High-throughput Screening of Small Molecule Libraries to Identify Novel Therapeutics for ARSACS Using Patient Cells – Dr. Paola Giunti and Dr. Suran Nethisinghe

This research consists of : 1. Establishing baseline measurements for our mitochondrial and cytoskeletal phenotypic readouts on the Perkin Elmer Opera Phenix High Content Screening platform. We will use tetramethylrhodamine methyl ester perchlorate (TMRM, a cationic fluorescent dye sequestered by healthy mitochondria) to measure basal mitochondrial membrane potential (ΔYm) as a read out of mitochondrial health in live cells. In parallel, fixed cells will be dual-stained by immunofluorescence for Tom20 and vimentin to examine the mitochondrial network and intermediate filament morphology, respectively. We will set out and optimise the parameters for the screen using ARSACS patient and control fibroblast lines. 2. Performing small molecule library screens of Reata Pharmaceutical’s compound library and BioAscent’s Phenotypic Toolbox. 3. Validating positive hits using traditional confocal, immunoblotting and luminescence assays. This will include examining NADH levels and calcium signalling on a CCD camera (14, 15). Respiration rate and oxygen consumption will be measured with a Clark-type oxygen electrode (16). These studies will provide mechanistic insight into the mode of action of these compounds. Duration: one year Grant: $50,000 Contacts Dr. Paola Giunti, Ataxia Centre, Department of Molecular Neuroscience UCL Institute of Neurology, Queen Square House Queen Square, London WC1N 3BG, United Kingdom Tel: +44 (0)20 3448 3153; Dr. Suran Nethisinghe, Ataxia Centre, Department of Molecular Neuroscience UCL Institute of Neurology, Queen Square House Queen Square, London WC1N 3BG, United Kingdom Tel: +44 (0)20 3448 3153; email: S.Nethisinghe@ucl.ac.uk