Neuropilin 1 Directly Interacts with Fer Kinase to Mediate Semaphorin 3A-induced Death of Cortical Neurons

Neuropilins (NRPs) are receptors for the major chemorepulsive axonal guidance cue semaphorins (Sema). The interaction of Sema3A/NRP1 during development leads to the collapse of growth cones. Here we show that Sema3A also induces death of cultured cortical neurons through NRP1. A specific NRP1 inhibitory peptide ameliorated Sema3A-evoked cortical axonal retraction and neuronal death. Moreover, Sema3A was also involved in cerebral ischemia-induced neuronal death. Expression levels of Sema3A and NRP1, but not NRP2, were significantly increased early during brain reperfusion following transient focal cerebral ischemia. NRP1 inhibitory peptide delivered to the ischemic brain was potently neuroprotective and prevented the loss of motor functions in mice. The integrity of the injected NRP1 inhibitory peptide into the brain remained unchanged, and the intact peptide permeated the ischemic hemisphere of the brain as determined using MALDI-MS-based imaging. Mechanistically, NRP1-mediated axonal collapse and neuronal death is through direct and selective interaction with the cytoplasmic tyrosine kinase Fer. Fer RNA interference effectively attenuated Sema3A-induced neurite retraction and neuronal death in cortical neurons. More importantly, down-regulation of Fer expression using Fer-specific RNA interference attenuated cerebral ischemia-induced brain damage. Together, these studies revealed a previously unknown function of NRP1 in signaling Sema3A-evoked neuronal death through Fer in cortical neurons. Neuropilins (NRPs) are receptors for the major chemorepulsive axonal guidance cue semaphorins (Sema). The interaction of Sema3A/NRP1 during development leads to the collapse of growth cones. Here we show that Sema3A also induces death of cultured cortical neurons through NRP1. A specific NRP1 inhibitory peptide ameliorated Sema3A-evoked cortical axonal retraction and neuronal death. Moreover, Sema3A was also involved in cerebral ischemia-induced neuronal death. Expression levels of Sema3A and NRP1, but not NRP2, were significantly increased early during brain reperfusion following transient focal cerebral ischemia. NRP1 inhibitory peptide delivered to the ischemic brain was potently neuroprotective and prevented the loss of motor functions in mice. The integrity of the injected NRP1 inhibitory peptide into the brain remained unchanged, and the intact peptide permeated the ischemic hemisphere of the brain as determined using MALDI-MS-based imaging. Mechanistically, NRP1-mediated axonal collapse and neuronal death is through direct and selective interaction with the cytoplasmic tyrosine kinase Fer. Fer RNA interference effectively attenuated Sema3A-induced neurite retraction and neuronal death in cortical neurons. More importantly, down-regulation of Fer expression using Fer-specific RNA interference attenuated cerebral ischemia-induced brain damage. Together, these studies revealed a previously unknown function of NRP1 in signaling Sema3A-evoked neuronal death through Fer in cortical neurons. IntroductionInjured central nervous system axons have a very limited capacity to regenerate due to the presence of a plethora of growth inhibitory ligands secreted from oligodendrocytes/myelin, reactive astrocytes, and fibroblasts in the damaged tissue (1.De 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 (228) Google Scholar, 2.Giger R.J. Pasterkamp R.J. Holtmaat A.J. Verhaagen J. Prog. Brain Res. 1998; 117: 133-149Crossref PubMed Google Scholar, 3.Pasterkamp R.J. De Winter F. Giger R.J. Verhaagen J. Prog. Brain Res. 1998; 117: 151-170Crossref PubMed Google Scholar, 4.Pasterkamp R.J. Giger R.J. Verhaagen J. Exp. Neurol. 