De novo CNV analysis implicates specific abnormalities of postsynaptic signalling complexes in the pathogenesis of schizophrenia.

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.

alpha-Isoform of calcium-calmodulin-dependent protein kinase II and postsynaptic density protein 95 differentially regulate synaptic expression of NR2A- and NR2B-containing N-methyl-d-aspartate receptors in hippocampus.

N-methyl-d-aspartate receptors (NMDARs) are critical determinants of bidirectional synaptic plasticity, however, studies of NMDAR function have been based primarily on pharmacological and electrophysiological manipulations, and it is still debated whether there are subunit-selective forms of long-term potentiation (LTP) and long-term depression (LTD). Here we provide ultrastructural analyses of axospinous synapses in cornu ammonis field 1 of hippocampus (CA1) stratum radiatum of transgenic mice with mutations to two key underlying postsynaptic density (PSD) proteins, postsynaptic density protein 95 (PSD-95) and the alpha-isoform of calcium-calmodulin-dependent protein kinase II (alphaCaMKII). Distribution profiles of synaptic proteins in these mice reveal very different patterns of subunit-specific NMDAR localization, which may be related to the divergent phenotypes of the two mutants. In PSD-95, Dlg, ZO-1/Dlg-homologous region (PDZ) 3-truncated mutant mice in which LTD could not be induced but LTP was found to be enhanced, we found a subtle, yet preferential displacement of synaptic N-methyl-d-aspartate receptor subunit 2B (NR2B) subunits in lateral regions of the synapse without affecting changes in the localization of N-methyl-d-aspartate receptor subunit 2A (NR2A) subunits. In persistent inhibitory alphaCaMKII Thr305 substituted with Asp in alpha-isoform of calcium-calmodulin kinase II (T305D) mutant mice with severely impaired LTP but stable LTD expression, we found a selective reduction of NR2A subunits at both the synapse and throughout the cytoplasm of the spine without any effect on the NR2B subunit. In an experiment of mutual exclusivity, neither PSD-95 nor alphaCaMKII localization was found to be affected by mutations to the corresponding PSD protein suggesting that they are functionally independent of the other in the regulation of NR2A- and NR2B-containing NMDARs preceding synaptic activity. Consequently, there may exist at least two distinct PSD-95 and alphaCaMKII-specific NMDAR complexes involved in mediating LTP and LTD through opposing signal transduction pathways in synapses of the hippocampus. The contrasting phenotypes of the PSD-95 and alphaCaMKII mutant mice further establish the prospect of an independent and, possibly, competing mechanism for the regulation of NMDAR-dependent bidirectional synaptic plasticity.

Supramolecular signalling complexes in the nervous system.

It is now apparent that multiprotein signalling complexes or "signalling machines" are responsible for orchestrating many complex signalling pathways in the cell. The synapse is a sub-cellular specialisation which transmits and converts patterns of electrical activity into cellular memory. This processing of electrical information is mediated by the protein components of the synapse. The organisation of synaptic proteins has been investigated over the last number of years using proteomic methods and with the application ofbioinformatics; a landscape of modular protein complexes at the synapse is emerging. Many share a common organisation centred on a receptor/channel, a protein scaffold, (in which the signalling molecules are localised) and membrane to cytoskeleton interactions. The use of PDZ-domain based protein scaffolds is a particularly common feature in the construction of neuronal protein complexes and the differential presence of these proteins in complexes can have functional consequences. Here we overview current proteomic methodologies for the analysis of multiprotein complexes. In addition, we describe the characterisation of a number of multiprotein complexes associated with ion channels (NMDAR, P2X7 and Kir2) and GPCRs (5-HT2A/5-HT2C, D2 and mGluR5) and discuss common their common components and organisation.

Dlgap1 knockout mice exhibit alterations of the postsynaptic density and selective reductions in sociability.

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.

Standardized experiments in mutant mice reveal behavioural similarity on 129S5 and C57BL/6J backgrounds.

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-standardized 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 standardized 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 < 0.05 threshold results in a reproducibility of 60% between identical experiments. Reproducibility was reduced in the presence of strain differences. We also identified a P-value threshold that maximized reproducibility of mutant phenotypes across strains. This study illustrates the value of standardized approaches for quantitative assessment of behavioural phenotypes and highlights approaches that may improve the translational value of mouse behavioural studies.

A novel role for cyclic guanosine 3',5'monophosphate signaling in synaptic plasticity: a selective suppressor of protein kinase A-dependent forms of long-term potentiation.

