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Nogo-A expression in mature oligodendrocytes of rat spinal cord in association with specific molecules
Neuroscience Letters 332 (2002) 37–40 www.elsevier.com/locate/neulet
Nogo-A expression in mature oligodendrocytes of rat spinal cord in association with specific molecules Masanori Taketomi a,b,*, Nagatoki Kinoshita c, Kazushi Kimura a, Masaaki Kitada a, Toru Noda a, Hiroaki Asou d, Takashi Nakamura b, Chizuka Ide a a
Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan Department of Orthopaedic and Musculoskeletal Surgery, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan c Cellular Morphogenesis, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan d Department of Neurobiology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
b
Received 19 July 2002; received in revised form 6 August 2002; accepted 9 August 2002
Abstract Nogo-A is known as an oligodendrocyte/myelin-associated molecule having an inhibitory effect on neurite outgrowth in the central nervous system. During development, starting from P21 Nogo-A was detected in the cytoplasm of mature oligodendrocytes with compact myelin sheaths in the rat spinal cord. COS7 cells transfected with recNogo-A displayed strong Nogo-A immunoreactivity in their cytoplasm as well as on the mitotic spindle. Nogo-A was not detected in membrane protein fractions from transfected plus biotinylated COS7 cells. Nogo-A was co-immunoprecipitated with a-tubulin and myelin basic protein (MBP) from rat brain tissue. These results show that Nogo-A is expressed in association with tubulin and MBP in the mature oligodendrocytes. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nogo-A; Oligodendrocyte; Myelin; Tubulin; Myelin basic protein; Development
Nerve regeneration in the adult mammalian central nervous system (CNS) is highly restricted. It is known that myelination progressively reduces the ability of neuronal regeneration in the CNS. Neuronal growth inhibitory proteins (NI35/250) have been identified in the myelin fraction from CNS tissue [2]. Recently, the bovine molecule corresponding to NI220 has been cloned, and named Nogo-A, -B, and -C [3,5,12]. Nogo-A, -B, and -C share a common domain (Nogo-66) in their carboxy-terminal region, which is flanked by two hydrophobic regions. Two hypotheses have been proposed for the topology of Nogo-A in terms of neurite outgrowth inhibitory activity. One hypothesis is that Nogo-66 is exposed to the extracellular space, and has an inhibitory effect on nerve regeneration [5]. The other hypothesis is that a region near the N-terminus is considered to be located extracellularly [3,12]. However, sequence analysis of Nogo has revealed no motif associated with extracellular functions such as cell adhesion or axonal contact [3]. It is reported that the nogo gene product is
* Corresponding author. Tel.: 1 81-75-753-4337; fax: 1 81-75751-7286. E-mail address: [email protected] (M. Taketomi).
located intracellularly rather than in association with the plasma membrane [7]. There have been a few detailed studies on the cellular and subcellular distribution of Nogo-A [6,15]. In the present study, by using polyclonal antibodies specific for Nogo-A, we examined the distribution of Nogo-A in oligodendrocytes during development in rat spinal cord. Nogo-A was detected only in the mature oligodendrocytes with compact myelin sheaths. The immunoelectron microscopy showed that Nogo-A was mainly localized within the cytoplasm, but not on the plasma membrane of oligodendrocytes or transfected COS7 cells. Immunoblot analysis also showed that Nogo-A was not present in the membrane protein fraction from transfected plus biotinylated COS7 cells. The mitotic spindle of transfected COS7 cells was strongly immunostained by the antiNogo-A antibody. Nogo-A was co-immunoprecipitated with a-tubulin from the COS7 lysates, and with myelin basic protein (MBP) as well as a-tubulin from the lysate of spinal cord and brain. Such findings suggest that Nogo-A is associated with tubulin and MBP in the cytoplasm of mature oligodendrocytes. The full-length sequence of Nogo-A (KIAA0886) was amplified by the polymerase chain reaction and ligated into the pEF-BOS-Myc to generate a plasmid encoding
