P13–

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15, but not at ~P21–27. (n = 10 (4) for WT and 10 (4) for KO at P13–15, and 8 (6) for WT and 16 (9) for KO at P21–27, *P < 0.05, ns, not significant, Student t-test). (E) NMDAR currents at P14 Shank2–/– SC-CA1 synapses are more sensitive to ifenprodil (GluN2B antagonist) relative to those at WT synapses, which, in combination with the data in (A), predicts a ~2.07-fold and ~1.06-fold increases in ifenprodil-sensitive GluN2B and ifenprodil-insensitive GluN2A components, respectively. Numbers 1 and 2

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in WT and KO traces indicate NMDAR currents before and after ifenprodil treatment, respectively, and grey lines in WT and KO traces indicate ifenprodil-sensitive (or

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GluN2B-dependent) component. Note that the decay constant of the residual NMDAREPSCs lacking the GluN2B component is comparable between genotypes. (n = 15 (9) for WT and 15 (7) for KO, **P < 0.01, ns, not significant, Student t-test).

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Figure 3. Early memantine treatment normalizes NMDAR function in the juvenile and adult Shank2–/– hippocampus.

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(A) Early memantine treatment (P7–21) normalizes the NMDA/AMPA ratio at juvenile (~P25–31) Shank2–/– SC-CA1 synapses. Young Shank2–/– mice were orally administered memantine (20 mg/kg) twice daily for 2 weeks during P7–21 followed by measurements of NMDA/AMPA ratio at ~P25–31. (n = 22 neurons from 9 mice for WT-

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V, 19 (6) for WT-M, 18 (9) for KO-V, and 13 (3) for KO-M, *P < 0.05, ns, not significant, two-way ANOVA with Holm-Sidak test).

(B and C) Early memantine treatment (P7–21) normalizes LTP induced by high-

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frequency stimulation at juvenile (~P25–31; B) and adult (> P56; C) Shank2–/– SC-CA1

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synapses. Numbers 1 and 2 indicate fEPSP slopes during the baseline and last 5-min recordings, respectively. (Juvenile, n = 12 slices from 6 mice for WT-V, 10 (5) for WT-M, 16 (8) for KO-V, and 14 (8) for KO-M; adult, n = 18 (9) for WT-V, 25 (12) for WT-M, 23 (9) for KO-V, and 20 (7) for KO-M, **P < 0.01, ***P < 0.001, ns, not significant, two-way ANOVA with Holm-Sidak test). (D) Early memantine treatment for a shorter period (P7–14; 20 mg/kg oral/day) normalizes the NMDA/AMPA ratio at ~P15 at Shank2–/– SC-CA1 synapses. (n = 14 33

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neurons from 8 mice for WT-V, 14 (4) for WT-M, 12 (7) for KO-V, and 11 (6) for KO-M, *P < 0.05, **P < 0.05, two-way ANOVA).

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Figure 4. Early memantine treatment improves social interaction in juvenile and adult Shank2–/– mice.

(A) Schematic depiction of early memantine treatment. Young Shank2–/– mice were

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orally administered memantine (20 mg/kg) twice daily for 2 weeks from P7 to P21,

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followed by measurements of social interactions at juvenile (P28–35) and adult (P56) stages.

(B) Early memantine treatment improves social interaction of juvenile Shank2–/– mice (P28–35), as indicated by time spent in social interaction in the direct interaction test. (n = 18 mice (WT-V/vehicle), 19 (WT-M/memantine), 29 (KO-V), and 23 (KO-M), *P < 0.05,

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**P < 0.01, two-way ANOVA with Bonferroni test). (C–E) Early memantine treatment improves social interaction of adult Shank2–/– mice (>

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P56), as indicated by time spent sniffing S1/O (stranger mouse/object; D) and the social preference index based on sniffing time (the numerical difference between the time

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spent sniffing S1 and O divided by their sum x 100) (E) in the three-chamber test. (n = 25 mice (WT-V), 24 (WT-M), 27 (KO-V), and 17 (KO-M), ***P < 0.001, Student’s t-test (D-WT/V and D-WT/M), Wilcoxon matched-pairs signed rank test (D-KO/V and D-KO/M), and Mann Whitney U test (E; two-way ANOVA was not performed because the social preference index is a normalized value)).

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Figure 5. Early D-cycloserine or late memantine treatment does not improve social interaction in adult Shank2–/– mice.

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(A) Schematic depiction of early D-cycloserine (DCS) treatment and behavioral tests. Young Shank2–/– mice were orally administered D-cycloserine (40 mg/kg) twice daily for two weeks during P7–21, followed by consecutive measurements of open-field

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hyperactivity and three-chamber social interaction during P56–70.

(B and C) Early D-cycloserine treatment fails to improve social interaction of adult

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Shank2–/– mice, as indicated by time sniffing S1/O and the social preference index based on sniffing time in the three-chamber test. (n = 21 mice for WT-V, 16 (WT-M), 21 (KO-V), and 11 (KO-M), ns, not significant, ***P < 0.001, ns, not significant, paired

(C).

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Student’s t-test or Wilcoxon matched pairs signed rank test (B), Mann Whitney U test

(D) Schematic depiction of late chronic memantine treatment and behavioral tests.

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Shank2–/– mice were orally administered memantine (20 mg/kg) twice daily for 2 weeks during P25–39, followed by consecutive measurements of three-chamber social

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interaction and open-field hyperactivity during P56–90. (E and F) Late chronic memantine treatment fails to improve social interaction in Shank2–/– mice, as indicated by time sniffing S1/O and the social preference index based on sniffing time in the three-chamber test. (n = 15 (WT-V), 14 (WT-M), 13 (KO-V), and 14 (KO-M), ns, not significant, ***P < 0.001, ns, not significant, paired Student’s ttest or Wilcoxon matched pairs signed rank test (E), unpaired Student’s t-test (F).

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Early Correction of N-methyl-D-aspartate Receptor Function Improves Autistic-like Social Behaviors in Adult Shank2–/– Mice

Supplementary Methods and Materials

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Animals

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Supplementary Information

All mice were housed and bred at the mouse facility of Korea Advanced Institute of

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Science and Technology (KAIST) and maintained according to the Animal Research Requirements of KAIST, except for Shank2–/– mice lacking exon 24 (corresponding to exon 15 in our nomenclature), which were bred and maintained at Duke University according to the approval of the Institutional Animal Care and Use Committee (IACUC).

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Mice were fed ad libitum under a 12-h light cycle. For every experiment performed in this research, heterozygous x heterozygous mating was used. Pups have been weaned after 21 days from birth, and 3–5 mice were grouped in each cage. The mice

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for behavioral assays were selected randomly and age and sex matched. All procedures were approved by the Committee of Animal Research at KAIST

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(KA2012_19). PCR genotyping of Shank2–/– mice lacking exons 6–7, 7, and 15 was performed using the following two sets of oligonucleotide primers: exons 6–7, set for WT allele (522 bp), forward, 5’-TGG TCT ATG TTC ATT GCA TGG-3’, reverse, 5’-ATT GGC TGG GTG AGA TAC ACC-3’; set for mutant allele (626 bp), forward, 5’- CCG ACT GCA TCT GCG TGT TC-3’, reverse, 5’-ATT GGC TGG GTG AGA TAC ACC-3’; exon 7, set for WT allele (698 bp) and mutant allele (299 bp), forward, 5´-TCC ATG GTT TCG GCA GAG CG-3´, reverse, 5’-CAG CAT CAT GAC AAT GTC TCC A-3´.

