In Vitro and in Vivo Characterization of a Novel Semaphorin 3A Inhibitor, SM-216289 or Xanthofulvin

Abstract

SM-216289 (xanthofulvin) isolated from the fermentation broth of a fungal strain, Penicillium sp. SPF-3059, was identified as a strong semaphorin 3A (Sema3A) inhibitor. Sema3A-induced growth cone collapse of dorsal root ganglion neurons in vitro was completely abolished in the presence of SM-216289 at levels less than 2 μm (IC50 = 0.16 μm). When dorsal root ganglion explants were co-cultured with Sema3A-producing COS7 cells in a collagen gel matrix, SM-216289 enabled neurites to grow toward the COS7 cells. SM-216289 diminished the binding of Sema3A to its receptor neuropilin-1 in vitro, suggesting a direct interference of receptor-ligand association. Moreover, our data suggest that SM-216289 interacted with Sema3A directly and blocked the binding of Sema3A to its receptor. We examined the efficacy of SM-216289 in vivo using a rat olfactory nerve axotomy model, in which strong Sema3A induction has been reported around regenerating axons. The regeneration of olfactory nerves was significantly accelerated by a local administration of SM-216289 in the lesion site, suggesting the involvement of Sema3A in neural regeneration as an inhibitory factor. SM-216289 is an excellent molecular probe to investigate the function of Sema3A, in vitro and in vivo, and may be useful for the treatment of traumatic neural injuries. SM-216289 (xanthofulvin) isolated from the fermentation broth of a fungal strain, Penicillium sp. SPF-3059, was identified as a strong semaphorin 3A (Sema3A) inhibitor. Sema3A-induced growth cone collapse of dorsal root ganglion neurons in vitro was completely abolished in the presence of SM-216289 at levels less than 2 μm (IC50 = 0.16 μm). When dorsal root ganglion explants were co-cultured with Sema3A-producing COS7 cells in a collagen gel matrix, SM-216289 enabled neurites to grow toward the COS7 cells. SM-216289 diminished the binding of Sema3A to its receptor neuropilin-1 in vitro, suggesting a direct interference of receptor-ligand association. Moreover, our data suggest that SM-216289 interacted with Sema3A directly and blocked the binding of Sema3A to its receptor. We examined the efficacy of SM-216289 in vivo using a rat olfactory nerve axotomy model, in which strong Sema3A induction has been reported around regenerating axons. The regeneration of olfactory nerves was significantly accelerated by a local administration of SM-216289 in the lesion site, suggesting the involvement of Sema3A in neural regeneration as an inhibitory factor. SM-216289 is an excellent molecular probe to investigate the function of Sema3A, in vitro and in vivo, and may be useful for the treatment of traumatic neural injuries. The adult central nervous system (CNS) 1The abbreviations used are: CNS, central nervous system; DRG, dorsal root ganglion; WGA-HRP, wheat germ agglutinin-horseradish peroxidase; PBS, phosphate-buffered saline; FBS, fetal bovine serum; AP, alkaline phosphatase; Sema3A, semaphorin 3A; NP-1, neuropilin-1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. in higher vertebrates shows only a limited capacity for anatomical and functional recovery after nerve injury. Many studies suggest that the limited capacity for nerve regeneration is due to the presence of axonal growth inhibitory components in the CNS tissue (1Fawcett J.W. Asher R.A. Brain Res. Bull. 1999; 49: 377-391Crossref PubMed Scopus (1486) Google Scholar, 2Qiu J. Cai D. Filbin M.T. Glia. 2000; 29: 166-174Crossref PubMed Scopus (100) Google Scholar, 3Horner P.J. Gage F.H. Nature. 2000; 407: 963-970Crossref PubMed Scopus (694) Google Scholar, 4Fournier A.E. Strittmatter S.M. Curr. Opin. Neurobiol. 2001; 11: 89-94Crossref PubMed Scopus (161) Google Scholar). A number of molecules that may contribute to this inhibition have been reported, such as myelin-associated glycoprotein, tenascins, and some chondroitin sulfate proteoglycans, etc. One of the in vitro activities of these molecules is growth cone collapse or retraction of growing neurites (1Fawcett J.W. Asher R.A. Brain Res. Bull. 1999; 49: 377-391Crossref PubMed Scopus (1486) Google Scholar, 2Qiu J. Cai D. Filbin M.T. Glia. 2000; 29: 166-174Crossref PubMed Scopus (100) Google Scholar, 3Horner P.J. Gage F.H. Nature. 