A p75 NTR and Nogo receptor complex mediates repulsive signaling by myelin-associated glycoprotein

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1 A p75 NTR nd Nogo reeptor omplex medites repulsive signling y myelin-ssoited glyoprotein Sott T. Wong 1, John R. Henley 1, Kevin C. Knning 2, Kuo-hu Hung 1, Mrk Bothwell 2 nd Mu-ming Poo 1 1 Division of Neuroiology, Deprtment of Moleulr nd Cell Biology, University of Cliforni, Berkeley, Cliforni 94720, USA 2 Deprtment of Physiology nd Biophysis, University of Wshington, Settle, Wshington 98195, USA The first two uthors ontriuted eqully to this work. Correspondene should e ddressed to M.P. (mpoo@ulink.erkeley.edu) Pulished online 11 Novemer 2002; doi: /nn975 Myelin-ssoited glyoprotein (MAG), n inhiitor of xon regenertion, inds with high ffinity to the Nogo-66 reeptor (NgR). Here we report tht the p75 neurotrophin reeptor (p75 NTR ) is oreeptor of NgR for MAG signling. In ultured humn emryoni kidney (HEK) ells expressing NgR, p75 NTR ws required for MAG-indued intrellulr C 2+ elevtion. Co-immunopreipittion showed n ssoition of NgR with p75 NTR tht n e disrupted y n ntiody ginst p75 NTR (NGFR5), nd extensive oexpression ws oserved in the developing rt nervous system. Furthermore, NGFR5 olished MAG-indued repulsive turning of Xenopus xonl growth ones nd C 2+ elevtion, oth in neurons nd in NgR/p75 NTR -expressing HEK ells. Thus we onlude tht p75 NTR is o-reeptor of NgR for MAG signling nd potentil therpeuti trget for promoting nerve regenertion. Myelin of the entrl nervous system ontins severl inhiitory ftors tht prevent xon regenertion fter injury 1 3. Among these ftors re myelin-ssoited glyoprotein (MAG) 4,5, Nogo- A 6,7 nd oligodendroyte myelin glyoprotein 8, ll of whih show high-ffinity inding to the Nogo-66 reeptor (NgR) As NgR lks ytoplsmi domin, MAG is thought to require oreeptor (s yet unidentified) for trnsmemrne signling fter inding to NgR. A reent study 13 shows tht MAG-dependent inhiition of neurite outgrowth nd tivtion of RhoA is redued in ultured dorsl root gnglion or ereellr neurons from p75 NTR knokout mie, suggesting the involvement of p75 NTR in MAG signling. As MAG did not diretly ind to p75 NTR, it ws proposed tht MAG inding to the gnglioside GT1 my tivte p75 NTR. However, the susequent disovery of MAG inding to NgR 10,11 led to the lterntive possiility tht MAG my tivte p75 NTR vi its intertion with NgR. Bsed on our previous finding tht MAG-dependent repulsion of xonl growth ones requires C 2+ signling 14, we used intrellulr C 2+ signls nd growth one repulsion s funtionl ssys to exmine whether p75 NTR medites MAG-dependent ellulr tions s o-reeptor of NgR. In the present study, we first showed tht extrellulr MAG ould rpidly indue rise in intrellulr C 2+ onentrtion ([C 2+ ] i ) in the growth one of ultured Xenopus spinl neurons. Using ultured HEK-293 ells expressing NgR nd p75 NTR, either lone or together, we found tht MAG-indued [C 2+ ] i elevtion required the oexpression of p75 NTR nd NgR. Coimmunopreipittion studies showed diret ssoition of p75 NTR nd NgR in memrne extrts from HEK-293 ells nd E17 rt ereell. Furthermore, this diret ssoition etween p75 NTR nd NgR n e disrupted y n ntiody 15 rised speifilly ginst the etodomin of p75 NTR (NGFR5). Immunostining of rt emryos lso reveled extensive oexpression of p75 NTR nd NgR in the developing nervous system. The funtionl signifine of the p75 NTR /NgR ssoition ws demonstrted y the finding tht NGFR5, whih ross-reted with Xenopus p75 NTR, ould olish oth the repulsive turning of Xenopus growth ones tht hd een indued y grdient of MAG nd y MAG-indued [C 2+ ] i elevtion. Finlly, removl of GPI-linked surfe proteins, whih presumly inlude Xenopus NgR, olished the MAG-indued [C 2+ ] i elevtion in the growth one. Tken together, these results identify p75 NTR s o-reeptor of NgR for MAG signling in ultured Xenopus spinl neurons nd in heterologous