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Authors would like to acknowledge the following funders: Motor Neurone Disease (MND) Association UK (Miles/Apr18/863-791), the Euan MacDonald Centre and Chief Scientist Office, The European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (695568 SYNNOVATE), Simons Foundation Autism Research Initiative (529085), and the Wellcome Trust (Technology Development grant 202932). Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder. Separate lines of evidence suggest that synapses and astrocytes play a role in the pathological mechanisms underlying ALS. Given that astrocytes make specialised contacts with some synapses, called tripartite synapses, we hypothesise that tripartite synapses could act as the fulcrum of disease in ALS. To test this hypothesis, we have performed an extensive microscopy-based investigation of synapses and tripartite synapses in the spinal cord of ALS model mice and post-mortem human tissue from ALS cases. We reveal widescale synaptic changes at the early symptomatic stages of the SOD1G93a mouse model. Super-resolution microscopy reveals that large complex postsynaptic structures are lost in ALS mice. Most surprisingly, tripartite synapses are selectively lost, while non-tripartite synapses remain in equal number to healthy controls. Finally, we also observe a similar selective loss of tripartite synapses in human post-mortem ALS spinal cords. From these data we conclude that tripartite synaptopathy is a key hallmark of ALS. Publisher PDF

The lifetime of proteins in synapses is important for their signaling, maintenance, and remodeling, and for memory duration. We quantified the lifetime of endogenous PSD95, an abundant postsynaptic protein in excitatory synapses, at single-synapse resolution across the mouse brain and lifespan, generating the Protein Lifetime Synaptome Atlas. Excitatory synapses have a wide range of PSD95 lifetimes extending from hours to several months, with distinct spatial distributions in dendrites, neurons, and brain regions. Synapses with short protein lifetimes are enriched in young animals and in brain regions controlling innate behaviors, whereas synapses with long protein lifetimes accumulate during development, are enriched in the cortex and CA1 where memories are stored, and are preferentially preserved in old age. Synapse protein lifetime increases throughout the brain in a mouse model of autism and schizophrenia. Protein lifetime adds a further layer to synapse diversity and enriches prevailing concepts in brain development, aging, and disease., Sarah Lempriere, Cathy McLaughlin, Emma Sigfridsson, Rand Dahan, Gabor Varga, Emily Robson, Theresa Wong, Bev Notman, Ian Hawes, Dimitra Koukaroudi, William Mungall, and Fabio De Moliner for technical assistance. Colin Davey for editing. Debbie Maizels for artwork. Trevor Robbins, Szu-Han Wang, Tim Bussey for comments on the manuscript. Funding: S.G.N.G.: The European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (695568 SYNNOVATE), Simons Foundation Autism Research Initiative (529085), and the Wellcome Trust (Technology Development Grant 202932). J.D.: Interdisciplinary Platform Cajal Blue Brain (CSIC, Spain). P.M.-S.: Spanish Ministerio de Ciencia e Innovación (IJCI-2016-27658). T.J.O.: National Institute of Mental Health Grant (R01MH060919-15). A.H.: Swiss National Science Foundation (grant 31003A_173125), the Swiss National Centre Competence in Research (NCCR) Synapsy (grant 51NF40-185897), and a gift from a private foundation with public interest through the International Foundation for Research in Paraplegia (chair Alain Rossier). For the purpose of open access, the author has applied a CC-BY public copyright license to any author accepted manuscript version arising from this submission.

Neurodevelopmental disorders of genetic origin delay the acquisition of normal abilities and cause disabling phenotypes. Spontaneous attenuation and even complete amelioration of symptoms in early childhood and adolescence occur in many disorders1–10, suggesting that brain circuits possess an intrinsic capacity to repair themselves. We examined the molecular composition of almost a trillion excitatory synapses on a brain-wide scale between birth and adulthood in mice carrying a mutation in the homeobox transcription factor Pax6, a neurodevelopmental disorder model11. Pax6 haploinsufficiency had no impact on total synapse number at any age. By contrast, the postnatal expansion of synapse diversity and acquisition of normal synaptome architecture were delayed in all brain regions, interfering with network and cognitive functions. Specific excitatory synapse types and subtypes were affected in two key developmental age-windows. These phenotypes were reversed within 2-3 weeks of onset, restoring synaptome architecture to its normal developmental trajectory. Synapse subtypes with high rates of protein turnover mediated these events. These results show synaptome remodelling confers resilience to neurodevelopmental disorders.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder. Separate lines of evidence suggest that synapses and astrocytes play a role in the pathological mechanisms underlying ALS. Given that astrocytes make specialised contacts with some synapses, called tripartite synapses, we hypothesise that tripartite synapses could act as the fulcrum of disease in ALS. To test this hypothesis, we have performed an extensive microscopy-based investigation of synapses and tripartite synapses in the spinal cord of ALS model mice and post-mortem human tissue from ALS cases. We reveal widescale synaptic changes at the early symptomatic stages of the SOD1G93a mouse model. Super-resolution microscopy reveals that large complex postsynaptic structures are lost in ALS mice. Most surprisingly, tripartite synapses are selectively lost while non-tripartite synapses remain in equal number to healthy controls. Finally, we also observe a similar selective loss of tripartite synapses in human post-mortem ALS spinal cords. From these data we conclude that tripartite synaptopathy is a key hallmark of ALS.