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 91 0- 2
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M. Taketomi et al. / Neuroscience Letters 332 (2002) 37–40
Fig. 1. (A) In the immunoblot, anti-Nogo-A antidody and anti-myc antibody show a MW of 220 K. Pre-incubation of the anti-Nogo-A antibody with peptide 472 abolished the band. (B) The anti-Nogo-A antibody immunostained the oligodendrocytes. (C) Pre-incubation of anti-Nogo-A antibody with peptide 472 abolishes the immunostaining. (D) A mitotic spindle in this transfected COS7 cell is immunostained by anti-Nogo-A antibody. (E) Anti-Nogo-A antibody stains the cytoplasm in a lace-like pattern. (F, I, M) Double-immunostaining with anti-Nogo-A antibody (red) and anti-MBP antibody (green). (G, J, N) Double-immunostaining with anti-Nogo-A antibody (red) and anti-a-tubulin antibody (green). (H, K, L, O, P) Conventional electron microscopy (arrowhead: inner loops; the area of double arrows and arrow: outer loops). P1: F–H; P12: I–L; P21: M–P. Scale bars: white bar, 100 mm; (H, K, O), 1 mm; (L, P), 100 nm.
Nogo-A fused to the Myc epitope (Myc -Nogo-A) at the N terminus. COS-7 cells were transfected by using a LipofectAMINE PLUS TM Reagent Kit (Invitrogen, Carlsbad, CA). The Nogo-A-specific peptide (Pep472, amino acids 623– 640) was used as an immunogen to generate an anti-Nogo antiserum in the rabbit, and the antibody was affinity-purified. The cervical spinal cords (C4) of male Wistar rats from postnatal day 1 (P1)–P21 and adult (over 8 weeks) were used. Perfusion, fixation and immunostaining were performed as previously described[8,14].The primary antibodies used were as follows: anti-Nogo-A antibody (3.25 mg/ml), Rip (mouse monoclonal antibody, Iowa Hybridoma Bank, Iowa, IA; 1:400), mouse monoclonal anti-MBP (epitopes 67–74) antibody (Sigma, St. Louis, MO; 1:1600), mouse monoclonal anti-a-tubulin antibody (Sigma, 1:1000) and anti-rabbit Ig antibody conjugated to Nanogold (Nanoprobe, Yaphank, NY; 1:100). Biotinylation of cell surface proteins was performed as described previously [10]. Immunoprecipitation assay was performed as described previously with anti-Nogo-A antibody, anti-atubulin antibody, anti-MBP antibody and rabbit IgG by using Protein G-Sepharose (Amersham, Buckinghamshire, UK) [13]. Immunoblot analysis was performed using antibodies as follows; 9E10 antibody as an anti-myc antibody (kindly donated by Prof. K. Kaibuchi, Nagoya Univ.,
Japan), anti-Nogo-A antibody, anti-a-tubulin antibody and anti-MBP antibody. The specificity of the Nogo-A antibody was examined by immunoblotting and immunocytochemistry. By the former means, anti-Nogo-A antibody and anti-myc antibody recognized a protein of molecular weight (MW , 220 KD) from the rat brain homogenate and from transfected COS7 cells (Fig. 1A). This MW coincided with that of Nogo-A. Preincubation of Nogo-A antibody with the peptide 472 abolished the staining completely both in immunoblots and in immunocytochemically stained sections of rat spinal cord, and in transfected COS-7 cells (Fig. 1A–C). We examined whether anti-Nogo-A antibody could specifically stain oligodendrocytes by immunocytochemistry. Both Nogo-A antibody and Rip (a marker of mature oligodendrocyte) were detected in the soma of the same oligodendrocytes by confocal laser scanning microscopy (data not shown) [4]. These data show that the anti-Nogo-A antibody is specific for Nogo-A. In transfected COS7 cells immunoreactive Nogo-A was found on mitotic spindles and distributed in a lace-like pattern in the cytoplasm (Fig. 1D,E). Electron microscopy showed that there were occasional axons surrounded by only one or two myelin lamellae at P1 (Fig. 1H). Nogo-A was detected only after permeabilization. An immunoreaction with anti-MBP antibody was already found in some developing myelin sheaths (Fig. 1F).
