REVIEW

Downstream effector molecules in successful peripheralnerve regeneration

Smriti Patodia & Gennadij Raivich

Received: 29 February 2012 /Accepted: 19 March 2012 /Published online: 15 May 2012# Springer-Verlag 2012

Abstract The robust axon regeneration that occurs follow-ing peripheral nerve injury is driven by transcriptional acti-vation of the regeneration program and by the expression ofa wide range of downstream effector molecules from neuro-peptides and neurotrophic factors to adhesion molecules andcytoskeletal adaptor proteins. These regeneration-associatedeffector molecules regulate the actin-tubulin machinery ofgrowth-cones, integrate intracellular signalling and stimula-tory and inhibitory signals from the local environment andtranslate them into axon elongation. In addition to the neuro-nally derived molecules, an important transcriptional com-ponent is found in locally activated Schwann cells andmacrophages, which release a number of cytokines, growthfactors and neurotrophins that support neuronal survival andaxonal regeneration and that might provide directional guid-ance cues towards appropriate peripheral targets. Thisreview aims to provide a comprehensive up-to-date accountof the transcriptional regulation and functional role of theseeffector molecules and of the information that they can giveus with regard to the organisation of the regenerationprogram.

Keywords Nerve regeneration . Peripheral nerve injury .

Downstream effector molecules . Adhesion molecules .

Cytoskeletal adaptor proteins

Introduction

Injury to peripheral nerves sets in motion a targeted programof neurite outgrowth, culminating in the re-innervation oftargets (skin, muscle, visceral organs, blood vessels) andrecovery of motor, sensory and autonomic function. Thisfunctionally successful regeneration depends both on theactivation of the intrinsic growth capacity of neurons andon the presence of a permissive environment with axonguidance cues (Chen et al. 2007). This is also clearlyemphasised by comparison with the centrally projectingcentral and primary sensory neurons, where a combinationof diminished intrinsic capacity for regeneration and height-ened presence of inhibitory factors of their extracellularenvironment results in poor regeneration (Buchli et al.2007; Bolsover et al. 2008; Gordon et al. 2009).

Chromatolysis and transcriptional changes

Axonal injury generates several major signalling cues to theneuronal cell body (Raivich and Makwana 2007), whichundergoes a “chromatolytic” reaction (Lieberman 1971).This includes the interruption of the normal flow of trophicsignals (Raivich et al. 1991; Zigmond and Sun 1997) andthe exposure of the transected axons to new signals in theextracellular environment (Sendtner et al. 1997), all ofwhich results in the retrograde transport of de novo activatedmolecules to the cell body within 12-24 h following injury(Hanz et al. 2003; Ben-Yaakov et al. 2012). Disruption ofthe tight ionic concentration gradient through membraneleaks also causes the rapid elevation of Ca2+ and cAMPlevels, transmitting successive injury-mediated action

S. Patodia :G. Raivich (*)Centre for Perinatal Brain Protection and Repair,University College London,Chenies Mews 86-96,London WC1E 6HX, UKe-mail: g.raivich@ucl.ac.uk

Cell Tissue Res (2012) 349:15–26DOI 10.1007/s00441-012-1416-6

potentials, which, in turn, activate a cascade of signallingpathways, including the mitogen-activated protein kinases(extracellular signal-regulated kinase [ERK], p38, c-JunN-terminal kinase [JNK]), Janus kinases (JAK)/signal trans-ducers and activators of transcription (STAT), Ras/Raf,phosphatidylinositol 3 kinase (PI3K)/AKT, abl, mammaliantarget of rapamycin (mTOR) and Mstb3 pathways (Chang etal. 2003; Lindwall and Kanje 2005; Chen et al. 2007;Michaelevski et al. 2010).

This activation of cytoplasmic signalling pathways israpidly followed by the appearance and nuclear transloca-tion of a host of transcription factors such as c-jun, jun D,ATF3 (cyclic AMP-dependent transcription factor), STAT3,P311, p53, CREB (cAMP response element-binding), NFAT(nuclear factor of activated T-cells), KLF (Krüppel-likefactor), Sox11 (sex-determining region Y box-containing 11)and C/EBP (CCAAT/enhancer binding protein) β, δ(Schwaiger et al. 2000; Mason et al. 2003; Raivich et al.2004; Nadeau et al. 2005; Di Giovanni et al. 2006; Jankowskiet al. 2009; Magoulas and Lopez-de Heredia 2010; Moore andGoldberg 2011; Ruff et al. 2012; Patodia and Raivich 2012).Transcription factors provide a vital link between injury-induced signals and downstream protein expression via generegulation. They rapidly condition the injured nerve and,within 1–4 days after injury, the neuronal perikaryon producesa plethora of regeneration associated genes (RAGs), anumbrella term including the vast number of genes that aredifferentially regulated during nerve regeneration and thatmight be involved in cell-cell signalling, axonal growth andsprouting and the activation of the non-neuronal cellularmilieu (Skene and Willard 1981; Verhaagen et al. 1986;Boeshore et al. 2004). A summary of signalling from earlysensors to cytoplasmic mediators to transcription factors andthe synthesis of effector molecules is shown in Fig. 1.