Aims Synaptic Ras GTPase‐activating protein 1 (SYNGAP1) regulates synaptic plasticity through AMPA receptor trafficking. SYNGAP1 mutations have been found in human patients with intellectual disability (ID) and autism spectrum disorder (ASD). Almost every individual with SYNGAP1‐related ID develops epilepsy, and approximately 50% have ASD. SYNGAP1‐related ID is estimated to account for at least 1% of ID cases. In mouse models with Syngap1 mutations, strong cognitive and affective dysfunctions have been reported, yet some findings are inconsistent across studies. To further understand the behavioral significance of the SYNGAP1 gene, we assessed various domains of behavior in Syngap1 heterozygous mutant mice using a behavioral test battery. Methods Male mice with a heterozygous mutation in the Syngap1 gene (Syngap1 −/+ mice) created by Seth Grant's group were subjected to a battery of comprehensive behavioral tests, which examined general health, and neurological screens, rotarod, hot plate, open field, light/dark transition, elevated plus maze, social interaction, prepulse inhibition, Porsolt forced swim, tail suspension, gait analysis, T‐maze, Y‐maze, Barnes maze, contextual and cued fear conditioning, and home cage locomotor activity. To control for type I errors due to multiple‐hypothesis testing, P‐values below the false discovery rate calculated by the Benjamini‐Hochberg method were considered as study‐wide statistically significant. Results Syngap1 −/+ mice showed increased locomotor activity, decreased prepulse inhibition, and impaired working and reference spatial memory, consistent with preceding studies. Impairment of context fear memory and increased startle reflex in Syngap1 mutant mice could not be reproduced. Significant decreases in sensitivity to painful stimuli and impaired motor function were observed in Syngap1 −/+ mice. Decreased anxiety‐like behavior and depression‐like behavior were noted, although increased locomotor activity is a potential confounding factor of these phenotypes. Increased home cage locomotor activity indicated hyperlocomotor activity not only in specific behavioral test conditions but also in familiar environments. Conclusion In Syngap1 −/+ mice, we could reproduce most of the previously reported cognitive and emotional deficits. The decreased sensitivity to painful stimuli and impaired motor function that we found in Syngap1 −/+ mice are consistent with the common characteristics of patients with SYNGAP‐related ID. We further confirmed that the Syngap1 heterozygote mouse recapitulates the symptoms of ID and ASD patients., In Syngap1 −/+ mice, a well‐known model of SYNGAP‐related ID, we could reproduce most of the previously reported cognitive and emotional deficits. The decreased sensitivity to painful stimuli and impaired motor function that we found in Syngap1 −/+ mice are consistent with the common characteristics of patients with SYNGAP‐related ID. We further confirmed that the Syngap1 heterozygote mouse recapitulates the symptoms of ID and ASD patients.

Glutamatergic synapses form the vast majority of connections within neuronal circuits but how these subcellular structures are molecularly organized within the mammalian brain is poorly understood. Conventional electron microscopy using chemically fixed, metal-stained tissue has identified a proteinaceous, membrane-associated thickening called the ‘postsynaptic density’ (PSD). Here, we combined mouse genetics and cryo-electron tomography to determine the 3D molecular architecture of fresh synapses in the adult forebrain. The native glutamatergic synapse lacked a PSD. Instead, a concentrated ‘synaptoplasm’ consisting of cytoskeletal elements, macromolecular complexes and membrane-bound organelles extended throughout the pre- and post-synaptic compartments. Snapshots of active processes gave insights into the architectural basis for synaptic remodeling. Clusters of 4-60 ionotropic glutamate receptors were positioned inside and outside the synaptic cleft. Together, these information-rich tomographic maps present a detailed molecular framework for the coordinated activity within mammalian brain synapses.

The postsynaptic terminal of vertebrate excitatory synapses contains a highly conserved multiprotein complex that comprises neurotransmitter receptors, cell-adhesion molecules, scaffold proteins and enzymes, which are essential for brain signalling and plasticity underlying behaviour. Increasingly, mutations in genes that encode postsynaptic proteins belonging to the PSD-95 protein complex, continue to be identified in neurodevelopmental disorders (NDDs) such as autism spectrum disorder, intellectual disability and epilepsy. These disorders are highly heterogeneous, sharing genetic aetiology and comorbid cognitive and behavioural symptoms. Here, by using genetically engineered mice and innovative touchscreen-based cognitive testing, we sought to investigate whether loss-of-function mutations in genes encoding key interactors of the PSD-95 protein complex display shared phenotypes in associative learning, updating of learned associations and reaction times. Our genetic dissection of mice with loss-of-function mutations in Syngap1, Nlgn3, Dlgap1, Dlgap2 and Shank2 showed that distinct components of the PSD-95 protein complex differentially regulate learning, cognitive flexibility and reaction times in cognitive processing. These data provide insights for understanding how human mutations in these genes lead to the manifestation of diverse and complex phenotypes in NDDs.

The postsynaptic terminal of vertebrate excitatory synapses contains a highly conserved multiprotein complex that comprises neurotransmitter receptors, cell-adhesion molecules, scaffold proteins and enzymes, which are essential for brain signalling and plasticity underlying behaviour. Increasingly, mutations in genes that encode postsynaptic proteins belonging to the PSD-95 protein complex, continue to be identified in neurodevelopmental disorders (NDDs) such as autism spectrum disorder, intellectual disability and epilepsy. These disorders are highly heterogeneous, sharing genetic aetiology and comorbid cognitive and behavioural symptoms. Here, by using genetically engineered mice and innovative touchscreen-based cognitive testing, we sought to investigate whether loss-of-function mutations in genes encoding key interactors of the PSD-95 protein complex display shared phenotypes in associative learning, updating of learned associations and reaction times. Our genetic dissection of mice with loss-of-function mutations in Syngap1, Nlgn3, Dlgap1, Dlgap2 and Shank2 showed that distinct components of the PSD-95 protein complex differentially regulate learning, cognitive flexibility and reaction times in cognitive processing. These data provide insights for understanding how human mutations in these genes lead to the manifestation of diverse and complex phenotypes in NDDs.

A recently reported rapid potentiation of NMDA receptors by Group I metabotropic glutamate receptors (mGluRIs) via a Homer protein link is distinct from the classical, relatively slow inhibitory G-protein-associated signaling triggered by mGluRI activation. The relationship between these two mechanisms remains unknown. Here, we focused on the mGluRI-dependent modulation of NMDAR response in hippocampal dentate gyrus granule cells and cerebellar granule cells of C57BL6-J mice and found that these two contrasting mechanisms overlap competitively on the time scale from hundreds of milliseconds to seconds, with the net effect depending on the cell type. At a shorter time interval (units of millisecond), the Homer-mediated signal from mGluRIs prevails, causing upregulation of NMDAR function, in both dentate gyrus granule cells and cerebellar granule cells. Our results shed light on the possible mechanisms of anti-schizophrenia drugs that disrupt Homer-containing protein link.SIGNIFICANCE STATEMENTHere we study modulation of NMDA receptors triggered by activation of metabotropic glutamate receptors Group I via two distinct pathways: classical G-protein signaling system and newly discovered high-speed modulatory mechanism associated with Homer-protein-containing direct molecular link. We found that these two contrasting mechanisms overlap competitively on the time scale from hundreds of milliseconds to seconds, with the net effect depending on the cell type. We have also found that both crosstalk mechanisms cause significant changes in synaptic strength and plasticity. Our results resolve an apparent discrepancy between earlier studies that demonstrated contradictive effects of Homer-containing protein link disruption on NMDA receptor signaling. On top of that, our data provide a plausible explanation for unclear action mechanisms of anti-schizophrenia drugs.