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Drug administration Memantine hydrochloride (Sigma, M9292; 20 mg/kg), or D-cycloserine (Sigma, C6880; 40 mg/kg), was orally administered to Shank2–/– mice twice a day with a pipette during P7–21. Memantine was also administered for a shorter period at an early stage (P7–

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14), for two weeks at a late stage (P25–39), or acutely at P56. To administer drugs more efficiently to mice older than P21, Nutella was coated at the end of the pipette tip. Memantine hydrochloride was dissolved in 0.1% saccharin based drinking water

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to reach 40 mg/ml. D-cycloserine was dissolved in 0.1% saccharin based drinking

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water to reach 80 mg/ml. Acute intraperitoneal injection of memantine (10 mg/kg) to mice isolated for four days was followed by a three-chamber test 30 min after the injection. These mice were subjected, after another 4-day isolation and switching of vehicle/drug treatment, to 3-chamber social interaction test, followed by an open field

Behavioral assays

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test the next day.

All the behavioral tests were conducted with at least 30-min habituation in the

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environment.

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experimental rooms/booths except for the open field test, which should be a novel

Three-chamber social interaction test Three-chamber social interaction assay, a test known to measure social approach behavior

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, was performed as previously described 3. Briefly, the subject mice were

isolated for four days before the assay. Three-chambered apparatus consisted of left, center, and right chambers with two entrances to the center chamber. The light intensity of the apparatus was 100~130 lux. Two empty containers were located in the corner of the left or right chamber. This assay consisted of three sessions. First, the

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subjects were allowed to freely explore all three chambers with empty containers in two side chambers for 10 min. Second, the subject mouse was allowed to explore the containers with a stranger mouse 1 (S1) or a novel inanimate object (O) for 10 min. Finally, the novel object was changed to a stranger mouse 2 (S2), and the subject

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mouse was allowed to explore the two strangers. During the interval between each session, the subject was gently guided to the center chamber and the two entrances were blocked. EthoVision XT 10 (Noldus) was used to analyze time spent in chamber

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or sniffing stranger/object during the first 5 min of the second and third sessions. Preference index was defined by 100 x (difference of time spent sniffing stranger

Direct social interaction test

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mouse vs. object)/(total sniffing time).

Direct social interactions were tested as previously described

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, with slight

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modifications. A gray test box (33 × 33 × 22 cm) with bedding under dim light (< 50 lux) was used to test direct social interaction. A juvenile male subject mouse (C57BL/6N; P28–35) with or without weaning and with or without 4-day isolation in home cages

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was habituated in a booth similar to the experimental booth at least for 30 min. An ageand sex-matched 129/Sv, or C3H/HeNHsd, wild-type stranger mouse was first placed

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in the test box and allowed to explore the box for habituation for 10 min. Next, a subject mouse was placed into the test box and allow to interact with the stranger mouse for 10 min. Direct interaction using mouse pairs under different genetic backgrounds was attempted to minimize the numbers of mice used for experiments, and was based on the previous result that inter-strain social interaction can be measured in the direct social interaction test 4. The recorded interactions were analyzed manually by researchers blind to genotypes and experimental conditions. Social interactions were defined as activities of a WT or Shank2–/– mouse with black coat color towards a target

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stranger mouse with agouti coat color, involving sniffing, following, mounting, allogrooming, huddling, and wrestling. Olfactory test

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The test procedure was slightly modified from the previous paper 6. Briefly, we exposed mice to Kim-wipes soaked with four different odors: water, banana, coffee, stranger male mice. Pieces of Kim-wipes were placed in 60-phi Petri dishes with nine small

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holes in the cover. A single mouse was placed in a new home cage, and an empty petri dish was introduced for 30 min before the experiment for habituation. Each odor

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was sequentially presented into the cage for 4 min. This four-odor set was presented to the mouse for three times with 1-min inter-session intervals. A skilled experimenter measured the sniffing time toward Petri dishes in a blind manner. Tube test

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This assay was performed as described previously 7. Before social dominance tests including the tube test, each mouse group (WT-V, WT-M, KO-V, and KO-M) was group housed for two weeks. We used transparent acryl tubes with 30-cm length and 3-cm

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inner diameter, a size that an adult mouse can just pass but usually cannot turn around.

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During two-day training sessions, each mouse was trained to pass through the tube in either direction for eight times. When the mice hesitated to move, they were gently pushed by a plastic bar. After the training sessions, three days of test sessions were performed. During the test sessions, animals went through three more training trials before the real test. For the test, two different mice were placed into the opposite ends of the test tube and released. The mouse first retreated from the tube was marked as a “loser”. Among six possible pairs between four cage-mates, two pairs were tested per day. Each mouse was ordered by its rank from 1 to 4.

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Urine-marking test The assay was performed as described previously 7. As mentioned above for the tube test, each mouse group (WT-V, WT-M, KO-V, and KO-M) was co-housed for two weeks before the urine-marking test. We designed a two-chambered cage (26 cm × 21 cm ×

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26 cm) that are partitioned by a transparent wall with holes. A filter paper was placed at the bottom of each compartment to mark the urinated area. Two different mice in a pair were placed on the opposite sides of the wall and allowed to freely interact through

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the holes of the wall for 2 hours. The filter paper with marked urine was visualized

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under UV light, and the urine marks of each side were scored in a blind manner. The mouse with more number and larger size of urine marks was designated as a “winner”. Similar to the tube test, each mouse was ranked between 1 and 4. Urine and tube tests were performed consecutively in a random order.

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Repetitive behaviors

Repetitive behaviors were measured using mice grouped in home cages. On the test day, the subject mice were allowed to freely move in a clean new cage under 100~120

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lux light intensity with bedding. Amounts of time spent in digging, self-grooming, and jumping during a single 10-min session recorded were measured manually. Grooming

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was defined as a mouse stroking or scratching its face or body area, or licking its body. Jumping was defined as both two hind limbs simultaneously pushing against the ground. Digging was defined as a mouse uses its head or forelimbs to dig out beddings. Experiments were performed in a double-blind manner, and repetitive behaviors were manually measured. Ultrasonic vocalization test To perform this experiment, female young adult mice (> 8 weeks) in the same genetic

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background (C57BL/6N) were used as a stranger/intruder. The subject male mice were isolated at least four days in home cages so that they recognize the cage as their own territories. A mouse in its home cage (100~120 lux) was habituated in the experimental booth for 5 min while recording basal USVs without a female

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intruder/stranger. Next, a female mouse was introduced into the cage, followed by recording courtship USVs of the subject mice for 5 min. Avisoft SASLab Pro software was used to analyze USVs. Spectrograms were generated with 256 Fourier

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transformation length, 75% overlap of temporal resolution and 45 kHz of lower cut-off

Elevated plus maze test

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frequency. USV calls longer than 2 ms were counted automatically.

The elevated plus maze (EPM) apparatus (center zone light intensity: 100~120 lux), located 50 cm above the ground, consisted of two open arms, two closed arms, and a

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neutral center zone with access to both arms. Closed arms were enclosed with 12 cmhigh walls. A subject mouse was gently introduced into the center zone and allowed to move freely in the open and closed arms for 8 min. The center point of the mouse

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body was used to determine the entries to open/closed arms, and entry duration and frequency were measured using EthoVision XT 10.

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Open field test

Open field test was performed using a 40 x 40 x 40 cm white acryl chamber (center zone light intensity: 100~120 lux). A subject mouse was gently introduced into the center zone of the chamber and recorded the movements for 1 hour. Locomotor activities and time spent in the center region of the open field arena were analyzed using EthoVision XT 10.

