Cell and Tissue Research

, Volume 349, Issue 1, pp 15–26

Downstream effector molecules in successful peripheral nerve regeneration

Authors

  • Smriti Patodia
    • Centre for Perinatal Brain Protection and RepairUniversity College London
    • Centre for Perinatal Brain Protection and RepairUniversity College London
Review

DOI: 10.1007/s00441-012-1416-6

Cite this article as:
Patodia, S. & Raivich, G. Cell Tissue Res (2012) 349: 15. doi:10.1007/s00441-012-1416-6

Abstract

The robust axon regeneration that occurs following peripheral nerve injury is driven by transcriptional activation of the regeneration program and by the expression of a wide range of downstream effector molecules from neuropeptides and neurotrophic factors to adhesion molecules and cytoskeletal adaptor proteins. These regeneration-associated effector molecules regulate the actin-tubulin machinery of growth-cones, integrate intracellular signalling and stimulatory and inhibitory signals from the local environment and translate them into axon elongation. In addition to the neuronally derived molecules, an important transcriptional component is found in locally activated Schwann cells and macrophages, which release a number of cytokines, growth factors and neurotrophins that support neuronal survival and axonal regeneration and that might provide directional guidance cues towards appropriate peripheral targets. This review aims to provide a comprehensive up-to-date account of the transcriptional regulation and functional role of these effector molecules and of the information that they can give us with regard to the organisation of the regeneration program.

Keywords

Nerve regenerationPeripheral nerve injuryDownstream effector moleculesAdhesion moleculesCytoskeletal adaptor proteins

Introduction

Injury to peripheral nerves sets in motion a targeted program of neurite outgrowth, culminating in the re-innervation of targets (skin, muscle, visceral organs, blood vessels) and recovery of motor, sensory and autonomic function. This functionally successful regeneration depends both on the activation of the intrinsic growth capacity of neurons and on the presence of a permissive environment with axon guidance cues (Chen et al. 2007). This is also clearly emphasised by comparison with the centrally projecting central and primary sensory neurons, where a combination of diminished intrinsic capacity for regeneration and heightened presence of inhibitory factors of their extracellular environment 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 the neuronal cell body (Raivich and Makwana 2007), which undergoes a “chromatolytic” reaction (Lieberman 1971). This includes the interruption of the normal flow of trophic signals (Raivich et al. 1991; Zigmond and Sun 1997) and the exposure of the transected axons to new signals in the extracellular environment (Sendtner et al. 1997), all of which results in the retrograde transport of de novo activated molecules to the cell body within 12-24 h following injury (Hanz et al. 2003; Ben-Yaakov et al. 2012). Disruption of the tight ionic concentration gradient through membrane leaks also causes the rapid elevation of Ca2+ and cAMP levels, transmitting successive injury-mediated action potentials, which, in turn, activate a cascade of signalling pathways, including the mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK], p38, c-Jun N-terminal kinase [JNK]), Janus kinases (JAK)/signal transducers and activators of transcription (STAT), Ras/Raf, phosphatidylinositol 3 kinase (PI3K)/AKT, abl, mammalian target of rapamycin (mTOR) and Mstb3 pathways (Chang et al. 2003; Lindwall and Kanje 2005; Chen et al. 2007; Michaelevski et al. 2010).

This activation of cytoplasmic signalling pathways is rapidly followed by the appearance and nuclear translocation 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-like factor), 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; Jankowski et al. 2009; Magoulas and Lopez-de Heredia 2010; Moore and Goldberg 2011; Ruff et al. 2012; Patodia and Raivich 2012). Transcription factors provide a vital link between injury-induced signals and downstream protein expression via gene regulation. They rapidly condition the injured nerve and, within 1–4 days after injury, the neuronal perikaryon produces a plethora of regeneration associated genes (RAGs), an umbrella term including the vast number of genes that are differentially regulated during nerve regeneration and that might be involved in cell-cell signalling, axonal growth and sprouting and the activation of the non-neuronal cellular milieu (Skene and Willard 1981; Verhaagen et al. 1986; Boeshore et al. 2004). A summary of signalling from early sensors to cytoplasmic mediators to transcription factors and the synthesis of effector molecules is shown in Fig. 1.
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Fig. 1