Summary Arc is an activity-regulated neuronal protein, but little is known about its interactions, assembly into multiprotein complexes, and role in human disease and cognition. We applied an integrated proteomic and genetic strategy by targeting a tandem affinity purification (TAP) tag and Venus fluorescent protein into the endogenous Arc gene in mice. This allowed biochemical and proteomic characterization of native complexes in wild-type and knockout mice. We identified many Arc-interacting proteins, of which PSD95 was the most abundant. PSD95 was essential for Arc assembly into 1.5-MDa complexes and activity-dependent recruitment to excitatory synapses. Integrating human genetic data with proteomic data showed that Arc-PSD95 complexes are enriched in schizophrenia, intellectual disability, autism, and epilepsy mutations and normal variants in intelligence. We propose that Arc-PSD95 postsynaptic complexes potentially affect human cognitive function., Graphical Abstract, Highlights • TAP tag and purification of endogenous Arc protein complexes from the mouse brain • PSD95 is the major Arc binding protein, and both assemble into 1.5-MDa supercomplexes • PSD95 is essential for recruitment of Arc to synapses • Mutations and genetic variants in Arc-PSD95 are linked to cognition, Fernández et al. use genetics and proteomics to study the Arc protein in the mouse brain. PSD95 recruits Arc to the synapse and assembles it into signaling complexes with neurotransmitter receptors and other proteins. Arc-PSD95 supercomplexes contain genetic variants previously linked to epilepsy, schizophrenia, intellectual disability, and IQ.

How synapses change molecularly during the lifespan and across all brain circuits is unknown. We analyzed the protein composition of billions of individual synapses from birth to old age on a brain-wide scale in the mouse, revealing a program of changes in the lifespan synaptome architecture spanning individual dendrites to the systems level. Three major phases were uncovered, corresponding to human childhood, adulthood and old age. An arching trajectory of synaptome architecture drives the differentiation and specialization of brain regions to a peak in young adults before dedifferentiation returns the brain to a juvenile state. This trajectory underscores changing network organization and hippocampal physiology that may account for lifespan transitions in intellectual ability and memory, and the onset of behavioral disorders.One sentence summaryThe synaptome architecture of the mouse brain undergoes continuous changes that organize brain circuitry across the lifespan.

In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1,000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons, the extent of synapse diversity across the brain is largely unknown. This is mainly due to the limitations of current techniques. Here, we report an efficient method for the purification of synaptic protein complexes, fusing a high‐affinity tag to endogenous PSD95 in specific cell types. We also developed a strategy, which enables the visualisation of endogenous PSD95 with fluorescent‐protein tag in Cre‐recombinase‐expressing cells. We demonstrate the feasibility of proteomic analysis of synaptic protein complexes and visualisation of these in specific cell types. We find that the composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. The results suggest that there might be differential interactions in the PSD95 complexes in different brain regions. We have detected differentially interacting proteins by comparing data sets from the whole hippocampus and the CA3 subfield of the hippocampus. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific brain regions and subsets of neuronal cells, but also provide an opportunity to better understand brain region‐ and cell‐type‐specific alterations associated with various psychiatric/neurological diseases. These newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses., We have developed an efficient method for the purification of synaptic protein complexes, fusing a high‐affinity tag to endogenous PSD95 in specific cell types. We also created a labelling strategy that enables visualisation of PSD95 with a fluorescent tag in Cre‐expressing cells. We demonstrated the feasibility of visualisation and proteomic analysis of synapse protein complexes in specific cell types. These newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses.

Dendritic spines are the postsynaptic sites that receive most of the excitatory synaptic inputs, and thus provide the structural basis for synaptic function. Here, we describe an accurate method for measurement and analysis of spine morphology based on structured illumination microscopy (SIM) and computational geometry in cultured neurons. Surface mesh data converted from SIM images were comparable to data reconstructed from electron microscopic images. Dimensional reduction and machine learning applied to large data sets enabled identification of spine phenotypes caused by genetic mutations in key signal transduction molecules. This method, combined with time-lapse live imaging and glutamate uncaging, could detect plasticity-related changes in spine head curvature. The results suggested that the concave surfaces of spines are important for the long-term structural stabilization of spines by synaptic adhesion molecules., We are currently short of methods that can extract objective parameters of dendritic spines useful for their categorization. Authors present in this study an automatic analytical pipeline for spine geometry using 3D-structured illumination microscopy, which can effectively extract many geometrical parameters of dendritic spines without bias and automatically categorize spine population based on their morphological features

How is the information encoded within patterns of nerve impulses converted into diverse behavioral responses? To address this question, we conducted the largest genetic study to date of the electrophysiological and behavioral properties of synapses. Postsynaptic responses to elementary patterns of activity in the hippocampal CA1 region were measured in 58 lines of mice carrying mutations in the principal classes of excitatory postsynaptic proteins. A combinatorial molecular mechanism was identified in which distinct subsets of proteins amplified or attenuated responses across timescales from milliseconds to an hour. The same mechanism controlled the diversity and magnitude of innate and learned behavioral responses. PSD95 supercomplex proteins were central components of this synaptic machinery. The capacity of vertebrate synapses to compute activity patterns increased with genome evolution and is impaired by disease-relevant mutations. We propose that this species-conserved molecular mechanism converts the temporally encoded information in nerve impulses into the repertoire of innate and learned behavior.

Although molecular mechanisms underpinning specific behaviors have been described, whether there are mechanisms that orchestrate a behavioral repertoire is unknown. To test if the postsynaptic proteome of excitatory synapses could impart such a mechanism we conducted the largest genetic study of mammalian synapses yet undertaken. A repertoire of sixteen innate and learned behaviors was assessed from 290,850 measures in 55 lines of mutant mice carrying targeted mutations in the principal classes of postsynaptic proteins. Each innate and learned behavior used a different combination of proteins. These combinations were comprised of proteins that amplified or attenuated the magnitude of each behavioral response. All behaviors required proteins found in PSD95 supercomplexes. We show the vertebrate increase in proteome complexity drove an expansion in behavioral repertoires and generated susceptibility to a wide range of diseases. Our results reveal a molecular mechanism that generates a versatile and complex behavioral repertoire that is central to human behavioral disorders.

