Sugar distributions on gangliosides guide the formation and stability of amyloid-β oligomers

Abstract

Aggregation of Aβ peptides is a key contributor to the etiology of Alzheimer's disease. Being intrinsically disordered, monomeric Aβ is susceptible to conformational excursions, especially in the presence of important interacting partners such as membrane lipids, to adopt specific aggregation pathways. Furthermore, components such as gangliosides in membranes and lipid rafts are known to play important roles in the adoption of pathways and the generation of discrete neurotoxic oligomers. Yet, what roles do carbohydrates on gangliosides play in this process remains unknown. Here, using GM1, GM3, and GD3 ganglioside micelles as models, we show that the sugar distributions and cationic amino acids within Aβ N-terminal region modulate oligomerization of Aβ temporally, and dictate the stability and maturation of oligomers. These results demonstrate the selectivity of sugar distributions on the membrane surface toward oligomerization of Aβ and thus implicate cell-selective enrichment of oligomers.

Keywords: Aggregation; Alzheimer's disease; Amyloid-β; Gangliosides; Oligomers.

Figure 1:

(a, b, and c) Chemical structures of GM1, GM3, and GD3 gangliosides respectively. (d) Sequences of wt-Aβ, AβK16A, AβH¹³ H¹⁴AA, and AβR5A.

Figure 2:

(a, b, c and d) Normalized ThT fluorescence kinetics of 25 μM wt-Aβ, K16A, H¹³H¹⁴ AA, and R5A mutants of Aβ respectively without (▼; control) or with 75μM GM1 (▄), GM3(●), or GD3 (▲) ganglioside lipid micelles buffered in 20mM tris buffer pH 8.00 with 50 mM NaCl and 50 μM ThT monitored with intermittent shaking for 16–18 h at 37°C in BioTeK synergy HTX 96-well plate reader. Data containing gangliosides in buffer without Aβ were used as blanks to correct prior to normalization.

Figure 3:

(a, b, c, and d) Partially denaturing SDS-PAGE immunoblots of 25 μM wt-Aβ, AβK16A, AβH¹³ H¹⁴ AA, and AβR5A, respectively, in the presence of 75μM GM1, GM3, or GD3 micelles or without micelles, i.e., control (C). Samples were incubated in 20 mM Tris pH 8.00 with 50 mM NaCl at 37 °C and centrifuged at 130,000g for 30 minutes at 4°C at intervals of after 3, 6, and 24 h. The total and supernatant samples were run on gel respectively and probed with either Ab5 monoclonal antibody that binds to the N-terminal of Aβ sequence for (a), (b), and (c) or Ab42.2 monoclonal antibody with a C-terminal epitope of Aβ42 for (d). All single arrows represent fibrils, double arrows represent high molecular weight species and triple arrows represent low molecular weight oligomers.

Figure 4:

AFM height images for 2–2.5 μM samples of Aβ oligomers generated in the presence of ganglioside micelles in 20 mM Tris pH 8.00. Reactions after 24h of incubation at 37 °C were spun at 136,000 g, and supernatant for each sample was spotted and imaged on a mica grid: (a-c) wt-Aβ, (d-f) K16A, (g-i) HHAA, and (j-l) R5A with GM1, GM3, and GD3 in columns left to right respectively. Scale bars represent 400 nm.

Figure 5:

Quantitative stability analysis of oligomers by SEC fractionation. SEC chromatograms (a-c) of the supernatants of wt-Aβ samples (25 μM) incubated in the presence of 75μM GM1 (—), GM3 (---), or GD3 (—) ganglioside micelles buffered in 20 mM Tris pH 8.0, 50 mM NaCl at 37 °C after centrifugation at 130,000×g for 30 min after 3h (a), 6h (b) and 24 h (c) of incubation at 37°C. The fractions were monitored by UV-Vis at 280 nm. The arrow indicates the void volume. (d-f) Bar diagram showing the corresponding integrated area under the peak for SEC chromatograms for GM1, GD3, or GM3 incubated samples.

Figure 6:

(a) SEC chromatogram for isolation of Aβ oligomers generated in the presence of 75μM GD3 ganglioside micelles buffered in tris pH-8.0 50 mM NaCl after incubation at 37 °C for 24 h and centrifugation at 130,000g for 30 min. (b) Immunoblots of SEC-isolated oligomer fraction 15–16 of Aβ oligomers generated in the presence of GD3 micelles in (a) probed by Ab5 monoclonal antibody. (c) DLS for fraction 16 of SEC-isolated Aβ oligomers (accumulation of a total of 12 scans) generated in the presence of GD3 gangliosides. (d) CD spectra (accumulation of 3 scans) of fraction 16 of SEC-fractionated Aβ oligomers generated in the presence of GD3 (—) or GM1(---) ganglioside micelles. (e) SEC chromatogram for isolation of Aβ oligomers generated in the presence of 75μM GM1 ganglioside micelle buffered in tris pH-8.0 with 50 mM NaCl after incubation at 37°C for 24 h and centrifugation at 130000g for 30 min. (f) Immunoblots of 15–16 from SEC-isolated Aβ oligomer fraction generated in the presence of GM1 micelles in (a) probed by Ab5 monoclonal antibody. (g) DLS for fraction 16 of SEC-isolated Aβ oligomers (accumulation of a total of 12 scans) generated in the presence of GM1 gangliosides.