Evolutionary expansion and anatomical specialization of synapse proteome complexity.

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.

G2Cdb: the Genes to Cognition database.

Neuroscience databases linking genes, proteins, (patho)physiology, anatomy and behaviour across species will be valuable in a broad range of studies of the nervous system. G2Cdb is such a neuroscience database aiming to present a global view of the role of synapse proteins in physiology and disease. G2Cdb warehouses sets of genes and proteins experimentally elucidated by proteomic mass spectroscopy of signalling complexes and proteins biochemically isolated from mammalian synapse preparations, giving an experimentally validated definition of the constituents of the mammalian synapse. Using automated text-mining and expert (human) curation we have systematically extracted information from published neurobiological studies in the fields of synaptic signalling electrophysiology and behaviour in knockout and other transgenic mice. We have also surveyed the human genetics literature for associations to disease caused by mutations in synaptic genes. The synapse proteome datasets that G2Cdb provides offer a basis for future work in synapse biology and provide useful information on brain diseases. They have been integrated in a such way that investigators can rapidly query whether a gene or protein is found in brain-signalling complex(es), has a phenotype in rodent models or whether mutations are associated with a human disease. G2Cdb can be freely accessed at http://www.genes2cognition.org.

A unified resource and configurable model of the synapse proteome and its role in 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.

Automated design of genomic Southern blot probes.

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.

Confirmed rare copy number variants implicate novel genes in schizophrenia.

Understanding how cognitive processes including learning, memory, decision making and ideation are encoded by the genome is a key question in biology. Identification of sets of genes underlying human mental disorders is a path towards this objective. Schizophrenia is a common disease with cognitive symptoms, high heritability and complex genetics. We have identified genes involved with schizophrenia by measuring differences in DNA copy number across the entire genome in 91 schizophrenia cases and 92 controls in the Scottish population. Our data reproduce rare and common variants observed in public domain data from >3000 schizophrenia cases, confirming known disease loci as well as identifying novel loci. We found copy number variants in PDE10A (phosphodiesterase 10A), CYFIP1 [cytoplasmic FMR1 (Fragile X mental retardation 1)-interacting protein 1], K(+) channel genes KCNE1 and KCNE2, the Down's syndrome critical region 1 gene RCAN1 (regulator of calcineurin 1), cell-recognition protein CHL1 (cell adhesion molecule with homology with L1CAM), the transcription factor SP4 (specificity protein 4) and histone deacetylase HDAC9, among others (see http://www.genes2cognition.org/SCZ-CNV). Integrating the function of these many genes into a coherent model of schizophrenia and cognition is a major unanswered challenge.

Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins.

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.

Expression screening and annotation of a zebrafish myoblast cDNA library.

To analyse the myogenic transcriptome and identify novel genes involved in muscle development in an in vivo context, we have constructed a muscle specific cDNA library from GFP-expressing myoblasts purified by fluorescent activated cell sorting of transgenic zebrafish embryos. We have generated 153,428 EST sequences from this library that have been clustered into consensi, mapped to the genome assembly Zv6 and analysed for protein homology. Expression analysis of a randomly picked sample of clones using whole mount in situ hybridisation, identified 30 genes that are expressed specifically within the myotome, one third of which represent novel sequences. These genes have been assigned to syn-expression groups. The sequencing of the myoblast enriched cDNA library has significantly increased the number of zebrafish ESTs, facilitating the prediction of new spliced transcripts in the genome assembly and providing a transcriptome of an in vivo myoblast cell.

Arc Requires PSD95 for Assembly into Postsynaptic Complexes Involved with Neural Dysfunction and Intelligence.

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.

Human post-mortem synapse proteome integrity screening for proteomic studies of postsynaptic complexes.

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.

Comparative study of human and mouse postsynaptic proteomes finds high compositional conservation and abundance differences for key synaptic proteins.

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.

Characterization of the proteome, diseases and evolution of the human postsynaptic density.

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.

Clustered coding variants in the glutamate receptor complexes of individuals with schizophrenia and bipolar disorder.

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.

Neurotransmitters drive combinatorial multistate postsynaptic density networks.

The mammalian postsynaptic density (PSD) comprises a complex collection of approximately 1100 proteins. Despite extensive knowledge of individual proteins, the overall organization of the PSD is poorly understood. Here, we define maps of molecular circuitry within the PSD based on phosphorylation of postsynaptic proteins. Activation of a single neurotransmitter receptor, the N-methyl-D-aspartate receptor (NMDAR), changed the phosphorylation status of 127 proteins. Stimulation of ionotropic and metabotropic glutamate receptors and dopamine receptors activated overlapping networks with distinct combinatorial phosphorylation signatures. Using peptide array technology, we identified specific phosphorylation motifs and switching mechanisms responsible for the integration of neurotransmitter receptor pathways and their coordination of multiple substrates in these networks. These combinatorial networks confer high information-processing capacity and functional diversity on synapses, and their elucidation may provide new insights into disease mechanisms and new opportunities for drug discovery.