Central nervous system (CNS) axons lose their intrinsic ability to regenerate upon maturity, whereas peripheral nervous system (PNS) axons do not. A key difference between these neuronal types is their ability to transport integrins into axons. Integrins can mediate PNS regeneration, but are excluded from adult CNS axons along with their Rab11 carriers. We reasoned that exclusion of the contents of Rab11 vesicles including integrins might contribute to the intrinsic inability of CNS neurons to regenerate, and investigated this by performing laser axotomy. We identify a novel regulator of selective axon transport and regeneration, the ARF6 guanine-nucleotide-exchange factor (GEF) EFA6 (also known as PSD). EFA6 exerts its effects from a location within the axon initial segment (AIS). EFA6 does not localise at the AIS in dorsal root ganglion (DRG) axons, and in these neurons, ARF6 activation is counteracted by an ARF GTPase-activating protein (GAP), which is absent from the CNS, ACAP1. Depleting EFA6 from cortical neurons permits endosomal integrin transport and enhances regeneration, whereas overexpressing EFA6 prevents DRG regeneration. Our results demonstrate that ARF6 is an intrinsic regulator of regenerative capacity, implicating EFA6 as a focal molecule linking the AIS, signalling and transport.This article has an associated First Person interview with the first author of the paper.

Centre of Reconstructive Neuroscience Institute of Experimental Medicine AVCR Prague Czech Republic

Institute of Life Science College of Medicine Swansea University Singleton Park Swansea SA2 8PP U K

John Van Geest Centre for Brain Repair Department of Clinical Neurosciences University of Cambridge Cambridge CB2 OPY U K

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Alther T. A., Domanitskaya E. and Stoeckli E. T. (2016). Calsyntenin 1-mediated trafficking of axon guidance receptors regulates the switch in axonal responsiveness at a choice point. Development 143, 994-1004. 10.1242/dev.127449 PubMed DOI

Andrews M. R., Soleman S., Cheah M., Tumbarello D. A., Mason M. R., Moloney E., Verhaagen J., Bensadoun J. C., Schneider B., Aebischer P. et al. (2016). Axonal localization of integrins in the CNS is neuronal type and age dependent. eNeuro 3, 0029-16.2016 10.1523/ENEURO.0029-16.2016 PubMed DOI PMC

Arnold D. B. (2009). Actin and microtubule-based cytoskeletal cues direct polarized targeting of proteins in neurons. Sci. Signal. 2, pe49 10.1126/scisignal.283pe49 PubMed DOI PMC

Ascano M., Richmond A., Borden P. and Kuruvilla R. (2009). Axonal targeting of Trk receptors via transcytosis regulates sensitivity to neurotrophin responses. J. Neurosci. 29, 11674-11685. 10.1523/JNEUROSCI.1542-09.2009 PubMed DOI PMC

Bentley M. and Banker G. (2016). The cellular mechanisms that maintain neuronal polarity. Nat. Rev. Neurosci. 17, 611-622. 10.1038/nrn.2016.100 PubMed DOI

Bodrikov V., Pauschert A., Kochlamazashvili G. and Stuermer C. A. O. (2017). Reggie-1 and reggie-2 (flotillins) participate in Rab11a-dependent cargo trafficking, spine synapse formation and LTP-related AMPA receptor (GluA1) surface exposure in mouse hippocampal neurons. Exp. Neurol. 289, 31-45. 10.1016/j.expneurol.2016.12.007 PubMed DOI

Bradke F., Fawcett J. W. and Spira M. E. (2012). Assembly of a new growth cone after axotomy: the precursor to axon regeneration. Nat. Rev. Neurosci. 13, 183-193. 10.1038/nrn3176 PubMed DOI

Britt D. J., Farías G. G., Guardia C. M. and Bonifacino J. S. (2016). Mechanisms of polarized organelle distribution in neurons. Frontier. Cell. Neurosci. 10, 88 10.3389/fncel.2016.00088 PubMed DOI PMC

Brown F. D., Rozelle A. L., Yin H. L., Balla T. and Donaldson J. G. (2001). Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic. J. Cell Biol. 154, 1007-1017. 10.1083/jcb.200103107 PubMed DOI PMC

Cheah M., Andrews M. R., Chew D. J., Moloney E. B., Verhaagen J., Fassler R. and Fawcett J. W. (2016). Expression of an activated integrin promotes long-distance sensory axon regeneration in the spinal cord. J. Neurosci. 36, 7283-7297. 10.1523/JNEUROSCI.0901-16.2016 PubMed DOI PMC

