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Molecular and Cellular Mechanisms of Axonal Regeneration After Spinal Cord Injury*

  • Erna A. van Niekerk
    Correspondence
    To whom correspondence should be addressed:Department of Neuroscience, University of California San Diego, La Jolla, CA 92093-0626. Tel.:858-534-8857;
    Affiliations
    Department of Neurosciences, University of California, San Diego, La Jolla, CA, 92093;
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  • Mark H. Tuszynski
    Affiliations
    Department of Neurosciences, University of California, San Diego, La Jolla, CA, 92093;

    Veterans Administration Medical Center, San Diego, CA 92161
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  • Paul Lu
    Affiliations
    Department of Neurosciences, University of California, San Diego, La Jolla, CA, 92093;

    Veterans Administration Medical Center, San Diego, CA 92161
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  • Jennifer N. Dulin
    Affiliations
    Department of Neurosciences, University of California, San Diego, La Jolla, CA, 92093;
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  • Author Footnotes
    * This work was supported by the Veterans Administration, the NIH (NS09881 and EB014986), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the Craig H. Neilsen Foundation and the California Institute for Regenerative Medicine. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    1 The abbreviations used are:CNScentral nervous systemPNSperipheral nervous systemDRGdorsal root ganglionSTATsignal transducer and activator of transcriptionHNFhepatocyte nuclear factorUSFupstream stimulatory factorSILACstable isotope labeling by amino acidsNIFneuroscience information networkECMextracellular matrixCSPGchondroitin sulfate proteoglycanNCAMneural cell adhesion moleculePSTpolysialyltransferase.
Open AccessPublished:December 22, 2015DOI:https://doi.org/10.1074/mcp.R115.053751
      Following axotomy, a complex temporal and spatial coordination of molecular events enables regeneration of the peripheral nerve. In contrast, multiple intrinsic and extrinsic factors contribute to the general failure of axonal regeneration in the central nervous system. In this review, we examine the current understanding of differences in protein expression and post-translational modifications, activation of signaling networks, and environmental cues that may underlie the divergent regenerative capacity of central and peripheral axons. We also highlight key experimental strategies to enhance axonal regeneration via modulation of intraneuronal signaling networks and the extracellular milieu. Finally, we explore potential applications of proteomics to fill gaps in the current understanding of molecular mechanisms underlying regeneration, and to provide insight into the development of more effective approaches to promote axonal regeneration following injury to the nervous system.
      During development, neurons extend axons throughout the nervous system, establishing connections with postsynaptic targets that are often located quite long distances away from their origin. The ability of these young neurons to robustly extend their axons is dramatically diminished in adulthood, and this reduced intrinsic growth capacity is a key mechanism underlying the inability of adult central nervous system (CNS)
      The abbreviations used are:
      CNS
      central nervous system
      PNS
      peripheral nervous system
      DRG
      dorsal root ganglion
      STAT
      signal transducer and activator of transcription
      HNF
      hepatocyte nuclear factor
      USF
      upstream stimulatory factor
      SILAC
      stable isotope labeling by amino acids
      NIF
      neuroscience information network
      ECM
      extracellular matrix
      CSPG
      chondroitin sulfate proteoglycan
      NCAM
      neural cell adhesion molecule
      PST
      polysialyltransferase.
      neurons to regenerate their axons following injury (
      • Blesch A.
      • Tuszynski M.H.
      Spinal cord injury: plasticity, regeneration, and the challenge of translational drug development.
      ,
      • Seijffers R.
      • Benowitz L.I.
      Intrinsic determinants of axon regeneration.
      ,
      • Goldberg J.L.
      • Klassen M.P.
      • Hua Y.
      • Barres B.A.
      Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion cells.
      ,
      • Plunet W.
      • Kwon B.K.
      • Tetzlaff W.
      Promoting axonal regeneration in the central nervous system by enhancing the cell body response to axotomy.
      ). The injured adult CNS is a nonpermissive environment for axon outgrowth because of the abundance of inhibitory proteins and glycoproteins (
      • Filbin M.T.
      Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS.
      ,
      • Busch S.A.
      • Silver J.
      The role of extracellular matrix in CNS regeneration.
      ,
      • Yiu G.
      • He Z.
      Glial inhibition of CNS axon regeneration.
      ), together with a deficiency of adequate trophic support (
      • Blesch A.
      • Fischer I.
      • Tuszynski M.H.
      Gene therapy, neurotrophic factors, and spinal cord regeneration.
      ,
      • Hollis 2nd, E.R.
      • Tuszynski M.H.
      Neurotrophins: potential therapeutic tools for the treatment of spinal cord injury.
      ,
      • Hendriks W.T.
      • Ruitenberg M.J.
      • Blits B.
      • Boer G.J.
      • Verhaagen J.
      Viral vector-mediated gene transfer of neurotrophins to promote regeneration of the injured spinal cord.
      ). In addition, intrinsic neuronal mechanisms initiating a growth program are limited in injured adult CNS neurons (
      • Fernandes K.J.
      • Fan D.P.
      • Tsui B.J.
      • Cassar S.L.
      • Tetzlaff W.
      Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation of GAP-43, tubulins, and neurofilament-M.
      ). Because of the failure of CNS axons to spontaneously regenerate, sensory, motor, autonomic, or cognitive deficits resulting from CNS injury are often permanent. Hence, there remains a great unmet need for therapeutic strategies to enhance regeneration of injured CNS axons and thereby improve function. Here, we will review key protein networks identified in the injured and regenerating axon and discuss progress in experimental approaches to promote repair of the injured nervous system.

      Axonal Regeneration in the Peripheral Nervous System

      Following axotomy, injured neurons of the peripheral nervous system (PNS) shift to a regenerative state. Injury signals are communicated to the soma, cell survival pathways are activated, and numerous regeneration-associated genes are up-regulated. This injury-induced cascade of events includes neurochemical changes, functional alterations in excitability, growth cone reconstruction, local protein synthesis, and multiple signaling pathways to activate axonal regeneration. In this section, we will review molecular mechanisms that underlie axon regeneration following peripheral nerve injury.

