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Neogenin-RGMa Signaling at the Growth Cone Is Bone Morphogenetic Protein-independent and Involves RhoA, ROCK, and PKC

2007; Elsevier BV; Volume: 282; Issue: 22 Linguagem: Inglês

10.1074/jbc.m610901200

1083-351X

Sabine Conrad, Harald Genth, Fred Hofmann, Ingo Just, Thomas Skutella,

Neurogenesis and neuroplasticity mechanisms

The repulsive guidance molecule RGMa has been shown to induce outgrowth inhibition of neurites by interacting with the transmembrane receptor neogenin. Here we show that RGMa-induced growth cone collapse is mediated by activation of the small GTPase RhoA, its downstream effector Rho kinase and PKC. In contrast to DRG cultures from neogenin-/- mice, in which no RGMa-mediated growth cone collapse and activation of RhoA occurred, treatment of wild type DRG neurites with soluble RGMa led to a marked activation of RhoA within 3 min followed by collapse, but left Rac1 and Cdc42 unaffected. Furthermore, preincubation of DRG axons with the bone morphogenetic protein (BMP) antagonist noggin had no effect on RGMa-mediated growth cone collapse, implying that the role of RGM in axonal guidance is neogenin- and not BMP receptor-dependent. Pretreatment with 1) C3-transferase, a specific inhibitor of the Rho GTPase; 2) Y-27632, a specific inhibitor of Rho kinase; and 3) Gö6976, the general PKC inhibitor, strongly inhibited the collapse rate of PC12 neurites. Growth cone collapse induced by RGMa was abolished by the expression of dominant negative RhoA, but not by dominant negative Rac1. In contrast to RGMa, netrin-1 induced no growth cone retraction but instead reduced RGMa-mediated growth cone collapse. These results suggest that activation of RhoA, Rho kinase, and PKC are physiologically relevant and important elements of the RGMa-mediated neogenin signal transduction pathway involved in axonal guidance. The repulsive guidance molecule RGMa has been shown to induce outgrowth inhibition of neurites by interacting with the transmembrane receptor neogenin. Here we show that RGMa-induced growth cone collapse is mediated by activation of the small GTPase RhoA, its downstream effector Rho kinase and PKC. In contrast to DRG cultures from neogenin-/- mice, in which no RGMa-mediated growth cone collapse and activation of RhoA occurred, treatment of wild type DRG neurites with soluble RGMa led to a marked activation of RhoA within 3 min followed by collapse, but left Rac1 and Cdc42 unaffected. Furthermore, preincubation of DRG axons with the bone morphogenetic protein (BMP) antagonist noggin had no effect on RGMa-mediated growth cone collapse, implying that the role of RGM in axonal guidance is neogenin- and not BMP receptor-dependent. Pretreatment with 1) C3-transferase, a specific inhibitor of the Rho GTPase; 2) Y-27632, a specific inhibitor of Rho kinase; and 3) Gö6976, the general PKC inhibitor, strongly inhibited the collapse rate of PC12 neurites. Growth cone collapse induced by RGMa was abolished by the expression of dominant negative RhoA, but not by dominant negative Rac1. In contrast to RGMa, netrin-1 induced no growth cone retraction but instead reduced RGMa-mediated growth cone collapse. These results suggest that activation of RhoA, Rho kinase, and PKC are physiologically relevant and important elements of the RGMa-mediated neogenin signal transduction pathway involved in axonal guidance. Neogenin-RGMa signaling at the growth cone is bone morphogenetic protein-independent and involves RhoA, ROCK, and PKC.Journal of Biological ChemistryVol. 285Issue 53PreviewPAGE 16430: Full-Text PDF Open Access During the development of the central and peripheral nervous system, target-derived axon extension is guided by attractive and repulsive diffusible or membrane-bound factors acting over short and long distances (1.Tessier-Lavigne M. Goodman C.S. Science. 