2787Research Article

IntroductionFailure of axon regeneration in the adult central nervous system(CNS) is due to both a muted response of CNS neurons to axoninjury and a CNS environment that is hostile to axon growth.Myelin-based proteins, such as Nogo, MAG and OMgp,together with chondroitin sulfate proteoglycans (CSPGs) inthe glial scar, are two major categories of CNS inhibitorymolecules demonstrated to impede axon regeneration afterinjury (for a review, see David and Lacroix, 2003). Data fromseveral studies demonstrate that Nogo, MAG and OMpg blockaxon growth by binding to their common receptor complex thatincludes NgR, p75/TROY and Lingo (Liu, B. P. et al., 2002;Mandemakers and Barres, 2005; Mi et al., 2004; Park et al.,2005). Recent studies indicate that the inhibitory effects ofthese myelin-based molecules are mediated by Rho activationdownstream of PKC (Sivasankaran et al., 2004), p75-TROY(Mandemakers and Barres, 2005; Park et al., 2005; Yamashitaet al., 2002), and probably EGFR activation induced bycalcium influx (Koprivica et al., 2005). As a result, inhibitionof these signaling mediators is able to antagonize the effects ofmyelin-based inhibitors. By contrast, the mechanisms bywhich CSPGs block axon growth are less clear because noCSPG receptor has been identified. Early studies suggested thatCSPGs may impede axon growth through interfering with

extracellular matrix (ECM)-integrin interactions (McKeon etal., 1995; Smith-Thomas et al., 1994). Indeed, endogenousupregulation as well as overexpression of integrin has beenshown to promote axon growth on CSPG (Condic, 2001;Condic et al., 1999). However, recent studies suggest thatCSPG may inhibit axon growth by similar mechanisms to thoseof Nogo, MAG and OMgp through Rho activation (Monnier etal., 2003; Sivasankaran et al., 2004).

Axon growth requires both gene transcription for thesynthesis of raw materials and coordinated assembly ofcytoskeletal elements in the extending axon. CNS inhibitorymolecules are thought to act primarily at the growth cone toshut down axon assembly. Axon assembly is a highly regulatedprocess strongly influenced by extracellular factors during bothdevelopment and successful peripheral nervous system (PNS)regeneration (Kuruvilla et al., 2004; Patel et al., 2000; Werneret al., 2000). Two well-studied groups of extracellular factorsthat mediate axon assembly are neuronal growth factors, suchas neurotrophins, and adhesion proteins, which include ECMproteins and cell adhesion molecules (CAMs). Recent in vitrostudies of highly purified neurons in defined media indicatethat either neurotrophins or ECM proteins alone induce onlylimited axon growth from either PNS or CNS neurons duringdevelopment (Goldberg et al., 2002; Lentz et al., 1999; Liu,

Chondroitin sulfate proteoglycans (CSPGs) and myelin-based inhibitors are the most studied inhibitory moleculesin the adult central nervous system. Unlike myelin-basedinhibitors, few studies have reported ways to overcome theinhibitory effect of CSPGs. Here, by using regeneratingadult dorsal root ganglion (DRG) neurons, we show thatchondroitin sulfate proteoglycans inhibit axon assembly bya different mechanism from myelin-based inhibitors.Furthermore, we show that neither Rho inhibition norcAMP elevation rescues extracellular factor-induced axonassembly inhibited by CSPGs. Instead, our data suggestthat CSPGs block axon assembly by interfering withintegrin signaling. Surprisingly, we find that nerve growthfactor (NGF) promotes robust axon growth of regeneratingDRG neurons over CSPGs. We have found that, unlikenaive neurons that require simultaneous activation of

neurotrophin and integrin pathways for axon assembly,either neurotrophin or integrin signaling alone is sufficientto induce axon assembly of regenerating neurons. Thus,our results suggest that the ability of NGF to overcomeCSPG inhibition in regenerating neurons is probably dueto the ability of regenerating neurons to assemble axonsusing an integrin-independent pathway. Finally, our datashow that the GSK-3��-APC pathway, previously shown tomediate developing axon growth, is also necessary for axonregeneration.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/119/13/2787/DC1

Key words: Axon regeneration, CSPG, Neurotrophin, Integrin

Summary

Neurotrophins support regenerative axon assemblyover CSPGs by an ECM-integrin-independentmechanismFeng-Quan Zhou1,2, Mark Walzer1, Yao-Hong Wu1, Jiang Zhou1, Shoukat Dedhar3 and William D. Snider1,*1Neuroscience Center, 8109 Neuroscience Research Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA2Departments of Orthopedic Surgery and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA3BC Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada*Author for correspondence (e-mail: wsnider@med.unc.edu)

Accepted 11 April 2006Journal of Cell Science 119, 2787-2796 Published by The Company of Biologists 2006doi:10.1242/jcs.03016

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R. Y. et al., 2002). However, neurotrophins and ECM togetherinduce robust axon growth (Goldberg et al., 2002; Liu, R. Y.et al., 2002), suggesting that coordinated activation ofneurotrophin and ECM-integrin signaling is necessary forefficient and long-distance axon extension. CNS inhibitorymolecules could block axon assembly by interfering with eithersignaling cascades that drive axon assembly induced byextracellular factors, or the intracellular machinery thatassembles axons, or both. Most previous studies have focusedon how CNS inhibitors affect the intracellular axon assemblymachinery in the absence of extracellular axon-promotingfactors. Less attention has been paid to the interaction betweenCNS inhibitors and axon growth-promoting signaling activatedby extracellular factors.