M. Taketomi et al. / Neuroscience Letters 332 (2002) 37–40
Fig. 2. Immunoelectron microscopy with anti Nogo-A antibody; (A) mature oligodendrocyte with compact myelin sheaths (preembedding method). (B) Myelinated axon (post-embedding method). (C) Transfected COS7 cell (arrows: cytoskeletal pattern). Scale bar: (A, B), 100 nm; (C), 1 mm.
However, Nogo-A was not found in any developing glial cells at this stage. We noted that Nogo-A was detected in the axons that were also positive for a-tubulin in the corticospinal tract at P1 (Fig. 1G). At P12, numerous axons were surrounded by myelin sheaths of an immature type having thick cytoplasmic inner and outer loops (Fig. 1K,L). The number of oligodendrocytes immunoreactive with antiMBP antibody increased, but immunoreactivity for NogoA was not found on those oligodendrocytes (Fig. 1I,J). At P21, almost all of the axons were surrounded by thick myelin sheaths of the mature type consisting of compact myelin with almost no cytoplasmic remnants of inner and outer loops, except for occasional outer tongues at the outer surface (Fig. 1O,P). Nogo-A immunostaining was found in the soma as well as in cellular processes of mature oligodendrocytes with compact myelin sheaths at this time. Some Nogo-A-positive oligodendrocytes were also positive for MBP and for a-tubulin (Fig. 1M,N). MBP has 4 isoforms, which differ from each other in expression during oligodendrocyte development. Isoforms of 21.5 KD and 17 KD are expressed in the early phase of myelinogenesis, whereas those of 14 KD and 18.5 KD are considered to be involved in compaction of the myelin sheath, as their level increases with oligodendrocyte maturation at the late stage of development [11]. Nogo-A is expressed in MBP-positive mature oligodendrocytes. By pre-embedding method for immunoelectron microscopy, Nogo-A was found only soma and process of oligodendrocyte but neither on the plasma
39
membrane nor in the myelin sheath (Fig. 2A). These findings were confirmed by the post-embedding method using ultrathin cryosections (Fig. 2B). This indicates that Nogo-A is not related to the myelin sheath itself, but may be involved in the formation and/or maintenance of the mature compact myelin sheath. Nogo-A was not detected in astrocytes at any developmental stages (data not shown). Nogo-A immunostaining displayed a cytoskeleton-like pattern in transfected COS7 cells by immunoelectron microscopy (Fig. 2C). These findings indicate that Nogo-A is not associated with the surface plasma membrane, but with some cytoskeletal components. Nogo-A was detected exclusively in the non-biotinylated proteins of the intracellular fraction (Fig. 3A). The fact that Nogo-A was not detected among the biotinylated proteins supports our contention that Nogo-A is not exposed on the surface of COS7 cells. Although Nogo-A has 2 hydrophobic regions, these data show that Nogo-A is localized in the cytoplasmic compartment, but not on the surface of the plasma membrane. The localization of NogoA in association with any specific cellular components was not determined in the present study, but the fact that mitotic spindles were immunostained by anti-Nogo-A suggests that Nogo-A is at least associated with microtubules of the spindle. a-tubulin from transfected COS7 cell lysates was precipitated with anti-Nogo-A antibody (Fig. 3B). Nogo-A was also detected in axons in the dorsal funiculus in the present
Fig. 3. (A) Immunoblot of transfected plus biotinylated COS7 cells lysates. (B) Immunoprecipitation of a-tubulin by antiNogo-A from a transfected COS7 cell lysate. (C) Immunoprecipitation of Nogo-A with a-tubulin, and MBP from rat CNS lysates.
