Profiling axonal mRNA transport

Methods Mol Biol. 2011;714:335-52. doi: 10.1007/978-1-61779-005-8_21.

Abstract

The importance of mRNA localization and localized protein synthesis to spatially modulate protein levels in distinct subcellular domains has increasingly been recognized in recent years. Axonal and dendritic processes of neurons represent separate functional domains of the cell that have shown the capacity to autonomously respond to extracellular stimuli through localized protein synthesis. With the vast distance often separating distal axons and dendrites from the neuronal cell body, these processes have provided an appealing and useful model system to study the mechanisms that drive mRNA localization and regulate localized mRNA translation. Here, we discuss the methodologies that have been used to isolate neuronal processes to purity, and provide an in-depth method for using a modified Boyden chamber to isolate axons from adult dorsal root ganglion neurons for analyses of axonal mRNA content. We further show how this method can be utilized to identify specific mRNAs whose transport into axons is altered in response to extracellular stimuli, providing a means to begin to understand how axonal protein synthesis contributes to the proper function of the neuron.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Axons / drug effects
  • Axons / metabolism*
  • Cell Separation / instrumentation
  • Cell Separation / methods*
  • Ganglia, Spinal / cytology
  • Ganglia, Spinal / drug effects
  • Mice
  • Microfluidic Analytical Techniques
  • Nerve Growth Factors / pharmacology
  • Polymerase Chain Reaction
  • RNA Transport* / drug effects
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • Reverse Transcription
  • Tissue Culture Techniques

Substances

  • Nerve Growth Factors
  • RNA, Messenger