In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons and synapses, the extent of synapse diversity across the brain is largely unknown, mainly owing to technical limitations. Combining mouse genetics and proteomics we have previously reported highly efficient methods for purification of synaptic protein complexes under native conditions. In that approach, tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD95 using gene targeting in mice. Here we report an approach that restricts tagging of endogenous PSD95 to cells expressing Cre recombinase. In addition, we developed a labelling strategy enabling visualization of endogenous PSD95 tagged by fluorescent proteins in Cre-expressing cells. We demonstrate the feasibility of proteomic characterisation of synapse proteomes and visualization of synapse proteins in specific cell types. We find that composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific subsets of regions/neuronal cells, but also provide an opportunity to better understand brain region-specific alterations associated with various psychiatric/neurological diseases. The newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will thus facilitate research on the molecular complexity of synapses.

The scaffold protein DLGAP1 is localized at the post-synaptic density (PSD) of glutamatergic neurons and is a component of supramolecular protein complexes organized by PSD95. Gain-of-function variants of DLGAP1 have been associated with obsessive-compulsive disorder (OCD), while haploinsufficient variants have been linked to autism spectrum disorder (ASD) and schizophrenia in human genetic studies. We tested male and female Dlgap1 wild type (WT), heterozygous (HT), and knockout (KO) mice in a battery of behavioral tests: open field, dig, splash, prepulse inhibition, forced swim, nest building, social approach, and sucrose preference. We also used biochemical approaches to examine the role of DLGAP1 in the organization of PSD protein complexes. Dlgap1 KO mice were most notable for disruption of protein interactions in the PSD, and deficits in sociability. Other behavioral measures were largely unaffected. Our data suggest that Dlgap1 knockout leads to PSD disruption and reduced sociability, consistent with reports of DLGAP1 haploinsufficient variants in schizophrenia and ASD.

Previous studies have hypothesized that diverse genetic causes of intellectual disability (ID) and autism spectrum disorders (ASDs) converge on common cellular pathways. Testing this hypothesis requires detailed phenotypic analyses of animal models with genetic mutations that accurately reflect those seen in the human condition (i.e., have structural validity) and which produce phenotypes that mirror ID/ASDs (i.e., have face validity). We show that SynGAP haploinsufficiency, which causes ID with co-occurring ASD in humans, mimics and occludes the synaptic pathophysiology associated with deletion of theFmr1gene.Syngap+/−andFmr1−/ymice show increases in basal protein synthesis and metabotropic glutamate receptor (mGluR)-dependent long-term depression that, unlike in their wild-type controls, is independent of new protein synthesis. Basal levels of phosphorylated ERK1/2 are also elevated inSyngap+/−hippocampal slices. Super-resolution microscopy reveals thatSyngap+/−andFmr1−/ymice show nanoscale alterations in dendritic spine morphology that predict an increase in biochemical compartmentalization. Finally, increased basal protein synthesis is rescued by negative regulators of the mGlu subtype 5 receptor and the Ras–ERK1/2 pathway, indicating that therapeutic interventions for fragile X syndrome may benefit patients with SYNGAP1 haploinsufficiency.SIGNIFICANCE STATEMENTAs the genetics of intellectual disability (ID) and autism spectrum disorders (ASDs) are unraveled, a key issue is whether genetically divergent forms of these disorders converge on common biochemical/cellular pathways and hence may be amenable to common therapeutic interventions. This study compares the pathophysiology associated with the loss of fragile X mental retardation protein (FMRP) and haploinsufficiency of synaptic GTPase-activating protein (SynGAP), two prevalent monogenic forms of ID. We show thatSyngap+/−mice phenocopyFmr1−/ymice in the alterations in mGluR-dependent long-term depression, basal protein synthesis, and dendritic spine morphology. Deficits in basal protein synthesis can be rescued by pharmacological interventions that reduce the mGlu5receptor–ERK1/2 signaling pathway, which also rescues the same deficit inFmr1−/ymice. Our findings support the hypothesis that phenotypes associated with genetically diverse forms of ID/ASDs result from alterations in common cellular/biochemical pathways.

PSD-95 is one of the most abundant proteins of the postsynaptic density of excitatory synapses. It functions as the backbone of protein supercomplexes that mediate signalling between membrane glutamate receptors and intracellular pathways. Homozygous deletion of the Dlg4 gene encoding PSD-95 was previously found to cause a profound impairment in operant and Pavlovian conditioning in Dlg4 −/− mice studied in touch screen chambers that precluded evaluation of PSD-95's role in shaping more subtle forms of learning and memory. In this study, using a battery of touch screen tests, we investigated cognitive behaviour of mice with a heterozygous Dlg4 mutation. We found that in contrast to learning deficits of Dlg4 −/− mice, Dlg4 +/− animals demonstrated enhanced performance in the Visual Discrimination, Visual Discrimination Reversal and Paired-Associates Learning touch screen tasks. The divergent directions of learning phenotypes observed in Dlg4 −/− and Dlg4 +/− mice also contrasted with qualitatively similar changes in the amplitude and plasticity of field excitatory postsynaptic potentials recorded in the CA1 area of hippocampal slices from both mutants. Our results have important repercussions for the studies of genetic models of human diseases, because they demonstrate that reliance on phenotypes observed solely in homozygous mice may obscure qualitatively different changes in heterozygous animals and potentially weaken the validity of translational comparisons with symptoms seen in heterozygous human carriers.

Background: Synaptic Ras-GTPase-activating protein 1 (SYNGAP1) is an abundant brain-specific protein localized at the postsynaptic density of mammalian excitatory synapses. SYNGAP1 functions as a crucial regulator of downstream intracellular signaling triggered by N-methyl-D-aspartate receptor activation. One of the most important signaling pathways regulated by SYNGAP1 is the Ras-Raf-MEK-ERK pathway. SYNGAP1 deficiency is associated with hyperphosphorylation of MEK and ERK kinases and with altered synaptic function in Syngap1(+/-) mice. Loss-of-function mutations in the SYNGAP1 gene have been documented in many human cognitive and neurological disorders. However, there are currently no approaches that reverse the phenotypes of SYNGAP1 deficiency. Methods: Using electrophysiological recordings of field responses in hippocampal slices, we examined if disturbances of synaptic physiology in the hippocampus of 7-8-month old Syngapr1(+/-) mice were sensitive to the effect of the MEK inhibitor PD-0325901 given orally for 6 days. Results: We found that in hippocampal slices from vehicle-treated Syngap1(+/-) mice, basal synaptic responses were higher and their long-term potentiation (LTP) was lower than in slices from wild-type littermates. Chronic administration of PD-0325901 normalized basal synaptic responses, but did not reverse LTP deficit. Conclusions: The differential sensitivity of basal synaptic transmission and LTP to MEK inhibition indicates that the effects of SYNGAP1 deficiency on these synaptic parameters are mediated by distinct pathways. Our findings also suggest that at least some physiological phenotypes of the germline Syngap1 mutation can be ameliorated by pharmacological treatment of adult animals. (C) 2018 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.