expression system, nd suggest tht interferene of p75 NTR signling my e useful therpeuti pproh in reversing the growth inhiition nd repulsive tion of MAG nd, perhps, other myelin ftors tht ind to NgR. RESULTS In the presene of mirosopi grdient of guidne ftors, isolted growth ones of ultured Xenopus spinl neurons show ttrtive or repulsive turning responses 16. A grdient of MAG n indue repulsive turning of the growth one 14,17, nd the response depends on the presene of extrellulr C 2+. This suggests tht C 2+ influx or [C 2+ ] i elevtion my medite the MAGindued growth inhiition or repulsive turning responses. Using 1302 nture neurosiene volume 5 no 12 deemer 2002

2 fluoresene rtio imging of C 2+ -sensitive inditors (Oregon Green BAPTA dextrn nd Fur Red; see Methods), we monitored [C 2+ ] i hnges in the Xenopus growth one. A rise in growth-one [C 2+ ] i ws oserved within minutes of the onset of the MAG grdient (Fig. 1). This finding is onsistent with the previous oservtions tht NogoA nd MAG ind to the sme reeptor (NgR); the inhiition nd ollpse of growth ones of dorsl root gnglion neurons indued y myelin-ssoited inhiitor ftor NI-35 (NogoA) is ompnied y rpid rise of [C 2+ ] i (ref. 18). The rise in [C 2+ ] i fter MAG inding to the ell surfe suggests tht C 2+ signls my provide sensitive ssy for the trnsmemrne signling of MAG. As the NgR is tthed to the ell surfe vi glyosylphosphtidyl-inositol (GPI) linkge 12, trnsmemrne oreeptor of NgR is presumly required for the MAG-NgR inding to trigger the C 2+ signl. A reent report 13 shows tht ereellr nd spinl sensory neurons from p75 NTR -defiient mie re insensitive to MAG. Although this suggested tht n intertion etween gnglioside GT1 nd the p75 NTR omplex might e suffiient to render ells responsive to MAG, the study used neurons tht express NgR onstitutively, leving open the possiility tht MAG exerts its tivity through NgR to tivte p75 NTR. To ssess the role of p75 NTR nd NgR in MAG-indued trnsmemrne signling, we used [C 2+ ] i elevtion s funtionl ssy for the MAG response. We trnsfeted HEK-293 ells with expression vetors for NgR nd humn p75 NTR (hp75 NTR ), either lone or together, nd performed fluoresene rtio imging of [C 2+ ] i in response to MAG. Bseline [C 2+ ] i ws reltively similr mong ll groups of trnsfeted ells. Ten minutes fter th pplition of MAG (1 µg/ml), ells expressing vetor ontrol, NgR or hp75 NTR lone showed no detetle hnges in [C 2+ ] i, wheres ells o-trnsfeted with hp75 NTR nd NgR onsistently showed mrked elevtion of [C 2+ ] i. Furthermore, ddition of the monolonl ntiody NGFR5 ompletely olished MAG-dependent C 2+ signls in o-trnsfeted ells (Fig. 2 Fig. 1. Clium signls t the growth one indued y n extrellulr grdient of MAG. () Fluoresene imges of the growth one of ultured Xenopus spinl neuron, whih ws o-injeted with the C 2+ - sensitive fluoresene inditors Oregon green-bapta nd Fur Red (see Methods). Imges depit green fluoresene t different times (in min) efore nd fter the onset of the MAG grdient. Sle r, 10 µm. () Exmple tres of [C 2+ ] i hnges tht were oserved t the growth one nd were indued y the presene of MAG grdient (mrked y the lk r). The fluoresene vlues mesured t eh time point were normlized to the men vlue otined during the 5-min period efore the onset of the MAG grdient for eh growth one. Normlized hnges in [C 2+ ] i levels t the growth one re shown y perentge hnges in the rtio of Oregon Green nd Fur Red fluoresene ( F/F) with time. () Summry of the MAG-indued [C 2+ ] i elevtion in the growth one for ll dt olleted s in ( MAG lone). Dt points represent men ± s.e.m. (n = 19). Also shown re dt olleted from experiments in whih funtion-loking ntiody ginst hp75 NTR (NGFR5, 5 µg/ml, n = 10) or mouse IgG ontrol ntiody (5 µg/ml, n = 7) ws dded in the medium (error rs were removed for lrity), or the neurons were treted with PI-PLC (1 unit/ml, n = 12) for >30 min efore C 2+ imging. The