Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system

BMC Neuroscience

Research Article

Erich M Ehlert¹  Joost Verhaagen¹  Ruben Eggers¹  Simone P Niclou² 

[1] Department of Neuroregeneration, Netherlands Institute for Neuroscience, an institute of the Royal Academy of Arts and Sciences, Amsterdam, the Netherlands;NorLux Neuro-Oncology Laboratory, Centre de Recherche Public Santé, Val Fleuri, Luxembourg, Luxembourg;

关键词: Green Fluorescent Protein;    Dorsal Root Ganglion;    Dorsal Root Ganglion Neuron;    Green Fluorescent Protein Expression;    Control shRNA;   

DOI  :  10.1186/1471-2202-11-20

 received in 2009-10-21, accepted in 2010-02-18,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundAfter 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).ResultsWe 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.ConclusionsRNAi 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.

【 授权许可】

Unknown   
© Ehlert et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.