There is clear evidence that local translation of proteins at distal regions of neurons is an important solution to the highly compartmentalized nature of these cells. Protein synthesis is well documented in dendrites where it serves to facilitate synaptogenesis and neuroplasticity at postsynaptic sites. However, less is known about local translation in axons. A growing body of work has shown that axonal protein synthesis is required for the development, maintenance, and plasticity of axons and presynaptic terminals and may underlie a number of neurodegenerative conditions. However, much of these studies have relied on cultured neuronal systems since axonal ribosomes are not abundant and the lack of reliable methods for isolating axonal compartments have made in vivo studies extremely challenging. The proposed study aims to develop a novel method that overcomes many of these obstacles to examine locally translated axonal mRNAs in distinct cell populations in vivo. This method is based on the translating ribosome affinity purification (TRAP) technique and will utilize viral vectors to deliver tagged ribosome proteins to the axons of genetically defined cell populations, allowing for the isolation of ribosome- associated transcripts. This viral strategy will then be used to perform a comprehensive comparative analysis of axonal translation across four distinct classes of projection neurons with distinct neurotransmitter profiles. Bioinformatics analysis will, for the first time, establish shared and cell type specific properties and regulation of axonal translated mRNAs. The development of this viral approach will provide the field with a simple and versatile method for profiling locally translated transcripts by axons in vivo and offer a strategy for measuring changes in axonal gene expression during development, plasticity, or neurodegenerative disorders.
Local translation of proteins in the axons of projection neurons likely plays an important role in the establishment, maintenance, and plasticity of presynaptic terminals, neurodegeneration in disease, as well as regeneration following injury. The proposed research will develop a novel and simple technique for isolating translated genes from the axons of specific neuron populations in the intact brain, which has traditionally been extremely technically challenging. By comparing results from four distinct classes of projection neurons, this project aims to elucidate fundamental properties of axonally-localized transcripts while establishing a method to ask how these genes are regulated in health and disease.
