Neurotransmitters Drive Combinatorial Multistate Postsynaptic Density Networks

Genes to Cognition, Wellcome Trust Sanger Institute, Cambridgeshire, UK.
Science Signaling (Impact Factor: 6.28). 02/2009; 2(68):ra19. DOI: 10.1126/scisignal.2000102
Source: PubMed


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.


Available from: Seth G N Grant
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    • "The fact that UCP2 is originally found as a proton transporter responsible for uncoupling of respiration from oxidative phosphorylation in mitochondria [39], [40] gives rise to an idea that cellular dysfunctions may involve a mechanism associated with the deteriorated energy synthesis after ΔΨ disruption mediated by the interaction between GluN1 subunit and UCP2 in cells with functional NMDAR channels permeable to Ca2+. Furthermore, the cytoplasmic C-terminal tail of NMDAR interacts with a variety of proteins within the postsynaptic density (PSD), notably the membrane-associated guanylate kinase family including PSD-95 [41], whereas PSD-95 plays an important role in NMDAR-mediated synaptic plasticity through a mechanism related to modulation of the localization, clustering and subunit expression profile [42]–[44]. Although the possibility that the interaction may be merely derived from the artificial expression of UCP2 and NMDAR subunits in non-neuronal cells is not ruled out, a recent study has demonstrated the expression of a Ca2+ transport protein with features similar to NMDAR in mitochondria of the rat brain [45]. "
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    ABSTRACT: We have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity by N-methyl-D-aspartate receptor (NMDAR) through a mechanism relevant to the increased mitochondrial Ca(2+) levels in HEK293 cells with acquired NMDAR channels. Here, we evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca(2+) levels in cultured murine neocortical neurons. In neurons exposed to glutamate or NMDA, a significant increase was seen in mitochondrial Ca(2+) levels determined by Rhod-2 at concentrations of 0.1 to 100 µM. Further addition of 250 mM ethanol significantly inhibited the increase by glutamate and NMDA in Rhod-2 fluorescence, while similarly potent inhibition of the NMDA-induced increase was seen after exposure to ethanol at 50 to 250 mM in cultured neurons. Lentiviral overexpression of UCP2 significantly accelerated the increase by NMDA in Rhod-2 fluorescence in neurons, without affecting Fluo-3 fluorescence for intracellular Ca(2+) levels. In neurons overexpressing UCP2, exposure to ethanol resulted in significantly more effective inhibition of the NMDA-induced increase in mitochondrial free Ca(2+) levels than in those without UCP2 overexpression, despite a similarly efficient increase in intracellular Ca(2+) levels irrespective of UCP2 overexpression. Overexpression of UCP2 significantly increased the number of dead cells in a manner prevented by ethanol in neurons exposed to glutamate. In HEK293 cells with NMDAR containing GluN2B subunit, more efficient inhibition was similarly induced by ethanol at 50 and 250 mM on the NMDA-induced increase in mitochondrial Ca(2+) levels than in those with GluN2A subunit. Decreased protein levels of GluN2B, but not GluN2A, subunit were seen in immunoprecipitates with UCP2 from neurons with brief exposure to ethanol at concentrations over 50 mM. Ethanol could inhibit the interaction between UCP2 and NMDAR channels to prevent the mitochondrial Ca(2+) incorporation and cell death after NMDAR activation in neurons.
    PLoS ONE 07/2013; 8(7):e69718. DOI:10.1371/journal.pone.0069718 · 3.23 Impact Factor
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    • "Compared with microarray based screening and phage display system, the mass spectrometry not only identifies phosphoproteins, but also pinpoints their phosphosites. There have been increasing attempts to develop mass spectrometrybased proteomic strategies for the kinase substrates exploration (Huang et al., 2007; Amanchy et al., 2008; Coba et al., 2009). "
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    ABSTRACT: Since the discovery of protein phosphorylation as an important modulator of many cellular processes, the involvement of protein kinases in diseases, such as cancer, diabetes, cardiovascular diseases, and central nervous system pathologies, has been extensively documented. Our understanding of many disease pathologies at the molecular level, therefore, requires the comprehensive identification of substrates targeted by protein kinases. In this review, we focus on recent techniques for kinase substrate identification in high throughput, in particular on genetic and proteomic approaches. Each method with its inherent advantages and limitations is discussed.
    04/2013; 8(2). DOI:10.1007/s11515-013-1257-z
    • "Biochemical assays. Postsynaptic density (PSD) and nuclear fractions from mouse hippocampus were prepared using a three-step protocol as previously described (Coba et al., 2009). In brief, tissue was homogenized in ice-cold buffer (10 mM Tris-HCl, pH 7.4/320 mM sucrose, 20 mM ␤-glycerol phosphate, 1 mM sodium orthovanadate, and 50 mM sodium fluoride) and protease inhibitors (Roche-Complete protease inhibitor mixture ). "
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    ABSTRACT: Traf2 and NcK interacting kinase (TNiK) contains serine-threonine kinase and scaffold domains and has been implicated in cell proliferation and glutamate receptor regulation in vitro. Here we report its role in vivo using 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.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2012; 32(40):13987-13999. DOI:10.1523/JNEUROSCI.2433-12.2012 · 6.34 Impact Factor
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