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In the hippocampus, locations associated with salient features are represented by a disproportionately large number of neurons, but the cellular and molecular mechanisms underlying this over-representation remain elusive. Using longitudinal calcium imaging in mice learning to navigate in virtual reality, we find that the over-representation of reward and landmark locations are mediated by persistent and separable subsets of neurons, with distinct time courses of emergence and differing underlying molecular mechanisms. Strikingly, we find that in mice lacking Shank2, an autism spectrum disorder (ASD)-linked gene encoding an excitatory postsynaptic scaffold protein, the learning-induced over-representation of landmarks was absent whereas the over-representation of rewards was substantially increased, as was goal-directed behavior. These findings demonstrate that multiple hippocampal coding processes for unique types of salient features are distinguished by a Shank2-dependent mechanism and suggest that abnormally distorted hippocampal salience mapping may underlie cognitive and behavioral abnormalities in a subset of ASDs.Using longitudinal two-photon calcium imaging in mice during virtual navigation, Sato et al. demonstrate that persistent and separable neuronal subsets mediate the hippocampal over-representation of reward and landmark locations. Learning-induced over-representation of landmarks is absent while rapid over-representation of rewards is enhanced, in a mouse model of autism lacking Shank2., This work was supported by Precursory Research for Embryonic Science and Technology (PRESTO) JPMJPR12A1 from the Japan Science and Technology Agency (JST), KAKENHI grants 24700403, 25116528, 26115530,17H05985, 19H04942, and 20H03550 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS), and a grant from the Senshin Medical Research Foundation to M.S.; RIKEN, NIH grant R01DA17310, KAKENHI grants 22110006, 16H01292, and 18H05434, a Human Frontier Science Programme grant, and Fujitsu Laboratories to Y.H.; KAKENHI grants 25830023, 15H01571, 17H05695, and 19K16293 to K.M.; KAKENHI grant 26870577 to T.T.; KAKENHI grant 15H04265 to T.F; KAKENHI grants 26115504, 15H05723, and 16H06536, and the program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from MEXT and the Japan Agency for Medical Research and Development (AMED) to J.N.; Regional Innovation Cluster Program grant (City Area Type, Central Saitama Area) from MEXT to J.N. and M.O.; and grants from the VW Foundation (ZN2632), BCCN (01GQ1005A and 01GQ1005B), DFG (CRC 1286, 889), the Ministry for Science and Culture of Lower Saxony, and the Max Planck Society to F.W. D.G.-D. is a recipient of the PhD fellowship BES-2013-064171 and a grant from the shortterm visit program EEBB-I-15-09552.

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders, characterized by early-onset deficits in social behavior and communication across multiple contexts, together with restricted, repetitive patterns of behavior, interests, or activities. ASD is among the most heritable neuropsychiatric conditions with heritability estimates higher than 80%, and while available evidence points to a complex set of genetic factors, the SHANK (also known as ProSAP) gene family has emerged as one of the most promising candidates. Several genetic Shank mouse models for ASD were generated, including Shank1 knockout mice. Behavioral studies focusing on the Shank1 knockout mouse model for ASD included assays for detecting ASD-relevant behavioral phenotypes in the following domains: (I) social behavior, (II) communication, and (III) repetitive and stereotyped patterns of behavior. In addition, assays for detecting behavioral phenotypes with relevance to comorbidities in ASD were performed, including but not limited to (IV) cognitive functioning. Here, we summarize and discuss behavioral and neuronal findings obtained in the Shank1 knockout mouse model for ASD. We identify open research questions by comparing such findings with the symptoms present in humans diagnosed with ASD and carrying SHANK1 deletions. We conclude by discussing the implications of the behavioral and neuronal phenotypes displayed by the Shank1 knockout mouse model for the development of future pharmacological interventions in ASD.

