- Email: [email protected]
Peptides derived from repulsive guidance molecule act as antagonists
Biochemical and Biophysical Research Communications 371 (2008) 501–504
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Peptides derived from repulsive guidance molecule act as antagonists Masayoshi Suda a, Katsuhiko Hata a,b, Aika Sawada b, Yuka Nakamura b, Takekazu Kubo a, Atsushi Yamaguchi a, Toshihide Yamashita a,b,* a b
Department of Neurobiology, Graduate School of Medicine, Chiba University, Japan Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e
i n f o
Article history: Received 4 April 2008 Available online 29 April 2008
Keywords: Axon growth Neurite Inhibitor Repulsive guidance molecule
a b s t r a c t Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system [T. Yamashita, B.K. Mueller, K. Hata, Neogenin and RGM signaling in the central nervous system, Curr. Opin. Neurobiol. 17 (2007) 29–34]. Functional studies in Xenopus and chick embryos have revealed the roles of RGM in axon guidance and laminar patterning, while those in mouse embryos have demonstrated its function in regulating the cephalic neural tube closure. Importantly, RGM inhibition enhanced the growth of injured axons and promoted functional recovery after spinal cord injury in rats. Here, we identified two RGMa-derived peptides that functioned as antagonists against RGMa. The peptides studied in vitro dose-dependently suppressed the neurite growth inhibition and growth cone collapse induced by RGMa. Thus, these peptides are promising reagents to treat injuries of the central nervous system. Ó 2008 Elsevier Inc. All rights reserved.
Repulsive guidance molecule (RGM) was originally identified as a membrane-bound protein with repulsive and growth cone collapse-inducing activities in the chick retinotectal system [2]. In the stripe assay, RGM was found to repel temporal retinal axons and collapse growth cones. The overexpression or downregulation of RGM in the chick tectum resulted in pathfinding and mapping errors, proving that the overexpression or downregulation of RGM has a negative effect on topographic mapping along the anterior–posterior axis of the chick tectum [3]. RGMa, one of the three vertebrate homologs of the chick RGM, is expressed in the inner molecular layer of the dentate gyrus; it repels the fibers of the entorhinal cortex, leading to their termination in the outer molecular layer of the gyrus [4]. Loss-of-function experiments have shown the RGMa-mediated restriction of the entorhinal fibers to the outer molecular layer. The RGMa-knockout mice show defects in neural tube closure at the cephalic level, and consequently possess an exencephalic phenotype with major morphological defects in the dorsal brain structures [5]. Thus, RGM appears to play important roles in the nervous system during the developmental stages. In addition to the wide variety of roles of this molecule during the developmental stages as described, both in vitro and in vivo experiments have demonstrated the role of RGMa in pathological state in the adult [6]. RGMa was upregulated around the lesion site in adult rats with a spinal cord injury (SCI) [6,7]. Local application
* Corresponding author. Address: Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Fax: +81 6 68793669. E-mail address: [email protected] (T. Yamashita). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.114
of a neutralizing antibody to RGMa significantly promoted axon regeneration of the corticospinal tract (CST) following SCI [6]. RGMa inhibition was demonstrated to enhance synapse formation in the spared CST proximal to the injured site after SCI [8]. Thus, RGMa is suggested to inhibit axon regeneration and synapse formation in the injured neurons following SCI. From a clinical point of view, it is important to develop reagents that can inhibit the action mediated by RGM, because these reagents can be used for developing promising therapeutic drugs to treat patients with injuries to the central nervous system. Here we developed two peptides from the rat RGMa sequence, and these peptides could dose-dependently block the effect of RGM on neurite growth. Materials and methods Synthesis of peptides. Peptides were synthesized by Custom Peptide Synthesis (Sigma Genosys). The amino acid sequences of the peptides were as follows. Pep1: FADGSKNGGDKHGA (rat RGMa, 226–239), and Pep2: DFQAFRANAESPRR (rat RGMa, 309–322). NEP1–40 was obtained from Sigma. Generation of RGMa-CHO cells. The Flp-In System (Invitrogen, Carlsbad, CA, USA) was used to generate the RGMa-expressing cells, according to the manufacturer’s recommendations. We generated an HA-RGMa fragment containing a signal peptide from the pSecTag2 vector by using 2 restriction endonucleases and ligated it into pcDNA5 FRT (Invitrogen). This construct (pcDNA5 FRT/Igkleader/HA/RGMa) and pOG44 were cotransfected into the Flp-in CHO cells, and stably expressing cells were generated after culture in a medium containing Hygromycin B
