The objective of the proposed research is to identify and characterize signaling mechanisms that lead to local mRNA translation in growth cones and developing axons. Guidance cues, such as Semaphorin 3A (Sema3A), mediate their effects on growth cones by activating signaling pathways that result in cytoskeletal reorganization. An emerging concept is that guidance cues, and other signaling molecules that act on growth cones, activate intracellular signaling pathways that result in intra-axonal mRNA translation, and this """"""""local translation"""""""" is a crucial mechanism by which developing axons can dynamically and rapidly regulate protein composition at specific sites. To identify signaling pathways that lead to local translation, we have performed screens to identify axonal mRNAs and we have developed assays to characterize local translation using live-cell imaging. Our recent studies have demonstrated that transcripts encoding RhoA, a major regulator of the neuronal cytoskeleton, are localized to developing axons and growth cones, and this localization is mediated by an axonal targeting element located in the RhoA 3'untranslated region (UTR). Sema3A induces intra-axonal translation of RhoA mRNA, and this local translation of RhoA is necessary for Sema3A-mediated growth cone collapse. As part of our overall goal to understand the intracellular signaling pathways that regulate the translation of mRNAs in growth cones and to identify the signaling mechanisms required for the actions of Sema3A and other guidance cues, the specific aims of this proposal are: (1) To identify signal transduction pathways regulating RhoA translation in growth cones. Experiments in Aim I describe pharmacologic and genetic approaches to probe the role of the p42/44 MARK and mTOR pathways in axonal RhoA translation using quantitative immunofluorescence and a live-cell fluorescent translational reporter assay. (2) To determine the role of the microRNA pathway in the regulation of RhoA translation. The microRNA machinery is present and functional in developing axons, and the RhoA mRNA S'UTR has a striking enrichment of predicted microRNA binding sites. Experiments in Aim II address the role of microRNAs in axonal RhoA translation. (3) To determine the role of FMRP in the local translation of RhoA in developing axons. RhoA mRNA binds the fragile X mental retardation protein, FMRP, and experiments in Aim III determine the role of FMRP in local RhoA translation. Together, the experiments in this proposal will advance our understanding of signaling by Sema3A, a prototypical guidance cue, as well as the regulation of RhoA, a protein that has crucial roles in neuronal morphogenesis. Furthermore, the experiments proposed here address several critical and emerging signaling pathways and significantly advance our understanding of their role in local translation in axons. We anticipate that our studies will have broad relevance to other neuronal activities that utilize local translation, including synaptogenesis and synaptic plasticity.
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