Cascade of cellular signalling in successful regeneration, from early sensors of axonal injury, to cytoplasmic signals, transcriptional activation and downstream effectors. Modified from Figure 4 in Raivich (2011)

Neuronal deletion of transcription factors has frequently shown reduced regenerative ability. For example, the removal of genes encoding c-Jun and STAT3 have been shown to strongly decrease the speed of axonal regeneration, persistently reduce target re-innervation, and delay functional recovery (Patodia et al. 2011; Ruff et al. 2012). A number of different downstream effector targets of these transcription factors are almost completely abolished, as seen in the case of neuronal c-jun deletion (Fig. 2). Interestingly, functional recovery tends to catch up after prolonged periods of regeneration, possibly because of rerouting and collateral sprouting, whereas anatomical reinnervation is still brought down; in cases of c-Jun and STAT3 deletion, the whisker hair pad, a major target of facial nerve regeneration, still only receives approximately a third of the normal number of axons at 3 months after nerve cut (Raivich et al. 2004; Patodia et al. 2011). Interestingly, the effects of STAT3 deletion are milder in the dorsal root ganglia (Bareyre et al. 2011; Ben-Yaakov et al. 2012), suggesting the presence of complementary pathways, alongside STAT3, in the peripheral sensory neurons.
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Fig. 2

Summary of selected downstream effector targets of neuronal c-Jun. Overview showing immunohistochemistry for the facial nucleus at 14 days after nerve cut in control mice (jun f/f; a, b, e, f, i, j, m, n, q, r) and in junΔS mutant (syn jun f7F; c, d, g, h, k, l, o, p, s, t) mice in which the floxed c-Jun gene was deleted with Cre under the control of the synapsin promoter. Columns 1, 3 Contralateral side (co). Columns 2, 4 Axotomised side (ax). After axotomy, jun f/f mice show a prominent increase in adhesion molecules CD44 (a-d) and β1 integrin subunit (e-h), in neuropeptides calcitonin gene-related peptide (CGRP; i-l) and galanin (Galn; m-p) and in transcription factor ATF3 (q-t); these changes are reduced or abolished in the junΔS mutants. Bar 0.2 mm. Reproduced from Figure 5 in Ruff et al. (2012)

Proregenerative effects have also been shown for p53 (Di Giovanni et al. 2006) and, to a lesser extent, for Sox11 and C/EBPδ (Jankowski et al. 2009; Magoulas and Lopez-de Heredia 2010). In the case of STAT3, strong pro-regenerative effects have also been observed when the upstream regulation of this transcription factor is modified. Inactivation of floxed neuronal SOCS3, an upstream inhibitor of STAT3, by using viral vector carrying cre recombinase elicits pronounced axonal regeneration in the central crushed optic nerve model (Sun et al. 2011). This effect can be blocked by the concurrent deletion of floxed STAT3. In a similar vein, deletion of gp130, the common neurokine receptor subunit that transduces the effects of interleukin-6 (IL6), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), etc. in the sympathetic neurons of the superior cervical ganglion abolishes up-regulation, phosphorylation and nuclear translocation of STAT3 after postgangionic axotomy, while also blocking most of the injury response (Hyatt Sachs et al. 2010).