      Axotomy: Cellular and Molecular Events after Nerve Injury

      An immediate influx of calcium occurs at an injured axon tip (
      • Bradke F.
      • Fawcett J.W.
      • Spira M.E.
      Assembly of a new growth cone after axotomy: the precursor to axon regeneration.
      ) that reaches above 1 mm in concentration (
      • Ziv N.E.
      • Spira M.E.
      Axotomy induces a transient and localized elevation of the free intracellular calcium concentration to the millimolar range.
      ). This drastic rise in intracellular calcium is necessary for triggering axon regeneration, as neurons in a calcium-free environment fail to initiate axon outgrowth (
      • Kamber D.
      • Erez H.
      • Spira M.E.
      Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone.
      ,
      • Ziv N.E.
      • Spira M.E.
      Localized and transient elevations of intracellular Ca2+ induce the dedifferentiation of axonal segments into growth cones.
      ). A first-wave retrograde injury signal transmits to the cell body through a back-propagating calcium wave, which is thought to lead to chromatin remodeling (
      • Cho Y.
      • Sloutsky R.
      • Naegle K.M.
      • Cavalli V.
      Injury-induced HDAC5 nuclear export is essential for axon regeneration.
      ). This is the first communication the injured tip has with the soma. Electron-micrograph images have shown that injury-induced vesicles occlude the cut ends of the axon, forming a plasmalemmal seal (
      • Eddleman C.S.
      • Bittner G.D.
      • Fishman H.M.
      Barrier permeability at cut axonal ends progressively decreases until an ionic seal is formed.
      ,
      • Spira M.E.
      • Benbassat D.
      • Dormann A.
      Resealing of the proximal and distal cut ends of transected axons: electrophysiological and ultrastructural analysis.
      ). As the cytoskeleton undergoes reconstruction, the rate of sealing is dependent on calcium-regulated proteins such as synaptotagmin, syntaxin, and synaptobrevin (
      • Yoo S.
      • Nguyen M.P.
      • Fukuda M.
      • Bittner G.D.
      • Fishman H.M.
      Plasmalemmal sealing of transected mammalian neurites is a gradual process mediated by Ca(2+)-regulated proteins.
      ). Following this initial burst of events, the soma undergoes chromatolysis, in which chromatin within the nucleus is dissipated and spread to the cell periphery as the cell body undergoes swelling. Excitatory inputs are mostly eliminated, leaving inhibitory inputs as the main communicators to the injured soma. This “synaptic stripping” is thought to involve glial cells and may prevent glutamate excitotoxicity (
      • Spejo A.B.
      • Oliveira A.L.
      Synaptic rearrangement following axonal injury: old and new players.
      ).
      A second wave of retrograde signaling, thought to comprise the main injury signal, occurs by 4–6 h postinjury (
      • Rishal I.
      • Fainzilber M.
      Axon-soma communication in neuronal injury.
      ). The complete scope of the temporal communication between the injured axon and soma is still not fully understood (
      • Saito A.
      • Cavalli V.
      Signaling over Distances.
      ). Local protein synthesis is necessary for the formation of the main retrograde signal, in which importins and vimentin are locally translated in the injured axon (
      • Yudin D.
      • Hanz S.
      • Yoo S.
      • Iavnilovitch E.
      • Willis D.
      • Gradus T.
      • Vuppalanchi D.
      • Segal-Ruder Y.
      • Ben-Yaakov K.
      • Hieda M.
      • Yoneda Y.
      • Twiss J.L.
      • Fainzilber M.
      Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve.
      ,
      • Perry R.B.
      • Doron-Mandel E.
      • Iavnilovitch E.
      • Rishal I.
      • Dagan S.Y.
      • Tsoory M.
      • Coppola G.
      • McDonald M.K.
      • Gomes C.
      • Geschwind D.H.
      • Twiss J.L.
      • Yaron A.
      • Fainzilber M.
      Subcellular knockout of importin beta1 perturbs axonal retrograde signaling.
      ). Perturbation of this signal attenuates the cellular response to injury (
      • Yudin D.
      • Hanz S.
      • Yoo S.
      • Iavnilovitch E.
      • Willis D.
      • Gradus T.
      • Vuppalanchi D.
      • Segal-Ruder Y.
      • Ben-Yaakov K.
      • Hieda M.
      • Yoneda Y.
      • Twiss J.L.
      • Fainzilber M.
      Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve.
      ,
      • Perry R.B.
      • Doron-Mandel E.
      • Iavnilovitch E.
      • Rishal I.
      • Dagan S.Y.
      • Tsoory M.
      • Coppola G.
      • McDonald M.K.
      • Gomes C.
      • Geschwind D.H.
      • Twiss J.L.
      • Yaron A.
      • Fainzilber M.
      Subcellular knockout of importin beta1 perturbs axonal retrograde signaling.
      ). The motor binding protein Jun-N-terminal kinase (JNK)-interacting protein JIP links axon vesicles to the injury signal and retrogradely transport back the injury signal along microtubules (
      • Abe N.
      • Almenar-Queralt A.
      • Lillo C.
      • Shen Z.
      • Lozach J.
      • Briggs S.P.
      • Williams D.S.
      • Goldstein L.S.
      • Cavalli V.
      Sunday driver interacts with two distinct classes of axonal organelles.
      ). As the membrane resting potential is restored, the axon may form either a retraction bulb or a new growth cone. Retraction bulbs are thought to be the nongrowing counterparts of growth cones, where growth failure is attributed to microtubule destabilization (
      • Ertürk A.
      • Hellal F.
      • Enes J.
      • Bradke F.
      Disorganized microtubules underlie the formation of retraction bulbs and the failure of axonal regeneration.
      ). Ertürk and colleagues showed that destabilizing microtubules both attenuated retraction bulb formation and axonal degeneration in PNS neurons, and enhanced the growth capacity of CNS neurons cultured on myelin (
      • Ertürk A.
      • Hellal F.
      • Enes J.
      • Bradke F.
      Disorganized microtubules underlie the formation of retraction bulbs and the failure of axonal regeneration.
      ). Within the axoplasm, calpain proteins are activated to cleave membrane spectrins, leading to brief proteolysis of the cytoskeleton (
      • Czogalla A.
      • Sikorski A.F.
      Spectrin and calpain: a “target” and a “sniper” in the pathology of neuronal cells.
      ). This is thought to restructure the microtubule ends, and is necessary for the transformation into a growth cone (
      • Gitler D.
      • Spira M.E.
      Real time imaging of calcium-induced localized proteolytic activity after axotomy and its relation to growth cone formation.
      ,
      • Gitler D.
      • Spira M.E.
      Short window of opportunity for calpain induced growth cone formation after axotomy of Aplysia neurons.
      ). As microtubules repolymerize, a brief reorientation of polarities form. Vesicular transport resumes, and selective traps for anterogradely transported vesicles are established (
      • Erez H.
      • Malkinson G.
      • Prager-Khoutorsky M.
      • De Zeeuw C.I.
      • Hoogenraad C.C.
      • Spira M.E.
      Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy.
      ). For example, Golgi-derived vesicles can only undergo anterograde transport and as microtubules reorient themselves with plus-end out orientation in the axon, these vesicles remain trapped at the growth cone, allowing for growth cone reconstruction.
      The newly formed growth cone begins to recycle material thereby generating an immediate supply, but local protein synthesis is ultimately necessary for growth cone formation (
      • Verma P.
      • Chierzi S.
      • Codd A.M.
      • Campbell D.S.
      • Meyer R.L.
      • Holt C.E.
      • Fawcett J.W.
      Axonal protein synthesis and degradation are necessary for efficient growth cone regeneration.
      ). Synthesis of proteins within the injured axon is a crucial event underlying regeneration, and improper regulation of this process can cause growth deficits (
      • Donnelly C.J.
      • Park M.
      • Spillane M.
      • Yoo S.
      • Pacheco A.
      • Gomes C.
      • Vuppalanchi D.
      • McDonald M.
      • Kim H.H.
      • Merianda T.T.
      • Gallo G.
      • Twiss J.L.
      Axonally synthesized beta-actin and GAP-43 proteins support distinct modes of axonal growth.
      ). Translation machinery and mRNAs are transported into the injured axon from the soma (
      • Yoo S.
      • van Niekerk E.A.
      • Merianda T.T.
      • Twiss J.L.
      Dynamics of axonal mRNA transport and implications for peripheral nerve regeneration.
      ). Schwann cells have also been shown to transfer polyribosomes to the axoplasm of desomatized axons, suggesting that Schwann cells can modify protein expression of injured axons (
      • Court F.A.
      • Hendriks W.T.
      • MacGillavry H.D.
      • Alvarez J.
      • van Minnen J.
      Schwann cell to axon transfer of ribosomes: toward a novel understanding of the role of glia in the nervous system.
      ,
      • Koenig E.
      • Martin R.
      • Titmus M.
      • Sotelo-Silveira J.R.
      Cryptic peripheral ribosomal domains distributed intermittently along mammalian myelinated axons.
      ).
      After growth cone formation, injured axons start to regenerate or sprout. Earlier studies have indicated that axonal regeneration or sprouting is accompanied by activation of genes associated with developmental axon growth (
      • Represa A.
      • Pollard H.
      • Moreau J.
      • Ghilini G.
      • Khrestchatisky M.
      • Ben-Ari Y.
      Mossy fiber sprouting in epileptic rats is associated with a transient increased expression of alpha-tubulin.
      ,
      • Bendotti C.
      • Pende M.
      • Samanin R.
      Expression of GAP-43 in the granule cells of rat hippocampus after seizure-induced sprouting of mossy fibers: in situ hybridization and immunocytochemical studies.
      ,
      • Mearow K.M.
      • Kril Y.
      • Gloster A.
      • Diamond J.
      Expression of NGF receptor and GAP-43 mRNA in DRG neurons during collateral sprouting and regeneration of dorsal cutaneous nerves.
      ). Indeed, neurite outgrowth has been correlated with the ability to express regeneration-associated genes that are normally expressed during development (
      • Skene J.H.
      Axonal growth-associated proteins.
      ); though there are distinctive regeneration programs associated with different modes of growth. Without a conditioning lesion, the dorsal root ganglion (DRG) neuron extends highly arborized neurites for a discrete period, a process that is dependent on new transcription. Within 24 h, this growth switches to elongation growth in which fewer, longer axons extend from the soma (
      • Smith D.S.
      • Skene J.H.
      A transcription-dependent switch controls competence of adult neurons for distinct modes of axon growth.
      ). In contrast, following a conditioning lesion, DRG neurons only exhibit elongation growth, which is dependent on translation for the formation of regenerating axons (
      • Twiss J.L.
      • Smith D.S.
      • Chang B.
      • Shooter E.M.
      Translational control of ribosomal protein L4 mRNA is required for rapid neurite regeneration.
      ). Together, these findings show that different modes of growth are associated with different patterns of protein expression. Key studies have revealed the importance of coordinated regulation of protein expression through the activation of specific signaling networks during axonal outgrowth and regeneration (see Table I). This emphasizes a potential for combined manipulation of multiple signaling pathways to activate robust regeneration programs. Indeed, there is a need to more fully understand the interactions between protein networks, and how these interactions impact axon growth and regeneration.
      Table ICentral regulators identified in axon growth after injury
      Target moleculeEffected pathwayBiological outcomeNeural system
      pTEN deletionmTOR signalingAccelerate axon outgrowth in PNS (
      • Christie K.J.
      • Webber C.A.
      • Martinez J.A.
      • Singh B.
      • Zochodne D.W.
      PTEN inhibition to facilitate intrinsic regenerative outgrowth of adult peripheral axons.
      )
      Dorsal root ganglion
      SOCS3 deletionJAK/STAT signalingSustained axon regeneration in vivo (
      • Smith P.D.
      • Sun F.
      • Park K.K.
      • Cai B.
      • Wang C.
      • Kuwako K.
      • Martinez-Carrasco I.
      • Connolly L.
      • He Z.
      SOCS3 deletion promotes optic nerve regeneration in vivo.
      )
      Retinal ganglion
      pTEN & SOCS3 co-deletionmTOR & JAK/STAT signalingSynergistic effects on regeneration in vivo (
      • Sun F.
      • Park K.K.
      • Belin S.
      • Wang D.
      • Lu T.
      • Chen G.
      • Zhang K.
      • Yeung C.
      • Feng G.
      • Yankner B.A.
      • He Z.
      Sustained axon regeneration induced by codeletion of PTEN and SOCS3.
      )
      Retinal ganglion
      PCAF expressionERK-mediated signalingPromotes acetylation of regeneration-associated promoters (
      • Puttagunta R.
      • Tedeschi A.
      • Soria M.G.
      • Hervera A.
      • Lindner R.
      • Rathore K.I.
      • Gaub P.
      • Joshi Y.
      • Nguyen T.
      • Schmandke A.
      • Laskowski C.J.
      • Boutillier A.L.
      • Bradke F.
      • Di Giovanni S.
      PCAF-dependent epigenetic changes promote axonal regeneration in the central nervous system.
      )
      Dorsal root ganglion
      c-myc expressionMyc signalingPromotes survival and axon regeneration (
      • Belin S.
      • Nawabi H.
      • Wang C.
      • Tang S.
      • Latremoliere A.
      • Warren P.
      • Schorle H.
      • Uncu C.
      • Woolf C.J.
      • He Z.
      • Steen J.A.
      Injury-induced decline of intrinsic regenerative ability revealed by quantitative proteomics.
      )
      Retinal ganglion
      c-AbI tyrosine kinase inhibitionBCR-ABL signalingTransient inhibition of axon growth in vitro (
      • Michaelevski I.
      • Medzihradszky K.F.
      • Lynn A.
      • Burlingame A.L.
      • Fainzilber M.
      Axonal transport proteomics reveals mobilization of translation machinery to the lesion site in injured sciatic nerve.
      )
      Dorsal root ganglion
      Akt inhibitionPI3K/Akt signalingInhibits axon growth in vitro (
      • Michaelevski I.
      • Medzihradszky K.F.
      • Lynn A.
      • Burlingame A.L.
      • Fainzilber M.
      Axonal transport proteomics reveals mobilization of translation machinery to the lesion site in injured sciatic nerve.
      )
      Dorsal root ganglion
      Atf3 expressionAtf3/CREB signalingIncreased neurite outgrowth in vitro (
      • Seijffers R.
      • Allchorne A.J.
      • Woolf C.J.
      The transcription factor ATF-3 promotes neurite outgrowth.
      )
      Dorsal root ganglion
      STAT3 phosphorylationSTAT3/gp130 signalingIncreased regeneration after injury (
      • Quarta S.
      • Baeumer B.E.
      • Scherbakov N.
      • Andratsch M.
      • Rose-John S.
      • Dechant G.
      • Bandtlow C.E.
      • Kress M.
      Peripheral nerve regeneration and NGF-dependent neurite outgrowth of adult sensory neurons converge on STAT3 phosphorylation downstream of neuropoietic cytokine receptor gp130.
      )
      Dorsal root ganglion
      Set-β phosphorylationCytoplasmic localization of set-βIncreased neurite outgrowth (
      • Trakhtenberg E.F.
      • Wang Y.
      • Morkin M.I.
      • Fernandez S.G.
      • Mlacker G.M.
      • Shechter J.M.
      • Liu X.
      • Patel K.H.
      • Lapins A.
      • Yang S.
      • Dombrowski S.M.
      • Goldberg J.L.
      Regulating set-beta's subcellular localization toggles its function between inhibiting and promoting axon growth and regeneration.
      )
      Retinal ganglion & hippocampal neurons
      Dock6 phosphorylationRAC1 signalingDecreased axon regeneration in vitro and in vivo (
      • Miyamoto Y.
      • Torii T.
      • Yamamori N.
      • Ogata T.
      • Tanoue A.
      • Yamauchi J.
      Akt and PP2A reciprocally regulate the guanine nucleotide exchange factor Dock6 to control axon growth of sensory neurons.
      )
      Dorsal root ganglion
      DLK phosphorylationSTAT3 & c-JunIncreased regeneration in motor and sensory neurons (
      • Shin J.E.
      • Cho Y.
      • Beirowski B.
      • Milbrandt J.
      • Cavalli V.
      • DiAntonio A.
      Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration.
      )
      Dorsal root ganglion
      Rho inhibitionRho signalingIncreased axon and neurite outgrowth in vitro (
      • Dergham P.
      • Ellezam B.
      • Essagian C.
      • Avedissian H.
      • Lubell W.D.
      • McKerracher L.
      Rho signaling pathway targeted to promote spinal cord repair.
      ,
      • Monnier P.P.
      • Sierra A.
      • Schwab J.M.
      • Henke-Fahle S.
      • Mueller B.K.
      The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar.
      • Borisoff J.F.
      • Chan C.C.
      • Hiebert G.W.
      • Oschipok L.
      • Robertson G.S.
      • Zamboni R.
      • Steeves J.D.
      • Tetzlaff W.
      Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates.
      )
      Cortical neurons, retinal ganglion, dorsal root ganglion

