Molecular mechanisms of anterograde transport

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DESCRIPTION (provided by applicant): Anterograde transport is crucial for maintenance of the synapse in neurons. Anterograde transport over long distances in neuronal processes is mediated by a combination of actin and microtubule tracks and molecular motors, kinesin, dynein and myosin. How cargo attaches to the correct motor to be transported to specific locations within the cell is a major question in neuronal physiology. Numerous studies suggest a role for amyloid precursor protein (APP), which when proteolyzed produces the toxic AB fragment of Alzheimer's disease. We have developed human herpes simplex virus 1, HSV, as a tool to discover molecular mechanisms of cargo-transport interactions. At different times in its life cycle, HSV travels in either the retrograde or the anterograde direction in neuronal processes. We reconstituted retrograde transport of HSV in the giant axon of the squid by injecting GFP-VP16 labeled HSV into axon and imaged transport by confocal microscopy. In this project, anterograde transport of GFP- and mRFP1- labeled HSV will be reconstituted in the giant axon. This assay will be used to discover the molecular basis for cargo-motor interactions. In Preliminary Results, we show that GFP-VP16-labeled HSV is also transported in the anterograde direction in the giant axon. Motile virus co-purifies with full length APP -- and extraction of APP removes motility. By coating fluorescent beads with peptide fragments of APP and injecting then into the axon, we discovered a 15 amino acid sequence sufficient to mediate transport of the beads. In this application, we will: (1) analyze the direction and velocity of HSV anterograde transport and identify the motor receptor(s) that the virus uses, (2) determine the peptide sequence of APP sufficient for anterograde motility using peptide-coated fluorescent beads injected into the giant axon, (3) determine how HSV acquires the motor receptor during its synthesis in cultured cells, and (4) discover whether HSV affects APP proteolysis into toxic fragments. Results from these studies will definitively identify the mechanism of HSV transport and the role of APP. Thus, these results will yield answers to universal questions on axonal transport, specific facts about herpes pathogenesis, and fundamental new information about the role of APP in Alzheimer's disease.