502
M. Suda et al. / Biochemical and Biophysical Research Communications 371 (2008) 501–504
(500 mg/ml; Invitrogen) for 2 weeks. The expression of HA-RGMa was confirmed by immunodetection with Western blotting and immunocytochemistry. Neurite outgrowth assay. The cerebellar granule cells obtained from postnatal (P7–9) rat pups were dissociated by trypsinizaion (0.25% trypsin in phosphate-buffered saline [PBS] for 15 min at 37 °C), resuspended in the serum-containing medium, triturated, and washed three times with PBS. The cultures were grown in a serum-free DMEM/F12 medium. The neurons were plated on confluent monolayers of either RGMa-CHO cells or control CHO cells in chamber slides (LabTek II; Nunc). For the neutralizing assay, Pep1 or Pep2 was added to the culture at various concentrations at the time when the coculture was commenced. The cells were fixed in 4% (w/v) paraformaldehyde and immunostained with a monoclonal antibody (TuJ1) that recognized the neuron-specific b tubulin III protein (1:1000; Covance). Subsequently, the length of the longest neurite of each b tubulin III-positive neuron was determined. Data were collected from 100 neurons in each experiment. Data are expressed as means ± SEM (n = 3). Growth cone collapse assay. Cortical neurons obtained from mice on embryonic days 18–20 were dissociated by trypsinization (0.25% trypsin in PBS for 15 min at 37 °C), resuspended in the serum-containing medium, triturated, and washed three times with PBS. The dissociated neurons were plated at 2.5 104 cells/cm2 in the slide chambers coated with poly-L-lysine in a 10% serumcontaining Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen), and L-glutamine (Nacalai Tesque). The cortical neurons were incubated for 12 h, and the cells were further incubated for 1 h in the serum-free Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) and L-glutamine (Nacalai Tesque). Thereafter, 1 lg/ml of recombinant RGMa (R&D systems) was added to the culture medium and incubated for 30 min. Where indicated, Pep1 or Pep2 was added to the culture at various concentrations 30 min before the RGM treatment and included throughout the assay. The cells were fixed with 0.5% glutaralde-
hyde 30 min after the treatment. To visualize F-actin in the growth cones, the fixed neurons were stained with rhodamine-conjugated phalloidin (Invitrogen). To quantify the collapsed growth cones, the neurons were imaged for a fixed exposure time and with fixed camera settings. Data were collected from 100 neurons in each experiment, and expressed as a percent of the number of the neurons calculated. Data are expressed as means ± SEM (n = 3). Statistical analyses. Two-way ANOVA followed by Scheffe’s multiple comparison test was performed to compare the average values of axon length and those of collapsed growth cones. P values of <0.05 were considered significant. Results The neutralizing peptides reduced neurite growth inhibition elicited by RGMa in vitro We have previously reported that RGMa inhibits neurite growth in the cerebellar granule neurons in vitro [6]. In the present study, we employed these neurons obtained from postnatal (P7–9) rat pups because they expressed neogenin, an RGMa receptor [6]. We cultured the cerebellar granule neurons on confluent monolayers of either CHO cells expressing rat RGMa (RGMa-CHO cells) or the control CHO cells for 24 h and assessed the neurite outgrowth rate. Neurite outgrowth was significantly inhibited when grown on RGMa-CHO cells than on control CHO cells (Figs. 1 and 2). Thereafter, we developed two peptides selected from the selected amino acid sequences of rat RGMa. The amino acid sequences of the peptides were as follows. Pep1, FADGSKNGGDKHGA (rat RGMa, 226– 239); and Pep2, DFQAFRANAESPRR (rat RGMa, 309–322). We investigated whether these peptides could neutralize the inhibitory activity of RGMa in the coculture assay (Figs. 1 and 2). The addition of Pep1 and Pep2 significantly reversed the inhibitory effect of RGMa in a dose-dependent manner. Pep1 at the concentration of 4 lM significantly reduced the inhibitory effect of RGMa,
Fig. 1. Pep1 and Pep2 reversed the inhibitory effect of RGMa on neurite growth. The membrane-bound form of RGMa inhibited neurite outgrowth, whereas incubation of the cells with Pep1 or Pep2 eliminated the effects of RGMa. The cerebellar neurons were cultured for 24 h on control CHO cells (control) or RGMa-CHO cells (RGMa) in the presence or absence of 10 lM Pep1 or Pep2. Scale bars: 100 lm.