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Laboras test Long-term (3-day) movements of mice, including locomotor, climbing, rearing, grooming, eating, and drinking activities were recorded and automatically analyzed using the Laboratory Animal Behavior Observation Registration and Analysis System

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(LABORAS; Metris). Mice were individually caged in a specialized LABORAS recording environment with LABORAS cages (home-cage like) for 72 consecutive

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hours and fed ad libitum. Electrophysiology

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For hippocampal/medial prefrontal cortex (mPFC) electrophysiology experiments, sagittal/coronal sections were prepared in ice-cold dissection buffer containing (in mM) 212 Sucrose, 25 NaHCO3, 10 D-glucose, 2 Na-pyruvate, 1.25 ascorbic acid, 1.25 NaH2PO4, 5 KCl, 3.5 MgSO4, 0.5 CaCl2 bubbled with 95% O2 5% CO2 gas. Brain

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sections (~300-μm thick) were prepared using Leica VT 1200 vibratome and used for whole-cell and field recordings (P13-15), 400-μm brain sections were used for juvenile (3 or 4 weeks) and adult field recording experiments. After sectioning, the slices were

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recovered for 30 min in normal ACSF at 32oC (in mM: 124 NaCl, 25 NaHCO3, 10 Glucose, 2.5 KCl, 1 NaH2PO4, 2 CaCl2, 2 MgSO4 oxygenated with 95% O2 5% CO2

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gas). Then, the slices were moved to ACSF at room temperature and additionally recovered for 30 min. Brain slices were moved and maintained in a submerged-type recording chamber perfused with 28oC ACSF (2 ml min-1). Recording and stimulus glass pipettes from borosilicate glass capillaries (Harvard Apparatus) were pulled using an electrode puller (Narishige). For extracellular recordings, the Schaffer collateral pathway in the hippocampus was stimulated every 20 sec with a pipette (0.3-0.5 MΩ) filled with

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normal ACSF. Stimulation intensity was tuned to yield a half-maximal response. A recording electrode (1 MΩ) filled with normal ACSF was located in the stratum radiatum region of the hippocampal CA1 subfield to record fEPSPs, which were amplified (Multiclamp 700B, Molecular Devices) and digitized (Digidata 1550,

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Molecular Devices) for measurements. The averaged value of three successive rise slopes (20-50%) of fEPSP was used as a representative value of each bin (1 min). To induce LTP, stable baseline fEPSPs were maintained at least for 20 min, followed by

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applying high-frequency stimulation (100 Hz, 1 s). The averaged values of rise slopes for last 5 min (55 min to 60 min from LTP induction) were used for multiple comparison

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among experimental groups. For the input-output curve, stimulation intensity was increased every three consecutive responses; 5, 10, 15, 20, 30, 40, 50, 60, 70, 80 (in μA).

Whole-cell recordings in the CA1 region of the hippocampus and the layer 2/3

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region of the medial prefrontal cortex were performed using a recording pipette (2.53.5 MΩ) filled with the internal solution for EPSC (in mM: 100 CsMeSO4, 10 TEA-Cl,

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8 NaCl, 10 HEPES, 5 QX-314-Cl, 2 Mg-ATP, 0.3 Na-GTP and 10 EGTA with pH 7.25, 295 mOsm) or IPSC (in mM: 115 CsCl, 10 TEA-Cl, 8 NaCl, 10 HEPES, 5 QX-314Cl,

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2 Mg-ATP, 0.3 Na-GTP, 10 EGTA with pH 7.35, 280 mOsm). The response was amplified (Multiclamp 700B, Molecular Devices) and digitized (Digidata 1550, Molecular Devices) for measurements. Evoked EPSCs were induced by stimulating the Schaffer collateral pathway or the layer 1 of the medial prefrontal cortex with bipolar pulses (10-40 mA, 0.15 ms duration) every 15 s, while adding picrotoxin (60 μM) to ACSF to inhibit GABAergic transmission. We monitored series resistance by measuring the peak amplitude of the capacitance currents in response to hyperpolarizing step pulses (5 mV, 40 ms). Only the cells with a change in < 20% were

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included in the analysis. To measure evoked AMPAR-mediated and NMDAR-mediated EPSCs, cell membrane voltages were clamped at -70 mV and +40 mV, respectively. To measure the NMDA/AMPA ratio, the average value of NMDAR currents 60 ms after the peak NMDAR currents was divided by the mean value of AMPAR current peak.

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We did not add an AMPAR blocker for experiments measuring the NMDA/AMPA ratio, except for those described in Figure S22 (see the figure legend for details), because the amount of AMPAR currents remaining at 60 ms after the peak of NMDAR currents

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is likely to be very small 8. In detail, 30 consecutive stable AMPAR current (above 100 pA) recordings at the holding potential of -70 mV were followed by at least 20

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consecutive NMDAR current measurements at +40 mV. To calculate tau (t) constant of NMDAR currents, each NMDAR trace was fitted with exponential curve formula; 𝑓𝑓(𝑡𝑡) = 𝐴𝐴𝑒𝑒 −𝑡𝑡/𝜏𝜏 + 𝐶𝐶 (𝑡𝑡 is time, 𝐶𝐶 is arbitrary constant, 𝐴𝐴 is the amplitude of NMDAR

current). For the quantification of ifenprodil effects, peak amplitudes of NMDAR

comparison.

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currents pharmacologically isolated by NBQX (10 mM) were used for genotype

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To measure paired-pulse facilitation in CA1 Schaffer collaterals and mPFC layer 2/3, paired pulses with 25, 50, 75, 100, 200, 300 ms inter-stimulus intervals were

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given three times and averaged, respectively. Miniature EPSCs and IPSCs were recorded for 2 min after maintaining stable

baseline for 5 min. In detail, TTX (1 μM) was added in ACSF to inhibit action potentials. For mEPSC recording, picrotoxin (60 μM) was added to ACSF. For mIPSC recording, NBQX (10 μM) and APV (50 μM) were added to ACSF to block AMPA and NMDA receptor-mediated currents. To measure NMDAR-mediated miniature EPSCs, first whole-cell patch clamp

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was done at -70 mV in normal aCSF. After 2 min stable baseline recording, ACSF with SrCl2 (in mM: 124 NaCl, 25 NaHCO3, 10 Glucose, 2.5 KCl, 1 NaH2PO4, 4 SrCl2, oxygenated with 95% O2 5% CO2 gas) including TTX (1μM), NBQX (10μM) and

stable baseline, the mEPSCs were recorded for 2 min. Antibodies

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picrotoxin (60μM) were applied to the slice for 8 min monitoring the baseline. After

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Antibodies against GluA1 (#1193), and GluA2 (#1195) have been previously described . The following antibodies were purchased from commercial sources: Shank2

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(Synaptic Systems); GluN1, PKCa (BD Transduction Laboratories); GluN2A, mGluR1, mGluR5, phospho-GluN2B (Ser 1303), phospho-GluN1 (Ser 896), GluN3A (Millipore); GluN2B (NeuroMab); CaMKIIα/β, ERK1/2, phospho-ERK1/2 (Thr 202, Tyr 204), p38, phospho-p38 (Thr 180, Tyr 182), phospho-PKA (Thr 197), phospho-PKCa (Ser 600),

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mTOR, phospho-mTOR (Ser 2448), phospho-GluN2B (Tyr 1472), CREB, phosphoCREB (Ser 133) (Cell Signaling); phospho-CaMKIIα/β (Thr 286), phospho-GluN2B (Ser 1480), phospho-GluN2B (Tyr 1336), PKA (Abcam), α-tubulin (Sigma).