1998; 153: 313-327Crossref PubMed Scopus (92) Google Scholar, 5.He Z. Koprivica V. Annu. Rev. Neurosci. 2004; 27: 341-368Crossref PubMed Scopus (182) Google Scholar, 6.Yiu G. He Z. Nat. Rev. Neurosci. 2006; 7: 617-627Crossref PubMed Scopus (1142) Google Scholar). Neurons must integrate this multitude of inhibitory molecular cues, generated as a result of cortical damage, into a functional response. More often than not the response is one of growth cone collapse, axonal retraction, and neuronal death. Therefore, chemorepulsive factors likely contribute either directly or indirectly to neuronal death in the injured adult brain (7.Deckwerth T.L. Johnson Jr., E.M. J. Cell Biol. 1993; 123: 1207-1222Crossref PubMed Scopus (515) Google Scholar, 8.Wakade T.D. Palmer K.C. McCauley R. Przywara D.A. Wakade A.R. J. Physiol. 1995; 488: 123-138Crossref PubMed Scopus (77) Google Scholar, 9.Hou S.T. Jiang S.X. Smith R.A. Int. Rev. Cell Mol. Biol. 2008; 267: 125-181Crossref PubMed Scopus (89) Google Scholar, 10.Raff M.C. Whitmore A.V. Finn J.T. Science. 2002; 296: 868-871Crossref PubMed Scopus (558) Google Scholar). Indeed, the expression of Sema3A, a major chemorepulsive factor, has been reported in several brain injury models, such as peripheral nerve injury, spinal cord injury, cerebral ischemia, and Alzheimer disease (1.De 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 (228) Google Scholar, 3.Pasterkamp R.J. De Winter F. Giger R.J. Verhaagen J. Prog. Brain Res. 1998; 117: 151-170Crossref PubMed Google Scholar, 11.Hou S.T. Keklikian A. Slinn J. O'Hare M. Jiang S.X. Aylsworth A. Biochem. Biophys. Res. Commun. 2008; 367: 109-115Crossref PubMed Scopus (40) Google Scholar, 12.Beck H. Acker T. Püschel A.W. Fujisawa H. Carmeliet P. Plate K.H. J. Neuropathol. Exp. Neurol. 2002; 61: 339-350Crossref PubMed Scopus (95) Google Scholar, 13.Fujita H. Zhang B. Sato K. Tanaka J. Sakanaka M. Brain Res. 2001; 914: 1-14Crossref PubMed Scopus (67) Google Scholar, 14.Pasterkamp R.J. Verhaagen J. Brain Res. Brain Res. Rev. 2001; 35: 36-54Crossref PubMed Scopus (114) Google Scholar, 15.Kaneko S. Iwanami A. Nakamura M. Kishino A. Kikuchi K. Shibata S. Okano H.J. Ikegami T. Moriya A. Konishi O. Nakayama C. Kumagai K. Kimura T. Sato Y. Goshima Y. Taniguchi M. Ito M. He Z. Toyama Y. Okano H. Nat. Med. 2006; 12: 1380-1389Crossref PubMed Scopus (315) Google Scholar, 16.Pasterkamp R.J. Giger R.J. Curr. Opin. Neurobiol. 2009; 19: 263-274Crossref PubMed Scopus (162) Google Scholar). In addition, Sema3A expression has been shown to increase vascular permeability, which may indirectly contribute to neuronal damage (17.Acevedo L.M. Barillas S. Weis S.M. Göthert J.R. Cheresh D.A. Blood. 2008; 111: 2674-2680Crossref PubMed Scopus (171) Google Scholar).The biological activities of Sema3A during development are complex and context-dependent. Although best known for its role as an axonal growth cone repellent, Sema3A also serves as a chemoattractant during cortical layer development by guiding the radial migration of layer II/III cortical neurons (18.Chen G. Sima J. Jin M. Wang K.Y. Xue X.J. Zheng W. Ding Y.Q. Yuan X.B. Nat. Neurosci. 2008; 11: 36-44Crossref PubMed Scopus (186) Google Scholar) and the growth of apical dendrites toward the pial surface (19.Polleux F. Morrow T. Ghosh A. Nature. 