M. Suda et al. / Biochemical and Biophysical Research Communications 371 (2008) 501–504
**
503
**
70
Nurite Length (µm)
60 50
*
*
+ 4 −
+ 10 −
*
** **
40 30 20 10
0 RGM Pep1 Pep2
− − −
+ − −
+ 1 −
− − −
+ − −
+ − 1
+ − 4
+ − (µm) 10 (µm)
Fig. 2. Dose-dependent effect of Pep1 and Pep2 on neurite growth. The mean length of the longest neurite of each neuron. Data are expressed as means ± SEM of three independent experiments. The cerebellar granule neurons were cultured for 24 h on the control CHO cells or CHO cells expressing HA-tagged RGMa in the presence or absence of Pep1 or Pep2. Both Pep1 and Pep2 suppressed the inhibitory effect of RGMa in a dose-dependent manner. RGM ( ), control CHO cells; RGM (+), RGM-CHO cells; and Pep1 or Pep2, RGM-CHO cells in the presence of Pep1 or Pep2 at the indicated concentrations. Data are represented as means ± SEM of three independent experiments. * p < 0.05, **p < 0.01; one-way ANOVA followed by Scheffe’s multiple comparison test.
whereas Pep2 at 1 lM efficiently inhibited it (Fig. 2). These results demonstrate that the peptides function as neutralizing reagents against RGMa in vitro, and the neutralizing activity of Pep2 was higher than that of Pep1. The neutralizing peptides reduced growth cone collapse induced by RGMa in vitro It has been suggested that some repulsive guidance cues cause growth cone collapse and F-actin reduction in growth cones [9,10]. The cortical neurons from E18 to 20 mice were employed because they express neogenin (data not shown). We cultured these cortical neurons for 13 h, and then the cells were further incubated for
30 min with or without 1 lg/ml of recombinant RGMa. In order to examine whether RGMa induces the growth cone collapse, we employed phalloidin staining of cultured cortical neurons 30 min after RGMa treatment. In the control neurons that were treated with a vehicle, phalloidin-labeled F-actin was clearly detected in a growth cone at the tip of a neurite; Tuj1 antibody was used for the visualization (Fig. 3). The phalloidin-labeled growth cone at the tip of a neurite collapsed 30 min after the RGMa treatment (Figs. 3 and 4). To determine the effect of the peptides, each peptide was added at various concentrations 30 min before the RGMa treatment. As expected, both the peptides blocked the collapse-inducing effect of RGMa in a dose-dependent manner (Figs. 3 and 4). Both Pep1 and Pep2 at concentrations of 4 lM significantly reduced the
Fig. 3. Peptide inhibitors suppress the growth cone collapse elicited by soluble RGMa. The addition of recombinant RGMa (1 lg/ml) for 30 min in the culture medium induced growth cone collapse of the cortical neurons, whereas preincubation of the cells with Pep1 or Pep2 eliminated the effects of RGMa. The cortical neurons were cultured for 13 h and treated with or without soluble RGMa (1 lg/ml). Where indicated, 10 lM Pep1 or Pep2 was added in the culture 30 min before the addition of RGMa. Scale bars: 10 lm.
504
M. Suda et al. / Biochemical and Biophysical Research Communications 371 (2008) 501–504
* *
Collapsed growth cones (%)
60 50
*
40
* **
30 20 10
0 RGM Pep1 Pep2
− − −
+ − −
+ 1 −
+ 4 −
+ 10 −
+ − 1
+ − 4
+ − (µm) 10 (µm)
Fig. 4. Dose-dependent inhibition of growth cone collapse induced by Pep1 and Pep2. Mean values (%) of the collapsed growth cones. Data were collected from 3 to 4 wells and expressed as a percent of the number of the cortical neurons calculated. Data were expressed as means ± SEM. RGM (+), RGMa (1 lg/ml); Pep1 or Pep2, pretreatment with Pep1 or Pep2 at the indicated concentrations. The data are represented as means ± SEM of three independent experiments. *p < 0.05, **p < 0.01; one-way ANOVA followed by Scheffe’s multiple comparison test.