2000; 407: 963-970Crossref PubMed Scopus (694) Google Scholar, 4Fournier A.E. Strittmatter S.M. Curr. Opin. Neurobiol. 2001; 11: 89-94Crossref PubMed Scopus (161) Google Scholar). Schwab and co-workers (5Caroni P. Schwab M.E. J. Cell Biol. 1988; 106: 1281-1288Crossref PubMed Scopus (768) Google Scholar, 6Chen M.S. Huber A.B. van der Haar M.E. Frank M. Schnell L. Spillmann A.A. Christ F. Schwab M.E. Nature. 2000; 403: 434-439Crossref PubMed Scopus (334) Google Scholar, 7GrandPré T. Nakamura F. Vartanian T. Strittmatter S.M. Nature. 2000; 403: 439-444Crossref PubMed Scopus (1024) Google Scholar) have identified a neurite growth inhibitor, NI35/250 or Nogo, in CNS myelin. Blocking the activity of NI35/250 by using a neutralizing antibody (IN-1) promoted re-growth of injured axons in vivo (8Bregman B.S. Kunkel-Bagden E. Schnell L. Dai H.N. Gao D. Schwab M.E. Nature. 1995; 378: 498-501Crossref PubMed Scopus (663) Google Scholar, 9Merkler D. Metz G.A. Raineteau O. Dietz V. Schwab M.E. Fouad K. J. Neurosci. 2001; 21: 3665-3673Crossref PubMed Google Scholar). These results suggest that CNS neurons possess growth ability and support the hypothesis that inhibition of endogenous axonal growth inhibitors might allow regeneration of injured axons in the adult CNS. Various molecules, such as semaphorins and ephrins, have also been shown to have inhibitory activity for axonal growth of neurons during development (10Tessier-Lavigne M. Goodman C.S. Science. 1996; 274: 1123-1133Crossref PubMed Scopus (2706) Google Scholar, 11Mueller B.K. Annu. Rev. Neurosci. 1999; 22: 351-388Crossref PubMed Scopus (379) Google Scholar, 12Dickson B.J. Science. 2003; 298: 1959-1964Crossref Scopus (1173) Google Scholar) and have been demonstrated to cause growth cone collapse or neurite repulsion activities in vitro. Among these molecules, semaphorins compose the largest gene family of axonal growth inhibitory molecules (13Kolodkin A.L. Matthes D.J. Goodman C.S. Cell. 1993; 75: 1389-1399Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 14Püschel A.W. Adams R.H. Betz H. Neuron. 1995; 14: 941-948Abstract Full Text PDF PubMed Scopus (333) Google Scholar, 15Luo Y. Shepherd I. Li J. Renzi M.J. Chang S. Raper J.A. Neuron. 1995; 14: 1131-1140Abstract Full Text PDF PubMed Scopus (217) Google Scholar). Previous studies (16Vactor D.V. Lorenz L.J. Curr. Biol. 1999; 9: R201-R204Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 17Nakamura F. Kalb R.G. Strittmatter S.M. J. Neurobiol. 2000; 44: 219-229Crossref PubMed Scopus (248) Google Scholar, 18Goshima Y. Sasaki Y. Nakayama T. Ito T. Kimura T. Jpn. J. Pharmacol. 2000; 82: 273-279Crossref PubMed Scopus (38) Google Scholar) have shown that some semaphorins play an important role in the development of the nervous system. In addition, some semaphorins are expressed in adult nervous tissues (19Luo Y. Raible D. Raper J.A Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (1023) Google Scholar, 20Inagaki S. Furuyama T. Iwahashi Y. FEBS Lett. 1995; 370: 269-272Crossref PubMed Scopus (57) Google Scholar, 21Encinas J.A. Kikuchi K. Chédotal A. de Castro F. Goodman C.S. Kimura T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2491-2496Crossref PubMed Scopus (35) Google Scholar, 22Kikuchi K. Chédotal A. Hanafusa H. Ujimasa Y. de Castro F. Goodman C.S. Kimura T. Mol. Cell. Neurosci. 1999; 13: 9-23Crossref PubMed Scopus (29) Google Scholar, 23De Winter F. Oudega M. Lankhorst A.J. Hamers F.P. Blits B. Ruitenberg M.J. Pasterkamp R.J. Gispen W.H. Verhaagen J. Exp. Neurol. 2002; 175: 61-75Crossref PubMed Scopus (232) Google Scholar), although their function(s) remain to be elucidated. Semaphorin 3A (Sema3A), a prototype member of the family in vertebrates (13Kolodkin A.L. Matthes D.J. Goodman C.S. Cell. 1993; 75: 1389-1399Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 19Luo Y. Raible D. Raper J.A Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (1023) Google Scholar), has been demonstrated to be an axonal growth inhibitory molecule against specific neural populations during development, both in vivo and in vitro (19Luo Y. Raible D. Raper J.A Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (1023) Google Scholar, 24Messersmith E.K. Leonardo E.D. Shatz C.J. Tessier-Lavigne M. Goodman C.S. Kolodkin A.L. Neuron. 