In this study, we investigated the effects of Nogo andaggrecan on axon assembly of adult dorsal root ganglion(DRG) neurons promoted by either nerve growth factor (NGF)treatment or a pre-conditioning lesion (PCL) in the presence ofECM laminin. We have found that Nogo has no effect on eitherNGF or PCL-induced axon assembly when DRG neurons arecultured on laminin. By contrast, aggrecan significantly blocksboth NGF and PCL-induced axon assembly in the presence oflaminin. Surprisingly, we found that the addition of NGFovercomes the inhibitory effect of aggrecan and induces robustaxon assembly of PCL neurons over CSPGs. We alsodiscovered that, unlike naive neurons that require simultaneousactivation of both growth factor and integrin pathways for axonassembly, either growth factor or ECM-integrin signaling aloneis sufficient to induce axon assembly of PCL neurons.Furthermore, NGF mediates axon assembly of PCL neurons bya distinct ECM-integrin independent pathway, suggesting thatNGF can overcome the inhibitory effect of aggrecan by

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bypassing the requirement of ECM-integrin signaling for axongrowth in PCL neurons. Finally, we show that the previouslyidentified GSK-3�–APC pathway is a convergent point foraxon assembly of PCL neurons induced by both NGF andECM-integrin. Taken together, our data provide new insightinto the mechanisms of CNS inhibitory molecules, and thussuggest a new direction to achieve axon regeneration over ahostile CNS environment.

ResultsDifferent effects of Nogo and aggrecan on axonassembly from adult DRG neuronsMost studies of CNS inhibitory molecules have examined theireffects on developing neurons in the absence of extracellularsignals that promote axon assembly (e.g. on poly-D-lysine inthe absence of neurotrophic factors). Since extracellular axon-promoting factors are required for efficient axon growth, itis of considerable interest to study the effects of inhibitorymolecules on adult, especially regenerating neurons, in thepresence of defined signals promoting axon growth (e.g. NGF,ECM, etc.). We first tested how Nogo affects axon assemblyfrom adult naive DRG neurons cultured on laminin in thepresence of NGF. Our results showed that Nogo had noinhibitory effect on NGF-induced axon assembly from adultnaive DRG neurons plated on laminin (Fig. 1A). Additionally,regenerating adult DRG neurons primed with conditioninglesions and plated on laminin were also able to overcome theinhibitory effect of Nogo (Fig. 1A). These results are consistentwith earlier studies showing that signaling events activated bylaminin can abolish the inhibitory effect of CNS myelins(David et al., 1995), and a more recent study that lamininoverrides the inhibitory effect of MAG (Laforest et al., 2005).

Fig. 1. Aggrecan and Nogo affect axon assembly fromadult DRG neurons differently in the presence ofextracellular axon promoting factors. (A) When adultDRG neurons (both naive and PCL neurons) werecultured on laminin (LM), treatment with the CNSmyelin inhibitor Nogo had no effect on axonassembly. (B) NGF-induced axon assembly from adultnaive DRG neurons plated on laminin was markedlyinhibited by aggrecan. (C,D) Quantification of theinhibitory effect of aggrecan (Agg.) on axon assemblyfrom adult naive neurons (C) and PCL neurons (D).Values are means ± s.e.m. *P

2789Neurotrophins support regenerative axon assembly over CSPG

In significant contrast, aggrecan, a component of CSPGs,significantly abolished NGF-induced axon assembly of naiveneurons cultured in the presence of laminin (Fig. 1B,C).Immunostaining (supplementary material Fig. S1) showed thatNGF-induced ERK phosphorylation was not affected byaggrecan, suggesting that ECM-integrin signaling is probablythe major target of CSPGs (McKeon et al., 1995). Further, PCLneurons plated on a mixture of laminin and aggrecan alsoexhibited minimal axon growth (Fig. 1D). To determine whetherother CSPGs had similar growth inhibitory effects we platedPCL neurons on a commercially available mixture of CSPGs.Similar inhibitory effects were observed when a mixture ofCSPGs was used instead of aggrecan (data not shown). Thesedata are in agreement with previous results that regeneratingneurons triggered by cell dissociation (axotomy) cannotovercome the inhibitory effect of CSPGs of the glial scar (Davieset al., 1997). Taken together, our results show that in the presenceof signals promoting axon growth, myelin-based inhibitors andCSPGs act through distinctly different pathways to interfere withlocal signaling events that regulate axon assembly.

NGF but not Rho inactivation stimulates robust axonassembly from PCL neurons on CSPGIt is unclear how CSPGs block axon assembly, because noCSPG receptor has been identified. Recent studies suggest thatCSPGs may inhibit basal axon assembly in the absence ofextracellular signals by activation of Rho, EGFR or PKC,similar to the mechanism of Nogo, MAG and OMgp(Koprivica et al., 2005; Monnier et al., 2003; Sivasankaran etal., 2004). In fact, treatment of neurons with pharmacologicalinhibitors of Rho, PKC or EGFR completely rescued the basalaxon assembly inhibited by CSPGs.

To examine whether similar mechanisms mediate theinhibitory effect of aggrecan on axon assembly of adult DRGneurons in the presence of axon-growth-promoting factors, wetreated adult naive neurons with Rho kinase inhibitor Y27632.We found that the Rho kinase inhibitor did not overcome theinhibitory effect of aggrecan (Fig. 2A). A similar result wasobserved when a dominant-negative RhoA construct wasexpressed in naive neurons (data not shown). Similarly,addition of Y27632 to PCL neurons plated on aggrecan did notrescue axon assembly (Fig. 2B). These results indicate thataxon assembly of adult neurons in the presence of extracellularfactors can be inhibited by CSPGs and that Rho inactivationalone is not sufficient to reverse this axon growth inhibition.Neurons treated with PKC or EGFR inhibitors also did notshow rescued axon assembly under similar experimentalsettings (data not shown). Moreover, previous studies havesuggested that elevation of cAMP levels may underlieperipheral injury-induced intrinsic changes of DRG neuronsthat allow axon assembly on myelin-based inhibitorymolecules (Neumann et al., 2002; Qiu et al., 2002). Wetherefore tested whether naive adult neurons in the presence ofelevated cAMP could overcome aggrecan and grow axons. Ourresults showed that treating cells with db-cAMP did notpromote axon assembly from naive adult neurons cultured onaggrecan (Fig. 2A). Taken together, these data further suggestthat CSPGs inhibit extracellular-signal-induced axon assemblyby different mechanisms to that of myelin-based inhibitors.

