Analysis of huntingtin aggregation by fluorescence and FRET microscopy

Institute of Molecular Biology (IMB) gGmbH, Core Facility Microscopy, Mainz, Germany. Huntington's disease is a hereditary movement disorder that is characterized by progressive neuronal cell death mainly in the cortex and striatum of the brain. It is caused by an unstable CAG repeat extension in the first exon of the IT-15 gene which encodes a protein called huntingtin (Htt). The trinucleotide expansion translates into an elongated polyglutamine (polyQ) stretch. A polyQ length of more than 35 glutamine residues is associated with the appearance of huntingtin aggregates and the development of the disease. The process of aggregation is not fully understood but its inhibition and its modulation provide an insight into the mechanisms leading to aggregate formation which might be a target for the treatment of the disease. Using CFP- and YFP-tagged huntingtin exon 1 fragments we established a cellular model that visualizes the process of huntingtin aggregation and in which the aggregates could be specifically detected by FRET microscopy (acceptor photobleaching and fluorescence lifetime microscopy). The time course of the aggregation process was investigated by image analysis.