Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with...

This work explores the heterogeneity of aggregation of polyglutamine fusion constructs in crude extracts of transgenic Caenorhabditis elegans animals. The work takes advantage of the recent technical advances in fluorescence detection for the analytical ultracentrifuge. Further, new sedimentation velocity methods, such as the multi-speed method for data capture and wide distribution analysis for data analysis, are applied to improve the resolution of the measures of heterogeneity over a wide range of sizes. The focus here is to test the ability to measure sedimentation of polyglutamine aggregates in complex mixtures as a prelude to future studies that will explore the effects of genetic manipulation and environment on aggregation and toxicity. Using sedimentation velocity methods, we can detect a wide range of aggregates, ranging from robust analysis of the monomer species through an intermediate and quite heterogeneous population of oligomeric species, and all the way up to detecting species that likely represent intact inclusion bodies based on comparison to an analysis of fluorescent puncta in living worms by confocal microscopy. Our results support the hypothesis that misfolding of expanded polyglutamine tracts into insoluble aggregates involves transitions through a number of stable intermediate structures, a model that accounts for how an aggregation pathway can lead to intermediates that can have varying toxic or protective attributes. An understanding of the details of intermediate and large-scale aggregation for polyglutamine sequences, as found in neurodegenerative diseases such as Huntington's Disease, will help to more precisely identify which aggregated species may be involved in toxicity and disease.