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study. It has been shown by in situ hybridization and immunocytochemistry that Nogo-A is expressed in neurons and the immunoreaction for Nogo-A is found in axons [6,7,15]. The density of the immunoblot band for Nogo-A was higher in the case of immunopreciptation with anti-a-tubulin than with anti-MBP. Therefore, tubulin might have stronger association with Nogo-A than does MBP (Fig. 3C). These data suspected Nogo-A expression would be associated with tubulin and MBP in mature oligodendrocytes. Moreover, this expression suggests that Nogo-A might be associated with microtubules in the axons. Our present results show that Nogo-A was expressed only in mature oligodendrocytes and that Nogo-A was localized in the cytoplasm, but not in the myelin sheath. These findings together with those showing that Nogo-A was precipitated with anti-a-tubulin or anti-MBP indicate that Nogo-A would be mainly a cytosolic component in association with tubulin and MBP. It is localized within the cytoplasm, probably in some relation with tubulin/microtubules. The mature oligodendrocytes responsible for the maintenance of CNS myelin sheaths contain abundant microtubules [17]. Microtubules are few in young oligodendrocytes in culture [1]. In fact, we could not detect a-tubulin in oligodendrocytes at P1~P12. But at P21, there was immunoreactivity for a-tubulin in oligodendrocytes. It is reported that microtubules in mature oligodendrocytes are more resistant to depolymerization than those in young ones [9,16]. On the other hand, MBP is an important constituent for myelin formation, especially in the maturation of the myelin sheath. MBP was precipitated from spinal cord and brain lysates. These data indicate that Nogo-A is strongly associated with tubulin of stable microtubules and MBP in the mature oligodendrocytes. The finding that the density of the Nogo-A band obtained by immunopreciptation with anti-a-tubulin was higher than obtained with anti-MBP indicates that Nogo-A is more intimately associated with a-tubulin. We speculate that Nogo-A might act for myelin sheath maturation in association with a-tubulin and MBP. [1] Barry, C., Pearson, C. and Barbarese, E., Morphological organization of oligodendrocyte processes during development in culture and in vivo, Dev. Neurosci., 18 (1996) 233–242. [2] Caroni, P. and Schwab, M.E., Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading, J. Cell Biol., 106 (1988) 1281–1288. [3] Chen, M.S., Huber, A.B., van der Haar, M.E., Frank, M., Schnell, L., Spillmann, A.A., Christ, F. and Schwab, M.E., Nogo-A is a myelin-associated neurite outgrowth inhibitor
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and an antigen for monoclonal antibody IN-1, Nature, 403 (2000) 434–439. Friedman, B., Hockfield, S., Black, J.A., Woodruff, K.A. and Waxman, S.G., In situ demonstration of mature oligodendrocytes and their processes: an immunocytochemical study with a new monoclonal antibody Rip, Glia, 2 (1989) 380–390. GrandPre, T., Nakamura, F., Vartanian, T. and Strittmatter, S.M., Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein, Nature, 403 (2000) 439–444. Huber, A.B., Weinmann, O., Brosamle, C., Oertle, T. and Schwab, M.E., Patterns of Nogo mRNA and protein expression in the developing and adult rat and after CNS lesions, J. Neurosci., 22 (2002) 3553–3567. Josephson, A., Widenfalk, J., Widmer, H.W., Olson, L. and Spenger, C., NOGO mRNA expression in adult and fetal human and rat nervous tissue and in weight drop injury, Exp. Neurol., 169 (2001) 319–328. Kitada, M., Chakrabortty, S., Matsumoto, N., Taketomi, M. and Ide, C., Differentiation of choroid plexus ependymal cells into astrocytes after grafting into the pre-lesioned spinal cord in mice, Glia, 36 (2001) 364–374. Kuhlmann-Krieg, S., Sommer, I. and Schachner, M., Ultrastructural features of cultured oligodendrocytes expressing stage-specific cell-surface antigens, Brain Res., 467 (1988) 269–280. Munir, M., Correale, D.M. and Robinson, M.B., Substrateinduced up-regulation of Na(1)-dependent glutamate