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.

Department of Biological Sciences University of South Carolina Columbia 29208 SC USA

Department of Functional Genomics Center for Neurogenomics and Cognitive Research VU University Amsterdam De Boelelaan 1085 1081 HV Amsterdam the Netherlands

John van Geest Centre for Brain Repair Department of Clinical Neurosciences University of Cambridge Cambridge UK

John van Geest Centre for Brain Repair Department of Clinical Neurosciences University of Cambridge Cambridge UK; Centre for Reconstructive Neuroscience Institute of Experimental Medicine Czech Academy of Sciences Prague Czech Republic

MRC Centre for Regenerative Medicine and MS Society Edinburgh Centre Edinburgh bioQuarter University of Edinburgh Edinburgh UK

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Chambers S.M. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 2009 doi: 10.1038/nbt.1529. PubMed DOI PMC

Chen M.S. Nogo-a is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature. 2000;403(6768):434–439. doi: 10.1038/35000219. PubMed DOI

Ching R.C., Kingham P.J. The role of exosomes in peripheral nerve regeneration. Neural Regen. Res. 2015;10(5):743–747. doi: 10.4103/1673-5374.156968. PubMed DOI PMC

Costigan M. Multiple chronic pain states are associated with a common amino acid–changing allele in KCNS1. Brain. 2010;133(9):2519–2527. doi: 10.1093/brain/awq195. PubMed DOI PMC

Deng S. Plexin-B2, but not plexin-B1, critically modulates neuronal migration and patterning of the developing nervous system in vivo. J. Neurosci. 2007 doi: 10.1523/JNEUROSCI.5381-06.2007. PubMed DOI PMC

Eva R., Andrews M.R., Franssen E.H.P. Intrinsic mechanisms regulating axon regeneration: an integrin perspective. Int. Rev. Neurobiol. 2012;106:75–104. doi: 10.1016/B978-0-12-407178-0.00004-1. PubMed DOI

Fawcett J.W., Verhaagen J. Intrinsic determinants of axon regeneration. Dev. Neurobiol. 2018;78(10):890–897. doi: 10.1002/dneu.22637. PubMed DOI

Fawcett J.W. Defeating inhibition of regeneration by scar and myelin components. Handb. Clin. Neurol. 2012;109:503–522. doi: 10.1016/B978-0-444-52137-8.00031-0. PubMed DOI

Geeven G. LLM3D: a log-linear modeling-based method to predict functional gene regulatory interactions from genome-wide expression data. Nucleic Acids Res. 2011;39(13):5313–5327. doi: 10.1093/nar/gkr139. PubMed DOI PMC

Geoffroy C.G., Zheng B. Myelin-associated inhibitors in axonal growth after CNS injury. Curr. Opin. Neurobiol. 2014;27:31–38. doi: 10.1016/j.conb.2014.02.012. PubMed DOI PMC

Giles A.C., Grill B. Roles of the HUWE1 ubiquitin ligase in nervous system development, function and disease. Neural Dev. 2020 doi: 10.1186/s13064-020-00143-9. PubMed DOI PMC

Gumy L.F. Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization. RNA. 2011;17(1):85–98. doi: 10.1261/rna.2386111. PubMed DOI PMC

Gumy L.F. New insights into mRNA trafficking in axons. Dev. Neurobiol. 2014;74(3):233–244. doi: 10.1002/dneu.22121. PubMed DOI

Györffy B.A. Local apoptotic-like mechanisms underlie complementmediated synaptic pruning. Proc. Natl. Acad. Sci. U. S. A. 2018 doi: 10.1073/pnas.1722613115. PubMed DOI PMC

Haas C., Fischer I. Human astrocytes derived from glial restricted progenitors support regeneration of the injured spinal cord. J. Neurotrauma. 2013;30(12):1035–1052. doi: 10.1089/neu.2013.2915. PubMed DOI PMC

Hayakawa K. Glial restricted precursors maintain their permissive properties after long-term expansion but not following exposure to pro-inflammatory factors. Brain Res. 2015;1629:113–125. doi: 10.1016/j.brainres.2015.10.022. PubMed DOI PMC

