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A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord

Nature Medicine volume 12, pages 1380–1389 (2006)Cite this article

Abstract

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell–mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.

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Figure 1: Expression of Sema3A after SCI.
Figure 2: Temporal and quantitative analysis of Sema3A expression after SCI compared with the expression of other representative axonal growth inhibitors.
Figure 3: SM-216289 is a strong and specific Sema3A inhibitor.
Figure 4: SM-216289 enhances axonal regeneration and reduces cavity volume in the injured spinal cord.
Figure 5: SM-216289 enhances axonal regeneration of NP-1–positive axons, including serotonergic (5-HT–positive) raphespinal tract axons, in the injured spinal cord.
Figure 6: SM-216289 induces regeneration of myelinated fibers and enhances functional recovery after SCI.

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Acknowledgements

We are grateful to L. Benowitz (Children's Hospital Boston); H. Fujisawa (Nagoya University); A.L. Kolodkin (Johns Hopkins University); and Y. Ihara and K. Mori (Tokyo University) for reagents. We also thank M. Dezawa, S. Kawabata, U. Uchida, Y. Sugiyama, F. Nakamura, T. Nagai, S. Miyao, T. Harada, K. Watanabe, H. Hanafusa, Y. Ujimasa, T. Yagi and G. Yiu for technical assistance. We are also grateful to T. Takahashi, H. Fujisawa and F. Murakami for their critical reading of the manuscript, to the members of the Okano Laboratory for their comments on the manuscript. This work was supported by grants from the Leading Project for Realization of Regenerative Medicine from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; the Japan Science and Technology Corporation (JST); and the General Insurance Association of Japan. This work was also supported by a Keio University special grant-in-aid for innovative collaborative research projects to H.O.; a Keio University grant-in-aid for encouragement of young medical scientists to S.K. and A.I.; and a grant-in-aid from the 21st Century COE Program of MEXT, Japan, to Keio University.

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Author notes

  1. Shinjiro Kaneko and Akio Iwanami: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan

    Shinjiro Kaneko, Akio Iwanami, Masaya Nakamura, Takeshi Ikegami & Yoshiaki Toyama

  2. Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan

    Shinjiro Kaneko, Akio Iwanami, Shinsuke Shibata, Hirotaka J Okano, Ayako Moriya & Hideyuki Okano

  3. Division of Neuroscience, Children's Hospital Boston, Harvard Medical School, 320 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Shinjiro Kaneko & Zhigang He

  4. Clinical Research Center, National Hospital Organization, Murayama Medical Center 2-37-1 Gakuen, Musashimurayama, Tokyo, 208-0011, Japan

    Akio Iwanami

  5. Dainippon Sumitomo Pharma Co. Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka, 554-0022, Japan

    Akiyoshi Kishino, Kaoru Kikuchi, Osamu Konishi, Chikao Nakayama, Kazuo Kumagai & Toru Kimura

  6. Department of Molecular Pharmacology and Neurobiology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Kanagawa, Japan

    Yasufumi Sato & Yoshio Goshima

  7. Department of Biochemistry, Tokyo University School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Masahiko Taniguchi

  8. Central Institute for Experimental Animals, 1430 Nogawa, Kawasaki, 216-0001, Kanagawa, Japan

    Mamoru Ito

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  1. Shinjiro Kaneko
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Contributions

S.K. wrote the manuscript, conducted all the SCI experiments (including preparation of the SCI model rats, and behavioral and histochemical characterizations), conducted in vitro assays (including growth cone collapse assay) and all the immunoblotting, and prepared the recombinant proteins. A.I. conducted rat SCI experiments as detailed above, conducted SCI experiments of Sema3a-deficient mice, and cowrote the manuscript. M.N. instructed S.K. and A.I. on the technical aspects of the SCI experiments, and cowrote the manuscript. A.K. conducted histochemical characterization of SCI model rats. K. Kikuchi conducted in vitro experiments (including collagen gel coculture, KB-cell cell growth assay and Schwann cell migration assay). S.S. conducted cell preparation for the in vitro migration assay. H.J.O. prepared the recombinant proteins. T.I. participated in SCI experiments including behavioral characterizations. A.M. prepared the recombinant semaphorin proteins. O.K participated in histochemical characterization of SCI model rats. C.N. organized the collaboration with Keio University at Dainippon Sumitomo. K. Kumagai prepared SM-216289 and examined its specificity of action in vitro. T.K. supervised the SM-216289 project at Dainippon Sumitomo. Y.S. and Y.G. examined the specificity of SM-216289's effect on Sema3A by growth cone collapse assay. M.T. provided and conducted the genetic diagnosis of Sema3a-deficient mice. M.I. conducted the in vitro fertilization of Sema3a-deficient mice. Z.H. provided advice on the experimental design. Y.T. supported and supervised the SCI experiments conducted at Keio University. H.O. supervised the whole project and cowrote the manuscript with S.K., A.I. and M.N.

Corresponding author

Correspondence to Hideyuki Okano.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Expression of NP-1 after SCI. (PDF 1266 kb)

Supplementary Fig. 2

SM-216289 strongly inhibits Sema3A in vivo in the injured spinal cord. (PDF 214 kb)

Supplementary Fig. 3

Effect of SM-216289 upon sensory neurons in the lesion site after SCI. (PDF 541 kb)

Supplementary Fig. 4

Expression of Sema3A receptor components in fibloblast cell line, L-929 cells. . (PDF 995 kb)

Supplementary Fig. 5

SM-216289 reduces apoptosis and enhances angiogenesis in the injured spinal cord. (PDF 632 kb)

Supplementary Fig. 6

SM-216289-induced functional recovery after SCI was attenuated by retransection or administration of 5, 7-DHT. (PDF 403 kb)

Supplementary Table 1

Pharmacological profile of SM-216289. Summary of IC50 values for receptor and ion channel binding assays, and enzyme and kinase inhibition tests. (PDF 1347 kb)

Supplementary Discussion (PDF 44 kb)

Supplementary Methods (PDF 254 kb)

Supplementary Note (PDF 123 kb)

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Kaneko, S., Iwanami, A., Nakamura, M. et al. A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord. Nat Med 12, 1380–1389 (2006). https://doi.org/10.1038/nm1505

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