G2C::Synapse Proteomics

Introduction

Over the past decade Professor Seth Grant's laboratory and the Genes to Cognition Programme, have pioneered and subsequently refined and developed the use of proteomics in the nervous system (neuroproteomics) as a tool to dissect the composition and function of the mammalian synapse.

These studies have significantly advanced our knowledge of the protein composition of brain synapses, focussing on the postsynaptic compartment, to date finding and reporting over two thousand distinct proteins that make up the complex receptor and signal transduction machinery of the excitatory postsynaptic nerve terminal 11780971.

Knowledge of this 'molecular catalogue' of synaptic components has been enormously useful to the neuroscience community as a whole, acting as starting point for many avenues of neurobiological and genetic research. We ourselves have employed this 'synaptic parts list' to apply systems biology approaches to model the function and organisation of the synapse 16738568, and initiate a large scale mouse knockout and phenotyping programme, to determine the role of individual proteins within the network of the glutamatergic NMDA receptor and postsynaptic density (PSD) complex 14630221. Integration of our model organism findings with those observed in humans in a clinical research genetics setting, has allowed us to build up an overall model of synaptic function in health and disease, and survey human brain diseases resulting from derangements in synaptic proteins see 22409856 which we aim to freely provide to the world via our web database, G2Cdb.

 

Receptor Complex Proteomics

Our first neuroproteomics papers combined immunoblotting and mass spectroscopy to characterise the protein composition of the the NMDA receptor complex 14630221; 11279284. This complex, known to play key a role in synaptic plasticity at excitatory synapse in the mammalian system was found to comprise of 77 proteins built up of receptor, adaptor, signalling, cytoskeletal and novel proteins. These studies substantially extended the repertoire of proteins involved in fast excitatory signalling in the mammalian brain, and drew initial attention to synapse-linked genetic disorders of the human nervous system.

This characterisation of the NMDA receptor complex was extended in a comprehensive study 16635246 reporting the proteomic characterisation of many of the receptor complexes of the postsynaptic terminal, including the NMDA, metabotropic glutamate receptor 5 (mGluR5) and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) receptor complexes, as well as the postsynaptic density (PSD). Utilizing improving mass spectroscopy instrumentation and techniques, we found we could now detect 186 proteins in the NMDA receptor (NRC/MASC) complex.

 

Phosphoproteomics

Protein phosphorylation and its regulation is widely recognised to involved in many, if not all aspects of nervous system function. These include neuronal development, nervous system signalling, synaptic plasticity and various pathological processes. Given the number of potential protein phosphorylation sites on even a single synaptic protein, elucidating how dynamic phosphoregulation contributes to say trans-synaptic signalling and brain processes in a complex as large as the NMDA receptor, using conventional low-throughput biochemical detection techniques is clearly impossible.

We embarked upon research to develop methods employing proteomic techniques to detect protein phosphorylation state at a very large scale. Combining metal-affinity chromatography with mass spectrometry allowed us characterise hundreds of known and novel phosphorylation sites in parallel from hundreds of synapse proteins 15572359; 16118397; 19401593.

 

Targeted TAP-tagging technology for protein isolation

Proteomic analysis of neuronal receptor complexes, as we have described above depends upon biochemical isolation of the complexes prior to their analysis. The harsh conditions and chemicals employed often employed for the isolation step will almost certainly alter and denature these protein complexes from their native state.

Taking the lead from yeast biologists, we genetically engineered protein affinity purification tags into proteins of key interest in mice, using stem cell manipulation technology. Using this tandem affinity purification (TAP) tagging technique we isolated the PSD-95 scaffolding protein complex, a core organiser of the NMDA receptor complex at the postsynaptic density 19455133. Under these conditions we found PSD-95 associated with ~ 300 proteins, substantially more than prior investigations, and significantly enriched in schizophrenia susceptibility genes.

 

Human Brain Proteomics

Our most recent proteomics studies have focussed on applying these neuroproteomic techniques to characterise neuronal receptor multi-protein complexes isolated from human brain tissue specimens. Our motivations for doing this include developing a better understanding of the causes of human psychiatric and neurological disorders, and also to shed light on human brain evolution, by using comparative genomics approaches.

Proteomic analysis of human PSDs was carried out using brain tissue obtained from patients undergoing brain surgery. The proteomic mass spectroscopy yielded 1461 proteins, each one encoded by a different gene found in human synapses as part of the PSD. This made it possible, for the first time, to systematically identify the diseases that affect human synapses. Additionally we found, by examining the evolutionary rates of these synaptic proteins, that the human PSD complex is highly evolutionarily constrained, more so than all cellular organelles examined, and indeed the human brain as a whole 21170055.

Comparing this set of human PSD proteins to those we identified from the PSD using identical proteomic methods and instrumentation from murine brain tissue, has allowed us to make a critical evaluation of the use of mice as experimental models for human brain function and disorders which focus on the synapse 23071613

 

References

  • Synaptopathies: diseases of the synaptome.

