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Direct comparison of protein components from human and mouse excitatory synapses is important for determining the suitability of mice as models of human brain disease and to understand the evolution of the mammalian brain. The postsynaptic density is a highly complex set of proteins organized into molecular networks that play a central role in behavior and disease. We report the first direct comparison of the proteome of triplicate isolates of mouse and human cortical postsynaptic densities. The mouse postsynaptic density comprised 1556 proteins and the human one 1461. A large compositional overlap was observed; more than 70% of human postsynaptic density proteins were also observed in the mouse postsynaptic density. Quantitative analysis of postsynaptic density components in both species indicates a broadly similar profile of abundance but also shows that there is higher abundance variation between species than within species. Well known components of this synaptic structure are generally more abundant in the mouse postsynaptic density. Significant inter-species abundance differences exist in some families of key postsynaptic density proteins including glutamatergic neurotransmitter receptors and adaptor proteins. Furthermore, we have identified a closely interacting set of molecules enriched in the human postsynaptic density that could be involved in dendrite and spine structural plasticity. Understanding synapse proteome diversity within and between species will be important to further our understanding of brain complexity and disease.

Genes encoding synaptic proteins are highly associated with neuronal disorders many of which show clinical co-morbidity. We integrated 58 published synaptic proteomic datasets that describe over 8000 proteins and combined them with direct protein–protein interactions and functional metadata to build a network resource that reveals the shared and unique protein components that underpin multiple disorders. All the data are provided in a flexible and accessible format to encourage custom use.

This study identifies proteins from the postsynaptic density (PSD) of human neocortex and finds that the PSD shows enrichment of genes involved in cognitive and affective phenotypes and that PSD mutations are associated with neurological and psychiatric disease. We isolated the postsynaptic density from human neocortex (hPSD) and identified 1,461 proteins. hPSD mutations cause 133 neurological and psychiatric diseases and were enriched in cognitive, affective and motor phenotypes underpinned by sets of genes. Strong protein sequence conservation in mammalian lineages, particularly in hub proteins, indicates conserved function and organization in primate and rodent models. The hPSD is an important structure for nervous system disease and behavior.

Current models of schizophrenia and bipolar disorder implicate multiple genes, however their biological relationships remain elusive. To test the genetic role of glutamate receptors and their interacting scaffold proteins, the exons of ten glutamatergic 'hub' genes in 1304 individuals were re-sequenced in case and control samples. No significant difference in the overall number of non-synonymous single nucleotide polymorphisms (nsSNPs) was observed between cases and controls. However, cluster analysis of nsSNPs identified two exons encoding the cysteine-rich domain and first transmembrane helix of GRM1 as a risk locus with five mutations highly enriched within these domains. A new splice variant lacking the transmembrane GPCR domain of GRM1 was discovered in the human brain and the GRM1 mutation cluster could perturb the regulation of this variant. The predicted effect on individuals harbouring multiple mutations distributed in their ten hub genes was also examined. Diseased individuals possessed an increased load of deleteriousness from multiple concurrent rare and common coding variants. Together, these data suggest a disease model in which the interplay of compound genetic coding variants, distributed among glutamate receptors and their interacting proteins, contribute to the pathogenesis of schizophrenia and bipolar disorders.