differenes etween seline C 2+ signls, defined s the 10-min period efore MAG pplition, nd tht oserved during the tretment period, defined s 5 15 min fter, were sttistilly signifint for MAG nd MAG + IgG (oth, P < 0.001), ut not for MAG + NGFR5 (P > 0.4) or MAG + PI-PLC (P > 0.2), y ANOVA nd Tukey s test. nd ). It is unlikely tht the NGFR5 effet ws due to nonspeifi ntiody inding to the ell surfe, s there ws n exess of ovine IgG (19 34 µg/ml) in the stndrd ulture medium for these ells throughout the experiments. The finding tht p75 NTR n trigger ytoplsmi C 2+ signls is onsistent with previous study 19 showing tht [C 2+ ] i rises in response to neurotrophins in firolsts trnsfeted with p75 NTR. To determine whether p75 NTR nd NgR n physilly intert in the memrne, we rried out immunopreipittion (IP) nd western lotting on soluilized memrne frtions from HEK-293 ells trnsfeted with (i) flg-tgged NgR (NgR-flg), (ii) hp75 NTR or (iii) oth. We used ntiodies ginst the NgRflg epitope nd the p75 NTR intrellulr domin to void potentil ntiody msking of reeptor domins involved in the NgR/p75 NTR intertion. Both NgR-flg nd p75 NTR were strongly expressed in the pproprite ells ut not in those trnsfeted with vetor ontrol (Fig. 3). Anti-flg or nti-p75 NTR ntiodies were used for IP of NgR or p75 NTR, respetively. Antiflg IPs were proed for the presene of either p75 NTR or NgR y western lotting with eh respetive ntiody (Fig. 3). We deteted o-ip of p75 NTR with NgR-flg in IPs from NgR- nd p75 NTR -oexpressing ells, ut not in ells expressing NgR-flg or empty vetor. Conversely, NgR-flg ws deteted in p75 NTR IPs from HEK-293 ells in whih oth NgR nd p75 NTR were oexpressed. Thus NgR nd p75 NTR n physilly ssoite with eh other. The ddition of NGFR5 ws le to disrupt the MAG-indued rise in [C 2+ ] i (Fig. 2 nd ), inditing tht this ntiody inding interferes with the ssoition of the two reeptors. Indeed, inresing NGFR5 onentrtions redued the NgR/p75 NTR ssoition (Fig. 3), wheres mouse IgG (100 µg/ml) hd no effet (Fig. 3, lst lne). These results support the ide tht the extrellulr domins of NgR nd p75 NTR intert to medite MAG signling. Is there similr intertion etween NgR nd p75 NTR in vivo? Co-IP experiments on freshly disseted ereell from rt pups (emryoni dy E17) showed tht p75 NTR nd NgR indeed ssoite t endogenous expression levels for these two reeptors (Fig. 4 nd ). We further exmined, y immunohistohemil nture neurosiene volume 5 no 12 deemer

3 Fig. 2. The p75 NTR medites MAG-dependent C 2+ signling in HEK- 293 ells. () Fluoresene imging of [C 2+ ] i in trnsfeted HEK-293 ells in the sene nd presene of MAG. Left, Fur Red fluoresene. Middle two pnels, Fluo-4 fluoresene efore (t 0 ) nd 10 min fter (t 10 ) th pplition of MAG (1 µg/ml), respetively. Right, differene of the fluoresene rtios (Fluo-4/Fur Red) t t 10 nd t 0. Control, HEK-293 ells trnsfeted with empty expression vetor. () Summry of perentge hnges in [C 2+ ] i. The men [C 2+ ] i levels over eh ell ws mesured y the rtio Fluo-4/Fur Red, nd the perentge hnge t t 10 reltive to t 0 ws lulted for eh ell. Dt represent men ± s.e.m. The numer ove eh r is the totl numer of ells mesured from t lest five seprte ultures. *P < 0.001, t-test. stining, whether p75 NTR nd NgR re oexpressed in neurons tht re known to e responsive to MAG. In the rt nervous system (E14.5), p75 NTR is widely expressed in neuronl nd nonneuronl tissues. However, ololiztion with NgR ws found to e exlusively neuronl, inluding the som of dorsl root gngli, their projetions to the dorsl horn of the spinl ord (Fig. 4 e) nd spinl ord motor neurons (Fig. 4f h). These results re onsistent with the reports tht emryoni spinl nd sensory neurons re responsive to MAG 20. As the NGFR5 ntiody olished MAG-indued C 2+ signling in HEK-293 ells trnsfeted with oth NgR nd hp75 NTR, we further exmined the effet of NGFR5 on the MAG-indued C 2+ signling nd repulsive turning of Xenopus