collapse-inducing effect of RGMa (Fig. 4). In this assay, there was no difference in the neutralizing activity between Pep1 and Pep2. However, the results of the neurite growth assay were consistent with those of the growth cone collapse assay. Finally, we assessed whether the neutralizing effect was specific to the two peptides. We employed a peptide antagonist, NEP1–40, which competes for binding of Nogo-66 to the Nogo receptor [11]. NEP1–40 at concentrations of 10 lM did not modulate the collapse-inducing effect of RGMa (RGM induced collapses of 86% growth cones in the absence of NEP1–40, and induced collapses of 86% growth cones in the presence of NEP1–40). The results of these two assays demonstrate that Pep1 and Pep2 function as antagonist peptides against the collapse-inducing effect and neurite growth inhibition mediated by RGMa. Discussion In this study, we identified two peptides that suppress the inhibitory effect of RGMa. We have previously shown that RGM inhibition promoted both axon growth and locomotor recovery following SCI; thus, the identified peptides have potential as therapeutic reagents to treat patients with SCI. RGM is a glycosylphosphatidylinositol-anchored glycoprotein, and it does not bear a significant homology with any other proteins. It contains a putative autoproteolytic or unstable cleavage site; an N-terminal signal peptide; an Arg-Gly-Asp site; a partial, structurally related, von Willebrand factor type D domain; and a hydrophobic domain of unknown function [1,12]. Both the peptides correspond to the sequences of unknown functions present
between the von Willebrand factor type D domain and the hydrophobic domain. We reported previously that the polyclonal antibody raised against Pep2 showed neutralizing activity toward RGMa-induced neurite growth inhibition and was used to treat SCI [1]. An infusion of this antibody in rats with SCI resulted in an enhanced locomotor recovery and axon growth [6]. Thus, the sequence between the von Willebrand factor type D domain and the hydrophobic domain may be critical for the signal transduction of RGMa. Our preliminary data suggested that neither of the peptides inhibited the binding of RGMa to the cell surface that expressed neogenin (data not shown). A possible mechanism of the action is that these peptides modulate the neogenin function by binding, thus rendering the cells insensitive to RGMa. However, since the peptides themselves had no effect on both neurite growth and growth cone collapse, the effects of the peptides are specific to RGMa binding to the receptor. The molecular mechanism of the action of these peptides is currently not known and should be elucidated in the future. Acknowledgments This work was supported by a Research Grant from the National Institute of Biomedical Innovation (05-12) and Grant-in-Aid for Young Scientists (S) from JSPS. References [1] T. Yamashita, B.K. Mueller, K. Hata, Neogenin and RGM signaling in the central nervous system, Curr. Opin. Neurobiol. 17 (2007) 29–34. [2] B. Stahl, B. Müller, Y.V. Boxberg, E.C. Cox, F. Bonhoeffer, Biochemical characterization of a putative axonal guidance molecule of the chick visual system, Neuron 5 (1990) 735–743. [3] E. Matsunaga, H. Nakamura, A. Chedotal, Repulsive guidance molecule plays multiple roles in neuronal differentiation and axon guidance, J. Neurosci. 26 (2006) 6082–6088. [4] H. Brinks, S. Conrad, J. Vogt, J. Oldekamp, A. Sierra, L. Deitinghoff, I. Bechmann, G. Alvarez-Bolado, B. Heimrich, P.P. Monnier, B.K. Mueller, T. Skutella, The repulsive guidance molecule RGMa is involved in the formation of afferent connections in the dentate gyrus, J. Neurosci. 24 (2004) 3862–3869. [5] V. Niederkofler, R. Salie, M. Sigrist, S. Arber, Repulsive guidance molecule (RGM) gene function is required for neural tube closure but not retinal topography in the mouse visual system, J. Neurosci. 24 (2004) 808–818. [6] K. Hata, M. Fujitani, Y. Yasuda, H. Doya, T. Saito, S. Yamagishi, B.K. Mueller, T. Yamashita, RGMa inhibition promotes axonal growth and recovery after spinal cord injury, J. Cell Biol. 173 (2006) 47–58. [7] J.M. Schwab, S. Conrad, P.P. Monnier, S. Julien, B.K. Mueller, H.J. Schluesener, Spinal cord injury-induced lesional expression pattern of the repulsive guidance molecule (RGM), Eur. J. Neurosci. 21 (2005) 1569–1576. [8] A. Kyoto, K. Hata, T. Yamashita, Synapse formation of the cortico-spinal axons is enhanced by RGMa inhibition after spinal cord injury, Brain Res. 1186 (2007) 74–86. [9] J. Fritsche, B.F. Reber, B. Schindelholz, C.E. Bandtlow, Differential cytoskeletal changes during growth cone collapse in response to hSema III and thrombin, Mol. Cell Neurosci. 14 (1999) 398–418. [10] G. Gallo, P.C. Letourneau, Regulation of growth cone actin filaments by guidance cues, J. Neurobiol. 58 (2004) 92–102. [11] T. GrandPre, S. Li, S.M. Strittmatter, Nogo-66 receptor antagonist peptide promotes axonal regeneration, Nature 417 (2002) 547–551. [12] P.P. Monnier, A. Sierra, P. Macchi, L. Deitinghoff, J.S. Andersen, M. Mann, M. Flad, M. Hornberger, B. Stahl, F. Bonhoeffer, B.K. Mueller, RGM is a repulsive guidance molecule for retinal axons, Nature 419 (2002) 392–395.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Peptides derived from repulsive guidance molecule act as antagonists Masayoshi Suda a, Katsuhiko Hata a,b, Aika Sawada b, Yuka Nakamura b, Takekazu Kubo a, Atsushi Yamaguchi a, Toshihide Yamashita a,b,* a b