1996; 274: 1123-1133Crossref PubMed Scopus (2706) Google Scholar). During axonal chemorepulsion, repulsive guidance cues hinder neurite outgrowth, turn growth cones away from a guidance cue, and in the case of growth cone collapse induce a dramatic retraction of the growth cone. Although the repulsive guidance molecules (RGM) 2The abbreviations used are: RGM, repulsive guidance molecule; DCC, deleted in colorectal carcinoma; RasGAP, Ras GTPase-activating protein; DRG, dorsal root ganglia; PKC, protein kinase C; NGF, nerve growth factor; BMP, bone morphogenetic protein; GST, glutathione S-transferase; GFP, green fluorescent protein. and their receptor neogenin have been identified as guidance factors (2.Monnier P.P. Sierra A. Macchi P. Deitinghoff L. Andersen J.S. Mann M. Flad M. Hornberger M.R. Stahl B. Bonhoeffer F. Mueller B.K. Nature. 2002; 419: 392-395Crossref PubMed Scopus (257) Google Scholar, 3.Brinks H. Conrad S. Vogt J. Oldekamp J. Sierra A. Deitinghoff L. Bechmann I. Varez-Bolado G. Heimrich B. Monnier P.P. Mueller B.K. Skutella T. J. 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Cell Biol. 2006; 173: 47-58Crossref PubMed Scopus (236) Google Scholar) demonstrated that neurite extension of cerebellar granule neurons was inhibited when these cells were plated on a monolayer of RGMa overexpressing cells and that this effect can be blocked with RhoA and Rho kinase inhibitors. Furthermore, increased amounts of GTP-RhoA were observed in these primary neurons after the addition of soluble RGMa. Neogenin, which is expressed by growing nerve cells in the developing vertebrate brain, consists of four immunoglobulin-like domains followed by six fibronectin type III domains, a transmembrane domain, and an intracellular domain (4.Rajagopalan S. Deitinghoff L. Davis D. Conrad S. Skutella T. Chedotal A. Mueller B.K. Strittmatter S.M. Nat. Cell Biol. 2004; 6: 756-762Crossref PubMed Scopus (227) Google Scholar, 8.Fitzgerald D.P. Seaman C. Cooper H.M. Dev. Dyn. 2006; 235: 1720-1725Crossref PubMed Scopus (26) Google Scholar, 9.Vielmetter J. Kayyem J.F. Roman J.M. Dreyer W.J. J. 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Growth Differ. 2004; 46: 481-486Crossref PubMed Scopus (57) Google Scholar, 12.Mehlen P. Cell Death Differ. 2005; 121003Crossref PubMed Scopus (8) Google Scholar, 13.Matsunaga E. Tauszig-Delamasure S. Monnier P.P. Mueller B.K. Strittmatter S.M. Mehlen P. Chedotal A. Nat. Cell Biol. 2004; 6: 749-755Crossref PubMed Scopus (223) Google Scholar, 14.Matsunaga E. Nakamura H. Chedotal A. J. Neurosci. 2006; 26: 6082-6088Crossref PubMed Scopus (104) Google Scholar). Recently Watanabe et al. (15.Watanabe K. Tamamaki N. Furuta T. Ackerman S.L. Ikenaka K. Ono K. Development. 2006; 133: 1379-1387Crossref PubMed Scopus (65) Google Scholar) reported finding no effect of netrin-1 on either axon attraction or repulsion of dorsal root ganglia (DRG) neurites, but did see a general suppression of axon outgrowth from DRG by netrin-1 after 24–48 h in co-culture experiments. In addition to the ligand-receptor interaction of RGMa and neogenin it has been shown that RGMa, RGMb (dragon), and RGMc (hemojevelin) act as BMP co-receptors (16.Samad T.A. Srinivasan A. Karchewski L.A. Jeong S.J. Campagna J.A. Ji R.R. Fabrizio D.A. Zhang Y. Lin H.Y. Bell E. Woolf C.J. J. Neurosci. 2004; 24: 2027-2036Crossref PubMed Scopus (93) Google Scholar, 17.Babitt J.L. Zhang Y. Samad T.A. Xia Y. Tang J. Campagna J.A. Schneyer A.L. Woolf C.J. Lin H.Y. J. Biol. Chem. 2005; 280: 29820-29827Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 18.Babitt J.L. Huang F.W. Wrighting D.M. Xia Y. Sidis Y. Samad T.A. Campagna J.A. Chung R.T. Schneyer A.L. Woolf C.J. Andrews N.C. Lin H.Y. Nat. Genet. 2006; 38: 531-539Crossref PubMed Scopus (846) Google Scholar). Although no functional data were provided it was hypothesized that RGM family members might increase the sensitivity of cells in which they are expressed to BMP stimulation, thereby allowing these cells to respond earlier or more robustly to low levels of BMP ligand (18.Babitt J.L. Huang F.W. Wrighting D.M. Xia Y. Sidis Y. Samad T.A. Campagna J.A. Chung R.T. Schneyer A.L. Woolf C.J. Andrews N.C. Lin H.Y. Nat. Genet. 