Surprisingly, when we added NGF to PCL neurons culturedon aggrecan, it significantly restored axon growth (Fig. 3A-E),

even though NGF was unable to initiate axon assembly in naiveneurons cultured on aggrecan (see Fig. 1B). The average axonlength and the percentage of neurons with axons are similar tothose of control neurons on laminin (Fig. 3D,E). Addition ofother neurotrophic factors, NT3 and GDNF, together with NGFfurther increased the percentage of neurons that extend axonsover aggrecan (data not shown). To determine whether theseeffects of NGF are through activation of the TrkA receptorfollowed by PI3K activation, neurons were treated with a TrkAreceptor antagonist K252a. Axon growth from neurons treatedwith NGF and K252a cultured on aggrecan was significantlydecreased. The phosphoinositide 3-kinase (PI3K) inhibitor,LY294002, had an even greater effect preventing NGF-inducedgrowth on aggrecan (Fig. 3D,E). These results clearlydemonstrated that adult regenerating neurons are able toovercome glial scar inhibitors when a neurotrophic pathway isactivated. Axon growth of PCL neurons on laminin has beenshown to be independent of transcription (Smith and Skene,1997). The NGF-induced axon growth of PCL neurons overaggrecan was also transcription independent, as thetranscription inhibitor DRB showed no inhibitory effect (Fig.3E). Finally, a �1 integrin function-blocking antibody showedno effect on NGF-induced axon growth of PCL neurons (Fig.3E), indicating that NGF induces axon growth over aggrecanby an integrin-independent pathway.

PCL allows adult DRG neurons to extend axons inresponse to either ECMs or NGF aloneIn order to determine how a conditioning lesion allowsneurotrophic factors to promote axon assembly of PCL neurons

Fig. 2. Neither acute elevation of cAMP nor inhibiting Rho kinaseactivity is sufficient to overcome the inhibitory effect of aggrecan onaxon assembly from adult DRG neurons. (A) Neither addition ofdbcAMP (1 mM) nor addition of Y27632 (25 �M) induced axongrowth from naive neurons cultured on aggrecan and laminin in thepresence of NGF. (B) Addition of the Rho kinase inhibitor Y27632had little effect on axon assembly from the PCL neurons cultured onaggrecan and laminin. *P

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but not naive neurons over CSPGs, we compared howextracellular signals mediate axon assembly from naive or PCLadult DRG neurons.

We took advantage of the fact that there is a 24-hour windowin which to study axon assembly from naive neurons. Longerculture times mimic axotomy effects and switch naive neuronsinto a regeneration mode similar to PCL neurons (Smith andSkene, 1997). Our results demonstrate that similar to the casefor embryonic DRG neurons (Liu, R. Y. et al., 2002), NGF andlaminin are both required as extracellular signals to mediateefficient axon assembly from naive adult DRG neurons(supplementary material Fig. S2). NGF or laminin alone wereunable to induce significant axon outgrowth in naive neuronscultured for 24 hours. Importantly, experiments with atranscription inhibitor verify that there is a requirement for NGFin axon growth that is independent of its effects on genetranscription.

We then examined how axon assembly of PCL neurons wasregulated. We found that no significant axon growth occurredin 24 hours when PCL neurons were cultured in the absenceof any extracellular factors (on poly-D-lysine in serum-freemedium). However, in significant contrast to naive neurons,when PCL neurons were plated in serum-free medium onlaminin, long-distance axon growth occurred from roughly45% of neurons (Fig. 4A-C). Presumably the other 55% of theDRG neurons that do not extend axons during the 24-hourperiod were either not axotomized by transection of the sciatic

nerve, or extend axons so slowly that significant growth doesnot occur over this time frame. Interestingly, other ECMs, suchas fibronection and tenascin C, were also able to induce axongrowth (data not shown). However, L1 CAM, a non-integrin-mediated adhesion protein could not support significant axongrowth from PCL neurons (supplementary material Fig. S3),suggesting a specific role of ECMs in mediating regenerativeaxon growth from adult DRG neurons. When we examinedPCL-induced axon assembly in the presence of the �1 integrinfunction-blocking antibody, it significantly impaired axonassembly induced by laminin, whereas the control IgG proteinhad little effect (Fig. 4D), indicating that ECMs induce axongrowth from PCL neurons through the activation of integrinsignaling. To rule out the possibility that neurotrophinssecreted by the neurons themselves mediate the axon assembly,the Trk inhibitor K252a was added to the medium. Resultsshowed that K252a had no effect on regenerative axon growth(Fig. 4C), even though the same concentration of K252asignificantly blocked NGF-mediated axon assembly. Together,these results indicate that axon growth of PCL neurons onlaminin is mediated by the activation of integrin signalingalone.

