Fibrillization of 40-Residue β-Amyloid Peptides in Membrane-Like Environments Leads to Different Fibril Structures and Reduced Molecular Polymorphisms

Abstract

The molecular-level polymorphism in β-Amyloid (Aβ) fibrils have recently been considered as a pathologically relevant factor in Alzheimer's disease (AD). Studies showed that the structural deviations in human-brain-seeded Aβ fibrils potentially correlated with the clinical histories of AD patients. For the 40-residue Aβ (Aβ₄₀) fibrils derived from human brain tissues, a predominant molecular structure was proposed based on solid-state nuclear magnetic resonance (ssNMR) spectroscopy. However, previous studies have shown that the molecular structures of Aβ ₄₀ fibrils were sensitive to their growth conditions in aqueous environments. We show in this work that biological membranes and their phospholipid bilayer mimics serve as environmental factors to reduce the structural heterogeneity in Aβ₄₀ fibrils. Fibrillization in the presence of membranes leads to fibril structures that are significantly different to the Aβ₄₀ fibrils grown in aqueous solutions. Fibrils grown from multiple types of membranes, including the biological membranes extracted from the rats' synaptosomes, shared similar ssNMR spectral features. Our studies emphasize the biological relevance of membranes in Aβ₄₀ fibril structures and fibrillization processes.

Keywords: biological membranes; structural polymorphisms; synaptic plasma membranes; β-amyloid fibrils.

Figure 1

(a) Representative Thin-Layer Chromatography (TLC) analysis of the membrane compositions of rats’ synaptic plasma membrane vesicles (rat synaptic plasma membrane vesicles (rSPMV), left side) and TLC for the standard lipids/cholesterol at different concentrations. (b) Standard curves for the quantifications of membrane compositions using TLC. (c) Plots of lipid compositions in rSPMV of different ages. Error bars were generated from three independent TLC analyses.

Figure 2

Thioflavin-T fluorescence traces (three repetitions for each membrane composition) for Aβ₄₀ fibrillization kinetics in the presence of different membrane models and a control that only contains the peptides.

Figure 3

Plot of the lag periods (t_lag, A) and growth rate constants (k, B) for Aβ₄₀ fibrillization in different membrane models. Error bars were generated from three repetitions of thioflavin-T (ThT) kinetic curves shown in Figure 2. Different colors are used to facilitate the presentation (different model membranes).

Figure 4

(a) Negatively stained transmission electron microscopy (TEM) images for Aβ₄₀ fibrils grown in the presence of MM1 (top), MM2 (middle), and BM2 (bottom) model membranes (MM1: the model bilayer with 100% phosphatidylcholine; MM2: the model bilayer with phosphatidylcholine/phosphatidylglycerol/cholesterol/sphingomyelin/ganglioside GM1 at molar ratio 1.00/1.00/1.33/1.00/0.10; BM2: rat synaptic plasma membrane vesicles with 12-month age). (b–d) Representative short-mixing (20 ms) 2D ¹³C–¹³C spin diffusion spectra for Aβ₄₀ fibrils formed with (b) MM1, (c) MM2, and (d) BM2 membrane models. The peptides were selectively uniformly labeled at residues G9, L17, A21, V24, N27, I32, G33, and M35.

Figure 5

Plot of the residue-specific ¹³C chemical shift deviation for membrane-associated Aβ₄₀ fibrils derived from MM1 (the fibrils grown from phosphatidylcholine (PC) model bilayers). The comparison contains two types of fibrils from current studies: those grown from the MM2 model bilayer (black) and those grown from the extracted synaptic membranes BM2 (red), and three fibrils reported in the literature: the three-fold Aβ₄₀ fibrils grown from synthetic peptides (green) [35], the first molecular structure from human-brain-seeded Aβ₄₀ fibril (blue) [6] and the reported predominant structure from multiple human brain tissue samples (purple) [8]. The dashed lines represent the thresholds of significant chemical shift difference for individual residues, by considering the ssNMR line widths.

Figure 6

Representative long-mixing (500 ms) 2D ¹³C–¹³C spin diffusion spectra for Aβ₄₀ fibrils (a) MM1, (b) MM2 and (c) BM2 membrane models. The same labeled samples were utilized as in Figure 4. The 1D slices highlight the inter-residue cross peaks between I32 and V24, G33, and M35, as well as between A21 and I32 in all samples.

Figure 7

Calcein leakage assay for measuring the membrane content leakage with Aβ₄₀ fibrillization in different membrane models. Insets show the rapid increase of calcein leakages within the first 10 h of incubation.

Figure 8

Quantitative analysis of membrane-bound Aβ₄₀ as a function of incubation time within the first 20 h. Different color-codings were utilized for different model membranes: black, PC; red, PC/chol; blue, PC/S; purple, PC/S/chol; green, PC/S/E; dark navy, PC/S/E/chol. Error bars represent the s.t.d. from three independent measurements.

Figure 9

CD spectra for total and free Aβ₄₀ peptides with different incubation times: (a,d) 0 h, (b,e) 5 h, (c,f) 10 h. Different color-codings were utilized for different model membranes: black, PC; red, PC/chol; blue, PC/S; purple, PC/S/chol; green, PC/S/E; dark navy, PC/S/E/chol.