transport activity, Neurochem. Int., 37 (2000) 147–162. Pedraza, L., Fidler, L., Staugaitis, S.M. and Colman, D.R., The active transport of myelin basic protein into the nucleus suggests a regulatory role in myelination, Neuron, 18 (1997) 579–589. Prinjha, R., Moore, S.E., Vinson, M., Blake, S., Morrow, R., Christie, G., Michalovich, D., Simmons, D.L. and Walsh, F.S., Inhibitor of neurite outgrowth in humans, Nature, 403 (2000) 383–384. Shirasu, M., Kimura, K., Kataoka, M., Takahashi, M., Okajima, S., Kawaguchi, S., Hirasawa, Y., Ide, C. and Mizoguchi, A., VAMP-2 promotes neurite elongation and SNAP25 A increases neurite sprouting in PC12 cells, Neurosci. Res., 37 (2000) 265–275. Tokuyasu, K.T., Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy, Histochem. J., 21 (1989) 163–171. Wang, X., Chun, S.J., Treloar, H., Vartanian, T., Greer, C.A. and Strittmatter, S.M., Localization of Nogo-A and Nogo-66 receptor proteins at sites of axon-myelin and synaptic contact, J. Neurosci., 22 (2002) 5505–5515. Wilson, R. and Brophy, P.J., Role for the oligodendrocyte cytoskeleton in myelination, J. Neurosci. Res., 22 (1989) 439–448. Wood, P.M. and Williams, A.K., Oligodendrocyte proliferation and CNS myelination in cultures containing dissociated embryonic neuroglia and dorsal root ganglion neurons, Brain Res., 314 (1984) 225–241.
Nogo-A expression in mature oligodendrocytes of rat spinal cord in association with specific molecules Masanori Taketomi a,b,*, Nagatoki Kinoshita c, Kazushi Kimura a, Masaaki Kitada a, Toru Noda a, Hiroaki Asou d, Takashi Nakamura b, Chizuka Ide a a
Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan Department of Orthopaedic and Musculoskeletal Surgery, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan c Cellular Morphogenesis, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan d Department of Neurobiology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
b
Received 19 July 2002; received in revised form 6 August 2002; accepted 9 August 2002
Abstract Nogo-A is known as an oligodendrocyte/myelin-associated molecule having an inhibitory effect on neurite outgrowth in the central nervous system. During development, starting from P21 Nogo-A was detected in the cytoplasm of mature oligodendrocytes with compact myelin sheaths in the rat spinal cord. COS7 cells transfected with recNogo-A displayed strong Nogo-A immunoreactivity in their cytoplasm as well as on the mitotic spindle. Nogo-A was not detected in membrane protein fractions from transfected plus biotinylated COS7 cells. Nogo-A was co-immunoprecipitated with a-tubulin and myelin basic protein (MBP) from rat brain tissue. These results show that Nogo-A is expressed in association with tubulin and MBP in the mature oligodendrocytes. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nogo-A; Oligodendrocyte; Myelin; Tubulin; Myelin basic protein; Development
Nerve regeneration in the adult mammalian central nervous system (CNS) is highly restricted. It is known that myelination progressively reduces the ability of neuronal regeneration in the CNS. Neuronal growth inhibitory proteins (NI35/250) have been identified in the myelin fraction from CNS tissue [2]. Recently, the bovine molecule corresponding to NI220 has been cloned, and named Nogo-A, -B, and -C [3,5,12]. Nogo-A, -B, and -C share a common domain (Nogo-66) in their carboxy-terminal region, which is flanked by two hydrophobic regions. Two hypotheses have been proposed for the topology of Nogo-A in terms of neurite outgrowth inhibitory activity. One hypothesis is that Nogo-66 is exposed to the extracellular space, and has an inhibitory effect on nerve regeneration [5]. The other hypothesis is that a region near the N-terminus is considered to be located extracellularly [3,12]. However, sequence analysis of Nogo has revealed no motif associated with extracellular functions such as cell adhesion or axonal contact [3]. It is reported that the nogo gene product is
* Corresponding author. Tel.: 1 81-75-753-4337; fax: 1 81-75751-7286. E-mail address: [email protected] (M. Taketomi).
