Neuronal dendrites are the initial post-synaptic interpreters and integrators of presynaptic information. Over the last several decades data has been generated from both in vitro and in vivo experiments that shows that dendrites increase in number and size in response to various behavioral and pharmacological manipulations of the experimental system. More recently mRNAs have been localized in dendrites. Further data has been generated that shows that these mRNAs can be translated locally, in the dendrite, in response to various types of modulators. Given these observations, it is disappointing that so little is known about the mechanism of mRNA transport to dendrites. This grant will examine aspects of regulated mRNA transport into the dendrites of rat hippocampal pyramidal neurons. In particular we will take a two pronged approach in which we characterize the identity and kinetics of mRNAs transported into dendrites in response to DHPG, BDNF and serum starvation using single dendrite mRNA amplification and mRNA analysis. Initially, we will use microarrays, differential display and in situ hybridization, to characterize the mRNA population that is transported into the dendrite. As an adjunct, we also develop the """"""""molecular beacon"""""""" technology for analysis of mRNA transport in vivo. Secondly, we will characterize the mRNA cargo's associated with particular mRNA binding proteins known to localize to dendrites. mRNA cargo characterization will be performed using standard mutagenesis, gel shift assays and UV cross-linking as well as with a novel technology called antibody-positioned RNA amplification (APRA) that was recently developed in our laboratory. APRA permits the identification and quantitation of mRNAs associated with any specific RNA-binding protein in fixed cells. We have used this assay to successfully define candidate RNA cargos for FMR1, an RNA-binding protein found in dendrites. The characterization of mRNA cargo's associated with particular RNA binding proteins should permit a determination of whether the distribution of RNAs bound to these binding proteins differs with different types of stimulation. Those mRNAs whose transport rates are similar, or those that bind to the same RNA-binding protein, wifi be candidates for sequence analysis in an effort to determine if a common structural feature (primary sequence or 2nd structure) exists within a class of mRNAs that is responsible for the targeting of these mRNAs to dendrites. These data will have impact upon how we think about post-synaptic involvement in regulation of the Hebbian synapse. It is conceivable that age-related changes in RNA transport and local protein synthesis may underlie aspects of dysfunction during the aging process.
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