In Vivo Investigations of AMPA receptor transport

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Project Summary/Abstract: The overall goal of this proposal is to investigate the poorly understood mechanisms controlling long distance AMPAR transport in delivery and removal of receptors for synaptic maintenance and plasticity. Excitatory neurotransmission mediated by glutamate and ionotropic glutamate receptors of the AMPA subtype (AMPAR) at synapses plays a central role in cognition. Tight regulation of the number and function of these receptors is, therefore, essential. Since synapses are often far away from the neuronal cell body, they are critically dependent on long-distance transport by microtubule-dependent molecular motors to provide a steady supply of AMPARs. The field of excitatory synaptic transmission has a detailed understanding of how cell- signaling pathways control local synaptic AMPAR trafficking but almost no understanding of how these synaptic signaling events control long-distance AMPAR transport. The major reason for this lack is technical: transport studies require powerful, high-speed microscopy in intact neuronal circuits. Direct observation and informative manipulation of transport in vivo is extremely difficult in vertebrates. We have pioneered real-time in vivo studies of AMPAR transport in intact neuronal circuits using the transparent model organism, C. elegans. Here we will test a new mechanistic framework for the regulated cellular distribution of AMPARs to synapses centered on the long-distance transport of receptors by molecular motors. Our model predicts that Kinesin-1 scaffolds (JIP1 and 3) are necessary for AMPAR transport and their assembly onto Kinesin-1 is dependent on neuronal activity, calcium and calcium calmodulin-dependent kinase 2 (CaMKII). In addition, we identify a modulator of transport, PTP-3A, that modifies export from the cell body and synaptic delivery ultimately affecting memory. Specific Aim 1 will determine how synaptic inputs at cell bodies and at dendrites modify calcium and AMPAR transport. Specific Aim 2 tests the hypothesis that synaptic activity leads to modification of the AMPAR transport complex conferring different export and synaptic delivery properties. Specific Aim 3 tests the hypothesis that PTP-3A the longest isoform of the receptor tyrosine phosphatase PTP-3, regulates AMPAR somatic export and synaptic delivery using 2 domains released by cleavage induced by neuronal activity. The experiments described in these aims will combine genetics, in vivo spinning disk dual channel microscopy, optogenetics, photobleaching and photoconversion, biochemistry and behavior analyses to elucidate the mechanisms of long- distance AMPAR transport regulation by synaptic signaling. Our studies will: 1) provide a new model for understanding the cellular mechanisms regulating synaptic function, 2) have broad impact on the understanding of cargo delivery and removal mechanisms by molecular motors applicable to multiple biological systems, and 3) improve understanding of AMPAR transport that could reveal novel therapeutic targets for modulating excitatory synaptic transmission in the context of human disease.