Background The SHANK gene family encodes for postsynaptic scaffolding proteins with an important role at glutamatergic synapses in the brain and has been linked to a spectrum of neurodevelopmental disorders. The schizophrenia associated microRNA miR-137 and the SHANK genes converge on several levels, (a) both are expressed at the synapse, (b) both influence neuronal development, and (c) both have a strong link to neurodevelopmental and neuropsychiatric disorders like intellectual disability, autism and schizophrenia. This compiled evidence raised the question if the SHANKs might be targets of miR-137. Methods The in silico analysis of all three SHANK genes identified a single, highly conserved binding site for miR-137 in the 3´UTR of SHANK2. Luciferase assays were carried out in a neuroblastoma cell line (SH-SY5Y) and in mouse primary hippocampal neurons to validate the putative binding site. MiR-137 was overexpressed or inhibited in hippocampal neurons and Shank2 expression was analyzed by qPCR and Western blot. In addition, we analyzed expression levels of experimentally validated miR-137 target genes in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia and control individuals using the RNA-seq data from the CommonMind Consortium. Results We can show that miR-137 targets the 3´UTR of SHANK2 in a site-specific manner. Overexpression of miR-137 significantly lowered endogenous Shank2 protein levels without detectable influence on mRNA level; conversely, the inhibition of miR-137 resulted in the increase of Shank2 protein in neuronal cells. To further support the link between miR-137 and schizophrenia, we compared the expression of mir-137 precursor and miR-137 target genes (including SHANK2) in the DLPFC of schizophrenia and control individuals. Almost one third (18/63; 29%) of validated miR-137 target genes showed significant expression differences in the DLPFC of schizophrenia individuals compared to controls and we propose that further targets (like SHANK2) may be regulated on protein level. Discussion In this study we identified SHANK2 as a novel direct target of miR-137. We discovered that physiological levels of miR-137 regulate SHANK2 expression and that the most likely mechanism of microRNA-mediated regulation is by repressing SHANK2 protein translation rather than mRNA degradation. Our study provides first evidence of a direct regulatory link between miR-137 and SHANK2 and supports the finding that miR-137 signaling is altered in schizophrenia.

Background Autism spectrum disorder (ASD) is characterized by persistent deficits in social behavior and communication, together with restricted and repetitive patterns of behavior. Several ASD candidate genes have been identified, including the SHANK gene family with its three family members SHANK1 , SHANK2 , and SHANK3 . Methods Typically, repetitive behavior in mouse models for ASD is assessed by measuring self-grooming behavior. The first aim of the current study was to assess repetitive behaviors in Shank1 −/− null mutant, Shank1 +/− heterozygous, and Shank1 +/+ wildtype littermate control mice by means of a comprehensive approach, including the assessment of self-grooming, digging behavior, and marble burying. The second aim was to establish a test paradigm that allows for assessing the effects of social context on the occurrence of repetitive behaviors in a genotype-dependent manner. To this aim, repetitive behaviors were repeatedly tested on three consecutive days in distinct social contexts, namely in presence or absence of social odors. Results Shank1 +/− heterozygous and to a lesser extent Shank1 −/− null mutant mice displayed slightly elevated levels of self-grooming behavior as adults, but not as juveniles, with genotype differences being most prominent in the social context. In contrast to elevated self-grooming behavior, marble burying was strongly reduced in adult Shank1 +/− heterozygous and Shank1 −/− null mutant mice across social contexts, as compared to adult Shank1 +/+ wildtype littermate controls. Conclusion The opposite effects of the Shank1 deletion on the two types of repetitive behaviors are in line with a number of studies on repetitive behaviors in other genetic Shank models.

Both erythropoietin (EPO) and carbamylated EPO (cEPO) have been shown to increase the length of neurites and spine density in neurons. However, the molecular mechanism underlying the EPO- and cEPO-induced neuronal differentiation has yet to be investigated. To address this issue, we investigated epigenetic modifications that regulate gene expression in neurons. Neurons treated with EPO or cEPO display an upregulation of E1A-binding protein (p300) and p300-mediated p53 acetylation, possibly increasing the transactivation activity of p53 on growth-associated protein 43 (GAP43). Treatment of cells with cEPO markedly increases spine formation and potentiates p300-mediated transactivation of PSD95, Shank2 and 3 compared to EPO. These results demonstrate that cEPO controls neuronal differentiation via acetylation of transcription factors and subsequent transactivation of target genes. These findings have important medical implications because cEPO is of interest in the development of therapeutic agents against neuropsychiatric disorders.

