Cholesterol Modifies Huntingtin Binding to, Disruption of, and Aggregation on Lipid Membranes

Abstract

Huntington's disease (HD) is an inherited neurodegenerative disease caused by abnormally long CAG-repeats in the huntingtin gene that encode an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ domains are directly correlated to disease-related htt aggregation. Htt is found highly associated with a variety of cellular and subcellular membranes that are predominantly comprised of lipids. Since cholesterol homeostasis is altered in HD, we investigated how varying cholesterol content modifies the interactions between htt and lipid membranes. A combination of Langmuir trough monolayer techniques, vesicle permeability and binding assays, and in situ atomic force microscopy were used to directly monitor the interaction of a model, synthetic htt peptide and a full-length htt-exon1 recombinant protein with model membranes comprised of total brain lipid extract (TBLE) and varying amounts of exogenously added cholesterol. As the cholesterol content of the membrane increased, the extent of htt insertion decreased. Vesicles containing extra cholesterol were resistant to htt-induced permeabilization. Morphological and mechanical changes in the bilayer associated with exposure to htt were also drastically altered by the presence of cholesterol. Disrupted regions of pure TBLE bilayers were grainy in appearance and associated with a large number of globular aggregates. In contrast, morphological changes induced by htt in bilayers enriched in cholesterol were plateau-like with a smooth appearance. Collectively, these observations suggest that the presence and amount of cholesterol in lipid membranes play a critical role in htt binding and aggregation on lipid membranes.

Figure 1

(A) Insertion of Nt¹⁷-Q₃₅-P₁₀-KK into lipid monolayers of varying TBLE:cholesterol ratios; expressed as % area increase at each surface pressure. (B) Calcein leakage, measured as relative fluorescence, from LUVs composed of different TBLE:cholesterol ratios exposed to Nt¹⁷-Q₃₅-P₁₀-KK (or buffer acting as a control) as a function of peptide:lipid ratio. Error bars indicate one standard deviation (n = 3).

Figure 2

Percent colorimetric response (%CR) of TBLE/PDA vesicles containing various amounts of cholesterol upon exposure to htt-exon1(51Q) plotted as a function of time. Error bars indicate one standard deviation (n = 3). The highlighted area indicates the time frame accessible with AFM experiments described later.

Figure 3

(A) Sequential AFM height and phase images taken in solution of supported TBLE bilayers exposed to htt-exon1(51Q). Blue arrows indicate oligomer-like structures. (B) AFM height images taken as a function of time demonstrating the rough, grainy morphological changes induced in a pure TBLE bilayer by htt-exon1(51Q). Blue lines correspond to the height profile presented directly below the images.

Figure 4

(A) Sequential AFM height and phase images taken in solution of supported TBLE bilayers containing 10% exogenously added cholesterol exposed to htt-exon1(51Q). (B) AFM height images taken as a function of time demonstrating the smooth morphological changes induced in a TBLE + 10% cholesterol bilayer by htt-exon1(51Q). Blue lines correspond to the height profile presented directly below the images.

Figure 5

Percent area of disrupted bilayer morphology associated with exposure of lipid bilayers containing various amounts of cholesterol to htt-exon1(51Q) presented as a function of time. Error bars indicate one standard deviation (n = 3).