A cell base system for compounds regulating tau splicing

描述（由申请人提供）： tau蛋白病，即由tau蛋白组成的不溶性神经元内聚集体的形成是几种神经退行性疾病的主要临床特征，包括与17号染色体相关的额颞叶痴呆和帕金森综合征（FTDP-17）。从TDP-17患者中鉴定tau基因突变直接将tau基因与这些疾病联系起来。这些突变中的几个改变外显子10的剪接，导致4 R/3R tau比率向有利于4 R tau的方向移动。我们最近开发了一种基于细胞的系统来鉴定可以调节SMN基因外显子7剪接的化合物和小分子。基于细胞的系统已经成功地进行了测试，并用于鉴定化合物。SMN基因的外显子7剪接和tau基因的外显子10剪接之间的相似性使我们相信，也可以为tau基因的外显子10剪接建立基于细胞的系统，并且可以鉴定化合物来调节外显子10剪接。本提案的目标是开发用于鉴定调节外显子10剪接的小分子的基于细胞的测定。这些分子可能用于校正神经退行性疾病（如FTDP-17）中4 R/3R tau亚型之间的比例。该项目的第一个目的是通过修饰用于tau剪接研究的已发表的小基因来产生小基因构建体，所述小基因与报告基因、荧光素酶连接，使得外显子10剪接的效率可以通过荧光素酶活性来测量。将使用RT-PCR和荧光素酶活性来验证瞬时转染到细胞系中后的这些构建体。同时，还将构建和测试预期将外显子10包含在其mRNA中的突变tau迷你基因。本项目的第二个目的是建立包含外显子10（突变体）或排除外显子10（正常）的微基因构建体的稳定细胞系。将通过RT-PCR和SR蛋白验证细胞系，SR蛋白已显示影响外显子10的剪接。然后将优化的细胞系用于高（低）通量筛选（HTS，LTS）。

DESCRIPTION (provided by applicant): Tauopathies, formation of insoluble intraneuronal aggregates made of tau protein are major clinical features of several neurodegenerative disorders including frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Identification of mutations in the tau gene from TDP-17 patients directly linked the tau gene to these diseases. Several of these mutations alter splicing of exon 10, resulting in shifting of 4R/3R tau ratios in favor of 4R tau. We have recently developed a cell-based system to identify compounds and small molecules that can regulate exon 7 splicing of the SMN genes. The cell- based system has been successfully tested and used for identification of compounds. Similarities between exon 7 splicing of the SMN gene and exon 10 splicing of the tau gene lead us to believe that cell-based systems can also be established for exon 10 splicing of the tau gene and compounds can be identified to regulate exon 10 splicing. The goal of this proposal is to develop the cell-based assays for identification of small molecules that modulate exon 10 splicing. These molecules are potentially used to correct the ratios between 4R/3R tau isoforms in neurodegenerative diseases such as FTDP-17. The first aim of this project is to generate mini-gene constructs by modifying published mini-genes used for tau splicing studies with a reporter gene, luciferase attached so that efficiency of exon 10 splicing can be measured by luciferase activities. RT-PCR and activities of luciferase will be used to validate these constructs after transient transfection into cell lines. At the same time, mutant tau mini-genes that are expected to include exon 10 into their mRNAs will be also constructed and tested. The second aim of this project will be to establish stable cell lines with exon 10 included (mutant) or exon 10 excluded (normal) mini-gene constructs. The cell lines will be validated by RT-PCR and by SR proteins, which have been shown to affect splicing of the exon 10. The optimized cell lines will be then used in high (low) throughput screens (HTS, LTS).