SHANK genes in autism: Defining therapeutic targets

Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Progress in Neuropsychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp

SHANK genes in autism: Defining therapeutic targets Adele Mossa, Federica Giona, Jessica Pagano, Carlo Sala, Chiara Verpelli

⁎

CNR Neuroscience Institute, Department of Biotechnology and Translational Medicine, University of Milan, Milan, Italy

A B S T R A C T The term “Shankopathies” identifies neurodevelopmental diseases, such as autism Spectrum Disorders (ASD), Intellectual Disability (ID), and schizophrenia (SCZ) caused by deletion or mutations of SHANK/ProSAP genes. The three SHANK genes code for a postsynaptic scaffold protein which has a main function of regulating synaptic formation, development and plasticity. The review summarizes the major genetic, molecular and electrophysiological studies that provide new information about the function of Shanks proteins and are prodromic in identifying therapeutic approaches, pharmacological targets for treating patients with SHANK deletions and mutations and eventually for other patients affected by neuropsychiatric and neurodevelopmental disorders.

1. Introduction The Shank/ProSAP (SH3-Ankirin and Prolin rich Synaptic Associated Protein) proteins are master scaffold proteins located in the postsynaptic density (PSD) of glutamatergic synapses end, encoded by the three genes SHANK1, SHANK2 and SHANK3. Much like many other synaptic genes, mutations in SHANK genes are strongly associated with Autism Spectrum Disorders (ASD) and syndromic forms of Intellectual Disability (ID) (Verpelli and Sala, 2012; Guilmatre et al., 2014). The first pathology identified in patients affected by SHANK gene mutations was the 22q13 deletion syndrome, now known as Phelan McDermid syndrome (PMS), a form of intellectual disability caused by Shank3 haploinsufficiency and often associated with ASD (Bonaglia et al., 2001; Phelan et al., 2001). In these recent years, further extensive analyses of patients with ASD have shown a significant number of mutations not only in SHANK3, but also in SHANK1 and SHANK2 genes (Durand et al., 2007; Leblond et al., 2014), which is a clear indication that Shank proteins are involved in regulating a common molecular pathway associated with ASD (Jiang and Ehlers, 2013). Even if the specific roles of the various Shank proteins in the pathogenesis of ASD and ID are still unclear, several in vitro and in vivo models have been developed to better dissect the molecular function of Shank proteins and to help to understand how their deletion or mutations play a role in the pathogenesis of ASD and ID. This review aims to summarize the most significant recent generated data that is helping to understand the function of Shank proteins and the possible new therapeutic targets and approaches that can

⁎

potentially be used to treat patients with ASD and ID caused by SHANK gene mutations or deletions. 2. Shank proteins: structure and function Shank proteins are master scaffold located in the postsynaptic density (PSD) of the glutamatergic synapses involved in different synaptic functions, such as spine morphogenesis, synapse formation, glutamate receptor trafficking and activity-dependent neuronal signaling (Boeckers et al., 1999a, 2002; Naisbitt et al., 1999; Tu et al., 1999; Sala et al., 2001, 2005; Gerrow et al., 2006; Verpelli et al., 2011). The function of Shank was first linked to dendritic spine formation because the overexpression of either Shank1 or Shank3 in neurons induce the formation and maturation of dendritic spines (Sala et al., 2001; Roussignol et al., 2005). Shank proteins are structurally composed of five conserved proteinprotein interaction motifs, from the N-terminal to the C-treminal: 5–6 ankyrin repeated domains (ANK), an Src Homology 3 (SH3), a PDZ domain, a proline-rich region (Pro) and a sterile alpha motif (SAM) domain. Using these domains, Shank proteins interact with several synaptic proteins including other scaffold molecules, glutamatergic receptors, signaling and cytoskeletal proteins (Ehlers, 1999; Sheng and Kim, 2000; Baron et al., 2006; Gundelfinger et al., 2006; Sala et al., 2015). From the several interactions, Shank proteins are able to indirectly bind both ionotropic and metabotropic synapse glutamate receptors; indeed NMDA-type glutamate receptors are linked to the Shank PDZ domain by GKAP/SAPAPs proteins which bind PSD-95-NMDAR complex (Naisbitt et al., 1999; Tu et al., 1999); metabotropic glutamate

Corresponding author at: CNR Neuroscience Institute, Via Vanvitelli 32, 20129 Milano, Italy. E-mail address: [email protected] (C. Verpelli).

https://doi.org/10.1016/j.pnpbp.2017.11.019 Received 5 September 2017; Received in revised form 14 November 2017; Accepted 18 November 2017 0278-5846/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Mossa, A., Progress in Neuropsychopharmacology & Biological Psychiatry (2017), https://doi.org/10.1016/j.pnpbp.2017.11.019

Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxx–xxx

A. Mossa et al.

different amount and ratio of Shank3 proteins splice variants that differentially regulate their function and morphology (Bockers et al., 2004; Falley et al., 2009; Wang et al., 2014).

receptor (mGluR) and inositol triphosphate receptor (IP3R) are linked to Shank protein complex by Homer proteins that bind to the prolinerich region (Tu et al., 1999; Sala et al., 2005). More indirectly AMPAtype glutamate receptors are linked to the Shank complex via the interaction of PSD-95 with Stargazin, a transmembrane AMPA receptor associated protein, but in the cerebellum, Shank PDZ domain directly interact with of the C-terminus of the glutamate receptor δ2 subunit (GluRδ2) (Uemura et al., 2004; Yasumura et al., 2008). It was well demonstrated that the SAM domains are involved in Shank multimerization as the SAM domains of Shank3 form regular stacks of helical fibers with the essential requirement ofZn2 + binding (Baron et al., 2006; Grabrucker et al., 2011). The binding site for Zn2 + is also present in the SAM Shank2 domain but not in Shank1. Interestingly, while the Shank1 localization to synapses is mediated by the PDZ domain binding to GKAP/SAPAP (Sala et al., 2001; Romorini et al., 2004), for both Shank2 and Shank3 the C-terminal domain, including the SAM domains, are required (Boeckers et al., 2005). Some Shank3 isoforms (see below) can also translocate into the nucleus in an activity dependent manner and control the transcription of a number of genes, including synaptic genes, through the modulation of hnRNP complex, however the function of these Shank3 protein nuclear complex remains to be clarified (Grabrucker et al., 2014). Interestingly the N-terminal domain of Shank1 and Shank3 are able to sequester active Rap1 and R-Ras and inhibit integrin receptor activation. This new role for Shank proteins has been well demonstrated in cell lines and in cultured hippocampal neurons (Lilja et al., 2017). The three SHANK genes can codify for several mRNA splice variants that generate multiple protein isoforms (Jiang and Ehlers, 2013). Most of these mRNAs are widely expressed in the mammalian brain by a generalized location for some, and area specific location for others (Wang et al., 2014). The mRNA for Shank1 has been found expressed at a high level in hippocampus, cortex, amygdala and cerebellar Purkinje cells (Zitzer et al., 1999; Bockers et al., 2004). Shank1 is also the only member of the family to be expressed in the hypothalamus, while it is almost absent in striatum. Interestingly Shank mRNAs are also present in the neuropil layer of the CA1 region of hippocampus with the highest dendritic expression of Shank1 mRNA, followed by Shank3 mRNA and a weaker level of Shank2 mRNA (Epstein et al., 2014) suggesting that local translation is an essential source of Shank protein at the synapse that might regulate activity-dependent plasticity. Regarding Shank1, a non-canonical initiation site essential for efficient translation dendritically localized mRNA has recently been described (Studtmann et al., 2014). While Shank1 is exclusively expressed in the brain, Shank2 is also expressed in the kidneys and the liver (Du et al., 1998; Lim et al., 1999); particularly copious in the hippocampus, striatum, cortex, the olfactory bulb and in Purkinje cells of the cerebellum. Shank3 mRNA expression in the brain overlaps with Shank2 mRNA distribution, except for the cerebellum where Shank3 expression is instead localized to granule cells (Boeckers et al., 1999b). Shank3 mRNA is also highly expressed in the heart and spleen (Lim et al., 1999). The SHANK3 gene is the most complex of the family because it contains six alternative promoters that can code for several mRNA splice variants (Durand et al., 2007; Wang et al., 2014). Moreover, the gene contains at least five CpG islands (CGIs) that display a brain region-specific DNA methylation pattern causing tissue-specific expression of different Shank3 isoforms (Ching et al., 2005; Beri et al., 2007; Maunakea et al., 2010). All of these different isoforms appear to be expressed in a neuronal type, developmental and activity dependent manner and might have different synaptic functions as some differentially regulate dendritic spine morphology (Wang et al., 2014). Thus, mRNAs and relative protein expression could be different among different brain areas and during different brain and synapse development. This leads us to speculate that each glutamatergic synapse might have a