2000; 404: 567-573Crossref PubMed Scopus (587) Google Scholar). In contrast, Sema3A is also important in stereotyped pruning of long hippocampal axon branches (20.Bagri A. Cheng H.J. Yaron A. Pleasure S.J. Tessier-Lavigne M. Cell. 2003; 113: 285-299Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), causing dorsal root ganglia axon retraction (21.Gallo G. J. Cell Sci. 2006; 119: 3413-3423Crossref PubMed Scopus (118) Google Scholar), and evoking apoptosis of sensory neurons (22.Shirvan A. Ziv I. Fleminger G. Shina R. He Z. Brudo I. Melamed E. Barzilai A. J. Neurochem. 1999; 73: 961-971Crossref PubMed Scopus (136) Google Scholar, 23.Shirvan A. Kimron M. Holdengreber V. Ziv I. Ben Shaul Y. Melamed S. Melamed E. Barzilai A. Solomon A.S. J. Biol. Chem. 2002; 277: 49799-49807Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 24.Gagliardini V. Fankhauser C. Mol. Cell Neurosci. 1999; 14: 301-316Crossref PubMed Scopus (81) Google Scholar) possibly through activating apoptotic pathways involving PlexinA3 receptor and mitogen-activated protein kinases (25.Campbell D.S. Holt C.E. Neuron. 2003; 37: 939-952Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 26.Ben-Zvi A. Manor O. Schachner M. Yaron A. Tessier-Lavigne M. Behar O. J. Neurosci. 2008; 28: 12427-12432Crossref PubMed Scopus (48) Google Scholar).The cellular receptors for semaphorins are neuropilins (NRP1 and NRP2) 2The abbreviations used are: NRP1 and -2neuropilins 1 and 2MALDI-MSImatrix-assisted laser desorption/ionization-mass spectrometry imagingTOFtime of flightMCAOmiddle cerebral artery occlusionPIpropidium iodideRNAiinhibitory RNASema3Asemaphorin 3ATTC2,3,5-triphenyltetrazolium chlorideTUNELterminal deoxynucleotidyltransferase dUTP nick end labelingDIVdays in vitroNMDAN-methyl-d-aspartic acidIPimmunoprecipitation. (27.He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 28.Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar). Structurally, both NRPs contain an extracellular domain of two CUB motifs, adjacent to two domains with homology to coagulation factors V and VIII; a MAM domain; a single transmembrane domain; and a short intracellular domain of 39 amino acids lacking any known signaling motifs (27.He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 28.Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 29.Chen H. Chédotal A. He Z. Goodman C.S. Tessier-Lavigne M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). The structural domain required for Sema3A/NRP1 interaction has been characterized (30.Antipenko A. Himanen J.P. van Leyen K. Nardi-Dei V. Lesniak J. Barton W.A. Rajashankar K.R. Lu M. Hoemme C. Püschel A.W. Nikolov D.B. Neuron. 2003; 39: 589-598Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and several specific polypeptides antagonizing Sema3A IgG domain and NRP1 MAM domain have been found to inhibit Sema3A/NRP1-mediated inhibition of axonal outgrowth in vitro (31.Williams G. Eickholt B.J. Maison P. Prinjha R. Walsh F.S. Doherty P. J. Neurochem. 2005; 92: 1180-1190Crossref PubMed Scopus (27) Google Scholar).NRP1 is a multifunctional receptor, which mediates activities of structurally distinct ligands during development of the heart, vasculature, and neuronal system (32.Gu C. Rodriguez E.R. Reimert D.V. Shu T. Fritzsch B. Richards L.J. Kolodkin A.L. Ginty D.D. Dev. Cell. 2003; 5: 45-57Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). An indication of the importance of NRP1 function in adult mice brain came from our recent discovery that NRP1 expression was transcriptionally regulated by the apoptosis-inducing transcription factor E2F1 (33.Jiang S.X. Sheldrick M. Desbois A. Slinn J. Hou S.T. Mol. Cell. Biol. 2007; 27: 1696-1705Crossref PubMed Scopus (37) Google Scholar). Activation of E2F1 causes neuronal death during cerebral ischemia (34.Hou S.T. Callaghan D. Fournier M.C. Hill I. Kang L. Massie B. Morley P. Murray C. Rasquinha I. Slack R. MacManus J.P. J. Neurochem. 