growth ones. First, we determined whether the nti-hp75 NTR ntiody NGFR5 ross-rets with Xenopus p75 NTR (xp75 NTR ). We trnsfeted HEK-293 ells with xp75 NTR, hp75 NTR or trka nd tested whether NGFR5 ould reognize the xp75 NTR in memrne frtions from these ells y western lotting. Indeed, we found tht NGFR5 identified oth xp75 NTR nd hp75 NTR in the ltter memrnes, ut not in memrnes from ells trnsfeted with vetor ontrol or trka (Fig. 5). Reognition of xp75 NTR y NGFR5 is further supported y the finding tht NGFR5 strongly immunostined the surfe of unpermelized HEK-293 ells expressing xp75 NTR, nd tht this stining ws olished y the etodomin peptide of hp75 NTR (Fig. 5). Hving estlished the ross-retivity of NGFR5 with xp75 NTR, we further exmined whether xp75 NTR is expressed in the Xenopus growth ones t the sme developmentl stge t whih we performed our C 2+ imging experiments (Fig. 1). NGFR5 immunostining of Xenopus spinl neurons reveled high level of xp75 NTR expression in the growth one (Fig. 5). Furthermore, we found tht NGFR5 inding to Xenopus spinl neurons ould disrupt MAG signling in the growth ones of these neurons. Bth ddition of NGFR5 ompletely olished MAG-indued elevtion of [C 2+ ] i in these growth ones, wheres similr tretment with nonspeifi ontrol IgG (5 µg/ml) hd no signifint effet (Fig. 1). Although we hve shown tht Xenopus neurons express p75 NTR, it remins unler whether Fig. 3. Assoition of NgR with hp75 NTR. () Western nlysis of totl ell lystes from HEK-293 ells trnsfeted with empty vetor, NgR-flg, hp75 NTR or NgR-flg + hp75 NTR. Immunolots (WB) were mde with M2 ma to flg (upper) or nti-p75 NTR ntiody rised ginst ytoplsmi domin of p75 NTR (lower). () Co-immunopreipittion (IP) with p75 NTR nd flg ntiodies on memrne preprtions mde from HEK-293 ells trnsfeted with empty vetor, NgRflg, hp75 NTR or NgR-flg + hp75 NTR nd immunolotted with either hp75 NTR or flg ntiodies. Arrowhed, IgG hevy hin. () NGFR5 pretretment n disrupt the NgR nd p75 NTR ssoition. HEK-293 ells otrnsfeted with oth NgR-flg nd humn p75 NTR were inuted for 1 h with indited onentrtions of NGFR5 or mouse IgG ontrol ntiody (fr right lne). Susequent immunopreipittion with immoilized, flgntiody Sephrose reveled deresing ssoition of p75 NTR with NgR with inresing ntiody onentrtion (top row), wheres there ws no signifint hnge in the mount of NgR-flg tht ws ound to the resin (ottom row). The mount of free p75 NTR inresed with higher NGFR5 onentrtions in the IP superntnts (middle row). The depited lots re representtive of t lest three independent experiments for eh study nture neurosiene volume 5 no 12 deemer 2002

4 d e n endogenous NgR ortholog is present in these ells. However, s it is well hrterized tht these neurons re sensitive to MAG 14,16,21 nd tht in mmmls MAG interts with high ffinity to the NgR 10,11 to exert its effets on p75 NTR, it is likely tht Xenopus neurons express NgR or NgR-like homolog. Consistent with the ft tht NgR is GPI-linked protein, we found tht tretment of the Xenopus neurons with the phosphoinositidespeifi phospholipse C (PI-PLC), n enzyme tht releses GPInhored proteins from the ell surfe, olished MAG-indued [C 2+ ] i elevtion (Fig. 1). Tken together, these results strongly support the ide tht p75 NTR nd GPI-linked surfe omponent, presumly Xenopus NgR, re essentil omponents for MAG-indued C 2+ signling in neurons. To determine whether p75 NTR -dependent MAG signling is funtionlly importnt in xonl growth regultion, we exmine the role of p75 NTR in the growth one turning response indued y n extrellulr grdient of MAG. In the stndrd turning ssy 22, grdient of MAG ws estlished ross the growth one y pulstile pplition of pioliters of onentrted solution of MAG (100 µg/ml) from miropipette, whih ws positioned t distne of 65 µm from the enter of the growth one, t n ngle of 45 with respet to the originl diretion of growth one extension. Within one hour fter