Department of Neurobiology, Graduate School of Medicine, Chiba University, Japan Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e
i n f o
Article history: Received 4 April 2008 Available online 29 April 2008
Keywords: Axon growth Neurite Inhibitor Repulsive guidance molecule
a b s t r a c t Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system [T. Yamashita, B.K. Mueller, K. Hata, Neogenin and RGM signaling in the central nervous system, Curr. Opin. Neurobiol. 17 (2007) 29–34]. Functional studies in Xenopus and chick embryos have revealed the roles of RGM in axon guidance and laminar patterning, while those in mouse embryos have demonstrated its function in regulating the cephalic neural tube closure. Importantly, RGM inhibition enhanced the growth of injured axons and promoted functional recovery after spinal cord injury in rats. Here, we identified two RGMa-derived peptides that functioned as antagonists against RGMa. The peptides studied in vitro dose-dependently suppressed the neurite growth inhibition and growth cone collapse induced by RGMa. Thus, these peptides are promising reagents to treat injuries of the central nervous system. Ó 2008 Elsevier Inc. All rights reserved.
Repulsive guidance molecule (RGM) was originally identified as a membrane-bound protein with repulsive and growth cone collapse-inducing activities in the chick retinotectal system [2]. In the stripe assay, RGM was found to repel temporal retinal axons and collapse growth cones. The overexpression or downregulation of RGM in the chick tectum resulted in pathfinding and mapping errors, proving that the overexpression or downregulation of RGM has a negative effect on topographic mapping along the anterior–posterior axis of the chick tectum [3]. RGMa, one of the three vertebrate homologs of the chick RGM, is expressed in the inner molecular layer of the dentate gyrus; it repels the fibers of the entorhinal cortex, leading to their termination in the outer molecular layer of the gyrus [4]. Loss-of-function experiments have shown the RGMa-mediated restriction of the entorhinal fibers to the outer molecular layer. The RGMa-knockout mice show defects in neural tube closure at the cephalic level, and consequently possess an exencephalic phenotype with major morphological defects in the dorsal brain structures [5]. Thus, RGM appears to play important roles in the nervous system during the developmental stages. In addition to the wide variety of roles of this molecule during the developmental stages as described, both in vitro and in vivo experiments have demonstrated the role of RGMa in pathological state in the adult [6]. RGMa was upregulated around the lesion site in adult rats with a spinal cord injury (SCI) [6,7]. Local application
* Corresponding author. Address: Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Fax: +81 6 68793669. E-mail address: [email protected] (T. Yamashita). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.114
of a neutralizing antibody to RGMa significantly promoted axon regeneration of the corticospinal tract (CST) following SCI [6]. RGMa inhibition was demonstrated to enhance synapse formation in the spared CST proximal to the injured site after SCI [8]. Thus, RGMa is suggested to inhibit axon regeneration and synapse formation in the injured neurons following SCI. From a clinical point of view, it is important to develop reagents that can inhibit the action mediated by RGM, because these reagents can be used for developing promising therapeutic drugs to treat patients with injuries to the central nervous system. Here we developed two peptides from the rat RGMa sequence, and these peptides could dose-dependently block the effect of RGM on neurite growth. Materials and methods Synthesis of peptides. Peptides were synthesized by Custom Peptide Synthesis (Sigma Genosys). The amino acid sequences of the peptides were as follows. Pep1: FADGSKNGGDKHGA (rat RGMa, 226–239), and Pep2: DFQAFRANAESPRR (rat RGMa, 309–322). NEP1–40 was obtained from Sigma. Generation of RGMa-CHO cells. The Flp-In System (Invitrogen, Carlsbad, CA, USA) was used to generate the RGMa-expressing cells, according to the manufacturer’s recommendations. We generated an HA-RGMa fragment containing a signal peptide from the pSecTag2 vector by using 2 restriction endonucleases and ligated it into pcDNA5 FRT (Invitrogen). This construct (pcDNA5 FRT/Igkleader/HA/RGMa) and pOG44 were cotransfected into the Flp-in CHO cells, and stably expressing cells were generated after culture in a medium containing Hygromycin B
