Report |
Address correspondence to Toshihide Yamashita, Dept. of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-68793221. Fax: 81-6-68793229. E-mail: tyama{at}anat2.med.osaka-u.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: p75; neurotrophin; myelin-associated glycoprotein; Rho; ganglioside
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Myelin-associated glycoprotein (MAG) is a well-characterized component of both the central nervous system and the peripheral nervous system myelin and is a potent inhibitor of axonal regeneration (McKerracher et al., 1994; Mukhopadhyay et al., 1994). It inhibits axonal outgrowth from adult dorsal root ganglion (DRG) and in postnatal ages of cerebellar, retinal, spinal, hippocampal, and superior cervical ganglion neurons. Although the molecular mechanism of action of MAG on neurons or the receptor that transmits the signals remained to be established, neurite growth inhibition by MAG is blocked if neurons are exposed to neurotrophins before encountering the inhibitor (Cai et al., 1999). We attempted to ask if p75NTR had some roles in transducing the inhibitory signals of MAG in neurons. The results we obtained were surprising, since p75NTR itself turned out to be a signal transducing element for MAG and neurotrophins. Association of p75NTR with ganglioside GT1b is also shown to provide a possible link between MAG and p75NTR.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
p75NTR has been shown to be required for the inhibition of axonal growth and target innervation of peripheral neurons in vivo and in vitro (Kimpinski et al., 1999; Kohn et al., 1999) and for suppression of hyperinnervation of cholinergic neurons in vivo (Yeo et al. 1997). It was reported recently that the growth of sympathetic axons within the myelinated portions of the cerebellum was greater in NGF transgenic mice lacking expression of p75NTR compared with those expressing p75NTR in vivo (Walsh et al., 1999). It may be a relevant finding supporting our data, since neurons carrying a mutation in the p75NTR gene are suggested to be refractory to inhibitory factors.
Signaling mechanisms of MAG on the neurons
Some neurons extend neurites rapidly when RhoA is inactivated, and neurite retraction occurs when RhoA is active (Davies, 2000). Previous study shows that inactivation of RhoA promoted axonal regeneration in vivo (Lehmann et al., 1999). Thus, to examine if activation of RhoA is necessary for modification of neurite outgrowth by MAG in our system, we employed the exoenzyme C3 transferase from Clostridium botulinum, which ADP ribosylates RhoA. The recombinant C3 transferase was introduced into the cytoplasm of DRG neurons by trituration. The C3 transferase completely abolished the effect of MAG on DRG neurons from wild-type mice (Fig. 2 A). These data are consistent with the previous report, suggesting RhoA is on the pathway of MAG signaling (Lehmann et al., 1999).
|
Regulation of RhoA activity when the proteins are artificially expressed may be difficult to detect in natural cells. Therefore, to see if RhoA activity is regulated by MAG in the cells expressing endogenous p75NTR postnatal cerebellar granule neurons were used, since these neurons also are sensitive to MAG with regard to neurite outgrowth. Consistent with the observation in transfected 293 cells, MAGFc activates RhoA in cerebellar granule neurons from wild-type mice (P9), which express abundant p75NTR (Fig. 3 A). This rapid activation was in contrast with the effect of NGF on the neurons, which is also mediated by p75NTR, since they don't express trkA (Fig. 3 B). RhoA activity (Fig. 3 C) and the effect of MAG on neurite outgrowth (unpublished data) seem to be saturated by MAG at the concentration of 25 µg/ml. Activation of RhoA by MAG was lost in the neurons from mice carrying a mutation in the p75NTR gene (Fig. 3 D). These data demonstrate that MAG activates RhoA by a p75NTR-dependent mechanism, thus inhibiting neurite outgrowth of postnatal cerebellar granule neurons.
|
Colocalization of p75NTR and MAG binding
MAG binds to neurons in a sialic aciddependent manner, but MAG's sialic acid binding site is distinct from its neurite inhibitory activity. The sialic aciddependent binding to MAG is not sufficient or necessary for MAG's inhibitory effect (Tang et al., 1997a). Therefore, it is possible that the binding partner and the signal transducing element for MAG may form a receptor complex. We assumed that the binding partner for MAG and p75NTR might interact in a cis manner. To test this hypothesis, the localization of p75NTR and MAG binding was assessed on the subcellular level. Binding of MAGFc was visualized by incubation with a fluorescent-tagged antihuman IgG. Fig. 4 shows binding of MAGFc to adult DRG neurons using confocal laser microscopy. MAGFc binding appears punctate. The same cells were stained with an anti-p75NTR antibody, and the distribution was assessed. p75NTR expression on the cell body was rather diffuse but that on the neurites showed fine speckled staining (Fig. 4 A, top). The vast majority of puncta for p75NTR immunoreactivity was colocalized with MAG binding. At high magnification, the colocalization was evident by the similar distribution of hot spots on the neuritic plasma membrane (Fig. 4 A, bottom). Binding of MAGFc was still observed in DRG neurons from mice carrying a mutation in the p75NTR gene (Fig. 4 B). These data demonstrate that the p75NTR and MAG binding colocalize.