      Contribution of Proteomic Studies to Axon Regeneration

      Proteomics can provide a sytematic approach for analysis of protein expression in a cell, tissue, or tissue systems under a given condition. As quantitative proteomics has begun taking center stage for relative and absolute quantification of protein species, it has fast tracked the discovery of biomarkers, drug target identification through protein–protein interactions, and improved diagnostics by altered protein expression. For example, individuals with a range in spinal cord injury (SCI) severity were shown to express different protein profiles within their cerebrospinal fluid (
      • Sengupta M.B.
      • Basu M.
      • Iswarari S.
      • Mukhopadhyay K.K.
      • Sardar K.P.
      • Acharyya B.
      • Mohanty P.K.
      • Mukhopadhyay D.
      CSF proteomics of secondary phase spinal cord injury in human subjects: perturbed molecular pathways post injury.
      ). Twenty-four hours postinjury, a variety of unique proteins were found to be up-regulated in the lesion epicenter, including 14–3-3 zeta/delta, HSP90α, peripherin, apolipoprotein A, and transferrin (
      • Chen A.
      • McEwen M.L.
      • Sun S.
      • Ravikumar R.
      • Springer J.E.
      Proteomic and phosphoproteomic analyses of the soluble fraction following acute spinal cord contusion in rats.
      ,
      • Kang S.K.
      • So H.H.
      • Moon Y.S.
      • Kim C.H.
      Proteomic analysis of injured spinal cord tissue proteins using 2-DE and MALDI-TOF MS.
      ). Unique phosphorylation events were also detected at the lesion site, including the phosphorylation of neurofilament light chain and fertuin-A. Further efforts to understand the proteomic profile in experimental SCI models has indicated that proteins altered after injury can be functionally categorized into metabolic homeostasis, stress response, protein and lipid degradation, development, neural survival and regeneration, and stimulus response (
      • Yan X.
      • Liu T.
      • Yang S.
      • Ding Q.
      • Liu Y.
      • Zhang X.
      • Que H.
      • Wei K.
      • Luo Z.
      • Liu S.
      Proteomic profiling of the insoluble pellets of the transected rat spinal cord.
      ,
      • Ding Q.
      • Wu Z.
      • Guo Y.
      • Zhao C.
      • Jia Y.
      • Kong F.
      • Chen B.
      • Wang H.
      • Xiong S.
      • Que H.
      • Jing S.
      • Liu S.
      Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury.
      ).
      In order to investigate changes associated with injury, transcriptional profiling was performed in the cell body of DRG neurons at various time points after peripheral nerve injury (
      • Michaelevski I.
      • Segal-Ruder Y.
      • Rozenbaum M.
      • Medzihradszky K.F.
      • Shalem O.
      • Coppola G.
      • Horn-Saban S.
      • Ben-Yaakov K.
      • Dagan S.Y.
      • Rishal I.
      • Geschwind D.H.
      • Pilpel Y.
      • Burlingame A.L.
      • Fainzilber M.
      Signaling to transcription networks in the neuronal retrograde injury response.
      ). Transcription factor binding site analysis revealed 26 transcription factor families associated with gene expression changes 18–28 h postinjury. These included STAT (signal transducer and activator of transcription), HNF (hepatocyte nuclear factor), USF (upstream stimulatory factor), Jun, Smad, SRF, and ERα transcription families. Combining transcriptional changes with proteomic changes by performing transcription factor enrichment and ExPlain driven network analysis, revealed several hub pathways. Pharmacological inhibition of these pathways, including Abl (Abelson proto-oncogene), which was identified as a major hub, reduced neurite outgrowth within 24 h, but did not show sustained inhibition over time. Inhibiting Akt showed marked reductions in axon outgrowth over 72 h, whereas p38 inhibition only had transient effects. Combinatorial inhibition of Abl and Akt failed to have a significant effect on neurite length, indicating these two networks may not have overlapping mechanisms.
      Other signaling proteins within the soma have been associated with regeneration. Following sciatic nerve crush, coordinated changes in expression were observed beginning 6 h postinjury. Some of these proteins included Atf3, neural peptide Y, Arginase I, and ankyrin repeat domain 1 (Ankrd1) (
      • Stam F.J.
      • MacGillavry H.D.
      • Armstrong N.J.
      • de Gunst M.C.
      • Zhang Y.
      • van Kesteren R.E.
      • Smit A.B.
      • Verhaagen J.
      Identification of candidate transcriptional modulators involved in successful regeneration after nerve injury.
      ). Indeed, when the transcription factor Atf3 was overexpressed, neurite outgrowth was enhanced; suggesting Atf3 is a central regulatory hub for regeneration (
      • Seijffers R.
      • Allchorne A.J.
      • Woolf C.J.
      The transcription factor ATF-3 promotes neurite outgrowth.
      ).
      In an effort to understand the proteome profile in injured axons, Fainzilber and colleagues identified 145 proteins associated with retrograde transport and 154 proteins associated with anterograde transport after injury (
      • Michaelevski I.
      • Medzihradszky K.F.
      • Lynn A.
      • Burlingame A.L.
      • Fainzilber M.
      Axonal transport proteomics reveals mobilization of translation machinery to the lesion site in injured sciatic nerve.
      ). They further identified the axon phospho-proteome profile, where 268 unique phosphorylation sites were associated with injury in the retrograde compartment of the axoplasm. These proteins could be functionally classified into signal transduction, guanosine triphosphate activity, microtubule-based transport, and metabolism (
      • Michaelevski I.
      • Segal-Ruder Y.
      • Rozenbaum M.
      • Medzihradszky K.F.
      • Shalem O.
      • Coppola G.
      • Horn-Saban S.
      • Ben-Yaakov K.
      • Dagan S.Y.
      • Rishal I.
      • Geschwind D.H.
      • Pilpel Y.
      • Burlingame A.L.
      • Fainzilber M.
      Signaling to transcription networks in the neuronal retrograde injury response.
      ). Most recently, Steen and colleagues used a tandem mass tag (TMT) proteomics approach to uncover c-myc as a central injury-response hub in retinal ganglion cells (
      • Belin S.
      • Nawabi H.
      • Wang C.
      • Tang S.
      • Latremoliere A.
      • Warren P.
      • Schorle H.
      • Uncu C.
      • Woolf C.J.
      • He Z.
      • Steen J.A.
      Injury-induced decline of intrinsic regenerative ability revealed by quantitative proteomics.
      ). Quantitative mass spectrometry revealed several pathways altered after retinal ganglion injury including p53, MAPK, NF-κB, and Huntington protein.
      To date, there have been a small number of published reports of proteomic analyses in injured spinal cord tissue (
      • Kang S.K.
      • So H.H.
      • Moon Y.S.
      • Kim C.H.
      Proteomic analysis of injured spinal cord tissue proteins using 2-DE and MALDI-TOF MS.
      ,
      • Ding Q.
      • Wu Z.
      • Guo Y.
      • Zhao C.
      • Jia Y.
      • Kong F.
      • Chen B.
      • Wang H.
      • Xiong S.
      • Que H.
      • Jing S.
      • Liu S.
      Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury.
      ,
      • Tsai M.C.
      • Shen L.F.
      • Kuo H.S.
      • Cheng H.
      • Chak K.F.
      Involvement of acidic fibroblast growth factor in spinal cord injury repair processes revealed by a proteomics approach.
      ,
      • Yan X.
      • Liu J.
      • Luo Z.
      • Ding Q.
      • Mao X.
      • Yan M.
      • Yang S.
      • Hu X.
      • Huang J.
      • Luo Z.
      Proteomic profiling of proteins in rat spinal cord induced by contusion injury.
      ,
      • Noor N.M.
      • Steer D.L.
      • Wheaton B.J.
      • Ek C.J.
      • Truettner J.S.
      • Dietrich W.D.
      • Dziegielewska K.M.
      • Richardson S.J.
      • Smith A.I.
      • VandeBerg J.L.
      • Saunders N.R.
      Age-dependent changes in the proteome following complete spinal cord transection in a postnatal South American opossum (Monodelphis domestica).
      ,
      • Saunders N.R.
      • Noor N.M.
      • Dziegielewska K.M.
      • Wheaton B.J.
      • Liddelow S.A.
      • Steer D.L.
      • Ek C.J.
      • Habgood M.D.
      • Wakefield M.J.
      • Lindsay H.
      • Truettner J.
      • Miller R.D.
      • Smith A.I.
      • Dietrich W.D.
      Age-dependent transcriptome and proteome following transection of neonatal spinal cord of Monodelphis domestica (South American grey short-tailed opossum).
      ). In these studies, 2 dimensional SDS-PAGE based mass spec identified only 61 proteins differentially expressed in injured spinal cord tissue, a relatively low number, which is not surprising because of the limited range of detection. More sensitive LC-MS/MS techniques have now allowed for a greater detection range, although mass spectrometry is not inherently quantitative in nature because of differences in proteolytic physicochemical properties (
      • Bantscheff M.
      • Schirle M.
      • Sweetman G.
      • Rick J.
      • Kuster B.
      Quantitative mass spectrometry in proteomics: a critical review.
      ). Hence, quantitative mass spec best compares each individual peptide between experiments. Stable isotope labeling by amino acids (SILAC) in cell culture has overcome these limitations where every protein contains at least one amino acid isotope tag, and labeled proteins are not dependent on peptide sequence. For in vivo applications this technique has been attempted (
      • Krijgsveld J.
      • Ketting R.F.
      • Mahmoudi T.
      • Johansen J.
      • Artal-Sanz M.
      • Verrijzer C.P.
      • Plasterk R.H.
      • Heck A.J.
      Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics.
      ,
      • Wu C.C.
      • MacCoss M.J.
      • Howell K.E.
      • Matthews D.E.
      • Yates 3rd, J.R.
      Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis.
      ). Isobaric mass tagging of peptides has overcome limitations of proteome quantification where the identity and relative abundance of peptide pairs can be investigated in up to eight different biological samples (
      • Hebert A.S.
      • Merrill A.E.
      • Bailey D.J.
      • Still A.J.
      • Westphall M.S.
      • Strieter E.R.
      • Pagliarini D.J.
      • Coon J.J.
      Neutron-encoded mass signatures for multiplexed proteome quantification.
      ). This is particularly useful when investigating protein changes over time or therapeutic treatments in the same experiment, for example. Detection of phosphorylated peptide- and glycosylated peptide techniques have also been developed, which delves into the post-translational modifications associated with disease and/or treatment such as SCI. Although quantitative proteomics has advanced rapidly in the past decade, limitations still remain. For example, in detecting the enrichment of proteins, only a small percentage of the proteome is detectable if enrichment detection programs factor in the entire genome as background, decreasing the number of identified protein species. In contrast to transcriptomic analyses that typically identify thousands of differentially expressed genes following SCI, (
      • Saunders N.R.
      • Noor N.M.
      • Dziegielewska K.M.
      • Wheaton B.J.
      • Liddelow S.A.
      • Steer D.L.
      • Ek C.J.
      • Habgood M.D.
      • Wakefield M.J.
      • Lindsay H.
      • Truettner J.
      • Miller R.D.
      • Smith A.I.
      • Dietrich W.D.
      Age-dependent transcriptome and proteome following transection of neonatal spinal cord of Monodelphis domestica (South American grey short-tailed opossum).
      ) quantitative proteomics cannot amplify proteins as a means to increase dynamic range.
      The development of bioinformatic tools that focus on proteomic-based discovery is key to further understand these complex biological mechanisms. First order analyses have generated many free web-based tools including David, GSEA, Kegg pathway analysis, and genemania to name a few. However, these analytic programs are gene-centric rather than protein-centric. Second and third order analysis requires hypothesis driven approaches to obtain reasonable subnetworks within a framework that would directly address the scientific question at hand. For neural systems, some of the best web-based tools for hypothesis-driven discoveries are stored at the neuroscience information framework (NIF; http://neuinfo.org/). NIF has been cataloging and surveying the tool and data landscape since 2008. It currently contains the largest inventory of searchable tools and data in neuroscience (
      • Cachat J.
      • Bandrowski A.
      • Grethe J.S.
      • Gupta A.
      • Astakhov V.
      • Imam F.
      • Larson S.D.
      • Martone M.E.
      A survey of the neuroscience resource landscape: perspectives from the neuroscience information framework.
      ).
      Of note, transcriptomics and proteomics approaches have been pursued largely independently of one another, with researchers operating under the general assumption that for the majority of genes, changes in levels of transcript directly correlate with expression of the protein product. However, recent studies have shown that the correlation between mRNA and protein expression may be low (
      • Haider S.
      • Pal R.
      Integrated analysis of transcriptomic and proteomic data.
      ). A gene's biological function may determine the relative stability of its transcript and protein levels; for example, genes that are involved in constitutive cellular processes, such as glycolysis and translation, exhibit high stability of both transcripts and protein products (
      • Schwanhausser B.
      • Busse D.
      • Li N.
      • Dittmar G.
      • Schuchhardt J.
      • Wolf J.
      • Chen W.
      • Selbach M.
      Global quantification of mammalian gene expression control.
      ). In contrast, genes associated with processing RNAs tend to have unstable transcripts but stable proteins, whereas genes that produce stable transcripts but unstable proteins tend to be extracellular in nature. Similar discrepancies can be expected for post-translational changes in proteins and their effect on transcriptomics. Accordingly, integration of transcriptomic and proteomic findings will need to take into account the nature of the type of cell under study, and the biochemical function that is being probed, to reconcile genetic and protein control.

      Importance of Proteomics in Identifying Mechanisms Involved in Regeneration

      Alterations in protein expression drive metabolic changes necessary for supported axonal growth. The cell's ability to activate these transcriptional and translational switches may account for the marked differences in regeneration observed in different classes of neurons. Following a peripheral injury to an axon in the sciatic nerve, regeneration is successful and neuromuscular junctions become reinnervated. However, injury to the central branch of a DRG axon root results in no regeneration (
      • Oblinger M.M.
      • Lasek R.J.
      A conditioning lesion of the peripheral axons of dorsal root ganglion cells accelerates regeneration of only their peripheral axons.
      ). However, the central branch of a DRG axon can successfully regenerate if its peripheral branch was first crushed days earlier (
      • Richardson P.M.
      • Issa V.M.
      Peripheral injury enhances central regeneration of primary sensory neurones.
      ,
      • Richardson P.M.
      • Verge V.M.
      Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection.
      ). This is known as the conditioning effect. These observations indicate that the intrinsic state of a neuron can readily alter its regenerative ability. This difference in growth capacity has been associated with signaling pathways activated after peripheral nerve injury and relies on the coordinated expression of proteins in the axons and their somata (
      • Donnelly C.J.
      • Park M.
      • Spillane M.
      • Yoo S.
      • Pacheco A.
      • Gomes C.
      • Vuppalanchi D.
      • McDonald M.
      • Kim H.H.
      • Merianda T.T.
      • Gallo G.
      • Twiss J.L.
      Axonally synthesized beta-actin and GAP-43 proteins support distinct modes of axonal growth.
      ,
      • Twiss J.L.
      • Smith D.S.
      • Chang B.
      • Shooter E.M.
      Translational control of ribosomal protein L4 mRNA is required for rapid neurite regeneration.
      ,
      • Stam F.J.
      • MacGillavry H.D.
      • Armstrong N.J.
      • de Gunst M.C.
      • Zhang Y.
      • van Kesteren R.E.
      • Smit A.B.
      • Verhaagen J.
      Identification of candidate transcriptional modulators involved in successful regeneration after nerve injury.
      ,
      • Costigan M.
      • Befort K.
      • Karchewski L.
      • Griffin R.S.
      • D'Urso D.
      • Allchorne A.
      • Sitarski J.
      • Mannion J.W.
      • Pratt R.E.
      • Woolf C.J.
      Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.
      ).

