Zhou Chun, Emadi Sharareh, Sierks Michael R, Messer Anne
Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
Mol Ther. 2004 Dec;10(6):1023-31. doi: 10.1016/j.ymthe.2004.08.019.
alpha-Synuclein (alpha-syn) has been identified as the major component of Lewy bodies that characterize neurodegenerative synucleinopathies, including Parkinson's disease. Overexpression of alpha-syn, and prefibrillar alpha-syn oligomers, has been implicated in these pathologies; therefore, prevention of prefibril accumulation, and inhibition of other aberrant effects of overexpressed alpha-syn, could provide novel treatments. Here, we have selected a human single-chan Fv (scFv) antibody, D10, that binds human monomeric wild-type alpha-syn. We demonstrate, by retargeting assays and coimmunoprecipitation, that the D10 scFv is a specific and efficient intracellular antibody (intrabody). By transfecting the D10 scFv gene into an HEK 293 cell line that overexpresses wild-type alpha-syn, we show that the D10 intrabody stabilizes detergent-soluble monomeric alpha-syn and inhibits the formation of detergent-insoluble high-molecular-weight alpha-syn species. In addition, the D10 intrabody ameliorates the decreased cell adhesion that characterizes the alpha-syn-overexpressing cells. Given the important role of alpha-syn pathology, and the facility with which intrabodies can be further engineered in vitro, anti-alpha-syn intrabodies may represent novel molecular therapeutics for synucleinopathies, with implications for other neurodegenerative disorders caused by misfolded accumulated proteins.
α-突触核蛋白（α-syn）已被确定为路易小体的主要成分，路易小体是神经退行性突触核蛋白病（包括帕金森病）的特征性表现。α-syn的过度表达以及原纤维前体α-syn寡聚体与这些病理过程有关；因此，预防原纤维前体的积累以及抑制过度表达的α-syn的其他异常作用可能提供新的治疗方法。在这里，我们选择了一种人单链Fv（scFv）抗体D10，它能与人单体野生型α-syn结合。我们通过重新靶向分析和免疫共沉淀证明，D10 scFv是一种特异性且高效的细胞内抗体（胞内抗体）。通过将D10 scFv基因转染到过表达野生型α-syn的HEK 293细胞系中，我们发现D10胞内抗体可稳定去污剂可溶的单体α-syn，并抑制去污剂不溶的高分子量α-syn物种的形成。此外，D10胞内抗体改善了α-syn过表达细胞所特有的细胞黏附能力下降的情况。鉴于α-syn病理的重要作用以及胞内抗体可在体外进一步改造的便利性，抗α-syn胞内抗体可能代表了突触核蛋白病的新型分子疗法，对由错误折叠的积累蛋白引起的其他神经退行性疾病也有意义。