located intracellularly rather than in association with the plasma membrane [7]. There have been a few detailed studies on the cellular and subcellular distribution of Nogo-A [6,15]. In the present study, by using polyclonal antibodies specific for Nogo-A, we examined the distribution of Nogo-A in oligodendrocytes during development in rat spinal cord. Nogo-A was detected only in the mature oligodendrocytes with compact myelin sheaths. The immunoelectron microscopy showed that Nogo-A was mainly localized within the cytoplasm, but not on the plasma membrane of oligodendrocytes or transfected COS7 cells. Immunoblot analysis also showed that Nogo-A was not present in the membrane protein fraction from transfected plus biotinylated COS7 cells. The mitotic spindle of transfected COS7 cells was strongly immunostained by the antiNogo-A antibody. Nogo-A was co-immunoprecipitated with a-tubulin from the COS7 lysates, and with myelin basic protein (MBP) as well as a-tubulin from the lysate of spinal cord and brain. Such findings suggest that Nogo-A is associated with tubulin and MBP in the cytoplasm of mature oligodendrocytes. The full-length sequence of Nogo-A (KIAA0886) was amplified by the polymerase chain reaction and ligated into the pEF-BOS-Myc to generate a plasmid encoding
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 91 0- 2
38
M. Taketomi et al. / Neuroscience Letters 332 (2002) 37–40
Fig. 1. (A) In the immunoblot, anti-Nogo-A antidody and anti-myc antibody show a MW of 220 K. Pre-incubation of the anti-Nogo-A antibody with peptide 472 abolished the band. (B) The anti-Nogo-A antibody immunostained the oligodendrocytes. (C) Pre-incubation of anti-Nogo-A antibody with peptide 472 abolishes the immunostaining. (D) A mitotic spindle in this transfected COS7 cell is immunostained by anti-Nogo-A antibody. (E) Anti-Nogo-A antibody stains the cytoplasm in a lace-like pattern. (F, I, M) Double-immunostaining with anti-Nogo-A antibody (red) and anti-MBP antibody (green). (G, J, N) Double-immunostaining with anti-Nogo-A antibody (red) and anti-a-tubulin antibody (green). (H, K, L, O, P) Conventional electron microscopy (arrowhead: inner loops; the area of double arrows and arrow: outer loops). P1: F–H; P12: I–L; P21: M–P. Scale bars: white bar, 100 mm; (H, K, O), 1 mm; (L, P), 100 nm.
Nogo-A fused to the Myc epitope (Myc -Nogo-A) at the N terminus. COS-7 cells were transfected by using a LipofectAMINE PLUS TM Reagent Kit (Invitrogen, Carlsbad, CA). The Nogo-A-specific peptide (Pep472, amino acids 623– 640) was used as an immunogen to generate an anti-Nogo antiserum in the rabbit, and the antibody was affinity-purified. The cervical spinal cords (C4) of male Wistar rats from postnatal day 1 (P1)–P21 and adult (over 8 weeks) were used. Perfusion, fixation and immunostaining were performed as previously described[8,14].The primary antibodies used were as follows: anti-Nogo-A antibody (3.25 mg/ml), Rip (mouse monoclonal antibody, Iowa Hybridoma Bank, Iowa, IA; 1:400), mouse monoclonal anti-MBP (epitopes 67–74) antibody (Sigma, St. Louis, MO; 1:1600), mouse monoclonal anti-a-tubulin antibody (Sigma, 1:1000) and anti-rabbit Ig antibody conjugated to Nanogold (Nanoprobe, Yaphank, NY; 1:100). Biotinylation of cell surface proteins was performed as described previously [10]. Immunoprecipitation assay was performed as described previously with anti-Nogo-A antibody, anti-atubulin antibody, anti-MBP antibody and rabbit IgG by using Protein G-Sepharose (Amersham, Buckinghamshire, UK) [13]. Immunoblot analysis was performed using antibodies as follows; 9E10 antibody as an anti-myc antibody (kindly donated by Prof. K. Kaibuchi, Nagoya Univ.,