3. SHANK genes and ASD Autism spectrum disorders (ASD) are a complex neurodevelopmental disorder defined by alteration in social communication and repetitive behavior. Chromosomal rearrangements, copy number variations and coding sequence variants, involving more than 100 genes, have been found in patients with ASD (Betancur, 2011). Many mutations have been detected in gene encoding for synaptic proteins and in particular, deletions, duplications and mutations in the three SHANK genes have been described in several patients with ASD (Berkel et al., 2010; Pinto et al., 2010; Jiang and Ehlers, 2013; Sala et al., 2015). Analyzing a large number of individuals with ASD, Leblond et al. found that mutations or disruptions in the SHANK gene family account for ~1% of all patients with ASD (Leblond et al., 2014). Furthermore, SHANK3 gene is the main gene associated with neuropsychiatric symptoms of patients with Phelan McDermid syndrome (PMS) (Bonaglia et al., 2001; Phelan et al., 2001). PMS is a complex neurodevelopmental disorder characterized by a significant expressive language delay, intellectual disability, hypotonia, minor craniofacial dysmorphisms, increased tolerance to pain and epilepsy (Phelan and McDermid, 2012). More than 80% of the diagnosed patients showed autism or autistic-like behavior (Betancur and Buxbaum, 2013). Deletions, ring chromosomes, interstitial deletions and unbalanced translocations, all causing Shank3 haploinsufficiency, were described in patients with PMS (Bonaglia et al., 2011). Typical phenotypic variability of the PMS was also identified in patients carrying SHANK3 de novo mutations, interstitial or terminal deletion (Durand et al., 2007; Boccuto et al., 2013; Leblond et al., 2014). Others studies propose that not only SHANK3 mutations, but also SHANK3 duplications are associated with ASD (Durand et al., 2007; Moessner et al., 2007) suggesting that SHANK3 gene dosage is essential for correct brain function. Another study showed an increased level of SHANK3 gene methylation in a cohort of patients with ASD underlying that also epigenetic dysregulation of SHANK3 gene may be associated to the pathogenesis of ASD (Zhu et al., 2014). Together these findings suggest that SHANK mutations, causing defects in synapses maturation and function, are strongly involved in the etiology of ASD. Importantly, there is also a correlation between the degree of cognitive impairment and the different Shanks mutations; patients with SHANK3 mutations are more severely affected than patients with SHANK2 and SHANK1 mutations (Leblond et al., 2014).Although more studies are needed to clarify the molecular basis of these differences, one explanation might be that each SHANK gene has a specific and temporal distribution that cannot be completely compensated by the two other genes. 4. ModelingShank mutations in transgenic mice Several Shank mutant mice have been generated to clarify the specific role of Shank proteins in brain and synapse development and function. 4.1. Shank1 The Shank1 mutant mouse was obtained by deletion of exons 14 and 15 of Shank1 leading to the loss of all Shank1 splice variant (Hung et al., 2008). Molecularly, Shank1 KO mice displayed a reduced expression ofGKAP/SAPAP and Homer, specifically in PSD fractions; there were fewer and smaller spines, and the thickness of the PSD in CA1 synapses was reduced in Shank1 KO mice. A decrease in basal synaptic transmission was observed in Shank1 KO mice while long-term potentiation (LTP) and long-term depression (LTD) in CA1 hippocampus were 2

Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxx–xxx

A. Mossa et al.

(Won et al., 2012), while reduced spine density and smaller, thinner PSD were reported in Shank2 (7) (Schmeisser et al., 2012). The strongest differences between the studies are in electrophysiology. Synaptic basal transmission was decreased only in Shank2 (7) mutant mice (Schmeisser et al., 2012) and decreased NMDAR-mediated synaptic function was described in Shank2 (6–7) (Won et al., 2012), while in Shank2 (7) NMDAR-mediated synaptic function increased (Schmeisser et al., 2012). Similar autistic-like behavioral traits, including excessive grooming, hyperactivity, reduced digging, altered ultrasonic vocalization and impaired social interaction, were observed in both models suggesting that the alterations in NMDA function are critical in inducing ASD behavior. Nevertheless, a study showed that the two Shank2 deletion mutants, Shank2 (6–7) and Shank2 (7), are different at the transcriptome level. In particular, GABAergic neurotransmission was impaired in Shank2 (6–7) KO mice but not in Shank2 (7) KO mice (Lim et al., 2017). Although further analysis is needed to better clarify these anomalies between the two mutant mice, the study demonstrates its importance as it suggests a new role of Shank2 in GABAergic transmission. Two recent studies showed that Shank2 is essential in regulating cerebellar excitatory synapse density and functions suggesting that absence of Shank2 in Purkinje cells is responsible for altered motor learning and coordination, as well as repetitive and anxiety-like behaviors (Ha et al., 2016; Peter et al., 2016).

Table 1 List of treatments and rescued (yes) or not rescued (no) behavioral and functional alterations in the Shank2 (6–7) KO mice. Shank2 (6–7) Not treated Hyperactivity Social interaction Social novelty Repeated jumping Anxiety-like behaviors Pup retrieval Morris WM Elevated zero-maze NMDA/AMPA ratio LTP LTD GABA synapses I/E balance

High Impaired Normal Present Present Impaired Impaired Impaired Reduces Reduced

CDPPB

D-serine

Yes No No No No

Yes No No No No

L838,417

Clioquinol

No No

Yes

No No Yes No

Yes Yes Yes

Yes Yes

Yes Yes

Reduced Reduced

Yes

unaffected. Behaviorally, Shank1 KO mice showed a reduction in sniffing during male-female interaction and a reduction in the interaction with novel mice compared to wild-type littermates (Silverman et al., 2011; Wöhr et al., 2011), as well as increased self-grooming behavior (Sungur et al., 2014). Consistent with an ASD-associated behavioral phenotype, male Shank1 KO mice performed less calls in malefemale interactions (Silverman et al., 2011; Wöhr et al., 2011; Wöhr, 2014). Curiously, Shank1 KO mice exhibited an enhanced acquisition of spatial memory despite having an impaired contextual fear memory (Hung et al., 2008) and a severe impairment in object recognition memory (Sungur et al., 2017) suggesting that Shank1 plays an essential role in the retention of memory.

4.3. Shank3 Given the complexity of the Shank3 gene, which is transcribed from six intragenic promoters, several Shank3 mutant lines were generated. Three independent models were generated targeting the exons 4–7 (Peça et al., 2011), exons 4–9 (Bozdagi et al., 2010; Wang et al., 2011) and the exon 9 (Lee et al., 2015b), all encoding for the ankyrin repeat domain. In one model, the exon 11, encoding the SH3 domain, was deleted (Schmeisser et al., 2012) while in two other models the exons 13–16 (Peça et al., 2011) or the exon 13 (Jaramillo et al., 2017) encoding the PDZ domain were disrupted. Two independent mutant lines were obtained targeting the exon 21 encoding the Proline-rich domain (Kouser et al., 2013; Bidinosti et al., 2016). In all these models some Shank3 isoforms were still present and only recently Wang et al. developed the Shank3 complete KO by the deletion of the protein-coding exons 4–22 (Wang et al., 2016). The complete knockout model displays

4.2. Shank2 Two independent mutant lines targeting exons 6 and 7 (Won et al., 2012) and exon 7 (Schmeisser et al., 2012), respectively, were generated to knock down Shank2; both models lack all Shank2 isoforms. Both studies showed similar protein, synaptic development and synaptic plasticity as well as some differences between the two Shank2 mutant mice (Table 1). In CA1 pyramidal neurons, no changes in spine density, length and thickness of PSD were described in Shank2 (6–7)

Table 2 List of treatments and rescued (yes) or not rescued (no) behavioral and functional alterations in the Shank3 (4–9), Shank3 (11), Shank3 (21) and Shank3 (4–22) KO mice. Shank3 (4–9) Not treated

