A cell base system for compounds regulating tau splicing

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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).