|
|
Dual signals elicited by p75NTR
p75NTR has been shown to bind more than just neurotrophins, such as CRNF (Fainzilber et al., 1996) or rabies virus glycoprotein (Tuffereau et al., 1998), but it is not known if these ligands trigger any signals through p75NTR. Thus, our findings demonstrating that p75NTR is a signal transducer not only for neurotrophins but also for MAG is intriguing. More interestingly, neurotrophins binding to p75NTR promote axonal outgrowth of neurons presumably by inhibiting RhoA activity (Yamashita et al., 1999), but MAG elicits the opposite effect via p75NTR on neurons by activating RhoA. This implies that p75NTR has dual signals as a transducing element. It is also important to note that essentially all adult neurons are sensitive to inhibition by MAG, whereas p75NTR has a restricted distribution. The identification and characterization of MAG signals have shed light on a previously unrecognized mechanism by which neurons respond to extracellular inhibitory molecules.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neurite outgrowth assay
DRGs were removed from adult mice and dissociated into single cells by incubation with 0.025% trypsin and 0.15% collagenase type I (Sigma-Aldrich) for 30 min at 37°C. For cerebellar neurons, the cerebella from two animals were combined in 5 ml of 0.025% trypsin, triturated, and incubated for 10 min at 37°C. DME containing 10% FCS was added, and the cells were centrifuged at 800 rpm. Neurons were plated in Sato medium (Cai et al., 1999) on poly-L-lysinecoated chamber slides. For outgrowth assays, plated cells were incubated for 24 h, fixed in 4% (wt/vol) paraformaldehyde, and immunostained with a monoclonal antibody (TuJ1) recognizing the neuron-specific ßtubulin III protein. Then, the length of the longest neurite or the total process outgrowth for each ßtubulin IIIpositive neuron was determined. Where indicated, recombinant rat MAGFc chimera (R&D Systems) was added to the medium after plating. The recombinant C3 transferase was introduced into the cytoplasm of the neurons before plating by trituration as described previously (Borasio et al., 1989).
Affinity precipitation of GTPRhoA
293 cells were transfected with pcDNA3 vectors containing wild-type NH2-terminally HA-tagged RhoA (Yamashita et al., 1999) and/or full-length human p75NTR by lipofection using Lipofectamine 2000 (GIBCO BRL). Cerebellar neurons from P9 mice were isolated as described previously (Cai et al., 1999). Cells were lysed in 50 mM Tris, pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, and 10 mM MgCl2 with leupeptin and aprotinin each at 10 µg/ml. Cell lysates were clarified by centrifugation at 13,000 g at 4°C for 10 min, and the supernatants were incubated with the 20 µg of GST-Rho binding domain of Rhotekin beads (Upstate Biotechnology) at 4°C for 45 min. The beads were washed four times with washing buffer (50 mM Tris, pH 7.5, containing 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2,10 µg/ml each of leupeptin and aprotinin). Bound Rho proteins were detected by Western blotting using a monoclonal antibody against RhoA (Santa Cruz Biotechnology).
MAGFc binding and immunocytochemistry
DRG neuron cultures were fixed in 1% paraformaldehyde in PBS for 30 min. They were then blocked in PBS containing 2% FCS. To localize the MAG binding molecule, MAGFc (5 µg/ml) and antihuman IgG (1µg/ml) were precomplexed for 30 min at room temperature before being added to the fixed and blocked DRG neurons (Turnley and Bartlett, 1999). To identify p75NTR, cells were permeabilized with 0.2% Triton X-100/PBS and then incubated overnight with polyclonal antibody to p75NTR (Promega) followed by an Alexa fluorTM 568labeled antirabbit IgG (Molecular Probes) for 1 h. The specificity of the antibodies was assessed by Western blot analysis of cells expressing the proteins, and control experiments of immunocytochemistry were performed by leaving out the primary antibodies.
Coprecipitation of recombinant p75NTR and GT1b
Recombinant human p75Fc chimera (1 µg; Genzyme-Techne) and 1 µg of purified ganglioside GT1b (>98% purity; Seikagaku Co.) were incubated in 200 µl 0.025% Tween 20/PBS for 2 h, and p75NTR was precipitated using protein A sepharose (Amersham Pharmacia Biotech). The resultant precipitates were electrophoretically transferred to polyvinylidene difluoride membranes after SDS-PAGE with 7% gels and were immunoblotted with anti-GT1b antibody (IgM; Seikagaku Co.) or anti-p75NTR antibody.