      Successful Peripheral Regeneration and Failure of Central Regeneration

      The failure of axonal regeneration in the CNS is partially attributed to the proteome profile of the CNS, especially the extracellular milieu surrounding the injured adult axons (Fig. 1). The lesion site forms cystic or trabeculated cavities to which axons are unable to attach for growth (
      • Jones L.L.
      • Sajed D.
      • Tuszynski M.H.
      Axonal regeneration through regions of chondroitin sulfate proteoglycan deposition after spinal cord injury: a balance of permissiveness and inhibition.
      ). These inhibitors fall into two classes: inhibitory molecules of the extracellular matrix (ECM), and inhibitory proteins associated with adult myelin. After SCI, reactive macrophages, oligodendrocyte precursor cells, and reactive astrocytes shift the nature of the ECM at the lesion site, releasing cytokines, myelin associated inhibitors, and chondroitin sulfate proteoglycan (CSPG) molecules that potently inhibit neurite growth (
      • Snow D.M.
      • Lemmon V.
      • Carrino D.A.
      • Caplan A.I.
      • Silver J.
      Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro.
      ,
      • Jones L.L.
      • Yamaguchi Y.
      • Stallcup W.B.
      • Tuszynski M.H.
      NG2 is a major chondroitin sulfate proteoglycan produced after spinal cord injury and is expressed by macrophages and oligodendrocyte progenitors.
      ,
      • Jones L.L.
      • Margolis R.U.
      • Tuszynski M.H.
      The chondroitin sulfate proteoglycans neurocan, brevican, phosphacan, and versican are differentially regulated following spinal cord injury.
      ). These inhibitory molecules are not present within the peripheral nervous system, where axon regeneration robustly occurs after injury.
      Figure thumbnail gr1
      Fig. 1.The inhibitory extracellular milieu of the central nervous system. Following spinal cord injury (SCI), inhibitory molecules act as barriers of regeneration. These inhibitors fall into two broad classes: inhibitory molecules of the extracellular matrix such as the chondroitin sulfate proteoglycans (CSPGs); and inhibitory proteins associated with adult myelin. Myelin-associated inhibitors include Nogo, oligodendrocyte-myelin glycoprotein (OMgp), myelin-associated glycoprotein (MAG), Netrin-1, Ephrins and others. They bind to their respective receptors on the surface of injured axons, activating downstream effectors leading to inhibited axon outgrowth.
      A second distinction between peripheral and central axons is microtubule stabilization. Following injury, most mammalian CNS axons retract, and only a few axons sprout for short distances. These axons exhibit dystrophic, swollen endings where axons are exposed to an inhibitory environment. They fail to initiate growth cones, leading to failure to regenerate (
      • Knoferle J.
      • Koch J.C.
      • Ostendorf T.
      • Michel U.
      • Planchamp V.
      • Vutova P.
      • Tonges L.
      • Stadelmann C.
      • Bruck W.
      • Bahr M.
      • Lingor P.
      Mechanisms of acute axonal degeneration in the optic nerve in vivo.
      ). Recent studies show that retraction bulbs in the CNS have a disorganized microtubule network, whereas growth cones in the PNS contain organized bundling of microtubules (
      • Ertürk A.
      • Hellal F.
      • Enes J.
      • Bradke F.
      Disorganized microtubules underlie the formation of retraction bulbs and the failure of axonal regeneration.
      ). In addition, disruption of microtubules in growth cones using pharmacological agents transforms them into retraction bulb-like structures. Hence, microtubule stabilization prevents the formation of retraction bulbs and enhances the growth capacity of CNS neurons both in vitro and in vivo (
      • Hellal F.
      • Hurtado A.
      • Ruschel J.
      • Flynn K.C.
      • Laskowski C.J.
      • Umlauf M.
      • Kapitein L.C.
      • Strikis D.
      • Lemmon V.
      • Bixby J.
      • Hoogenraad C.C.
      • Bradke F.
      Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury.
      ).
      A third mechanism that may influence regeneration is a potential lack of the localization of ribosomes and mRNAs in CNS axons. Local protein synthesis and mRNA localization has been closely associated with successful peripheral nerve regeneration (
      • Vogelaar C.F.
      • Gervasi N.M.
      • Gumy L.F.
      • Story D.J.
      • Raha-Chowdhury R.
      • Leung K.M.
      • Holt C.E.
      • Fawcett J.W.
      Axonal mRNAs: characterization and role in the growth and regeneration of dorsal root ganglion axons and growth cones.
      ). Ribosomes actively translate proteins at the peripheral injury site that both propagate signals back to the soma and supply building blocks for new growth cone formation (
      • Verma P.
      • Chierzi S.
      • Codd A.M.
      • Campbell D.S.
      • Meyer R.L.
      • Holt C.E.
      • Fawcett J.W.
      Axonal protein synthesis and degradation are necessary for efficient growth cone regeneration.
      ,
      • Yoo S.
      • van Niekerk E.A.
      • Merianda T.T.
      • Twiss J.L.
      Dynamics of axonal mRNA transport and implications for peripheral nerve regeneration.
      ,
      • Goldshmit Y.
      • McLenachan S.
      • Turnley A.
      Roles of Eph receptors and ephrins in the normal and damaged adult CNS.
      ). Equivalent mechanisms have not been extensively reported in the CNS, and we remain unaware of their potential contribution to CNS axon regeneration (
      • Twiss J.L.
      • Fainzilber M.
      Ribosomes in axons–scrounging from the neighbors?.
      ). A mere lack of these local mechanisms in the injured axon tip could limit the cell's ability to successfully regenerate in the CNS, and the adequacy of this local response requires further study.
      Lastly, the intrinsic cellular pathways that are activated after injury differ between peripheral and central neurons. This was first hypothesized when injury to the central axons of DRG neurons failed to activate regeneration programs. Injury to the peripheral axons of DRG neurons activates regeneration-associated genes and results in robust axonal outgrowth (
      • Richardson P.M.
      • Verge V.M.
      Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection.
      ). Successful regeneration is coincident with prolonged expression of genes including CAP-23 and GAP-43 (
      • Mason M.R.
      • Lieberman A.R.
      • Grenningloh G.
      • Anderson P.N.
      Transcriptional upregulation of SCG10 and CAP-23 is correlated with regeneration of the axons of peripheral and central neurons in vivo.
      ). Similarly, the up-regulation of regeneration-associated genes has been observed in corticospinal neurons following a proximal intracortical injury, but not a distal spinal axotomy (
      • Mason M.R.
      • Lieberman A.R.
      • Anderson P.N.
      Corticospinal neurons up-regulate a range of growth-associated genes following intracortical, but not spinal, axotomy.
      ). Overexpression of GAP-43 showed improved regeneration in the PNS, but no long-distance regeneration in the CNS (
      • Holtmaat A.J.
      • Dijkhuizen P.A.
      • Oestreicher A.B.
      • Romijn H.J.
      • Van der Lugt N.M.
      • Berns A.
      • Margolis F.L.
      • Gispen W.H.
      • Verhaagen J.
      Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular fiber growth.
      ,
      • Holtmaat A.J.
      • Hermens W.T.
      • Sonnemans M.A.
      • Giger R.J.
      • Van Leeuwen F.W.
      • Kaplitt M.G.
      • Oestreicher A.B.
      • Gispen W.H.
      • Verhaagen J.
      Adenoviral vector-mediated expression of B-50/GAP-43 induces alterations in the membrane organization of olfactory axon terminals in vivo.
      ,
      • Klein R.L.
      • McNamara R.K.
      • King M.A.
      • Lenox R.H.
      • Muzyczka N.
      • Meyer E.M.
      Generation of aberrant sprouting in the adult rat brain by GAP-43 somatic gene transfer.
      ,
      • Buffo A.
      • Holtmaat A.J.
      • Savio T.
      • Verbeek J.S.
      • Oberdick J.
      • Oestreicher A.B.
      • Gispen W.H.
      • Verhaagen J.
      • Rossi F.
      • Strata P.
      Targeted overexpression of the neurite growth-associated protein B-50/GAP-43 in cerebellar Purkinje cells induces sprouting after axotomy but not axon regeneration into growth-permissive transplants.
      ). Targeting other regeneration-associated genes individually has had marginal effects on outgrowth (
      • Werner A.
      • Willem M.
      • Jones L.L.
      • Kreutzberg G.W.
      • Mayer U.
      • Raivich G.
      Impaired axonal regeneration in alpha7 integrin-deficient mice.
      ,
      • Holmes F.E.
      • Mahoney S.
      • King V.R.
      • Bacon A.
      • Kerr N.C.
      • Pachnis V.
      • Curtis R.
      • Priestley J.V.
      • Wynick D.
      Targeted disruption of the galanin gene reduces the number of sensory neurons and their regenerative capacity.
      ), illustrating that manipulating individual proteins may be insufficient to achieve robust, long-distance axonal regeneration. It is therefore likely that targeting protein regulatory networks may present a more potent strategy to activate neuronal growth programs.

      Signaling Networks Associated with Regeneration

      Several signaling pathways have been shown to play a role in outgrowth after injury. For example, both the PI3K and ERK pathways are essential for axon assembly (
      • Atwal J.K.
      • Massie B.
      • Miller F.D.
      • Kaplan D.R.
      The TrkB-Shc site signals neuronal survival and local axon growth via MEK and P13-kinase.
      ). When inhibitors to both pathways were applied to the axon compartment, axon extension was abolished (
      • Atwal J.K.
      • Massie B.
      • Miller F.D.
      • Kaplan D.R.
      The TrkB-Shc site signals neuronal survival and local axon growth via MEK and P13-kinase.
      ).
      The PI3K pathway has been linked to neurotrophin-induced axonal branching where axon turning toward an NGF gradient is PI3K dependent (
      • Gallo G.
      • Letourneau P.C.
      Localized sources of neurotrophins initiate axon collateral sprouting.
      ,
      • Ming G.
      • Song H.
      • Berninger B.
      • Inagaki N.
      • Tessier-Lavigne M.
      • Poo M.
      Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance.
      ). PI3K-Akt regulates local protein synthesis in the axon through the mTOR pathway, where adult CNS neurons require mTOR signaling for axon regeneration (
      • Verma P.
      • Chierzi S.
      • Codd A.M.
      • Campbell D.S.
      • Meyer R.L.
      • Holt C.E.
      • Fawcett J.W.
      Axonal protein synthesis and degradation are necessary for efficient growth cone regeneration.
      ,
      • Park K.K.
      • Liu K.
      • Hu Y.
      • Smith P.D.
      • Wang C.
      • Cai B.
      • Xu B.
      • Connolly L.
      • Kramvis I.
      • Sahin M.
      • He Z.
      Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway.
      ,
      • Liu K.
      • Lu Y.
      • Lee J.K.
      • Samara R.
      • Willenberg R.
      • Sears-Kraxberger I.
      • Tedeschi A.
      • Park K.K.
      • Jin D.
      • Cai B.
      • Xu B.
      • Connolly L.
      • Steward O.
      • Zheng B.
      • He Z.
      PTEN deletion enhances the regenerative ability of adult corticospinal neurons.
      ). Quantitative mass spectrometry identified syntaxin13 as a protein locally synthesized by activated mTOR in the axon (
      • Cho Y.
      • Di Liberto V.
      • Carlin D.
      • Abe N.
      • Li K.H.
      • Burlingame A.L.
      • Guan S.
      • Michaelevski I.
      • Cavalli V.
      Syntaxin13 expression is regulated by mammalian target of rapamycin (mTOR) in injured neurons to promote axon regeneration.
      ). Knockdown of syntaxin13 in culture prevented axon growth and regeneration. Interestingly, within the regenerating axon, PI3K is only activated at the distal tip, and its signaling is conveyed downstream through the inactivation of glycogen synthase kinase 3β (GSK3β) (
      • Zhou F.Q.
      • Zhou J.
      • Dedhar S.
      • Wu Y.H.
      • Snider W.D.
      NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC.
      ,
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B.
      ). In adult DRG neurons, GSK3β inhibition leads to enhanced neurite outgrowth (
      • Jones D.M.
      • Tucker B.A.
      • Rahimtula M.
      • Mearow K.M.
      The synergistic effects of NGF and IGF-1 on neurite growth in adult sensory neurons: convergence on the PI 3-kinase signaling pathway.
      ). In hippocampal neurons, inactivation of GSK3β is crucial for axon specification and growth through the phosphorylation of CRMP-2 (
      • Jiang H.
      • Guo W.
      • Liang X.
      • Rao Y.
      Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3beta and its upstream regulators.
      ,
      • Yoshimura T.
      • Arimura N.
      • Kawano Y.
      • Kawabata S.
      • Wang S.
      • Kaibuchi K.
      Ras regulates neuronal polarity via the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway.
      ,
      • Yoshimura T.
      • Kawano Y.
      • Arimura N.
      • Kawabata S.
      • Kikuchi A.
      • Kaibuchi K.
      GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity.
      ). In cerebellar granule neurons, GRMP-2 is localized to dendritic branches, where its phosphorylation by GSK3β inhibits dendritic growth (
      • Tan M.
      • Ma S.
      • Huang Q.
      • Hu K.
      • Song B.
      • Li M.
      GSK-3alpha/beta-mediated phosphorylation of CRMP-2 regulates activity-dependent dendritic growth.
      ).
      GSK3β is widely expressed within the adult brain and is regulated through Akt as well as ILK, and PKC (
      • Kanzaki M.
      • Mora S.
      • Hwang J.B.
      • Saltiel A.R.
      • Pessin J.E.
      Atypical protein kinase C (PKCzeta/lambda) is a convergent downstream target of the insulin-stimulated phosphatidylinositol 3-kinase and TC10 signaling pathways.
      ,
      • Shen J.Y.
      • Yi X.X.
      • Xiong N.X.
      • Wang H.J.
      • Duan X.W.
      • Zhao H.Y.
      GSK-3beta activation mediates Nogo-66-induced inhibition of neurite outgrowth in N2a cells.
      ,
      • Delcommenne M.
      • Tan C.
      • Gray V.
      • Rue L.
      • Woodgett J.
      • Dedhar S.
      Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase.
      ). Inactivation of GSK3β stimulates axon elongation on inhibitory substrates in adult neurons, and induced corticospinal axon sprouting after injury (
      • Dill J.
      • Wang H.
      • Zhou F.
      • Li S.
      Inactivation of glycogen synthase kinase 3 promotes axonal growth and recovery in the CNS.
      ). Slit2 signaling inhibits neurite outgrowth through GSK3β phosphorylation in adult DRG neurons (
      • Byun J.
      • Kim B.T.
      • Kim Y.T.
      • Jiao Z.
      • Hur E.M.
      • Zhou F.Q.
      Slit2 inactivates GSK3beta to signal neurite outgrowth inhibition.
      ). Therefore, GSK3β has been negatively linked to axon growth and sprouting. The role of GSK3β in axon growth is, however, controversial (
      • Gobrecht P.
      • Leibinger M.
      • Andreadaki A.
      • Fischer D.
      Sustained GSK3 activity markedly facilitates nerve regeneration.
      ). Interestingly, GSK3β phosphorylation of MAP1B acts as a molecular switch to regulate microtubule dynamics in growing axons. Further, the MAPK pathway is involved in NGF mediated activation of TrkA, through phosphorylation of GSK3β during development (
      • Goold R.G.
      • Gordon-Weeks P.R.
      The MAP kinase pathway is upstream of the activation of GSK3beta that enables it to phosphorylate MAP1B and contributes to the stimulation of axon growth.
      ). Therefore, distinct molecular switches control developmental growth, which may be perceived as inhibitory in the adult neuron.
      A few MAP kinases are involved in repair mechanisms (
      • Hollis 2nd, E.R.
      • Jamshidi P.
      • Low K.
      • Blesch A.
      • Tuszynski M.H.
      Induction of corticospinal regeneration by lentiviral trkB-induced Erk activation.
      ,
      • Hammarlund M.
      • Nix P.
      • Hauth L.
      • Jorgensen E.M.
      • Bastiani M.
      Axon regeneration requires a conserved MAP kinase pathway.
      ). In dual leucine zipper kinase (DLK) knockout mice, neurons did not respond to a preconditioned lesion, suggesting this MAPKK kinase acts as a key mediator between the injured axon and somatic response to injury (
      • Shin J.E.
      • Cho Y.
      • Beirowski B.
      • Milbrandt J.
      • Cavalli V.
      • DiAntonio A.
      Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration.
      ). Extracellular signal regulated kinase (ERK) phosphorylation is an essential component of the retrograde injury signal, and is thought to transmit information from the site of injury to the soma (
      • Perlson E.
      • Hanz S.
      • Ben-Yaakov K.
      • Segal-Ruder Y.
      • Seger R.
      • Fainzilber M.
      Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve.
      ). ERK is thought to regulate local protein translation after injury, and may play a role in axon outgrowth (
      • Zhou F.Q.
      • Snider W.D.
      Intracellular control of developmental and regenerative axon growth.
      ). Growth cone collapse in response to semaphorin 3A is also regulated by ERK-mediated local protein synthesis (
      • Campbell D.S.
      • Holt C.E.
      Apoptotic pathway and MAPKs differentially regulate chemotropic responses of retinal growth cones.
      ). In adult DRG neurons, inhibition of RhoA stimulates neurite outgrowth, even in the presence of inhibitory substrates (
      • Fu Q.
      • Hue J.
      • Li S.
      Nonsteroidal anti-inflammatory drugs promote axon regeneration via RhoA inhibition.
      ). Local protein synthesis and ERK signaling is required for axon resensitization during chemotactic guidance (
      • Ming G.L.
      • Wong S.T.
      • Henley J.
      • Yuan X.B.
      • Song H.J.
      • Spitzer N.C.
      • Poo M.M.
      Adaptation in the chemotactic guidance of nerve growth cones.
      ), suggesting it may play a role in axon assembly (
      • Atwal J.K.
      • Singh K.K.
      • Tessier-Lavigne M.
      • Miller F.D.
      • Kaplan D.R.
      Semaphorin 3F antagonizes neurotrophin-induced phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase signaling: a mechanism for growth cone collapse.
      ). ERK phosphorylation is induced by neurotrophins, netrin (
      • Forcet C.
      • Stein E.
      • Pays L.
      • Corset V.
      • Llambi F.
      • Tessier-Lavigne M.
      • Mehlen P.
      Netrin-1-mediated axon outgrowth requires deleted in colorectal cancer-dependent MAPK activation.
      ), semaphorin 7A (
      • Pasterkamp R.J.
      • Peschon J.J.
      • Spriggs M.K.
      • Kolodkin A.L.
      Semaphorin 7A promotes axon outgrowth through integrins and MAPKs.
      ), and cell adhesion molecules (
      • Schmid R.S.
      • Pruitt W.M.
      • Maness P.F.
      A MAP kinase-signaling pathway mediates neurite outgrowth on L1 and requires Src-dependent endocytosis.
      ). PI3 Kinase and MEK kinase is necessary for NGF-induced axon growth in sympathetic neurons, whereas MEK-signaling is required for BDNF/TrkB mediated axon elongation (
      • Atwal J.K.
      • Massie B.
      • Miller F.D.
      • Kaplan D.R.
      The TrkB-Shc site signals neuronal survival and local axon growth via MEK and P13-kinase.
      ,
      • Kuruvilla R.
      • Ye H.
      • Ginty D.D.
      Spatially and functionally distinct roles of the PI3-K effector pathway during NGF signaling in sympathetic neurons.
      ). The MAP kinase pathway may therefore play a central role linking the extracellular environment to intracellular mediated responses.
      STAT3 phosphorylation through gp130 signaling has also shown to increase regeneration after injury (
      • Quarta S.
      • Baeumer B.E.
      • Scherbakov N.
      • Andratsch M.
      • Rose-John S.
      • Dechant G.
      • Bandtlow C.E.
      • Kress M.
      Peripheral nerve regeneration and NGF-dependent neurite outgrowth of adult sensory neurons converge on STAT3 phosphorylation downstream of neuropoietic cytokine receptor gp130.
      ), whereas phosphorylation of cytoplasmic set-β abolished its inhibitory role on neurite outgrowth (
      • Trakhtenberg E.F.
      • Wang Y.
      • Morkin M.I.
      • Fernandez S.G.
      • Mlacker G.M.
      • Shechter J.M.
      • Liu X.
      • Patel K.H.
      • Lapins A.
      • Yang S.
      • Dombrowski S.M.
      • Goldberg J.L.
      Regulating set-beta's subcellular localization toggles its function between inhibiting and promoting axon growth and regeneration.
      ). Phosphorylation profiles in the injured cell have also been associated with inhibiting growth. The phosphorylation of Dock6, a guanine nucleotide exchange factor, inhibited its activity, which suppressed axon regeneration after injury in vitro and in vivo (
      • Miyamoto Y.
      • Torii T.
      • Yamamori N.
      • Ogata T.
      • Tanoue A.
      • Yamauchi J.
      Akt and PP2A reciprocally regulate the guanine nucleotide exchange factor Dock6 to control axon growth of sensory neurons.
      ). Identifying key signaling networks associated with axon regeneration allows us to experimentally manipulate these pathways, unveiling potential therapies for regeneration.