2000; 75: 91-100Crossref PubMed Scopus (103) Google Scholar, 35.MacManus J.P. Jian M. Preston E. Rasquinha I. Webster J. Zurakowski B. J. Cereb. Blood Flow Metab. 2003; 23: 1020-1028Crossref PubMed Scopus (34) Google Scholar). In this study, the neuroprotective effects of blocking NRP1 interaction with Sema3A were investigated and the cytoplasmic tyrosine kinase Fer was determined as a downstream effecter for NRP1-mediated death signal transduction.DISCUSSIONThe present study revealed two very important findings: 1) the interaction between the chemorepulsive guidance molecule Sema3A and its receptor NRP1 is important in neuronal death both in cultured cortical neurons and during cerebral ischemia and 2) NRP1 directly interacts with the cytoplasmic non-receptor tyrosine kinase Fer to mediate neurite damage and neuronal death.Although Sema/NRP's functions during development have been established R.J. Giger R.J. Curr. Opin. Neurobiol. 2009; 19: 263-274Crossref PubMed Scopus (162) Google Scholar, G. Sima J. Jin M. Wang K.Y. Xue X.J. Zheng W. Ding Y.Q. Yuan X.B. Nat. Neurosci. 2008; 11: 36-44Crossref PubMed Scopus (186) Google Scholar, F. Morrow T. Ghosh A. Nature. 2000; 404: 567-573Crossref PubMed Scopus (587) Google Scholar, S. Yuan X.B. Biochem. Biophys. Res. Commun. 2007; PubMed Scopus Google Scholar), functions in the injured adult are to Although several studies have semaphorins in a of Alzheimer motor and by cerebral ischemia R.J. Giger R.J. Curr. Opin. Neurobiol. 2009; 19: 263-274Crossref PubMed Scopus (162) Google Scholar, Winter F. Holtmaat A.J. Verhaagen J. Exp. Med. Biol. 2002; PubMed Google Scholar), direct neuronal death from of sensory neurons (22.Shirvan A. Ziv I. Fleminger G. Shina R. He Z. Brudo I. Melamed E. Barzilai A. J. Neurochem. 1999; 73: 961-971Crossref PubMed Scopus (136) Google Scholar, 24.Gagliardini V. Fankhauser C. Mol. Cell Neurosci. 1999; 14: 301-316Crossref PubMed Scopus (81) Google Scholar, D.S. Holt C.E. Neuron. 2003; 37: 939-952Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 26.Ben-Zvi A. Manor O. Schachner M. Yaron A. Tessier-Lavigne M. Behar O. J. Neurosci. 2008; 28: 12427-12432Crossref PubMed Scopus (48) Google Scholar). An to Sema3A has been shown to from death following nerve in A. Kimron M. Holdengreber V. Ziv I. Ben Shaul Y. Melamed S. Melamed E. Barzilai A. Solomon A.S. J. Biol. Chem. 2002; 277: 49799-49807Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and blocking Sema3A using a selective also nerve of spinal cord in adult mice S. Iwanami A. Nakamura M. Kishino A. Kikuchi K. Shibata S. Okano H.J. Ikegami T. Moriya A. Konishi O. Nakayama C. Kumagai K. Kimura T. Sato Y. Goshima Y. Taniguchi M. Ito M. He Z. Toyama Y. Okano H. Nat. Med. 2006; 12: 1380-1389Crossref PubMed Scopus (315) Google Scholar). is that Sema3A may a neuronal death. is not is Sema3A function is NRP1 or NRP2, and specific intracellular are to mediate In the present study, we used a peptide blocking Sema3A/NRP1 interaction to show both in cultured cortical neurons and in ischemic The following from this study a direct of Sema3A/NRP1 interaction with the death of adult cortical 1) Sema3A protein axonal growth cone collapse, axonal damage, and neuronal death to both and 2) NRP1 inhibitory of Sema3A/NRP1 prevented neuronal death. importantly, Sema3A/NRP1 expression increased ischemia and the NRP1 inhibitory are neuroprotective to the brain during ischemia, the Sema3A/NRP1 to the of to ischemic neuronal death. the expression of the present study the of Sema3A/NRP1 The long of blocking Sema3A/NRP1 interaction in ischemic to MALDI-MS-based molecular in its as a to