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Synaptic membrane fraction

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Whole brains of Shank2–/– mice and WT littermates at postnatal day 14 and 21 were briefly homogenized in ice-cold brain homogenization buffer (0.32 M sucrose, 10 mM HEPES, pH7.4, 2 mM EDTA, 2 mM EGTA, protease inhibitors, phosphatase inhibitors). The homogenates were centrifuged at 1200 g for 10 min, and the supernatants were further centrifuged at 12000 g for 10 min. The pellet from each brain was resuspended in the brain homogenization buffer and used as a synaptic or crude membrane (P2) fraction.

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In situ hybridization Mouse brain sections (12 mm thick) at embryonic day 18 (E18) and postnatal days (P0, P7, P14, P21, and P42) were prepared using a cryostat (Leica CM 1950).

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Hybridization probe specific for mouse Shank2 mRNA was generated using the following constructs: pGEM®T Easy vector containing nt 4608~4932 of Shank2 (NM_001113373.2). Antisense riboprobes were prepared by RNA polymerase

RNA-Seq library preparation and sequencing

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transcription using a Riboprobe System (Promega) in the presence of a-35S-UTP.

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Mouse brains were immersed in RNAlater solution (Ambion, AM7020) to stabilize RNA. RNA extraction, library preparation, cluster generation, and sequencing were performed by Macrogen Inc. (Seoul, Korea). RNA samples for sequencing were prepared using a TruSeq RNA Sample Prep Kit v2 (Illumina) according to the

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manufacturer’s instructions. An Illumina’s HiSeq 2000 were used for sequencing to generate 101-bp paired-end reads. Image analysis and base calling were performed with the standard Illumina software (RTA, Real Time Analysis v1.18). The BCL (base

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calls) binary was converted into FASTQ utilizing Illumina package bcl2fastq (v1.8.4).

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RNA-Seq analysis

The accession series number for the RNA-Seq data is GSE82066. The raw reads were aligned to the Mus musculus genome (GRCm38) using the spliced junction mapper HISAT2

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(version 2.1.0, default options). The gene-level read counts were created

from the aligned reads by HTSeq

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and differential gene expression analysis was

carried out using R/Bioconductor DEseq2 package (v1.19.11) 12. The normalized read counts were computed by dividing the raw read counts by size factors and fit to a negative binomial distribution. The sequencing batch effect was adjusted between

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sample groups (see GEO sample annotation) by modeling DESeq2 design formula (design = ~batch + condition). The P-values were first corrected by applying empirical estimation of the null distribution using the R fdrtool (v.1.2.15) package and then were adjusted for multiple testing with the Benjamini–Hochberg correction. Genes with an

differentially expressed. Gene set enrichment analysis (GSEA)

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adjusted P-value less than 0.05 and with |fold-change| > 2 were considered

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The GSEA software (gsea2-2.2.4.jar) (http://software.broadinstitute.org/gsea)

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used to determine whether a priori-defined gene sets showed statistically significant differences between different groups of samples. Enrichment analysis was performed using GSEAPreranked module on gene set collections H (Hallmark gene sets; 50 gene sets), C2 (curated; 4,731 gene sets), C3 (TFT, transcription factor targets; 615 gene sets), and C5 (GO; 5917 gene sets) downloaded from Molecular Signature Database

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(MSigDB) v6.0 (http://software.broadinstitute.org/gsea/msigdb). GSEAPreranked is applied using the list of all expressed genes ranked by the fold change multiplied by

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the inverse of the p-value. At the top and bottom of the list are genes with the strongest up- and down-regulation respectively. All the recommended default settings with 1,000

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permutations and a classic scoring scheme were used. The False Discovery Rate (FDR) is estimated to control the false positive finding of given Normalized Enrichment Score (NES) by comparing the tails of the observed and null distributions derived from 1000 gene set permutations. The FDR (q-value) is adjusted for gene set size but not between different comparisons. The gene sets with FDR less than 0.05 were considered significantly enriched.

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Hippocampal mRNA extraction Preparation of hippocampal mRNAs was performed using RNeasy® kit (QIAGEN).

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Quantitative PCR (qPCR) cDNAs was synthesized using TOPscriptTM cDNA synthesis kit (Enzynomics, EZ005). qPCR was performed using SsoAdvancedTMSYBR® Green Supermix (BIORAD,1708882AP), CFX96TM Real-Time system, and the following primers:

Shank2 Ex8-11 (5+6) Shank2 ex 12-13 (7+8) Shank2 ex5’-8 (9+10)

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Structural modeling

CTTCACTCAACAGGCTGGGTG CTCTCCGATGCTCAGAACTTTGAC TGGTTCCCAGCTGAGTGTGTG CCAAAGCCCTCGTTGTCCTTC AGCCAGAAAGAAAGCTCCCC GGCTTTGTCCACGAGTTCCT TCCAAGCCCTCCAGGACTGC CGAGGGAGGCCCAGAAATGG GCTGTGATGATGAGCGTCCCCG CTGTGGCCCACCTTGACGAC

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Shank2 ex4-6 (3+4)

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

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Shank2 ex2-4 (1+2)

For homology-based molecular modeling of the Shank2 PDZ domain in complex with

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the C-terminal PDZ-binding motif (PBM) of SAPAP3, the crystal structure of the mouse Shank3 PDZ domain with the extra amino acid sequence in the immediate upstream in complex with the PBM of mouse SAPAP3 (PDB ID: 5IZU) was used as a template 14

. The homology model of the Shank2a PDZ domain (1-131 aa) was built using

SWISS-MODEL modeling web server 15. To generate the extended binding interfaces between the modeled mouse Shank2a PDZ domain and the SAPAP3 PBM, the modeled Shank2a PDZ domain was superimposed to the mouse Shank3 PDZ domain (PDB ID: 5IZU). All structural images were generated using PyMOL software 16. 13

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Statistical analysis Experimenters were blind to the genotype and gender of the animal tested. Before analysis, all data file names were randomized (both genotype and gender). Statistical analyses of behavioral results were performed using unpaired Student t-test or Mann-

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Whitney U test for the comparison of unpaired WT and KO data, paired Student t-test or Wilcoxon matched-pairs signed rank test for the comparison of paired WT and KO data, and two-way ANOVA for drug-rescue data followed by performing multiple

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comparisons if gene x drug interaction is significant. In non-behavioral experiments,

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the data from male and female mice at the juvenile or younger stage (< P28) were pooled, based on NMDAR functions (NMDA/AMPA ratio and NMDAR-dependent LTP) that are similar in males and females. Exact numbers of male and female mice used in the experiments, and details of statistical results, including main effects and p values

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for all possible post-hoc comparisons, are indicated in Supplementary Table S6.

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Supplementary Figures

Figure S1. Distinct enrichment of KO(Shank2–/–)/WT-P14 and KO/WT-P25 gene

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lists in target gene sets in the C2 category (curated gene sets). (A and B) GSEA using two query gene lists from KO/WT-P14 and KO/WT-P25 ranked by differential expression and target gene sets in the C2 category (curated gene sets). Green and violet colors in NES indicate positive and negative enrichments, respectively; blue colors indicate target gene sets that overlap between P14 and P25. (n = 6 mice for WT and KO at P14 and P25; ***FDR < 0.001; see Supplementary Table S3 for details).

