The functional component of the nervous system, neurons, are the largest cells in the body. Animals have somehow evolved mechanisms to maintain these huge cells. While most neuronal proteins are provided to distant cellular regions (i.e., dendritic and axonal compartments) by transport from the cell body, it has become clear in recent years that some proteins are produced locally. Localized protein synthesis has been best characterized in dendrites where activity appears to regulate local post-synaptic protein synthesis. Several lines of evidence indicate that translation also occurs in the axonal compartment. While this has been best documented in invertebrate species, several laboratories have reported that vertebrate neurons can locally synthesize proteins in developing axons. We have recently shown that regenerating axons of adult rat neurons locally synthesize proteins during regeneration. In both the developing and regenerating neurons, intra-axonal translation contributes to the structure of the growing axon by providing a locally renewable source of cytoskeleton. Most studies of localized neuronal protein synthesis have been restricted to cultured neurons. In situ hybridization and immunofluorescence have been used to confirm that axons contain RNA and translation machinery in vivo. However, there are no other methods to visualize RNA transport in real tissues. In situ hybridization suffers from low resolution and provides no clue of where the mRNA is utilized. The objective of this proposal is to develop a mouse model in which we can visualize RNA trafficking and local protein synthesis in vivo. Here we will generate transgenic mice where localization of a reporter is driven by a well-characterized cis-element of beta-actin mRNA. Beta-actin is the most abundant mRNA that we have identified in regenerating axons and it also extends into developing axons. We will initially determine the elements necessary for localization of beta-actin mRNA in cultured rat and mouse sensory neurons. We will then use this information to generate mice whose neurons express chimeric mRNAs with the axonal-localization motif. If successful, these animals will allow us to analyze RNA trafficking and local protein synthesis in vivo. Furthermore, we will be able to determine if local protein synthesis occurs in axons of CNS neurons that we cannot culture from adult animals (e.g., spinal motor neurons) and whether we can utilize exogenous agents to increase local axonal protein synthesis for expediting axonal regeneration.
| Donnelly, Christopher J; Willis, Dianna E; Xu, Mei et al. (2011) Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity. EMBO J 30:4665-77 |
| Willis, Dianna E; Xu, Mei; Donnelly, Christopher J et al. (2011) Axonal Localization of transgene mRNA in mature PNS and CNS neurons. J Neurosci 31:14481-7 |
| Wang, Wenlan; van Niekerk, Erna; Willis, Dianna E et al. (2007) RNA transport and localized protein synthesis in neurological disorders and neural repair. Dev Neurobiol 67:1166-82 |
| Fainzilber, Mike; Twiss, Jeffery L (2006) Tracking in the Wlds--the hunting of the SIRT and the luring of the Draper. Neuron 50:819-21 |
| Willis, Dianna E; Twiss, Jeffery L (2006) The evolving roles of axonally synthesized proteins in regeneration. Curr Opin Neurobiol 16:111-8 |
| Twiss, Jeffery L; van Minnen, Jan (2006) New insights into neuronal regeneration: the role of axonal protein synthesis in pathfinding and axonal extension. J Neurotrauma 23:295-308 |