Although N-terminal phosphorylation of c-Jun plays an important role in modifying transcriptional activity and elicits strong effects in stroke, ischaemia and excitotoxicty, its effects on regeneration are less clear. Unlike complete neuronal deletion, with a strikingly persistent defect in target reinnervation and neuronal cell death (Raivich et al. 2004), the deletion of the more upstream Jun N-terminal kinases only shows a moderate transient effect on functional recovery (Ruff et al. 2012). Likewise, the replacement of the serine 63 and 73 and the threonine 91 and 93 phosphoacceptor residues with alanines only reproduces some of the atrophy and delayed functional recovery (S. Patodia, A. Behrens and G. Raivich, unpublished; Ruff et al. 2012). However, upstream signals leading to c-Jun chemical modification are not confined to the N-terminus: glycogen-synthase-kinase (GSK) phosphorylation of threonine 239, activity that is inhibited by ERK and PI3K signals, enhances Jun ubiquitination and degradation (Morton et al. 2003; Wei et al. 2005) and thus, in the presence of ERK or PI3K activation, Jun would become more stable. Activating effects on Jun function have also been observed with the p300 acetylation of lysine 268 and 271 (Vries et al. 2001; Wang et al 2006). Either of these or an additional upstream signal-mediated modification might play a role in Jun regeneration signalling but this will need to be demonstrated in vivo by selective amino-acid replacements, similar to the exchange of the N-terminal serines and threonines.

Overall, because transcription factors are able to bind to promoters of many different target genes, they can switch on/off the expression of a diverse range of downstream effector molecules. An overview of the transcriptional regulation of the expression and function of downstream effector molecules in peripheral nerve regeneration is shown in Table 1. In this review, we will focus on the roles and regulatory mechanisms of such identified downstream effectors in successful peripheral nerve regeneration, specifically axonal adhesion molecules, chemoattractant signaling, cell surface-cytoskeletal adaptors and neuropeptides but also neurotrophins produced in the injured nerve. Here, recent advances in cre/loxP technology permit cell-type and/or time-specific genetic knockouts (Sauer 1998; Akira 2000) and have begun to provide useful insights into their upstream transcriptional regulation and downstream function in orchestrating complex axon growth and regenerative responses.
Table 1

Overview of the transcriptional regulation of expression and function of downstream effector molecules in successful peripheral nerve regeneration (STAT signal transducer and activator of transcription 3, ATF3 cyclic AMP-dependent transcription factor, N neuronal, SC Schwann cell, NMJ neuromuscular junction, increase)

Downstream effectors (DE)

Transcription factors driving DE

Expression after axotomy

Effects of DE deletion on nerve regeneration

References

Adhesion molecules

  α7β1 Integrin

c-jun (N), STAT3 (N)

Global α7 deletion strongly reduces regenerative 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 of transplanted central noradrenergic neurons

Nagy et al. 1998

Lin and Chan 2003

Errors in retinal axonal growth in development

Raivich et al. 2004

Patodia et al. 2011

However, no data regarding effects on regeneration, as yet

Ruff et al. 2012

  DINE (damage-induced   neuronal endopeptidase)

ATF3, c-jun, STAT3

Reduced sprouting at developing NMJ

Kiryu-Seo et al. 2008

However, no data regarding effects on regeneration, 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 after injury

Raivich et al. 2004

Holmes et al. 2005

Patodia et al. 2011

Ruff et al. 2012

  CGRP (calcitonin   gene-related peptide)

c-jun (N), STAT3 (N)

Reduced number of axons crossing from 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 from proximal to distal stump

Parrinello et al. 2010

Cytoskeletal adaptors

  CAP23 (cytoskeleton-  associated protein 23)

c-jun (N)

Reduced neurite outgrowth and target re-innervation

Anderson et al. 2006;

J. Verhaagen, Y. Mattson and G. Raivich, unpublished

  GAP43 (neuromodulin)

C/EBPβ (N)

Abnormal developmental path-finding

Strittmatter et al. 1995

However, no data regarding effects on regeneration, as yet

Nadeau et al. 2005

Neurotrophins

  BDNF (brain-derived   neurotrophic factor)

c-jun (SC)

No data regarding effects on regeneration, as yet

Meyer et al. 1992

  GDNF (glial-cell-derived   neurotrophic factor)

c-jun (SC)

