Department of Neuroregeneration, Netherlands Institute for Neuroscience, an institute of the Royal Academy of Arts and Sciences, Amsterdam, the Netherlands.
BMC Neurosci. 2010 Feb 18;11:20. doi: 10.1186/1471-2202-11-20.
After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2).
We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents.
RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.
脊髓损伤后，轴突再生受到损伤环境中多种抑制分子的抑制。在损伤部位及其周围，髓鞘相关抑制剂、硫酸软骨素蛋白聚糖（CSPGs）和几种轴突导向分子，包括分泌型（class 3）Semaphorin 的所有成员，都有表达。干扰多种抑制信号可能会增强之前报道的阻断单一分子的有益效果。RNA 干扰（RNAi）是一种可以同时沉默多个基因表达的工具。在这项研究中，我们旨在利用腺相关病毒（AAV）介导的短发夹 RNA（shRNA）表达来靶向 Semaphorin class 3 信号的所有受体，即神经纤毛蛋白 1（Npn-1）和神经纤毛蛋白 2（Npn-2）。
我们成功地生成了可以在神经元细胞系中敲低 Npn-1 和 Npn-2 的 shRNA。当在大鼠背根神经节（DRG）中注射表达 Npn-2 特异性 shRNA 的 AAV5 病毒载体颗粒时，我们检测到 Npn-2 mRNA 的大量敲低。然而，出乎意料的是，AAV1 介导的 Npn-2 shRNA 和对照 shRNA 在红核中的表达导致了组织不良反应和神经元变性。观察到的毒性是剂量依赖性的，与对照 GFP 表达的 AAV 载体不同，这表明 shRNA 是导致毒性的原因。
RNAi 是一种在体外和体内敲低神经元细胞中 Semaphorin 受体表达的强大工具。然而，当 shRNA 在中枢神经系统神经元中高水平表达时，它们会引发组织不良反应，导致神经元降解。