Self-grooming Activity in open field Social interaction Social novelty Avoidance behavior/marble burying Morris WM Morris WM reverse Rotarod test Instrumental learning NMDA/AMPA ratio NMDAR-EPSC LTP LTD AMPA currents/mEPSC mGlu5 dependent NMDAR potentiation mGlu5 dependent Ca2 + intracellular release Homer1b/c synaptic localization Synaptic GluN1 and GluN2A

Shank3 (11) IGF1

Shank3 (21)

Shank3 (4–22)

Not treated

CDPPB

Not treated

TAT-p-cofilin peptide

CLK2 inhibitor TG003

Not treated

CDPPB

MPEP

High

Yes

High

Yes

Yes

High Low

No No

Yes Yes

Impaired Impaired

Yes Yes

Impaired

Yes

Yes

Impaired

Yes

Reduced Reduced

Yes Yes

Reduced

Yes

Reduced Normal Impaired Impaired

No

Yes

Yes Reduced Reduced

Reduced

Yes

Reduced

Yes Impaired Impaired

Yes Yes

Reduced

Yes

Reduced

Yes

Yes Yes

3

Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxx–xxx

A. Mossa et al.

lines by inserting a single guanine nucleotide (G) at cDNA position 3680 (InsG3680 mutation) in the first and changing an arginine 1117 to a stop codon (R1117X mutation) in the second. The two mutant lines showed both specific and shared molecular, functional, synaptic and behavioral abnormalities (Zhou et al., 2016) suggesting that mutation in the same gene may lead to mutation-specific behaviors. As a duplication of the SHANK3 gene was found in patients diagnosed with neuropsychiatric conditions, a Shank3 overexpression model was generated in which the expression of Shank3 was increased to 50% more than the wild type (Han et al., 2013). These animals exhibited synaptic dysfunction and manic-like behavior, but no increase in repetitive behavior that seemed specifically due to Shank3 absence.

Table 3 List of treatments and rescued (yes) or not rescued (no) behavioral and functional alterations in the rat Shank3 KO and hiPSCs-derived neurons from PMS patients.

Long-term social recognition memory Contextual fear conditioning Attentionally demanding task Spontaneous EPSCs Amplitude of evoked AMPA- and NMDA-EPSCs LTP mGlu LTD

Rat Shank3 KO

hiPSCs-derived neurons from PMS patients

Not treated

Oxitocin

Not treated

IGF1

SC79 or TG003

Impaired

Yes

Reduced

Yes

Yes

Reduced

Yes

Yes

Reduced Reduced

Yes Yes

Yes Yes

Normal Impaired

Impaired Impaired

Yes

5. Modeling SHANK mutations with human induced pluripotent stem cells (HiPSCs) A critical point in studying neurological diseases is the generation of valid in vivo and in vitro models. Although genetically modified mice are a strong model for studying the pathophysiology of ASD and to clarify the specific role played by mutated proteins in different brain areas, there are considerable evolutionary differences between the human and mouse physiology that encumber the evaluation of the extent to which mouse models can fully represent human disease pathophysiology. HiPSCs can be obtained from human fibroblasts or other somatic cells by the overexpression of specific genes (Takahashi and Yamanaka, 2006; Farra et al., 2012); these cells can be further differentiated into neurons and glial cells (Marchetto et al., 2010). The use of neuron-derived hiPSCs provides the opportunity to test cellular and molecular phenotypes in human neurons and to correlate them with phenotypes found in mice models. Moreover, considering the large number of SHANK variants detected in ASD and/or ID patients so far, and the heterogeneity in terms of deletion size found in PMS patients, hiPSCs promise major advances in the study of specific SHANK mutations (Table 3). iPSC-derived neurons from MS patients showed reduced expression of Shank3 and impaired excitatory synaptic transmission (Shcheglovitov et al., 2013; Bidinosti et al., 2016). Staining of synaptic proteins revealed a decrease in both pre- and post-synaptic puncta, suggesting that the neurons had fewer synapses (Shcheglovitov et al., 2013). Moreover, a second study described a decrease in Homer1b/c puncta in PMS patient differentiated cortical neurons confirming the data obtained analyzing Shank3 KO mice (Vicidomini et al., 2017). Another study, using a proteomic approach, showed impaired Akt activity in PMS iPSC-derived patient neurons both in basal condition and after BDNF stimulation (Bidinosti et al., 2016) To specifically study the role of Shank3 in human neuron differentiation and function Yi et al. produced heterozygous and homozygous human neurons lacking SHANK3, but no other genes that are often deleted in PMS patients. SHANK3 mutated neurons showed impaired hyperpolarization-activated cation (Ih) channels that causes alterations in neuronal morphology and synaptic connectivity (Yi et al., 2016). Darville et al. used neurons differentiated from iPSCs derived from ASD individuals heterozygous for SHANK3 null mutations to screen more than 200 pharmacological agents. Using a qPCR-based high throughput screening (HTS) method they found increased expression of Shank3 mRNA in neurons treated with lithium and valproic acid (Darville et al., 2016). Lithium was subsequently administered to one of the ASD patients whose cells were used in the screening study and, after one year of treatment, a decrease in autistic behavior was observed. Although a randomized double blind clinical trial against placebo is needed in order to validate the effect of litium in PMS patients, this study showed the power of using neuronal cells derived from hiPSCs for high-throughput drug screening.

Yes Yes

severe impairments in behavioral and synaptic traits; notably the phenotypic differences in most of the other Shank3 mutant mice were not dramatic (Table 2). Despite the fact that the paradigms used to characterize the Shank3 mutant lines were completely different, all the mutants displayed autistic-like behaviors including increased repetitive behaviors, reduced social interaction and motor coordination deficits. Learning and memory were different among individual lines with the Shank3 mutant supporting the hypothesis that different Shank3 isoforms play different roles in brain functions. On the molecular level, a major overlapping phenotype of various Shank3 mutants is the reduction of AMPA receptor subunits (Bozdagi et al., 2010; Peça et al., 2011; Wang et al., 2011; Schmeisser et al., 2012; Kouser et al., 2013) and of Homer1b/c at synapses (Peça et al., 2011; Wang et al., 2011, 2016; Vicidomini et al., 2017). The high level of Shank3 expression in striatum might explain the evidence suggesting that striatal circuitries are dysfunctional in absence of Shank3 (Peça et al., 2011; Wang et al., 2016; Vicidomini et al., 2017). Wang et al. recently showed that several striatal synaptic functions are selectively impaired in the striatopallidal D2-type MSNs in a line of Shank3 KO mice (Wang et al., 2017). The data is corroborated by a study revealing that a loss of Shank3 caused a developmental circuit defect leading to abnormal corticostriatal maturation (Peixoto et al., 2016). Homer1b/c expression strongly decreased in striatum of Shank3 KO mice leading to the disruption of mGlu5-Homer complex that is essential for mediating mGlu5 intracellular signaling after glutamate activation (Mei et al., 2016; Wang et al., 2016; Vicidomini et al., 2017). Morphologically striatal neurons presented a decreased spine density and length and PSD thickness (Collingridge et al., 2010; Peça et al., 2011; Mei et al., 2016; Wang et al., 2016). Notably, restoring Shank3 expression in adult mice helped to rescue Homer synaptic levels and increase dendritic spines in the dorsal striatum leading to a recovery of the repetitive grooming behavior and social deficit (Mei et al., 2016). Interestingly, the maniac-like behavior found in the Shank3 overexpressing mice (Han et al., 2013) is associated with abnormal striatal mTORC1 activity strengthening the important role played by Shank3 in mediating striatal function (Lee et al., 2017). Beside the deletions of SHANK3, which are clearly associated with neurodevelopmental disorders (Wilson et al., 2003), a variety of mutations in the SHANK3 gene have been reported in patients with ASD (Durand et al., 2007; Moessner et al., 2007; Gauthier et al., 2009, 2010). Thus, to clarify how single mutations specifically affect synaptic development and function is essential to characterize Shank3 mutant mice with single mutations. Zhou et al. generated two mutant mice 4

Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxx–xxx

A. Mossa et al.

Fig. 1. The cartoon shows a summary of possible therapeutic intervention for rescuing synaptic and behavioral defects induced by Shank2 and Shank3 mutation/deletion. (A) For Shank2 enhancing NMDAR function by direct NMDAR activation with D-cycloserine or indirect NMDAR stimulation with the mGlu5 positive allosteric modulator CDPPB or clioquinol, a Zn chelator and ionophore, improved social interaction (Won et al., 2012; Lee et al., 2015a). Spatial memory deficits, but not social deficits, can be also rescued in adult Shank2 (6–7) KO mice, with the GABAA receptor alpha 2 positive allosteric modulator L438,417 (Lim et al., 2017) (not shown). (B) For Shank3 different approaches have been used to reverse the phenotype. mGlu5 positive agonist CDPPB ameliorates ASD-like behavior in the Shank3(11) KO mice (Vicidomini et al., 2017) and partially rescued instrumental learning in Shank3Δe4–22−/− mice (Wang et al., 2016). In another mouse model, CLK2 inhibitor was able to decrease self-grooming and recover normal preference for social interaction in the Shank3(21) KO, in which CLK2 expression was increased (Bidinosti et al., 2016). Using a genetic approach, Mei et al. showed that restoration of Shank3 expression in adult mice was able to rescue social interaction deficit and repetitive grooming behavior (Mei et al., 2016). Notably all these studies rescued behavioral phenotypes in adult rodents, suggesting the possibility of treatment for patients with SHANK mutations or deletion during adulthood.Two clinical studies showed that intraperitoneal administration of Insulin Grown Factor-1 (IGF-1) or intranasal insulin administration were associated with improvement of social impairment and restrictive behavior (Kolevzon et al., 2014; Zwanenburg et al., 2016).