2006; 38: 531-539Crossref PubMed Scopus (846) Google Scholar). It remains open whether BMPs could be involved in mediating the RGMa role in axonal guidance and growth cone collapse. Each guidance molecule has to activate a cascade of intracellular effectors that eventually results in a cytoskeletal rearrangement underlying the guidance of axon extension or repulsion (19.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar, 20.Huber A.B. Kolodkin A.L. Ginty D.D. Cloutier J.F. Annu. Rev. Neurosci. 2003; 26: 509-563Crossref PubMed Scopus (634) Google Scholar). There is a great deal of evidence indicating that axon guidance cue signaling involves the action of proteins belonging to the Rho family of small GTP-binding proteins, key regulators of actin cytoskeletal dynamics. Rho family GTPases orchestrate actin filament assembly and disassembly by controlling actin polymerization, branching, and depolymerization. These GTPases are thus likely to function as key mediators linking the guidance signal to cytoskeletal dynamics and controlling the organization of actin filaments. Indeed, marked changes in the morphology, motility, and pathfinding of axons have been observed after perturbation of the Rho family GTPases in vitro and in vivo (21.Mueller B.K. Annu. Rev. Neurosci. 1999; 22: 351-388Crossref PubMed Scopus (379) Google Scholar, 22.Mueller B.K. Mack H. Teusch N. Nat. Rev. Drug Discov. 2005; 4: 387-398Crossref PubMed Scopus (527) Google Scholar). In general, these studies suggest that Rac and Cdc42 are positive regulators that promote neurite extension, whereas Rho is a negative regulator that causes the inhibition or collapse of growth cones. Several known guidance factors, including netrins, slits, semaphorins, ephrins, receptor tyrosine phosphatases, neurotrophins, cell adhesion receptors, myelin-associated receptors, and also neogenin have been shown to regulate intracellular Rho GTPase activity (4.Rajagopalan S. Deitinghoff L. Davis D. Conrad S. Skutella T. Chedotal A. Mueller B.K. Strittmatter S.M. Nat. Cell Biol. 2004; 6: 756-762Crossref PubMed Scopus (227) Google Scholar, 7.Hata K. Fujitani M. Yasuda Y. Doya H. Saito T. Yamagishi S. Mueller B.K. Yamashita T. J. Cell Biol. 2006; 173: 47-58Crossref PubMed Scopus (236) Google Scholar, 22.Mueller B.K. Mack H. Teusch N. Nat. Rev. Drug Discov. 2005; 4: 387-398Crossref PubMed Scopus (527) Google Scholar). We therefore theorized that different Rho GTPases might have distinct roles in the RGMa guidance signal's mediation of growth cone collapse. To analyze the signal transduction machinery in primary sensory neurons and PC12 cells underlying RGMa-neogenin-induced collapse, we focused on PKC, the small GTPase Rho and its downstream effectors, and serine/threonine Rho kinase. In several cell systems, such as fibroblasts, neuroblastoma cells, neuron-like PC12 cells, and primary neurons, activation of Rho leads to rapid growth cone collapse, neurite retraction, or neurite growth inhibition. These responses are prevented by a specific inhibitor of Rho and the bacterial exoenzyme C3-transferase; C3-transferase ADP ribosylates RhoA, -B, and -C (but not Cdc42 and Rac) at Asn-41, thereby inhibiting these GTPases (23.Aktories K. Just I. Curr. Top. Microbiol. Immunol. 2005; 291: 113-145PubMed Google Scholar, 24.Sekine A. Fujiwara M. Narumiya S. J. Biol. Chem. 1989; 264: 8602-8605Abstract Full Text PDF PubMed Google Scholar). One of the downstream targets of active GTP-bound Rho is the Rho-associated kinase ROCK (25.Redowicz M.J. Arch. Biochem. Biophys. 1999; 364: 122-124Crossref PubMed Scopus (24) Google Scholar), which is specifically inhibited by the pyridine derivative Y-27632 (26.Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2555) Google Scholar). In this study, we analyzed the signal transduction of RGMa-mediated growth cone collapse. Using neogenin knock-out mice we found strong evidence that the interaction of RGMa and neogenin is specific and important in axon repulsion. We could exclude BMP signaling in this context. We used primary sensory neurons and differentiated PC12 cells to examine the effects of expressing mutant forms of Rho and Rac1 in growth cone collapse induced by RGMa. Pull-down assays showed that RhoA mediates chemorepulsion, but not Rac1 or Cdc42. We found that PKC is involved in addition to the RhoA pathway and that regulation of each of these can trigger repulsive behavior mediated by the neogenin receptor. Experiments with netrin-1 indicated that the repulsive effect of RGMa could be silenced by preincubation with netrin-1. Neogenin Mutants−Mice homozygous for neogenin were kindly provided by Dr. M. Tessier-Lavigne (Genentech). In Situ Hybridization−For in situ hybridization experiments, E14.5 BL6 mice embryos were frozen over liquid nitrogen and embedded in OCT. Hybridization was performed with digoxygenin-labeled antisense riboprobes, corresponding to coding frames of mouse RGMa and neogenin cDNA (27.Oldekamp J. Kramer N. Varez-Bolado G. Skutella T. Gene Expr. Patterns. 2004; 4: 283-288Crossref PubMed Scopus (66) Google Scholar, 28.Schmidtmer J. Engelkamp D. Gene Expr. Patterns. 2004; 4: 105-110Crossref PubMed Scopus (81) Google Scholar), on sagittal cryostat sections (20 μm). Dorsal Root Ganglia and PC12 Cell Culture−For differentiation and neurite outgrowth a single cell culture of dorsal root cells was prepared from E12 wild type or neogenin-/- mutant embryos and cultured for 48 h on laminin-coated 12-well plates in Neurobasal medium supplemented with 5% fetal calf serum, B27, glutamine, mitose inhibitors, and 50 ng/ml NGF. To analyze RGMa-induced growth cone collapse in more detail, we also used PC12 cells seeded in 12-well tissue culture plates coated with laminin (20 μg/ml) and cultivated in 500 μl of RMPI 1640 media supplemented with 1% fetal calf serum, 1% l-glutamine, and 1% penicillin/streptomycin for 12 h at 37 °C in aCO2 incubator. 50 ng/ml of NGF was then added to the culture medium and the cells were incubated for an additional 48 h. PCR of DRG, PC12 Cell cDNA, and Embryonic Mouse Brain cDNA−The following primer combinations were used to amplify RGMa, RGMb, BMP-2, and BMP-4: RGMa, 5′-tgcaaaatcctcaagtg, 5′-cggcggcggcggcggccacggctc; RGMb, 5′-ggtgattgccaacagcc, 5′-catccctacatcccccacagctgc; BMP-2, 5′-aagaagccgtggaggaactt, 5′-tgacgcttttctcgtttgtg; BMP-4, 5′-tcttcaacctcagcagcatcc, 5′-ccaatcattccagcccacg. Coimmunoprecipitation of Clustered RGMa and the Extracellular Domain of Neogenin−HEK293 cell supernatant containing neogenin-AP (neogenin in AP-tag vector) or supernatant containing RGMa-Fc (RGMa in pigtail plus vector) was concentrated with Nanosep 10K columns. Samples containing equal amounts of control supernatants (AP-tag alone) or supernatants containing neogenin-AP were mixed with samples containing clustered RGMa-Fc. These mixtures were incubated at room temperature for 2 h. Then an equal volume of an immunoprecipitation buffer (20 mm Tris, pH 8.0, 140 mm NaCl, 0.5 mm EDTA, and 2% Nonidet P-40) was added to each mixture, and the samples were centrifuged at 15,000 × g for 15 min at 4 °C. Supernatants were recovered and mixed with monoclonal AP-Sepharose beads (GenHunter), and the samples were incubated under stirring at 4 °C for 2 h. The samples were centrifuged again at 5,000 × g for 1 min, and the immune complexes were washed twice with immunoprecipitation buffer and once with phosphate-buffered saline. Then the beads were mixed with SDS sample buffer and boiled for 2 min at 100 °C. Immunoprecipitated proteins were separated using SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblot analysis with antibodies to AP-TAG and RGM protein. Immunohistochemistry−After fixation with 4% paraformaldehyde, the PC12 cells were stained with monoclonal antibody specific for RhoA and a polyclonal antibody for neogenin (both from Santa Cruz, Biotechnology). As negative control we used a specific blocking peptide for the neogenin antibody (Santa Cruz, Biotechnology); for RhoA the primary antibody was omitted in the control. An alkaline phosphatase-conjugated antibody (Dianova) was used as a secondary antibody, and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrate. PC12 cells were also stained with Alexa 488-phalloidin (Molecular Probes). The In Situ Cell Death Detection Kit (Roche) and caspase-3 immunostaining (Cell Signaling Technology, Roche) were used for the analysis of cell death. Collapse Assay−RGMa-Fc was generated in a pigtail plus vector (Novagen) by inserting the mouse RGMa coding sequence (27.Oldekamp J. Kramer N. Varez-Bolado G. Skutella T. Gene Expr. Patterns. 