Coimmunoprecipitation experiments
Cells were lysed on ice for 20 min with lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 25 µg/ml leupeptin, and 25 µg/ml aprotinin). The lysates were centrifuged at 13,000 g for 20 min, and the supernatants were collected. They were then incubated with the anti-GT1b antibody or an anti-HA antibody (for transfected HA-p75NTR) overnight followed by incubation with the antimouse IgM antibody (for GT1b). Immunocomplex or MAGFc was collected with protein A sepharose (Amersham Pharmacia Biotech). The suspension was centrifuged at 1,000 g for 5 min. The pellets were washed four times with lysis buffer and subjected to SDS-PAGE followed by immunoblot analysis.
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
Submitted: 4 February 2002
Revised: 29 March 2002
Accepted: 29 March 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Buck, C.R., H.J. Martinez, I.B. Black, and M.V. Chao. 1987. Developmentally regulated expression of the nerve growth factor receptor gene in the periphery and brain. Proc. Natl. Acad. Sci. USA. 84:30603063.[Abstract]
Davies, A.M. 2000. Neurotrophins: neurotrophic modulation of neurite growth. Curr. Biol. 10:R198R200.[CrossRef][Medline]
Ernfors, P., A. Henschen, L. Olson, and H. Persson. 1989. Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons. Neuron. 2:16051613.[Medline]
Fainzilber, M., A.B. Smit, N.I. Syed, W.C. Wildering, P..M. Hermann, R.C. van der Schors, C. Jimenez, K.W. Li, J. van Minnen, A.G. Bulloch, C.F. Ibanez, and W.P. Geraerts. 1996. CRNF, a molluscan neurotrophic factor that interacts with the p75 neurotrophin receptor. Science. 274:15401543.
Kohn, J., R.S. Aloyz, J.G. Toma, M. Haak-Frendscho, and F.D. Miller. 1999. Functionally antagonistic interactions between the TrkA and p75 neurotrophin receptors regulate sympathetic neuron growth and target innervation. J. Neurosci. 19:53935408.
Lee, K.F., E. Li, L.J. Huber, S.C. Landis, A.H. Sharpe, M.V. Chao, and R. Jaenisch. 1992. Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell. 69:737749.[Medline]
Lehmann, M., A. Fournier, I. Selles-Navarro, P. Dergham, A. Sebok, N. Leclerc, G. Tigyi, and L. McKerracher. 1999. Inactivation of Rho signaling pathway promotes CNS axon regeneration. J. Neurosci. 19:75377547.
Mukhopadhyay, G., P. Doherty, F.S. Walsh, P.R. Crocker, and M.T. Filbin. 1994. A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration. Neuron. 13:757767.[Medline]
Ren, X.D., W.B. Kiosses, and M.A. Schwartz. 1999. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskelton. EMBO J. 18:578585.
Roux, P.P., M.A. Colicos, P.A. Barker, and T.E. Kennedy. 1999. p75 neurotrophin receptor expression is induced in apoptotic neurons after seizure. J. Neurosci. 19:68876896.
Sheikh, K.A., J. Sun, Y. Liu, H. Kawai, T.O. Crawford, R.L. Proia, J.W. Griffin, and R.L. Schnaar. 1999. Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects. Proc. Natl. Acad. Sci. USA. 96:75327537.
Tang, S., Y.J. Shen, M.E. DeBellard, G. Mukhopadhyay, J.L. Salzer, P.R. Crocker, and M.T. Filbin. 1997a. Myelin-associated glycoprotein interacts with neurons via a sialic acid binding site at ARG118 and a distinct neurite inhibition site. J. Cell Biol. 138:13551366.
Tuffereau, C., J. Benejean, D. Blondel, B. Kieffer, and A. Flamand. 1998. Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus. EMBO J. 17:72507259.
Vinson, M., P.J. Strijbos, A. Rowles, L. Facci, S.E. Moore, D.L. Simmons, and F.S. Walsh. 2001. Myelin-associated glycoprotein interacts with ganglioside GT1b. A mechanism for neurite outgrowth inhibition. J. Biol. Chem. 276:2028020285.
Walsh, G.S., K.M. Krol, K.A. Crutcher, and M.D. Kawaja. 1999. Enhanced neurotrophin-induced axon growth in myelinated portions of the CNS in mice lacking the p75 neurotrophin receptor. J. Neurosci. 19:41554168.
Yan, Q., and E.M. Johnson, Jr. 1988. An immunohistochemical study of the nerve growth factor receptor in developing rats. J. Neurosci. 8:34813498.[Abstract]
Yang, L.J., C.B. Zeller, N.L. Shaper, M. Kiso, A. Hasegawa, R.E. Shapiro, and R.L. Schnaar. 1996. Gangliosides are neuronal ligands for myelin-associated glycoprotein. Proc. Natl. Acad. Sci. USA. 93:814818.
Yeo, T.T., J. Chua-Couzens, L.L. Butcher, D.E. Bredesen, J.D. Cooper, J.S. Valletta, W.C. Mobley, and F.M. Longo. 1997. Absence of p75NTR causes increased basal forebrain cholinergic neuron size, choline acetyltransferase activity, and target innervation. J. Neurosci. 17:75947605.
Related Article