Increased neuronal cell death

X. Fontana et al., in preparation

Reduced functional recovery and speed of regeneration

  Artemin

c-jun (SC)

Increased neuronal cell death

X. Fontana et al., in preparation

Reduced functional recovery and speed of regeneration

  LIF (leukemia   inhibitory factor)

c-jun (SC)

No data regarding effects on regeneration, as yet

Curtis et al. 1994

  NGF (nerve growth   factor)

c-fos (SC)

No data regarding effects on regeneration, as yet

Hengerer et al. 1990

Adhesion molecules

Adhesion molecules enable the interaction of the cell surface of the axonal growth cone with the adjacent bands of Büngner, which consist in denervated Schwann cells and with the inner lining of the neural tube basal membrane; both structures act as a scaffold for the growing axon (Grumet 1991; Shapiro et al. 2007). Signaling between adhesion molecules on axonal and Schwann cell surfaces is frequently bi-directional (Quintes et al. 2010; Fricker and Bennett 2011) but, in order to simplify this for the purpose of axonal regeneration, it is helpful to concentrate on axonal molecules that act as receptors for axonal guidance cues from Schwann cells and the associated extracellular matrix.

Regenerating neurons up-regulate a variety of adhesion molecules. These include integrins that can serve as receptors for 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 expression of CD44, a receptor for hyaluronic acid (Jones et al. 2000) and of 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 molecules involved in homophilic binding such as ninjurin (nerve injury-induced protein; Araki and Milbrandt 2000), gicerin/CD146, a heterophilic receptor for the neurite outgrowth factor (Taira et al. 1994) and adhesion molecule FLTR3, which can form complexes with receptors for fibroblast growth factor (FGF) and potentiate FGF signalling (Bottcher et al. 2004). On the functional side, administration of exogenous galectin-1 promotes neurite outgrowth, whereas its removal by antibody neutralisation or through gene deletion decreases the speed of neurite outgrowth (Horie and Kadoya 2000; McGraw et al. 2004). Removal of β2-microglobulin produces a moderate 20 % reduction in the ability of regenerating motor axons to cross the proximal to distal gap after nerve cut (Oliveira et al. 2004).

Finally, regenerating axons also contain an assortment of degradative enzymes including urokinase and plasminogen activator (PA), metalloproteinases (MMP) 2, 3 and 9 (Demestre et al. 2004; Shubayev and Myers 2004) and damage-induced neuronal endopeptidase (DINE; Kiryu-Seo et al. 2000). Activation of MMP2 and MMP9 is partially dependent on the presence of tissue PA (tPA) and/or urokinase PA (UPA) systems (Siconolfi and Seeds 2003) and the deletion of tPA and/or UPA interferes with functional recovery after peripheral nerve injury (Siconolfi and Seeds 2001). As with chondroitinase (Graham et al. 2007; Hattori et al. 2008; Udina et al. 2010), the administration of exogenous MMP2 has been shown to degrade chondroitin sulphate proteoglycans and to improve recovery and anatomical reinnervation following nerve transection; furthermore, in the presence of saturating concentrations of MMP2, the co-administration of chondrotinase ABC does not produce any additional improvement, suggesting that both enzymes attack the same target (Zuo et al. 1998a, 1998b). Interestingly, although regenerating sympathetic neurons show an increase in MMP2 protein activity, no comparative increase occurs in MMP2 mRNA (Leone et al. 2005). However, the axotomised dorsal root ganglion neurons show injury-caused down-regulation of TIMP2, an inhibitor of MMP2 (Huang et al. 2011) and a similar downward trend is seen in the facial motor nucleus (G. Raivich, M.R. Mason, J. Verhaagen, unpublished), suggesting a regeneration-induced disinhibition of MMP2.