      Identification of Post-Translational Modifications in the Injured, Degenerating, and Regenerating Environment

      Post-translational modifications (PTMs) of proteins are also crucial modulators of the neuron's ability to respond to its environment. During glaucomatous neurodegeneration, the 14–3-3 family of proteins have been shown to regulate apoptosis in a phosphorylation dependent manner (
      • Yang X.
      • Luo C.
      • Cai J.
      • Pierce W.M.
      • Tezel G.
      Phosphorylation-dependent interaction with 14-3-3 in the regulation of bad trafficking in retinal ganglion cells.
      ). Holzbaur and colleagues showed a decrease in phospho-proteins associated with dynein retrograde signaling, including phospho-Trk receptors and phosho-ERK1/2 signaling proteins (
      • Perlson E.
      • Jeong G.B.
      • Ross J.L.
      • Dixit R.
      • Wallace K.E.
      • Kalb R.G.
      • Holzbaur E.L.
      A switch in retrograde signaling from survival to stress in rapid-onset neurodegeneration.
      ), suggesting that post-translational modifications play a role in ALS neurodegeneration. Intraneuronal post-translational modifications can therefore modulate the cell's homeostasis and response to extracellular cues. The activity of synaptic proteins has also been shown to be phosphorylation-dependent (
      • Vinters H.V.
      Emerging concepts in Alzheimer's disease.
      ,
      • Ahmed S.
      • Wittenmayer N.
      • Kremer T.
      • Hoeber J.
      • Kiran Akula A.
      • Urlaub H.
      • Islinger M.
      • Kirsch J.
      • Dean C.
      • Dresbach T.
      Mover is a homomeric phospho-protein present on synaptic vesicles.
      ,
      • Piccini A.
      • Perlini L.E.
      • Cancedda L.
      • Benfenati F.
      • Giovedi S.
      Phosphorylation by PKA and Cdk5 mediates the early effects of synapsin III in neuronal morphological maturation.
      ,
      • Jang S.S.
      • Royston S.E.
      • Xu J.
      • Cavaretta J.P.
      • Vest M.O.
      • Lee K.Y.
      • Lee S.
      • Jeong H.G.
      • Lombroso P.J.
      • Chung H.J.
      Regulation of STEP61 and tyrosine-phosphorylation of NMDA and AMPA receptors during homeostatic synaptic plasticity.
      ). Specifically, a novel motif of serine/threonine-glutamate ([S/T]-Q) containing substrates has recently been identified to localize predominantly in dendrites, synapses and the soma (
      • Siddoway B.
      • Hou H.
      • Yang H.
      • Petralia R.
      • Xia H.
      Synaptic activity bidirectionally regulates a novel sequence-specific S-Q phosphoproteome in neurons.
      ). Phosphoproteomics identified 150 proteins containing this motif, thought to bidirectionally regulate synaptic activity. Other post-translational modifications have also been shown to regulate synaptic plasticity. Sumoylation of the GluA1 AMPAR subunit is required for its surface expression during long-term potentiation (LTP) (
      • Jaafari N.
      • Konopacki F.A.
      • Owen T.F.
      • Kantamneni S.
      • Rubin P.
      • Craig T.J.
      • Wilkinson K.A.
      • Henley J.M.
      SUMOylation is required for glycine-induced increases in AMPA receptor surface expression (ChemLTP) in hippocampal neurons.
      ). Decreased sumoylation showed a marked reduction in surface expression of AMPAR, emphasizing the role of SUMO in synaptic plasticity. Sumoylation of the transcription factor MeCP2 also leads to transcriptional repression, known to play a role in synaptic development (
      • Cheng J.
      • Huang M.
      • Zhu Y.
      • Xin Y.J.
      • Zhao Y.K.
      • Huang J.
      • Yu J.X.
      • Zhou W.H.
      • Qiu Z.
      SUMOylation of MeCP2 is essential for transcriptional repression and hippocampal synapse development.
      ). Directional transport of proteins in axons has also been shown to be SUMO dependent (
      • van Niekerk E.A.
      • Willis D.E.
      • Chang J.H.
      • Reumann K.
      • Heise T.
      • Twiss J.L.
      Sumoylation in axons triggers retrograde transport of the RNA-binding protein La.
      ); and sumoylation and phosphorylation have further shown to have synergistic effects (
      • Chamberlain S.E.
      • Gonzalez-Gonzalez I.M.
      • Wilkinson K.A.
      • Konopacki F.A.
      • Kantamneni S.
      • Henley J.M.
      • Mellor J.R.
      SUMOylation and phosphorylation of GluK2 regulate kainate receptor trafficking and synaptic plasticity.
      ).
      Proteins within the environment can also modulate post-translational modifications within the injured and regenerating axon. A recent study employed isobaric tag (iTRAQ) labeling for quantitative proteomics to identify changes in the phosphoproteome of primary cerebellar granular neurons induced by culturing with inhibitory CSPGs (
      • Yu P.
      • Pisitkun T.
      • Wang G.
      • Wang R.
      • Katagiri Y.
      • Gucek M.
      • Knepper M.A.
      • Geller H.M.
      Global analysis of neuronal phosphoproteome regulation by chondroitin sulfate proteoglycans.
      ). Over 100 significantly altered phosphopeptides were identified, reflecting differential phosphorylation of cytoskeletal proteins, DNA- and RNA-binding proteins, and transcription factors, among others. Post-translational modifications within the extrinsic milieu have also been shown to influence the dynamics of injured axons. Phosphorylation of the adhesion protein galectin-3 on the heparin sulfate proteoglycan (HSPG) substrate interacts with L1-NCAM on hippocampal axons, promoting axonal branching via local actin destabilization (
      • Diez-Revuelta N.
      • Velasco S.
      • Andre S.
      • Kaltner H.
      • Kubler D.
      • Gabius H.J.
      • Abad-Rodriguez J.
      Phosphorylation of adhesion- and growth-regulatory human galectin-3 leads to the induction of axonal branching by local membrane L1 and ERM redistribution.
      ). Hence, phosphorylation of this extracellular protein acts as a highly functionally relevant molecular switch modulating complex axon dynamics. Recently, Ghosh and colleagues found that grafted Schwann cells expressing polysialyltransferase (PST) to modify polysialic acid residues on neural cell adhesion molecule (NCAM) displayed an enhanced ability to associate with and support very modest growth of injured corticospinal axons (
      • Ghosh M.
      • Tuesta L.M.
      • Puentes R.
      • Patel S.
      • Melendez K.
      • El Maarouf A.
      • Rutishauser U.
      • Pearse D.D.
      Extensive cell migration, axon regeneration, and improved function with polysialic acid-modified Schwann cells after spinal cord injury.
      ).
      Post-translational modifications play a critical role in the regulation of protein activity and biological signaling activity. Quantitative proteomics have the advantage to detect phospho-peptides, sumoylated-peptides, ubiquitinated-peptides, and palmitoylated-peptides to name a few (
      • Galisson F.
      • Mahrouche L.
      • Courcelles M.
      • Bonneil E.
      • Meloche S.
      • Chelbi-Alix M.K.
      • Thibault P.
      A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells.
      ,
      • Mommen G.P.
      • van de Waterbeemd B.
      • Meiring H.D.
      • Kersten G.
      • Heck A.J.
      • de Jong A.P.
      Unbiased selective isolation of protein N-terminal peptides from complex proteome samples using phospho tagging (PTAG) and TiO(2)-based depletion.
      ). As more researchers employ these techniques, greater understanding of post-translational modifications related to disease will arise. At this juncture, combining high-throughput techniques and disease models will advance our understanding of spinal cord injury and help determine better therapeutic strategies (
      • Hart G.W.
      • Ball L.E.
      Post-translational modifications: a major focus for the future of proteomics.
      ).

      Experimental Strategies to Promote Axonal Regeneration

      In order to identify therapeutic targets that can promote neural repair and functional recovery following CNS injury, it is important to understand neuron-intrinsic and -extrinsic pathways underlying axonal regeneration (
      • Afshari F.T.
      • Kappagantula S.
      • Fawcett J.W.
      Extrinsic and intrinsic factors controlling axonal regeneration after spinal cord injury.
      ).

      Stimulating the Endogenous Growth State of the Neuron

      Experimental approaches to enhance neuron-intrinsic growth capacity have largely been informed by the study of molecular mechanisms active in regeneration-enabled neuronal populations. Here we review several studies in which key proteins have been identified in these populations and successfully manipulated to enhance axonal regeneration in models of CNS injury.