The Shank family comprises three core postsynaptic scaffold proteins of excitatory synapses in the mammalian brain: Shank1, Shank2 and Shank3. Since mutations in all three human SHANK genes are linked to neuropsychiatric disorders such as autism and schizophrenia, Shank mutant mice serve as corresponding in vivo model systems. Besides intriguing alterations in behavior, dysfunction of glutamatergic synapses has emerged as a pathological hallmark among several Shank mutant lines. However, there is very limited knowledge of the underlying pathomechanisms. Therefore, precise neurobiological evaluation of morphological, molecular and electrophysiological phenotypes in Shank mutants is crucially needed. In this brief review, I will focus on the Shank mutant mouse lines we have generated so far and discuss how they might help us to develop translational treatment studies in the future.

International audience; Mouse ultrasonic vocalisations have been often used as a paradigm to extrapolate vocal communication defects observed in patients with autism spectrum disorders (ASD). The role of these vocalisations as well as their development, structure and informational content, however, remain largely unknown. In the present study, we characterised in depth the emission of pup and adult ultrasonic vocalisations of wild-type mice and their ProSAP1/Shank2(-/-) littermates lacking a synaptic scaffold protein mutated in ASD. We hypothesised that the vocal behaviour of ProSAP1/Shank2(-/-) mice not only differs from the vocal behaviour of their wild-type littermates in a quantitative way, but also presents more qualitative abnormalities in temporal organisation and acoustic structure. We first quantified the rate of emission of ultrasonic vocalisations, and analysed the organisation of vocalisations sequences using Markov models. We subsequently measured duration and peak frequency characteristics of each ultrasonic vocalisation, to characterise their acoustic structure. In wild-type mice, we found a high level of organisation in sequences of ultrasonic vocalisations, suggesting a communicative function in this complex system. Very limited significant sex-related variations were detected in their usage and acoustic structure, even in adult mice. In adult ProSAP1/Shank2(-/-) mice, we found abnormalities in the call usage and the structure of ultrasonic vocalisations. Both ProSAP1/Shank2(-/-) male and female mice uttered less vocalisations with a different call distribution and at lower peak frequency in comparison with wild-type littermates. This study provides a comprehensive framework to characterise abnormalities of ultrasonic vocalisations in mice and confirms that ProSAP1/Shank2(-/-) mice represent a relevant model to study communication defects.

Since we first identified the PLC-β isozyme, enormous studies have been conducted to investigate the functional roles of this protein (Min et al., 1993; Suh et al.,1988). It is now well-known that the four PLC-β subtypes are major effector molecules in GPCR-mediated signaling, especially for intracellular Ca2+ signaling. Nonetheless, it is still poorly understood why multiple PLC-β subtype exist. Most cells express multiple subtypes of PLC-β in different combinations, and each subtype is involved in somewhat different signaling pathways. Therefore, studying the differential roles of each PLC-β subtype is a very interesting issue. In this regard, we focus here on PDZ domain proteins which are novel PLC-β interacting proteins. As scaffolders, PDZ domain proteins recruit various target proteins ranging from membrane receptors to cytoskeletal proteins to assemble highly organized signaling complexes; this can give rise to efficiency and diversity in cellular signaling. Because PLC-β subtypes have different PDZ-binding motifs, it is possible that they are engaged with different PDZ domain proteins, and in turn participate in distinct physiological responses. To date, several PDZ domain proteins, such as the NHERF family, Shank2, and Par-3, have been reported to selectively interact with certain PLC-β subtypes and GPCRs. Systematic predictions of potential binding partners also suggests differential binding properties between PLC-β subtypes. Furthermore, we elucidated parallel signaling processes for multiple PLC-β subtypes, which still perform distinct functions resulting from differential interactions with PDZ domain proteins within a single cell. Therefore, these results highlight the novel function of PDZ domain proteins as intermediaries in subtype-specific role of PLC-β in GPCR-mediated signaling. Future studies will focus on the physiological meanings of this signaling complex formation by different PDZ domain proteins and PLC-β subtypes. It has been observed for a long time that the expression of certain PLC-β subtype fluctuates during diverse physiological conditions. For example, the expression of PLC-β1 is selectively increased during myoblast and adipocyte differentiation (Faenza et al., 2004; O'Carroll et al., 2009). Likewise, PLC-β2 is highly up-regulated during breast cancer progression and plays a critical role in cell migration and mitosis (Bertagnolo et al., 2007). Although PLC-β3 is selectively down-regulated in neuroendocrine tumors, the expression of PLC-β1 is increased in small cell lung carcinoma (Stalberg et al., 2003; Strassheim et al., 2000). In our hypothetical model, it is most likely that up- and down regulation of certain PLC-β subtypes are due to their selective coupling with specific GPCR-mediated signaling, implicated in these pathophysiologic conditions. Therefore, better understanding of selective coupling between PLC-β subtypes, PDZ domain proteins, and GPCRs will shed light on new prognosis and therapy of diverse diseases, and provide potential targets for drug development.