So far, three from the above group of adhesion molecules up-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 vivo downstream targets of the regeneration-associated transcription factors mentioned in the above section. Deletion of neuronal c-Jun or STAT3 interferes with the up-regulation of CD44 and of the α7 and β1 integrin subunits (Raivich et al. 2004; Patodia et al. 2011; Ruff et al. 2012). Preliminary in vivo data show the same regulatory pattern for DINE (S. Kiryu-Seo, H. Kiyama, G. Raivich, unpublished); in vitro, DINE expression in neuronal cell cultures is strongly up-regulated by STAT3, in combination with an ATF3/c-Jun fusion hybrid (Kiryu-Seo et al. 2008). The data regarding the neuronal functions of DINE and CD44 come from in vitro, explantation and developmental studies. Thus, antibody inhibition of CD44 reduces neurite outgrowth of transplanted central noradrenergic neurons (Nagy et al. 1998) and creates multiple errors in retinal axonal growth trajectory through the optic chiasm (Lin and Chan 2003). In the case of DINE, the global deletion of its function following an insertion of loxP sites into its gene is associated with a massive reduction in terminal sprouting of motor axons at the neuromuscular junction (Nagata et al. 2010). Unfortunately, these floxed DINE animals coincidentally suffer from perinatal lethality, precluding the direct examination of DINE effect in the adult by using the Cre/lox system.

In the case of the α7β1 integrin, global deletion of the α7 subunit causes a strong reduction of approximately 40 % in the speed of adult motor axon regeneration in the facial nerve model, with a commensurate delay in the reinnervation of its main peripheral target, namely the whisker pad (Werner et al. 2000). Similarly, deletion of the α7 integrin subunit also abolishes the ex vivo conditioning effect; the explantation of axotomised wild-type sensory ganglia in vitro leads to brisk neurite outgrowth on laminin, whereas the outgrowth is weaker and delayed when using previously uninjured ganglia (Ekstrom et al. 2003).

Overall, the integrins are a large family of heterodimeric transmembrane glycoproteins composed of specifically paired α and β subunits. In addition to cell adhesion, migration and axonal outgrowth, integrin signalling is important for non-neuronal and neuronal survival (Previtali et al. 2001; Chen et al. 2007; Lemons and Condic 2008; Tucker and Mearow 2008). Although some α subunits can only dimerise with single β subunits (α1, α2, α3, α5, α7 and 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 partner multiple β subunits (Previtali et al. 2001). Many of the β1-pairing α subunits (α1, α4, α7, α9, etc.) have been shown to promote axonal regeneration in vitro and following forced expression in vivo (Toyota et al. 1990; Vogelezang et al. 2001; Snider et al. 2002; Andrews et al. 2009), raising the expectation that the complete removal of β1 will produce a more severe phenotype. However, preliminary results involving the neuron-specific deletion of β1 in the facial nerve model suggest that the phenotype is roughly on par with that observed for the global α7 deletion (S. Patodia, X. Santos and G. Raivich, unpublished). This coincides with the finding that α7 is the main neuronal α subunit that appears up-regulated following axotomy (Kloss et al. 1999; Werner et al. 2000; Vogelezang et al. 2001; Andrews et al. 2009).

Neuropeptides

Axon transection frequently causes increased expression for a variety of neuropeptides. For example, a pronounced increase of calcitonin gene-related peptide (CGRP), galanin and to a lesser extent pituitary adenylate cyclase activating peptide (PACAP) occurs in subpopulations of transected cranial and spinal motoneurons (Moore 1989; Raivich et al. 1995). Sensory dorsal root ganglion neurons show an up-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-regulated in every group of neurons: some, such as substance P or CGRP in the axotomised sensory dorsal root ganglia or NPY in the sympathetic neurons, actually show decreased levels (Dumoulin et al. 1991; Habecker et al. 2009), similar to the down-regulation of enzymes and transporter systems for adrenergic and cholinergic neurotransmitters in the sympathetic and motor neurons (Zigmond and Sun 1997; Kalla et al. 2001). Recent studies point to decreased expression of the Hand2 transcription factor causing the post-axotomy down-regulation for components of the adrenergic system (Pellegrino et al. 2011); similar pathways might thus be involved in the down-regulation of some neuropeptides in injured neurons.