Neuronal deletion of transcription factors has frequentlyshown reduced regenerative ability. For example, the removalof genes encoding c-Jun and STAT3 have been shown tostrongly decrease the speed of axonal regeneration, persistentlyreduce target re-innervation, and delay functional recovery(Patodia et al. 2011; Ruff et al. 2012). A number of differentdownstream effector targets of these transcription factors arealmost completely abolished, as seen in the case of neuro-nal c-jun deletion (Fig. 2). Interestingly, functional recoverytends to catch up after prolonged periods of regeneration,possibly because of rerouting and collateral sprouting,whereas anatomical reinnervation is still brought down; incases of c-Jun and STAT3 deletion, the whisker hair pad, amajor target of facial nerve regeneration, still only receivesapproximately a third of the normal number of axons at3 months after nerve cut (Raivich et al. 2004; Patodia et al.2011). Interestingly, the effects of STAT3 deletion aremilder in the dorsal root ganglia (Bareyre et al. 2011;

Ben-Yaakov et al. 2012), suggesting the presence of com-plementary pathways, alongside STAT3, in the peripheralsensory neurons.

Proregenerative effects have also been shown for p53(Di Giovanni et al. 2006) and, to a lesser extent, forSox11 and C/EBPδ (Jankowski et al. 2009; Magoulasand Lopez-de Heredia 2010). In the case of STAT3,strong pro-regenerative effects have also been observedwhen the upstream regulation of this transcription factoris modified. Inactivation of floxed neuronal SOCS3, anupstream inhibitor of STAT3, by using viral vector car-rying cre recombinase elicits pronounced axonal regener-ation in the central crushed optic nerve model (Sun et al.2011). This effect can be blocked by the concurrentdeletion of floxed STAT3. In a similar vein, deletion ofgp130, the common neurokine receptor subunit thattransduces the effects of interleukin-6 (IL6), leukemiainhibitory factor (LIF), ciliary neurotrophic factor(CNTF), etc. in the sympathetic neurons of the superiorcervical ganglion abolishes up-regulation, phosphorylationand nuclear translocation of STAT3 after postgangionicaxotomy, while also blocking most of the injury response(Hyatt Sachs et al. 2010).

Although N-terminal phosphorylation of c-Jun plays animportant role in modifying transcriptional activity and elic-its strong effects in stroke, ischaemia and excitotoxicty, itseffects on regeneration are less clear. Unlike complete neu-ronal deletion, with a strikingly persistent defect in targetreinnervation and neuronal cell death (Raivich et al. 2004),the deletion of the more upstream Jun N-terminal kinasesonly shows a moderate transient effect on functional recov-ery (Ruff et al. 2012). Likewise, the replacement of theserine 63 and 73 and the threonine 91 and 93 phosphoac-ceptor residues with alanines only reproduces some of theatrophy and delayed functional recovery (S. Patodia, A.Behrens and G. Raivich, unpublished; Ruff et al. 2012).However, upstream signals leading to c-Jun chemical mod-ification are not confined to the N-terminus: glycogen-synthase-kinase (GSK) phosphorylation of threonine 239,activity that is inhibited by ERK and PI3K signals, enhancesJun ubiquitination and degradation (Morton et al. 2003; Weiet al. 2005) and thus, in the presence of ERK or PI3Kactivation, Jun would become more stable. Activatingeffects on Jun function have also been observed with thep300 acetylation of lysine 268 and 271 (Vries et al. 2001;Wang et al 2006). Either of these or an additional upstreamsignal-mediated modification might play a role in Junregeneration signalling but this will need to be demonstratedin vivo by selective amino-acid replacements, similar to theexchange of the N-terminal serines and threonines.