1995; 14: 949-959Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 25Taniguchi M. Yuasa S. Fujisawa H. Naruse I. Saga S. Mishina M. Yagi T. Neuron. 1997; 19: 519-530Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 26Polleux F. Giger R.J. Ginty D.D. Kolodkin A.L. Ghosh A. Science. 1998; 282: 1904-1906Crossref PubMed Scopus (222) Google Scholar). Even in the adult, specific subtypes of neurons are responsive to the repulsive activity of Sema3A. Trigeminal sensory axons innervating the cornea are repulsed by Sema3A derived from a Sema3A expression plasmid introduced into the eye of rabbits by gene gun transfection (27Tanelian D.L. Barry M.A. Johnston S.A. Le T. Smith G.M. Nat. Med. 1997; 3: 1398-1401Crossref PubMed Scopus (116) Google Scholar). In addition, in vitro experiments have shown that adult DRG neurons are sensitive to Sema3A (28Reza J.N. Gavazzi I. Cohen J. Mol. Cell. Neurosci. 1999; 14: 317-326Crossref PubMed Scopus (58) Google Scholar, 29Owesson C. Pizzy J. Tonge D. Exp. Neurol. 2000; 165: 394-398Crossref PubMed Scopus (9) Google Scholar). Recently, Verhaagen and co-workers (23De Winter F. Oudega M. Lankhorst A.J. Hamers F.P. Blits B. Ruitenberg M.J. Pasterkamp R.J. Gispen W.H. Verhaagen J. Exp. Neurol. 2002; 175: 61-75Crossref PubMed Scopus (232) Google Scholar, 30Pasterkamp R.J. De Winter F. Holtmaat A.J. Verhaagen J. J. Neurosci. 1998; 18: 9962-9976Crossref PubMed Google Scholar, 31Pasterkamp R.J. Giger R.J. Ruitenberg M.-J. Holtmaat A.J. De Wit J. De Winter F. Verhaagen J. Mol. Cell. Neurosci. 1999; 13: 143-166Crossref PubMed Scopus (279) Google Scholar, 32Pasterkamp R.J. Anderson P.N. Verhaagen J. Eur. J. Neurosci. 2001; 13: 457-471Crossref PubMed Scopus (121) Google Scholar) have demonstrated that mRNA for Sema3A is induced in the glial scar formed at the injury site of CNS tissue in several adult animal models, including olfactory nerve axotomy and spinal cord injury. These results suggest the possibility that induced Sema3A in glial scars may suppress the re-growth of injured CNS axons. In order to investigate the function of semaphorin in vitro and in vivo, a specific inhibitor for the molecule would be very useful. In this report, we describe the identification of a novel semaphorin inhibitor, xanthofulvin or SM-216289, which was isolated from a fungus. In addition, we characterize the activity of SM-216289 in vitro and in vivo. Production of SM-216289 by of Penicillium sp. for the fermentation and of SM-216289 K. Kikuchi K. Kimura T. I. J. 2003; PubMed Scopus Google Scholar). the fermentation broth of Penicillium sp. that was for at with at was to the and the which were with and The were and by a of gel and to Sema3A was into the expression A.W. Adams R.H. Betz H. Neuron. 1995; 14: 941-948Abstract Full Text PDF PubMed Scopus (333) Google Scholar, A. Kimura T. K. Nakamura T. FEBS Lett. 1993; PubMed Scopus Google Scholar). The expression plasmid was into COS7 cells using the transfection as in the after the was and used for the growth cone collapse after of Sema3A The growth cone collapse was as by (19Luo Y. Raible D. Raper J.A Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (1023) Google Scholar). DRG explants derived from or rat were for in fetal bovine and nerve growth in with and In order to the Sema3A inhibitory activity of SM-216289, the was to the Sema3A. A of was to the to of by a the DRG explants were with and growth were a to the of Sema3A by (19Luo Y. Raible D. Raper J.A Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (1023) Google Scholar), to and of Sema3A and in our Cell growth was to the of COS7 cells were a and the was to the at of growth was using the Cell to the in experiments were as S. A. Scopus Google Scholar), with a COS7 were as COS7 cells in a were with of or the plasmid using after the cells were and in of of of the were the of a and for T. de Tessier-Lavigne M. Cell. Full Text PDF PubMed Scopus Google Scholar). COS7 and DRG explants derived from were co-cultured in collagen gel by a of The collagen gel the and the DRG was for 2 in and nerve growth with or we was the growth cone collapse the inhibitor was with the of Sema3A. In the 3A was with the inhibitor for used to the DRG DRG was for with a of the DRG was with a of Sema3A in the Sema3A was used as a for receptor binding A to of Sema3A was into the of the alkaline gene in the M. P. 2000; PubMed Scopus Google Scholar) in order to a at the of the A for the was into the expression was expressed in COS7 cells that were with the expression plasmid using was from the by as by T. Nakamura F. Strittmatter S.M. J. Neurosci. 