    Grant SG

    Genes to Cognition Programme, Centre for Clinical Brain Sciences, The University of Edinburgh, Chancellors Building, 47 Little France Crescent, Edinburgh EH16 4SB, United Kingdom. seth.grant@ed.ac.uk

    The human synapse proteome is a highly complex collection of proteins that is disrupted by hundreds of gene mutations causing over 100 brain diseases. These synaptic diseases, or synaptopathies, cause major psychiatric, neurological and childhood developmental disorders through mendelian and complex genetic mechanisms. The human postsynaptic proteome and its core signaling complexes built by the assembly of receptors and enzymes around Membrane Associated Guanylate Kinase (MAGUK) scaffold proteins are a paradigm for systematic analysis of synaptic diseases. In humans, the MAGUK Associated Signaling Complexes are mutated in Autism, Schizophrenia, Intellectual Disability and many other diseases, and mice carrying orthologous mutations show relevant cognitive, social, motoric and other phenotypes. Diseases with similar phenotypes and symptom spectrums arise from disruption of complexes and interacting proteins within the synapse proteome. Classifying synaptic disease phenotypes with genetic and proteome data provides a new brain disease classification system based on molecular etiology and pathogenesis.

    Funded by: Medical Research Council: G06706B; Wellcome Trust

    Current opinion in neurobiology 2012;22;3;522-9

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

    Bayés A, Collins MO, Croning MD, van de Lagemaat LN, Choudhary JS and Grant SG

    Molecular Physiology of the Synapse Laboratory, Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau, UAB, Barcelona, Catalonia, Spain. ABayesP@santpau.cat

    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.

    Funded by: Medical Research Council: G0802238; Wellcome Trust

    PloS one 2012;7;10;e46683

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

    Bayés A, van de Lagemaat LN, Collins MO, Croning MD, Whittle IR, Choudhary JS and Grant SG

    Genes to Cognition Programme, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire, UK.

    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.

    Funded by: Chief Scientist Office: CZB/4/486; Medical Research Council: G0802238, G0802238(89569); Wellcome Trust: 066717, 077155

    Nature neuroscience 2011;14;1;19-21

  • Neurotransmitters drive combinatorial multistate postsynaptic density networks.

    Coba MP, Pocklington AJ, Collins MO, Kopanitsa MV, Uren RT, Swamy S, Croning MD, Choudhary JS and Grant SG

    Genes to Cognition, Wellcome Trust Sanger Institute, Cambridgeshire, UK.

    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.

    Funded by: Medical Research Council: G90/93; Wellcome Trust: 066717

    Science signaling 2009;2;68;ra19

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

    Fernández E, Collins MO, Uren RT, Kopanitsa MV, Komiyama NH, Croning MD, Zografos L, Armstrong JD, Choudhary JS and Grant SG

    Genes to Cognition Programme, The Wellcome Trust Sanger Institute, Cambridge, UK.

    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.

    Funded by: Wellcome Trust

    Molecular systems biology 2009;5;269

  • Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome.

    Collins MO, Husi H, Yu L, Brandon JM, Anderson CN, Blackstock WP, Choudhary JS and Grant SG

    Genes to Cognition, The Wellcome Trust Sanger Institute, Hinxton, UK.

    Characterization of the composition of the postsynaptic proteome (PSP) provides a framework for understanding the overall organization and function of the synapse in normal and pathological conditions. We have identified 698 proteins from the postsynaptic terminal of mouse CNS synapses using a series of purification strategies and analysis by liquid chromatography tandem mass spectrometry and large-scale immunoblotting. Some 620 proteins were found in purified postsynaptic densities (PSDs), nine in AMPA-receptor immuno-purifications, 100 in isolates using an antibody against the NMDA receptor subunit NR1, and 170 by peptide-affinity purification of complexes with the C-terminus of NR2B. Together, the NR1 and NR2B complexes contain 186 proteins, collectively referred to as membrane-associated guanylate kinase-associated signalling complexes. We extracted data from six other synapse proteome experiments and combined these with our data to provide a consensus on the composition of the PSP. In total, 1124 proteins are present in the PSP, of which 466 were validated by their detection in two or more studies, forming what we have designated the Consensus PSD. These synapse proteome data sets offer a basis for future research in synaptic biology and will provide useful information in brain disease and mental disorder studies.

    Funded by: Wellcome Trust

    Journal of neurochemistry 2006;97 Suppl 1;16-23

  • The proteomes of neurotransmitter receptor complexes form modular networks with distributed functionality underlying plasticity and behaviour.

    Pocklington AJ, Cumiskey M, Armstrong JD and Grant SG

    School of Informatics, Edinburgh University, Edinburgh, UK.