a and to the integrity and of the injected NRP1 inhibitory peptide A. E. A. Chem. 2008; PubMed Scopus Google Scholar, D. M. Dev. 2005; PubMed Scopus Google Scholar, M. P. Nat. Med. 2001; 7: PubMed Scopus Google Scholar). this we that NRP1 inhibitory peptide was to the following cerebral ischemia, through the increased of the as by the of signal in the of the NRP1 inhibitory peptide permeated a of the brain both in the and the which with the of damage. a direct of the integrity and of the injected NRP1 peptide and the that the in the NRP1 inhibitory was due to the presence of the the of in the of hippocampal F. G. J. D. G. V. J. Neurosci. 2006; 23: PubMed Scopus Google Scholar). the in vitro is that NRP1 inhibitory was of The that the NRP1 inhibitory Sema3A protein any neuronal importantly, of NRP1 inhibitory peptide or Sema3A protein not into neurons as shown in the that NRP1-mediated neuronal death may involved in with studies to for downstream of which are not known to The cytoplasmic domain of NRP1 is and is that the cytoplasmic domain of NRP1 is to mediate functional to Sema3A L. Cell Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). an for using a we found that Fer kinase was with the NRP1 cytoplasmic blocking Sema3A/NRP1 interaction with the specific inhibitory peptide Fer to the NRP1 cytoplasmic The in Fer with NRP1 cytoplasmic domain with Together, these the of NRP1 in downstream signal through Fer. down-regulation of Fer expression is neuroprotective Sema3A and cerebral ischemia as shown in The that dorsal root neurons are not to Sema3A Z. K. Dev. Biol. 2007; 7: PubMed Scopus Google Scholar) a role of Fer in neuronal response to study, for the that Fer is an important of the death signal in adult cortical neurons. this as to the downstream for neuronal and are response we have previously are important in ischemic neuronal death S.T. Keklikian A. Slinn J. O'Hare M. Jiang S.X. Aylsworth A. Biochem. Biophys. Res. Commun. 2008; 367: 109-115Crossref PubMed Scopus (40) Google Scholar, S.X. Sheldrick M. Desbois A. Slinn J. Hou S.T. Mol. Cell. Biol. 2007; 27: 1696-1705Crossref PubMed Scopus (37) Google Scholar, S.T. Jiang S.X. Desbois A. D. J. L. L. J. J. Neurosci. 2006; PubMed Scopus Google the present study a role for Sema3A/NRP1 in the death of adult cortical neurons through direct and selective interaction with the cytoplasmic non-receptor tyrosine kinase Fer. Sema3A/NRP1 interaction is neuroprotective during a of as of the damage response in neuronal death. Although the long of blocking Sema3A/NRP1 to this interaction a to receptors for development of brain damage. IntroductionInjured central nervous system axons have a very limited capacity to regenerate due to the presence of a plethora of growth inhibitory ligands secreted from oligodendrocytes/myelin, reactive astrocytes, and fibroblasts in the damaged tissue (1.De 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 (228) Google Scholar, 2.Giger R.J. Pasterkamp R.J. Holtmaat A.J. Verhaagen J. Prog. Brain Res. 1998; 117: 133-149Crossref PubMed Google Scholar, 3.Pasterkamp R.J. De Winter F. Giger R.J. Verhaagen J. Prog. Brain Res. 1998; 117: 151-170Crossref PubMed Google Scholar, 4.Pasterkamp R.J. Giger R.J. Verhaagen J. Exp. Neurol. 1998; 153: 313-327Crossref PubMed Scopus (92) Google Scholar, 5.He Z. Koprivica V. Annu. Rev. Neurosci. 2004; 27: 341-368Crossref PubMed Scopus (182) Google Scholar, 6.Yiu G. He Z. Nat. Rev. Neurosci. 