502
M. Suda et al. / Biochemical and Biophysical Research Communications 371 (2008) 501–504
(500 mg/ml; Invitrogen) for 2 weeks. The expression of HA-RGMa was confirmed by immunodetection with Western blotting and immunocytochemistry. Neurite outgrowth assay. The cerebellar granule cells obtained from postnatal (P7–9) rat pups were dissociated by trypsinizaion (0.25% trypsin in phosphate-buffered saline [PBS] for 15 min at 37 °C), resuspended in the serum-containing medium, triturated, and washed three times with PBS. The cultures were grown in a serum-free DMEM/F12 medium. The neurons were plated on confluent monolayers of either RGMa-CHO cells or control CHO cells in chamber slides (LabTek II; Nunc). For the neutralizing assay, Pep1 or Pep2 was added to the culture at various concentrations at the time when the coculture was commenced. The cells were fixed in 4% (w/v) paraformaldehyde and immunostained with a monoclonal antibody (TuJ1) that recognized the neuron-specific b tubulin III protein (1:1000; Covance). Subsequently, the length of the longest neurite of each b tubulin III-positive neuron was determined. Data were collected from 100 neurons in each experiment. Data are expressed as means ± SEM (n = 3). Growth cone collapse assay. Cortical neurons obtained from mice on embryonic days 18–20 were dissociated by trypsinization (0.25% trypsin in PBS for 15 min at 37 °C), resuspended in the serum-containing medium, triturated, and washed three times with PBS. The dissociated neurons were plated at 2.5 104 cells/cm2 in the slide chambers coated with poly-L-lysine in a 10% serumcontaining Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen), and L-glutamine (Nacalai Tesque). The cortical neurons were incubated for 12 h, and the cells were further incubated for 1 h in the serum-free Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) and L-glutamine (Nacalai Tesque). Thereafter, 1 lg/ml of recombinant RGMa (R&D systems) was added to the culture medium and incubated for 30 min. Where indicated, Pep1 or Pep2 was added to the culture at various concentrations 30 min before the RGM treatment and included throughout the assay. The cells were fixed with 0.5% glutaralde-
hyde 30 min after the treatment. To visualize F-actin in the growth cones, the fixed neurons were stained with rhodamine-conjugated phalloidin (Invitrogen). To quantify the collapsed growth cones, the neurons were imaged for a fixed exposure time and with fixed camera settings. Data were collected from 100 neurons in each experiment, and expressed as a percent of the number of the neurons calculated. Data are expressed as means ± SEM (n = 3). Statistical analyses. Two-way ANOVA followed by Scheffe’s multiple comparison test was performed to compare the average values of axon length and those of collapsed growth cones. P values of <0.05 were considered significant. Results The neutralizing peptides reduced neurite growth inhibition elicited by RGMa in vitro We have previously reported that RGMa inhibits neurite growth in the cerebellar granule neurons in vitro [6]. In the present study, we employed these neurons obtained from postnatal (P7–9) rat pups because they expressed neogenin, an RGMa receptor [6]. We cultured the cerebellar granule neurons on confluent monolayers of either CHO cells expressing rat RGMa (RGMa-CHO cells) or the control CHO cells for 24 h and assessed the neurite outgrowth rate. Neurite outgrowth was significantly inhibited when grown on RGMa-CHO cells than on control CHO cells (Figs. 1 and 2). Thereafter, we developed two peptides selected from the selected amino acid sequences of rat RGMa. The amino acid sequences of the peptides were as follows. Pep1, FADGSKNGGDKHGA (rat RGMa, 226– 239); and Pep2, DFQAFRANAESPRR (rat RGMa, 309–322). We investigated whether these peptides could neutralize the inhibitory activity of RGMa in the coculture assay (Figs. 1 and 2). The addition of Pep1 and Pep2 significantly reversed the inhibitory effect of RGMa in a dose-dependent manner. Pep1 at the concentration of 4 lM significantly reduced the inhibitory effect of RGMa,
Fig. 1. Pep1 and Pep2 reversed the inhibitory effect of RGMa on neurite growth. The membrane-bound form of RGMa inhibited neurite outgrowth, whereas incubation of the cells with Pep1 or Pep2 eliminated the effects of RGMa. The cerebellar neurons were cultured for 24 h on control CHO cells (control) or RGMa-CHO cells (RGMa) in the presence or absence of 10 lM Pep1 or Pep2. Scale bars: 100 lm.