Japan), anti-Nogo-A antibody, anti-a-tubulin antibody and anti-MBP antibody. The specificity of the Nogo-A antibody was examined by immunoblotting and immunocytochemistry. By the former means, anti-Nogo-A antibody and anti-myc antibody recognized a protein of molecular weight (MW , 220 KD) from the rat brain homogenate and from transfected COS7 cells (Fig. 1A). This MW coincided with that of Nogo-A. Preincubation of Nogo-A antibody with the peptide 472 abolished the staining completely both in immunoblots and in immunocytochemically stained sections of rat spinal cord, and in transfected COS-7 cells (Fig. 1A–C). We examined whether anti-Nogo-A antibody could specifically stain oligodendrocytes by immunocytochemistry. Both Nogo-A antibody and Rip (a marker of mature oligodendrocyte) were detected in the soma of the same oligodendrocytes by confocal laser scanning microscopy (data not shown) [4]. These data show that the anti-Nogo-A antibody is specific for Nogo-A. In transfected COS7 cells immunoreactive Nogo-A was found on mitotic spindles and distributed in a lace-like pattern in the cytoplasm (Fig. 1D,E). Electron microscopy showed that there were occasional axons surrounded by only one or two myelin lamellae at P1 (Fig. 1H). Nogo-A was detected only after permeabilization. An immunoreaction with anti-MBP antibody was already found in some developing myelin sheaths (Fig. 1F).
M. Taketomi et al. / Neuroscience Letters 332 (2002) 37–40
Fig. 2. Immunoelectron microscopy with anti Nogo-A antibody; (A) mature oligodendrocyte with compact myelin sheaths (preembedding method). (B) Myelinated axon (post-embedding method). (C) Transfected COS7 cell (arrows: cytoskeletal pattern). Scale bar: (A, B), 100 nm; (C), 1 mm.
However, Nogo-A was not found in any developing glial cells at this stage. We noted that Nogo-A was detected in the axons that were also positive for a-tubulin in the corticospinal tract at P1 (Fig. 1G). At P12, numerous axons were surrounded by myelin sheaths of an immature type having thick cytoplasmic inner and outer loops (Fig. 1K,L). The number of oligodendrocytes immunoreactive with antiMBP antibody increased, but immunoreactivity for NogoA was not found on those oligodendrocytes (Fig. 1I,J). At P21, almost all of the axons were surrounded by thick myelin sheaths of the mature type consisting of compact myelin with almost no cytoplasmic remnants of inner and outer loops, except for occasional outer tongues at the outer surface (Fig. 1O,P). Nogo-A immunostaining was found in the soma as well as in cellular processes of mature oligodendrocytes with compact myelin sheaths at this time. Some Nogo-A-positive oligodendrocytes were also positive for MBP and for a-tubulin (Fig. 1M,N). MBP has 4 isoforms, which differ from each other in expression during oligodendrocyte development. Isoforms of 21.5 KD and 17 KD are expressed in the early phase of myelinogenesis, whereas those of 14 KD and 18.5 KD are considered to be involved in compaction of the myelin sheath, as their level increases with oligodendrocyte maturation at the late stage of development [11]. Nogo-A is expressed in MBP-positive mature oligodendrocytes. By pre-embedding method for immunoelectron microscopy, Nogo-A was found only soma and process of oligodendrocyte but neither on the plasma
39
membrane nor in the myelin sheath (Fig. 2A). These findings were confirmed by the post-embedding method using ultrathin cryosections (Fig. 2B). This indicates that Nogo-A is not related to the myelin sheath itself, but may be involved in the formation and/or maintenance of the mature compact myelin sheath. Nogo-A was not detected in astrocytes at any developmental stages (data not shown). Nogo-A immunostaining displayed a cytoskeleton-like pattern in transfected COS7 cells by immunoelectron microscopy (Fig. 2C). These findings indicate that Nogo-A is not associated with the surface plasma membrane, but with some cytoskeletal