6. Therapeutic approaches

activation with D-cycloserine and indirect NMDAR stimulation with the mGlu5 positive allosteric modulator CDPPB improved social interaction, but failed to rescue impaired pup retrieval, jumping, anxiety-like behaviors, and hyperactivity in the Shank2 (6–7) mutant mice (Won et al., 2012). Another study rescued social deficits treating Shank2 (6–7) KO mice with clioquinol that indirectly potentiate NMDA function by mobilizing zinc (Lee et al., 2015a). The results of both these studies suggest that reduced NMDAR function may selectively underlie the social deficit in the Shank2 (6–7) KO mice. As an imbalance of I/E ratio was suggested as one of the

The availability of several Shank mutant mice and the generation of patient specific iPSCs contributed to a better understanding of the role of the Shank proteins in synaptic functions, opening the way to identify molecular pathways that can provide novel therapeutic targets to treat patients with SHANK mutations (Fig. 1). For SHANK2 mutations, interventional studies have only been performed in Shank2 (6–7) mutant mice (Won et al., 2012; Lim et al., 2017) (Table 1). Enhancing NMDAR function by direct NMDAR 5

Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxx–xxx

A. Mossa et al.

also on different patho-mechanims causing the disease. For this reason, a better identification of these different patho-mechanims in the mice model could help to develop novel therapeutic treatment, with a combination of drugs, that might be able to rescue all the different core features of the autistic and ID manifestations present in the patients.

mechanisms causing behavioral deficits in several ASD mouse models (Chao et al., 2010; Han et al., 2012, 2013; Jurgensen and Castillo, 2015) and in line with the reduced GABA neurotransmission that was found in Shank2 (6–7) KO mice, Lim et al. treated adult Shank2 (6–7) KO mice with the GABAA receptor alpha 2 positive allosteric modulator L438,417, reversing spatial memory deficits but not social deficits (Lim et al., 2017). Different approaches have been used to reverse the phenotype due to Shank3 absence including pharmacological treatments and genetic restoration of Shank3 expression (Table 2). Two independent studies showed mGlu5 receptor as a potential pharmacological target to rescue synaptic and behavioral alteration in Shank3 KO mice (Wang et al., 2016; Vicidomini et al., 2017). The first study found that the mGlu5 positive agonist CDPPB ameliorate ASDlike behavior in Shank3(11) KO mouse (Vicidomini et al., 2017). The second study showed that CDPPB treatment partially rescued instrumental learning but exacerbated grooming in Shank3Δe4–22−/− mice (Wang et al., 2016). Although the results of these studies seem partially discordant, it is important to note that the authors used two different mutant mice and that the paradigms used to characterize these two Shank3 mutant lines were not exactly the same. As Bidinosti et al. found increased CDC-like kinase 2 (CLK2) expression in Shank3 KO cortical neuron, they treated Shank3(21) KO with a specific CLK2 inhibitor that was able to decreased self-grooming, recover normal preference for social interaction and rescue both AMPA currents and synaptic GluN1 and GluN2A (Bidinosti et al., 2016). A recent study showed that oxytocin treatment ameliorates both the long-term social memory and attention deficits found in Shank3 KO rats (Harony-Nicolas et al., 2017). In these rats, oxcitoxin was also able to rescue impaired hippocampal LTP and mGlu receptor dependent LTD (Harony-Nicolas et al., 2017) (Table 3). Using a genetic approach, Mei et al. showed that restoration of Shank3 expression in adult mice was able to rescue social interaction deficit and repetitive grooming behavior (Mei et al., 2016). Notably all these studies rescued behavioral phenotypes in adult rodents, suggesting the possibility of treatment for patients with SHANK mutations or deletion during adulthood. Only two clinical studies have been conducted on PMS patients using both indirect therapeutic approaches. The first study, which included nine PMS children, showed that 12 weeks of intraperitoneal administration of Insulin Grown Factor-1 (IGF-1) were associated with an improvement of social impairment and restrictive behavior (Kolevzon et al., 2014). A phase-2 study is currently ongoing in the USA to validate the effect of IGF-1 in patients with PMS (www.ClinicalTrials. gov ID: NCT01970345). The second study evaluated the effect of intranasal insulin administration in a group of 25 PMS patients. Despite the fact that the final data was not evident enough to reach statistical significance, the study suggests a beneficial effect of insulin on psychomotor and behavioral development, especially in children older than 3 years of age (Zwanenburg et al., 2016).