Overall, because transcription factors are able to bind topromoters of many different target genes, they can switch

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on/off the expression of a diverse range of downstreameffector molecules. An overview of the transcriptional reg-ulation of the expression and function of downstream effec-tor molecules in peripheral nerve regeneration is shown inTable 1. In this review, we will focus on the roles andregulatory mechanisms of such identified downstream effec-tors in successful peripheral nerve regeneration, specificallyaxonal adhesion molecules, chemoattractant signaling, cellsurface-cytoskeletal adaptors and neuropeptides but alsoneurotrophins produced in the injured nerve. Here, recentadvances in cre/loxP technology permit cell-type and/ortime-specific genetic knockouts (Sauer 1998; Akira 2000)and have begun to provide useful insights into theirupstream transcriptional regulation and downstream func-tion in orchestrating complex axon growth and regenerativeresponses.

Adhesion molecules

Adhesion molecules enable the interaction of the cell surfaceof the axonal growth conewith the adjacent bands of Büngner,which consist in denervated Schwann cells and with the innerlining of the neural tube basal membrane; both structures actas a scaffold for the growing axon (Grumet 1991; Shapiro et

al. 2007). Signaling between adhesion molecules on axonaland Schwann cell surfaces is frequently bi-directional(Quintes et al. 2010; Fricker and Bennett 2011) but, in orderto simplify this for the purpose of axonal regeneration, it ishelpful to concentrate on axonal molecules that act as recep-tors for axonal guidance cues from Schwann cells and theassociated extracellular matrix.

Regenerating neurons up-regulate a variety of adhesionmolecules. These include integrins that can serve as receptorsfor the matrix and cell surface molecules such as laminin,fibronectin and paxillin (Kloss et al. 1999; Werner et al.2000; Vogelezang et al. 2001, 2007; Ekstrom et al. 2003;Wallquist et al. 2004; Gardiner et al. 2005). The expressionof CD44, a receptor for hyaluronic acid (Jones et al. 2000) andof galectin-1, a lectin receptor for the galactoside side chains(Horie and Kadoya 2000; Akazawa et al. 2004), increases.Regenerating axons express cell multimodal surface mole-cules involved in homophilic binding such as ninjurin (nerveinjury-induced protein; Araki and Milbrandt 2000), gicerin/CD146, a heterophilic receptor for the neurite outgrowthfactor (Taira et al. 1994) and adhesion molecule FLTR3,which can form complexes with receptors for fibroblastgrowth factor (FGF) and potentiate FGF signalling (Bottcheret al. 2004). On the functional side, administration of exoge-nous galectin-1 promotes neurite outgrowth, whereas its

Fig. 1 Cascade of cellularsignalling in successfulregeneration, from early sensorsof axonal injury, to cytoplasmicsignals, transcriptionalactivation and downstreameffectors. Modified from Figure4 in Raivich (2011)

Cell Tissue Res (2012) 349:15–26 17

removal by antibody neutralisation or through gene deletiondecreases the speed of neurite outgrowth (Horie and Kadoya2000; McGraw et al. 2004). Removal of β2-microglobulinproduces a moderate 20 % reduction in the ability of regener-ating motor axons to cross the proximal to distal gap afternerve cut (Oliveira et al. 2004).

Finally, regenerating axons also contain an assortment ofdegradative enzymes including urokinase and plasminogenactivator (PA), metalloproteinases (MMP) 2, 3 and 9(Demestre et al. 2004; Shubayev and Myers 2004) anddamage-induced neuronal endopeptidase (DINE; Kiryu-Seoet al. 2000). Activation of MMP2 and MMP9 is partiallydependent on the presence of tissue PA (tPA) and/or urokinasePA (UPA) systems (Siconolfi and Seeds 2003) and the dele-tion of tPA and/or UPA interferes with functional recoveryafter peripheral nerve injury (Siconolfi and Seeds 2001). Aswith chondroitinase (Graham et al. 2007; Hattori et al. 2008;Udina et al. 2010), the administration of exogenous MMP2has been shown to degrade chondroitin sulphate proteogly-cans and to improve recovery and anatomical reinnervationfollowing nerve transection; furthermore, in the presence ofsaturating concentrations of MMP2, the co-administration of

chondrotinase ABC does not produce any additional improve-ment, suggesting that both enzymes attack the same target(Zuo et al. 1998a, 1998b). Interestingly, although regeneratingsympathetic neurons show an increase in MMP2 proteinactivity, no comparative increase occurs in MMP2 mRNA(Leone et al. 2005). However, the axotomised dorsal rootganglion neurons show injury-caused down-regulation ofTIMP2, an inhibitor of MMP2 (Huang et al. 2011) and asimilar downward trend is seen in the facial motor nucleus(G. Raivich, M.R. Mason, J. Verhaagen, unpublished), sug-gesting a regeneration-induced disinhibition of MMP2.