    Neuronal synapses play fundamental roles in information processing, behaviour and disease. Neurotransmitter receptor complexes, such as the mammalian N-methyl-D-aspartate receptor complex (NRC/MASC) comprising 186 proteins, are major components of the synapse proteome. Here we investigate the organisation and function of NRC/MASC using a systems biology approach. Systematic annotation showed that the complex contained proteins implicated in a wide range of cognitive processes, synaptic plasticity and psychiatric diseases. Protein domains were evolutionarily conserved from yeast, but enriched with signalling domains associated with the emergence of multicellularity. Mapping of protein-protein interactions to create a network representation of the complex revealed that simple principles underlie the functional organisation of both proteins and their clusters, with modularity reflecting functional specialisation. The known functional roles of NRC/MASC proteins suggest the complex co-ordinates signalling to diverse effector pathways underlying neuronal plasticity. Importantly, using quantitative data from synaptic plasticity experiments, our model correctly predicts robustness to mutations and drug interference. These studies of synapse proteome organisation suggest that molecular networks with simple design principles underpin synaptic signalling properties with important roles in physiology, behaviour and disease.

    Funded by: Medical Research Council: G90/93; Wellcome Trust

    Molecular systems biology 2006;2;2006.0023

  • Robust enrichment of phosphorylated species in complex mixtures by sequential protein and peptide metal-affinity chromatography and analysis by tandem mass spectrometry.

    Collins MO, Yu L, Husi H, Blackstock WP, Choudhary JS and Grant SG

    The Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK. moc@sanger.ac.uk

    Reversible protein phosphorylation mediated by kinases, phosphatases, and regulatory molecules is an essential mechanism of signal transduction in living cells. Although phosphorylation is the most intensively studied of the several hundred known posttranslational modifications on proteins, until recently the rate of identification of phosphorylation sites has remained low. The use of tandem mass spectrometry has greatly accelerated the identification of phosphorylation sites, although progress was limited by difficulties in phosphoresidue enrichment techniques. We have improved upon existing immobilized metal-affinity chromatography (IMAC) techniques for capturing phosphopeptides, to selectively purify phosphoproteins from complex mixtures. Combinations of phosphoprotein and phosphopeptide enrichment were more effective than current single phosphopeptide purification approaches. We have also implemented iterative mass spectrometry-based scanning techniques to improve detection of phosphorylated peptides in these enriched samples. Here, we provide detailed instructions for implementing and validating these methods together with analysis by tandem mass spectrometry for the study of phosphorylation at the mammalian synapse. This strategy should be widely applicable to the characterization of protein phosphorylation in diverse tissues, organelles, and in cell culture.

    Science's STKE : signal transduction knowledge environment 2005;2005;298;pl6

  • Proteomic analysis of in vivo phosphorylated synaptic proteins.

    Collins MO, Yu L, Coba MP, Husi H, Campuzano I, Blackstock WP, Choudhary JS and Grant SG

    Division of Neuroscience, University of Edinburgh, Edinburgh EH8 9JZ, UK.

    In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.

    The Journal of biological chemistry 2005;280;7;5972-82

  • Systems biology in neuroscience: bridging genes to cognition.

    Grant SG

    Division of Neuroscience, 1 George Square, Edinburgh EH8 9JZ, UK. seth.grant@ed.ac.uk

    Systems biology is a new branch of biology aimed at understanding biological complexity. Genomic and proteomic methods integrated with cellular and organismal analyses allow modelling of physiological processes. Progress in understanding synapse composition and new experimental and bioinformatics methods indicate the synapse is an excellent starting point for global systems biology of the brain. A neuroscience systems biology programme, organized as a consortium, is proposed.

    Current opinion in neurobiology 2003;13;5;577-82

  • Proteomics of multiprotein complexes: answering fundamental questions in neuroscience.

    Grant SG and Husi H

    Dept of Neuroscience, University of Edinburgh, UK. seth.grant@ed.ac.uk

    Proteomics tools offer new ways to analyse networks of proteins that control important neurobiological phenomena such as learning and memory. In this review, we discuss how a combined proteomic, pharmacological and genetic approach reveals that multiprotein complexes process neural information and encode memories. Simultaneous analysis of multiple proteins enables the development of new concepts and approaches for neuroscience research.

    Trends in biotechnology 2001;19;10 Suppl;S49-54

  • Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain.

    Husi H and Grant SG

    Department of Neuroscience, University of Edinburgh, Edinburgh, UK.

    Neurotransmitter receptors in vivo are linked to intracellular adaptor proteins and signalling molecules driving downstream pathways. Methods for physical isolation are essential to answer fundamental questions about the size, structure and composition of in vivo complexes and complement the widely used yeast 2-hybrid method. The N-methyl-D-aspartate receptor (NMDAR) binds postsynaptic density 95 (PSD-95) protein; both are required for synaptic plasticity and learning and participate in other important pathophysiological functions. Here we describe the development and optimization of novel methods for large-scale isolation of NMDAR--PSD-95 complexes from mouse brain including immunoaffinity, immunoprecipitation, ligand-affinity and immobilized PSD-95 binding peptides. Short PDZ binding peptides modelled on NMDAR subunits were shown to isolate NMDAR complexes. Gel filtration indicated the native NMDAR--PSD-95 complexes were 2000 kDa, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a complexity suggesting a huge network of both structural components and signalling enzymes. These methods can be used to define the structure of the complexes at different synapses and in mice carrying gene mutations as well as new tools for drug discovery.

    Journal of neurochemistry 2001;77;1;281-91

© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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