Acknowledgments This work was supported by Comitato Telethon Fondazione Onlus grant no. GGP16131 (to C.V.). References Baron, M.K., Boeckers, T.M., Vaida, B., Faham, S., Gingery, M., Sawaya, M.R., Salyer, D., Gundelfinger, E.D., Bowie, J.U., 2006. An architectural framework that may lie at the core of the postsynaptic density. Science 311, 531–535. Beri, S., Tonna, N., Menozzi, G., Bonaglia, M.C., Sala, C., Giorda, R., 2007. DNA methylation regulates tissue-specific expression of Shank3. J. Neurochem. 101 (5), 1380–1391. Berkel, S., Marshall, C.R., Weiss, B., Howe, J., Roeth, R., Moog, U., Endris, V., Roberts, W., Szatmari, P., Pinto, D., Bonin, M., Riess, A., Engels, H., Sprengel, R., Scherer, S.W., Rappold, G.A., 2010. Mutations in the SHANK2 synaptic scaffolding gene in autism spectrum disorder and mental retardation. Nat. Genet. 42, 489–491. Betancur, C., 2011. Etiological heterogeneity in autism spectrum disorders: more than 100 genetic and genomic disorders and still counting. Brain Res. 1380, 42–77. Betancur, C., Buxbaum, J.D., 2013. SHANK3 haploinsufficiency: a "common" but underdiagnosed highly penetrant monogenic cause of autism spectrum disorders. Mol. Autism 4, 17. Bidinosti, M., Botta, P., Krüttner, S., Proenca, C.C., Stoehr, N., Bernhard, M., Fruh, I., Mueller, M., Bonenfant, D., Voshol, H., Carbone, W., Neal, S.J., McTighe, S.M., Roma, G., Dolmetsch, R.E., Porter, J.A., Caroni, P., Bouwmeester, T., Lüthi, A., Galimberti, I., 2016. CLK2 inhibition ameliorates autistic features associated with SHANK3 deficiency. Science 351, 1199–1203. Boccuto, L., Lauri, M., Sarasua, S.M., Skinner, C.D., Buccella, D., Dwivedi, A., Orteschi, D., Collins, J.S., Zollino, M., Visconti, P., Dupont, B., Tiziano, D., Schroer, R.J., Neri, G., Stevenson, R.E., Gurrieri, F., Schwartz, C.E., 2013. Prevalence of SHANK3 variants in patients with different subtypes of autism spectrum disorders. Eur. J. Hum. Genet. 21, 310–316. Bockers, T.M., Segger-Junius, M., Iglauer, P., Bockmann, J., Gundelfinger, E.D., Kreutz, M.R., Richter, D., Kindler, S., Kreienkamp, H.J., 2004. Differential expression and dendritic transcript localization of Shank family members: identification of a dendritic targeting element in the 3′ untranslated region of Shank1 mRNA. Mol. Cell. Neurosci. 26, 182–190. Boeckers, T.M., Kreutz, M.R., Winter, C., Zuschratter, W., Smalla, K.H., Sanmarti-Vila, L., Wex, H., Langnaese, K., Bockmann, J., Garner, C.C., Gundelfinger, E.D., 1999a. Proline-rich synapse-associated protein-1/cortactin binding protein 1 (ProSAP1/ CortBP1) is a PDZ-domain protein highly enriched in the postsynaptic density. J. Neurosci. 19, 6506–6518. Boeckers, T.M., Winter, C., Smalla, K.H., Kreutz, M.R., Bockmann, J., Seidenbecher, C., Garner, C.C., Gundelfinger, E.D., 1999b. Proline-rich synapse-associated proteins ProSAP1 and ProSAP2 interact with synaptic proteins of the SAPAP/GKAP family. Biochem. Biophys. Res. Commun. 264, 247–252. Boeckers, T.M., Bockmann, J., Kreutz, M.R., Gundelfinger, E.D., 2002. ProSAP/Shank proteins - a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease. J. Neurochem. 81, 903–910. Boeckers, T.M., Liedtke, T., Spilker, C., Dresbach, T., Bockmann, J., Kreutz, M.R., Gundelfinger, E.D., 2005. C-terminal synaptic targeting elements for postsynaptic density proteins ProSAP1/Shank2 and ProSAP2/Shank3. J. Neurochem. 92, 519–524. Bonaglia, M.C., Giorda, R., Borgatti, R., Felisari, G., Gagliardi, C., Selicorni, A., Zuffardi, O., 2001. Disruption of the ProSAP2 gene in a t(12;22)(q24.1;q13.3) is associated with the 22q13.3 deletion syndrome. Am. J. Hum. Genet. 69, 261–268. Bonaglia, M.C., Giorda, R., Beri, S., De Agostini, C., Novara, F., Fichera, M., Grillo, L., Galesi, O., Vetro, A., Ciccone, R., Bonati, M.T., Giglio, S., Guerrini, R., Osimani, S., Marelli, S., Zucca, C., Grasso, R., Borgatti, R., Mani, E., Motta, C., Molteni, M., Romano, C., Greco, D., Reitano, S., Baroncini, A., Lapi, E., Cecconi, A., Arrigo, G., Patricelli, M.G., Pantaleoni, C., D'Arrigo, S., Riva, D., Sciacca, F., Dalla Bernardina, B., Zoccante, L., Darra, F., Termine, C., Maserati, E., Bigoni, S., Priolo, E., Bottani, A., Gimelli, S., Bena, F., Brusco, A., di Gregorio, E., Bagnasco, I., Giussani, U., Nitsch, L., Politi, P., Martinez-Frias, M.L., Martínez-Fernández, M.L., Martínez Guardia, N., Bremer, A., Anderlid, B.M., Zuffardi, O., 2011. Molecular mechanisms generating and stabilizing terminal 22q13 deletions in 44 subjects with Phelan/McDermid syndrome. PLoS Genet. 7, e1002173. Bozdagi, O., Sakurai, T., Papapetrou, D., Wang, X., Dickstein, D.L., Takahashi, N., Kajiwara, Y., Yang, M., Katz, A.M., Scattoni, M.L., Harris, M.J., Saxena, R., Silverman, J.L., Crawley, J.N., Zhou, Q., Hof, P.R., Buxbaum, J.D., 2010. Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication. Mol. Autism 1, 15. Chao, H.T., Chen, H., Samaco, R.C., Xue, M., Chahrour, M., Yoo, J., Neul, J.L., Gong, S., Lu, H.C., Heintz, N., Ekker, M., Rubenstein, J.L., Noebels, J.L., Rosenmund, C., Zoghbi, H.Y., 2010. Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes. Nature 468, 263–269. Ching, T.T., Maunakea, A.K., Jun, P., Hong, C., Zardo, G., Pinkel, D., Albertson, D.G.,

7. Conclusion In recent years, the availability of several Shank mutant rodents and the generation of patient specific iPSCs have been helping to fully clarify the function of Shank proteins in the correct neuronal and synapses development. Some possible drug targets have also been identified, but there is still a considerable way to go before we are able to translate these findings to therapies for patients. The patients with SHANK gene mutations show a wide range of the core features of autistic and ID clinical manifestations that might depend on the different type of SHANK gene mutations, or by the presence of additional mutations in other genes that can ether aggravate or rescue the clinical manifestation. Thus, patients might need a different combination of drug treatments or therapeutic approaches depending 6

Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxx–xxx

A. Mossa et al.

Tavassoli, T., Chaplin, W., Soorya, L., Buxbaum, J.D., 2014. A pilot controlled trial of insulin-like growth factor-1 in children with Phelan-McDermid syndrome. Mol. Autism 5, 54. Kouser, M., Speed, H.E., Dewey, C.M., Reimers, J.M., Widman, A.J., Gupta, N., Liu, S., Jaramillo, T.C., Bangash, M., Xiao, B., Worley, P.F., Powell, C.M., 2013. Loss of predominant Shank3 isoforms results in hippocampus-dependent impairments in behavior and synaptic transmission. J. Neurosci. 33, 18448–18468. Leblond, C.S., Nava, C., Polge, A., Gauthier, J., Huguet, G., Lumbroso, S., Giuliano, F., Stordeur, C., Depienne, C., Mouzat, K., Pinto, D., Howe, J., Lemière, N., Durand, C.M., Guibert, J., Ey, E., Toro, R., Peyre, H., Mathieu, A., Amsellem, F., Rastam, M., Gillberg, I.C., Rappold, G.A., Holt, R., Monaco, A.P., Maestrini, E., Galan, P., Heron, D., Jacquette, A., Afenjar, A., Rastetter, A., Brice, A., Devillard, F., Assouline, B., Laffargue, F., Lespinasse, J., Chiesa, J., Rivier, F., Bonneau, D., Regnault, B., Zelenika, D., Delepine, M., Lathrop, M., Sanlaville, D., Schluth-Bolard, C., Edery, P., Perrin, L., Tabet, A.C., Schmeisser, M.J., Boeckers, T.M., Coleman, M., Sato, D., Szatmari, P., Scherer, S.W., Rouleau, G.A., Betancur, C., Leboyer, M., Gillberg, C., Delorme, R., Bourgeron, T., 2014. Meta-analysis of SHANK mutations in autism Spectrum disorders: a gradient of severity in cognitive impairments. PLoS Genet. 10, e1004580. Lee, E.J., Lee, H., Huang, T.N., Chung, C., Shin, W., Kim, K., Koh, J.Y., Hsueh, Y.P., Kim, E., 2015a. Trans-synaptic zinc mobilization improves social interaction in two mouse models of autism through NMDAR activation. Nat. Commun. 6, 7168. Lee, J., Chung, C., Ha, S., Lee, D., Kim, D.Y., Kim, H., Kim, E., 2015b. Shank3-mutant mice lacking exon 9 show altered excitation/inhibition balance, enhanced rearing, and spatial memory deficit. Front. Cell. Neurosci. 9, 94. Lee, Y., Kim, S.G., Lee, B., Zhang, Y., Kim, Y., Kim, S., Kim, E., Kang, H., Han, K., 2017. Striatal transcriptome and interactome analysis of Shank3-overexpressing mice reveals the connectivity between Shank3 and mTORC1 signaling. Front. Mol. Neurosci. 10, 201. Lilja, J., Zacharchenko, T., Georgiadou, M., Jacquemet, G., De Franceschi, N., Peuhu, E., Hamidi, H., Pouwels, J., Martens, V., Nia, F.H., Beifuss, M., Boeckers, T., Kreienkamp, H.J., Barsukov, I.L., Ivaska, J., 2017. SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras. Nat. Cell Biol. 19, 292–305. Lim, S., Naisbitt, S., Yoon, J., Hwang, J.I., Suh, P.G., Sheng, M., Kim, E., 1999. Characterization of the Shank family of synaptic proteins. Multiple genes, alternative splicing, and differential expression in brain and development. J. Biol. Chem. 274, 29510–29518. Lim, C.S., Kim, H., Yu, N.K., Kang, S.J., Kim, T., Ko, H.G., Lee, J., Yang, J.E., Ryu, H.H., Park, T., Gim, J., Nam, H.J., Baek, S.H., Wegener, S., Schmitz, D., Boeckers, T.M., Lee, M.G., Kim, E., Lee, J.H., Lee, Y.S., Kaang, B.K., 2017. Enhancing inhibitory synaptic function reverses spatial memory deficits in Shank2 mutant mice. Neuropharmacology 112, 104–112. Marchetto, M.C., Carromeu, C., Acab, A., Yu, D., Yeo, G.W., Mu, Y., Chen, G., Gage, F.H., Muotri, A.R., 2010. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143, 527–539. Maunakea, A.K., Nagarajan, R.P., Bilenky, M., Ballinger, T.J., D'Souza, C., Fouse, S.D., Johnson, B.E., Hong, C., Nielsen, C., Zhao, Y., Turecki, G., Delaney, A., Varhol, R., Thiessen, N., Shchors, K., Heine, V.M., Rowitch, D.H., Xing, X., Fiore, C., Schillebeeckx, M., Jones, S.J., Haussler, D., Marra, M.A., Hirst, M., Wang, T., Costello, J.F., 2010. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257. Mei, Y., Monteiro, P., Zhou, Y., Kim, J.A., Gao, X., Fu, Z., Feng, G., 2016. Adult restoration of Shank3 expression rescues selective autistic-like phenotypes. Nature 530, 481–484. Moessner, R., Marshall, C.R., Sutcliffe, J.S., Skaug, J., Pinto, D., Vincent, J., Zwaigenbaum, L., Fernandez, B., Roberts, W., Szatmari, P., Scherer, S.W., 2007. Contribution of SHANK3 mutations to autism spectrum disorder. Am. J. Hum. Genet. 81, 1289–1297. Naisbitt, S., Kim, E., Tu, J.C., Xiao, B., Sala, C., Valtschanoff, J., Weinberg, R.J., Worley, P.F., Sheng, M., 1999. Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin. Neuron 23, 569–582. Peça, J., Feliciano, C., Ting, J.T., Wang, W., Wells, M.F., Venkatraman, T.N., Lascola, C.D., Fu, Z., Feng, G., 2011. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472, 437–442. Peixoto, R.T., Wang, W., Croney, D.M., Kozorovitskiy, Y., Sabatini, B.L., 2016. Early hyperactivity and precocious maturation of corticostriatal circuits in Shank3B(−/−) mice. Nat. Neurosci. 19, 716–724. Peter, S., Ten Brinke, M.M., Stedehouder, J., Reinelt, C.M., Wu, B., Zhou, H., Zhou, K., Boele, H.J., Kushner, S.A., Lee, M.G., Schmeisser, M.J., Boeckers, T.M., Schonewille, M., Hoebeek, F.E., De Zeeuw, C.I., 2016. Dysfunctional cerebellar Purkinje cells contribute to autism-like behaviour in Shank2-deficient mice. Nat. Commun. 7, 12627. Phelan, K., McDermid, H.E., 2012. The 22q13.3 deletion syndrome (Phelan-McDermid syndrome). Mol. Syndromol. 2, 186–201. Phelan, M.C., Rogers, R.C., Saul, R.A., Stapleton, G.A., Sweet, K., McDermid, H., Shaw, S.R., Claytor, J., Willis, J., Kelly, D.P., 2001. 22q13 deletion syndrome. Am. J. Med. Genet. 101, 91–99. Pinto, D., Pagnamenta, A.T., Klei, L., Anney, R., Merico, D., Regan, R., Conroy, J., Magalhaes, T.R., Correia, C., Abrahams, B.S., Almeida, J., Bacchelli, E., Bader, G.D., Bailey, A.J., Baird, G., Battaglia, A., Berney, T., Bolshakova, N., Bölte, S., Bolton, P.F., Bourgeron, T., Brennan, S., Brian, J., Bryson, S.E., Carson, A.R., Casallo, G., Casey, J., Chung, B.H., Cochrane, L., Corsello, C., Crawford, E.L., Crossett, A., Cytrynbaum, C., Dawson, G., de Jonge, M., Delorme, R., Drmic, I., Duketis, E., Duque, F., Estes, A., Farrar, P., Fernandez, B.A., Folstein, S.E., Fombonne, E., Freitag, C.M., Gilbert, J., Gillberg, C., Glessner, J.T., Goldberg, J., Green, A., Green, J., Guter, S.J., Hakonarson, H., Heron, E.A., Hill, M., Holt, R., Howe, J.L., Hughes, G., Hus, V., Igliozzi, R., Kim,

Fridlyand, J., Mao, J.H., Shchors, K., Weiss, W.A., Costello, J.F., 2005. Epigenome analyses using BAC microarrays identify evolutionary conservation of tissue-specific methylation of SHANK3. Nat. Genet. 37, 645–651. Collingridge, G.L., Peineau, S., Howland, J.G., Wang, Y.T., 2010. Long-term depression in the CNS. Nat. Rev. Neurosci. 11, 459–473. Darville, H., Poulet, A., Rodet-Amsellem, F., Chatrousse, L., Pernelle, J., Boissart, C., Héron, D., Nava, C., Perrier, A., Jarrige, M., Cogé, F., Millan, M.J., Bourgeron, T., Peschanski, M., Delorme, R., Benchoua, A., 2016. Human pluripotent stem cell-derived cortical neurons for high throughput medication screening in autism: a proof of concept study in SHANK3 Haploinsufficiency syndrome. EBioMedicine 9, 293–305. Du, Y., Weed, S.A., Xiong, W.-C., Marshall, T.D., Parsons, J.T., 1998. Identification of a novel cortactin SH3 domain-binding protein and its localization to growth cones of cultured neurons. Mol. Cell. Biol. 18, 5838–5851. Durand, C.M., Betancur, C., Boeckers, T.M., Bockmann, J., Chaste, P., Fauchereau, F., Nygren, G., Rastam, M., Gillberg, I.C., Anckarsater, H., Sponheim, E., GoubranBotros, H., Delorme, R., Chabane, N., Mouren-Simeoni, M.C., de Mas, P., Bieth, E., Roge, B., Heron, D., Burglen, L., Gillberg, C., Leboyer, M., Bourgeron, T., 2007. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nat. Genet. 39, 25–27. Ehlers, M.D., 1999. Synapse structure: glutamate receptors connected by the shanks. Curr. Biol. 9, R848–850. Epstein, I., Tushev, G., Will, T.J., Vlatkovic, I., Cajigas, I.J., Schuman, E.M., 2014. Alternative polyadenylation and differential expression of Shank mRNAs in the synaptic neuropil. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 369, 20130137. Falley, K., Schütt, J., Iglauer, P., Menke, K., Maas, C., Kneussel, M., Kindler, S., Wouters, F.S., Richter, D., Kreienkamp, H.J., 2009. Shank1 mRNA: Dendritic transport by kinesin and translational control by the 5'untranslated region. Traffic 10, 844–857. Farra, N., Zhang, W.B., Pasceri, P., Eubanks, J.H., Salter, M.W., Ellis, J., 2012. Rett syndrome induced pluripotent stem cell-derived neurons reveal novel neurophysiological alterations. Mol. Psychiatry 17 (12), 1261–1271. Gauthier, J., Spiegelman, D., Piton, A., Lafreniere, R.G., Laurent, S., St-Onge, J., Lapointe, L., Hamdan, F.F., Cossette, P., Mottron, L., Fombonne, E., Joober, R., Marineau, C., Drapeau, P., Rouleau, G.A., 2009. Novel de novo SHANK3 mutation in autistic patients. Am. J. Med. Genet. B Neuropsychiatr. Genet. 150B, 421–424. Gauthier, J., Champagne, N., Lafrenière, R.G., Xiong, L., Spiegelman, D., Brustein, E., Lapointe, M., Peng, H., Côté, M., Noreau, A., Hamdan, F.F., Addington, A.M., Rapoport, J.L., Delisi, L.E., Krebs, M.O., Joober, R., Fathalli, F., Mouaffak, F., Haghighi, A.P., Néri, C., Dubé, M.P., Samuels, M.E., Marineau, C., Stone, E.A., Awadalla, P., Barker, P.A., Carbonetto, S., Drapeau, P., Rouleau, G.A., Team, S.D., 2010. De novo mutations in the gene encoding the synaptic scaffolding protein SHANK3 in patients ascertained for schizophrenia. Proc. Natl. Acad. Sci. U. S. A. 107, 7863–7868. Gerrow, K., Romorini, S., Nabi, S.M., Colicos, M.A., Sala, C., El-Husseini, A., 2006. A preformed complex of postsynaptic proteins is involved in excitatory synapse development. Neuron 49, 547–562. Grabrucker, A.M., Knight, M.J., Proepper, C., Bockmann, J., Joubert, M., Rowan, M., Nienhaus, G.U., Garner, C.C., Bowie, J.U., Kreutz, M.R., Gundelfinger, E.D., Boeckers, T.M., 2011. Concerted action of zinc and ProSAP/Shank in synaptogenesis and synapse maturation. EMBO J. 30, 569–581. Grabrucker, S., Proepper, C., Mangus, K., Eckert, M., Chhabra, R., Schmeisser, M.J., Boeckers, T.M., Grabrucker, A.M., 2014. The PSD protein ProSAP2/Shank3 displays synapto-nuclear shuttling which is deregulated in a schizophrenia-associated mutation. Exp. Neurol. 253, 126–137. Guilmatre, A., Huguet, G., Delorme, R., Bourgeron, T., 2014. The emerging role of SHANK genes in neuropsychiatric disorders. Dev. Neurobiol. 74, 113–122. Gundelfinger, E.D., Boeckers, T.M., Baron, M.K., Bowie, J.U., 2006. A role for zinc in postsynaptic density asSAMbly and plasticity? Trends Biochem. Sci. 31, 366–373. Ha, S., Lee, D., Cho, Y.S., Chung, C., Yoo, Y.E., Kim, J., Lee, J., Kim, W., Kim, H., Bae, Y.C., Tanaka-Yamamoto, K., Kim, E., 2016. Cerebellar Shank2 regulates excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors. J. Neurosci. 36, 12129–12143. Han, S., Tai, C., Westenbroek, R.E., Yu, F.H., Cheah, C.S., Potter, G.B., Rubenstein, J.L., Scheuer, T., de la Iglesia, H.O., Catterall, W.A., 2012. Autistic-like behaviour in Scn1a +/− mice and rescue by enhanced GABA-mediated neurotransmission. Nature 489, 385–390. Han, K., Holder, J.L., Schaaf, C.P., Lu, H., Chen, H., Kang, H., Tang, J., Wu, Z., Hao, S., Cheung, S.W., Yu, P., Sun, H., Breman, A.M., Patel, A., Lu, H.C., Zoghbi, H.Y., 2013. SHANK3 overexpression causes manic-like behaviour with unique pharmacogenetic properties. Nature 503, 72–77. Harony-Nicolas, H., Kay, M., Hoffmann, J.D., Klein, M.E., Bozdagi-Gunal, O., Riad, M., Daskalakis, N.P., Sonar, S., Castillo, P.E., Hof, P.R., Shapiro, M.L., Baxter, M.G., Wagner, S., Buxbaum, J.D., 2017. Oxytocin improves behavioral and electrophysiological deficits in a novel Shank3-deficient rat. elife 6. Hung, A.Y., Futai, K., Sala, C., Valtschanoff, J.G., Ryu, J., Woodworth, M.A., Kidd, F.L., Sung, C.C., Miyakawa, T., Bear, M.F., Weinberg, R.J., Sheng, M., 2008. Smaller dendritic spines, weaker synaptic transmission, but enhanced spatial learning in mice lacking Shank1. J. Neurosci. 28, 1697–1708. Jaramillo, T.C., Speed, H.E., Xuan, Z., Reimers, J.M., Escamilla, C.O., Weaver, T.P., Liu, S., Filonova, I., Powell, C.M., 2017. Novel Shank3 mutant exhibits behaviors with face validity for autism and altered striatal and hippocampal function. Autism Res. 10, 42–65. Jiang, Y.H., Ehlers, M.D., 2013. Modeling autism by SHANK gene mutations in mice. Neuron 78, 8–27. Jurgensen, S., Castillo, P.E., 2015. Selective dysregulation of hippocampal inhibition in the mouse lacking autism candidate gene CNTNAP2. J. Neurosci. 35, 14681–14687. Kolevzon, A., Bush, L., Wang, A.T., Halpern, D., Frank, Y., Grodberg, D., Rapaport, R.,

