Discovery of highly toxic synuclein sequence variants

描述（由申请人提供）：本项目旨在筛选帕金森氏病神经母细胞瘤细胞系模型系统中通过易出错PCR创建的α -突触核蛋白突变体文库。α -突触核蛋白cDNA序列变体将被单独纯化（高通量格式），并在多孔板的单个孔中转染到细胞中。中等通量筛选（约104个序列）将鉴定出相对于野生型α -突触核蛋白产生高度神经毒性表型（例如氧化应激、细胞凋亡）的突变体。随后对这些高毒性突变体的体外聚集研究将使我们能够确认或排除突触核蛋白的各种寡聚形式（原纤维、孔结构和原纤维）具有细胞毒性，有助于我们理解聚集在PD中的作用，验证药物靶点，并为未来的研究提供方向。此外，基于多孔板的PD模型系统和分析将直接用于筛选新的PD候选药物的化合物库。最后，极毒序列变异本身将是有价值的工具，可以使野生型α -突触核蛋白刺激的细胞毒性途径更加明显，并将使PD的转基因动物模型更加健壮和表型正确成为可能。

DESCRIPTION (provided by applicant): This project seeks to screen a library of alpha-synuclein mutants, created via error-prone PCR, in a neuroblastoma cell-line model system of Parkinson's disease. Alpha-Synuclein cDNA sequence variants will be individually purified (high throughput format) and transfected into cells in individual wells of multiwell plates. A medium-throughput screen (ca. 104 sequences) will then identify those mutants which produce a highly neurotoxic phenotype (e.g., oxidative stress, apoptosis), relative to wild type alpha-synuclein. Subsequent in vitro aggregation studies of these highly toxic mutants will enable us to confirm or rule out various oligomeric forms (protofibril, pore structure, and fibril) of synuclein as cytotoxic, aiding our understanding of the role of aggregation in PD, validating drug targets, and providing direction for future research. Additionally, the multiwell plate based model system and assays of PD will be directly useful for screening compound libraries for new PD drug candidates. Finally, the extremely toxic sequence variants will be valuable tools in of themselves that may make more apparent the cytotoxic pathways stimulated by wild type alpha-synuclein and will make possible more robust and phenotypically correct transgenic animal models of PD.