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Figure S2. GSEA plots for the KO/WT-P14 and KO/WT-P25 ranked input gene lists from Shank2–/– mice and highly enriched target gene sets. (A-H) GSEA plots for the KO/WT-P14 and KO/WT-P25 ranked input gene lists enriched for target gene sets (see Fig. 1D,E); C5CC_GO_Postsynapse (A and B),

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C5CC_GO_Excitatory synapse (C and D), C2_KEGG_Long term depression (E and F), and C2_KEGG_Long term potentiation (G and H). The enrichment score reflects the extent to which the genes in a target gene set falls at the top or bottom of the input

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gene list in this case ranked by the extent of up and down regulations. Note that the

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enriched genes from a particular gene set tend to fall more on the left side of the KO/WT-P14 list, relative to those more evenly distributed in the KO/WT-P25 list, leading to higher enrichment scores (n = 6 mice for P14 and P25; see Fig. 1D,E, and

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Tables S3 and S4 for further details).

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Figure S3. Normal basal synaptic transmission and paired-pulse facilitation at

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P14 Shank2–/– SC-CA1 synapses.

(A) Normal basal synaptic transmission at P14 Shank2–/– SC-CA1 synapses, as shown by fEPSP slopes plotted against fiber volley amplitudes. (n = 16 slices from 6 mice for

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WT and 19 (8) for KO at P13–15, ns, not significant, repeated measure of two-way

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ANOVA with Holm-Sidak test).

(B) Normal paired-pulse facilitation at P14 Shank2–/– SC-CA1 synapses, as shown by fEPSP slopes plotted against inter-stimulus intervals. (n = 9 slices from 3 mice for WT and KO at P13–15, ns, not significant, repeated measure of two-way ANOVA with Holm-Sidak test).

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Figure S4. Normal mEPSCs and mIPSCs in P14 Shank2–/– CA1 pyramidal cells. (A) Normal mEPSC frequency and amplitude in P14 Shank2–/– CA1 pyramidal neurons. (n = 21 neurons from 3 mice for WT and 23 (4) for KO, ns, not significant, Mann-

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Whitney U test).

(B) Normal mIPSC frequency and amplitude in P14 Shank2–/– CA1 pyramidal neurons.

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(n = 16 (3) neurons for WT and 15 (3) for KO, ns, not significant, Student t-test).

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Figure S5. Gender does not affect the NMDA/AMPA ratio in Shank2–/– mice at P14 and P25.

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(A and B) NMDAR function is enhanced at Shank2–/– hippocampal SC-CA1 synapses at ~P13–15, but reduced at ~P21–27 both in males or females, as shown by the NMDA/AMPA ratio. Note that the data in Fig. 2a,b were segregated into those from

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males and females to determine potential sexual dimorphism. (n = 6 neurons from 4

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mice for WT-male and 9 (4) for KO-male, 12 (7) for WT-female, and 7 (7) for KO-female at P13–15, and 7 (5) for WT-male, 4 (2) for KO-male, 6 (3) for WT-female, and 6 (4) for KO-female at P21–27, *P < 0.05, Student t-test).

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Figure S6. Enhanced and suppressed NMDAR functions in the P14 and P25

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Shank2–/– mPFC.

(A and B) NMDAR function at Shank2–/– layer II/III pyramidal synapses is enhanced at ~P13–15, but reduced at ~P21–27, as shown by the NMDA/AMPA ratio. (n = 11 neurons from 4 mice for WT and 9 (4) for KO at P13–15, and 13 (6) for WT and 11 (7)

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for KO at P21–27, *P < 0.05, Student t-test).

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Figure S7. Normal amplitude, but reduced frequency, of mEPSCs and normal paired-pulse facilitation in the P14 Shank2–/– mPFC.

(A) mEPSC amplitude and frequency in P14 Shank2–/– mPFC layer II/III pyramidal

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neurons. (n = 18 neurons from 4 mice for WT and 19 (4) for KO, *P < 0.05, ns, not significant, Mann-Whitney U test for frequency, and Student t-test for amplitude).

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(B) Normal paired-pulse facilitation in P14 Shank2–/– mPFC layer II/III pyramidal neurons. (n = 7 neurons from 3 mice for WT and 5 (2) for KO, ns, not significant,

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repeated measures two-way ANOVA with Holm-Sidak test).

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Figure S8. Total and phosphorylation levels of glutamate receptors and signaling molecules in P14 and P21 Shank2–/– brains. (A–D) Whole-brain crude synaptosomes from P14 and P21 WT and Shank2–/– (KO) mice were immunoblotted for total and phosphorylated glutamate receptor subunits and signaling proteins. KO band/a-tubulin signals were normalized to WT/a-tubulin values. (n = 4~8 mice for WT and KO, ns, not significant, *P < 0.05, **P < 0.01, one sample t-test). Note that some of the immunoblot results here, including that for GluN1, are different from our previous results 3 for reasons, including that the two experiments 23

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used mice at different ages (we do not know the exact ages of the mice used in the previous study other than an age range [3–4 weeks]), mice of different male-female ratios (2 + 2 in the previous study vs. 2 + 4, i.e. for GluN1, in the present study), mice at different generations (genetic backgrounds might have drifted), mice bred in two

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different methods (cervical dislocation vs isoflurane).

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very different environments (institutions in different cities), or mice sacrificed using

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Figure S9. Early memantine treatment has no effect on mEPSCs in Shank2–/– hippocampal CA1 pyramidal neurons.

(A–D) mEPSC frequency and amplitude in the CA1 pyramidal neurons in WT and

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Shank2–/– mice (P26–33) treated with early memantine or vehicle (P7–21). Examples of currents traces (b). (n = 15 neurons from 4 mice for WT-V, 16 (3) for WT-M, 16 (3)

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for KO-V, and 15 (3) for KO-M, two-way ANOVA for (c and d)).

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Figure S10. Gender does not affect NMDAR-dependent LTP and its memantine-

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dependent rescue in Shank2–/– mice.

(A and B) NMDAR-dependent LTP is suppressed at Shank2–/– SC-CA1 synapses at ~P56–84, and memantine-dependent rescues of NMDAR-dependent LTP are similar

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in males and females. Note that the data in Fig. 3C were segregated into those from

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males and females to determine potential sexual dimorphism. (n = 10 slices from 5 mice for WT-V, 14 (8) for WT-M, 18 (7) for KO-V and 15 (5) for KO-M in males, and 8 (4) for WT-V, 11 (4) for WT-M, 5 (2) for KO-V and 5 (2) for KO-M in females, *P < 0.05, ***P < 0.001, ns, not significant, two-way ANOVA with Holm-Sidak test).

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Figure S11. Early memantine treatment for a shorter period (P7–14) does not

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affect mEPSCs in the Shank2–/– hippocampus. (A and B) Early memantine treatment for a shorter period (P7–14) has no effect on

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mEPSCs in Shank2–/– CA1 pyramidal neurons (P13–15 (n = 17 neurons from 3 mice

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for WT-V, 11 (2) for KO-V, and 14 (3) for KO-M, Kruskal-Wallis test).

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Figure S12. Early memantine treatment improves direct social interaction in juvenile Shank2–/– mice tested in the absence of isolation and before weaning. (A) Early memantine treatment improves social interaction of juvenile Shank2–/– mice

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(P28–35) in the direct social interaction test, as indicated by time spent in social interaction. Young Shank2–/– mice were orally administered memantine (20 mg/kg) twice daily for 2 weeks from P7 to P21, followed by measurement of direct social

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interactions at juvenile (P28–35) stages in the absence of social isolation and weaning.