2004; 4: 283-288Crossref PubMed Scopus (66) Google Scholar, 28.Schmidtmer J. Engelkamp D. Gene Expr. Patterns. 2004; 4: 105-110Crossref PubMed Scopus (81) Google Scholar) without the signal peptide or glycosylphosphatidylinositol anchor and amplifying by PCR with the HindIII and XbaI sites. RGMa-Fc was stably transfected into HEK293 cells, and the supernatants with the fusion protein were further processed for collapse experiments. The proteins were clustered with an anti-human IgG1-Fc antibody (Calbiochem) to induce a stronger collapsing activity of the RGMa fusion protein. 500 μl of medium containing 60 μl of RGMa-pigtail protein clustered (RGMa-pI + IgG) were used for the collapse experiments; pigtail protein alone (pI + IgG) served as the control. In all experiments, a baseline was set to check the vitality of the differentiated DRGs or PC12 cells. RGMa-pI + IgG or pI + IgG was added 5 min after the beginning of the collapse assay, and every experiment was observed and recorded for 30 min with a Live Cell Imaging System (Zeiss) at a magnification of ×20. The collapsed and non-collapsed growth cones (n = 100) were counted in each culture, and the data were analyzed with Microsoft Excel. Data are expressed here as mean ± S.E. For toxin A experiments, the cells were treated with 5–10 nm toxin A and incubated for 2 h before start of the collapse experiments after addition of RGMa-pI + IgG. For C3-transferase experiments, 500 μl of medium containing 15 μl of C2–C3 (300 ng/ml) was added to the PC12 cell cultures 5 h before collapse induction by RGMa-pI + IgG. Rho kinase was inhibited by adding 100 nm Y-27632 Rho kinase inhibitor (Calbiochem) to the cultures 30 min before the RGMa-pI + IgG collapse experiments. For inhibition of protein kinase C, the cells were pretreated for 24 h with 20 μl of 10 nm to 10 μm PKC inhibitor (Calbiochem). Direct BMP-receptor activation was blocked by preincubating the BMP antagonist noggin at 100 ng/ml for 1 h. Netrin-1 Experiments−300 ng/ml netrin-1 (R&D Systems) was preincubated for 30 min or incubated in parallel with RGMa-Fc for collapse experiments with PC12 cells. For transfection with AMAXA, the genes for wild-type normal active (RhoA WT; Rac1 WT), dominant negative (RhoA N19, Rac1 T17N), or constitutive active (RhoA V14; Rac1 G12V) RhoA or Rac1 were cloned into pEGFP-C1 eukaryotic expression vector. After transfection the cells were plated on laminin-coated wells as described above, followed by the collapse assay. Glutathione S-Transferase (GST) Fusion Protein Pull-down Assay−This assay is based on the capability of GST-Rhotekin and GST-PAK1 to bind to GTP-bound Rho, Cdc42, and Rac1, respectively. The fusion proteins were prepared after induction with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside and bound to glutathione-Sepharose beads 4B (GE Healthcare). PC12 cells were plated on laminin-coated 12-well dishes. After 24 h, RGMa-pI + IgG (40 μg/ml) or pI + IgG (40 μg/ml) was added to the cultures, and the cells were lysed with GST-FISH buffer after 3, 6, 9, and 12 min of incubation. The lysates were bound to the prepared beads, and aliquots were taken to determine total amounts of Rho, Cdc42, and Rac1 by immunodetection on Western blots using monoclonal RhoA, Cdc42, and Rac1-specific antibodies (Santa Cruz Biotechnology). Total RhoA was used for quantification of the protein contents. For the quantification of Rho and PKC kinase activities, we used non-radioactive enzyme-linked immunosorbent assay kits for Rho (Cyclex Co., Ltd.) and PKC (Stressgene). Kinase activities were inhibited with 100 nm of the specific inhibitor for Rho and 2 μm of the