Regeneration capacity is reduced as CNS axons mature. Using laser-mediated axotomy, proteomics and puromycin-based tagging of newly-synthesized proteins in a human embryonic stem cell-derived neuron culture system that allows isolation of axons from cell bodies, we show here that efficient regeneration in younger axons (d45 in culture) is associated with local axonal protein synthesis (local translation). Enhanced regeneration, promoted by co-culture with human glial precursor cells, is associated with increased axonal synthesis of proteins, including those constituting the translation machinery itself. Reduced regeneration, as occurs with the maturation of these axons by d65 in culture, correlates with reduced levels of axonal proteins involved in translation and an inability to respond by increased translation of regeneration promoting axonal mRNAs released from stress granules. Together, our results provide evidence that, as in development and in the PNS, local translation contributes to CNS axon regeneration.
Bibliographical noteFunding Information:
Grants supporting this study were EMBO ALTF1436-2015 (SvE), MS Society UK Research Grant 79 (CffC and SvE), Neurostemcellrepair FP7 (CffC), Wellcome Trust Investigator award 104783/Z/14/Z (CffC). This study was furthermore made possible by funding from National Institutes of Health to JLT (R01-NS117821) and South Carolina Spinal Cord Injury Research fund to PKS (2019-PD-02). Alex von Kriegsheim at the IGMM Mass Spectrometry facility (University of Edinburgh) carried out the identification of newly synthesized peptides following axonal injury, for which the ?Wellcome Trust (Multiuser Equipment Grant, 208402/Z/17/Z)? was used to buy Lumos. Lisa Imrie and Dr. Thierry Le Bihan at the EdinOmics research facility at the University of Edinburgh carried out the mass spectrometry experiments and analyses on axonal samples of different ages. The authors also want to thank Tissue Culture (SCRM) and Imaging (CRM and CALM at QMRI) facility staff for their support and expertise, in particular Dr Trudi Gillespie, Dr Rachel Verdon, and Dr Bertrand Vernay.
© 2021 The Author(s)
Copyright 2021 Elsevier B.V., All rights reserved.
- Axon regeneration
- Human stem cells
- In vitro live imaging
- Local translation