7

Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxx–xxx

A. Mossa et al.

Uemura, T., Mori, H., Mishina, M., 2004. Direct interaction of GluRdelta2 with Shank scaffold proteins in cerebellar Purkinje cells. Mol. Cell. Neurosci. 26, 330–341. Verpelli, C., Sala, C., 2012. Molecular and synaptic defects in intellectual disability syndromes. Curr. Opin. Neurobiol. 22, 530–536. Verpelli, C., Dvoretskova, E., Vicidomini, C., Rossi, F., Chiappalone, M., Schoen, M., Di Stefano, B., Mantegazza, R., Broccoli, V., Boeckers, T.M., Dityatev, A., Sala, C., 2011. Importance of shank3 in regulating metabotropic glutamate receptor 5 (mGluR5) expression and signaling at synapses. J. Biol. Chem. 286 (40), 34839–34850. Vicidomini, C., Ponzoni, L., Lim, D., Schmeisser, M.J., Reim, D., Morello, N., Orellana, D., Tozzi, A., Durante, V., Scalmani, P., Mantegazza, M., Genazzani, A.A., Giustetto, M., Sala, M., Calabresi, P., Boeckers, T.M., Sala, C., Verpelli, C., 2017. Pharmacological enhancement of mGlu5 receptors rescues behavioral deficits in SHANK3 knock-out mice. Mol. Psychiatry 22, 689–702. Wang, X., McCoy, P.A., Rodriguiz, R.M., Pan, Y., Je, H.S., Roberts, A.C., Kim, C.J., Berrios, J., Colvin, J.S., Bousquet-Moore, D., Lorenzo, I., Wu, G., Weinberg, R.J., Ehlers, M.D., Philpot, B.D., Beaudet, A.L., Wetsel, W.C., Jiang, Y.H., 2011. Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3. Hum. Mol. Genet. 20, 3093–3108. Wang, X., Xu, Q., Bey, A.L., Lee, Y., Jiang, Y.H., 2014. Transcriptional and functional complexity of Shank3 provides a molecular framework to understand the phenotypic heterogeneity of SHANK3 causing autism and Shank3 mutant mice. Mol. Autism 5, 30. Wang, X., Bey, A.L., Katz, B.M., Badea, A., Kim, N., David, L.K., Duffney, L.J., Kumar, S., Mague, S.D., Hulbert, S.W., Dutta, N., Hayrapetyan, V., Yu, C., Gaidis, E., Zhao, S., Ding, J.D., Xu, Q., Chung, L., Rodriguiz, R.M., Wang, F., Weinberg, R.J., Wetsel, W.C., Dzirasa, K., Yin, H., Jiang, Y.H., 2016. Altered mGluR5-Homer scaffolds and corticostriatal connectivity in a Shank3 complete knockout model of autism. Nat. Commun. 7, 11459. Wang, W., Li, C., Chen, Q., van der Goes, M.S., Hawrot, J., Yao, A.Y., Gao, X., Lu, C., Zang, Y., Zhang, Q., Lyman, K., Wang, D., Guo, B., Wu, S., Gerfen, C.R., Fu, Z., Feng, G., 2017. Striatopallidal dysfunction underlies repetitive behavior in Shank3-deficient model of autism. J. Clin. Invest. 127, 1978–1990. Wilson, H.L., Wong, A.C., Shaw, S.R., Tse, W.Y., Stapleton, G.A., Phelan, M.C., Hu, S., Marshall, J., McDermid, H.E., 2003. Molecular characterisation of the 22q13 deletion syndrome supports the role of haploinsufficiency of SHANK3/PROSAP2 in the major neurological symptoms. J. Med. Genet. 40, 575–584. Wöhr, M., 2014. Ultrasonic vocalizations in shank mouse models for autism spectrum disorders: detailed spectrographic analyses and developmental profiles. Neurosci. Biobehav. Rev. 43, 199–212. Wöhr, M., Roullet, F.I., Hung, A.Y., Sheng, M., Crawley, J.N., 2011. Communication impairments in mice lacking Shank1: reduced levels of ultrasonic vocalizations and scent marking behavior. PLoS One 6, e20631. Won, H., Lee, H.R., Gee, H.Y., Mah, W., Kim, J.I., Lee, J., Ha, S., Chung, C., Jung, E.S., Cho, Y.S., Park, S.G., Lee, J.S., Lee, K., Kim, D., Bae, Y.C., Kaang, B.K., Lee, M.G., Kim, E., 2012. Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function. Nature 486, 261–265. Yasumura, M., Uemura, T., Yamasaki, M., Sakimura, K., Watanabe, M., Mishina, M., 2008. Role of the internal Shank-binding segment of glutamate receptor delta2 in synaptic localization and cerebellar functions. Neurosci. Lett. 433, 146–151. Yi, F., Danko, T., Botelho, S.C., Patzke, C., Pak, C., Wernig, M., Südhof, T.C., 2016. Autism-associated SHANK3 haploinsufficiency causes Ih channelopathy in human neurons. Science 352, aaf2669. Zhou, Y., Kaiser, T., Monteiro, P., Zhang, X., Van der Goes, M.S., Wang, D., Barak, B., Zeng, M., Li, C., Lu, C., Wells, M., Amaya, A., Nguyen, S., Lewis, M., Sanjana, N., Zhang, M., Zhang, F., Fu, Z., Feng, G., 2016. Mice with Shank3 mutations associated with ASD and schizophrenia display both shared and distinct defects. Neuron 89, 147–162. Zhu, L., Wang, X., Li, X.L., Towers, A., Cao, X., Wang, P., Bowman, R., Yang, H., Goldstein, J., Li, Y.J., Jiang, Y.H., 2014. Epigenetic dysregulation of SHANK3 in brain tissues from individuals with autism spectrum disorders. Hum. Mol. Genet. 23, 1563–1578. Zitzer, H., Honck, H.H., Bachner, D., Richter, D., Kreienkamp, H.J., 1999. Somatostatin receptor interacting protein defines a novel family of multidomain proteins present in human and rodent brain. J. Biol. Chem. 274, 32997–33001. Zwanenburg, R.J., Bocca, G., Ruiter, S.A., Dillingh, J.H., Flapper, B.C., van den Heuvel, E.R., van Ravenswaaij-Arts, C.M., 2016. Is there an effect of intranasal insulin on development and behaviour in Phelan-McDermid syndrome? A randomized, doubleblind, placebo-controlled trial. Eur. J. Hum. Genet. 24, 1696–1701.