specific inhibitor for PKC. The kinase activities were measured at a wavelength of 450 nm. Statistical Analysis of the Different Groups−Statistics were derived by one-way analysis of variance with at least 3 independent experiments performed in each experimental setting. In all analyses, differences were considered statistically significant at p = 0.01. The criteria for collapsed DRG and PC12 neurites were a total loss of growth cones and retraction of the neurites. Images were collected with the Zeiss Live Cell Imaging System and AxioVision analysis software. RGMa Induces Growth Cone Collapse in Neogenin-expressing Primary Dorsal Root Ganglion Cells−Nonradioactive in situ hybridization showed that neogenin is expressed by dorsal root ganglia at E14.5, but RGMa is not (Fig. 1A). Furthermore, coimmunoprecipitation assays confirmed that neogenin and RGMa interact directly (Fig. 1, B and C). In a first step, we analyzed the growth cone collapse activity of RGMa with primary dorsal root ganglion cells from wild-type and neogenin knock-out mutants (Fig. 2, A–D). In the in vitro assay, the growth cones of NGF-stimulated dorsal root ganglion cells started to collapse 3–9 min after the addition of RGMa-Fc to the culture medium (Fig. 2, A and D). No effect was observed after the application of Fc-control protein (Fig. 2, B and D). Furthermore, no RGMa-induced growth cone collapse was observed in DRG cells prepared from neogenin-/- mutants (Fig. 2C,D). The RGMa protein was clustered with a specific antibody against the anti-human IgG1-Fc domain. In comparison to unclustered RGMa, which also induced growth cone retraction in PC12 cells (data not shown), the clustered form induced a faster and stronger effect. To determine whether neogenin directly regulates Rho GTPase activity, we carried out biochemical assays with the DRG cultures and observed a marked activation of RhoA in wild-type but not in neogenin-/- mutants within 3 min after the application of clustered RGMa (Fig. 2E). Furthermore, no activation of Rac1 and Cdc42 was observed in wild-type DRGs (data not shown). BMPs Are Not Involved in RGMa-mediated Growth Cone Collapse of DRGs−The next experimental question concerned the involvement of the BMP pathway in the RGMa-induced collapse response. Because it has been shown that RGMa, RGMb (dragon), and RGMc (hemojevelin) act as BMP-2 and BMP-4 co-receptors under certain conditions (16.Samad T.A. Srinivasan A. Karchewski L.A. Jeong S.J. Campagna J.A. Ji R.R. Fabrizio D.A. Zhang Y. Lin H.Y. Bell E. Woolf C.J. J. Neurosci. 2004; 24: 2027-2036Crossref PubMed Scopus (93) Google Scholar, 17.Babitt J.L. Zhang Y. Samad T.A. Xia Y. Tang J. Campagna J.A. Schneyer A.L. Woolf C.J. Lin H.Y. J. Biol. Chem. 2005; 280: 29820-29827Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 18.Babitt J.L. Huang F.W. Wrighting D.M. Xia Y. Sidis Y. Samad T.A. Campagna J.A. Chung R.T. Schneyer A.L. Woolf C.J. Andrews N.C. Lin H.Y. Nat. Genet. 2006; 38: 531-539Crossref PubMed Scopus (846) Google Scholar), we tested whether BMPs could be involved in mediating RGMa-mediated growth cone collapse. PCR analysis revealed that DRG and PC12 cells express BMP-2, but not BMP-4 (Fig. 3A). Next, we tested whether the BMP antagonist noggin influences RGMa-induced growth cone collapse. We found no case in which the RGMa-induced collapse response could be influenced by noggin in primary DRG neurites (Fig. 3B) or PC12 cells (data not shown). This is consistent with the results of neogenin mutant experiments and implies that neogenin rather than a BMP receptor is necessary to mediate the RGMa signal to the cellular pathway to initiate the collapse response in the primary neurons. We believe that the results are strong evidence that neogenin plays a role in RGMa-induced signaling events. Expression of Neogenin and Rho GTPases but Not RGMs in PC12 Neurons−We selected PC12 cells to study growth cone collapse induced by RGMa because, as confirmed by PCR analysis, these cells constitutively express neogenin but not RGMa or RGMb (Fig. 4A). To analyze the role