Several of these up-regulated neuropeptides have also been recently identified as in vivo downstream targets of the regeneration-associated transcription factors. Deletion of neuronal c-Jun or STAT3 interferes with the up-regulation of CGRP and galanin in facial motoneurons (Raivich et al. 2004; Patodia et al. 2011; Ruff et al. 2012). In sympathetic neurons, targeted deletion of the core neurokine receptor gp130, which transduces signals for IL6, LIF, CNTF and other neurokines, show drastically lowered post-axotomy expression for VIP, galanin and PACAP but does not affect the up-regulation of cholecystokinin (Habecker et al. 2009). Deletion of gp130 also abolishes the nuclear appearance of phosphorylated STAT3, suggesting that this transcription factor is also involved in the normally occurring post-axotomy up-regulation of galanin and associated neuropeptides.

At the functional level, the deletion of galanin or its type 2 receptor expressed in neurons results in a 35 % reduction in rate of peripheral nerve regeneration after sciatic nerve crush (Holmes et al. 2000) and modulates pain transmission (Holmes et al. 2005). In the case of CGRP, the local inhibition of intra-axonal CGRP synthesis with short interfering RNA (siRNA) strongly reduces the number of axons growing in the conduit between proximal and distal nerve stumps (Toth et al. 2009). Similar inhibitory effects have also been observed by blocking the CGRP receptor expressed on neighbouring Schwann cells, via siRNA against the CGRP receptor activity modifying protein-1, suggesting that the locally expressed peptide is used to recruit Schwann cell cooperation for neurite outgrowth (Raivich et al. 1992; Toth et al. 2009). In the case of PACAP, homozygous deletion of the gene reduces initial neurite outgrowth in the first 24 h after injury, with a moderate retarding effect (1-2 days) on the reinnervation of the peripheral target (Armstrong et al. 2008). Reverse experiments, with local application of PACAP and galanin, have shown that either peptide will strongly augment the peripheral branching of regenerating axons, increasing the number of neurons regenerating into each of the identified rami of the facial nerve by up to five-fold (Suarez et al. 2006). However, these pro-sprouting effects are actually associated with an impaired outcome, underscoring the importance of misrouting as a major impediment to the resumption of coordinated functional activity.

Guidance signalling

Guidance signals (through cell surface contact or diffusible signals mediating attraction or repulsion) play an important role by binding to receptors on growth cone surfaces, triggering secondary signals and steering axon extensions in the correct direction (Ide 1996; Giger et al. 2010). Overall, many different families of guidance molecules have been found, including the semaphorins and their receptors; the neuropilins and plexins (Tessier-Lavigne and Goodman 1996; Huber et al. 2003), ephrins (Tessier-Lavigne 2002; Koeberle and Bähr 2004; Parrinello et al. 2010), slits (Koeberle and Bähr 2004) or inhibitory myelin components such as MAG (myelin-associated glycoprotein) can activate Rho via p75 neurotrophin receptor (p75NTR; Yamashita et al. 2002).

Functionally, the deletion of neuropilin-2 reduces the density of the light neurofilament-positive axons in the distal nerve and interferes with functional recovery (Lindholm et al. 2004; Bannerman et al. 2008). Axotomy induces the expression of Bex1, an intracellular adaptor molecule that interacts with p75NTR to reduce neurite outgrowth inhibition by myelin inhibitors (Lindholm et al. 2004). Global deletion of Bex1 reduces the number of regenerating axons crossing the sciatic nerve crush site by >50 % and impedes functional recovery (Khazaei et al. 2010). Interestingly, the deletion of p75 does not affect neuronal survival or the speed of axonal regeneration in the facial nerve model, although p75 null mice show an enhanced neuroinflammatory response compared with their wild-type littermates (Gschwendtner et al. 2003).