Chen L., Wang Z., Ghosh-Roy A., Hubert T., Yan D., O'Rourke S., Bowerman B., Wu Z., Jin Y. and Chisholm A. D. (2011). Axon regeneration pathways identified by systematic genetic screening in C. elegans. Neuron 71, 1043-1057. 10.1016/j.neuron.2011.07.009 PubMed DOI PMC

Chiba K., Araseki M., Nozawa K., Furukori K., Araki Y., Matsushima T., Nakaya T., Hata S., Saito Y., Uchida S. et al. (2014). Quantitative analysis of APP axonal transport in neurons: role of JIP1 in enhanced APP anterograde transport. Mol. Biol. Cell 25, 3569-3580. 10.1091/mbc.E14-06-1111 PubMed DOI PMC

Choi S., Ko J., Lee J. R., Lee H. W., Kim K., Chung H. S., Kim H. and Kim E. (2006). ARF6 and EFA6A regulate the development and maintenance of dendritic spines. J. Neurosci. 26, 4811-4819. 10.1523/JNEUROSCI.4182-05.2006 PubMed DOI PMC

Derrien V., Couillault C., Franco M., Martineau S., Montcourrier P., Houlgatte R. and Chavrier P. (2002). A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions. J. Cell Sci. 115, 2867-2879. PubMed

Eva R., Dassie E., Caswell P. T., Dick G., ffrench-Constant C., Norman J. C. and Fawcett J. W. (2010). Rab11 and its effector Rab coupling protein contribute to the trafficking of beta 1 integrins during axon growth in adult dorsal root ganglion neurons and PC12 cells. J. Neurosci. 30, 11654-11669. 10.1523/JNEUROSCI.2425-10.2010 PubMed DOI PMC

Eva R., Crisp S., Marland J. R. K., Norman J. C., Kanamarlapudi V., ffrench-Constant C. and Fawcett J. W. (2012). ARF6 directs axon transport and traffic of integrins and regulates axon growth in adult DRG neurons. J. Neurosci. 32, 10352-10364. 10.1523/JNEUROSCI.1409-12.2012 PubMed DOI PMC

Fitch M. T. and Silver J. (2008). CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure. Exp. Neurol. 209, 294-301. 10.1016/j.expneurol.2007.05.014 PubMed DOI PMC

Franssen E. H. P., Zhao R.-R., Koseki H., Kanamarlapudi V., Hoogenraad C. C., Eva R. and Fawcett J. W. (2015). Exclusion of integrins from CNS axons is regulated by Arf6 activation and the AIS. J. Neurosci. 35, 8359-8375. 10.1523/JNEUROSCI.2850-14.2015 PubMed DOI PMC

Gardiner N. J. (2011). Integrins and the extracellular matrix: key mediators of development and regeneration of the sensory nervous system. Dev. Neurobiol. 71, 1054-1072. 10.1002/dneu.20950 PubMed DOI

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

Gomis-Rüth S., Stiess M., Wierenga C. J., Meyn L. and Bradke F. (2014). Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits. Nat. Protoc. 9, 1028-1037. 10.1038/nprot.2014.069 PubMed DOI

Guo X., Farías G. G., Mattera R. and Bonifacino J. S. (2016). Rab5 and its effector FHF contribute to neuronal polarity through dynein-dependent retrieval of somatodendritic proteins from the axon. Proc. Natl. Acad. Sci. USA 113, E5318-E5327. 10.1073/pnas.1601844113 PubMed DOI PMC

Hirst J., Lui W. W. Y., Bright N. A., Totty N., Seaman M. N. J. and Robinson M. S. (2000). A family of proteins with gamma-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome. J. Cell Biol. 149, 67-80. 10.1083/jcb.149.1.67 PubMed DOI PMC

Hung C. O. Y. and Coleman M. P. (2016). KIF1A mediates axonal transport of BACE1 and identification of independently moving cargoes in living SCG neurons. Traffic 17, 1155-1167. 10.1111/tra.12428 PubMed DOI PMC

Koseki H., Donegá M., Lam B. Y. H., Petrova V., Yeo G. S. H., Kwok J. C. F., van Erp S., ffrench-Constant C., Eva R. I. and Fawcett J. W. (2017). Selective Rab11 transport and the intrinsic regenerative ability of CNS axons. Elife 6, e26956 10.7554/eLife.26956 PubMed DOI PMC

Kuijpers M., van de Willige D., Freal A., Chazeau A., Franker M. A., Hofenk J., Rodrigues R. J. C., Kapitein L. C., Akhmanova A., Jaarsma D. et al. (2016). Dynein regulator NDEL1 controls polarized cargo transport at the axon initial segment. Neuron 89, 461-471. 10.1016/j.neuron.2016.01.022 PubMed DOI