So far, three from the above group of adhesion moleculesup-regulated after injury, namely DINE (Kiryu-Seo et al.2008), CD44 (Jones et al. 2000) and the α7β1 integrin(Werner et al. 2000), have also been identified as in vivodownstream targets of the regeneration-associated transcrip-tion factors mentioned in the above section. Deletion ofneuronal c-Jun or STAT3 interferes with the up-regulationof CD44 and of the α7 and β1 integrin subunits (Raivich etal. 2004; Patodia et al. 2011; Ruff et al. 2012). Preliminaryin vivo data show the same regulatory pattern for DINE (S.Kiryu-Seo, H. Kiyama, G. Raivich, unpublished); in vitro,

Fig. 2 Summary of selecteddownstream effector targets ofneuronal c-Jun. Overviewshowing immunohistochemistryfor the facial nucleus at 14 daysafter nerve cut in control mice(jun f/f; a, b, e, f, i, j, m, n, q, r)and in junΔS mutant (syn junf7F; c, d, g, h, k, l, o, p, s, t) micein which the floxed c-Jun genewas deleted with Cre under thecontrol of the synapsin promoter.Columns 1, 3 Contralateral side(co). Columns 2, 4 Axotomisedside (ax). After axotomy, jun f/fmice show a prominent increasein adhesion molecules CD44(a-d) and β1 integrin subunit(e-h), in neuropeptides calcitoningene-related peptide (CGRP; i-l)and galanin (Galn; m-p) and intranscription factor ATF3 (q-t);these changes are reduced orabolished in the junΔS mutants.Bar 0.2 mm. Reproduced fromFigure 5 in Ruff et al. (2012)

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Table 1 Overview of the transcriptional regulation of expression andfunction of downstream effector molecules in successful peripheral nerveregeneration (STAT signal transducer and activator of transcription 3,

ATF3 cyclic AMP-dependent transcription factor, N neuronal, SCSchwann cell, NMJ neuromuscular junction, ↑ increase)

Downstreameffectors (DE)

Transcriptionfactors driving DE

Expressionafter axotomy

Effects of DE deletion on nerveregeneration

References

Adhesion molecules

α7β1 Integrin c-jun (N), STAT3(N)

↑ Global α7 deletion strongly reducesregenerative speed after nerve crush

Werner et al. 2000

Raivich et al. 2004

Similar effects with neuronal β1 deletion Patodia et al. 2011

S. Patodia, C. Santos and G.Raivich, unpublished

CD44 c-jun (N), STAT3(N)

↑ Reduced neurite outgrowth oftransplanted central noradrenergicneurons

Nagy et al. 1998

Lin and Chan 2003

Errors in retinal axonal growth indevelopment

Raivich et al. 2004

Patodia et al. 2011However, no data regarding effectson regeneration, as yet Ruff et al. 2012

DINE (damage-inducedneuronalendopeptidase)

ATF3, c-jun,STAT3

↑ Reduced sprouting at developing NMJ Kiryu-Seo et al. 2008

However, no data regarding effects onregeneration, as yet

Nagata et al. 2010

Neuropeptides

Galanin c-jun (N), STAT3(N)

↑ Reduced rate of regeneration Holmes et al. 2000

Modulation of pain transmission afterinjury

Raivich et al. 2004

Holmes et al. 2005

Patodia et al. 2011

Ruff et al. 2012

CGRP (calcitoningene-related peptide)

c-jun (N), STAT3(N)

↑ Reduced number of axons crossingfrom proximal to distal stump

Raivich et al. 2004

Toth et al. 2009

Patodia et al. 2011

Ruff et al. 2012

Guidance molecules

Ephrin-B Sox-2 (SC) ↑ Disordered axonal outgrowth fromproximal to distal stump

Parrinello et al. 2010

Cytoskeletal adaptors

CAP23 (cytoskeleton-associated protein 23)

c-jun (N) ↑ Reduced neurite outgrowth and targetre-innervation

Anderson et al. 2006;

J. Verhaagen, Y. Mattson andG. Raivich, unpublished

GAP43 (neuromodulin) C/EBPβ (N) ↑ Abnormal developmental path-finding Strittmatter et al. 1995

However, no data regarding effects onregeneration, as yet

Nadeau et al. 2005

Neurotrophins

BDNF (brain-derivedneurotrophic factor)

c-jun (SC) ↑ No data regarding effects onregeneration, as yet

Meyer et al. 1992

GDNF (glial-cell-derivedneurotrophic factor)

c-jun (SC) ↑ Increased neuronal cell death X. Fontana et al., in preparationReduced functional recovery and speedof regeneration