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K + channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K + channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease. Synopsis Systems biology has the potential to explain physiological processes as emergent properties of sets of genes and proteins. Beyond simple cellular systems, the challenge of delivering systems biology into the intact and freely behaving animal will require new methods. Currently, the most widely used approach is immunoprecipitation of the target protein and its associate binding proteins. This method suffers from the drawbacks of single step purification strategies that include a high level of non‐specific background proteins amongst other limitations. To overcome these limitations we demonstrate that the tandem affinity purification (TAP) technology originally developed in yeast (Rigaut et al , 1999 ), when combined with gene targeting, can be used to efficiently isolate highly specific complexes from mouse. The ‘targeted TAP tagging’ strategy combines the two major advantages of each system. The first advantage is that the insertion of two tags into the protein of interest allows two consecutive purification steps that facilitate the recovery of protein complexes with high confidence and decreases the recovery of non‐specific proteins or weak interactors. The second advantage, conferred by targeting the endogenous gene, is that the tagged protein is expressed under its natural regulatory mechanisms. We have designed an endogenous TAP targeting strategy to isolate complexes from mouse brain excitatory synapses. The brain is the most complex organ from a cellular and molecular perspective and thus an ideal model to explore the TAP method. Post Synaptic Density 95 (PSD‐95/Dlg4) is an adaptor protein comprised of PDZ, SH3 and GK domains and is expressed in the postsynaptic terminal of excitatory synapses where it organizes signaling from neurotransmitter receptors to downstream pathways (Kornau et al , 1995 ; Hunt et al , 1996 ; Tu et al , 1999 ; Husi et al , 2000 ; Nehring et al , 2000 ; Dosemeci et al , 2007 ; Carlisle et al , 2008 ). Mice carrying a knockout mutation in PSD‐95 show it is essential for synaptic plasticity and a range of important behaviours (Migaud et al , 1998 ; El‐Husseini et al , 2000 ; Beique et al , 2006 ). Here, a new TAP tag was fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function (Figure 2 ). As a result of four independent tandem purifications and mass spectrometry analysis, we were able to define PSD‐95 core complexes with high sensitivity and reproducibility. The four purifications show an average of 125±19 proteins, having 118 proteins (94%) common in at least three of four replicates. This reproducibility rate is among the highest rate reported for systematic protein complex isolation. To further validate this interaction data we compared it to information from public datasets. Of the 118 proteins, 22% were proteins that directly bind PSD‐95 and 18% were proteins not previously found in other PSD‐95 analysis. All together, these data show robust reproducibility and sensitivity of this method for purifying synaptic complexes. These PSD‐95 core complexes comprise key functional components of synapses including the glutamate neurotransmitter receptors, K + channels, scaffolding and signaling proteins. These complexes contain ionotropic glutamate receptors of the NMDA, AMPA and kainate subtypes as well as major K + channels that together are the major postsynaptic constituents responsible for synaptic transmission and shaping the postsynaptic electrophysiological response to presynaptic input (Watanabe et al , 2002 ; Chen et al , 2006 ; Kim et al , 2007 ). We believe that this is the first method that has allowed the robust copurification of these proteins. To explore functional organization using network models, we manually curated interactions (Pocklington et al , 2006 ) and the UniHi database ( http://www.mdc‐berlin.de/unihi ) to identify 119 interactions between 50 proteins (excluding self‐interactions) of the PSD‐95 core complexes. Network clustering of the interacting proteins showed 40 out of the 50 proteins formed a large connected component (major connected component, MCC) and a modular structure that was segregated into 5 clusters referred to as cluster a (Cla) to cluster e (Cle) (Figure 5A ). In addition to the 5 MCC clusters, 2 further disconnected clusters (‘Clf’ and ‘Clg’) were found. Of great interest is the location and proximity of the receptors and channels responsible for the postsynaptic depolarization and subsequent action potential generation. All NMDA, AMPA and kainate glutamate receptors were restricted to Cla and Clb and the voltage‐dependent K + channels were found in Cla and Clc (entirely comprised of K + channels). It therefore appears that Cla, Clb and Clc are enriched with membrane proteins responsible for electrical properties of the postsynaptic terminal. The central role of PSD‐95 was supported by calculation of the shortest path from each protein to every other protein and PSD‐95 showed the lowest. Annotation of clusters with human disease associations revealed that multiple disorders map onto the network with a highly significant correlation of schizophrenia within the glutamate receptor clusters ( P <10−6). 20 genes involved in schizophrenia were significantly associated with the clusters Cla and Clb that contains all the glutamate receptors and MAGUK/Dlg proteins (Figure 5B ). Mapping the primary interactors of these schizophrenia proteins recruited many other proteins found in the other modules of the network. This suggests that the overall network and its different modules are a substrate for schizophrenia, and not simply the glutamate receptors, as was generally considered in the ‘glutamate hypothesis’ of schizophrenia (Greene, 2001 ; Coyle, 2006 ; Lisman et al , 2008 ). This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease. TAP tagged mice are a valuable resource and useful for a wide range of physiological studies and whole animal studies. A novel approach for isolating native protein complexes from mouse tissues using gene targeting of tandem affinity tags is presented. A protein core complex from brain synapses comprising principal electrophysiological and signalling components for synaptic transmission and synaptic plasticity was isolated. The protein interaction network shows clusters of functionally distinct proteins and schizophrenia susceptibility genes. This targeted TAP tagging method has general application to all types of protein complexes in the mouse and will be particularly useful for analysing molecular networks and systems biology in the intact animal.