To determine whether other growth factor signaling pathwayscan mediate axon assembly from PCL neurons, we screenedvarious polypeptide growth factors in the absence of integrinactivation. When NGF was added to the medium, we saw asignificant increase in the percentage of PCL neurons that were

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Fig. 3. Activation of neurotrophin signaling is ableto overcome the inhibitory effect of aggrecan onaxon assembly from PCL neurons. (A,B) Laminin-induced axon assembly from PCL neurons (A) wasmarkedly inhibited by aggrecan (B). (C) Addition ofNGF overcame the inhibitory effect of aggrecan onaxon assembly from PCL neurons. Magnification,4�. (D,E) Quantification of the stimulatory effect ofNGF on axon assembly from PCL neurons culturedon aggrecan. Note that the rescue effect of NGFdepends on the activities of TrkA and PI3K,mediators of the NGF signaling, but is independentof integrin signaling and transcription (E).*P

2791Neurotrophins support regenerative axon assembly over CSPG

able to extend long axons on poly-D-lysine (Fig. 4A). Thepercentage of neurons with axons includes the neurons thatrespond to both axotomy and NGF. In addition to NGF, NT3 andGDNF were also capable of promoting axon assembly from PCLneurons (data not shown), whereas the addition of IGF or EGFthat also act through receptor tyrosine kinases did not supportaxon growth from these neurons (Fig. 4E). Additionally, thegrowth-promoting effect of NGF was TrkA dependent becauseK252a significantly attenuated NGF-induced axon assembly(Fig. 4E). When axon length was measured, we found that NGFcould induce axon assembly from the PCL neurons to the sameextent as that induced by laminin alone (Fig. 4F). Both integrinactivation and NGF stimulation have been shown to inactivateRhoA (Arthur and Burridge, 2001; Yamashita et al., 1999),however when regenerating neurons were treated with a Rhokinase inhibitor Y27632 alone, little axon assembly was inducedwhen compared with NGF or laminin treatments (Fig. 4F). Thisresult suggests that inactivation of RhoA by itself is not sufficientto mediate the axon-promoting effects of integrin or NGFstimulation.

Since ECM-integrin is a major target of CSPGs, together ourdata suggest that the ability of NGF to promote axon assemblyof PCL neurons over CSPGs is due to the fact that PCL allowsneurons to extend axons in an ECM-integrin independentmanner in response to NGF.

NGF and ECM-integrin signaling mediate axon assemblyof PCL neurons by divergent signaling pathwaysWe next determined whether growth factor or integrinactivation require the same downstream signaling mediators topromote axon assembly of PCL neurons. We first examined aseries of pharmacological inhibitors to determine their effectson axon assembly of PCL neurons. We found that NGF-mediated axon assembly of PCL neurons requires classicalPI3K and PKC activities (Fig. 5A). In significant contrast,ECM-integrin-mediated axon assembly from PCL neurons isindependent of PI3K, the major signaling mediator ofneurotrophins, but requires atypical PKC and Src activity (Fig.5B). To confirm the fact that integrin-mediated axon assemblyfrom PCL neurons does not depend on PI3K, we overexpresseda dominant-negative form of PI3K, previously shown to blockNGF-induced axon assembly from embryonic neurons(Markus et al., 2002b), in PCL neurons. Results showed thatdominant-negative PI3K had no effect on axon assembly fromPCL neurons induced by laminin (Fig. 5C). We also testedwhether Raf, another major signaling mediator of NGF, isrequired for ECM-integrin induced axon assembly of PCLneurons by expressing a dominant-negative form of C-Raf thatis sufficient to block NGF-induced axon assembly (Markus etal., 2002b). We found that Raf activation was not required forECM-integrin mediated axon assembly from PCL neurons

Fig. 4. Either laminin or NGF alone is sufficient tomediate axon assembly from PCL neurons. (A) PCLneurons were unable to extend axons when cultured onpoly-D-lysine (PDL) in the absence of extracellular cues.Neurons plated on laminin-coated coverslips extendedaxons robustly. (B) Neurons did not extend axons whencultured on poly-D-lysine-coated coverslips in serum-freemedium. Addition of NGF induced robust axon assemblyfrom the PCL neurons on poly-D-lysine. Magnification,4�. (C) Quantification of laminin-induced axon assemblyfrom PCL neurons. Please note that the addition oflaminin (S, 20 �g/ml) directly to the culture mediuminduced a similar extent of axon assembly to that ofcoated laminin (C). Laminin-induced axon growth wasindependent of the neurotrophin receptor Trk activity.*P

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(Fig. 5D). Finally, the requirement of Src for integrin-mediatedaxon growth was confirmed with a dominant-negative Srcconstruct (Fig. 5E). These results indicate that neurotrophinsand ECMs use distinct signaling mechanisms to mediate axonassembly of PCL neurons. Consistent with these functionaldata, western blot results showed that PI3K and ERK pathwayswere not activated in PCL neurons upon laminin stimulation(Fig. 6G). Together, these results suggest that, in PCL neurons,ECM-integrin mediates axon assembly by different pathwaysto NGF. As a result, CSPGs can specifically block ECM-integrin-induced axon assembly while having no effect onNGF-mediated axon assembly of PCL neurons.

Previously we showed that integrin-linked kinase (ILK)mediates NGF-induced axon assembly from embryonic DRGneurons downstream of PI3K (Zhou et al., 2004). We thereforeexamined whether ILK activity was also required forregenerative axon growth from adult PCL neurons. Applicationof a specific ILK kinase inhibitor KP74728 significantly blockedboth NGF and laminin-induced axon assembly from PCLneurons (Fig. 5A,B), indicating that ILK is a convergingsignaling mediator in these neurons. Consistent with animportant role of ILK, the protein level of ILK was significantlyincreased in DRGs after peripheral axotomy (data not shown).