M. Suda et al. / Biochemical and Biophysical Research Communications 371 (2008) 501–504
**
503
**
70
Nurite Length (µm)
60 50
*
*
+ 4 −
+ 10 −
*
** **
40 30 20 10
0 RGM Pep1 Pep2
− − −
+ − −
+ 1 −
− − −
+ − −
+ − 1
+ − 4
+ − (µm) 10 (µm)
Fig. 2. Dose-dependent effect of Pep1 and Pep2 on neurite growth. The mean length of the longest neurite of each neuron. Data are expressed as means ± SEM of three independent experiments. The cerebellar granule neurons were cultured for 24 h on the control CHO cells or CHO cells expressing HA-tagged RGMa in the presence or absence of Pep1 or Pep2. Both Pep1 and Pep2 suppressed the inhibitory effect of RGMa in a dose-dependent manner. RGM ( ), control CHO cells; RGM (+), RGM-CHO cells; and Pep1 or Pep2, RGM-CHO cells in the presence of Pep1 or Pep2 at the indicated concentrations. Data are represented as means ± SEM of three independent experiments. * p < 0.05, **p < 0.01; one-way ANOVA followed by Scheffe’s multiple comparison test.
whereas Pep2 at 1 lM efficiently inhibited it (Fig. 2). These results demonstrate that the peptides function as neutralizing reagents against RGMa in vitro, and the neutralizing activity of Pep2 was higher than that of Pep1. The neutralizing peptides reduced growth cone collapse induced by RGMa in vitro It has been suggested that some repulsive guidance cues cause growth cone collapse and F-actin reduction in growth cones [9,10]. The cortical neurons from E18 to 20 mice were employed because they express neogenin (data not shown). We cultured these cortical neurons for 13 h, and then the cells were further incubated for
30 min with or without 1 lg/ml of recombinant RGMa. In order to examine whether RGMa induces the growth cone collapse, we employed phalloidin staining of cultured cortical neurons 30 min after RGMa treatment. In the control neurons that were treated with a vehicle, phalloidin-labeled F-actin was clearly detected in a growth cone at the tip of a neurite; Tuj1 antibody was used for the visualization (Fig. 3). The phalloidin-labeled growth cone at the tip of a neurite collapsed 30 min after the RGMa treatment (Figs. 3 and 4). To determine the effect of the peptides, each peptide was added at various concentrations 30 min before the RGMa treatment. As expected, both the peptides blocked the collapse-inducing effect of RGMa in a dose-dependent manner (Figs. 3 and 4). Both Pep1 and Pep2 at concentrations of 4 lM significantly reduced the
Fig. 3. Peptide inhibitors suppress the growth cone collapse elicited by soluble RGMa. The addition of recombinant RGMa (1 lg/ml) for 30 min in the culture medium induced growth cone collapse of the cortical neurons, whereas preincubation of the cells with Pep1 or Pep2 eliminated the effects of RGMa. The cortical neurons were cultured for 13 h and treated with or without soluble RGMa (1 lg/ml). Where indicated, 10 lM Pep1 or Pep2 was added in the culture 30 min before the addition of RGMa. Scale bars: 10 lm.
504
M. Suda et al. / Biochemical and Biophysical Research Communications 371 (2008) 501–504
* *
Collapsed growth cones (%)
60 50
*
40
* **
30 20 10
0 RGM Pep1 Pep2
− − −
+ − −
+ 1 −
+ 4 −
+ 10 −
+ − 1
+ − 4
+ − (µm) 10 (µm)
Fig. 4. Dose-dependent inhibition of growth cone collapse induced by Pep1 and Pep2. Mean values (%) of the collapsed growth cones. Data were collected from 3 to 4 wells and expressed as a percent of the number of the cortical neurons calculated. Data were expressed as means ± SEM. RGM (+), RGMa (1 lg/ml); Pep1 or Pep2, pretreatment with Pep1 or Pep2 at the indicated concentrations. The data are represented as means ± SEM of three independent experiments. *p < 0.05, **p < 0.01; one-way ANOVA followed by Scheffe’s multiple comparison test.
