Sedimentation Velocity Analysis with Fluorescence Detection of Mutant Huntingtin Exon 1 Aggregation in Drosophila melanogaster and Caenorhabditis elegans

Abstract

At least nine neurodegenerative diseases that are caused by the aggregation induced by long tracts of glutamine sequences have been identified. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington’s disease. Sedimentation velocity with fluorescence detection is applied to perform a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in Drosophila melanogaster and Caenorhabditis elegans. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and semidenaturing detergent agarose gel electrophoresis, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble inclusion bodies. Differences in aggregation in the two animal model systems are noted, possibly because of differences in levels of expression of glutamine-rich sequences. An increased level of aggregation is shown to correlate with increased toxicity for both animal models. Co-expression of the human Hsp70 in D. melanogaster showed some mitigation of aggregation and toxicity, correlating best with inclusion body formation. The comparative study emphasizes the value of the analytical ultracentrifuge equipped with fluorescence detection as a useful and rigorous tool for in situ aggregation analysis to assess commonalities in aggregation across animal model systems.

Solution‐Based Determination of Dissociation Constants for the Binding of Aβ42 to Antibodies

“…The determined values may offer additional information for the application of these antibodies. AUC and MST are also capable of analyzing challenging samples such as tissue lysates, blood serum or other biological liquids, [21] which envisions the possible translation of the approach to physiological samples. However, solution based methods also have some limitations.…”

Lipid Membranes Influence the Ability of Small Molecules To Inhibit Huntingtin Fibrillization

“…(14)(15)(16) htt can form a heterogeneous mixture of oligomers (15,(17)(18)(19) that have been observed in a variety of HD models. (20)(21)(22)(23)(24) Due to the apparent role of protein aggregation in amyloid diseases, a number of small molecules that inhibit or modify amyloid formation have been identified. (25,26) Two specific polyphenols, (−)-epigallocatechin 3-gallate (EGCG) and curcumin, have been extensively studied due to their ability to generically inhibit amyloid formation.…”

“…For example, changes in pH, , metal ions, and preparatory protocols influence aggregate structure and morphology. In particular, HD is caused by an expanded polyglutamine domain that must surpass a critical threshold of ∼35 repeat units, − and the extent of htt fibrillization correlates directly with an increase in polyQ length. − htt can form a heterogeneous mixture of oligomers ,− that have been observed in a variety of HD models. − …”