In most cases, the in vivo transcriptional regulation for these molecules in neurons has not been clearly delineated. However, some of the Schwann cell function in creating axonal guidance scaffolds is under the control of post-traumatically expressed transcription factor Sox-2, induced by fibroblast ephrin-B signalling. Acting on the Schwann cell EphB2-receptor, the fibroblast ligand induces Sox2 and, in a Sox2-dependent manner, N-cadherin required for sorting Schwann cells into cell cords that link proximal and distal nerve stump (Parrinello et al. 2010). Interference with this process through EphB2 deletion or blocking antibodies produces a significantly more disordered axonal outgrowth in the gap region between the proximal and distal part of the nerve.

Growth cone and cytoskeletal adaptors

Appearing at the tip of growing axons, the growth cones are specialised quasi-autonomous structures that are responsible for growth, path-finding and recognition of targets after nerve injury. In agreement with their navigatory function, they are heavily decorated with adhesion molecules and receptors for chemoattractive and repulsive signals, as discussed above. Inside the growth cone, cytoskeletal adaptors play a crucial role in mediating connections between the cell surface and cytoskeletal actin-microtubule core of the growing 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, namely GAP43/neuromodulin, myristoylated alanine-rich C kinase substrate (MARCKS) and cytoskeleton-associated protein 23 (CAP23; Skene and Willard 1981; Verhaagen et al. 1986; Frey et al. 2000; Bomze et al. 2001). GAP43 and CAP23 are among the most abundant proteins in axonal growth cones (Goslin and Banker 1990; Bomze et al. 2001). These proteins co-distribute with phosphoinositol-4, 5-diphosphate (PIP2) at the semi-crystalline plasmalemmal raft regions and modify raft-recruitment of signalling molecules such as src (Laux et al. 2000), bind to acidic phospholipids such as PIP2, calcium/calmodulin, protein kinase C and actin filaments in a mutually exclusive manner, alter actin cytoskeleton polymerisation, organisation and disassembly and translate receptor-mediated calcium fluxes into signals guiding growth cone activity (Skene 1990; Ide 1996; Laux et al. 2000; Henley and Poo 2004; Kulbatski et al. 2004). In vitro, the depletion of GAP43 does not impair nerve growth factor (NGF)-elicited neurite outgrowth but leads to poorer adhesion, unstable lamellar extensions devoid of local F-actin, reduced branching and enhanced sensitivity to inhibitory stimuli (Aigner and Caroni 1995). In vivo, the global deletion of GAP43 interferes with normal developmental pathfinding (Strittmatter et al. 1995) and that of CAP23 with inactivity-induced sprouting at the neuromuscular synapse (Frey et al. 2000). Overexpression of GAP43 or CAP23 induces excessive neuromuscular sprouting (Caroni et al. 1997) and the combined overexpression of both adaptor components (GAP43 and CAP23) strongly enhances neurite growth from peripheral sensory neurons into the injured adult mouse spinal cord, with little regeneration being observed if only one of the two components is overexpressed (Bomze et al. 2001).

After axotomy, the transcriptional up-regulation of the mRNAs encoding GAP43 and CAP23 is biphasic and is down-regulated after target reconnection (Mason et al. 2002). In the case of GAP43, the early phase (24 h after axotomy) is c/EBPβ-independent but is followed by a c/EBPβ-dependent later phase, with an almost complete disappearance of increased GAP43 mRNA in c/EBPβ null mutants at day 3 (Nadeau et al. 2005). In the case of CAP23, up-regulation in the first 24 h is unaffected but the second phase of 4-14 days is reduced in the absence of neuronal c-Jun (J. Verhaagen, Y. Mattson and G. Raivich, unpublished observations). Since the long-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 CAP23 deletion (Caroni et al. 1997; Frey et al. 2000). Recent data obtained by using the neuron-specific deletion of the floxed CAP23 gene with the adenoassociated virus carrying Cre-recombinase (Anderson et al. 2006) or by selective neuronal expression of Cre under the synapsin promoter syn::cre (S. Patodia and P.N. Anderson, unpublished) reveal an approximately 50 % reduction in the reinnervation of the whisker pad, the main target of regenerating facial nerve fibers (Werner et al. 2000).