Summary Synapses are found in vast numbers in the brain and contain complex proteomes. We developed genetic labeling and imaging methods to examine synaptic proteins in individual excitatory synapses across all regions of the mouse brain. Synapse catalogs were generated from the molecular and morphological features of a billion synapses. Each synapse subtype showed a unique anatomical distribution, and each brain region showed a distinct signature of synapse subtypes. Whole-brain synaptome cartography revealed spatial architecture from dendritic to global systems levels and previously unknown anatomical features. Synaptome mapping of circuits showed correspondence between synapse diversity and structural and functional connectomes. Behaviorally relevant patterns of neuronal activity trigger spatiotemporal postsynaptic responses sensitive to the structure of synaptome maps. Areas controlling higher cognitive function contain the greatest synapse diversity, and mutations causing cognitive disorders reorganized synaptome maps. Synaptome technology and resources have wide-ranging application in studies of the normal and diseased brain., Graphical Abstract, Highlights • Single-synapse-resolution maps of postsynaptic proteins across the whole mouse brain • Systematic catalogs and anatomical mapping of synapse types and subtypes • Synapse diversity is organized into a synaptome map architecture • Disease-relevant mutations reprogram synaptome maps, Synapse molecular composition and diversity was systematically mapped across the whole mouse brain. A molecular logic orchestrates the spatial architecture of synapse diversity from individual dendrites to the systems level. Synaptome architecture and function is important for cognition and disease.

Aberrant NMDA receptor (NMDAR) activity contributes to several neurological disorders, but direct antagonism is poorly tolerated therapeutically. The GluN2B cytoplasmic C-terminal domain (CTD) represents an alternative therapeutic target since it potentiates excitotoxic signaling. The key GluN2B CTD-centred event in excitotoxicity is proposed to involve its phosphorylation at Ser-1303 by Dapk1, that is blocked by a neuroprotective cell-permeable peptide mimetic of the region. Contrary to this model, we find that excitotoxicity can proceed without increased Ser-1303 phosphorylation, and is unaffected by Dapk1 deficiency in vitro or following ischemia in vivo. Pharmacological analysis of the aforementioned neuroprotective peptide revealed that it acts in a sequence-independent manner as an open-channel NMDAR antagonist at or near the Mg2+ site, due to its high net positive charge. Thus, GluN2B-driven excitotoxic signaling can proceed independently of Dapk1 or altered Ser-1303 phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.17161.001

Association studies implicate multiple PDZ domain protein (MPDZ/MUPP1) sequence and/or expression in risk for alcoholism in humans and ethanol withdrawal (EW) in mice, but confirmation has been hindered by the dearth of targeted genetic models. We report the creation of transgenic (MPDZ-TG) and knockout heterozygote (Mpdz(+/-) ) mice, with increased (2.9-fold) and decreased (53%) target expression, respectively. Both models differ in EW compared with wild-type littermates (P ≤ 0.03), providing compelling evidence for an inverse relationship between Mpdz expression and EW severity. Additionally, ethanol consumption is reduced up to 18% (P = 0.006) in Mpdz(+/-) , providing the first evidence implicating Mpdz in ethanol self-administration.

The origins and evolution of higher cognitive functions, including complex forms of learning, attention and executive functions, are unknown. A potential mechanism driving the evolution of vertebrate cognition early in the vertebrate lineage (550 million years ago) was genome duplication and subsequent diversification of postsynaptic genes. Here we report, to our knowledge, the first genetic analysis of a vertebrate gene family in cognitive functions measured using computerized touchscreens. Comparison of mice carrying mutations in each of the four Dlg paralogs showed that simple associative learning required Dlg4, whereas Dlg2 and Dlg3 diversified to have opposing functions in complex cognitive processes. Exploiting the translational utility of touchscreens in humans and mice, testing Dlg2 mutations in both species showed that Dlg2's role in complex learning, cognitive flexibility and attention has been highly conserved over 100 million years. Dlg-family mutations underlie psychiatric disorders, suggesting that genome evolution expanded the complexity of vertebrate cognition at the cost of susceptibility to mental illness.

Behavioural analysis of mice carrying engineered mutations is widely used to identify roles of specific genes in components of the mammalian behavioural repertoire. The reproducibility and robustness of phenotypic measures has become a concern that undermines the use of mouse genetic models for translational studies. Contributing factors include low individual study power, non-standardised behavioural testing, failure to address confounds and differences in genetic background of mutant mice. We have examined the importance of these factors using a statistically robust approach applied to behavioural data obtained from three mouse mutations on 129S5 and C57BL/6J backgrounds generated in a standardised battery of five behavioural assays. The largest confounding effect was sampling variation, which partially masked the genetic background effect. Our observations suggest that strong interaction of mutation with genetic background in mice in innate and learned behaviours is not necessarily to be expected. We found composite measures of innate and learned behaviour were similarly impacted by mutations across backgrounds. We determined that, for frequently-used group sizes, a single retest of a significant result conforming to the commonly used p

How neuronal proteomes self-organize is poorly understood because of their inherent molecular and cellular complexity. Here, focusing on mammalian synapses we use blue-native PAGE and ‘gene-tagging’ of GluN1 to report the first biochemical purification of endogenous NMDA receptors (NMDARs) directly from adult mouse brain. We show that NMDARs partition between two discrete populations of receptor complexes and ~1.5 MDa supercomplexes. We tested the assembly mechanism with six mouse mutants, which indicates a tripartite requirement of GluN2B, PSD93 and PSD95 gate the incorporation of receptors into ~1.5 MDa supercomplexes, independent of either canonical PDZ-ligands or GluN2A. Supporting the essential role of GluN2B, quantitative gene-tagging revealed a fourfold molar excess of GluN2B over GluN2A in adult forebrain. NMDAR supercomplexes are assembled late in postnatal development and triggered by synapse maturation involving epigenetic and activity-dependent mechanisms. Finally, screening the quaternary organization of 60 native proteins identified numerous discrete supercomplexes that populate the mammalian synapse., Seventh Framework Programme (European Commission) (grant agreement no. HEALTH-F2-2009-241498), Wellcome Trust (London, England), Wellcome Trust Sanger Institute, National Institute of Mental Health (U.S.)