Hayakawa K., Haas C., Fischer I. Examining the properties and therapeutic potential of glial restricted precursors in spinal cord injury. Neural Regen. Res. 2016;11(4):529–533. doi: 10.4103/1673-5374.180725. PubMed DOI PMC

Ji S.-J., Jaffrey S.R. Axonal transcription factors: novel regulators of growth cone-to-nucleus signaling. Dev. Neurobiol. 2013;74(3):245–258. doi: 10.1002/dneu.22112. PubMed DOI PMC

Jin Y., Shumsky J.S., Fischer I. Axonal regeneration of different tracts following transplants of human glial restricted progenitors into the injured spinal cord in rats. Brain Res. 2018;1686:101–112. doi: 10.1016/j.brainres.2018.01.030. PubMed DOI PMC

Kar A.N., Lee S.J., Twiss J.L. Expanding axonal Transcriptome brings new functions for Axonally synthesized proteins in health and disease. Neuroscientist. 2017 doi: 10.1177/1073858417712668. 107385841771266. PubMed DOI PMC

Keirstead H.S. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J. Neurosci. 2005;25(19):4694–4705. doi: 10.1523/JNEUROSCI.0311-05.2005. PubMed DOI PMC

Kirkeby A. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep. 2012;1(6):703–714. doi: 10.1016/j.celrep.2012.04.009. PubMed DOI

Koseki H. Selective rab11 transport and the intrinsic regenerative ability of CNS axons. eLife. 2017;6 doi: 10.7554/eLife.26956. PubMed DOI PMC

Li D., Field P.M., Raisman G. Failure of axon regeneration in postnatal rat Entorhino-hippocampal slice Coculture is due to maturation of the axon, not that of the pathway or target. Eur. J. Neurosci. 1995;7(6):1164–1171. doi: 10.1111/j.1460-9568.1995.tb01106.x. PubMed DOI

Livesey M.R. Maturation and electrophysiological properties of human pluripotent stem cell-derived oligodendrocytes. Stem Cells. 2016;34(4):1040–1053. doi: 10.1002/stem.2273. PubMed DOI PMC

Ma T.C., Willis D.E. What makes a RAG regeneration associated? Front. Mol. Neurosci. 2015 doi: 10.3389/fnmol.2015.00043. PubMed DOI PMC

Mi H. Large-scale gene function analysis with the PANTHER classification system. Nat. Protoc. 2013;8(8):1551–1566. doi: 10.1038/nprot.2013.092. PubMed DOI PMC

Michaelevski I. Signaling to transcription networks in the neuronal retrograde injury response. Sci. Signal. 2010;3(130) doi: 10.1126/scisignal.2000952. ra53-ra53. PubMed DOI PMC

Michalski A. Mass spectrometry-based proteomics using Q exactive, a high-performance benchtop quadrupole orbitrap mass spectrometer. Mol. Cell. Proteomics. 2011;10(9) doi: 10.1074/mcp.M111.011015. M111.011015. PubMed DOI PMC

Nieuwenhuis B. PI 3-kinase delta enhances axonal PIP 3 to support axon regeneration in the adult CNS. EMBO Mol. Med. 2020 doi: 10.15252/emmm.201911674. PubMed DOI PMC

Pacheco A. Mechanism and role of the intra-axonal Calreticulin translation in response to axonal injury. Exp. Neurol. 2020 doi: 10.1016/j.expneurol.2019.113072. PubMed DOI PMC

Painter M.W. Diminished Schwann cell repair responses underlie age-associated impaired axonal regeneration. Neuron. 2014 doi: 10.1016/j.neuron.2014.06.016. PubMed DOI PMC

Preitner N. IMP2 axonal localization, RNA interactome, and function in the development of axon trajectories. Development (Cambridge) 2016 doi: 10.1242/dev.128348. PubMed DOI PMC

Prinjha R. Inhibitor of neurite outgrowth in humans. Nature. 2000;403(6768):383–384. doi: 10.1038/35000287. PubMed DOI