the onset of the grdient, gret mjority of Xenopus spinl neurons showed repulsive turning of the growth one (Fig. 5d), nd no onsistent turning ws oserved when MAG ws omitted in the pipette solution (Fig. 5e). However, ddition of NGFR5 (5 µg/ml) in the th olished the repulsive turning indued y the MAG grdient f g h Fig. 4. Nogo Reeptor nd p75 NTR re oexpressed in the developing nervous system. (, ) Co-immunopreipittion of p75 NTR nd NgR in E17 rt ereellum. Using speifi ntiody ginst the NgR, p75 NTR is o-immunopreipitted with the NgR (, lne 1), ut not with mouse IgG ontrol (, lne 2). Immunopreipittion with p75 NTR ntiody shows the reiprol intertion with NgR (, lne 3), suggesting tht the two proteins intert in vivo. Cereellr extrts were immunopreipitted with the denoted ntiodies (mrked ove lnes) nd then western lotted (WB) with the indited ntiodies. Arrowhed, IgG hevy hin. ( e) Immunohistohemistry of sgittl setion of n E14.5 rt emryo shows tht the expression of p75 NTR () nd Nogo R (d) is ololized in motor neurons of the spinl ord (SC) nd dorsl root gngli (DRG). Note tht p75 NTR is expressed independently of NgR in non-neurl ells suh s Shwnn ells surrounding sensory xon trts (Sw. sheth), ut ololizes extensively in neurons (e). Sle r, 250 µm; dorsl is up, nterior is left. (f h) Confol imges of spinl motor neurons (oxed res in e) re shown t higher mgnifition. Sle r, 50 µm. A ler overlp etween p75 NTR (f) nd NgR (g) shown in the merged imge (h). MN, motor neurons; R, developing ris; DH inn., xons from the DRG innervting the dorsl horn; DH Proj., projetion from DRG into dorsl horn of spinl ord. (Fig. 5f), wheres ddition of ontrol mouse IgG (5 µg/ml) hd no effet. The verge turning ngle in the MAG grdient ws signifintly negtive, nd ws redued to ner zero y the presene of NGFR5. The verge neurite extension did not differ signifintly under the vrious onditions (Fig. 5g nd h). These results showed tht interfering with the intertion etween NgR nd p75 NTR y speifi inding of NGFR5 to p75 NTR (Fig. 3) indeed loked MAG-indued repulsive growth one turning. DISCUSSION The p75 NTR is memrne protein tht signls in response to the inding of proneurotrophins or to lower-ffinity inding of mture neurotrophins It my onvey trnsmemrne signls either independently or s o-reeptor of the Trk fmily of tyrosine reeptor kinses, whih re preferentilly tivted y mture neurotrophins 26. There is growing evidene tht p75 NTR hs key role in xon guidne during the development of the nervous system 25,26. Axon pthfinding errors of p75 NTR -expressing neurons re prominent mong the phenotypes oserved in p75 NTR mutnt mie, inluding mistrgeting of symptheti nd ortil suplte xons 27,28. Asene of the p75 NTR lters the pttern of sympthosensory sprouting in the trigeminl gngli of mie overexpressing nerve growth ftor 29. It remins to e determined whether these p75 NTR -medited ehviors re predominntly regulted y neurotrophins or y myelin-ssoited ftors suh s MAG nd NogoA. The o-distriution of immunoretivity for p75 NTR nd NgR in motor neurons of ventrl emryoni rt spinl ord nd in primry sensory neurons of dorsl root gngli suggests tht funtionl p75 NTR /NgR omplexes re likely to e present in these emryoni neurons. The presene of p75 NTR in these neuronl popultions throughout emryoni development is onsistent with previous reports (see ref. 30). Although expression of NgR in the rodent nervous system hs minly een found in neurons of dult nimls 31, the presene of funtionl NgR in emryoni rodent spinl ord neurons nd dorsl root gnglion neurons is indited y reports tht emryoni spinl neurons 32 nd neworn dorsl root gnglion neurons 5 re responsive to MAG. Neurite outgrowth of these emryoni neurons is stimulted y MAG, whih hs n inhiitory effet on mture neurons. We posit, however, tht prtiiption of distint reeptor system need not e nture neurosiene volume 5 no 12 deemer