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(n = 17 mice (WT-V/vehicle), 18 (WT-M/memantine), 15 (KO-V), and 19 (KO-M), *P < 0.05, ***P < 0.001, ns, not significant, two-way ANOVA with Holm-Sidak test).

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Figure S13. Early memantine treatment improves social approach but has no

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effect on social novelty in adult Shank2–/– mice in the three-chamber test. (A and B) Early memantine treatment improves social approach of adult Shank2–/– mice (>P56) in the three-chamber test, as shown by log10(Social/Object) values and time spent sniffing (S1/O). (n = 25 mice for WT-V, 24 for WT-M, 27 for KO-V, and 17

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for KO-M, **P < 0.01, ***P < 0.001, ns, not significant, two-way ANOVA with HolmSidak test for (A; there was a significant genotype x drug interaction) and three-way ANOVA with Bonferroni test for (B; main effect and interaction p values are indicated

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in the figure)). The log10(Social/Object) values were derived from the raw S1/O data to

used for two-way ANOVA 17.

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perform two-ANOVA analysis, based on the previous report that ratio values can be

(C and D) Early memantine treatment improves social interaction of adult Shank2–/– mice (> P56) in the three-chamber social interaction test, as shown by time spent in

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chamber with S1/O (C; not time spent sniffing) and the social preference index (D). (n = 25 mice (WT-V), 24 (WT-M), 27 (KO-V), and 17 (KO-M), ***P < 0.001, ns, not significant, paired Student’s t-test (C), and unpaired Student’s t-test for (D; two-way

value)).

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(E and F) Early memantine treatment (P7–21) has no effect on social novelty recognition of Shank2–/– mice (>P56) in the three-chamber test, as shown by the time spent sniffing S2/S1 (novel stranger/familiar stranger; E) and the social preference index based on sniffing time (F). (n = 15 mice for WT-V, 16 for WT-M, 16 for KO-V, and 10 for KO-M, *P < 0.05, ***P < 0.001, ns, not significant, paired Student’s t-test or Wilcoxon matched pairs signed rank test for (E), and unpaired Student’s test or Mann Whitney U test for (F).

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Figure S14. Shank2–/– mice display social submissiveness that is not associated with social interaction and is resistant to early memantine treatment in Shank2–/– mice.

(A) Early memantine treatment has no effect on olfactory function in Shank2–/– mice at

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P56. Young Shank2–/– mice were orally administered memantine (20 mg/kg) twice daily for 2 weeks during P7–21, followed by measurement of olfactory function during P56–70. (n = 20 mice for WT-V, 21 for WT-M, 21 for KO-V, and 22 for KO-M, ns, not

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significant, two-way ANOVA). Two-way ANOVA for each olfactory cues was performed for biologically better interpretations, which did not yield any significant genotype effect,

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drug effect, or genotype x drug interaction. (B and C) Shank2–/– mice show submissiveness to WT mice in both tube and urine tests, as supported by the significant genotype effects. Note that early memantine treatment has no effect on social ranking in Shank2–/– mice at P56, as supported by the lack of main drug effects. Young Shank2–/– mice early memantine treated during P7–21 were subjected to two different social dominance tests during P56–90. (n = 11 mice for WT-V, WT-M, KO-V, and KO-M, **P < 0.01, ns, not significant, two-way

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ANOVA; post hoc comparisons were not performed due to the lack of significant genotype x drug interaction). (D) Lack of correlation between social interaction (direct social interaction test) and number of wins (tube test) in WT and Shank2–/– mice. These mice represent a subset

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of the mice for which we presented the data in Figure S12 and Figure S14B-C. This subset went through both direct social interaction and tube tests. Specifically, these mice were maintained under clear mixed genotype housing (MGH) starting from ~P7

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after PCR genotyping and subjected to the direct social interaction test before weaning

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and without social isolation ~P28–33, followed by weaning, additional MGH for more than two weeks, and the tube test. The colored dots indicate individual mice in distinct experimental groups. (n = 8 mice for WT-V, 8 for WT-M, 8 for KO-V, and 8 for KO-M, Spearman’s correlation).

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(E) Lack of correlation between social interaction (direct social interaction test) and number of wins (tube test) in WT and Shank2–/– mice. Note that the mice who won three times in the tube test (highest ranking) and those who never won (lowest ranking)

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show comparable levels of direct social interaction. These mice were selected from the mice shown in (D). (n = 8 mice for 3 times and 7 for 0 time, ns, not significant,

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Mann-Whitney U test).

(F and G) Lack of correlation between social interaction (direct social interaction test) and number of wins (tube test) in sub-group comparison of W-V and KO-V mice, or KO-V and KO-M mice, derived from the mice mentioned above (D). (n = 8 mice for WT-V, 8 for WT-M, 8 for KO-V, and 8 for KO-M, ns, not significant, Spearman’s correlation).

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Figure S15. Early memantine treatment modestly improves hyperactivity in the

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open-field test in adult Shank2–/– mice, and habitation dampens the memantine-

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dependent improvement.

(A–C) Early memantine treatment partially improves hyperactivity of adult Shank2–/– mice (P56) in the open-field test (a novel environment). (n = 11 mice for WT-V, 11 for WT-M, 13 for KO-V, and 12 for KO-M, *P < 0.05, ***P < 0.001, ns, not significant, twoway ANOVA with Holm-Sidak (normal distribution) or Bonferroni (non-normal distribution) test for (B) and two-way ANOVA with Holm-Sidak test for (C)). (D–G) Habituation dampens the effect of early memantine treatment on hyperactivity in adult Shank2–/– mice (P56) in an independent open-field test performed for two

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consecutive days. Shank2–/– mice early memantine treated during P7–21 were subjected to open-field tests during P56–68 for two consecutive days. Note that the difference between memantine-treated and vehicle-treated Shank2–/– mice on day 2 became no longer significant, as compared with that on day 1 (E and G), suggestive

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of the influence of habituation. These results, which are not as strong as that from an independent experiment mentioned above (A–C), led to the collective conclusion that memantine moderately improves hyperactivity in Shank2–/– mice. (n = 19 mice for WT-

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V, 20 for WT-M, 20 for KO-V, and 22 for KO-M, *P < 0.05, ***P < 0.001, ns, not significant, two-way ANOVA with Holm-Sidak test (D; post hoc comparisons were only

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performed for the 10-min block that show significant genotype x drug interaction), twoway ANOVA for (e; genotype x drug interaction P > 0.05), two-way ANOVA with

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Bonferroni test (F), and two-way ANOVA for (G; genotype x drug interaction P > 0.05).

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Figure S16. Early memantine treatment does not improve hyperactivity in adult Shank2–/– mice in the Laboras test (a familiar environment).

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(A and B) Locomotor activity of Shank2–/– mice (2–3 months) early memantine treated

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during P7–21 were subjected to the Laboras test, where mouse movements in Laboras cages (a home cage-like environment) were measured for three consecutive days. Note that the locomotor activities of memantine-treated and vehicle-treated Shank2–/– mice on day 2 and 3 are comparable, suggesting that memantine has no effect on hyperactivity in a sufficiently familiar environment. Light-off phases are shown in shades. (n = 23 mice for WT-V, 20 for WT-M, 24 for KO-V, and 26 for KO-M, ***P < 0.001, two-way ANOVA for (B)). ANOVA was not performed for (A).

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Figure S17. Early memantine treatment does not improve reduced USVs, enhanced repetitive behaviors, or enhanced anxiolytic-like behaviors in adult Shank2–/– mice.