of the Rho GTPase in RGMa-induced collapse of PC12 cells, we next used immunohistochemistry to determine whether RhoA and neogenin proteins are expressed by these cells (Fig. 4, B and C). Differentiated PC12 cells growing on a laminin substrate were fixed after 48 h of NGF treatment and stained with neogenin- and RhoA-specific antibodies. Neogenin and RhoA were strongly detected in the cell bodies, the growth cones, and to a lesser extent in the neurites of PC12 cells as well (Fig. 4, B, B/1, C, and C/1). The growth cones of the PC12 cells also stained positive with Alexa 488-phalloidin (Fig. 4D). RGMa Induces Growth Cone Collapse in PC12 Cells−We used differentiated PC12 cells as a cell culture model to analyze RGMa-induced growth cone collapse in more detail. During live-cell imaging, clustered RGMa induced a 85–95% collapse of PC12 cells (Fig. 5, B and C), and retraction of the growth cones occurred ∼3–6 min after the addition of 4.8 μg/ml of RGMa to the culture medium. We observed no unspecific, spontaneously collapsed PC12 cells in time-lapse experiments after the addition of the RGMa-Fc protein, and the onset of growth cone retraction was stable in the same time frame. In contrast, the addition of the clustered control protein did not induce collapse (Fig. 5, A and C). Toxin A, C2–C3, Rho Inhibitor, and Neogenin Antibody Block RGMa-induced Growth Cone Collapse−To test whether Rho GTPases are involved in RGMa-induced growth cone collapse via the neogenin receptor, we first examined the effect of toxin A, an inhibitor of Rho GTPases (Fig. 5C; supplementary Fig. S1A). Toxin A completely abolished the repulsive effect of clustered RGMa on PC12 growth cones. The neurites did not show retraction, leaving growth cones with long, hair-like protrusions. The collapse-inducing activity of clustered RGMa was also abolished after PC12 cells were incubated with C2–C3 (Fig. 5C; supplementary Fig. 1B), whereas individual components of the fusion toxin (C2II or C3) did not block the RGMa-induced collapse of PC12 cells (data not shown). The fraction of collapse-resistant PC12 cells increased from 5 to 10% in control cultures to 80% in C2–C3-treated cultures, and PC12 cells advanced despite the presence of RGMa. Similar results were obtained after inhibition of ROCK by the Rho inhibitor Y-27632. Two hours before the addition of RGMa, PC12 cells were treated with 10 mm Y-27632 (Fig. 5C; supplementary Fig. 1C). Inhibition of ROCK reduced the collapse-inducing activity of RGMa significantly, and the number of non-collapsed PC12 cells increased from 5–10 to 85–90%. To investigate the functional interaction between RGMa and neogenin, we further treated the PC12 culture with an antibody directed against the ectodomain of neogenin. This pre-treatment with the functional antibody blocked the collapse of growth cones by 75–80% (Fig. 5C; supplementary Fig. 1D). PKC Is Involved in Neogenin Signal Transduction−In a parallel approach, we also tested whether PKC is involved in the neogenin-mediated signal transduction pathway. PKC comprises a family of serine/threonine protein kinases that are widely distributed in a variety of tissues with high concentrations in neuronal tissues. We tested Gö6976, a general PKC inhibitor that influences all isoforms in their ability to inhibit RGMa-induced growth cone collapse of PC12 cells. Dose-response studies (10 nm to 10 μm) were carried out with Gö6976 (data not shown), and the strongest effects were seen with 100 nm. Treatment with Gö6976 significantly reduced neurite growth cone collapse after the addition of clustered RGMa (Fig. 5C; supplementary Fig. 1E). Quantification of neurite retraction outgrowth suggested that the effect of this PKC inhibitor was comparable with that of Y27632, a ROCK inhibitor that also blocks RGMa-induced neurite retraction. Expression of Dominant Negative RhoA, but Not Rac1 Inhibits RGMa-induced Growth Cone Collapse−To further examine the role of Rho family GTPases in mediating the growth cone collapse induced by RGMa, we expressed fusion proteins of normal