5 Fig. 5. MAG-indued growth one repulsion is olished y the p75 NTR ntiody NGFR5. ( ) The NGFR5 reognizes xp75 NTR. () Ntive gel eletrophoresis shows tht Xenopus p75 NTR (xp75) nd humn p75 NTR (hp75) were reognized y NGFR5 in memrne preprtions derived from HEK ells expressing eh ortholog of p75 NTR, wheres memrnes from ontrol or trkaexpressing ells were not reognized. () HEK ells expressing xp75 NTR were speifilly immunostined with the NGFR5 ntiody (middle), wheres vetor ontrol ells (CS2+) were not (left). Preinution with hp75 NTR etodomin frgment (hp75 ECD, 10 µg/ml) effetively neutrlized the immunoretivity (right). Sle r, 20 µm. () NGFR5 reognized p75 NTR in Xenopus spinl neurons. A representtive Xenopus spinl neuron (top two pnels) ws immunostined with NGFR5 nd nlyzed for p75 NTR immunoretivity. White ox indites the re mgnified ( 2) in the pnel diretly elow to show the growth one (GC) fluoresene. Preinution of the ntiody with the hp75 NTR etodomin frgment neutrlized the stining in these neurons (lower pir), suggesting speifi intertion of NGFR5 with xp75 NTR. Sle rs, 20 µm; CB, ell ody. (d) Imges of the growth one of ultured Xenopus spinl neuron t the onset (left) nd the end (right) of 1-h exposure to MAG grdient (mrked y the rrow). Superimposed tres depit smples of the trjetory of neurite extension during d e f the 1-h period. The origin ws the enter of the growth one t the onset of the ssy nd the originl diretion of extension ws ligned vertilly. Sle rs, 10 µm. (e) Similr to (d), exept tht MAG ws omitted from the pipette solution. (f) Similr to (e), exept tht the NGFR5 ma (5 µg/ml) ws dded in the ulture efore the MAG grdient ws pplied. (g) The distriution of turning ngles is shown in umultive perentge plot. For eh experimentl ondition, ngulr positions of ll growth ones t the end of 1-h exposure to the MAG grdient re plotted. The dt points represent the perentge of growth ones with turning ngles given ngulr vlue. Dt re from ll experiments similr to those illustrted in (d f) nd from ontrol experiments in whih mouse IgG (5 µg/ml) ws dded in the ulture insted of NGFR5. The differenes in the distriution of turning ngles etween dt for the g h MAG grdient in the presene or sene of the NGFR5 were sttistilly signifint (P < 0.01; Kolmogorov-Smirnov test), wheres the differenes in the distriution etween the NGFR5 ntiody tretment nd the medi ontrol were not signifint (n.s., P > 0.5). (h) Summry of verge turning ngles nd neurite extension for the sme dt set s tht in (g). invoked euse elevted AMP levels n swith repulsive/inhiitory responses to ttrtive/stimultory responses indued y MAG 14,33. Studies of Xenopus spinl neurons in ulture 14,16 show tht n extrellulr grdient of MAG n trigger either ttrtive or repulsive turning of the growth one, depending on the ytoplsmi level of AMP, with high levels of AMP fvoring ttrtion nd low levels fvoring repulsion. Growth ttrtion or repulsion usully reflets n symmetri filopodil extension or retrtion t the growth one, respetively, so it my depend on mehnisms tht re similr to those tht regulte growth stimultion or inhiition. Indeed, elevtion of AMP n lso overome neurite growth inhiition y myelin in vitro 33 nd promote xon regenertion in vivo 34,35. Similr AMP-dependent onversion of turning responses 21,36 nd reversl of growth inhiition 37 were lso oserved in ultured neurons in response to neurotrophins. It is possile tht the repulsive/inhiitory tion of oth MAG nd neurotrophins is medited through p75 NTR, nd the effiy of p75 NTR signling is regulted y AMP or its downstrem effetors. Mny dptor proteins tht ind to the ytoplsmi domin of p75 NTR hve een identified 25, inluding RhoA, Rho-fmily GTPse known to regulte ytoskeletl strutures 38 nd to medite neurite growth inhiition nd growth one ollpse Intivtion of the Rho signling pthwy pprently promotes CNS xon regenertion 40. As neurotrophin inding to p75 NTR n lso intivte RhoA nd promote xonl outgrowth 43, p75 NTR my