1997; PubMed Google Scholar). In order to NP-1, of A. T. S. Fujisawa H. J. Neurobiol. 1996; 29: PubMed Scopus Google Scholar) was into the expression COS7 cells were with the after the cells were in and for The cells were with in FBS, to a receptor binding as by and P. Cell. Full Text PDF PubMed Scopus Google Scholar). In order to the of SM-216289 the binding of to or DRG the was to the cells to in of to the cells were after the binding and using and as the cells were by and activity derived from was at using as P. Cell. Full Text PDF PubMed Scopus Google Scholar). of or DRG neurons was as T. Nakamura F. Strittmatter S.M. J. Neurosci. 1997; PubMed Google Scholar). of animal and treatment were to the local of the animal were with The olfactory was by the of the the olfactory using a The olfactory nerves were by of a the and the olfactory Brain Res. PubMed Scopus Google Scholar). the SM-216289 was and into the injury site at a using a and an of the olfactory nerve was as or after the the were with and their of the olfactory wheat germ agglutinin-horseradish in was into H. Exp. Brain Res. PubMed Scopus Google Scholar). the were and with The olfactory was and at μm were from the olfactory and was into a of the olfactory nerve and the were with in for the the were The number of the from the of the olfactory was as the nerve regeneration for of a Sema3A SM-216289, from the Penicillium sp. a strong Sema3A inhibitory activity in the broth of a fungal strain, SPF-3059, which was isolated from and identified as a to the of the of the fungal to the of the The molecular of SM-216289, as by was and the molecular was The was by and was to be to xanthofulvin which was isolated from a of as in in a K. Kikuchi K. Kimura T. I. J. 2003; PubMed Scopus Google Scholar). SM-216289 a Sema3A growth cone collapse in DRG The growth cone collapse was completely abolished in the presence of SM-216289, at less than 2 was 0.16 μm in growth cone The inhibition was also rat DRG neurons = μm). SM-216289 a inhibitory to and Sema3A, as as to that from These that the inhibitory was When COS7 cells were in the presence of of SM-216289, growth inhibition was at μm of SM-216289 is than higher than the for growth cone collapse SM-216289 a growth cone collapse by Sema3A is a and at the of the neurites J. T. Raper J.A. J. Cell Biol. 1993; PubMed Scopus Google Scholar), of growing neurites to of Sema3A the of the neurite growth a of is to be an important of axonal during neural SM-216289 has the to the of growing axons by Sema3A, we a collagen gel system. When neurons are co-cultured in a collagen gel very to cells repulsive or molecules, such as a semaphorin or growing neurites the and their of growth S. A. Scopus Google Scholar). shown in an of COS7 cells and DRG were co-cultured in a collagen gel for 2 with or SM-216289 in the growth the neurons in the DRG their neurites in the neurites from the COS7 co-cultured with in the presence of or μm SM-216289, the repulsion was growing neurites were in the of although their was in to the and μm SM-216289, neurites were from the DRG and were from the shown in the of neurites growing from COS7 was the DRG and the DRG suggesting that SM-216289 neurite When COS7 cells were with the expression plasmid mRNA expression for Sema3A was by SM-216289 These results that SM-216289 the activity of Sema3A during the and the repulsive activity of Sema3A. SM-216289 Sema3A order to the of of SM-216289, we examined the of the of the explants with SM-216289 the of Sema3A in the growth cone collapse In the the activity of Sema3A was of the SM-216289 When SM-216289 and Sema3A were to DRG