Lazo O. M., Gonzalez A., Ascano M., Kuruvilla R., Couve A. and Bronfman F. C. (2013). BDNF regulates Rab11-mediated recycling endosome dynamics to induce dendritic branching. J. Neurosci. 33, 6112-6122. 10.1523/JNEUROSCI.4630-12.2013 PubMed DOI PMC

Leterrier C., Vacher H., Fache M.-P., d'Ortoli S. A., Castets F., Autillo-Touati A. and Dargent B. (2011). End-binding proteins EB3 and EB1 link microtubules to ankyrin G in the axon initial segment. Proc. Natl. Acad. Sci. USA 108, 8826-8831. 10.1073/pnas.1018671108 PubMed DOI PMC

Li J., Ballif B. A., Powelka A. M., Dai J., Gygi S. P. and Hsu V. W. (2005). Phosphorylation of ACAP1 by Akt regulates the stimulation-dependent recycling of integrin beta1 to control cell migration. Dev. Cell 9, 663-673. 10.1016/j.devcel.2005.09.012 PubMed DOI

Lindner R., Puttagunta R. and Di Giovanni S. (2013). Epigenetic regulation of axon outgrowth and regeneration in CNS injury: the first steps forward. Neurotherapeutics 10, 771-781. 10.1007/s13311-013-0203-8 PubMed DOI PMC

Liu K., Tedeschi A., Park K. K. and He Z. (2011). Neuronal intrinsic mechanisms of axon regeneration. Annu. Rev. Neurosci. 34, 131-152. 10.1146/annurev-neuro-061010-113723 PubMed DOI

Luton F., Klein S., Chauvin J. P., Le Bivic A., Bourgoin S., Franco M. and Chardin P. (2004). EFA6, exchange factor for ARF6, regulates the actin cytoskeleton and associated tight junction in response to E-cadherin engagement. Mol. Biol. Cell 15, 1134-1145. 10.1091/mbc.E03-10-0751 PubMed DOI PMC

Macia E., Chabre M. and Franco M. (2001). Specificities for the small G proteins ARF1 and ARF6 of the guanine nucleotide exchange factors ARNO and EFA6. J. Biol. Chem. 276, 24925-24930. 10.1074/jbc.M103284200 PubMed DOI

Macia E., Partisani M., Favard C., Mortier E., Zimmermann P., Carlier M.-F., Gounon P., Luton F. and Franco M. (2008). The pleckstrin homology domain of the Arf6-specific exchange factor EFA6 localizes to the plasma membrane by interacting with phosphatidylinositol 4,5-bisphosphate and F-actin. J. Biol. Chem. 283, 19836-19844. 10.1074/jbc.M800781200 PubMed DOI

Montagnac G., Sibarita J.-B., Loubéry S., Daviet L., Romao M., Raposo G. and Chavrier P. (2009). ARF6 Interacts with JIP4 to control a motor switch mechanism regulating endosome traffic in cytokinesis. Curr. Biol. 19, 184-195. 10.1016/j.cub.2008.12.043 PubMed DOI

Moore D. L. and Goldberg J. L. (2011). Multiple transcription factor families regulate axon growth and regeneration. Dev. Neurobiol. 71, 1186-1211. 10.1002/dneu.20934 PubMed DOI PMC

Myers J. P., Santiago-Medina M. and Gomez T. M. (2011). Regulation of axonal outgrowth and pathfinding by integrin-ECM interactions. Dev. Neurobiol. 71, 901-923. 10.1002/dneu.20931 PubMed DOI PMC

Nguyen M. K., Kim C. Y., Kim J. M., Park B. O., Lee S., Park H. and Heo W. D. (2016). Optogenetic oligomerization of Rab GTPases regulates intracellular membrane trafficking. Nat. Chem. Biol. 12, 431-436. 10.1038/nchembio.2064 PubMed DOI

Padovani D., Folly-Klan M., Labarde A., Boulakirba S., Campanacci V., Franco M., Zeghouf M. and Cherfils J. (2014). EFA6 controls Arf1 and Arf6 activation through a negative feedback loop. Proc. Natl. Acad. Sci. U.S.A. 111, 12378-12383. 10.1073/pnas.1409832111 PubMed DOI PMC

Park K. K., Liu K., Hu Y., Smith P. D., Wang C., Cai B., Xu B., Connolly L., Kramvis I., Sahin M. et al. (2008). Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science 322, 963-966. 10.1126/science.1161566 PubMed DOI PMC

Pernet V. and Schwab M. E. (2014). Lost in the jungle: new hurdles for optic nerve axon regeneration. Trends Neurosci. 37, 381-387. 10.1016/j.tins.2014.05.002 PubMed DOI