C., Klauck, S.M., Kolevzon, A., Korvatska, O., Kustanovich, V., Lajonchere, C.M., Lamb, J.A., Laskawiec, M., Leboyer, M., Le Couteur, A., Leventhal, B.L., Lionel, A.C., Liu, X.Q., Lord, C., Lotspeich, L., Lund, S.C., Maestrini, E., Mahoney, W., Mantoulan, C., Marshall, C.R., McConachie, H., McDougle, C.J., McGrath, J., McMahon, W.M., Merikangas, A., Migita, O., Minshew, N.J., Mirza, G.K., Munson, J., Nelson, S.F., Noakes, C., Noor, A., Nygren, G., Oliveira, G., Papanikolaou, K., Parr, J.R., Parrini, B., Paton, T., Pickles, A., Pilorge, M., Piven, J., Ponting, C.P., Posey, D.J., Poustka, A., Poustka, F., Prasad, A., Ragoussis, J., Renshaw, K., Rickaby, J., Roberts, W., Roeder, K., Roge, B., Rutter, M.L., Bierut, L.J., Rice, J.P., Salt, J., Sansom, K., Sato, D., Segurado, R., Sequeira, A.F., Senman, L., Shah, N., Sheffield, V.C., Soorya, L., Sousa, I., Stein, O., Sykes, N., Stoppioni, V., Strawbridge, C., Tancredi, R., Tansey, K., Thiruvahindrapduram, B., Thompson, A.P., Thomson, S., Tryfon, A., Tsiantis, J., Van Engeland, H., Vincent, J.B., Volkmar, F., Wallace, S., Wang, K., Wang, Z., Wassink, T.H., Webber, C., Weksberg, R., Wing, K., Wittemeyer, K., Wood, S., Wu, J., Yaspan, B.L., Zurawiecki, D., Zwaigenbaum, L., Buxbaum, J.D., Cantor, R.M., Cook, E.H., Coon, H., Cuccaro, M.L., Devlin, B., Ennis, S., Gallagher, L., Geschwind, D.H., Gill, M., Haines, J.L., Hallmayer, J., Miller, J., Monaco, A.P., Nurnberger, J.I., Paterson, A.D., Pericak-Vance, M.A., Schellenberg, G.D., Szatmari, P., Vicente, A.M., Vieland, V.J., Wijsman, E.M., Scherer, S.W., Sutcliffe, J.S., Betancur, C., 2010. Functional impact of global rare copy number variation in autism spectrum disorders. Nature 466, 368–372. Romorini, S., Piccoli, G., Jiang, M., Grossano, P., Tonna, N., Passafaro, M., Zhang, M.J., Sala, C., 2004. A functional role of postsynaptic density-95-guanylate kinase-associated protein complex in regulating shank assembly and stability to synapses. J. Neurosci. 24, 9391–9404. Roussignol, G., Ango, F., Romorini, S., Tu, J.C., Sala, C., Worley, P.F., Bockaert, J.R.L., Fagni, L., 2005. Shank expression is sufficient to induce functional dendritic spine synapses in aspiny neurons. J. Neurosci. 25, 3560–3570. Sala, C., Piech, V., Wilson, N.R., Passafaro, M., Liu, G.S., Sheng, M., 2001. Regulation of dendritic spine morphology and synaptic function by Shank and Homer. Neuron 31, 115–130. Sala, C., Roussignol, G., Meldolesi, J., Fagni, L., 2005. Key role of the postsynaptic density scaffold proteins shank and homer in the functional architecture of Ca2 + homeostasis at dendritic spines in hippocampal neurons. J. Neurosci. 25, 4587–4592. Sala, C., Vicidomini, C., Bigi, I., Mossa, A., Verpelli, C., 2015. Shank synaptic scaffold proteins: keys to understanding the pathogenesis of autism and other synaptic disorders. J. Neurochem. 135, 849–858. Schmeisser, M.J., Ey, E., Wegener, S., Bockmann, J., Stempel, A.V., Kuebler, A., Janssen, A.L., Udvardi, P.T., Shiban, E., Spilker, C., Balschun, D., Skryabin, B.V., Dieck, S., Smalla, K.H., Montag, D., Leblond, C.S., Faure, P., Torquet, N., Le Sourd, A.M., Toro, R., Grabrucker, A.M., Shoichet, S.A., Schmitz, D., Kreutz, M.R., Bourgeron, T., Gundelfinger, E.D., Boeckers, T.M., 2012. Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2. Nature 486, 256–260. Shcheglovitov, A., Shcheglovitova, O., Yazawa, M., Portmann, T., Shu, R., Sebastiano, V., Krawisz, A., Froehlich, W., Bernstein, J.A., Hallmayer, J.F., Dolmetsch, R.E., 2013. SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients. Nature 503, 267–271. Sheng, M., Kim, E., 2000. The Shank family of scaffold proteins. J. Cell Sci. 113, 1851–1856. Silverman, J.L., Turner, S.M., Barkan, C.L., Tolu, S.S., Saxena, R., Hung, A.Y., Sheng, M., Crawley, J.N., 2011. Sociability and motor functions in Shank1 mutant mice. Brain Res. 1380, 120–137. Studtmann, K., Olschläger-Schütt, J., Buck, F., Richter, D., Sala, C., Bockmann, J., Kindler, S., Kreienkamp, H.J., 2014. A non-canonical initiation site is required for efficient translation of the dendritically localized Shank1 mRNA. PLoS One 9, e88518. Sungur, A., Vörckel, K.J., Schwarting, R.K., Wöhr, M., 2014. Repetitive behaviors in the Shank1 knockout mouse model for autism spectrum disorder: developmental aspects and effects of social context. J. Neurosci. Methods 234, 92–100. Sungur, A., Jochner, M.C.E., Harb, H., Kılıç, A., Garn, H., Schwarting, R.K.W., Wöhr, M., 2017. Aberrant cognitive phenotypes and altered hippocampal BDNF expression related to epigenetic modifications in mice lacking the post-synaptic scaffolding protein SHANK1: Implications for autism spectrum disorder. Hippocampus 27, 906–919. Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. Tu, J.C., Xiao, B., Naisbitt, S., Yuan, J.P., Petralia, R.S., Brakeman, P., Doan, A., Aakalu, V.K., Lanahan, A.A., Sheng, M., Worley, P.F., 1999. Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins. Neuron 23, 583–592.

8