Silvio O. Conte Center
Projects and Cores > Project 2
Project Leader: Jim Eberwine
The molecular biology of the glial component of the tripartite synapse has been woefully neglected. It is clear that the interactions of the post-synaptic and pre-synaptic sites of neurons balance with those of the astrocyte to produce a dynamic functional synapse. We are extending our previous work on neuronal dendrites to determine the capacity of astrocytic processes to locally control mRNA movement and protein translation. These studies are divided into two specific aims with the first Specific Aim devoted to characterization of the mRNA expression profile in astrocytic processes and the associated cell soma. This is being accomplished using the single cell aRNA nucleic acid amplification procedure coupled with micro- and macro-array analysis. The second Specific Aim assesses the ability of neuronal stimulation to modulate translation of mRNAs in astrocytic processes. These experiments utilize primary cultures of mouse astrocytes as vehicles for the transfection of mRNAs that, upon translation, give rise to detectable proteins.
Astrocytic processes are mechanically microdissected and various mRNAs transfected followed by fluorescent and immunodetection of protein locally translated from transfected mRNAs. Subsequent experiments will use intact astrocytes that have been transfected with caged mRNA that inhibits the mRNA translation. Upon in vivo laser uncaging the mRNA can be translated at the site of uncaging. Such experiments in combination with siRNA knock-down of selective gene expression will also permit an analysis of the functional significance of astrocytic process synthesized proteins.
Eventually we will determine the complement of mRNAs that are localized to astrocytes and astrocytic processes. Further we will determine whether astrocytic processes can translate these localized mRNAs as well as the spatiotemporal aspects of translation in this subcellular region. Finally, using RNA transfection methodologies, we will determine whether neuronal activity modulates astrocytic process mRNA translation, and using novel caged mRNAs in concert with siRNA knock-down experiments, determine whether astrocytic translation of mRNAs can modulate synaptic activity.