title: C3-induced release of neurotrophic factors from Schwann cells - potential mechanism behind its regeneration promoting activity.
abstract: Previous studies revealed a peripheral nerve regeneration (PNR)(1) promoting activity of Clostridium botulinum C3(2) exoenzyme or a 26(mer) C-terminal peptide fragment covering amino acids 156-181 (C3(156-181)),(3) when delivered as one-time injection at the lesion site. The current study was performed to 1) investigate if prolonged availability of C3 and C3(156-181) at the lesion site can further enhance PNR in vivo and to 2) elucidate effects of C3 and C3(156-181) on Schwann cells (SCs)(4)in vitro. For in vivo studies, 10 mm adult rat sciatic nerve gaps were reconstructed with the epineurial pouch technique or autologous nerve grafts. Epineurial pouches were filled with a hydrogel containing i) vehicle, ii) 40 μM C3 or iii) 40 μM C3(156-181). Sensory and motor functional recovery was monitored over 12 weeks and the outcome of PNR further analyzed by nerve morphometry. In vitro, we compared gene expression profiles (microarray analysis) and neurotrophic factor expression (western blot analysis) of untreated rat neonatal SCs with those treated with C3 or C3(156-181) for 72 h. Effects on neurotrophic factor expression levels were proven in adult human SCs. Unexpectedly, prolonged delivery of C3 and C3(156-181) at the lesion site did not increase the outcome of PNR. Regarding the potential mechanism underlying their previously detected PNR promoting action, however, 6 genes were found to be commonly altered in SCs upon treatment with C3 or C3(156-181). We demonstrate significant down-regulation of genes involved in glutamate uptake (Eaac1,(5)Grin2a(6)) and changes in neurotrophic factor expression (increase of FGF-2(7) and decrease of NGF(8)). Our microarray-based expression profiling revealed novel C3-regulated genes in SCs possibly involved in the axonotrophic (regeneration promoting) effects of C3 and C3(156-181). Detection of altered neurotrophic factor expression by C3 or C3(156-181) treated primary neonatal rat SCs and primary adult human SCs supports this hypothesis.
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Electronic address: rohrbeck.astrid@mh-hannover.de.
authors.forename: Astrid
authors.initials: A
authors.lastname: Rohrbeck
authors.affiliation: Institute for Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, D-30167 Hannover, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Stahl
authors.affiliation: Institute for Integrative Neuroanatomy, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Höltje
authors.affiliation: Institute of Neuroanatomy, Hannover Medical School and Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Hettwer
authors.affiliation: Institute for Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, D-30167 Hannover, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Lindner
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Hagemann
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Pich
authors.affiliation: Institute of Neuroanatomy, Hannover Medical School and Carl-Neuberg-Str. 1, D-30625 Hannover, Germany; Center for Systems Neuroscience (ZSN), Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Electronic address: haastert-talini.kirsten@mh-hannover.de.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Haastert-Talini
mesh_terms: D036002:ADP Ribose Transferases / Q000494:pharmacology*; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology*; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D005260:Female; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051379:Mice; D020932:Nerve Growth Factor / Q000378:metabolism; D009416:Nerve Regeneration / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D012583:Schwann Cells / Q000166:cytology / Q000187:drug effects*; D012584:Sciatic Nerve / Q000187:drug effects* / Q000378:metabolism
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D051100:Glial Cell Line-Derived Neurotrophic Factor; D020932:Nerve Growth Factor; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins
keywords: C3 exoenzyme; Gene expression; Mass spectrometry; Microarray; Peripheral nerve regeneration; Schwann cells