Sapru Mohan K, Yates Jonathan W, Hogan Shea, Jiang Lixin, Halter Jeremy, Bohn Martha C
Department of Pediatrics, Neurobiology Program, Children's Memorial Research Center, Feinberg School of Medicine, Northwestern University, 2300 Children's Plaza, Box 209, Chicago, IL 60614, USA.
Exp Neurol. 2006 Apr;198(2):382-90. doi: 10.1016/j.expneurol.2005.12.024. Epub 2006 Feb 7.
Human alpha-synuclein overexpression and its toxic accumulation in neurons or glia are known to play key roles in the pathogenesis of Parkinson's disease and other related neurodegenerative synucleinopathies. Several single point mutations in the alpha-synuclein gene, as well as gene duplication and triplication, have been linked to familial Parkinson's disease. Moreover, genetic variability of the alpha-synuclein gene promoter is associated with idiopathic Parkinson's disease. Silencing of the human alpha-synuclein gene by vector-based RNA interference (RNAi) is a promising therapeutic approach for synucleinopathies. Here, we report identification of a 21-nucleotide sequence in the coding region of human alpha-synuclein that constitutes an effective target for robust silencing by RNAi and demonstrate allele-specific silencing of the A53T mutant of human alpha-synuclein. Furthermore, we have developed a plasmid vector-based RNAi for silencing of human alpha-synuclein in vitro. Lastly, using a dual cassette lentivirus that co-expresses an alpha-synuclein-targeting small hairpin RNA (shRNA) and enhanced green fluorescent protein (EGFP) as a marker gene, we demonstrate effective silencing of endogenous human alpha-synuclein in vitro in the human dopaminergic cell line SH-SY5Y and also of experimentally expressed human alpha-synuclein in vivo in rat brain. Our results demonstrate potent silencing of human alpha-synuclein expression in vitro and in vivo by viral vector-based RNAi and provide the tools for developing effective gene silencing therapeutics for synucleinopathies, including Parkinson's disease.
已知人类α-突触核蛋白的过表达及其在神经元或神经胶质细胞中的毒性积累在帕金森病和其他相关神经退行性突触核蛋白病的发病机制中起关键作用。α-突触核蛋白基因中的几个单点突变，以及基因重复和三倍体，都与家族性帕金森病有关。此外，α-突触核蛋白基因启动子的遗传变异性与特发性帕金森病有关。通过基于载体的RNA干扰（RNAi）沉默人类α-突触核蛋白基因是一种有前景的突触核蛋白病治疗方法。在这里，我们报告在人类α-突触核蛋白编码区鉴定出一个21个核苷酸的序列，它构成了RNAi有效沉默的靶点，并证明了人类α-突触核蛋白A53T突变体的等位基因特异性沉默。此外，我们开发了一种基于质粒载体的RNAi用于体外沉默人类α-突触核蛋白。最后，使用共表达靶向α-突触核蛋白的小发夹RNA（shRNA）和增强型绿色荧光蛋白（EGFP）作为标记基因的双盒慢病毒，我们证明了在体外人多巴胺能细胞系SH-SY5Y中内源性人类α-突触核蛋白的有效沉默，以及在大鼠脑内实验表达的人类α-突触核蛋白的有效沉默。我们的结果证明了基于病毒载体的RNAi在体外和体内对人类α-突触核蛋白表达的有效沉默，并为开发针对包括帕金森病在内的突触核蛋白病的有效基因沉默疗法提供了工具。