We have shown previously that NGF can mediate axonassembly by the regulation of localized inactivation of GSK-3� and the interaction of APC with microtubules downstreamof ILK and PI3K. Similar localization of inactivated GSK-3�and APC was observed at the distal tips of regenerating axons(Zhou et al., 2004), suggesting that the GSK-3�–APC pathwaymay also play an important role in regulating regenerative axonassembly in PCL neurons. To investigate whether inactivationof GSK-3� and the subsequent APC-microtubule interactions

are necessary for axon assembly of PCL neurons plated onlaminin, we expressed constructs that interfere with thispathway to determine how they affect laminin-induced axonassembly of PCL neurons. Results showed that expression ofa control EGFP construct had little effect on axon assembly(Fig. 6A). By contrast, overexpression of a mutant GSK-3�,GSK-3�(S9A), which cannot be phosphorylated at Ser9,significantly blocked regenerative axon growth induced bylaminin (Fig. 6B,E). This result indicates that inactivation ofGSK-3� by phosphorylation is necessary for laminin to induceaxon assembly from the PCL neurons. Consistent with thisidea, plating PCL neurons on laminin increased GSK-3�phosphorylation, whereas laminin alone was not sufficient toinduce GSK-3� phosphorylation in naive and embryonic DRGneurons in the absence of NGF (Fig. 6G). Finally, we examinedwhether the interaction between APC and microtubules is alsorequired for integrin-mediated axon assembly. To interrupt thisinteraction, we overexpressed a mutant EB1 (C-EB1) that cansequester endogenous APC (Zhou et al., 2004). We found thatC-EB1 almost completely abolished integrin-induced axonassembly from regenerating neurons (Fig. 6C,F), whereas C-EB1 lacking the APC binding domain had little effect (Fig. 6D-F). Together, these results indicate that the GSK-3�–APCpathway is a converging point for both ECM-integrin- andNGF-induced regenerative axon growth of PCL neurons.

DiscussionIn this study, we examined how two CNS inhibitory molecules,Nogo and aggrecan, affect axon assembly of naive andregenerating adult DRG neurons in the presence ofextracellular factors that promote axon assembly. Under theseexperimental settings, we find that Nogo and aggrecan affect

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Fig. 5. ECM-integrin and neurotrophin signaling mediate axon assembly from PCL neurons by different signal pathways. (A) NGF-inducedaxon assembly from the PCL neurons was significantly inhibited by specific inhibitors of PI3K (LY294002, 20 �M), classical PKC (Go6976,200 nM), general PKC (BIS, 10 �M), Src (PP1, 10 �M) and ILK (KP74728, ILKi, 20 �M). *P

2793Neurotrophins support regenerative axon assembly over CSPG

axon assembly of adult DRG neurons quite differently. Ourdata suggest that aggrecan interferes with an ECM-integrin-mediated extracellular signal that promotes axon assembly(Fig. 7). Thus, CSPGs inhibit axon assembly mediated byECM-integrin signaling, which is necessary for axon assemblyof developing and naive adult DRG neurons. However, a pre-conditioning lesion induces intrinsic changes in neurons so thatPCL neurons are able to support ECM-integrin-independentaxon assembly. As a result, neurotrophins can bypass theECM-integrin signaling and promote axon assembly of PCLneurons in the presence of CSPGs (Fig. 7). Finally, we showthat both ECM-integrin and NGF promote axon assembly ofPCL neurons by a conserved GSK-3�–APC pathway.

CSPGs inhibit axon regeneration by a differentmechanism to myelin-based inhibitors in the presence ofaxon-growth-promoting factorsAxon extension is controlled by intracellular machinery thatassembles cytoskeletal elements and membrane componentsinto new axons. An intracellular signaling cascade activated byextracellular factors is necessary to power the machinery anddrive axon assembly, even though the basal activities ofintracellular signaling molecules (e.g. kinases, secondmessengers, etc.) can mediate limited axon extension in theabsence of extracellular factors. Recent studies suggest thatCSPGs inhibit basal axon growth in the absence ofextracellular signals by a similar mechanism to that of Nogo,MAG and OMgp (Monnier et al., 2003; Sivasankaran et al.,2004; Koprivica et al., 2005). In this study, we examined theeffects of Nogo and aggrecan on axon assembly of adult DRGneurons in the presence of defined extracellular axon growthfactors, including neurotrophins and ECMs. Our results clearlydemonstrate that Nogo and aggrecan have different effects onaxon assembly of adult DRG neurons.

First, when adult DRG neurons were cultured on laminin,

Nogo had no inhibitory effect on axon assembly induced byeither NGF or PCL. Since acute NGF treatment of DRG neuronswas unable to overcome the inhibitory effect of MAG whenneurons were cultured on poly-D-lysine (Cai et al., 1999), ourresult suggests that laminin acts to antagonize the effect of Nogo.Indeed, a previous observation has shown that laminin is able toovercome the inhibitory effect of CNS myelins (David et al.,1995). Moreover, a recent study has also demonstrated thatlaminin is able to override the inhibitory effect of MAG byregulation of Rac activity (Laforest et al., 2005).