explants the activity of Sema3A was completely the was after Sema3A, inhibition was that Sema3A may with SM-216289, Sema3A from binding to the receptor. In order to SM-216289 directly Sema3A, we a as the inhibitory of SM-216289 was its In our in which SM-216289 and Sema3A were to the μm SM-216289 Sema3A inhibition was at μm SM-216289 In the Sema3A was with μm of SM-216289 and for A of the was to a of in which DRG explants were a of Sema3A and SM-216289 which was to that used in the μm SM-216289 in inhibition of Sema3A in the the activity of Sema3A was significantly in the The the experiments is that in the Sema3A into with a higher of SM-216289 is also by the In this a DRG was with μm SM-216289 by with a of to a of μm inhibition of Sema3A was in this be that the of SM-216289 and Sema3A in the was the in these SM-216289 Sema3A in an SM-216289 of Sema3A to order to the of of SM-216289 we the of SM-216289 the binding of Sema3A to neuropilin-1 an of the Sema3A receptor Tessier-Lavigne M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A.L. D.V. Giger R.J. Ginty D.D. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. M. T. M. Y. Yagi T. Fujisawa H. Neuron. 1997; 19: Full Text Full Text PDF PubMed Scopus Google Scholar). We alkaline Sema3A for the binding experiments P. Cell. Full Text PDF PubMed Scopus Google Scholar). T. Nakamura F. Strittmatter S.M. J. Neurosci. 1997; PubMed Google Scholar), a growth cone activity = than the Sema3A SM-216289 the activity of at a higher (IC50 = The binding of to in the presence or of SM-216289 was examined by using COS7 cells that were shown in to and the binding was significantly by the presence of of the binding by alkaline activity that the inhibitory of SM-216289 was The of SM-216289 direct Sema3A and which is of Sema3A receptor T. A. Nakamura F. Y. Kalb R.G. Fujisawa H. Strittmatter S.M. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), be examined Sema3A to T. A. Nakamura F. Y. Kalb R.G. Fujisawa H. Strittmatter S.M. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). binding of to DRG neurons was also by SM-216289 at a SM-216289 the activity the expression of These results suggest that SM-216289 the activity of Sema3A by its receptor binding SM-216289 in in a R.J. De Winter F. Holtmaat A.J. Verhaagen J. J. Neurosci. 1998; 18: 9962-9976Crossref PubMed Google Scholar) have demonstrated that axotomy of the olfactory nerve to an induction of Sema3A mRNA at the injured is expressed the olfactory induced Sema3A may of olfactory axons R.J. De Winter F. Holtmaat A.J. Verhaagen J. J. Neurosci. 1998; 18: 9962-9976Crossref PubMed Google Scholar). using this animal model, we examined the in vivo efficacy of olfactory nerve SM-216289 was and the and the olfactory of at the of a using or after the olfactory nerves were with wheat germ agglutinin-horseradish H. Exp. Brain Res. PubMed Scopus Google Scholar), and the of olfactory nerve was by the shown in the of was to a from the of olfactory nerves with 2 the the regeneration for was the in was the of and significantly at and be that the regeneration were that the the of the neurites the the SM-216289 has a strong Sema3A inhibitory the of olfactory nerve regeneration in this may due to the inhibition of Sema3A by We isolated SM-216289 from the broth of Penicillium sp. SPF-3059, and we identified this as a Sema3A inhibitor. SM-216289 Sema3A from growth and growing neurites in vitro. The binding of Sema3A to its receptor was abolished by SM-216289, which may be to direct with Sema3A. Moreover, SM-216289 promoted nerve regeneration in vivo in a rat olfactory nerve axotomy shown in growth cone collapse was completely in the presence of 2 μm SM-216289 (IC50 = 0.16 μm). higher were to the repulsive activity of Sema3A in the gel and μm or The in the inhibitory for growth cone collapse and neurite repulsive activity may be in of the of the Sema3A to a receptor and growth cone collapse or the neurite and may the neurite is to grow in the presence of Sema3A, its activity to be and completely blocked during the by a of inhibitor as with that for the growth cone collapse the local of Sema3A in the collagen gel may be higher than the of Sema3A used for the collapse this