Artemin c-jun (SC) ↑ Increased neuronal cell death X. Fontana et al., in preparationReduced functional recovery and speedof regeneration

LIF (leukemiainhibitory factor)

c-jun (SC) ↑ No data regarding effects onregeneration, as yet

Curtis et al. 1994

NGF (nerve growthfactor)

c-fos (SC) ↑ No data regarding effects on regeneration,as yet

Hengerer et al. 1990

Cell Tissue Res (2012) 349:15–26 19

DINE expression in neuronal cell cultures is strongly up-regulated by STAT3, in combination with an ATF3/c-Junfusion hybrid (Kiryu-Seo et al. 2008). The data regardingthe neuronal functions of DINE and CD44 come from invitro, explantation and developmental studies. Thus, anti-body inhibition of CD44 reduces neurite outgrowth of trans-planted central noradrenergic neurons (Nagy et al. 1998)and creates multiple errors in retinal axonal growth trajec-tory through the optic chiasm (Lin and Chan 2003). In thecase of DINE, the global deletion of its function following aninsertion of loxP sites into its gene is associatedwith amassivereduction in terminal sprouting of motor axons at the neuro-muscular junction (Nagata et al. 2010). Unfortunately, thesefloxed DINE animals coincidentally suffer from perinatallethality, precluding the direct examination of DINE effect inthe adult by using the Cre/lox system.

In the case of the α7β1 integrin, global deletion of the α7subunit causes a strong reduction of approximately 40 % inthe speed of adult motor axon regeneration in the facialnerve model, with a commensurate delay in the reinnerva-tion of its main peripheral target, namely the whisker pad(Werner et al. 2000). Similarly, deletion of the α7 integrinsubunit also abolishes the ex vivo conditioning effect; theexplantation of axotomised wild-type sensory ganglia invitro leads to brisk neurite outgrowth on laminin, whereasthe outgrowth is weaker and delayed when using previouslyuninjured ganglia (Ekstrom et al. 2003).

Overall, the integrins are a large family of heterodimerictransmembrane glycoproteins composed of specificallypaired α and β subunits. In addition to cell adhesion,migration and axonal outgrowth, integrin signalling isimportant for non-neuronal and neuronal survival (Previtaliet al. 2001; Chen et al. 2007; Lemons and Condic 2008;Tucker and Mearow 2008). Although some α subunits canonly dimerise with single β subunits (α1, α2, α3, α5, α7and α8 only with β1; αL, αD, αM and αX only with β2;αIIb only with β3), others such as α4 (β1, β7), α6 (β1,β4), α9 (β1, β8) and αV (β1, β3, β5, β6, β8) can partnermultiple β subunits (Previtali et al. 2001). Many of the β1-pairing α subunits (α1, α4, α7, α9, etc.) have been shownto promote axonal regeneration in vitro and followingforced expression in vivo (Toyota et al. 1990; Vogelezanget al. 2001; Snider et al. 2002; Andrews et al. 2009), raisingthe expectation that the complete removal of β1 will pro-duce a more severe phenotype. However, preliminary resultsinvolving the neuron-specific deletion of β1 in the facialnerve model suggest that the phenotype is roughly on parwith that observed for the global α7 deletion (S. Patodia, X.Santos and G. Raivich, unpublished). This coincides withthe finding that α7 is the main neuronal α subunit thatappears up-regulated following axotomy (Kloss et al.1999; Werner et al. 2000; Vogelezang et al. 2001; Andrewset al. 2009).

Neuropeptides

Axon transection frequently causes increased expression fora variety of neuropeptides. For example, a pronouncedincrease of calcitonin gene-related peptide (CGRP), galaninand to a lesser extent pituitary adenylate cyclase activatingpeptide (PACAP) occurs in subpopulations of transectedcranial and spinal motoneurons (Moore 1989; Raivich etal. 1995). Sensory dorsal root ganglion neurons show anup-regulation of galanin, vasculointestinal peptide (VIP)and neuropeptide Y (NPY; Xu et al. 1990; Noguchi et al.1993); sympathetic neurons demonstrate an increase in VIP,galanin and substance P. Not every peptide is up-regulatedin every group of neurons: some, such as substance P orCGRP in the axotomised sensory dorsal root ganglia orNPY in the sympathetic neurons, actually show decreasedlevels (Dumoulin et al. 1991; Habecker et al. 2009), similarto the down-regulation of enzymes and transporter systemsfor adrenergic and cholinergic neurotransmitters in the sym-pathetic and motor neurons (Zigmond and Sun 1997; Kallaet al. 2001). Recent studies point to decreased expression ofthe Hand2 transcription factor causing the post-axotomydown-regulation for components of the adrenergic system(Pellegrino et al. 2011); similar pathways might thus beinvolved in the down-regulation of some neuropeptides ininjured neurons.