Two genome duplications early in the vertebrate lineage expanded gene families, including GluN2 subunits of the NMDA receptor. Diversification between the four mammalian GluN2 proteins occurred primarily at their intracellular C-terminal domains (CTDs). To identify shared ancestral functions and diversified subunit-specific functions, we exchanged the exons encoding the GluN2A (also known as Grin2a) and GluN2B (also known as Grin2b) CTDs in two knock-in mice and analyzed the mice's biochemistry, synaptic physiology, and multiple learned and innate behaviors. The eight behaviors were genetically separated into four groups, including one group comprising three types of learning linked to conserved GluN2A/B regions. In contrast, the remaining five behaviors exhibited subunit-specific regulation. GluN2A/B CTD diversification conferred differential binding to cytoplasmic MAGUK proteins and differential forms of long-term potentiation. These data indicate that vertebrate behavior and synaptic signaling acquired increased complexity from the duplication and diversification of ancestral GluN2 genes.

Summary It is currently unclear whether the GluN2 subtype influences NMDA receptor (NMDAR) excitotoxicity. We report that the toxicity of NMDAR-mediated Ca2+ influx is differentially controlled by the cytoplasmic C-terminal domains of GluN2B (CTD2B) and GluN2A (CTD2A). Studying the effects of acute expression of GluN2A/2B-based chimeric subunits with reciprocal exchanges of their CTDs revealed that CTD2B enhances NMDAR toxicity, compared to CTD2A. Furthermore, the vulnerability of forebrain neurons in vitro and in vivo to NMDAR-dependent Ca2+ influx is lowered by replacing the CTD of GluN2B with that of GluN2A by targeted exon exchange in a mouse knockin model. Mechanistically, CTD2B exhibits stronger physical/functional coupling to the PSD-95-nNOS pathway, which suppresses protective CREB activation. Dependence of NMDAR excitotoxicity on the GluN2 CTD subtype can be overcome by inducing high levels of NMDAR activity. Thus, the identity (2A versus 2B) of the GluN2 CTD controls the toxicity dose-response to episodes of NMDAR activity., Highlights ► The CTD of GluN2B promotes excitotoxicity better than that of GluN2A ► GluN2 CTD subtype differences are seen in both WT and chimeric 2A/2B subunits ► The GluN2B CTD couples to a prodeath PSD-95/nNOS-dependent CREB shut-off pathway, Martel et al. find that the two subtypes (2A versus 2B) of the GluN2 C-terminal domain differentially couple to the CREB shut-off pathway, causing distinct effects on NMDA receptor-mediated neuronal death both in vitro and in vivo.

The SH3 domain of postsynaptic density 95 mediates inflammatory pain through phosphatidylinositol-3-kinase recruitment Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here the authors report that the SH3 domain of the scaffold protein PSD-95 binds a lipid signaling enzyme, PI3K-C2a, that mediates inflammatory sensitization. The results show that different types of behavioural plasticity are mediated by specific domains of PSD-95 and suggest that PI3K-C2a is a potential drug target for inflammatory pain., Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino-acid substitution in the polyproline-binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol-3-kinase-C2α to the SH3-binding site of PSD95. In wild-type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.

Understanding the mechanisms whereby information encoded within patterns of action potentials is deciphered by neurons is central to cognitive psychology. The multiprotein complexes formed by NMDA receptors linked to synaptic membrane-associated guanylate kinase (MAGUK) proteins including synapse-associated protein 102 (SAP102) and other associated proteins are instrumental in these processes. Although humans with mutations in SAP102 show mental retardation, the physiological and biochemical mechanisms involved are unknown. Using SAP102 knock-out mice, we found specific impairments in synaptic plasticity induced by selective frequencies of stimulation that also required extracellular signal-regulated kinase signaling. This was paralleled by inflexibility and impairment in spatial learning. Improvement in spatial learning performance occurred with extra training despite continued use of a suboptimal search strategy, and, in a separate nonspatial task, the mutants again deployed a different strategy. Double-mutant analysis of postsynaptic density-95 and SAP102 mutants indicate overlapping and specific functions of the two MAGUKs. Thesein vivodata support the model that specific MAGUK proteins couple the NMDA receptor to distinct downstream signaling pathways. This provides a mechanism for discriminating patterns of synaptic activity that lead to long-lasting changes in synaptic strength as well as distinct aspects of cognition in the mammalian nervous system.

The development of ordered connections or “maps” within the nervous system is a common feature of sensory systems and is crucial for their normal function. NMDA receptors are known to play a key role in the formation of these maps; however, the intracellular signaling pathways that mediate the effects of glutamate are poorly understood. Here, we demonstrate that SynGAP, a synaptic Ras GTPase activating protein, is essential for the anatomical development of whisker-related patterns in the developing somatosensory pathways in rodent forebrain. Mice lacking SynGAP show only partial segregation of barreloids in the thalamus, and thalamocortical axons segregate into rows but do not form whisker-related patches. In cortex, layer 4 cells do not aggregate to form barrels. InSyngap+/−animals, barreloids develop normally, and thalamocortical afferents segregate in layer 4, but cell segregation is retarded. SynGAP is not necessary for the development of whisker-related patterns in the brainstem. Immunoelectron microscopy for SynGAP from layer 4 revealed a postsynaptic localization with labeling in developing postsynaptic densities (PSDs). Biochemically, SynGAP associates with the PSD in a PSD-95-independent manner, andPsd-95−/−animals develop normal barrels. These data demonstrate an essential role for SynGAP signaling in the activity-dependent development of whisker-related maps selectively in forebrain structures indicating that the intracellular pathways by which NMDA receptor activation mediates map formation differ between brain regions and developmental stage.

