Studying C9orf72 dipeptide repeat polypeptide aggregation using an analytical ultracentrifuge equipped with...

Sedimentation velocity, using an analytical ultracentrifuge equipped with fluorescence detection, and electrophoresis methods are used to study aggregation of proteins in transgenic animal model systems. Our previous work validated the power of this approach in an analysis of mutant huntingtin aggregation. We demonstrate that this method can be applied to another neurodegenerative disease studying the aggregation of three dipeptide repeats (DPRs) produced by aberrant translation of mutant c9orf72 containing large G₄C₂ hexanucleotide repeats. These repeat expansions are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We analyzed the aggregation patterns of (Gly-Pro)₄₇, (Gly-Ala)₅₀, and (Gly-Arg)₅₀ fused to fluorescent proteins in samples prepared from D. melanogaster, and (Gly-Ala)₅₀ in C. elegans, using AU-FDS and SDD-AGE. Results suggest that (GP)₄₇ is largely monomeric. In contrast, (GA)₅₀ forms both intermediate and large-scale aggregates. (GR)₅₀ is partially monomeric with some aggregation noted in SDD-AGE analysis. The aggregation of this DPR is likely to represent co-aggregated states with DNA and/or RNA. The power of these methods is the ability to gather data on aggregation patterns and characteristics in animal model systems, which may then be used to interpret the mitigation of aggregation through genetic or molecular therapeutic interventions.