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

Abstract

Several diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease (HD), are associated with specific proteins aggregating and depositing within tissues and/or cellular compartments. The aggregation of these proteins is characterized by the formation of extended, β-sheet rich fibrils, termed amyloid. In addition, a variety of other aggregate species also form, including oligomers and protofibrils. Specifically, HD is caused by the aggregation of the huntingtin (htt) protein that contains an expanded polyglutamine domain. Due to the link between protein aggregation and disease, small molecule aggregation inhibitors have been pursued as potential therapeutic agents. Two such small molecules are epigallocatechin 3-gallate (EGCG) and curcumin, both of which inhibit the fibril formation of several amyloid-forming proteins. However, amyloid formation is a complex process that is strongly influenced by the protein's environment, leading to distinct aggregation pathways. Thus, changes in the protein's environment may alter the effectiveness of aggregation inhibitors. A well-known modulator of amyloid formation is lipid membranes. Here, we investigated if the presence of lipid vesicles altered the ability of EGCG or curcumin to modulate htt aggregation and influence the interaction of htt with lipid membranes. The presence of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine or total brain lipid extract vesicles prevented the curcumin from inhibiting htt fibril formation. In contrast, EGCG's inhibition of htt fibril formation persisted in the presence of lipids. Collectively, these results highlight the complexity of htt aggregation and demonstrate that the presence of lipid membranes is a key modifier of the ability of small molecules to inhibit htt fibril formation.

Figure 1.

ThT aggregation assays for htt-exon1(46Q) in the presence of (A) curcumin or (B) EGCG. The htt-exon1(46Q) concentration was 20 μM. (C) The initial rate of aggregation and (D) the relative maximum fluorescence were determined with respect to the htt control. Normalization was performed with respect to the htt control. Analyses shown in panels C and D were determined as averages over all trials (shown in Figure S2). Error bars are provided for every sixth data point (30 min) and represent the standard error of the mean. One asterisk represents a p value of <0.05, and two asterisks represent a p value of <0.01.

Figure 2.

AFM analysis of the impact of curcumin or EGCG on htt-exon1(46Q) aggregation (5:1 small molecule:htt ratio). (A) Representative AFM images of 20 μM htt-exon1(46Q) incubated alone, with curcumin, or with EGCG as a function of time. The color map is the same for all images (see the top color bar). The insets in the AFM images of htt incubated with EGCG use a reduced color map (see bottom, labeled color bar) to make the shorter fibrils in these samples easier to see. (B) Image analysis of the number of oligomers per unit area as a function of time. (C) Height histograms of oligomers observed in AFM images as a function of time. (D) Image analysis of the number of fibrils per unit area as a function of time. (E) Height histograms of fibrils observed in AFM images as a function of time. For panels B and D, error bars represent the standard deviation, one asterisk represents a p value of <0.05, and two asterisks represent a p value of <0.01 using a Student’s t test.

Figure 3.

AFM images comparing the morphology of htt-exon1(46Q) fibrils formed (A) in the absence of small molecules, (B) with curcumin, or (C) with EGCG. In panel C, the boxed insets correspond to the second color bar to better visualize the fibril structures associated with htt incubated with EGCG. The color lines in each image correspond to the height profiles directly below each image.

Figure 4.

ThT aggregation assays for htt-exon1(46Q) aggregated in the presence of (A) POPC or (B) TBLE lipid vesicles. The htt-exon1(46Q) concentration was 20 μM. Curcumin and EGCG were added at a 5:1 small molecule:htt molar ratio. The initial rate of aggregation in the presence of (C) POPC or (D) TBLE vesicles was made relative to the aggregation rate of htt with the lipid vesicles in the absence of the small molecules. Finally, the relative maximum fluorescence values of all conditions in the presence of (E) POPC or (F) TBLE lipid vesicles at the end of the 18 h kinetic run are compared. Analyses shown in panels C–F were determined as averages over all trials (shown in Figure S2). Error bars are provided for every sixth data point (30 min) and represent the standard error of the mean. One asterisk represents a p value of <0.05, and two asterisks represent a p value of <0.01.

Figure 5.

AFM analysis of the impact of curcumin or EGCG on htt-exon1(46Q) aggregation (5:1 small molecule:htt ratio) in the presence of POPC lipid vesicles (20:1 lipid:protein ratio). (A) Representative AFM images of 20 μM htt-exon1(46Q) incubated with POPC with no small molecules, with curcumin, or with EGCG as a function of time. The color map is the same for all images. (B) Image analysis of the number of oligomers per unit area as a function of time. (C) Height histograms of oligomers observed in AFM images as a function of time. (D) Image analysis of the number of fibrils per unit area as a function of time. Arrows indicate that no fibrils were observed. (E) Height histograms of fibrils observed in AFM images as a function of time. As fibrils were not observed for htt-exon1(46Q) incubations with POPC and EGCG, this condition is not presented in the fibril histograms. For panels B and D, error bars represent the standard deviation, one asterisk represents a p value of <0.05, and two asterisks represent a p value of <0.01 using a Student’s t test.

Figure 6.

AFM analysis of the impact of curcumin or EGCG on htt-exon1(46Q) aggregation (5:1 small molecule:htt ratio) in the presence of TBLE vesicles (20:1 lipid:protein ratio). (A) Representative AFM images of 20 μM htt-exon1(46Q) incubated with TBLE with no small molecules, with curcumin, or with EGCG as a function of time. The color map is the same for all images. (B) Image analysis of the number of oligomers per unit area as a function of time. (C) Height histograms of oligomers observed in AFM images as a function of time. (D) Image analysis of the number of fibrils per unit area as a function of time. Arrows indicate that no fibrils were observed. (E) Height histograms of fibrils observed in AFM images as a function of time. As fibrils were not observed for incubations of htt-exon1(46Q) with TBLE and EGCG, this condition is not presented in the fibril histograms. For panels B and D, error bars represent the standard deviation, one asterisk represents a p value of <0.05, and two asterisks represent a p value <0.01 using a Student’s t test.

Figure 7.

AFM images comparing the morphology of htt-exon1(46Q) fibrils formed in the absence of small molecules, with curcumin, or with EGCG for incubations performed in the presence of (A) POPC or (B) TBLE vesicles. The color lines in each image correspond to the height profiles directly below each image.

Figure 8.

Lipid binding assays for (A) PDA/POPC vesicles exposed to either curcumin or EGCG, (B) PDA/POPC vesicles exposed to htt-exon1(46Q) with either curcumin or EGCG, (C) PDA/TBLE vesicles exposed to either curcumin or EGCG, and (D) PDA/TBLE vesicles exposed to htt-exon1(46Q) with either curcumin or EGCG. Error bars are provided for every sixth data point and represent the standard error of the mean.