components. Nogo-A was detected exclusively in the non-biotinylated proteins of the intracellular fraction (Fig. 3A). The fact that Nogo-A was not detected among the biotinylated proteins supports our contention that Nogo-A is not exposed on the surface of COS7 cells. Although Nogo-A has 2 hydrophobic regions, these data show that Nogo-A is localized in the cytoplasmic compartment, but not on the surface of the plasma membrane. The localization of NogoA in association with any specific cellular components was not determined in the present study, but the fact that mitotic spindles were immunostained by anti-Nogo-A suggests that Nogo-A is at least associated with microtubules of the spindle. a-tubulin from transfected COS7 cell lysates was precipitated with anti-Nogo-A antibody (Fig. 3B). Nogo-A was also detected in axons in the dorsal funiculus in the present
Fig. 3. (A) Immunoblot of transfected plus biotinylated COS7 cells lysates. (B) Immunoprecipitation of a-tubulin by antiNogo-A from a transfected COS7 cell lysate. (C) Immunoprecipitation of Nogo-A with a-tubulin, and MBP from rat CNS lysates.
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study. It has been shown by in situ hybridization and immunocytochemistry that Nogo-A is expressed in neurons and the immunoreaction for Nogo-A is found in axons [6,7,15]. The density of the immunoblot band for Nogo-A was higher in the case of immunopreciptation with anti-a-tubulin than with anti-MBP. Therefore, tubulin might have stronger association with Nogo-A than does MBP (Fig. 3C). These data suspected Nogo-A expression would be associated with tubulin and MBP in mature oligodendrocytes. Moreover, this expression suggests that Nogo-A might be associated with microtubules in the axons. Our present results show that Nogo-A was expressed only in mature oligodendrocytes and that Nogo-A was localized in the cytoplasm, but not in the myelin sheath. These findings together with those showing that Nogo-A was precipitated with anti-a-tubulin or anti-MBP indicate that Nogo-A would be mainly a cytosolic component in association with tubulin and MBP. It is localized within the cytoplasm, probably in some relation with tubulin/microtubules. The mature oligodendrocytes responsible for the maintenance of CNS myelin sheaths contain abundant microtubules [17]. Microtubules are few in young oligodendrocytes in culture [1]. In fact, we could not detect a-tubulin in oligodendrocytes at P1~P12. But at P21, there was immunoreactivity for a-tubulin in oligodendrocytes. It is reported that microtubules in mature oligodendrocytes are more resistant to depolymerization than those in young ones [9,16]. On the other hand, MBP is an important constituent for myelin formation, especially in the maturation of the myelin sheath. MBP was precipitated from spinal cord and brain lysates. These data indicate that Nogo-A is strongly associated with tubulin of stable microtubules and MBP in the mature oligodendrocytes. The finding that the density of the Nogo-A band obtained by immunopreciptation with anti-a-tubulin was higher than obtained with anti-MBP indicates that Nogo-A is more intimately associated with a-tubulin. We speculate that Nogo-A might act for myelin sheath maturation in association with a-tubulin and MBP. [1] Barry, C., Pearson, C. and Barbarese, E., Morphological organization of oligodendrocyte processes during development in culture and in vivo, Dev. Neurosci., 18 (1996) 233–242. [2] Caroni, P. and Schwab, M.E., Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading, J. Cell Biol., 106 (1988) 1281–1288. [3] Chen, M.S., Huber, A.B., van der Haar, M.E., Frank, M., Schnell, L., Spillmann, A.A., Christ, F. and Schwab, M.E., Nogo-A is a myelin-associated neurite outgrowth inhibitor
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