Pre- and post-synaptic targeting of synaptic molecules is depending upon specific targeting signals that are encoded within defined regions of the respective protein. For the post-synaptic scaffolding proteins of excitatory synapses, ProSAP1/Shank2 and ProSAP2/Shank3 this targeting information is located within about 460aa of the C-terminus. We found the C-terminal targeting signal to be bipartite composed of a 135aa stretch and the SAM (sterile alpha motif) domain embedding a relatively large variable spacer region. Based on this we developed a new GFP vector system called pSDTarget to easily clone proteins of interest as GFP fusion proteins flanked by a bipartite targeting signal for post-synaptic densities (PSDs) of excitatory synapses. The targeting signal has been derived from the PSD scaffolding protein ProSAP1/Shank2. In hippocampal neuron culture we could effectively localize and attach i.e. Glutathion-S-transferase (GST) at PSDs of excitatory synapses already during early synaptogenesis. Moreover, Gephyrin, an important scaffold molecule of inhibitory post-synapses was succesfully targeted to excitatory synapses followed by the subsequent recruitment of GABAergic receptors leading to hybrid synaptic contacts. In light of the role of specific protein domains for plastic changes of the post-synaptic compartment or investigations focusing on synaptogenesis, signalling and/or transsynaptic crosstalk this vector system provides a powerful and innovative tool for the functional analysis of molecular mechanisms and structural changes in a small but well defined neuronal compartment.

Glutamate receptor (GluR) delta2 selectively expressed in cerebellar Purkinje cells plays a central role in cerebellar long-term depression (LTD), motor learning, and formation of parallel fiber synapses. By yeast two-hybrid screening, we identified members of the Shank family of scaffold proteins as major GluRdelta2-interacting molecules. GluRdelta2 bound directly to the PDZ domain of Shank proteins through an internal motif in the carboxyl-terminal putative cytoplasmic domain. Shank1 and Shank2 proteins as well as GluRdelta2 proteins were localized in the dendritic spines of cultured Purkinje cells. Anti-GluRdelta2 antibodies immunoprecipitated Shank1, Shank2, Homer, and metabotropic GluR1alpha proteins from the synaptosomal membrane fractions of cerebella. Furthermore, Shank2 interacted with GRIP1 in the cerebellum. These results suggest that through Shank1 and Shank2, GluRdelta2 interacts with the metabotropic GluR1alpha, the AMPA-type GluR, and the inositol 1,4,5-trisphosphate receptor (IP3R) that are essential for cerebellar LTD.

The postsynaptic density is the ultrastructural entity containing the neurotransmitter reception apparatus of excitatory synapses in the brain. A recently identified family of multidomain proteins termed Src homology 3 domain and ankyrin repeat-containing (Shank), also known as proline-rich synapse-associated protein/somatostatin receptor-interacting protein, plays a central role in organizing the subsynaptic scaffold by interacting with several synaptic proteins including the glutamate receptors. We used the N-terminal ankyrin repeats of Shank1 and -3 to search for interacting proteins by yeast two-hybrid screening and by affinity chromatography. By cDNA sequencing and mass spectrometry the cytoskeletal protein α-fodrin was identified as an interacting molecule. The interaction was verified by pull-down assays and by coimmunoprecipitation experiments from transfected cells and brain extracts. Mapping of the interacting domains of α-fodrin revealed that the highly conserved spectrin repeat 21 is sufficient to bind to the ankyrin repeats. Both interacting partners are coexpressed widely in the rat brain and are colocalized in synapses of hippocampal cultures. Our data indicate that the Shank1 and -3 family members provide multiple independent connections between synaptic glutamate receptor complexes and the cytoskeleton.

