The effects of membrane physicochemical properties on huntingtin membrane association and downstream aggregation

Huntington’s Disease (HD) is a fatal neurodegenerative disorder caused by an expanded glutamine repeat region (polyQ) within the huntingtin protein (htt). As a result of the expanded polyQ domain, htt associates into a variety of toxic aggregate species. The polyQ domain of htt is flanked at the N-terminal end by 17 amino acids (Nt17) that adopt an amphipathic α-helical structure in the presence of binding partners such as lipid membranes. In addition to comprising a lipid binding domain, the Nt17 amphipathic α -helix has been directly implicated in htt aggregation initiation via self-association with other Nt17 α -helices. Due to this, htt/lipid interaction likely has a large impact on the rate and extent of htt aggregate formation, with potential implications for HD pathogenesis. The studies presented here focus on elucidating the effect of membrane physicochemical properties on htt membrane association and downstream htt aggregation. In order to measure membrane association, a method of normalizing polydiacetylene (PDA) lipid binding assays was developed to enable the direct comparison of various molecules’ binding affinity for different lipid systems. Then, the normalized PDA assay was utilized to determine if small molecule aggregation inhibitors influence the interaction of htt with pure and physiologically relevant lipid systems, and thioflavin-T (ThT) assays and atomic force microscopy (AFM) were used to monitor if the interaction altered the ability of the small molecules to inhibit htt aggregation. While both small molecules altered htt-membrane association, EGCG remained an effective aggregation inhibitor while curcumin no longer inhibited htt fibrillization in the presence of either lipid system. These results highlight the complex relationship between htt-membrane association, downstream aggregation, and the ability of small molecules to inhibit htt aggregation in a cellular environment. Subsequent studies utilized ThT, AFM, polydiacetylene (PDA) assays, and native MS to determine how altering membrane physicochemical properties by changing the headgroup or the tail of lipids influences htt aggregation and membrane affinity. Our results indicate that increasing the negative charge of lipid headgroups disrupts the insertion of Nt17 into the bilayer, which in turn decreases membrane disruption and results in a localization effect that increases htt fibrillization. Also, when varying the degree of unsaturation in the lipid tail, the trend of htt aggregation in the presence of each lipid system is different than the trend of association between htt and the vesicles, indicating that membrane properties alter the mechanism of downstream htt aggregation. Further investigation of the interaction between Nt17 and lipid membranes with molecular dynamics (MD) simulations reveal that a combination of the hydrophobic amino acid and available membrane defect sizes influence the orientation of Nt17 on bilayers during the initial stages of interaction. Collectively, these results highlight how the properties of lipid membranes modulate htt-membrane interactions and htt aggregation mechanisms