The experiments of this proposal seek to define some of the cellular and molecular mechanisms of synapse growth and modification in the mature mammalian CNS. The project is based on a model system (the rat's dentate gyrus) where denervation induces a growth of presynaptic terminals in the denervated zone (terminal proliferation) which reinnervate the denervated dentate granule cells (the phenomenon of sprouting). The hope is that an understanding of how neurons elaborate new terminals and synapses in response to denervation may lead to clues to promote regeneration of cut axons where it normally does not occur (for example, after spinal cord injury). The present experiments are based on three discoveries: 1) that there is an increase in incorporation of protein precursors in the denervated neuropil of the dentate gyrus during the early stages of sprouting (which presumably reflects and increase in the production of some protein(s) in the denervated zone), 2) that protein synthetic machinery (polyribosomes) are selectively localized in the base of dendritic spines of granule cells of the dentate gyrus and other spine-bearing neurons, and 3) that there is a dramatic increase in the number of polyribosomes under spines during the early phases of sprouting, at the time of the increases in protein synthesis. The working hypotheses are A) that the polyribosomes produce protein(s) related to the synapse (synaptic protein, cytoskeletal protein, or protein for local release at the site of synaptic contact), B) that these protein(s) are particularly crucial during periods of synaptic growth and modification, and may even be responsible for inducing growth of presynaptic elements (a growth factor produced and released at the synapse?), and C) that the synthetic activity of these polyribosomes is likely to be regulated by synaptic activity. We will explore these hypotheses by I) characterizing the nature of the polyribosomes under spines, defining their associations with other cytoplasmic organelles and their interactions with the cytoskeleton, II) Defining how protein synthesis in dendrites and RNA synthesis and transport into dendrites is regulated (particularly by synaptic activity and during periods of lesion-induced growth), III) Defining the biochemical nature of the proteins produced by polyribosomes under spines, IV) defining the biochemical nature of the proteins produced to a greater extent during reinnervation (if different than normal), and V) Defining the molecular architecture of the cytoskeleton of the dendritic spine, and the alterations in this molecular architecture during spine deterioration and renovation.
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