Neurotrophins, growth factors and cytokines

Peripheral nerve injury causes a massive increase in the synthesis and/or availability of a variety of neurotrophic and growth-promoting factors (Frostick et al. 1998; Raivich and 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 neurotrophic factor (GDNF; Naveilhan et al. 1997), insulin-like growth factors-1/2 (IGF1, IGF2; Kanje et al. 1989; Glazner et al. 1993), basic FGF (Jungnickel et al. 2004), vascular endothelial 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 et al. 1995; Hirota et al. 1996) and transforming growth factor-β1 (Lindholm et al. 1992).

On the functional side, exogenous application of IGF1 and IGF2 enhances the pinch-test-determined speed of axonal regeneration, whereas their antibody-mediated inhibition reduces 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 lacking NT4/5 display decreased ingrowth by regenerating axons of wild-type animals (English et al. 2005). The absence of the Schwann cell CNTF in CNTF null mice also appears to delay the appearance of phosphorylated STAT3 and its nuclear translocation in neuronal cell bodies (Kirsch et al. 2003).

Studies of the transcriptional regulation of induced neurotrophin and growth factor expression in peripheral nerve have concentrated particularly on the role of c-Fos and c-Jun. Sciatic nerve lesions cause a rapid local increase in c-fos and c-jun mRNA followed, within hours, by a peritrauma increase in NGF mRNA (Hengerer et al. 1990), with endoneurial fibroblasts forming a primary site of NGF synthesis (Heumann et al. 1987; Lindholm et al. 1988). That these two sets of changes, namely the up-regulation of c-Fos and the later expression of NGF, are probably linked has been shown by an elegant set of cell culture experiments. Here, heavy-metal-induced overexpression of c-Fos in fibroblasts from transgenic mice with metallothionein-promoter-driven Fos results in a rapid up-regulation of c-Jun and NGF, with the c-Fos/Jun heterodimer binding to the AP1 site in the first intron of the NGF gene, an event that is critical for NGF mRNA transcription (Hengerer et al. 1990).

Recent studies have also underscored the importance of the c-Jun counterpart. Here, the Schwann-cell-specific deletion of floxed c-Jun by using p0 promoter driven Cre (p0::cre) produces a massive increase in facial motoneuron cell death, with a commensurate reduction in peripheral target reinnervation and in functional recovery (X. Fontana et al., in preparation). Follow-on analysis of neurotrophin and growth factor expression in the injured sciatic nerves of p0::cre jun flox/flox mice (abbreviated as p0:jun) has revealed a strong deficit in many of the normally up-regulated trophic factors, including GDNF, Artemin, BDNF and LIF. Interestingly, the administration of recombinant GDNF and Artemin to these p0:jun mice substantially reduces post-traumatic neuronal cell death and improves functional recovery, underscoring the importance of Schwann cell c-Jun in providing trophic support for injured and regenerating neurons.

Concluding remarks

The current summary provides an overview of the sequence of events starting from injury signals via transcription factors and the appearance of downstream effectors involved in driving axonal regeneration and functional recovery. Amongst these effectors, adhesion and guidance molecules, cytoskeletal adaptors, neuropeptides and trophic factors all appear to be tangibly involved. In many cases, the direct injury-associated transcriptional regulation is still unclear; for example, little is known about the factors involved in the down-regulation of the sensitivity to inhibitory and chemorepulsive stimuli in peripheral regeneration, even though evidence exists for the presence of such regeneration blockers. Nevertheless, recent advances obtained by using cell-type-specific deletion of transcription factors enable more and more pieces of the puzzle to be joined together, making visible broad lines of an identified chain of activities contributing to recovery and underscoring the role of these transcription factors as master-switches of the regeneration program.

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© Springer-Verlag 2012