Huntington’s disease is a neurodegenerative disease caused by an expansion of the trinucleotide CAG repeat region in the HTT gene beyond 40 repeats. The expanded polyglutamine tract causes huntingtin (Htt) protein to misfold into β-sheet structures and aggregate within the cell. Characterizing the pathway of aggregation is critical to understanding the mechanism of Huntington’s disease and identifying therapeutic targets. However, the smallest species on the aggregation pathway are between 1 and 80 nm in size, with larger aggregates containing a great deal of fine structural detail below the diffraction limit of ~200 nm. To resolve these aggregates in vitro, we used direct stochastic optical reconstruction microscopy (dSTORM). Previously, we found that Htt exon 1 aggregation proceeded from the formation of ~80 nm globular species to ~1 μm-long fibers that increased in width, but not length, over time with the addition of more huntingtin monomers. This pattern of aggregation differs in significant ways from that of the proteins associated with Alzheimer’s and Parkinson’s diseases. To further investigate the Htt aggregation pathway and the factors principally responsible for determining aggregate structure, we have characterized the sensitivity of aggregation to preparation conditions. Samples prepared under static conditions had a greater degree of heterogeneity in the size distribution of globular species compared to agitated samples, and the average size of the agitated globular species was larger than that of the static globular species. The agitated samples also had decreased numbers of mature ~1 μm fibers when compared to static samples. We hypothesize that agitation both promoted aggregation by increasing molecular collisions and retarded growth by destabilizing aggregates. In the future, we plan to investigate the effects of other conditions such as temperature and molecular crowding on aggregation.

Characterization of the huntingtin aggregation pathway via single-molecule and super-resolution fluorescence microscopy

253rd National Meeting (2017)

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