Patterns of neural activity mediated by N-methyl-D-aspartate (NMDA) receptors are known to play important roles in development of the central nervous system. However, the signalling pathways downstream from NMDA receptors that are critical for normal neuronal development are not yet clearly understood. NMDA receptors interact with various signalling proteins via scaffolding proteins, which are important in adult neuronal and behavioural plasticity. For example, the NR2B subunits of the NMDA receptor interact with postsynaptic density 95 (PSD-95), which in turn binds to synaptic ras GTPase-activating protein (SynGAP). Interestingly, the developmental phenotype of mice carrying null mutations in these genes differ. NR2B and SynGAP homozygote mice die within the first week of birth whereas PSD-95 homozygote mice survive to adulthood. We therefore examined the expression patterns of PSD-95 and SynGAP genes from embryonic stages to adult using lacZ (beta-galactosidase) marker gene knock-in mice. Dramatic changes of expression were observed throughout development in brain and other tissues. Although SynGAP binds PSD-95, both genes had distinct, as well as overlapping expression. SynGAP expression peaked at times of synaptogenesis and developmental plasticity in contrast to PSD-95, which was expressed throughout the brain from early embryonic stages. Furthermore, SynGAP showed a more spatially restricted pattern as illustrated by its restriction to forebrain in contrast to PSD-95, which was also found in mid- and hindbrain. These data support the model that synaptic signalling complexes are heterogeneous and individual components show temporal and spatial specificity during development.

We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreERT2). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.

At excitatory synapses, the postsynaptic scaffolding protein postsynaptic density 95 (PSD-95) couples NMDA receptors (NMDARs) to the Ras GTPase-activating protein SynGAP. The close association of SynGAP and NMDARs suggests that SynGAP may have an important role in NMDAR-dependent activation of Ras signaling pathways, such as the MAP kinase pathway, and in synaptic plasticity. To explore this issue, we examined long-term potentiation (LTP), p42 MAPK (ERK2) signaling, and spatial learning in mice with a heterozygous null mutation of the SynGAP gene (SynGAP(−/+)). In SynGAP(−/+) mutant mice, the induction of LTP in the hippocampal CA1 region was strongly reduced in the absence of any detectable alteration in basal synaptic transmission and NMDAR-mediated synaptic currents. Although basal levels of activated ERK2 were elevated in hippocampal extracts from SynGAP(−/+)mice, NMDAR stimulation still induced a robust increase in ERK activation in slices from SynGAP(−/+) mice. Thus, although SynGAP may regulate the ERK pathway, its role in LTP most likely involves additional downstream targets. Consistent with this, the amount of potentiation induced by stimulation protocols that induce an ERK-independent form of LTP were also significantly reduced in slices from SynGAP(−/+) mice. An elevation of basal phospho-ERK2 levels and LTP deficits were also observed in SynGAP(−/+)/H-Ras(−)(/)(−)double mutants, suggesting that SynGAP may normally regulate Ras isoforms other than H-Ras. A comparison of SynGAP and PSD-95 mutants suggests that PSD-95 couples NMDARs to multiple downstream signaling pathways with very different roles in LTP and learning.

Schizophrenia is a common disease with a complex aetiology, probably involving multiple and heterogeneous genetic factors. Here, by analysing the exome sequences of 2,536 schizophrenia cases and 2,543 controls, we demonstrate a polygenic burden primarily arising from rare (less than 1 in 10,000), disruptive mutations distributed across many genes. Particularly enriched gene sets include the voltage-gated calcium ion channel and the signalling complex formed by the activity-regulated cytoskeleton-associated scaffold protein (ARC) of the postsynaptic density, sets previously implicated by genome-wide association and copy-number variation studies. Similar to reports in autism, targets of the fragile X mental retardation protein (FMRP, product of FMR1) are enriched for case mutations. No individual gene-based test achieves significance after correction for multiple testing and we do not detect any alleles of moderately low frequency (approximately 0.5 to 1 per cent) and moderately large effect. Taken together, these data suggest that population-based exome sequencing can discover risk alleles and complements established gene-mapping paradigms in neuropsychiatric disease., National Human Genome Research Institute (U.S.) (Grant U54HG003067)

Summary The CYFIP1/SRA1 gene is located in a chromosomal region linked to various neurological disorders, including intellectual disability, autism, and schizophrenia. CYFIP1 plays a dual role in two apparently unrelated processes, inhibiting local protein synthesis and favoring actin remodeling. Here, we show that brain-derived neurotrophic factor (BDNF)-driven synaptic signaling releases CYFIP1 from the translational inhibitory complex, triggering translation of target mRNAs and shifting CYFIP1 into the WAVE regulatory complex. Active Rac1 alters the CYFIP1 conformation, as demonstrated by intramolecular FRET, and is key in changing the equilibrium of the two complexes. CYFIP1 thus orchestrates the two molecular cascades, protein translation and actin polymerization, each of which is necessary for correct spine morphology in neurons. The CYFIP1 interactome reveals many interactors associated with brain disorders, opening new perspectives to define regulatory pathways shared by neurological disabilities characterized by spine dysmorphogenesis., Highlights • BDNF-Rac1 signaling redistributes CYFIP1 between eIF4E and WRC • FRET demonstrates that BDNF-Rac1 signaling alters CYFIP1 conformation • CYFIP1-eIF4E-WRC regulates ARC synthesis, actin polymerization, and spine morphology • The CYFIP1 interactome is enriched in genes implicated in neurological disorders, CYFIP1 plays a dual role in inhibiting local protein synthesis and favoring actin remodeling. De Rubeis et al. show that BDNF-driven synaptic signaling releases CYFIP1 from the translational inhibitory complex, triggering translation of target mRNAs and shifting CYFIP1 into the WAVE regulatory complex.

Recombinant DNA technology has enabled the large scale production of human hemoglobin in bacteria and yeast. This has opened up a way to produce a hemoglobin-based blood substitute which could replace conventional blood transfusion in some situations. Using our understanding of the structure-function relationships and evolutionary history of hemoglobin it has been possible to improve the oxygen transport properties of the molecule and solve a number of problems associated with the use of natural hemoglobin as a cell-free blood substitute.