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(A) Number of USV calls in WT and Shank2–/– mice (> P56) treated early (P7–21) with

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memantine or vehicle. (n = 8 mice for WT-V, 6 for WT-M, 6 for KO-V, and 7 for KO-M, ***P < 0.001, ns, not significant, Mann Whitney U test or Student t-test). (B–D) Repetitive behaviors (digging, self-grooming, and jumping) in memantine- and vehicle-treated (P7–21) WT and Shank2–/– mice (> P56). (n = 13 mice for WT-V, 13 for WT-M, 13 for KO-V, and 17 for KO-M, *P < 0.05, ***P < 0.001, two-way ANOVA). (E and F) Elevated plus maze behaviors in memantine- and vehicle-treated (P7–21) WT and Shank2–/– mice (> P56). (n = 16 mice for WT-V, 15 for WT-M, 16 for KO-V, and 18 for KO-M, ***P < 0.001, two-way ANOVA). 36

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(G) Time spent in the center region of the open-field arena in memantine- and vehicletreated (P7–21) WT and Shank2–/– mice (> P56). (n = 11 mice for WT-V, 11 for WT-M,

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13 for KO-V, and 12 for KO-M, two-way ANOVA).

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Figure S18. Early D-cycloserine treatment does not improve hyperactivity in adult Shank2–/– mice.

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(A–C) Early D-cycloserine treatment fails to improve hyperactivity of adult Shank2–/– mice in the open-field test. (n = 16 mice (WT-V), 9 (WT-M), 13 (KO-V), and 9 (KO-M),

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***P < 0.001, two-way ANOVA for (C)). ANOVA was not performed for (B).

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Figure S19. Early D-cycloserine treatment of WT mice during P7–21 suppresses NMDAR function at the juvenile stage.

(A and B) Early D-cycloserine treatment during P7–21 in WT mice (A) induces a decrease in the NMDA/AMPA ratio at the juvenile stage (~P21–25) (B). Young WT

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mice were orally administered D-cycloserine (40 mg/kg) twice daily for 2 weeks during P7–21 followed by measurements of the NMDA/AMPA ratio at SC-CA1 synapses at ~P21–25. (n = 14 neurons from 4 mice for control, 12 (5) for DCS, *P < 0.05, Student’s

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(C) Early D-cycloserine treatment during P7–21 in WT mice induces a decrease in mEPSC frequency but no effect on mEPSC amplitude at the juvenile stage (~P23–25). Young WT mice were orally administered D-cycloserine (40 mg/kg) twice daily for 2 weeks during P7–21 followed by measurements of mEPSCs in CA1 pyramidal neurons at ~P23–35. (n = 15 neurons from 3 mice for control, 14 (3) for DCS, *P < 0.05, ns, not significant, Mann Whitney U test).

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Figure S20. Late chronic memantine treatment does not improve hyperactivity in Shank2–/– mice.

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(A–C) Late chronic memantine treatment fails to improve hyperactivity of adult Shank2–/– mice in the open-field test. (n = 14 mice for WT-V, 11 for WT-M, 8 for KO-V,

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for (B).

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and 11 for KO-M, ***P < 0.001, two-way ANOVA for (C)). ANOVA was not performed

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Figure S21. Late acute memantine treatment does not improve social interaction or hyperactivity in Shank2–/– mice. (A) Schematic depiction of late acute memantine treatment. Shank2–/– mice were administered memantine acutely (10 mg/kg; intraperitoneal) at a later stage (~P56), followed by measurements of three-chamber social interaction and open-field hyperactivity 30 min after injection. (B and C) Late acute memantine treatment fails to improve social interaction in

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Shank2–/– mice in the three-chamber social interaction test, as indicated by time sniffing S1/O and the social preference index based on sniffing time. (n = 11 mice for WT-V, 10 for WT-M, 15 for KO-V, and 15 for KO-M, **P < 0.01, ***P < 0.001, ns, not significant, paired Student’s t-test or Wilcoxon matched pairs signed rank test for (B),

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and unpaired Student’s t-test or Mann Whitney U test for (C; two-way ANOVA was not performed because the social preference index is a normalized value)).

(D and E) Late acute memantine treatment fails to improve hyperactivity of adult

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Shank2–/– mice in the open-field test. (n = 7 mice (WT-V), 9 (WT-M), 9 (KO-V), and 8

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(KO-M), ***P < 0.001, two-way ANOVA for (D)). ANOVA was not performed for (E).

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Figure S22. Shank2–/– mice lacking exon 24 show suppressed NMDAR function at ~P21 but normal NMDAR function at ~P14.

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(A and B) Example traces of evoked NMDAR-EPSCs (at +40 mV) and AMPAR-EPSCs (at -70 mV) at Shank2–/– hippocampal SC-CA1 synapses at P14–15 (A) and P21–22 (B). Represented traces were obtained from WT (black) and Shank2–/– (red) mice.

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Both NMDAR- and AMPAR-mediated currents were elicited by 0.25 ms duration and 300 mA stimulation intensity in the same cell. Evoked AMPAR-EPSC recordings were

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carried out in the presence of the GABAA receptor antagonist picrotoxin (60 mM) at the holding potential of -70 mV; then NMDAR-EPSC recordings were followed by DNQX (20 mM) treatment and with the same stimulation pulse at the holding potential of +40 mV. (C) NMDA/AMPA ratio at WT and Shank2–/– SC-CA1 synapses at P14–15 and P21– 22. (P14–15, n = 14 neurons from 3 mice for WT and 15 (3) for KO; P21–22, n = 14 (3) for WT and 15 (3) for KO; *P < 0.05 [p = 0.0204], ns, not significant [p = 0.2643],

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unpaired Student t-test). (D) NMDAR-mediated EPSCs at WT and Shank2–/– SC-CA1 synapses at P14–15 and P21–22. (P14–15, n = 14 neurons from 3 mice for WT and 15 (3) for KO; P21–22, n = 14 (3) for WT and 15 (3) for KO; *P < 0.05 [p = 0. 0146], ns, not significant [p = 0.1265],

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unpaired Student t-test).

(E) AMPAR-mediated EPSCs at WT and Shank2–/– SC-CA1 synapses at P14–15 and

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P21–22. (P14–15, n = 14 cells from 3 mice for WT and 15 (3) for KO; P21–22, n = 14 (3) for WT and 15 (3) for KO; ns, not significant [p = 0.5100 for 14–15 and 0.4932 for

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P21–22], unpaired Student t-test).

(F) Decay kinetics of NMDAR-mediated EPSCs at WT and Shank2–/– SC-CA1 synapses at P14–15 and P21–22. (P14–15, n = 14 neurons from 3 mice for WT and 15 (3) for KO; P21–22, n = 14 (3) for WT and 15 (3) for KO; ns, not significant [p =

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0.4640 for 14–15 and 0.6517 for P21–22], unpaired Student t-test).

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Figure S23. Shank2–/– mice lacking exon 7 show enhanced NMDAR function at

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~P24 but normal NMDAR function at ~P14. (A) Normal NMDA/AMPA ratio at Shank2–/– SC-CA1 synapses at P13–15. (n = 9 neurons from 3 mice for WT and 8 neurons from 3 mice for KO, ns, not significant, Student t-test).

(B) Increased NMDA/AMPA ratio at Shank2–/– SC-CA1 synapses at P21–28. (n = 7 neurons from 3 mice for WT and 8 neurons from 3 mice for KO, **P < 0.01, Student ttest).

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(C) Normal mEPSCs in Shank2–/– CA1 pyramidal neurons at P21–28. (n = 14 neurons

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from 4 mice for WT and 10 (4) for KO; *P < 0.05, ns, not significant, Student t-test).