At excitatory synapses onto hippocampal CA1 pyramidal cells, activation of cyclic AMP-dependent protein kinase and subsequent down-regulation of protein phosphatases has a crucial role in the induction of long-term potentiation by low-frequency patterns of synaptic stimulation. Because the second messenger cyclic guanosine 3',5'monophosphate can regulate the activity of different forms of the cyclic AMP degrading enzyme phosphodiesterase, we examined whether increases in cyclic guanosine 3',5'monophosphate can modulate long-term potentiation induction in the mouse hippocampal CA1 region through effects on cyclic AMP signaling. Using the cyclic guanosine 3',5'monophosphate-specific phosphodiesterase inhibitor zaprinast or the nitric oxide donor S-nitroso-D,L-penicillamine to elevate cyclic guanosine 3',5'monophosphate levels we found that increases in cyclic guanosine 3',5'monophosphate strongly inhibit the induction of long-term potentiation by low-frequency patterns of synaptic stimulation where protein kinase A activation is required for long-term potentiation induction. In contrast, zaprinast and S-nitroso-D,L-penicillamine had no effect on the induction of long-term potentiation by high-frequency patterns of synaptic stimulation that induce long-term potentiation in a protein kinase A-independent manner. Directly activating protein kinase A with the phosphodiesterase-resistant cyclic AMP analog 8-Br-cAMP, blocking all phosphodiesterases with 3-isobutyl-1-methylxanthine, or inhibiting protein phosphatases rescued long-term potentiation induction in zaprinast-treated slices. Together, these results suggest that increases in cyclic guanosine 3',5'monophosphate inhibit long-term potentiation by activating phosphodiesterases that interfere with the protein kinase A-mediated suppression of protein phosphatases needed for long-term potentiation induction. Consistent with the notion that this cyclic guanosine 3',5'monophosphate-mediated inhibitory pathway is recruited by some patterns of synaptic activity, blocking cyclic guanosine 3',5'monophosphate production strongly facilitated the induction of long-term potentiation by long trains of theta-frequency synaptic stimulation. Together, our results indicate that increases in cyclic guanosine 3',5'monophosphate can act as a long-term potentiation suppressor mechanism that selectively constrains the induction of protein kinase A-dependent forms of long-term potentiation induced by low-frequency patterns of synaptic stimulation.

Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: normal electrophysiological and morphological properties in vitro.

BACKGROUND: Genetically manipulated embryonic stem (ES) cell derived neurons (ESNs) provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK), which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice. RESULTS: Mouse ES cells carrying homozygous null mutations (FAK-/-) were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases. CONCLUSION: FAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.

ATP from synaptic terminals and astrocytes regulates NMDA receptors and synaptic plasticity through PSD-95 multi-protein complex.

Recent studies highlighted the importance of astrocyte-secreted molecules, such as ATP, for the slow modulation of synaptic transmission in central neurones. Biophysical mechanisms underlying the impact of gliotransmitters on the strength of individual synapse remain, however, unclear. Here we show that purinergic P2X receptors can bring significant contribution to the signalling in the individual synaptic boutons. ATP released from astrocytes facilitates a recruitment of P2X receptors into excitatory synapses by Ca(2+)-dependent mechanism. P2X receptors, co-localized with NMDA receptors in the excitatory synapses, can be activated by ATP co-released with glutamate from pre-synaptic terminals and by glia-derived ATP. An activation of P2X receptors in turn leads to down-regulation of postsynaptic NMDA receptors via Ca(2+)-dependent de-phosphorylation and interaction with PSD-95 multi-protein complex. Genetic deletion of the PSD-95 or P2X4 receptors obliterated ATP-mediated down-regulation of NMDA receptors. Impairment of purinergic modulation of NMDA receptors in the PSD-95 mutants dramatically decreased the threshold of LTP induction and increased the net magnitude of LTP. Our findings show that synergistic action of glia- and neurone-derived ATP can pre-modulate efficacy of excitatory synapses and thereby can have an important role in the glia-neuron communications and brain meta-plasticity.

Bridging the translational divide: identical cognitive touchscreen testing in mice and humans carrying mutations in a disease-relevant homologous gene.

Development of effective therapies for brain disorders has been hampered by a lack of translational cognitive testing methods. We present the first example of using the identical touchscreen-based cognitive test to assess mice and humans carrying disease-related genetic mutations. This new paradigm has significant implications for improving how we measure and model cognitive dysfunction in human disorders in animals, thus bridging the gap towards effective translation to the clinic.

Human cognitive ability is influenced by genetic variation in components of postsynaptic signalling complexes assembled by NMDA receptors and MAGUK proteins.