serve s i-diretionl swith in setting the growth one ehvior in response to externl stimuli, depending on its downstrem tions on Rho GTPses. For exmple, MAG-NgR intertion with p75 NTR my tivte RhoA, leding to growth inhiition/repulsion, wheres neurotrophin-trk inding to p75 NTR my result in intivtion of RhoA nd growth stimultion/ttrtion. Although it remins unler how ytosoli C 2+ prtiiptes in MAG signl trnsdution, the present study hs defined reeptor omplex required for MAG-indued C 2+ signling nd repulsive growth one turning, whih is likely to involve RhoA-dependent ytoskeletl rerrngements 13,43. As o-reeptor of NgR, p75 NTR now emerges s key plyer not only for regulting neuronl development 26 nd poptosis 44, ut lso for regulting the inhiition of xon regenertion indued y myelin-ssoited ftors. METHODS Culture preprtions. Cultures of Xenopus spinl neurons were prepred from the neurl tue tissue of 1-d old Xenopus emryos y methods previously desried 45,46 nd used for experiments etween h fter plting t room temperture (20 22 C). All nimls were used ording to pproved protools (Animl Cre nd Use Committee, UC Berkeley). The ulture medium onsisted of 86% (v/v) Leiovitz medium (GIBCO, Githersurg, Mrylnd) ontining 4 mm Hepes, 0.8% (v/v) 1306 nture neurosiene volume 5 no 12 deemer 2002

6 fetl ovine serum (HyClone, Logn, Uth) nd 13.2% (v/v) sline solution (10 mm D-gluose, 5 mm Sodium pyruvte, 1.26 mm CCl 2, 1.34 mm N 2 HPO 4, 0.44 mm NH 2 PO 4 nd 4 mm HEPES; ph 7.5). The experiments on Xenopus neurons were rried out in serum-free ulture medium. The HEK ell ultures were mintined in D-MEM (Gio) with 10% FBS. MAG-F ws purhsed from R&D Systems (Minnepolis, Minnesot). The humn p75 NTR etodomin (p75 ECD) ws isolted from A875 ell onditioned medi nd purified y ffinity hromtogrphy over NGFR5 ma-sephrose. Bound etodomin ws eluted with glyine (ph 2.2) nd quikly neutrlized in Tris-HCl (ph 7.4). The onentrtion of purified p75 NTR ECD ws determined using the Brdford Protein Assy (Piere Biotehnology, Rokford, Illinois). The integrity of p75 NTR ECD ws onfirmed y western lot using NGFR5 nd visulized y ECL s single nd. Clium imging. For ultured Xenopus spinl neurons, the ells were miroinjeted with Oregon green 488 BAPTA-1 onjugted to dextrn, s well s with Fur Red (Moleulr Proes, Eugene, Oregon). Clium imging ws rried out using Lei onfol imging system (TCS SP) y methods desried previously 47,48. Exittion ws t 488 nm, nd the Oregon Green BAPTA dextrn nd Fur Red emission signls were olleted t nm nd nm, respetively. Fluoresene nd trnsmission imges ( pixels; voxel size, 207 nm) were olleted simultneously every 30 s nd nlyzed using Imge J softwre (Ntionl Institutes of Helth; The men fluoresene intensity ws mesured over fixed squre re tht overed the entire growth one throughout the mesurement period. Rtios of the normlized Oregon Green nd Fur Red fluoresene dt were used to ontrol for flututions in growth one thikness or hnges in fol plne tht my hve ourred during experiments. HEK-293 ells were trnsfeted with NgR, p75 NTR or oth with lipofetmine 2,000 (Invitrogen Life Tehnologies, Crlsd, Cliforni) ording to the supplier s reommendtions nd plted onto imging dishes (Mttek, Ashlnd, Msshusetts). The trnsfeted ells were loded with the fluoresene C 2+ inditors Fluo4 nd Fur Red. Imges were olleted for 15 min efore nd 25 min fter the ddition of MAG (1 µg/ml) t 30-s intervls nd nlyzed y rtio imging s desried ove for the Xenopus growth one. A funtion-loking monolonl ntiody ginst the etodomin of p75 NTR (NGFR5, L Vision, Fremont, Cliforni) ws dded to the th where indited. Immunopreipittion (IP) nd western lot nlysis. HEK-293 ells, trnsfeted with p75 NTR, NgR-flg or oth, were hrvested in ie-old homogeniztion uffer (50 mm Tris, 150 mm NCl, 5% surose; ph 7.6). Cells were homogenized, nd the lystes were lered of nulei nd unroken ells y entrifugtion for 10 min t 500g. The superntnts were entrifuged