Rohrbeck Astrid, Stahl Frank, Höltje Markus, Hettwer Timo, Lindner Patrick, Hagemann Sandra, Pich Andreas, Haastert-Talini Kirsten
Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
Institute for Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, D-30167 Hannover, Germany.
Neurochem Int. 2015 Nov;90:232-45. doi: 10.1016/j.neuint.2015.09.007. Epub 2015 Sep 28.
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.
先前的研究表明，肉毒杆菌C3外切酶或覆盖氨基酸156 - 181的26（聚体）C末端肽片段（C3(156 - 181)）具有促进周围神经再生（PNR）的活性，(3)当在损伤部位一次性注射给药时。本研究旨在：1）研究损伤部位C3和C3(156 - 181)的长期可用性是否能在体内进一步增强PNR，以及2）阐明C3和C3(156 - 181)对体外雪旺细胞（SCs）的影响。(4)对于体内研究，采用神经外膜囊技术或自体神经移植修复成年大鼠10毫米的坐骨神经缺损。神经外膜囊填充有含以下成分的水凝胶：i）赋形剂，ii）40μM C3或iii）40μM C3(156 - 181)。在12周内监测感觉和运动功能恢复情况，并通过神经形态计量学进一步分析PNR的结果。在体外，我们比较了未处理的新生大鼠SCs与用C3或C3(156 - 181)处理72小时的SCs的基因表达谱（微阵列分析）和神经营养因子表达（蛋白质印迹分析）。在成人SCs中证实了对神经营养因子表达水平的影响。出乎意料的是，在损伤部位长期递送C3和C3(156 - 181)并没有增加PNR的结果。然而，关于它们先前检测到的促进PNR作用的潜在机制，发现有6个基因在用C3或C3(156 - 181)处理后的SCs中发生了共同改变。我们证明了参与谷氨酸摄取的基因（Eaac1,(5)Grin2a(6)）显著下调以及神经营养因子表达的变化（FGF - 2增加(7)和NGF减少(8)）。我们基于微阵列的表达谱分析揭示了SCs中可能参与C3和C3(156 - 181)轴突营养（促进再生）作用的新的C3调节基因。用C3或C3(156 - 181)处理的原代新生大鼠SCs和原代成人SCs中神经营养因子表达改变的检测支持了这一假设。