Puertollano R., Randazzo P. A., Presley J. F., Hartnell L. M. and Bonifacino J. S. (2001). The GGAs promote ARF-dependent recruitment of clathrin to the TGN. Cell 105, 93-102. 10.1016/S0092-8674(01)00299-9 PubMed DOI

Raemaekers T., Peric A., Baatsen P., Sannerud R., Declerck I., Baert V., Michiels C. and Annaert W. (2012). ARF6-mediated endosomal transport of Telencephalin affects dendritic filopodia-to-spine maturation. EMBO J. 31, 3252-3269. 10.1038/emboj.2012.182 PubMed DOI PMC

Randazzo P. A., Miura K., Nie Z., Orr A., Theibert A. B. and Kearns B. G. (2001). Cytohesins and centaurins: mediators of PI 3-kinase regulated Arf signaling. Trends Biochem. Sci. 26, 220-221. 10.1016/S0968-0004(01)01806-0 PubMed DOI

Rasband M. N. (2010). The axon initial segment and the maintenance of neuronal polarity. Nat. Rev. Neurosci. 11, 552-562. 10.1038/nrn2852 PubMed DOI

Sannerud R., Declerck I., Peric A., Raemaekers T., Menendez G., Zhou L., Veerle B., Coen K., Munck S., De Strooper B. et al. (2011). ADP ribosylation factor 6 (ARF6) controls amyloid precursor protein (APP) processing by mediating the endosomal sorting of BACE1. Proc. Natl. Acad. Sci. USA 108, E559-E568. 10.1073/pnas.1100745108 PubMed DOI PMC

Santy L. C. and Casanova J. E. (2001). Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J. Cell Biol. 154, 599-610. 10.1083/jcb.200104019 PubMed DOI PMC

Schwab M. E. and Strittmatter S. M. (2014). Nogo limits neural plasticity and recovery from injury. Curr. Opin. Neurobiol. 27, 53-60. 10.1016/j.conb.2014.02.011 PubMed DOI PMC

Sheehan D., Ray G. S., Calhoun B. C. and Goldenring J. R. (1996). A somatodendritic distribution of Rab11 in rabbit brain neurons. Neuroreport 7, 1297-1300. 10.1097/00001756-199605170-00016 PubMed DOI

Silver J., Schwab M. E. and Popovich P. G. (2014). Central nervous system regenerative failure: role of oligodendrocytes, astrocytes, and microglia. Cold Spring Harb. Perspect. Biol. 7, a020602 10.1101/cshperspect.a020602 PubMed DOI PMC

Solis G. P., Hulsbusch N., Radon Y., Katanaev V. L., Plattner H. and Stuermer C. A. O. (2013). Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking. Mol. Biol. Cell 24, 2689-2702. 10.1091/mbc.E12-12-0854 PubMed DOI PMC

Stepanova T., Slemmer J., Hoogenraad C. C., Lansbergen G., Dortland B., De Zeeuw C. I., Grosveld F., van Cappellen G., Akhmanova A. and Galjart N. (2003). Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein). J. Neurosci. 23, 2655-2664. PubMed PMC

Steuble M., Diep T.-M., Schatzle P., Ludwig A., Tagaya M., Kunz B. and Sonderegger P. (2012). Calsyntenin-1 shelters APP from proteolytic processing during anterograde axonal transport. Biol. Open 1, 761-774. 10.1242/bio.20121578 PubMed DOI PMC

Suzuki A., Arikawa C., Kuwahara Y., Itoh K., Watanabe M., Watanabe H., Suzuki T., Funakoshi Y., Hasegawa H. and Kanaho Y. (2010). The scaffold protein JIP3 functions as a downstream effector of the small GTPase ARF6 to regulate neurite morphogenesis of cortical neurons. FEBS Lett. 584, 2801-2806. 10.1016/j.febslet.2010.05.020 PubMed DOI

van Bergeijk P., Adrian M., Hoogenraad C. C. and Kapitein L. C. (2015). Optogenetic control of organelle transport and positioning. Nature 518, 111-114. 10.1038/nature14128 PubMed DOI PMC

Wang J., Morita Y., Mazelova J. and Deretic D. (2012). The Arf GAP ASAP1 provides a platform to regulate Arf4- and Rab11-Rab8-mediated ciliary receptor targeting. EMBO J. 31, 4057-4071. 10.1038/emboj.2012.253 PubMed DOI PMC

Wojnacki J. and Galli T. (2016). Membrane traffic during axon development. Dev. Neurobiol. 76, 1185-1200. 10.1002/dneu.22390 PubMed DOI

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