The goal of this research is to understand the roles in regulating synapse formation, function and plasticity of neurotrophins, their receptors trkA, B, and C and their downstream signaling molecules. To examine the role of neurotrophins in synapse formation, we will generate DNA constructs encoding fusion proteins in which constituents of different elements of the synapse (release zone, synaptic vesicle), such as VAMP, synapsin I, and the Ca channel beta subunit are fused to Green Fluorescent Protein (GFP). These will be injected into Xenopus embryo cells whose descendents include retinal ganglion cells. After grafting of """"""""tagged"""""""" retinal anlage from these to fresh embryos, retinal axon and synapse development will be studied in the optic tectum. Effects on synapse formation of neurotrophins and dominant negative constructs of the BDNF receptor trkB will be studied. Signaling pathways important in differentiation will be determined by injecting trkA constructs defective in interactions with individual downstream signaling molecules and determining effects of exogenous NGF. After insertion of the GFP chimeras into a defective Adenovirus, infections of murine retina will permit examination of synapse development in normal and neurotrophin-deficient mutant mice. The second project will seek to understand the molecular bases of neurotrophin regulation of exocytosis, observed by others at the Xenopus neuromuscular junction and Schaeffer collateral CA 1 synapses in the hippocampus. We will determine effects of NGF application on synaptic transmission at the Xenopus NMJ after injection of precursors with trkA. To dissect downstream signaling pathways, trkA receptors defective in activation of specific signalling pathways will be used in the same model. To further delineate a pathway, effects of dominant negative constructs of downstream signalling molecules, such PI-3 kinase and ras, will be examined. Efforts will be made to extend this analysis will be extended to a biochemically amenable system using PC12 cells expressing receptors with signalling pathway deficiencies where effects of neurotrophin application on phosphorylation of proteins associated with the exocytotic apparatus will be examined and functional consequences can be assessed.
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