By contrast, aggrecan significantly blocked axon assemblyunder the same experimental conditions. Furthermore, in NGF-induced axon growth, aggrecan did not affect ERK activation,the major signaling pathway downstream of NGF. This resultindicates that NGF signaling is not a major target of aggrecanand implies that ECM-integrin signaling is probably the majortarget of aggrecan (Carulli et al., 2005; Smith-Thomas et al.,1994). This idea is further supported by the result that aggrecanblocked laminin-induced axon assembly of PCL neurons, aslaminin-integrin activation is the only extracellular signal thatmediates axon assembly under such conditions. The inhibitoryeffect of aggrecan shown here is unlikely to be due to its abilityto directly interfere with the basic intracellular axon assemblymachinery through Rho activation (see Discussion below).Together, the different functional effects of Nogo and aggrecanon axon assembly shown in this study suggest that myelin-based inhibitors and CSPGs inhibit axon assembly by differentmechanisms. This difference between myelin-based inhibitorsand CSPGs only becomes apparent when axon assembly isinduced by defined extracellular axon growth factors,especially the ECMs. The effect of myelin-based inhibitors canbe overcome by ECM-integrin activation, whereas CSPGs actto block ECM-integrin signaling (Fig. 7).

Second, both inactivation of Rho and elevation of cAMPhave been used to overcome the inhibitory effect of myelin-

Fig. 6. The GSK-3�–APC pathway is required forintegrin-induced regenerative axon assembly fromPCL neurons. (A) Overexpression of EGFP had noeffect on integrin-mediated axon assembly.(B) Expression of the constitutively activatedGSK-3� mutant (yellow) significantly blockedaxon assembly. (C) Axon assembly was abolishedin neurons expressing C-EB1 (yellow), a mutantprotein that interferes interactions between APCand microtubule plus ends. (D) Axon assembly inneurons expressing C-EB1�APC was not affected.Arrows indicate transfected cells that express GFP.Axons of transfected neurons are yellow.Neurofilament staining (red) reveals axons ofuntransfected cells. Magnification, 4�.(E,F) Quantification of axon assembly defect inneurons expressing the active GSK-3� mutant orthe EB1 mutant (*P

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based inhibitors (Fournier et al., 2003; Neumann et al., 2002;Qiu et al., 2002). We showed that these two treatments did notsignificantly rescue axon assembly of adult neurons plated onaggrecan, further indicating that CSPGs block axon assemblyvia different mechanisms from myelin-based inhibitors. Ourinterpretation of these conflicting results is that the Rho-mediated inhibitory effect mainly serves as a ‘brake’ thatinhibits the intracellular axon assembly machinery (Fig. 7).Therefore, Rho inactivation alone is not sufficient to drive axonassembly in the absence of axon growth-promoting signals(e.g. NGF, laminin). We hypothesize that the effect of Rhoinactivation in promoting axon assembly on CSPGdemonstrated in previous studies from Monnier et al. (Monnieret al., 2003) and Sivasankaran et al. (Sivasankaran et al., 2004)are due to the fact that neurons used in those studies exhibitlimited degree of basal axon growth that is ECM-integrinindependent. Taken together, we believe our data show thatalthough CSPGs may impede basic axon assembly via Rhoactivation, they inhibit axon growth mainly through blockingintegrin signaling (Fig. 7).

Finally, it is worth mentioning that neurons primed with PCLhave been shown to overcome the inhibitory effect of myelin-based inhibitors (Neumann et al., 2002; Qiu et al., 2002).However, in our study, laminin-induced axon assembly of PCLneurons was still inhibited by aggrecan, further indicating thatCSPGs act differently from myelin-based inhibitors to blockaxon assembly. This result is also consistent with in vivoobservations that transplanted adult DRG neurons (mimicking

PCL) can support robust axon growth over spinal cord myelinbut stop at the boundary of CSPGs (Davies et al., 1997).

Either integrin or neurotrophin signaling can induce axonassembly in PCL neuronsIt is well established that peripheral axotomy induces intrinsicchanges in adult DRG neurons that lead to a robust axonregenerative response (for a review, see Snider et al., 2002).Although numerous studies have addressed changes in geneexpression induced by axotomy (Costigan et al., 2002; Tanabeet al., 2003), little attention has been paid to axotomy-inducedchanges in the signaling events that mediate axon assembly. Inthis study, we show that in striking contrast to the situation indeveloping and naive neurons, which require simultaneousactivation of integrin and growth factor signaling for axongrowth, integrin activation by ECM alone is sufficient to inducerobust axon assembly of PCL neurons. Since integrin signalingby itself is not sufficient to induce efficient axon assembly inthe absence of neurotrophic peptides in developing neurons(for a review, see Goldberg, 2003), our study suggests that adifferent integrin-mediated pathway may specifically controlregenerative axon assembly.

More importantly, we have demonstrated that robust axonassembly from PCL neurons is induced when NGF signalingis activated in the absence of ECM-induced integrin activation.Thus, when PCL neurons are plated on a neutral substrate,addition of NGF triggers axon growth similar in extent to thatwhich occurs when PCL neurons are plated on laminin. ThisECM-integrin-independent axon assembly is also uniqueto regenerating neurons. As a result of these changes,regenerating axons are presumably more adaptive to the localsurface molecules, which may be markedly different fromthose encountered in the developing nervous system. How pre-conditioning lesion induces such changes is unclear.Interestingly, a similar growth phenomenon has been observedwith cancer cells. Normal cells require both growth factorsignaling and attachment to the ECM (integrin activation) forgrowth. By contrast, cancer cells are able to grow in suspensionindependently of integrin activation by ECMs (for a review, seeGiancotti and Ruoslahti, 1999). The idea of parallels betweensignaling in regeneration and malignant transformation issupported by recent gene-profiling studies, in which a group ofcancer-related genes was identified to be upregulated after aperipheral nerve injury (Cameron et al., 2003).