Grant and colleagues used comparative proteomics and genomics to examine the evolution of the postsynaptic density and MAGUK-associated signaling complexes implicated in learning and memory. They found conservation of synaptic components amidst diverse species, but also found species-specific adaptation and increased signaling complexity in vertebrates. Understanding the origins and evolution of synapses may provide insight into species diversity and the organization of the brain. Using comparative proteomics and genomics, we examined the evolution of the postsynaptic density (PSD) and membrane-associated guanylate kinase (MAGUK)-associated signaling complexes (MASCs) that underlie learning and memory. PSD and MASC orthologs found in yeast carry out basic cellular functions to regulate protein synthesis and structural plasticity. We observed marked changes in signaling complexity at the yeast-metazoan and invertebrate-vertebrate boundaries, with an expansion of key synaptic components, notably receptors, adhesion/cytoskeletal proteins and scaffold proteins. A proteomic comparison of Drosophila and mouse MASCs revealed species-specific adaptation with greater signaling complexity in mouse. Although synaptic components were conserved amongst diverse vertebrate species, mapping mRNA and protein expression in the mouse brain showed that vertebrate-specific components preferentially contributed to differences between brain regions. We propose that the evolution of synapse complexity around a core proto-synapse has contributed to invertebrate-vertebrate differences and to brain specialization.

Background Synapses are fundamental components of brain circuits and are disrupted in over 100 neurological and psychiatric diseases. The synapse proteome is physically organized into multiprotein complexes and polygenic mutations converge on postsynaptic complexes in schizophrenia, autism and intellectual disability. Directly characterising human synapses and their multiprotein complexes from post-mortem tissue is essential to understanding disease mechanisms. However, multiprotein complexes have not been directly isolated from human synapses and the feasibility of their isolation from post-mortem tissue is unknown. Results Here we establish a screening assay and criteria to identify post-mortem brain samples containing well-preserved synapse proteomes, revealing that neocortex samples are best preserved. We also develop a rapid method for the isolation of synapse proteomes from human brain, allowing large numbers of post-mortem samples to be processed in a short time frame. We perform the first purification and proteomic mass spectrometry analysis of MAGUK Associated Signalling Complexes (MASC) from neurosurgical and post-mortem tissue and find genetic evidence for their involvement in over seventy human brain diseases. Conclusions We have demonstrated that synaptic proteome integrity can be rapidly assessed from human post-mortem brain samples prior to its analysis with sophisticated proteomic methods. We have also shown that proteomics of synapse multiprotein complexes from well preserved post-mortem tissue is possible, obtaining structures highly similar to those isolated from biopsy tissue. Finally we have shown that MASC from human synapses are involved with over seventy brain disorders. These findings should have wide application in understanding the synaptic basis of psychiatric and other mental disorders.

Background Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to experimentally validate a number of these automated designs by Southern blotting. The majority of probes we tested performed well confirming our in silico prediction methodology and the general usefulness of the software for automated genomic Southern probe design. Conclusions Software and supplementary information are freely available at: http://www.genes2cognition.org/software/southern_blot

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.