Several of these up-regulated neuropeptides have alsobeen recently identified as in vivo downstream targets ofthe regeneration-associated transcription factors. Deletion ofneuronal c-Jun or STAT3 interferes with the up-regulation ofCGRP and galanin in facial motoneurons (Raivich et al.2004; Patodia et al. 2011; Ruff et al. 2012). In sympatheticneurons, targeted deletion of the core neurokine receptorgp130, which transduces signals for IL6, LIF, CNTF andother neurokines, show drastically lowered post-axotomyexpression for VIP, galanin and PACAP but does not affectthe up-regulation of cholecystokinin (Habecker et al. 2009).Deletion of gp130 also abolishes the nuclear appearance ofphosphorylated STAT3, suggesting that this transcription factoris also involved in the normally occurring post-axotomyup-regulation of galanin and associated neuropeptides.

At the functional level, the deletion of galanin or its type2 receptor expressed in neurons results in a 35 % reductionin rate of peripheral nerve regeneration after sciatic nervecrush (Holmes et al. 2000) and modulates pain transmission(Holmes et al. 2005). In the case of CGRP, the local inhibi-tion of intra-axonal CGRP synthesis with short interferingRNA (siRNA) strongly reduces the number of axons grow-ing in the conduit between proximal and distal nerve stumps(Toth et al. 2009). Similar inhibitory effects have also beenobserved by blocking the CGRP receptor expressed onneighbouring Schwann cells, via siRNA against the CGRPreceptor activity modifying protein-1, suggesting that the

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locally expressed peptide is used to recruit Schwann cellcooperation for neurite outgrowth (Raivich et al. 1992; Tothet al. 2009). In the case of PACAP, homozygous deletion ofthe gene reduces initial neurite outgrowth in the first 24 hafter injury, with a moderate retarding effect (1-2 days) onthe reinnervation of the peripheral target (Armstrong et al.2008). Reverse experiments, with local application of PACAPand galanin, have shown that either peptide will stronglyaugment the peripheral branching of regenerating axons,increasing the number of neurons regenerating into each ofthe identified rami of the facial nerve by up to five-fold(Suarez et al. 2006). However, these pro-sprouting effectsare actually associated with an impaired outcome, under-scoring the importance of misrouting as a major impedi-ment to the resumption of coordinated functional activity.

Guidance signalling

Guidance signals (through cell surface contact or diffusiblesignals mediating attraction or repulsion) play an importantrole by binding to receptors on growth cone surfaces, trig-gering secondary signals and steering axon extensions in thecorrect direction (Ide 1996; Giger et al. 2010). Overall,many different families of guidance molecules have beenfound, including the semaphorins and their receptors; theneuropilins and plexins (Tessier-Lavigne and Goodman1996; Huber et al. 2003), ephrins (Tessier-Lavigne 2002;Koeberle and Bähr 2004; Parrinello et al. 2010), slits(Koeberle and Bähr 2004) or inhibitory myelin componentssuch as MAG (myelin-associated glycoprotein) can activateRho via p75 neurotrophin receptor (p75NTR; Yamashitaet al. 2002).

Functionally, the deletion of neuropilin-2 reduces thedensity of the light neurofilament-positive axons in the distalnerve and interferes with functional recovery (Lindholm etal. 2004; Bannerman et al. 2008). Axotomy induces theexpression of Bex1, an intracellular adaptor molecule thatinteracts with p75NTR to reduce neurite outgrowth inhibi-tion by myelin inhibitors (Lindholm et al. 2004). Globaldeletion of Bex1 reduces the number of regenerating axonscrossing the sciatic nerve crush site by >50 % and impedesfunctional recovery (Khazaei et al. 2010). Interestingly, thedeletion of p75 does not affect neuronal survival or the speedof axonal regeneration in the facial nerve model, although p75null mice show an enhanced neuroinflammatory responsecompared with their wild-type littermates (Gschwendtneret al. 2003).