Furthermore, our studies also demonstrate that, in PCLneurons, ECM-integrin and NGF mediate axon assembly bydifferent signaling pathways. ECM-integrin-induced axonassembly of PCL neurons does not depend on PI3K and Raf-ERK activities, which are major mediators of NGF signaling(for a review, see Markus et al., 2002a). However, by using aconstitutively active GSK-3� construct and overexpressing amutant APC binding protein, we demonstrate that the GSK-3�–APC pathway, previously shown to mediate NGF-inducedaxon assembly, is also crucial for integrin-mediatedregenerative axon assembly of PCL neurons. This resultindicates that both ECM-integrin and NGF need to convergeonto the GSK-3�–APC pathway to mediate axon assembly ofPCL neurons. Indeed, we show that laminin alone is sufficientto induce GSK-3� phosphorylation of PCL neurons, furthersupporting the idea that ECM-integrin and NGF mediate axonassembly independently of each other in PCL neurons. Given

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Fig. 7. A proposed model for regulation of axon assembly of adultDRG neurons. To promote efficient axon growth, extracellular axongrowth promoting factors are required. Adult naive neurons requirethe activation of both integrin and neurotrophin signaling pathwayssimultaneously for efficient axon growth. Both the CNS myelin-based inhibitor Nogo and glial scar-based inhibitor CSPGs caninhibit basal axon assembly by activation of Rho. However, CSPGsand myelin-based inhibitors act differently to affect axon growthwhen the extracellular axon growth promoting factors are present.CSPGs can interfere with ECM-integrin signaling, thus preventingextracellular factors from stimulating axon growth. On the contrary,activation of ECM-integrin pathway is able to antagonize theinhibitory effect of the myelin-based inhibitors. Peripheral nerve-injury-mediated conditioning lesion separates the neurotrophin andECM-integrin pathways, thus allowing either ECM-integrin signalingor neurotrophin signaling to mediate axon assembly independently.As a result, neurotrophins can promote robust axon assembly of PCLneurons over CSPGs by bypassing the requirement of integrinactivation for axon assembly.

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the importance of GSK-3�–APC pathway in mediating axonassembly of regenerating neurons, it will be interesting in thefuture to investigate how it is regulated downstream of integrinor neurotrophins in PCL neurons and whether it is the targetof different CNS inhibitors. However, these results do notnecessarily mean that the ILK–GSK-3� pathway is the onlypathway that mediates axon growth downstream of both NGFand laminin-integrin signaling.

Neurotrophins mediate ‘regenerative’ axon assembly onCSPGs by bypassing ECM-integrin signalingAlthough much effort has been devoted to finding ways topromote axon regeneration over inhibitory CNS molecules,few studies have successfully induced robust axon assemblyover CSPGs, the major components of the glial scar. The mainreason is due to limited knowledge of mechanisms thatunderlie the inhibitory effect of CSPGs. Although recentstudies suggest that CSPGs may use the same molecularmechanisms as myelin-based inhibitors, in this studytreatments shown to overcome the myelin-based inhibitorsfailed to rescue axon growth induced by extracellular axon-promoting factors. By contrast, we show here, for the first time,that neurotrophins are able to induce long axon growth of PCLneurons in the presence of CSPGs. The rescue effect of NGFwas significant when either axon length or percentage ofneurons with axons was measured. Since our results, togetherwith previous observations, suggest that CSPGs can inhibitaxon assembly by interfering with ECM-integrin signaling, oneway to overcome its inhibitory effect is to induce axonassembly by pathways independent of ECM-integrin signaling.Indeed, we find that in PCL neurons, either ECM-integrin orNGF can induce axon assembly independently of each otherby distinct signaling pathways. As a result, neurotrophins areable to bypass the ECM-integrin signaling to induce axonassembly over CSPGs. Therefore, approaches that directlyactivate integrin signaling may be a potential way to promoteCNS axon regeneration over glial scar.

Another way to overcome the inhibitory effect of CSPGsmay be to increase the expression of integrins and thus theirresponsiveness to ECMs (Condic, 2001; Condic et al., 1999).Interestingly, several integrins are upregulated after sciaticnerve injury in DRG neurons (Wallquist et al., 2004). However,our data that PCL neurons were able to grow long axons in theabsence of any ECMs (i.e. on poly-D-lysine) suggest thatincreases in responsiveness to ECMs may not explain theconditioning lesion effect. Instead, our results suggest that theactivation of signaling mediators localized downstream ofintegrin at the convergent point with growth factor signaling,such as Src, ILK and FAK-Pyk2 (Ivankovic-Dikic et al., 2000),might underlie the ability of NGF to promote axon assemblyof PCL neurons on poly-D-lysine or CSPGs.

We show here that GSK-3� inhibition is necessary for bothNGF and ECM-integrin signaling in PCL neurons to mediateaxon assembly, suggesting GSK-3� as a key regulator of axonregeneration. In addition, inhibition of GSK-3� has also beenimplicated in CNS regeneration by inducing multiple axonsfrom hippocampal neurons (Jiang et al., 2005). However,global application of GSK-3� inhibitors on adult DRGneurons was unable to promote axon assembly over CSPGs(unpublished data), indicating that GSK-3� inactivation aloneis not sufficient to overcome the inhibitory effect of CSPGs, or

a localized inactivation of GSK-3� is required (Zhou et al.,2004). Indeed, NGF-induced activation and inactivation ofGSK-3� simultaneously is necessary to ensure optimal axongrowth by regulation of different microtubule-binding proteins(for a review, see Zhou and Snider, 2005).

In conclusion, our data indicate that CSPGs and myelin-based inhibitors block axon regeneration by differentmechanisms in the presence of extracellular axon-growth-promoting factors. In addition to acting through the samepathway as Nogo by Rho activation, CSPGs may also interferewith extracellular axon growth promoting signaling activatedby ECM-integrin. After a peripheral conditioning lesion, adultDRG neurons change their intrinsic properties, thus allowingeither NGF or ECMs to induce axon assembly by distinctpathways. As a result, NGF is able to overcome the inhibitoryeffect of CSPGs by bypassing the ECM-integrin signaling inPCL neurons. Our results suggest a novel approach toovercome CSPG inhibitory influences, namely, the directactivation of signaling pathways downstream of integrin.