Differences in general cognitive ability (intelligence) account for approximately half of the variation in any large battery of cognitive tests and are predictive of important life events including health. Genome-wide analyses of common single-nucleotide polymorphisms indicate that they jointly tag between a quarter and a half of the variance in intelligence. However, no single polymorphism has been reliably associated with variation in intelligence. It remains possible that these many small effects might be aggregated in networks of functionally linked genes. Here, we tested a network of 1461 genes in the postsynaptic density and associated complexes for an enriched association with intelligence. These were ascertained in 3511 individuals (the Cognitive Ageing Genetics in England and Scotland (CAGES) consortium) phenotyped for general cognitive ability, fluid cognitive ability, crystallised cognitive ability, memory and speed of processing. By analysing the results of a genome wide association study (GWAS) using Gene Set Enrichment Analysis, a significant enrichment was found for fluid cognitive ability for the proteins found in the complexes of N-methyl-D-aspartate receptor complex; P=0.002. Replication was sought in two additional cohorts (N=670 and 2062). A meta-analytic P-value of 0.003 was found when these were combined with the CAGES consortium. The results suggest that genetic variation in the macromolecular machines formed by membrane-associated guanylate kinase (MAGUK) scaffold proteins and their interaction partners contributes to variation in intelligence.

SynGAP isoforms exert opposing effects on synaptic strength.

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.

A general basis for cognition in the evolution of synapse signaling complexes.

Beneath the complexity of the human brain are molecular principles shaped by evolution explaining the origins of the behavioral repertoire. The role of the nervous system is to provide a repertoire of behaviors allowing the animal to respond and adapt to changing environments during the course of its life. Multiprotein complexes in the postsynaptic terminal of synapses control adaptive and cognitive processes in metazoan nervous systems. These multiprotein complexes are organized into molecular networks that detect and respond to patterns of neural activity. Combinations of proteins are used to build different complexes and pathways producing great diversity. These complexes evolved from an ancestral core set of proteins controlling adaptive behaviors in unicellular organisms known as the protosynapse. Later expansion in numbers and interactions resulted in more complex synapses in invertebrates and vertebrates. The resultant combinatorial complexity has contributed to the neuroanatomical, neurophysiological, and behavioral diversity in these species. Mutations in genes encoding the complexes result in many human diseases of the nervous system. This general mechanism of cognition provides a useful template for studying evolution of behavior in all animals.

Integrating synapse proteomics with transcriptional regulation.

The mammalian postsynaptic proteome (PSP) comprises a highly interconnected set of approximately 1,000 proteins. The PSP is organized into macromolecular complexes that have a modular architecture defined by protein interactions and function. Signals initiated by neurotransmitter receptors are integrated by these complexes and their constituent enzymes to orchestrate multiple downstream cellular changes, including transcriptional regulation of genes at the nucleus. Genome wide transcriptome studies are beginning to map the sets of genes regulated by the synapse proteome. Conversely, understanding the transcriptional regulation of genes encoding the synapse proteome will shed light on synapse formation. Mutations that disrupt synapse signalling complexes result in cognitive impairments in mice and humans, and recent evidence indicates that these mutation change gene expression profiles. We discuss the need for global approaches combining genetics, transcriptomics and proteomics in order to understand cognitive function and disruption in diseases.

The synapse proteome and phosphoproteome: a new paradigm for synapse biology.

Synapse proteomics has recently resulted in a quantum leap in knowledge of the protein composition of brain synapses and its phosphorylation. We now have the first draft picture of the synapse, comprising approximately 1000 proteins. This is not matched by available methods of functional analysis either in reduced systems or in whole animals. Fewer than 20% of synapse proteome proteins have a known function in the nervous system. A concerted effort is required to establish new technical approaches before we can understand the diversity of functions conferred by the synapse proteome on the synapse, the neuron and the animal. This review will highlight this change in knowledge and discuss current technical and interpretative limitations challenged by synapse proteomics.

Organization of brain complexity--synapse proteome form and function.

Proteomic study of the synapse has generated an extensive list of molecular components, revealing one of the most complex functional systems currently known to cell biology. While fundamental to neural information processing, behaviour and disease, the molecular organisation of the synapse and its relation to higher-level function has yet to be clearly understood. Neurotransmitter receptor complexes, such as the N-methyl-D-aspartate receptor complex (NRC/MASC), are major components of the synaptic proteome. We have recently completed a detailed study of MASC, its functional organisation and involvement in behaviour and disease. This pointed to simple design principles underlying synaptic organisation. Drawing together the results of proteomic and analytical study, we sketch out a model for synaptic functional organisation.