Monitoring translation in synaptic fractions using a ribosome profiling strategy

Konstanze Simbriger, Inês S. Amorim, Kleanthi Chalkiadaki, Gilliard Lach, Seyed Mehdi Jafarnejad, Arkady Khoutorsky, Christos G. Gkogkas

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Abstract

Background
The aim of this study was to develop a method to study genome-wide local translation in biochemically isolated synaptic fractions (synaptoneurosomes). This methodology is of particular interest for neurons, due to the cardinal role of local translational control in neuronal sub-compartments, such as dendrites, for plasticity, learning, memory, and for disorders of the nervous system.

New method
We combined established methods for purifying synaptoneurosomes with translational profiling (ribosome profiling), a method that employs unbiased next generation sequencing to simultaneously assess transcription and translation in a single sample.

Results
The two existing methods are compatible to use in combination and yield high quality sequencing data, which are specific to synaptic compartments. This new protocol provides an easy to implement workflow, which combines biochemical isolation of synaptoneurosomes of varying levels of purity (crude or Percoll gradient purified) with the use of a commercial kit to generate sequencing libraries.

Comparison with existing methods
Compared to previous studies of the synaptic translatome, our method shows less contamination with non-neuronal cell types or non-synaptic compartments, increasing the specificity of the data obtained.

Conclusions
Combining the isolation of functional synaptic units with ribosome profiling offers a powerful tool to study local translation in synaptic compartments both in health and disease.
Original languageEnglish
Article number108456
Pages (from-to)108456
Number of pages7
JournalJournal of Neuroscience Methods
Volume329
Early online date11 Oct 2019
DOIs
Publication statusPublished - 01 Jan 2020

Bibliographical note

Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.

Keywords

  • Local translation
  • Ribosome profiling
  • Synapse
  • Synaptoneurosomes
  • mRNA translation

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