In most cases, the in vivo transcriptional regulation forthese molecules in neurons has not been clearly delineated.However, some of the Schwann cell function in creatingaxonal guidance scaffolds is under the control of post-traumatically expressed transcription factor Sox-2, induced

by fibroblast ephrin-B signalling. Acting on the Schwanncell EphB2-receptor, the fibroblast ligand induces Sox2 and,in a Sox2-dependent manner, N-cadherin required for sort-ing Schwann cells into cell cords that link proximal anddistal nerve stump (Parrinello et al. 2010). Interference withthis process through EphB2 deletion or blocking antibodiesproduces a significantly more disordered axonal outgrowthin the gap region between the proximal and distal part ofthe nerve.

Growth cone and cytoskeletal adaptors

Appearing at the tip of growing axons, the growth cones arespecialised quasi-autonomous structures that are responsiblefor growth, path-finding and recognition of targets afternerve injury. In agreement with their navigatory function,they are heavily decorated with adhesion molecules andreceptors for chemoattractive and repulsive signals, as dis-cussed above. Inside the growth cone, cytoskeletal adaptorsplay a crucial role in mediating connections between the cellsurface and cytoskeletal actin-microtubule core of the grow-ing axons (Baas and Ahmad 2001; Ellezam et al. 2002;Zhang et al. 2003; Bouquet et al. 2004; Madura et al. 2004).

One important family of adaptor molecules is the “GMC”family, which includes “integral” membrane proteins, namelyGAP43/neuromodulin, myristoylated alanine-rich C kinasesubstrate (MARCKS) and cytoskeleton-associated protein 23(CAP23; Skene andWillard 1981; Verhaagen et al. 1986; Freyet al. 2000; Bomze et al. 2001). GAP43 and CAP23 areamong the most abundant proteins in axonal growth cones(Goslin and Banker 1990; Bomze et al. 2001). These proteinsco-distribute with phosphoinositol-4, 5-diphosphate (PIP2) atthe semi-crystalline plasmalemmal raft regions and modifyraft-recruitment of signalling molecules such as src (Laux etal. 2000), bind to acidic phospholipids such as PIP2, calcium/calmodulin, protein kinase C and actin filaments in a mutuallyexclusive manner, alter actin cytoskeleton polymerisation,organisation and disassembly and translate receptor-mediatedcalcium fluxes into signals guiding growth cone activity (Skene1990; Ide 1996; Laux et al. 2000; Henley and Poo 2004;Kulbatski et al. 2004). In vitro, the depletion of GAP43 doesnot impair nerve growth factor (NGF)-elicited neurite out-growth but leads to poorer adhesion, unstable lamellar exten-sions devoid of local F-actin, reduced branching and enhancedsensitivity to inhibitory stimuli (Aigner and Caroni 1995). Invivo, the global deletion of GAP43 interferes with normaldevelopmental pathfinding (Strittmatter et al. 1995) and thatof CAP23 with inactivity-induced sprouting at the neuromus-cular synapse (Frey et al. 2000). Overexpression of GAP43 orCAP23 induces excessive neuromuscular sprouting (Caroni etal. 1997) and the combined overexpression of both adaptorcomponents (GAP43 and CAP23) strongly enhances neurite

Cell Tissue Res (2012) 349:15–26 21

growth from peripheral sensory neurons into the injuredadult mouse spinal cord, with little regeneration being observedif only one of the two components is overexpressed (Bomzeet al. 2001).

After axotomy, the transcriptional up-regulation of themRNAs encoding GAP43 and CAP23 is biphasic and isdown-regulated after target reconnection (Mason et al. 2002).In the case of GAP43, the early phase (24 h after axotomy) isc/EBPβ-independent but is followed by a c/EBPβ-dependentlater phase, with an almost complete disappearance ofincreased GAP43 mRNA in c/EBPβ null mutants at day 3(Nadeau et al. 2005). In the case of CAP23, up-regulation in thefirst 24 h is unaffected but the second phase of 4-14 days isreduced in the absence of neuronal c-Jun (J. Verhaagen, Y.Mattson and G. Raivich, unpublished observations). Since thelong-term postnatal survival of GAP43 null mice is rare(Strittmatter et al. 1995; but see Donovan et al. 2002),studies in the adult have been limited to the CAP23deletion (Caroni et al. 1997; Frey et al. 2000). Recent dataobtained by using the neuron-specific deletion of the floxedCAP23 gene with the adenoassociated virus carrying Cre-recombinase (Anderson et al. 2006) or by selective neuro-nal expression of Cre under the synapsin promoter syn::cre(S. Patodia and P.N. Anderson, unpublished) reveal anapproximately 50 % reduction in the reinnervation of thewhisker pad, the main target of regenerating facial nervefibers (Werner et al. 2000).