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Figure S24. Similarly reduced amplitudes of NMDAR-mEPSCs in two Shank2–/– mouse lines in different genetic backgrounds (C57BL/6N and C57BL/6J). (A and B) Reduced amplitude but normal frequency of NMDAR-mediated mEPSCs in Shank2–/– CA1 pyramidal neurons from mice in two different genetic backgrounds (C57BL/6N and C57BL/6J) at P21–27. We attempted the new genetic background (C57BL/6J) because the two backgrounds have previously been shown to affect various mouse phenotypes, including synaptic properties, caused by a point mutation

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in the Cyfip2 gene

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. (C57BL/6N, n = 25 neurons from 4 mice for WT and 20 (4) for

KO; C57BL/6J, n = 15 (3) for WT and 14 (5) for KO; *P < 0.05, ns, not significant, Mann-Whitney U test for (6N-frequency and 6J-amplitude) and Student t-test for (6N-

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amplitude and 6J-frequency)).

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Figure S25. Schematic depiction of differential splice variants of Shank2 in

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Shank2–/– mice lacking exon 6–7 and exon 7. (A) Schematic depiction of the structure of the Shank2 gene and its two splice variants

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(short Shank2a and long Shank2b). Note that the short Shank2a splice variant is generated by alternative transcriptional initiation starting from a less known Shank2aspecific exon (termed here exon 5’) located between exons 5 and 6 in the Shank2 gene. The target locations of the primers used for RT-qPCR reactions are indicated. (B) Schematic of the Shank2b and Shank2a splice variants in Shank2–/– mice lacking exon 7 (termed Shank2-dEx7 mice). Note that near full-length Shank2b and Shank2a splice variants lacking the exon 7 region are produced by transcription, which are decayed by ~75% and markedly increased in amounts, respectively (see the specific

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RT-qPCR results in Figure S26). Note that the exon 7 deletion may lead to a premature protein truncation in both Shank2a and Shank2b proteins, and a marked increase in the levels of a short peptide (57 aa-long) derived from Shank2a. (C) Schematic of the Shank2b and Shank2a splice variants in Shank2–/– mice lacking

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exons 6–7 (termed Shank2-dEx6–7 mice). Note that a near full-length Shank2b splice variant lacking exons 6–7 is produced, similar to the case of exon 7 deletion, whereas the Shank2a splice variant is not produced at all because the exon 5’ (the first exon in

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the Shank2a transcript) is deleted together with exons 6 and 7 during homologous

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recombination by the gene-targeting construct (see the RT-qPCR results below).

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Figure S26. RT-qPCR results supporting differential splice variants of Shank2 in

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Shank2–/– mice lacking exon 6–7 and exon 7.

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(A) Examples of the results of RT-qPCR performed using hippocampal samples from Shank2-dEx7 and Shank2-dEx6–7 mice (P25–28) and primers 3 + 4 and 9 + 10. Note that the levels of RT-qPCR products amplified using primers 3 + 4 are decreased (~25% of WT levels) in Shank2-dEx7 mice but almost completely disappeared in Shank2-dEx6–7 mice (see also the quantification of the results in panel B), indicative of the decay of the Shank2b variant in Shank2-dEx7 mice and support the lack of exon 6 in the Shank2b variant in Shank2-dEx6–7 mice. In contrast, RT-qPCR products from primers 9 + 10 are strongly increased in Shank2-dEx7 mice (~6-fold; see also the

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quantification in panel B), whereas they are markedly decreased in Shank2-dEx6–7 mice caused by the loss of the first exon (exon 5’), suggesting that there is a marked difference in the levels of the Shank2a splice variant in the two mouse lines. (B and C) Quantification (B) and summary (C) of the results of RT-qPCRs performed

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using hippocampal samples from Shank2-dEx7 and Shank2-dEx6–7 mice (P25–28) and various combinations of primers indicated in Figure S25. The results of RTqPCRs using additional primer combinations can be interpreted in the following ways:

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the results with primers 3 + 4 indicate the decay of the Shank2b variant in Shank2-

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dEx7 mice and support the lack of exon 6 in the Shank2b variant in Shank2-dEx6–7 mice; the results with primers 5 + 6, or 7 + 8, indicate the decay of the Shank2b variant as well as a large abnormal increase of the Shank2a variant in Shank2-dEx7 mice, as mentioned above, and support the decay of the Shank2b variant in Shank2-dEx6–7 mice. (n = 4 mice for WT and KO, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant,

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nd, not detected, Student t-test).

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Figure S27. Molecular modeling of the Shank2 PDZ domain in complex with the C-terminal tail of SAPAP3 suggests that the short truncated peptide derived from the Shank2a splice variant in Shank2-dEx7 mice may compete with the fulllength Shank2 PDZ domain for SAPAP3 binding. (A and B) Molecular modeling of the mouse Shank2 PDZ domain in complex with the C-terminal PDZ-binding motif of SAPAP3 (A) and an alignment of the amino acid (aa) sequences of mouse Shank3 and Shank2 sequences in the PDZ domains and the aa 53

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stretches in the immediate upstream regions (termed N-PDZ or N-terminal extended PDZ) (B), which (those of Shank3) have been shown to form a tight complex with the extended C-terminal PDZ-binding motif (E-PBM) of SAPAP3

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. Note that the E-PBM

of SAPAP (blue) binds to three prominent sub-structures in the N-PDZ domain of

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Shank2 (pink), including the bN1 and bN2 strands in the N-terminal extended region, the BC loop located between bB and bC, and the PBM-binding pocket formed by bA*, bB*, and a2*. Note also that the short truncated peptide of Shank2a mimicking the N-

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terminal part of the N-PDZ domain of the mouse Shank2 might compete with the full-

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length N-PDZ domain of Shank2 for SAPAP3-PBM binding. Asterisks denote second molecules in the domain swapped dimer model. In the sequence alignment of Shank2/3 PDZ domains, identical and similar residues indicated by red filled boxes and red letters in open boxes, respectively. The secondary structures corresponding to a-helix, b-sheet, and b turns are indicated by squiggles, black arrows, and TT,

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respectively. The residues in the PDZ domain involved in the formation and binding for the C-terminal carboxylate of SAPAP3 (X1-F1-G-F2, X: any amino acids; F:

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hydrophobic residues; G: Gly) and the second hydrophobic pocket in the PDZ domain are indicated by green and red triangles, respectively. The magenta and green solid

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lines above the aa sequences indicates the short truncated peptide derived from Shank2a in Shank2-dEx7 mice and the full-length Shank2a PDZ domain, respectively. The dotted lines indicate unmodeled regions due to the lack of sufficient similarity for homology modeling. The indicated secondary structures and aa sequence alignment were acquired using the web-based software ESPript version 3.0 19.

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Supplementary Tables (see separate Excel files) Table S1. All RNA-Seq data from WT and Shank2–/– mice at P14 and P25. Table S2. (A) DEGs from naïve P14 KO (Shank2–/–) and WT mice. (B) Summary of normalized read counts of DEGs for P14. (C) DEGs from naïve P25 KO and WT

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mice. (D) Summary of normalized read counts of DEGs for P25. (E) Summary of RNA-Seq mapping results from naïve P14 and P25 Shank2–/– and WT mice.

Table S3. GSEA using KO/WT query gene lists at P14 and P25 and C2 target gene

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Table S4. GSEA using KO/WT query gene lists at P14 and P25 and C5 target gene

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Table S5. Summary of electrophysiological results from Shank2–/– mice lacking exon 6–7.

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Table S6. Statistical results.

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