      Intrinsic Mechanisms of Axon Regeneration: The Conditioning Lesion

      Enhanced spontaneous regeneration of PNS neurons is because of the activation of intrinsic signaling networks, in combination with a growth-permissive extrinsic environment, which together allow robust regeneration of injured peripheral axons (
      • Hoffman P.N.
      A conditioning lesion induces changes in gene expression and axonal transport that enhance regeneration by increasing the intrinsic growth state of axons.
      ). Though permissive extrinsic cues are important determinants contributing to successful regeneration of the DRG axon through the peripheral nerve (
      • Bosse F.
      Extrinsic cellular and molecular mediators of peripheral axonal regeneration.
      ), the injured DRG neuron presents a unique model with which to investigate the cell-intrinsic growth programs activated by axotomy. In the conditioning lesion paradigm, lesion of the peripheral, but not central DRG axon induces a cascade of signaling events in the axon and neuronal soma that enhances the intrinsic growth state of the neuron (
      • Smith D.S.
      • Skene J.H.
      A transcription-dependent switch controls competence of adult neurons for distinct modes of axon growth.
      ). Notably, this conditioning lesion also enhances regeneration of the injured central axon branch if performed prior to, or shortly after the central lesion (
      • Richardson P.M.
      • Issa V.M.
      Peripheral injury enhances central regeneration of primary sensory neurones.
      ,
      • Blesch A.
      • Lu P.
      • Tsukada S.
      • Alto L.T.
      • Roet K.
      • Coppola G.
      • Geschwind D.
      • Tuszynski M.H.
      Conditioning lesions before or after spinal cord injury recruit broad genetic mechanisms that sustain axonal regeneration: superiority to camp-mediated effects.
      ).
      Much work has been done to characterize the genetic programs recruited by conditioning lesion in the DRG neurons. Injured DRG neurons rapidly activate a transcriptional program of hundreds of genes as early as 1 day postinjury, and the majority of these exhibit sustained expression patterns by 2 weeks postinjury (
      • Blesch A.
      • Lu P.
      • Tsukada S.
      • Alto L.T.
      • Roet K.
      • Coppola G.
      • Geschwind D.
      • Tuszynski M.H.
      Conditioning lesions before or after spinal cord injury recruit broad genetic mechanisms that sustain axonal regeneration: superiority to camp-mediated effects.
      ,
      • Costigan M.
      • Befort K.
      • Karchewski L.
      • Griffin R.S.
      • D'Urso D.
      • Allchorne A.
      • Sitarski J.
      • Mannion J.W.
      • Pratt R.E.
      • Woolf C.J.
      Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.
      ) (
      • Smith D.S.
      • Skene J.H.
      A transcription-dependent switch controls competence of adult neurons for distinct modes of axon growth.
      ,
      • Blesch A.
      • Lu P.
      • Tsukada S.
      • Alto L.T.
      • Roet K.
      • Coppola G.
      • Geschwind D.
      • Tuszynski M.H.
      Conditioning lesions before or after spinal cord injury recruit broad genetic mechanisms that sustain axonal regeneration: superiority to camp-mediated effects.
      ,
      • Chong M.S.
      • Reynolds M.L.
      • Irwin N.
      • Coggeshall R.E.
      • Emson P.C.
      • Benowitz L.I.
      • Woolf C.J.
      GAP-43 expression in primary sensory neurons following central axotomy.
      ,
      • Greenberg S.G.
      • Lasek R.J.
      Neurofilament protein synthesis in DRG neurons decreases more after peripheral axotomy than after central axotomy.
      ,
      • Seijffers R.
      • Mills C.D.
      • Woolf C.J.
      ATF3 increases the intrinsic growth state of DRG neurons to enhance peripheral nerve regeneration.
      ,
      • Jankowski M.P.
      • McIlwrath S.L.
      • Jing X.
      • Cornuet P.K.
      • Salerno K.M.
      • Koerber H.R.
      • Albers K.M.
      Sox11 transcription factor modulates peripheral nerve regeneration in adult mice.
      ,
      • MacGillavry H.D.
      • Stam F.J.
      • Sassen M.M.
      • Kegel L.
      • Hendriks W.T.
      • Verhaagen J.
      • Smit A.B.
      • van Kesteren R.E.
      NFIL3 and cAMP response element-binding protein form a transcriptional feedforward loop that controls neuronal regeneration-associated gene expression.
      ,
      • Stam F.J.
      • MacGillavry H.D.
      • Armstrong N.J.
      • de Gunst M.C.
      • Zhang Y.
      • van Kesteren R.E.
      • Smit A.B.
      • Verhaagen J.
      Identification of candidate transcriptional modulators involved in successful regeneration after nerve injury.
      ,
      • Zou H.
      • Ho C.
      • Wong K.
      • Tessier-Lavigne M.
      Axotomy-induced Smad1 activation promotes axonal growth in adult sensory neurons.
      ). Because this gene expression program is not activated upon central axotomy, many groups have endeavored to promote CNS axon regeneration via the exogenous expression of regeneration associated genes or their upstream regulators (
      • Seijffers R.
      • Benowitz L.I.
      Intrinsic determinants of axon regeneration.
      ,
      • Liu K.
      • Tedeschi A.
      • Park K.K.
      • He Z.
      Neuronal intrinsic mechanisms of axon regeneration.
      ).

      Transcription Factors: Coordinators of Regeneration-Associated Gene Expression Programs

      Several studies have focused on manipulating transcription factors, regulators of intraneuronal growth programs, that can coordinate the simultaneous expression of multiple regeneration associated genes.
      In the spinal cord, regeneration and sprouting of dorsal column sensory axons has successfully been enhanced by overexpression or activation of transcription factors such as CREB (
      • Gao Y.
      • Deng K.
      • Hou J.
      • Bryson J.B.
      • Barco A.
      • Nikulina E.
      • Spencer T.
      • Mellado W.
      • Kandel E.R.
      • Filbin M.T.
      Activated CREB is sufficient to overcome inhibitors in myelin and promote spinal axon regeneration in vivo.
      ), RARβ2 (
      • Wong L.F.
      • Yip P.K.
      • Battaglia A.
      • Grist J.
      • Corcoran J.
      • Maden M.
      • Azzouz M.
      • Kingsman S.M.
      • Kingsman A.J.
      • Mazarakis N.D.
      • McMahon S.B.
      Retinoic acid receptor beta2 promotes functional regeneration of sensory axons in the spinal cord.
      ), STAT3 (
      • Bareyre F.M.
      • Garzorz N.
      • Lang C.
      • Misgeld T.
      • Buning H.
      • Kerschensteiner M.
      In vivo imaging reveals a phase-specific role of STAT3 during central and peripheral nervous system axon regeneration.
      ), Smad1 (
      • Parikh P.
      • Hao Y.
      • Hosseinkhani M.
      • Patil S.B.
      • Huntley G.W.
      • Tessier-Lavigne M.
      • Zou H.
      Regeneration of axons in injured spinal cord by activation of bone morphogenetic protein/Smad1 signaling pathway in adult neurons.
      ), and SnoN (
      • Do J.L.
      • Bonni A.
      • Tuszynski M.H.
      SnoN facilitates axonal regeneration after spinal cord injury.
      ) in DRG neurons. The corticospinal tract (CST), the most important motor projection in humans, is especially refractory to efforts to promote its regeneration following injury. Partial regeneration or sprouting of the lesioned CST has recently been reported via overexpression of KLF7 (
      • Blackmore M.G.
      • Wang Z.
      • Lerch J.K.
      • Motti D.
      • Zhang Y.P.
      • Shields C.B.
      • Lee J.K.
      • Goldberg J.L.
      • Lemmon V.P.
      • Bixby J.L.
      Krüppel-like Factor 7 engineered for transcriptional activation promotes axon regeneration in the adult corticospinal tract.
      ), p53 (
      • Floriddia E.M.
      • Rathore K.I.
      • Tedeschi A.
      • Quadrato G.
      • Wuttke A.
      • Lueckmann J.M.
      • Kigerl K.A.
      • Popovich P.G.
      • Di Giovanni S.
      p53 regulates the neuronal intrinsic and extrinsic responses affecting the recovery of motor function following spinal cord injury.
      ), STAT3 (
      • Lang C.
      • Bradley P.M.
      • Jacobi A.
      • Kerschensteiner M.
      • Bareyre F.M.
      STAT3 promotes corticospinal remodeling and functional recovery after spinal cord injury.
      ), and Sox11 (
      • Wang Z.
      • Reynolds A.
      • Kirry A.
      • Nienhaus C.
      • Blackmore M.G.
      Overexpression of sox11 promotes corticospinal tract regeneration after spinal injury while interfering with functional recovery.
      ), and by conditional deletion or systemic antagonism of PTEN (
      • Liu K.
      • Lu Y.
      • Lee J.K.
      • Samara R.
      • Willenberg R.
      • Sears-Kraxberger I.
      • Tedeschi A.
      • Park K.K.
      • Jin D.
      • Cai B.
      • Xu B.
      • Connolly L.
      • Steward O.
      • Zheng B.
      • He Z.
      PTEN deletion enhances the regenerative ability of adult corticospinal neurons.
      ,
      • Ohtake Y.
      • Park D.
      • Abdul-Muneer P.M.
      • Li H.
      • Xu B.
      • Sharma K.
      • Smith G.M.
      • Selzer M.E.
      • Li S.
      The effect of systemic PTEN antagonist peptides on axon growth and functional recovery after spinal cord injury.
      ) in CST motor neurons. Though fewer transcriptional regulators have been assessed in retinal ganglion cell (RGC) regeneration paradigms, it is known that KLF family members regulate regeneration in the optic nerve (
      • Moore D.L.
      • Blackmore M.G.
      • Hu Y.
      • Kaestner K.H.
      • Bixby J.L.
      • Lemmon V.P.
      • Goldberg J.L.
      KLF family members regulate intrinsic axon regeneration ability.
      ,
      • Apara A.
      • Goldberg J.L.
      Molecular mechanisms of the suppression of axon regeneration by KLF transcription factors.
      ), and that specific KLFs have antagonistic roles in modulating RGC growth cone dynamics (
      • Steketee M.B.
      • Oboudiyat C.
      • Daneman R.
      • Trakhtenberg E.
      • Lamoureux P.
      • Weinstein J.E.
      • Heidemann S.
      • Barres B.A.
      • Goldberg J.L.
      Regulation of intrinsic axon growth ability at retinal ganglion cell growth cones.
      ). Additional work has shown that subcellular localization of the transcription factor Set-β plays a regulatory role in regeneration (
      • Trakhtenberg E.F.
      • Wang Y.
      • Morkin M.I.
      • Fernandez S.G.
      • Mlacker G.M.
      • Shechter J.M.
      • Liu X.
      • Patel K.H.
      • Lapins A.
      • Yang S.
      • Dombrowski S.M.
      • Goldberg J.L.
      Regulating set-beta's subcellular localization toggles its function between inhibiting and promoting axon growth and regeneration.
      ). Perhaps the most impressive findings to date arise from work showing that deletion of PTEN (
      • Park K.K.
      • Liu K.
      • Hu Y.
      • Smith P.D.
      • Wang C.
      • Cai B.
      • Xu B.
      • Connolly L.
      • Kramvis I.
      • Sahin M.
      • He Z.
      Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway.
      ), SOCS3 (
      • Smith P.D.
      • Sun F.
      • Park K.K.
      • Cai B.
      • Wang C.
      • Kuwako K.
      • Martinez-Carrasco I.
      • Connolly L.
      • He Z.
      SOCS3 deletion promotes optic nerve regeneration in vivo.
      ,
      • Hellstrom M.
      • Muhling J.
      • Ehlert E.M.
      • Verhaagen J.
      • Pollett M.A.
      • Hu Y.
      • Harvey A.R.
      Negative impact of rAAV2 mediated expression of SOCS3 on the regeneration of adult retinal ganglion cell axons.
      ), or both (
      • Park K.K.
      • Liu K.
      • Hu Y.
      • Smith P.D.
      • Wang C.
      • Cai B.
      • Xu B.
      • Connolly L.
      • Kramvis I.
      • Sahin M.
      • He Z.
      Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway.
      ,
      • Sun F.
      • Park K.K.
      • Belin S.
      • Wang D.
      • Lu T.
      • Chen G.
      • Zhang K.
      • Yeung C.
      • Feng G.
      • Yankner B.A.
      • He Z.
      Sustained axon regeneration induced by codeletion of PTEN and SOCS3.
      ) substantially enhances optic nerve regeneration.

