Messenger RNAs are present in the dendrites of hippocampal neurons, and are probably used for local protein synthesis in response to synaptic activity during long-term plasticity. To begin to understand the mechanisms underlying dendritic mRNA targeting, we have cloned a gene from a rat hippocampal cDNA library that is related to Drosophila staufen, a gene critical for the asymmetric targeting of specific mRNAs in oocytes and neuroblasts. In situ hybridization experiments indicate that rat staufen is expressed in the hippocampus, including the CA1-CA3 and dentate gyrus regions. Preliminary results of in vitro RNA binding assays suggest that the recombinant rat Staufen protein can bind to multiple mRNAs present in the dendrites of hippocampal neurons, including CamKII, TrkB and Arc. Transfection of hippocampal neurons with a GFP-Staufen fusion construct resulted in a punctate GFP pattern in dendrites, suggesting a dendritic distribution for rat Staufen. Immunohistochemistry suggests a similar distribution. RNA staining experiments on GFP-Staufen expressing neurons revealed that signals for GFP and RNA co-localize. These results are consistent with the hypothesis that rat Staufen is associated with dendritic RNAs in vivo and is may be involved in their targeting to dendritic compartments.

We are also interested in visualizing, directly, the synthesis of proteins in dendrites. It appears that the 3' untranslated region (3'UTR) of some localized mRNAs contains a signal that allows for transport of some mRNAs to the dendrite. To this end, we have constructed a cDNA construct encoding a destabilized variant of EGFP flanked by the 5' and 3' UTR regulatory regions of the CamKII- gene. The cDNA constructs were transfected into cultured hippocampal neurons using Biolistics. Our preliminary results demonstrate a different fluorescence distribution between neurons transfected with un-targeted vs. targeted GFP (e.g. +/- the 3'UTR). Cells expressing the GFP-3'UTR construct exhibit a punctate GFP distribution within the processes as compared to an apparently diffusion-limited distribution seen in cells expressing the control GFP construct, suggesting local synthesis of GFP. In addition, neurons transfected with a construct containing the 5' UTR exhibit lower integrated levels of fluorescence suggesting a translational regulatory role of the 5'UTR. We are currently examining the effects of synaptic activity and plasticity on translation of the localized GFP message.