Discovery of highly toxic synuclein sequence variants

发现高毒性突触核蛋白序列变体

• 批准号: 7013561
• 负责人: PETER T LANSBURY
• 金额: $ 8.54万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7013561
• 关键词: Epstein Barr virus; Parkinson' s disease; alpha synuclein; cell line; complementary DNA; cytotoxicity; genetic library; genetic screening; high throughput technology; mutant; neuroblastoma; neurons; neurotoxicology; nucleic acid sequence; point mutation; recombinant proteins; technology /technique development; transfection /expression vector

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.

描述（由申请人提供）：该项目旨在筛选帕金森病神经母细胞瘤细胞系模型系统中通过易错PCR创建的α-突触核蛋白突变体库。α-突触核蛋白cDNA序列变体将被单独纯化（高通量格式）并转染到多孔板的各个威尔斯孔中的细胞中。一个中等通量的屏幕（约。104个序列）然后将鉴定产生高度神经毒性表型的那些突变体（例如，氧化应激、凋亡）。这些高毒性突变体的后续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体形式（原纤维，孔结构和原纤维）具有细胞毒性，帮助我们理解聚集在PD中的作用，验证药物靶点，并为未来的研究提供方向。此外，基于多孔板的模型系统和PD测定将直接用于筛选新的PD候选药物的化合物库。最后，极端毒性的序列变体本身将是有价值的工具，其可以使野生型α-突触核蛋白刺激的细胞毒性途径更明显，并且将使PD的更稳健和表型正确的转基因动物模型成为可能。