Sahoo P.K. Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration. Nat. Commun. 2018;9(1):3358. doi: 10.1038/s41467-018-05647-x. PubMed DOI PMC

Sahoo P.K. A Ca 2+-dependent switch activates axonal casein kinase 2α translation and drives G3BP1 granule disassembly for axon regeneration. Curr Biol. 2020;30(24):4882–4895. doi: 10.1016/j.cub.2020.09.043. Epub 2020 Oct 15. PubMed DOI PMC

Scheib J.L., Höke A. An attenuated immune response by Schwann cells and macrophages inhibits nerve regeneration in aged rats. Neurobiol. Aging. 2016 doi: 10.1016/j.neurobiolaging.2016.05.004. PubMed DOI

Segel M. Niche stiffness underlies the ageing of central nervous system progenitor cells. Nature. 2019 doi: 10.1038/s41586-019-1484-9. PubMed DOI PMC

Shakhbazau A. Demyelination induces transport of ribosome-containing vesicles from glia to axons: evidence from animal models and MS patient brains. Mol. Biol. Rep. 2016;43(6):495–507. doi: 10.1007/s11033-016-3990-2. PubMed DOI

Sharp J. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants improve recovery after cervical spinal cord injury. Stem Cells (Dayton, Ohio) 2010;28(1):152–163. doi: 10.1002/stem.245. PubMed DOI PMC

Shigeoka T. Dynamic axonal translation in developing and mature visual circuits. Cell. 2016;166(1):181–192. doi: 10.1016/j.cell.2016.05.029. PubMed DOI PMC

Shigeoka T. On-site ribosome Remodeling by locally synthesized ribosomal proteins in axons. Cell Rep. 2019 doi: 10.1016/j.celrep.2019.11.025. PubMed DOI PMC

Smith C.L. GAP-43 mRNA in growth cones is associated with HuD and ribosomes. J. Neurobiol. 2004 doi: 10.1002/neu.20038. PubMed DOI

Sotelo J.R. Glia to axon RNA transfer. Dev. Neurobiol. 2014;74(3):292–302. doi: 10.1002/dneu.22125. PubMed DOI

Springer J.E. Rapid Calpain I activation and cytoskeletal protein degradation following traumatic spinal cord injury: attenuation with Riluzole Pretreatment. J. Neurochem. 1997;69(4):1592–1600. doi: 10.1046/j.1471-4159.1997.69041592.x. PubMed DOI

Stam F.J. Identification of candidate transcriptional modulators involved in successful regeneration after nerve injury. Eur. J. Neurosci. 2007 doi: 10.1111/j.1460-9568.2007.05597.x. PubMed DOI

Terenzio M. Locally translated mTOR controls axonal local translation in nerve injury. Science. 2018;359(6382):1416–1421. doi: 10.1126/science.aan1053. PubMed DOI PMC

Turriziani B. On-beads digestion in conjunction with data-dependent mass spectrometry: a shortcut to quantitative and dynamic interaction proteomics. Biology. 2014 doi: 10.3390/biology3020320. PubMed DOI PMC

van Kesteren R.E. A gene network perspective on axonal regeneration. Front. Mol. Neurosci. 2011 doi: 10.3389/fnmol.2011.00046. PubMed DOI PMC

Verma P. Axonal protein synthesis and degradation are necessary for efficient growth cone regeneration. J. Neurosci. 2005;25(2):331–342. doi: 10.1523/JNEUROSCI.3073-04.2005. PubMed DOI PMC

Zheng J.Q. A functional role for intra-axonal protein synthesis during axonal regeneration from adult sensory neurons. J. Neurosci. 2001;21(23):9291–9303. http://www.ncbi.nlm.nih.gov/pubmed/11717363 Available at. (Accessed: 6 March 2017) PubMed PMC

Zou Y. Developmental decline in neuronal regeneration by the progressive change of two intrinsic timers. Science (New York, N.Y.) 2013;340(6130):372–376. doi: 10.1126/science.1231321. PubMed DOI PMC

Axonal Organelles as Molecular Platforms for Axon Growth and Regeneration after Injury