Lipid headgroups alter huntingtin aggregation on membranes

Abstract

Huntington's Disease is a fatal neurodegenerative disorder caused by expansion of a glutamine repeat region (polyQ) beyond a critical threshold within exon1 of the huntingtin protein (htt). As a consequence of polyQ expansion, htt associates into a variety of aggregate species that are thought to underlie cellular toxicity. Within cells, htt associates with numerous membranous organelles and surfaces that exert influence on the aggregation process. In particular, the first 17 amino acids at the N-terminus of htt (Nt17) serve as a lipid-binding domain that is intrinsically disordered in bulk solution but adopts an amphipathic α-helical structure upon binding membranes. Beyond this, Nt17 is implicated in initiating htt fibrillization. As the interaction between Nt17 and lipid membranes is likely influenced by lipid properties, the impact of lipid headgroups on htt-exon1 aggregation, membrane activity, and the ability to form protein:lipid complexes was determined. Htt-exon1 with a disease-length polyQ domain (46Q) was exposed to lipid vesicles comprised of lipids with either zwitterionic (POPC and POPE) or anionic (POPG and POPS) headgroups. With zwitterionic head groups, large lipid to peptide ratios were required to have a statistically significant impact on htt aggregation. Anionic lipids enhanced htt fibrillization, even at low lipid:protein ratios, and this was accompanied by changes in aggregate morphology. Despite the larger impact of anionic lipids, htt-exon1(46Q) was more membrane active with zwitterionic lipid systems. The ability of Nt17 to form complexes with lipids was also mediated by lipid headgroups as zwitterionic ionic lipids more readily associated with multimeric forms of Nt17 in comparison with anionic lipids. Collectively, these results highlight the complexity of htt/membrane interactions and the resulting impact on the aggregation process.

Keywords: Amyloid; Atomic force microscopy; Electrospray ionization-mass spectrometry; Huntington's disease; Lipid membranes; Polyglutamine.

Figure 1.

ThT aggregation assays for htt-exon1(46Q) in the presence of (a) POPC, (b) POPE, (c) POPG, or (d) POPS lipid vesicles. Htt concentration was 20 μM, and the lipid to htt molar ratio was 5:1, 10:1, or 20:1. The aggregation rate for each condition is represented by the (e) slope relative to the control assay with no lipids, which is set to 100. The (f) relative maximum fluorescence for each condition was calculated in the same way, setting the no lipid condition to 100. In panels a-d, error bars are provided for every sixth data point (30 min) and represent the standard error of the mean (SEM). Analyses shown in panels e and f were determined as averages over all trials, and values are normalized as a percentage with respect to the htt control in the absence of lipid. Error bars represent SEM. * represents a p value <0.05, and ** represent a p value <0.01 using a Student’s t-test.

Figure 2.

Representative AFM images of 20 μM htt-exon1(46Q) incubated with (a) no lipid, (b) POPC vesicles, (c) POPE vesicles, (d) POPG vesicles, or (e) POPS vesicles at various time points. The ratio of lipid to htt was 20:1. The colormap and scale bar is applicable to all images.

Figure 3.

AFM analysis of the impact of different lipid systems on htt-exon1(46Q) aggregation. (a) Analysis of the number of fibrils formed in the presence of each lipid system as a function of time. (b) The percent area of each image covered by fibrils at the 8 h time point for each condition. (c) Height histograms of fibrils formed in the presence of different lipid systems. (d) Image analysis of oligomers per unit area as a function of time for each condition. Correlation plots of the height and diameter of each oligomer and histograms of these morphological features at 1 h and 8 h in the presence of (e) no lipid, (f) POPC, (g) POPE, (h) POPG, and (i) POPS vesicles. For panels a, b, and d, error bars represent the SEM, * represents a p value <0.05, and ** represent a p value <0.01 using a Student’s t-test.

Figure 4.

Representative AFM images of a unique fibril morphology formed as a result of htt-exon1(46Q) incubation with POPG lipid vesicles.

Figure 5.

PDA/Lipid binding assays for (a) POPC, (b) POPE, (c) POPG, or (d) POPS exposed to 5, 10, or 20 μM htt-exon1(46Q). (e) The maximum % CR for each condition after 18 h. (f) Direct comparison of the kinetic lipid binding assay for each lipid system when exposed to 20 μM htt. Error bars are provided for every sixth data point and represent the SEM.

Figure 6.

Mass spectrum showing the ions obtained after ESI of Nt17 with (a) POPC, (b) POPE, (c) POPG, or (d) POPS lipid vesicles after 5 h of co-incubation. Insets represent expanded regions in which specific observed ions are present.

Figure 7.

The relative abundance of ions observed with POPC, POPE, POPG, or POPS lipids and Nt17 (a) monomers, (b) dimers, (c) trimers, or (d) tetramers. The total relative abundance of complexes formed with each Nt17 species is presented (top), along with the breakdown of relative abundance as a function of lipid content (bottom) for each system. Error bars represent the SEM.