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
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