2006; 7: 617-627Crossref PubMed Scopus (1142) Google Scholar). Neurons must integrate this multitude of inhibitory molecular cues, generated as a result of cortical damage, into a functional response. More often than not the response is one of growth cone collapse, axonal retraction, and neuronal death. Therefore, chemorepulsive factors likely contribute either directly or indirectly to neuronal death in the injured adult brain (7.Deckwerth T.L. Johnson Jr., E.M. J. Cell Biol. 1993; 123: 1207-1222Crossref PubMed Scopus (515) Google Scholar, 8.Wakade T.D. Palmer K.C. McCauley R. Przywara D.A. Wakade A.R. J. Physiol. 1995; 488: 123-138Crossref PubMed Scopus (77) Google Scholar, 9.Hou S.T. Jiang S.X. Smith R.A. Int. Rev. Cell Mol. Biol. 2008; 267: 125-181Crossref PubMed Scopus (89) Google Scholar, 10.Raff M.C. Whitmore A.V. Finn J.T. Science. 2002; 296: 868-871Crossref PubMed Scopus (558) Google Scholar). Indeed, the expression of Sema3A, a major chemorepulsive factor, has been reported in several brain injury models, such as peripheral nerve injury, spinal cord injury, cerebral ischemia, and Alzheimer disease (1.De 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 (228) Google Scholar, 3.Pasterkamp R.J. De Winter F. Giger R.J. Verhaagen J. Prog. Brain Res. 1998; 117: 151-170Crossref PubMed Google Scholar, 11.Hou S.T. Keklikian A. Slinn J. O'Hare M. Jiang S.X. Aylsworth A. Biochem. Biophys. Res. Commun. 2008; 367: 109-115Crossref PubMed Scopus (40) Google Scholar, 12.Beck H. Acker T. Püschel A.W. Fujisawa H. Carmeliet P. Plate K.H. J. Neuropathol. Exp. Neurol. 2002; 61: 339-350Crossref PubMed Scopus (95) Google Scholar, 13.Fujita H. Zhang B. Sato K. Tanaka J. Sakanaka M. Brain Res. 2001; 914: 1-14Crossref PubMed Scopus (67) Google Scholar, 14.Pasterkamp R.J. Verhaagen J. Brain Res. Brain Res. Rev. 2001; 35: 36-54Crossref PubMed Scopus (114) Google Scholar, 15.Kaneko S. Iwanami A. Nakamura M. Kishino A. Kikuchi K. Shibata S. Okano H.J. Ikegami T. Moriya A. Konishi O. Nakayama C. Kumagai K. Kimura T. Sato Y. Goshima Y. Taniguchi M. Ito M. He Z. Toyama Y. Okano H. Nat. Med. 2006; 12: 1380-1389Crossref PubMed Scopus (315) Google Scholar, 16.Pasterkamp R.J. Giger R.J. Curr. Opin. Neurobiol. 2009; 19: 263-274Crossref PubMed Scopus (162) Google Scholar). In addition, Sema3A expression has been shown to increase vascular permeability, which may indirectly contribute to neuronal damage (17.Acevedo L.M. Barillas S. Weis S.M. Göthert J.R. Cheresh D.A. Blood. 2008; 111: 2674-2680Crossref PubMed Scopus (171) Google Scholar).The biological activities of Sema3A during development are complex and context-dependent. Although best known for its role as an axonal growth cone repellent, Sema3A also serves as a chemoattractant during cortical layer development by guiding the radial migration of layer II/III cortical neurons (18.Chen G. Sima J. Jin M. Wang K.Y. Xue X.J. Zheng W. Ding Y.Q. Yuan X.B. Nat. Neurosci. 2008; 11: 36-44Crossref PubMed Scopus (186) Google Scholar) and the growth of apical dendrites toward the pial surface (19.Polleux F. Morrow T. Ghosh A. Nature. 2000; 404: 567-573Crossref PubMed Scopus (587) Google Scholar). In contrast, Sema3A is also important in stereotyped pruning of long hippocampal axon branches (20.Bagri A. Cheng H.J. Yaron A. Pleasure S.J. Tessier-Lavigne M. Cell. 2003; 113: 285-299Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), causing dorsal root ganglia axon retraction (21.Gallo G. J. Cell Sci. 2006; 119: 3413-3423Crossref PubMed Scopus (118) Google Scholar), and evoking apoptosis of sensory neurons (22.Shirvan A. Ziv I. Fleminger G. Shina R. He Z. Brudo I. Melamed E. Barzilai A. J. Neurochem. 1999; 73: 961-971Crossref PubMed Scopus (136) Google Scholar, 23.Shirvan A. Kimron M. Holdengreber V. Ziv I. Ben Shaul Y. Melamed S. Melamed E. Barzilai A. Solomon A.S. J. Biol. Chem. 2002; 277: 49799-49807Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 24.Gagliardini V. Fankhauser C. Mol. Cell Neurosci. 