The Shank family of proteins interacts with NMDA receptor and metabotropic glutamate receptor complexes in the postsynaptic density (PSD). Targeted to the PSD by a PDZ-dependent mechanism, Shank promotes the maturation of dendritic spines and the enlargement of spine heads via its ability to recruit Homer to postsynaptic sites. Shank and Homer cooperate to induce accumulation of IP3 receptors in dendritic spines and formation of putative multisynapse spines. In addition, postsynaptic expression of Shank enhances presynaptic function, as measured by increased minifrequency and FM4-64 uptake. These data suggest a central role for the Shank scaffold in the structural and functional organization of the dendritic spine and synaptic junction.

Shank1, Shank2, and Shank3 constitute a family of proteins that may function as molecular scaffolds in the postsynaptic density (PSD). Shank directly interacts with GKAP and Homer, thus potentially bridging the N-methyl-d-aspartate receptor-PSD-95-GKAP complex and the mGluR-Homer complex in synapses (Naisbitt, S., Kim, E., Tu, J. C., Xiao, B., Sala, S., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Neuron 23, 569–582; 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., and Worley, P. F. (1999) Neuron 23, 583–592). Shank contains multiple domains for protein-protein interaction including ankyrin repeats, an SH3 domain, a PSD-95/Dlg/ZO-1 domain, a sterile α motif domain, and a proline-rich region. By characterizingShank cDNA clones and RT-PCR products, we found that there are four sites for alternative splicing in Shank1 and another four sites in Shank2, some of which result in deletion of specific domains of the Shank protein. In addition, the expression of the splice variants is differentially regulated in different regions of rat brain during development. Immunoblot analysis of Shank proteins in rat brain using five different Shank antibodies reveals marked heterogeneity in size (120–240 kDa) and differential spatiotemporal expression. Shank1 immunoreactivity is concentrated at excitatory synaptic sites in adult brain, and the punctate staining of Shank1 is seen in developing rat brains as early as postnatal day 7. These results suggest that alternative splicing in the Shank family may be a mechanism that regulates the molecular structure of Shank and the spectrum of Shank-interacting proteins in the PSDs of adult and developing brain.

We have recently isolated a novel proline-rich synapse-associated protein-1 (ProSAP1) that is highly enriched in postsynaptic density (PSD). A closely related multidomain protein, ProSAP2, shares a highly conserved PDZ (PSD-95/discs-large/ZO-1) domain (80% identity), a ppI domain that mediates the interaction with cortactin, and a C-terminal SAM (sterile alpha-motif) domain. In addition, ProSAP2 codes for five ankyrin repeats and a SH3 (Src homology 3) domain. Transcripts for both proteins are coexpressed in many regions of rat brain, but show a distinct expression pattern in the cerebellum. Using the PDZ domains of ProSAP1 and 2 as bait in the yeast two-hybrid system, we isolated several clones of the SAPAP/GKAP (SAP90/PSD-95-associated protein/guanylate kinase-associated protein) family. The association of the proteins was verified by coimmunoprecipitation and cotransfection in HEK cells. Therefore, proteins of the ProSAP family represent a novel link between SAP90/PSD-95 bound membrane receptors and the cytoskeleton at glutamatergic synapses of the central nervous system.

Shank is a recently described family of postsynaptic proteins that function as part of the NMDA receptor–associated PSD-95 complex (Naisbitt et al., 1999 [this issue of Neuron ]). Here, we report that Shank proteins also bind to Homer. Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors and inositol trisphosphate receptors, thereby coupling these receptors in a signaling complex. A single Homer-binding site is identified in Shank, and Shank and Homer coimmunoprecipitate from brain and colocalize at postsynaptic densities. Moreover, Shank clusters mGluR5 in heterologous cells in the presence of Homer and mediates the coclustering of Homer with PSD-95/GKAP. Thus, Shank may cross-link Homer and PSD-95 complexes in the PSD and play a role in the signaling mechanisms of both mGluRs and NMDA receptors.

NMDA receptors are linked to intracellular cytoskeletal and signaling molecules via the PSD-95 protein complex. We report a novel family of postsynaptic density (PSD) proteins, termed Shank, that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton.