The long term goal of this project is to define the cellular mechanisms of dendritic transport, especially the mechanisms responsible for the selective dendritic transport of RNA. While the sorting and transport of mRNA are ubiquitous biological processes, they are of particular importance in neurons, where they are thought to contribute to the development and plasticity of postsynaptic sites. Previous studies have demonstrated that dendrites, but not axons, have the capacity for RNA transport and have defined some of the mRNAs that undergo dendritic transport. The present proposal focuses on three representative dendritic mRNAs, those that encode CAMII kinase, MAP2b, and Arc. Defective herpesvirus vectors will be used to express tagged constructs of these mRNAs in cultured rat hippocampal neurons in order to study their targeting and transport. One experiment will use deletion analysis to define a minimum targeting sequence that is sufficient to direct an exogenous message to the dendrites. The mechanisms that prevent most neuronal mRNAs from entering dendrites will also be examined. A second experiment will use in situ hybridization at different times after the expression of tagged constructs to define the rates of transport of CAMII kinase, MAP2b, and Arc mRNAs and to determine if their transport is regulated by neuronal activity. Selective inhibitors and antisense inhibition of motor protein expression will be used to test the hypothesis that dendritic mRNA transport is mediated by minus-end directed, microtubule-based motors.
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