All globins consist of eight helices and interconnecting loops except alpha hemoglobin subunits which lack the D-helix due to deletion of five consecutive residues. Previous site-directed mutagenesis work suggested that this deletion is a neutral modification in hemoglobin with respect to equilibrium O2 binding [Komiyama, N. H., Shih, T.-B., Looker, D., Tame, J., & Nagai, K. (1991) Nature 352, 349-351]. To examine the role of the D-helix in myoglobin, we have measured the O2 and CO binding and hemin dissociation properties of recombinant sperm whale myoglobin mutants in which residues 52-56 have been deleted, Mb(-D), replaced by five alanines, Mb(Ala52-56), and substituted with four alanines and a methionine, Mb(Ala52-55Met56). Crystal structures of aquometMb(-D) and aquometMb(Ala52-55Met56) were determined to 2.0 A resolution and show that the conformation of the distal pocket is little affected by removal of the D-helix or mutations in this region. As a result, these mutations have little effect on O2 and CO binding. Diffuse electron density is observed in the region between the C- and E-helices of Mb(-D), indicating a highly mobile or heterogeneous conformation in this portion of the tertiary structure. This flexibility provides an explanation for the 50-fold higher rate of hemin loss from Mb(-D) as compared to that from wild-type myoglobin. Hemin loss from Mb(Ala52-56) is also rapid. In contrast, Mb(Ala52-55Met56) shows a well-defined D-helix and has a rate of hemin loss identical to that of wild-type holoprotein [corrected].(ABSTRACT TRUNCATED AT 250 WORDS)

Traf2 and NcK interacting kinase (TNiK) contains serine-threonine kinase and scaffold domains and has been implicated in cell proliferation and glutamate receptor regulationin vitro. Here we report its rolein vivousing mice carrying a knock-out mutation. TNiK binds protein complexes in the synapse linking it to the NMDA receptor (NMDAR) via AKAP9. NMDAR and metabotropic receptors bidirectionally regulate TNiK phosphorylation and TNiK is required for AMPA expression and synaptic function. TNiK also organizes nuclear complexes and in the absence of TNiK, there was a marked elevation in GSK3β and phosphorylation levels of its cognate phosphorylation sites on NeuroD1 with alterations in Wnt pathway signaling. We observed impairments in dentate gyrus neurogenesis in TNiK knock-out mice and cognitive testing using the touchscreen apparatus revealed impairments in pattern separation on a test of spatial discrimination. Object-location paired associate learning, which is dependent on glutamatergic signaling, was also impaired. Additionally, TNiK knock-out mice displayed hyperlocomotor behavior that could be rapidly reversed by GSK3β inhibitors, indicating the potential for pharmacological rescue of a behavioral phenotype. These data establish TNiK as a critical regulator of cognitive functions and suggest it may play a regulatory role in diseases impacting on its interacting proteins and complexes.

Alternative promoter usage and alternative splicing enable diversification of the transcriptome. Here we demonstrate that the function of Synaptic GTPase-Activating Protein (SynGAP), a key synaptic protein, is determined by the combination of its amino-terminal sequence with its carboxy-terminal sequence. 5′ rapid amplification of cDNA ends and primer extension show that different N-terminal protein sequences arise through alternative promoter usage that are regulated by synaptic activity and postnatal age. Heterogeneity in C-terminal protein sequence arises through alternative splicing. Overexpression of SynGAP α1 versus α2 C-termini-containing proteins in hippocampal neurons has opposing effects on synaptic strength, decreasing and increasing miniature excitatory synaptic currents amplitude/frequency, respectively. The magnitude of this C-terminal-dependent effect is modulated by the N-terminal peptide sequence. This is the first demonstration that activity-dependent alternative promoter usage can change the function of a synaptic protein at excitatory synapses. Furthermore, the direction and degree of synaptic modulation exerted by different protein isoforms from a single gene locus is dependent on the combination of differential promoter usage and alternative splicing., Synaptic GTPase-activating protein, SynGAP, is a postsynaptic signalling protein that can regulate synaptic function. McMahon et al. express different SynGAP isoforms in neurons and find that the effect on synaptic strength depends on alternative promoter usage and alternative splicing of the C-terminus.

To access publisher's full text version of this article click on the hyperlink at the bottom of the page A small number of rare, recurrent genomic copy number variants (CNVs) are known to substantially increase susceptibility to schizophrenia. As a consequence of the low fecundity in people with schizophrenia and other neurodevelopmental phenotypes to which these CNVs contribute, CNVs with large effects on risk are likely to be rapidly removed from the population by natural selection. Accordingly, such CNVs must frequently occur as recurrent de novo mutations. In a sample of 662 schizophrenia proband-parent trios, we found that rare de novo CNV mutations were significantly more frequent in cases (5.1% all cases, 5.5% family history negative) compared with 2.2% among 2623 controls, confirming the involvement of de novo CNVs in the pathogenesis of schizophrenia. Eight de novo CNVs occurred at four known schizophrenia loci (3q29, 15q11.2, 15q13.3 and 16p11.2). De novo CNVs of known pathogenic significance in other genomic disorders were also observed, including deletion at the TAR (thrombocytopenia absent radius) region on 1q21.1 and duplication at the WBS (Williams-Beuren syndrome) region at 7q11.23. Multiple de novos spanned genes encoding members of the DLG (discs large) family of membrane-associated guanylate kinases (MAGUKs) that are components of the postsynaptic density (PSD). Two de novos also affected EHMT1, a histone methyl transferase known to directly regulate DLG family members. Using a systems biology approach and merging novel CNV and proteomics data sets, systematic analysis of synaptic protein complexes showed that, compared with control CNVs, case de novos were significantly enriched for the PSD proteome (P=1.72 × 10⁻⁶. This was largely explained by enrichment for members of the N-methyl-D-aspartate receptor (NMDAR) (P=4.24 × 10⁻⁶) and neuronal activity-regulated cytoskeleton-associated protein (ARC) (P=3.78 × 10⁻⁸) postsynaptic signalling complexes. In an analysis of 18 492 subjects (7907 cases and 10 585 controls), case CNVs were enriched for members of the NMDAR complex (P=0.0015) but not ARC (P=0.14). Our data indicate that defects in NMDAR postsynaptic signalling and, possibly, ARC complexes, which are known to be important in synaptic plasticity and cognition, play a significant role in the pathogenesis of schizophrenia. Janssen Research Foundation Stanley Medical Research Institute Merck Genome Research Foundation Herman Foundation Medical Research Council (MRC) G0800509 Centre Grants, the National Institutes of Mental Health (USA) CONTE: 2 P50 MH066392-05A1 EU (EU-GEI) EU HEALTH-2007-2.2.1-10-223423 PIAG-GA-2008-218251 241995 242498 242167 Genes to Cognition Program Wellcome Trust MRC G06706B EMBO European Commission info:eu-repo/grantAgreement/EC/FP7/218251