Neurotrophins, growth factors and cytokines

Peripheral nerve injury causes a massive increase in thesynthesis and/or availability of a variety of neurotrophicand growth-promoting factors (Frostick et al. 1998; Raivichand Makwana 2007). These include neurotrophin-3 (NT3;Terenghi 1999) and NT4/5 (English et al. 2005), NGF(Heumann et al. 1987), brain-derived neurotrophic factor(BDNF; Meyer et al. 1992), glial-cell-derived neurotrophicfactor (GDNF; Naveilhan et al. 1997), insulin-like growthfactors-1/2 (IGF1, IGF2; Kanje et al. 1989; Glazner et al.1993), basic FGF (Jungnickel et al. 2004), vascular endo-thelial growth factor (Islamov et al. 2004), LIF (Curtis et al.1994; Haas et al. 1999), CNTF (Kirsch et al. 2003),interleukin-1 (IL1; Lindholm et al. 1988), IL6 (Murphy etal. 1995; Hirota et al. 1996) and transforming growth factor-β1 (Lindholm et al. 1992).

On the functional side, exogenous application of IGF1and IGF2 enhances the pinch-test-determined speed of axo-nal regeneration, whereas their antibody-mediated inhibitionreduces this speed (Kanje et al. 1989; Glazner et al. 1993);IGF1 is also partly responsible for peripheral conditioning(Kanje et al. 1991). Peripheral nerve grafts from mice lack-ing NT4/5 display decreased ingrowth by regenerating

axons of wild-type animals (English et al. 2005). Theabsence of the Schwann cell CNTF in CNTF null mice alsoappears to delay the appearance of phosphorylated STAT3and its nuclear translocation in neuronal cell bodies (Kirschet al. 2003).

Studies of the transcriptional regulation of induced neu-rotrophin and growth factor expression in peripheral nervehave concentrated particularly on the role of c-Fos andc-Jun. Sciatic nerve lesions cause a rapid local increase inc-fos and c-jun mRNA followed, within hours, by a peri-trauma increase in NGF mRNA (Hengerer et al. 1990), withendoneurial fibroblasts forming a primary site of NGF syn-thesis (Heumann et al. 1987; Lindholm et al. 1988). Thatthese two sets of changes, namely the up-regulation of c-Fosand the later expression of NGF, are probably linked hasbeen shown by an elegant set of cell culture experiments.Here, heavy-metal-induced overexpression of c-Fos infibroblasts from transgenic mice with metallothionein-promoter-driven Fos results in a rapid up-regulation ofc-Jun and NGF, with the c-Fos/Jun heterodimer binding tothe AP1 site in the first intron of the NGF gene, an eventthat is critical for NGF mRNA transcription (Hengereret al. 1990).

Recent studies have also underscored the importance ofthe c-Jun counterpart. Here, the Schwann-cell-specific dele-tion of floxed c-Jun by using p0 promoter driven Cre (p0::cre) produces a massive increase in facial motoneuron celldeath, with a commensurate reduction in peripheral targetreinnervation and in functional recovery (X. Fontana et al.,in preparation). Follow-on analysis of neurotrophin andgrowth factor expression in the injured sciatic nerves ofp0::cre jun flox/flox mice (abbreviated as p0:jun) hasrevealed a strong deficit in many of the normally up-regulated trophic factors, including GDNF, Artemin, BDNFand LIF. Interestingly, the administration of recombinantGDNF and Artemin to these p0:jun mice substantiallyreduces post-traumatic neuronal cell death and improvesfunctional recovery, underscoring the importance ofSchwann cell c-Jun in providing trophic support for injuredand regenerating neurons.

Concluding remarks

The current summary provides an overview of the sequenceof events starting from injury signals via transcription fac-tors and the appearance of downstream effectors involved indriving axonal regeneration and functional recovery.Amongst these effectors, adhesion and guidance molecules,cytoskeletal adaptors, neuropeptides and trophic factors allappear to be tangibly involved. In many cases, the directinjury-associated transcriptional regulation is still unclear;for example, little is known about the factors involved in the

22 Cell Tissue Res (2012) 349:15–26

down-regulation of the sensitivity to inhibitory and chemo-repulsive stimuli in peripheral regeneration, even thoughevidence exists for the presence of such regeneration blockers.Nevertheless, recent advances obtained by using cell-type-specific deletion of transcription factors enable more and morepieces of the puzzle to be joined together, making visiblebroad lines of an identified chain of activities contributing torecovery and underscoring the role of these transcriptionfactors as master-switches of the regeneration program.

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