Cell and Molecular Screens of Polyglutamine Toxicity

University Of California San Francisco, San Francisco CA

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Abstract

DESCRIPTION (provided by applicant): The long-term objectives of this application are to design cell-based systems for high throughput screening of compounds that will modify the toxicity and abnormal processing associated with polyglutamine proteins. The specific aims of the project are: 1. Establish neural precursor cell lines with regulated gene expression of polyglutamine proteins suitable for high throughput applications. a. Express of polyglutamine-flourescent proteins for FRET-based studies of aggregation. b. Express of native polyglutamine proteins for cell viability studies. 2. Establish a fluorescent plate reader-based assay to monitor expanded polyglutamine protein aggregation. a. Establish FPR-based assays of protein aggregation based on FRET. b. Establish FPR-based assays of cell toxicity. The project is directly related to drug development for treatment and prevention of human neurodegenerative disease due to polyglutamine expansion proteins. The research design has two basic elements: development of appropriate cell models using inducible promoter systems in neural precursor cells; development of systems to monitor cell viability and abnormal processing using a fluorescent plate reader. Methods for cell development include culture and transfection of neural cell lines, establishment of inducible cell expression systems using fluroscence activated cell sorting to isolate cells with regulated expression, and characterization of responses to induced expression of polyglutamine proteins. Methods for FPR development are based on detection of FRET signal from polyglutamine proteins with abnormal aggregation, and detection of fluorescent signals in viable and dead cells, using expression of fluorescent proteins and detection dyes. To corroborate FPR results, standard methods of cell imaging and protein biochemistry shall be employed.