Identification of the Neogenin-Binding Site on the Repulsive Guidance Molecule A

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the chick retinotectal system. RGMa, one of the 3 isoforms found in mammals, is involved in laminar patterning, cephalic neural tube closure, axon guidance, and inhibition of axonal regeneration. In addition to its roles in the nervous system, RGMa plays a role in enhancing helper T-cell activation. Binding of RGM to its receptor, neogenin, is considered necessary to transduce these signals; however, information on the binding of RGM to neogenin is limited. Using co-immunoprecipitation studies, we have identified that the RGMa region required for binding to neogenin contains amino acids (aa) 259–295. Synthesized peptide consisting of aa 284–293 directly binds to the extracellular domain (ECD) of recombinant neogenin, and addition of this peptide inhibits RGMa-induced growth cone collapse in mouse cortical neurons. Thus, we propose that this peptide is a promising lead in finding reagents capable of inhibiting RGMa signaling

神経因性疼痛の発症にWnt/βカテニンシグナルが関与する

京都大学0048新制・課程博士博士(医学)甲第18149号医博第3869号新制||医||1002(附属図書館)31007京都大学大学院医学研究科医学専攻(主査)教授 福田 和彦, 教授 渡邉 大, 教授 河野 憲二学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Temporal changes in cell marker expression and cellular infiltration in a controlled cortical impact model in adult male C57BL/6 mice.

BACKGROUND: Traumatic injury to the central nervous system (CNS) triggers a robust inflammatory response that leads to axonal damage and secondary degeneration of spared tissue. In contrast, some immune responses have neuroprotective effects. However, detailed information regarding the dynamics of immune responses after traumatic CNS injury is still unavailable. METHODS: In the present study, changes in the immune cells present in the injured brain, spleen, and cervical lymph nodes (CLNs), which are draining lymphatic organs from the CNS, were analyzed after controlled cortical impact (CCI) by flow cytometry and immunohistochemistry. RESULTS: The number of neutrophils and macrophages that infiltrated the injured brain immediately increased 1 d post-injury and declined rapidly thereafter. In the injured brain, resident microglia showed a bimodal increase during the first week and in the chronic phase (≥3 weeks) after injury. Increase in the Iba-1(+) microglia/macrophages was observed around the injured site. Morphologic analysis showed that Iba-1(+) cells were round at 1 week, whereas those at 3 weeks were more ramified. Furthermore, CD86(+)/CD11b(+) M1-like microglia increased at 4 weeks after CCI, whereas CD206(+)/CD11b(+) M2-like microglia increased at 1 week. These results suggest that different subsets of microglia increased in the acute and chronic phases after CCI. Dendritic cells and T cells increased transiently within 1 week in the injured brain. In the CLNs and the spleen, T cells showed dynamic changes after CCI. In particular, the alteration in the number of T cells in the CLNs showed a similar pattern, with a 1-week delay, to that of microglia in the injured brain. CONCLUSION: The data from this study provide useful information on the dynamics of immune cells in CNS injuries

RGMa aa 284–293 are critical for the interaction with neogenin.

<p>(A) Sequence alignment of the synthesized RGMa peptide. Peptides 1–6 were designed within aa 257–310, which is the region required for the interaction between RGMa and neogenin as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032791#pone-0032791-g001" target="_blank">Fig. 1</a>. (B) RGMa peptide 4 (aa 284–293) directly binds to neogenin's ECD. Relative binding of each RGMa peptide to ELISA plates coated with neogenin-ECD-Fc is shown. p75NTR-Fc was used as a control. Results (%) are shown as the mean ± SEM of 3 independent experiments (**<i>P</i><0.01, *<i>P</i><0.05).</p

RGMa peptide 4 (aa 284–293) inhibits RGMa-induced growth cone collapse.

<p>(A) Effects of RGMa peptide 4 on RGMa-induced growth cone collapse. Cortical neurons were treated with RGMa and/or RGMa peptides for 30 min. The results were quantified from 3 independent experiments, and the percentage of collapsed growth cones is shown (**<i>P</i><0.01). Pep4, peptide 4; PepA, Peptide A; PepB, peptide B. (B) Representative images of growth cones. Red: Phalloidin, Green: Tuj1. Scale bar: 10 µm.</p

The RGMa domain required for binding to neogenin is within aa 259–295.

<p>(A and C) Schematic representation of RGMa and its deletion mutants are shown with their domain structures. Arrowhead shows potential cleavage site. SP: signal peptide, vWD: von Willebrand factor type-D domain, HD: hydrophobic domain, GPI: GPI-anchor. (B, D, and E) Co-immunoprecipitation of full-length neogenin-VSV-G with the deletion mutants of RGMa-Myc. HEK293T cells were transiently transfected with the indicated plasmids. Cell lysates were immunoprecipitated with the anti-VSV-G antibody. The immunoprecipitates (IP) and cell lysates (Lysates) were analyzed by western blotting with anti-Myc and anti-VSV-G antibodies.</p

Neutrophils and macrophages in the spleen after CCI.

<p>A: Dot plots of immune cells in the spleen for live cell analysis. B: Representative cytometry data for neutrophils in the spleen at the indicated days after CCI. C: Representative cytometry data for macrophages in the spleen at the indicated days after CCI. D: Graph illustrating quantitative data for neutrophils and macrophages in the CLNs after CCI. <i>n</i> = 3–5 for each experiment. Neutrophils: ** <i>p</i><0.01 at 28 dpi compared with no injury.</p

Neutrophils and macrophages in the CLNs after CCI.

<p>A: Dot plots of immune cells in the CLNs for live cell analysis. B: Representative cytometry data for neutrophils in the CLNs at the indicated days after CCI. C: Representative cytometry data for CD45⁺/CD11b⁺ macrophages in the CLNs at the indicated days after CCI. D: Graph illustrating quantitative data for neutrophils and macrophages in the CLNs after CCI. <i>n</i> = 3–4 for each experiment. Neutrophils: ** <i>p</i><0.01 at 1 and 14 dpi compared with no injury; macrophages: ** <i>p</i><0.01 at 7, 14, and 21 dpi compared with 1 dpi.</p

T cells and DCs in the cervical lymph nodes

<p>(<b>CLNs</b>) <b>after CCI.</b> A: Representative cytometry data for T cells in the CLNs at the indicated days after CCI. B: Graph illustrating quantitative data for T cells in the CLNs after CCI. C: Representative cytometry data for DCs in the CLNs at the indicated days after CCI. D: Graph illustrating quantitative data for DCs in the CLNs after CCI. (B, D) <i>n</i> = 3–4 for each experiment. T cells: * <i>p</i><0.05 at 28 dpi compared with 1 dpi; DCs: ** <i>p</i><0.01 at 3 dpi compared with no injury, * <i>p</i><0.05 at 7, 14, and 21 dpi compared with no injury.</p