collapse-inducing effect of RGMa (Fig. 4). In this assay, there was no difference in the neutralizing activity between Pep1 and Pep2. However, the results of the neurite growth assay were consistent with those of the growth cone collapse assay. Finally, we assessed whether the neutralizing effect was specific to the two peptides. We employed a peptide antagonist, NEP1–40, which competes for binding of Nogo-66 to the Nogo receptor [11]. NEP1–40 at concentrations of 10 lM did not modulate the collapse-inducing effect of RGMa (RGM induced collapses of 86% growth cones in the absence of NEP1–40, and induced collapses of 86% growth cones in the presence of NEP1–40). The results of these two assays demonstrate that Pep1 and Pep2 function as antagonist peptides against the collapse-inducing effect and neurite growth inhibition mediated by RGMa. Discussion In this study, we identified two peptides that suppress the inhibitory effect of RGMa. We have previously shown that RGM inhibition promoted both axon growth and locomotor recovery following SCI; thus, the identified peptides have potential as therapeutic reagents to treat patients with SCI. RGM is a glycosylphosphatidylinositol-anchored glycoprotein, and it does not bear a significant homology with any other proteins. It contains a putative autoproteolytic or unstable cleavage site; an N-terminal signal peptide; an Arg-Gly-Asp site; a partial, structurally related, von Willebrand factor type D domain; and a hydrophobic domain of unknown function [1,12]. Both the peptides correspond to the sequences of unknown functions present
between the von Willebrand factor type D domain and the hydrophobic domain. We reported previously that the polyclonal antibody raised against Pep2 showed neutralizing activity toward RGMa-induced neurite growth inhibition and was used to treat SCI [1]. An infusion of this antibody in rats with SCI resulted in an enhanced locomotor recovery and axon growth [6]. Thus, the sequence between the von Willebrand factor type D domain and the hydrophobic domain may be critical for the signal transduction of RGMa. Our preliminary data suggested that neither of the peptides inhibited the binding of RGMa to the cell surface that expressed neogenin (data not shown). A possible mechanism of the action is that these peptides modulate the neogenin function by binding, thus rendering the cells insensitive to RGMa. However, since the peptides themselves had no effect on both neurite growth and growth cone collapse, the effects of the peptides are specific to RGMa binding to the receptor. The molecular mechanism of the action of these peptides is currently not known and should be elucidated in the future. Acknowledgments This work was supported by a Research Grant from the National Institute of Biomedical Innovation (05-12) and Grant-in-Aid for Young Scientists (S) from JSPS. References [1] T. Yamashita, B.K. Mueller, K. Hata, Neogenin and RGM signaling in the central nervous system, Curr. Opin. Neurobiol. 17 (2007) 29–34. [2] B. Stahl, B. Müller, Y.V. Boxberg, E.C. Cox, F. Bonhoeffer, Biochemical characterization of a putative axonal guidance molecule of the chick visual system, Neuron 5 (1990) 735–743. [3] E. Matsunaga, H. Nakamura, A. Chedotal, Repulsive guidance molecule plays multiple roles in neuronal differentiation and axon guidance, J. Neurosci. 26 (2006) 6082–6088. [4] H. Brinks, S. Conrad, J. Vogt, J. Oldekamp, A. Sierra, L. Deitinghoff, I. Bechmann, G. Alvarez-Bolado, B. Heimrich, P.P. Monnier, B.K. Mueller, T. Skutella, The repulsive guidance molecule RGMa is involved in the formation of afferent connections in the dentate gyrus, J. Neurosci. 24 (2004) 3862–3869. [5] V. Niederkofler, R. Salie, M. Sigrist, S. Arber, Repulsive guidance molecule (RGM) gene function is required for neural tube closure but not retinal topography in the mouse visual system, J. Neurosci. 24 (2004) 808–818. [6] K. Hata, M. Fujitani, Y. Yasuda, H. Doya, T. Saito, S. Yamagishi, B.K. Mueller, T. Yamashita, RGMa inhibition promotes axonal growth and recovery after spinal cord injury, J. Cell Biol. 173 (2006) 47–58. [7] J.M. Schwab, S. Conrad, P.P. Monnier, S. Julien, B.K. Mueller, H.J. Schluesener, Spinal cord injury-induced lesional expression pattern of the repulsive guidance molecule (RGM), Eur. J. Neurosci. 21 (2005) 1569–1576. [8] A. Kyoto, K. Hata, T. Yamashita, Synapse formation of the cortico-spinal axons is enhanced by RGMa inhibition after spinal cord injury, Brain Res. 1186 (2007) 74–86. [9] J. Fritsche, B.F. Reber, B. Schindelholz, C.E. Bandtlow, Differential cytoskeletal changes during growth cone collapse in response to hSema III and thrombin, Mol. Cell Neurosci. 14 (1999) 398–418. [10] G. Gallo, P.C. Letourneau, Regulation of growth cone actin filaments by guidance cues, J. Neurobiol. 58 (2004) 92–102. [11] T. GrandPre, S. Li, S.M. Strittmatter, Nogo-66 receptor antagonist peptide promotes axonal regeneration, Nature 417 (2002) 547–551. [12] P.P. Monnier, A. Sierra, P. Macchi, L. Deitinghoff, J.S. Andersen, M. Mann, M. Flad, M. Hornberger, B. Stahl, F. Bonhoeffer, B.K. Mueller, RGM is a repulsive guidance molecule for retinal axons, Nature 419 (2002) 392–395.