t 100,000g for 25 min, nd pelleted memrnes were soluilized in RIPA uffer (Amershm Biosienes, Pistwy, New Jersey). Protein A Sephrose (Amershm) ws preinuted with polylonl p75 NTR ntiody (PAS-75, Promeg, Mdison, Wisonsin). The lrified RIPA extrts were then sujeted to IP for 2 h t 4 C with either PAS-75 or grose ovlently onjugted to the M2 monolonl flg epitope ntiody (Sigm, St. Louis, Missouri). The pellets were wshed twie with ie-old RIPA, extrted with oiling Lemmeli uffer, nd then seprted over 7.5% SDS-PAGE gel. Proteins were trnsferred to PVDF (Millipore, Bedford, Msshusetts), nd the presene of p75 NTR or NgR ws exmined y western lotting y ECL (Amershm) ording to the mnufturer s reommendtion. Ntive PAGE ws rried out s ove exept tht SDS nd DTT were omitted from the smple nd running uffers. Xenopus p75 NTR in CS2+ hs een desried previously 49. For in vivo IP experiments, ereell from eight E17 rt pup rins were disseted. Memrnes were prepred s desried for HEK-293 ells. Speifi ntiodies ginst NgR (Alph Dignostis Interntionl, Sn Antonio, Texs) nd p75 NTR (Promeg) were used to perform IP nd western lots on this preprtion. For the NGFR5 titrtion experiments, the denoted onentrtions of NGFR5 or ontrol mouse IgG ntiody were inuted with HEK ells trnsfeted with oth NgR-flg nd hp75 NTR for 1 h in HEK-293 ell ulture medi. Cells were wshed twie with ie-old PBS nd then prepred nd nlyzed s desried ove. Superntnt frtions were normlized for protein nd nlyzed diretly y western lot for p75 NTR. Immunohistohemistry. Biseted rt emryos were immersion-fixed in 10% formlin, proessed through 30% surose grdient, emedded in OCT, nd ryostt-setioned t 14 µm. Setions were loked in 5% milk/tbs/0.2% triton for 1 h nd immunostined with 2.5 µg/ml IgG192 (mouse nti-rt p75 NTR ) nd 2.5 µg/ml rit nti humn NgR (Alph Dignostis) t room temperture for 3 h. After extensive wshing in TBS, primry ntiodies were deteted using Alex 488 got nti-mouse (rt pre-dsored, Moleulr Proes) diluted 1:750, nd CY3 donkey nti-rit (Jkson ImmunoReserh, West Grove, Pennsylvni) diluted 1:500. Immunoytohemistry. Isolted Xenopus spinl neurons from stge 22 emryos growing in ulture for 14 h or HEK-293 ells trnsfeted with CS2+ or xp75 NTR were fixed for 20 min with serum-free ulture medium ontining 2% (v:v) formldehyde nd 4% (wt:v) surose. Cells were then rinsed nd inuted with serum-free ulture medium ontining 5 µg/ml NGFR5 ma to hp75 NTR lone or fter pre-inuting with 10 µg/ml p75 etodomin. Stining ws deteted using got ntimouse ntiody oupled to Alex 488 (2 µg/ml, Moleulr Proes). Cells were then mounted in ProLong ntifde regent (Moleulr Proes) nd sujeted to onfol mirosopy. Growth one turning ssy. Mirosopi grdients of MAG were produed y methods previously desried 22,50. Repetitive ejetion of pioliters of solution ontining MAG (100 µg/ml) ws mde y miropipette (tip opening 1 µm). A stndrd pressure pulse (3 psi, durtion 20 ms) ws pplied to the pipette t 2 Hz with pulse genertor (SD9, Grss Instruments, Quiny, Msshusetts). Phse-ontrst mirosopi imges of neurites were reorded with CCD mer (Toshi IK-541RA). For ssying growth one turning, the tip of the miropipette ws pled 65 µm wy from the enter of the growth one nd t n ngle of 45 with respet to the diretion of initil diretion of neurite extension, whih ws determined y the lst 10-µm segment of the neurite. The entire trjetory of the neurite t the end of the 1-h period ws mesured with digitizer. The turning ngle ws defined y the ngle etween the originl diretion of neurite extension nd stright line onneting the positions of the enter of the growth one t the eginning nd the end of ssy. Only those growth ones with extensions tht were >5 µm over the 1-h period were used for nlysis. Aknowledgments We thnk K. 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Turning of nerve growth ones indued y neurotrnsmitters. Nture 368, (1994) nture neurosiene volume 5 no 12 deemer 2002