in the local of Sema3A is growing neurites an of inhibitory molecules are by a J. B. F. J. Scholar). in a higher Sema3A SM-216289 to suppress the Sema3A we the of SM-216289 to suppress Sema3A in the collagen gel The binding of of Sema3A and alkaline to both cells and DRG neurons was by the would only binding of Sema3A to also to the receptor that and SM-216289 was to binding of to the receptor than was to suppress growth cone collapse of Sema3A. may be due to that was used for the binding of Sema3A. In with (19Luo Y. Raible D. Raper J.A Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (1023) Google Scholar, T. Nakamura F. Strittmatter S.M. J. Neurosci. 1997; PubMed Google Scholar), the specific activity of for growth cone collapse was than that of Sema3A in our experiments a higher of SM-216289 was to the activity of = was a the of SM-216289 to binding of and the for growth cone collapse by The shows activity than Sema3A is The and the Sema3A may its for the as as the inhibitor. In our μm of the was with Sema3A in the growth cone collapse activity was inhibition was μm was to DRG in direct of SM-216289 with Sema3A was for Sema3A is by The of of SM-216289 be of this the reported R.H. M. A. Betz H. A.W. J. 1997; PubMed Scopus Google Scholar) were in our and the molecular of by was by with our results suggest that SM-216289 directly with Sema3A to its In the the inhibition was collapse in that the inhibition was and that a of the was after the was to the DRG explants and to a was by an in which the activity of Sema3A was from by an inhibitory of SM-216289 after against SM-216289 also efficacy in vivo as as in vitro Sema3A nerve axotomy Sema3A mRNA expression in cells the and the olfactory regenerating axons in this animal R.J. De Winter F. Holtmaat A.J. Verhaagen J. J. Neurosci. 1998; 18: 9962-9976Crossref PubMed Google Scholar). Sema3A to be of the of a antibody for is to that Sema3A is in the injured shown in olfactory nerve regeneration was significantly SM-216289 was directly the injury of the to be the of administration was to the and to the of the We the of the in our system by a using a in of 2 of administration of the the was in the olfactory the nerve regeneration is to the inhibition of Sema3A by be that the of SM-216289 to semaphorins to be that SM-216289 at higher with Sema3A Recently, A. M. V. I. Y. S. E. A. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) reported that ganglion cells from nerve would be very to the antibody regeneration of olfactory The that for is to the recovery of function after axons the olfactory with neurons may be a and functional is to that the regenerating axons their is the to support the hypothesis that the of Sema3A activities in glial scar tissue the regeneration of axons in adult olfactory neurons are the regenerating axons in the olfactory of the CNS. in CNS nerve injury models, the regeneration of injured nerves may be by Sema3A expressed in glial scars (23De Winter F. Oudega M. Lankhorst A.J. Hamers F.P. Blits B. Ruitenberg M.J. Pasterkamp R.J. Gispen W.H. Verhaagen J. Exp. Neurol. 2002; 175: 61-75Crossref PubMed Scopus (232) Google Scholar, 30Pasterkamp R.J. De Winter F. Holtmaat A.J. Verhaagen J. J. Neurosci. 1998; 18: 9962-9976Crossref PubMed Google Scholar, 31Pasterkamp R.J. Giger R.J. Ruitenberg M.-J. Holtmaat A.J. De Wit J. De Winter F. Verhaagen J. Mol. Cell. Neurosci. 1999; 13: 143-166Crossref PubMed Scopus (279) Google Scholar, 32Pasterkamp R.J. Anderson P.N. Verhaagen J. Eur. J. Neurosci. 2001; 13: 457-471Crossref PubMed Scopus (121) Google Scholar). Sema3A, in scar tissue is the to the regeneration of CNS SM-216289 might re-growth of the axons. SM-216289 is the Sema3A inhibitor both in vitro and in vivo efficacy and is an excellent molecular probe to investigate the of Sema3A In addition, SM-216289 may be useful as a to neural in of some of an involvement of Sema3A in the inhibition of regeneration of CNS axons after traumatic injury. studies the of SM-216289 CNS regeneration using animal with such as spinal cord We are to K. for the the olfactory axotomy and also to J. T. H. A. L. and J. A. Raper for their of Sema3A and Sema3A We Y. Ujimasa and H. Hanafusa for their excellent