      Epigenetic Mechanisms of Axon Regeneration

      As epigenetic regulation of gene expression becomes more fully understood, the prospect that the epigenetic state of the neuron might significantly influence its intrinsic growth capacity becomes more compelling (
      • Trakhtenberg E.F.
      • Goldberg J.L.
      Epigenetic regulation of axon and dendrite growth.
      ,
      • Lindner R.
      • Puttagunta R.
      • Di Giovanni S.
      Epigenetic regulation of axon outgrowth and regeneration in CNS injury: the first steps forward.
      ). In the last few years, experimental manipulation of chromatin dynamics in injury models has provided valuable insight into epigenetic influence on axon regeneration.
      Gaub and colleagues showed that pan-inhibition of the histone deacetylases (HDACs) with the drug trichostatin A (TSA) promotes neurite outgrowth in vitro by enhancing activity of the histone acetyltransferases (HATs), CBP/p300 and PCAF (
      • Gaub P.
      • Tedeschi A.
      • Puttagunta R.
      • Nguyen T.
      • Schmandke A.
      • Di Giovanni S.
      HDAC inhibition promotes neuronal outgrowth and counteracts growth cone collapse through CBP/p300 and P/CAF-dependent p53 acetylation.
      ). This approach increased acetylation not only of histones but also of the transcription factor p53, resulting in increased expression of p53 target genes including the classical regeneration associated genes, GAP-43 and SCG10. HDAC inhibition was also shown to enhance sensory axon regeneration in the lesioned mouse spinal cord (
      • Finelli M.J.
      • Wong J.K.
      • Zou H.
      Epigenetic regulation of sensory axon regeneration after spinal cord injury.
      ). Cavalli and colleagues revealed that HDAC5 becomes exported from the nucleus following peripheral nerve injury, resulting in histone hyperacetylation and activation of a proregenerative gene expression program (
      • Cho Y.
      • Sloutsky R.
      • Naegle K.M.
      • Cavalli V.
      Injury-induced HDAC5 nuclear export is essential for axon regeneration.
      ). Expression of the histone deacetylase SIRT1 was also shown to contribute to regeneration of conditioned peripheral nerve (
      • Liu C.M.
      • Wang R.Y.
      • Saijilafu
      • Jiao Z.X.
      • Zhang B.Y.
      • Zhou F.Q.
      MicroRNA-138 and SIRT1 form a mutual negative feedback loop to regulate mammalian axon regeneration.
      ). Most recently, Di Giovanni's group reported that peripheral nerve injury, via ERK-mediated retrograde signaling, induces PCAF-dependent acetylation of histone H3K9 at the promoters of regeneration associated genes, triggering a transcriptional regeneration program (
      • Puttagunta R.
      • Tedeschi A.
      • Soria M.G.
      • Hervera A.
      • Lindner R.
      • Rathore K.I.
      • Gaub P.
      • Joshi Y.
      • Nguyen T.
      • Schmandke A.
      • Laskowski C.J.
      • Boutillier A.L.
      • Bradke F.
      • Di Giovanni S.
      PCAF-dependent epigenetic changes promote axonal regeneration in the central nervous system.
      ). Notably, overexpression of PCAF in DRG neurons was sufficient to enhance regeneration of ascending sensory axons in the injured spinal cord, suggesting that PCAF is a central epigenetic component of the conditioning lesion effect. Together, these findings implicate a role for epigenetics in determining the intrinsic neuronal growth state.
      In summary, the intrinsic mechanisms governing axon regeneration are complex and can be modulated by a host of factors acting at subcellular locations from the chromatin to the growth cone. Much of our current understanding is based on transcriptomic mechanisms associated with regeneration, where most of these studies have been done in the peripheral nervous system. A broader understanding of protein changes in response to injury and therapy is needed. Quantitative proteomics will allow us to develop a global view of the injured spinal cord's proteome. Not only will it reveal the active biological condition, but also the magnitude and complexity imparted by protein regulation and respective modifications (
      • Hart G.W.
      • Ball L.E.
      Post-translational modifications: a major focus for the future of proteomics.
      ). Additional perspective may be gained by characterizing the intrinsic proteomic mechanisms driving compensatory sprouting of both injured and intact CNS projections following injury (
      • Raineteau O.
      • Schwab M.E.
      Plasticity of motor systems after incomplete spinal cord injury.
      ). This includes corticospinal (
      • Weidner N.
      • Ner A.
      • Salimi N.
      • Tuszynski M.H.
      Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury.
      ,
      • Hill C.E.
      • Beattie M.S.
      • Bresnahan J.C.
      Degeneration and sprouting of identified descending supraspinal axons after contusive spinal cord injury in the rat.
      ,
      • Rosenzweig E.S.
      • Courtine G.
      • Jindrich D.L.
      • Brock J.H.
      • Ferguson A.R.
      • Strand S.C.
      • Nout Y.S.
      • Roy R.R.
      • Miller D.M.
      • Beattie M.S.
      • Havton L.A.
      • Bresnahan J.C.
      • Edgerton V.R.
      • Tuszynski M.H.
      Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury.
      ), rubrospinal (
      • Siegel C.S.
      • Fink K.L.
      • Strittmatter S.M.
      • Cafferty W.B.
      Plasticity of intact rubral projections mediates spontaneous recovery of function after corticospinal tract injury.
      ), reticulospinal, and propriospinal axons (
      • Filli L.
      • Engmann A.K.
      • Zorner B.
      • Weinmann O.
      • Moraitis T.
      • Gullo M.
      • Kasper H.
      • Schneider R.
      • Schwab M.E.
      Bridging the gap: a reticulo-propriospinal detour bypassing an incomplete spinal cord injury.
      ,
      • Tan A.M.
      • Chakrabarty S.
      • Kimura H.
      • Martin J.H.
      Selective corticospinal tract injury in the rat induces primary afferent fiber sprouting in the spinal cord and hyperreflexia.
      ). Finally, exploring the identified signaling networks activated within injured CNS neurons that regenerate into permissive cellular grafts discussed below may yield novel insight into the molecular switches that enable growth of an axon previously incapable of regeneration.

      Modifying the Injured Environment

      The injured, adult CNS axon is faced with an extracellular milieu very different from that of the developing CNS and the PNS. The increased permissiveness of the peripheral nerve environment first gained appreciation from early studies showing robust regeneration of central axons into grafted peripheral nerve “bridges” following CNS injury (
      • David S.
      • Aguayo A.J.
      Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats.
      ). In subsequent decades, a plethora of work has identified specific components of the CNS environment that inhibit regeneration of central axons. Here we will briefly highlight key efforts to shape the injured CNS environment in order to enhance regeneration.

      Myelin-Associated Inhibitors

      Myelin-associated inhibitors are a group of proteins that inhibit regeneration of injured adult axons within the CNS (Fig. 1) (
      • Filbin M.T.
      Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS.
      ,
      • Geoffroy C.G.
      • Zheng B.
      Myelin-associated inhibitors in axonal growth after CNS injury.
      ). The classical myelin-associated inhibitors, Nogo (
      • Huber A.B.
      • Schwab M.E.
      Nogo-A, a potent inhibitor of neurite outgrowth and regeneration.
      ), OMgp (
      • Kottis V.
      • Thibault P.
      • Mikol D.
      • Xiao Z.C.
      • Zhang R.
      • Dergham P.
      • Braun P.E.
      Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth.
      ), and MAG (
      • Domeniconi M.
      • Zampieri N.
      • Spencer T.
      • Hilaire M.
      • Mellado W.
      • Chao M.V.
      • Filbin M.T.
      MAG induces regulated intramembrane proteolysis of the p75 neurotrophin receptor to inhibit neurite outgrowth.
      ), bind to transmembrane receptors on the axon (
      • Wang K.C.
      • Kim J.A.
      • Sivasankaran R.
      • Segal R.
      • He Z.
      P75 interacts with the Nogo receptor as a coreceptor for Nogo, MAG, and OMgp.
      ,
      • Domeniconi M.
      • Cao Z.
      • Spencer T.
      • Sivasankaran R.
      • Wang K.
      • Nikulina E.
      • Kimura N.
      • Cai H.
      • Deng K.
      • Gao Y.
      • He Z.
      • Filbin M.
      Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth.
      ,
      • Wong S.T.
      • Henley J.R.
      • Kanning K.C.
      • Huang K.H.
      • Bothwell M.
      • Poo M.M.
      A p75(NTR) and Nogo receptor complex mediates repulsive signaling by myelin-associated glycoprotein.
      ), which modulates axon growth. We have also identified netrin-1 as a novel Myelin-associated inhibitor in the spinal cord (
      • Low K.
      • Culbertson M.
      • Bradke F.
      • Tessier-Lavigne M.
      • Tuszynski M.H.
      Netrin-1 is a novel myelin-associated inhibitor to axon growth.
      ). Though experimental deletions of these myelin-associated inhibitors or their receptors have thus far shown a limited effect on axonal regeneration (
      • Geoffroy C.G.
      • Zheng B.
      Myelin-associated inhibitors in axonal growth after CNS injury.
      ), it is clear that OMgp and Nogo modulate axon sprouting following SCI (
      • Lee J.K.
      • Geoffroy C.G.
      • Chan A.F.
      • Tolentino K.E.
      • Crawford M.J.
      • Leal M.A.
      • Kang B.
      • Zheng B.
      Assessing spinal axon regeneration and sprouting in Nogo-, MAG-, and OMgp-deficient mice.
      ).

      Neutralizing the Inhibitory ECM

      Following injury to the CNS, the lesion site becomes surrounded by reactive scar tissue, and axons interacting with this glial scar form dystrophic end bulbs and fail to regenerate; this process is reviewed in detail elsewhere (
      • Busch S.A.
      • Silver J.
      The role of extracellular matrix in CNS regeneration.
      ,
      • Fitch M.T.
      • Silver J.
      CNS injury, glial scars, and inflammation: inhibitory extracellular matrices and regeneration failure.
      ,
      • Sharma K.
      • Selzer M.E.
      • Li S.
      Scar-mediated inhibition and CSPG receptors in the CNS.
      ,
      • Cregg J.M.
      • DePaul M.A.
      • Filous A.R.
      • Lang B.T.
      • Tran A.
      • Silver J.
      Functional regeneration beyond the glial scar.
      ). Chondroitin sulfate proteoglycans (CSPGs), a class of proteins with sulfated glycosaminoglycan (CS-GAG) moieties, are deposited by macrophages, microglia, and reactive astroglia, and encompass a major component of this inhibitory environment from very early to chronic stages after injury (
      • Bradbury E.J.
      • Moon L.D.
      • Popat R.J.
      • King V.R.
      • Bennett G.S.
      • Patel P.N.
      • Fawcett J.W.
      • McMahon S.B.
      Chondroitinase ABC promotes functional recovery after spinal cord injury.
      ). Though the inhibitory nature of these proteins has long been appreciated, the axonal receptors that bind them have only recently begun to be identified. One of these, receptor protein tyrosine phosphatase sigma (PTPσ), has been successfully targeted in experimental studies to promote axon regeneration in the optic nerve (
      • Sapieha P.S.
      • Duplan L.
      • Uetani N.
      • Joly S.
      • Tremblay M.L.
      • Kennedy T.E.
      • Di Polo A.
      Receptor protein tyrosine phosphatase sigma inhibits axon regrowth in the adult injured CNS.
      ) and the spinal cord (
      • Shen Y.
      • Tenney A.P.
      • Busch S.A.
      • Horn K.P.
      • Cuascut F.X.
      • Liu K.
      • He Z.
      • Silver J.
      • Flanagan J.G.
      PTPsigma is a receptor for chondroitin sulfate proteoglycan, an inhibitor of neural regeneration.
      ,
      • Lang B.T.
      • Cregg J.M.
      • DePaul M.A.
      • Tran A.P.
      • Xu K.
      • Dyck S.M.
      • Madalena K.M.
      • Brown B.P.
      • Weng Y.L.
      • Li S.
      • Karimi-Abdolrezaee S.
      • Busch S.A.
      • Shen Y.
      • Silver J.
      Modulation of the proteoglycan receptor PTPsigma promotes recovery after spinal cord injury.
      ). Notably, Silver and colleagues successfully promoted regeneration of serotonergic projections and enhanced functional recovery via systemic delivery of a mimetic peptide, presenting an attractive strategy for potential clinical translation. The Nogo receptors NgR1 and NgR3 were also identified as CSPG receptors by Giger's group, who showed significant regeneration of injured optic nerve axons in NgR1−/− and NgR3−/− animals (
      • Dickendesher T.L.
      • Baldwin K.T.
      • Mironova Y.A.
      • Koriyama Y.
      • Raiker S.J.
      • Askew K.L.
      • Wood A.
      • Geoffroy C.G.
      • Zheng B.
      • Liepmann C.D.
      • Katagiri Y.
      • Benowitz L.I.
      • Geller H.M.
      • Giger R.J.
      NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans.
      ).
      Aside from inhibiting the activity of CSPG receptors, others have utilized the enzyme chondroitinase ABC (ChABC) to degrade CS-GAG moieties from CSPGs, rendering the scar environment less inhibitory to axon growth (
      • Bradbury E.J.
      • Carter L.M.
      Manipulating the glial scar: chondroitinase ABC as a therapy for spinal cord injury.
      ). These efforts have led to several reports of improved axon growth and, in some cases, functional gains in models of partial rodent spinal cord injury (
      • Bradbury E.J.
      • Moon L.D.
      • Popat R.J.
      • King V.R.
      • Bennett G.S.
      • Patel P.N.
      • Fawcett J.W.
      • McMahon S.B.
      Chondroitinase ABC promotes functional recovery after spinal cord injury.
      ,
      • Starkey M.L.
      • Bartus K.
      • Barritt A.W.
      • Bradbury E.J.
      Chondroitinase ABC promotes compensatory sprouting of the intact corticospinal tract and recovery of forelimb function following unilateral pyramidotomy in adult mice.
      ,
      • Barritt A.W.
      • Davies M.
      • Marchand F.
      • Hartley R.
      • Grist J.
      • Yip P.
      • McMahon S.B.
      • Bradbury E.J.
      Chondroitinase ABC promotes sprouting of intact and injured spinal systems after spinal cord injury.
      ,
      • Cafferty W.B.
      • Bradbury E.J.
      • Lidierth M.
      • Jones M.
      • Duffy P.J.
      • Pezet S.
      • McMahon S.B.
      Chondroitinase ABC-mediated plasticity of spinal sensory function.
      ,
      • Zhao R.R.
      • Muir E.M.
      • Alves J.N.
      • Rickman H.
      • Allan A.Y.
      • Kwok J.C.
      • Roet K.C.
      • Verhaagen J.
      • Schneider B.L.
      • Bensadoun J.C.
      • Ahmed S.G.
      • Yanez-Munoz R.J.
      • Keynes R.J.
      • Fawcett J.W.
      • Rogers J.H.
      Lentiviral vectors express chondroitinase ABC in cortical projections and promote sprouting of injured corticospinal axons.
      ).