1999; 14: 301-316Crossref PubMed Scopus (81) Google Scholar) possibly through activating apoptotic pathways involving PlexinA3 receptor and mitogen-activated protein kinases (25.Campbell D.S. Holt C.E. Neuron. 2003; 37: 939-952Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 26.Ben-Zvi A. Manor O. Schachner M. Yaron A. Tessier-Lavigne M. Behar O. J. Neurosci. 2008; 28: 12427-12432Crossref PubMed Scopus (48) Google Scholar).The cellular receptors for semaphorins are neuropilins (NRP1 and NRP2) 2The abbreviations used are: NRP1 and -2neuropilins 1 and 2MALDI-MSImatrix-assisted laser desorption/ionization-mass spectrometry imagingTOFtime of flightMCAOmiddle cerebral artery occlusionPIpropidium iodideRNAiinhibitory RNASema3Asemaphorin 3ATTC2,3,5-triphenyltetrazolium chlorideTUNELterminal deoxynucleotidyltransferase dUTP nick end labelingDIVdays in vitroNMDAN-methyl-d-aspartic acidIPimmunoprecipitation. (27.He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 28.Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar). Structurally, both NRPs contain an extracellular domain of two CUB motifs, adjacent to two domains with homology to coagulation factors V and VIII; a MAM domain; a single transmembrane domain; and a short intracellular domain of 39 amino acids lacking any known signaling motifs (27.He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 28.Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 29.Chen H. Chédotal A. He Z. Goodman C.S. Tessier-Lavigne M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). The structural domain required for Sema3A/NRP1 interaction has been characterized (30.Antipenko A. Himanen J.P. van Leyen K. Nardi-Dei V. Lesniak J. Barton W.A. Rajashankar K.R. Lu M. Hoemme C. Püschel A.W. Nikolov D.B. Neuron. 2003; 39: 589-598Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and several specific polypeptides antagonizing Sema3A IgG domain and NRP1 MAM domain have been found to inhibit Sema3A/NRP1-mediated inhibition of axonal outgrowth in vitro (31.Williams G. Eickholt B.J. Maison P. Prinjha R. Walsh F.S. Doherty P. J. Neurochem. 2005; 92: 1180-1190Crossref PubMed Scopus (27) Google Scholar).NRP1 is a multifunctional receptor, which mediates activities of structurally distinct ligands during development of the heart, vasculature, and neuronal system (32.Gu C. Rodriguez E.R. Reimert D.V. Shu T. Fritzsch B. Richards L.J. Kolodkin A.L. Ginty D.D. Dev. Cell. 2003; 5: 45-57Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). An indication of the importance of NRP1 function in adult mice brain came from our recent discovery that NRP1 expression was transcriptionally regulated by the apoptosis-inducing transcription factor E2F1 (33.Jiang S.X. Sheldrick M. Desbois A. Slinn J. Hou S.T. Mol. Cell. Biol. 2007; 27: 1696-1705Crossref PubMed Scopus (37) Google Scholar). Activation of E2F1 causes neuronal death during cerebral ischemia (34.Hou S.T. Callaghan D. Fournier M.C. Hill I. Kang L. Massie B. Morley P. Murray C. Rasquinha I. Slack R. MacManus J.P. J. Neurochem. 2000; 75: 91-100Crossref PubMed Scopus (103) Google Scholar, 35.MacManus J.P. Jian M. Preston E. Rasquinha I. Webster J. Zurakowski B. J. Cereb. Blood Flow Metab. 2003; 23: 1020-1028Crossref PubMed Scopus (34) Google Scholar). In this study, the neuroprotective effects of blocking NRP1 interaction with Sema3A were investigated and the cytoplasmic tyrosine kinase Fer was determined as a downstream effecter for NRP1-mediated death signal

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