Materials and MethodsReagentsLY294001, Wortmannin, K252a, Y27632, BIS, Go6976 and PP1 were fromCalbiochem (San Diego, CA). NGF was obtained from Harlan Bioproducts(Indianapolis, IN). The specific ILK inhibitor KP-074728 is an analog of thepreviously reported KP-392 (Tan et al., 2004). Aggrecan, IGF, EGF and db-cAMPwere from Sigma (St Louis, MO). Transcription inhibitor DRB was from MPBiomedicals (Irvine, CA). Mouse anti-neurofilament antibody SMI-31 was fromSternberger/Meyer Immunochemicals (Jarrettsville, MD). Anti-�1 integrin (CD29,clone Ha2/5) was from BD Pharmingen (San Diego, CA). Phospho-GSK-3�,phospho-ERK1/2 and phospho-Akt (Ser473) were all from Cell SignalingTechnology (Beverly, MA). L1 cell adhesion molecule is from R&D Systems(Minneapolis, MN). All fluorescence-tagged secondary antibodies were fromMolecular Probes (Eugene, OR). Nogo66 peptide was a generous gift from StephenStrittmatter (Yale University, New Haven, CT).

Dominant-negative PI3K, dominant-negative C-Raf, GSK-3�(S9A), C-EB1 andC-EB1�APC constructs were generated as described previously (Markus et al.,2002b; Zhou et al., 2004). Dominant-negative Src was a generous gift from DavidShalloway (Cornell University, Ithaca, NY).

Cell culture and immunocytochemistryL4, L5 and L6 DRGs were dissected from 8- to 12-week-old adult naive or pre-conditioning lesioned (Liu and Snider, 2001) CF-1 mice and digested withcollagenase (1 mg/ml) for 90 minutes followed by trypsin-EDTA (0.05%) for 15minutes at 37°C. The DRGs were then washed three times with plating medium(MEM with L-glutamine and 1� penicillin/streptomycin) plus 5% fetal calf serumand dissociated with a 1 ml pipette tip in plating medium. Glass coverslips werecoated with poly-D-lysine (100 �g/ml), laminin (10 �g/ml) with or withoutAggrecan (52 �g/ml; Sigma) for 60 minutes before plating. DRGs were plated andcultured at 37°C in serum-free MEM containing N2 supplement. Embryonic DRGculture for western blot study was as described in (Zhou et al., 2004).

After 20-24 hours of culture, neurons were fixed in 4% paraformaldehyde for 20minutes, washed in PBS and blocked in blocking solution (2% BSA, 0.1% TritonX-100 and 0.1% sodium azide in PBS) for 60 minutes. Coverslips were incubatedwith primary monoclonal anti-neurofilament antibody SMI-31 (1:200) for 1 hour,washed and incubated with goat anti-mouse Alexa Flour 594-conjugated secondaryfor 1 hour, washed in distilled H2O and attached to slides with Mowiol antifademounting media.

Western blotDissociated adult PCL neurons or embryonic neurons were plated on culture dishescoated with poly-D-lysine or laminin in the absence of NGF. After culture for 3-4hours, neurons were lysed with lysis buffer and prepared for western blot assay. Forembryonic neurons, cells cultured on laminin were also treated with NGF for 30minutes before cell lysis.

Gene transfection and pharmacologyDRG neurons were transfected with various DNA constructs using theelectroporation technique from Amaxa (Cologne, Germany). The transfectionprocedure was according to the Amaxa protocols for mouse neurons. Briefly,dissociated neurons were spun down to remove the supernatant completely andresuspended in 100 �l specified Amaxa electroporation buffer with 10-20 �g

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plasmid DNA. Suspended cells were then transferred to a 2.0 mm cuvette andeletroporated with an Amaxa NucleofectorTM apparatus. After electroporation, cellswere immediately transferred to the desired volume of culture medium and platedonto coated coverslips. After neurons fully attached to the substrates (2-4 hours),the medium was changed to remove the remnant transfection buffer.

All growth factors and pharmacological reagents were added directly to theneuronal culture medium at indicated concentration at the time of plating. For anti-integrin antibody treatment, cells were incubated with anti-beta1 integrin (20 �g/ml)at 4°C for 30 minutes before plating. Cells were then fixed for analysis followingovernight culture. For each plasmid and pharmacological treatment, at least threeindependent experiments were conducted.

Image analysis and statisticsImages were taken with Spot imaging software and a CCD camera (DiagnosticInstruments) attached to a Nikon Eclipse microscope. A 4� objective (0.45 NA)was used to record neurons with axons. 10-12 images were acquired for eachcoverslip. All image analysis was done with IPLab software. To quantify thepercentages of neurons that grow axons, all cells of each experimental conditionwere recorded by the camera. The number of neurons with axons longer than twocell bodies was then counted. To measure axon length, the first 50-60 neurons withaxons in each condition were selected for measurement. The longest axon of eachneuron was traced manually and the length was then calculated.

All data were reported as mean ± s.e.m., and an unpaired Student’s t-test wasused to determine the significance of the data between groups. For multiple groupcomparison, a one-way ANOVA was used followed by a t-test post-hoc analysis.

We would like to thank Sam Snider for help in the data analysis,and You-Jun Chen for help with western blot experiments. This studywas supported by a Spinal Cord Research Foundation fellowship (toF.Q.Z.) and NIH grants NS031768 and NS050968 (to W.D.S.).

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