      Providing Permissive Substrates for CNS Axon Regeneration

      Strategies to modulate the extrinsic environment to promote regeneration are not limited to the neutralization of inhibitory factors. Recent work from our group has shown the powerful potential of transplanting permissive neural stem cells (NSCs) into the lesioned spinal cord to promote host axonal regeneration (
      • Lu P.
      • Wang Y.
      • Graham L.
      • McHale K.
      • Gao M.
      • Wu D.
      • Brock J.
      • Blesch A.
      • Rosenzweig E.S.
      • Havton L.A.
      • Zheng B.
      • Conner J.M.
      • Marsala M.
      • Tuszynski M.H.
      Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.
      ,
      • Lu P.
      • Woodruff G.
      • Wang Y.
      • Graham L.
      • Hunt M.
      • Wu D.
      • Boehle E.
      • Ahmad R.
      • Poplawski G.
      • Brock J.
      • Goldstein L.S.
      • Tuszynski M.H.
      Long-distance axonal growth from human induced pluripotent stem cells after spinal cord injury.
      ). Following neural stem cell transplantation into sites of severe SCI, we observed that injured supraspinal host axons penetrated grafts for distances up to 2 mm, and formed synaptic connections with grafted neurons. Moreover, these grafts placed in sites of SCI attenuate the reactive glial scar surrounding the lesion (
      • Lu P.
      • Wang Y.
      • Graham L.
      • McHale K.
      • Gao M.
      • Wu D.
      • Brock J.
      • Blesch A.
      • Rosenzweig E.S.
      • Havton L.A.
      • Zheng B.
      • Conner J.M.
      • Marsala M.
      • Tuszynski M.H.
      Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.
      ). Though the molecular mechanisms supporting CNS axon regeneration into permissive neural stem cell grafts are just beginning to be explored, it is plausible that grafted cells provide a combination of trophic support for regeneration as well as permissive extracellular matrix and/or cell adhesion molecules that attract host axonal regeneration and synapse formation with graft-derived neurons. Ongoing work in our laboratory is focused on characterizing the molecular factors in these permissive cell grafts that support regeneration of host axons. These findings have opened the door to a promising new strategy to overcome the inhibitory extrinsic environment and provide new postsynaptic targets via the reconstitution of spinal cord tissue with neural stem cells.

      Stimulating Growth of the Injured Axon: Growth Factors and Diffusible Factors

      The success of peripheral nerve regeneration is attributed in part to the presence of neurotrophins secreted by Schwann cells in gradients that support regeneration throughout the peripheral nerve milieu (
      • Lu P.
      • Tuszynski M.H.
      Growth factors and combinatorial therapies for CNS regeneration.
      ,
      • Chen Z.L.
      • Yu W.M.
      • Strickland S.
      Peripheral regeneration.
      ). These proteins provide trophic support for peripheral neuron survival and axon growth; in contrast, trophic factors are not secreted in temporal and spatial gradients to enable regeneration in the adult injured CNS. We will briefly summarize studies utilizing exogenous growth factor delivery to support regeneration of injured CNS populations.

      Growth Factor Effects on Spinal Cord Axon Regeneration

      Spinal cord axons can be induced to regenerate by the provision of appropriate gradients of neurotrophins delivered exogenously either by genetically modified growth factor-secreting cell grafts (
      • Johnson Jr., E.M.
      • Tuszynski M.H.
      Neurotrophic Factors.
      ). Early work from our group showed that NGF-expressing fibroblasts grafted into sites of acute and chronic SCI support extensive growth of central supraspinal and sensory axons, establishing that adult CNS axons retain the ability to regenerate if appropriate gradients of growth factors are provided (
      • Tuszynski M.H.
      • Peterson D.A.
      • Ray J.
      • Baird A.
      • Nakahara Y.
      • Gage F.H.
      Fibroblasts genetically modified to produce nerve growth factor induce robust neuritic ingrowth after grafting to the spinal cord.
      ,
      • Tuszynski M.H.
      • Gabriel K.
      • Gage F.H.
      • Suhr S.
      • Meyer S.
      • Rosetti A.
      Nerve growth factor delivery by gene transfer induces differential outgrowth of sensory, motor, and noradrenergic neurites after adult spinal cord injury.
      ). Administration of NT-3 also promotes growth of injured sensory axons in vivo (
      • Bradbury E.J.
      • Khemani S.
      • Von R.
      • King
      • Priestley J.V.
      • McMahon S.B.
      NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord.
      ,
      • Ramer M.S.
      • Priestley J.V.
      • McMahon S.B.
      Functional regeneration of sensory axons into the adult spinal cord.
      ,
      • Lu P.
      • Jones L.L.
      • Snyder E.Y.
      • Tuszynski M.H.
      Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury.
      ,
      • Taylor L.
      • Jones L.
      • Tuszynski M.H.
      • Blesch A.
      Neurotrophin-3 gradients established by lentiviral gene delivery promote short-distance axonal bridging beyond cellular grafts in the injured spinal cord.
      ). Work by our group showed the ability of NT-3 gradients to enable guidance and reinnervation of appropriate brainstem targets by lesioned, ascending sensory axons (
      • Alto L.T.
      • Havton L.A.
      • Conner J.M.
      • Hollis 2nd, E.R.
      • Blesch A.
      • Tuszynski M.H.
      Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury.
      ). BDNF also promotes regeneration of multiple injured spinal cord projections, including rubrospinal (
      • Liu Y.
      • Kim D.
      • Himes B.T.
      • Chow S.Y.
      • Schallert T.
      • Murray M.
      • Tessler A.
      • Fischer I.
      Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function.
      ,
      • Kobayashi N.R.
      • Fan D.P.
      • Giehl K.M.
      • Bedard A.M.
      • Wiegand S.J.
      • Tetzlaff W.
      BDNF and NT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stimulate GAP-43 and Talpha1-tubulin mRNA expression, and promote axonal regeneration.
      ) and reticulospinal axons (
      • Jin Y.
      • Fischer I.
      • Tessler A.
      • Houle J.D.
      Transplants of fibroblasts genetically modified to express BDNF promote axonal regeneration from supraspinal neurons following chronic spinal cord injury.
      ). We observed growth of cerulospinal, serotonergic, CGRP+, and ChAT+ axons into BDNF-secreting MSC grafts, showing that BDNF exerts trophic effects on multiple axon populations (
      • Lu P.
      • Jones L.L.
      • Tuszynski M.H.
      BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury.
      ).

      Growth Factor effects on the Injured Corticospinal Projection

      Efforts to elicit growth of injured corticospinal axons using neurotrophic factors have been largely unimpressive. An early study by our group showed the failure of brain-derived neurotrophic factor (BDNF) to influence corticospinal axon growth even as BDNF supports survival of axotomized corticospinal neurons (
      • Lu P.
      • Blesch A.
      • Tuszynski M.H.
      Neurotrophism without neurotropism: BDNF promotes survival but not growth of lesioned corticospinal neurons.
      ); however, this may be because the BDNF receptor TrkB was not trafficked from the cell body into the axon. Indeed, viral-mediated TrkB overexpression in the corticospinal soma enhanced regeneration of a modest number of corticospinal axons into BDNF-expressing grafts placed in a subcortical lesion, but not in sites of SCI (
      • Hollis 2nd, E.R.
      • Jamshidi P.
      • Löw K.
      • Blesch A.
      • Tuszynski M.H.
      Induction of corticospinal regeneration by lentiviral trkB-induced Erk activation.
      ). Neurotrophin-3 (NT-3) also enhances corticospinal sprouting (
      • Schnell L.
      • Schneider R.
      • Kolbeck R.
      • Barde Y.A.
      • Schwab M.E.
      Neurotrophin-3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesion.
      ), resulting in partial functional recovery after SCI (
      • Grill R.
      • Murai K.
      • Blesch A.
      • Gage F.H.
      • Tuszynski M.H.
      Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury.
      ). In a primate spinal cord injury model, corticospinal axons also failed to penetrate grafts of BDNF/NT-3 secreting fibroblasts, although lesioned brainstem projections did regenerate in to the grafts (
      • Brock J.H.
      • Rosenzweig E.S.
      • Blesch A.
      • Moseanko R.
      • Havton L.A.
      • Edgerton V.R.
      • Tuszynski M.H.
      Local and remote growth factor effects after primate spinal cord injury.
      ). Together, these findings show that provision of BDNF or NT-3 is not a sufficient strategy to promote regeneration of corticospinal axons; rather, it is likely that more permissive substrates, such as neural stem cell transplant, in combination with intrinsic manipulations will be needed to achieve robust regeneration of this notoriously regeneration-deficient projection.

      The Role of Neurotrophins in the Injured Optic Nerve

      It has been well documented that trophic factors promote the survival of retinal ganglion cells (RGCs) following optic nerve injury (
      • Harvey A.R.
      • Ooi J.W.
      • Rodger J.
      Neurotrophic factors and the regeneration of adult retinal ganglion cell axons.
      ); however, the ability of growth factor treatment to support regeneration of retinal ganglion cell axons is less clear. Treatment with ciliary neurotrophic factor (CNTF) enhanced axonal regeneration after optic nerve injury into peripheral nerve grafts (
      • Cui Q.
      • Yip H.K.
      • Zhao R.C.
      • So K.F.
      • Harvey A.R.
      Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons.
      ). Viral delivery of CNTF to the RGC soma was also shown to increase survival of retinal ganglion cells after optic nerve injury, and to promote regeneration of injured axons for several millimeters (
      • Leaver S.G.
      • Cui Q.
      • Plant G.W.
      • Arulpragasam A.
      • Hisheh S.
      • Verhaagen J.
      • Harvey A.R.
      AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.
      ,
      • Leaver S.G.
      • Cui Q.
      • Bernard O.
      • Harvey A.R.
      Cooperative effects of bcl-2 and AAV-mediated expression of CNTF on retinal ganglion cell survival and axonal regeneration in adult transgenic mice.
      ).
      The multitude of intrinsic and extrinsic factors influencing axon regeneration does not act in isolation. It is almost impossible to consider the effect of an intrinsic manipulation of a CNS axon without regard to how that manipulation may affect the axon's response to factors in the environment. For example, delivery of a transcription factor known to promote axon regeneration likely does not simply switch the neuron into a growth state regardless of environmental cues. Rather, such factors most likely act in synchrony, modulating the axon's cadre of growth factors, proteins, myelin- and ECM-associated molecules. It is likely that combinatorial treatments encompassing multiple intrinsic and/or extrinsic manipulations will be needed in order to promote robust axonal regeneration of injured CNS neurons. Proteomics and its post-translational modifications have the potential to unveil how proteins interact with one another in response to injury, broadening our understanding of which signaling networks are at play. It can also reveal cellular communication, which will provide insight into what determines a suitable permissive substrate for growth.

      Extracellular Signals in the Environment

      Extracellular matrix molecules (ECM) provide a molecular scaffold in the extra-cellular environment. The predominant ECM molecules present in the CNS consist of tenascin-R and hyaluronic acid (
      • Galtrey C.M.
      • Kwok J.C.
      • Carulli D.
      • Rhodes K.E.
      • Fawcett J.W.
      Distribution and synthesis of extracellular matrix proteoglycans, hyaluronan, link proteins, and tenascin-R in the rat spinal cord.
      ,
      • Jager C.
      • Lendvai D.
      • Seeger G.
      • Bruckner G.
      • Matthews R.T.
      • Arendt T.
      • Alpar A.
      • Morawski M.
      Perineuronal and perisynaptic extracellular matrix in the human spinal cord.
      ). Attached to the core protein backbones of these ECM molecules are sugar side chains that generate distinct species of glycoproteins and proteoglycans. These species in turn interact with axons, providing either permissive or repulsive binding that can alternately support axon growth and guidance.
      One of the major classes of cell adhesion molecules that regulate axon growth during development and regeneration is the integrin receptor family. Integrin receptors located on the axon growth cone communicate with the ECM. Integrin mediated signals are generated through the nonreceptor tyrosine kinases, such as focal adhesion kinase (FAK) and Src. FAK activity is required for growth cone point contacts, which results in rapid neurite outgrowth (
      • Robles E.
      • Gomez T.M.
      Focal adhesion kinase signaling at sites of integrin-mediated adhesion controls axon pathfinding.
      ). The Rho family GTPases Rac1 and RhoA also regulates the maturation of point contacts downstream of integrins (
      • Rottner K.
      • Hall A.
      • Small J.V.
      Interplay between Rac and Rho in the control of substrate contact dynamics.
      ). Interestingly, CNS inhibitory molecules inactivate integrins, where forced activation can abolish inhibition (
      • Tan C.L.
      • Kwok J.C.
      • Patani R.
      • Ffrench-Constant C.
      • Chandran S.
      • Fawcett J.W.
      Integrin activation promotes axon growth on inhibitory chondroitin sulfate proteoglycans by enhancing integrin signaling.
      ). Some of the most refractory adult CNS neurons have shown an absence of localized integrins in their axons (
      • Eva R.
      • Fawcett J.
      Integrin signaling and traffic during axon growth and regeneration.
      ). Manipulation of integrins in vivo for promotion of axonal regeneration could therefore be a potential therapeutic strategy.

      Summary

      Substantial progress has been made in understanding the basic mechanisms underlying the refractory state of the injured spinal cord to regeneration. But further progress is needed to promote understanding of basic mechanisms that can drive CNS axon regeneration. Quantitative proteomics has the potential to provide a more complete understanding of protein networks that will be necessary to enhance axon regeneration.

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