Researchers have discovered that risk genes and associated molecular networks for autism spectrum disorder (ASD) and other psychiatric disorders participate in synapse development and function. Disorder risk, therefore, lies in the principle elements of neural communication, providing a focus for studies proposed in this competing renewal application. Advanced synaptic proteomics and functional experiments will bring a new understanding of the receptor tyrosine kinase c-MET (MET) and its interacting partners, which we recently discovered include other proteins implicated in ASD risk. Experiments will address a major knowledge gap in determining the mechanisms through which MET and members of its interactome contribute to early synapse development in circuits that are vulnerable in ASD. The proposed studies build upon new discoveries made during the current grant period: 1) MET is enriched in developing axons and synapses during the peak of synaptogenesis in rodent and primate forebrain; 2) genetic deletion of Met disrupts dendritic and spine architecture and neocortical interlaminar excitatory drive; 3) the functional promoter variant of MET alters human social-emotional circuit activation, network connectivity and the structural integrity of select fiber tracts; 4) MET transcription is regulated by proteins implicatd in other neurodevelopmental disorders, including Rett Syndrome and language delay; and 5) MET interacts directly with developmentally important synaptic proteins, including ?-catenin. The proposed experiments will 1) determine the MET synaptic protein interactome by co-immunoprecipiation/mass spectrometry (Aim 1), 2) measure changes in pre- and postsynaptic protein expression, including MET-interacting partners, in neocortex and striatum in Met-null compared to wild type mice, using unbiased global isobaric tagging for relative and absolute quantitation (iTRAQ)-based proteomics and targeted high resolution/accurate mass (HR/AM) proteomics (Aim 2), and 3) determine the role of the MET interactome, including ??catenin, on synapse development in vitro (Aims 1 & 2) and functional maturation of neocortical circuits in vivo (Aim 3). Specifically, experiments in vitro will use siRNA to disrupt the pre- and postsynaptic MET interactome and measure the impact on synaptogenesis and vesicle clustering. In utero electroporation in vivo will manipulate expression of postsynaptic interactome proteins, followed by functional mapping of input to layer 5 cortico-striatal neurons and measures of spine size and density. This research program provides a translational understanding of the proteins involved in synapse development related to circuit dysfunction. There will be high impact by integrating novel proteomics technologies with cell and circuit functional outcomes for determining mechanisms that underlie typical and atypical synapse development in the neocortex.
There is an enrichment of risk genes for psychiatric disorders that code for proteins involved in the development and maturation of synapses. The MET receptor tyrosine kinase is enriched at synapses and has been shown to increase autism risk due to a mutation that disrupts the normal regulation of gene expression. The proposed studies combine technically advanced approaches in proteomics, and neuronal and electrophysiological functional assays to determine how the MET protein interactome influences the receptor's functioning in synapse maturation and ultimately impacts the function of circuits in the neocortex.
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