Dual Effects of Presynaptic Membrane Mimetics on α-Synuclein Amyloid Aggregation

Abstract

Aggregation of intrinsically disordered α-synuclein (αSN) under various conditions is closely related to synucleinopathies. Although various biological membranes have shown to alter the structure and aggregation propensity of αSN, a thorough understanding of the molecular and mechanical mechanism of amyloidogenesis in membranes remains unanswered. Herein, we examined the structural changes, binding properties, and amyloidogenicity of three variations of αSN mutants under two types of liposomes, 1,2-Dioleoyl-sn-glycero-3-Phosphocholine (DOPC) and presynaptic vesicle mimetic (Mimic) membranes. While neutrally charged DOPC membranes elicited marginal changes in the structure and amyloid fibrillation of αSNs, negatively charged Mimic membranes induced dramatic helical folding and biphasic amyloid generation. At low concentration of Mimic membranes, the amyloid fibrillation of αSNs was promoted in a dose-dependent manner. However, further increases in the concentration constrained the fibrillation process. These results suggest the dual effect of Mimic membranes on regulating the amyloidogenesis of αSN, which is rationalized by the amyloidogenic structure of αSN and condensation-dilution of local αSN concentration. Finally, we propose physicochemical properties of αSN and membrane surfaces, and their propensity to drive electrostatic interactions as decisive factors of amyloidogenesis.

Keywords: Parkinson’s disease; amyloid fibril; electrostatic interaction; helical structure; intermolecular interaction; membrane mimetic; presynaptic vesicle; α-Synuclein.

FIGURE 1

Effects of model membranes on the structure and amyloid formation of αSNs. (A–L) Conformational transitions and fibrillation kinetics of αSN₁₂₉ (A–D), αSN_130CF (E–H), and αSN_A53T (I–L) in the absence and presence of Mimic (left) and DOPC membranes (right). Far-UV CD spectra of αSN₁₂₉ (A,C), αSN_130CF (E,G), and αSN_A53T (I,K) before (A,E,I) and after (C,G,K) incubation were acquired. (B,F,J) Fibrillation kinetics of αSN₁₂₉ (B), αSN_130CF (F), and αSN_A53T (J) were monitored by the ThT fluorescence assay. Raw data averaged from three separate samples are shown as closed circles. Solid lines represent the fit curves. Schematic representations of αSN₁₂₉, αSN_130CF, and αSN_A53T are displayed above the corresponding data. The N-terminal region (NTR), the non-amyloid β component (NAC) region, and the C-terminal region (CTR) are colored in blue, grey, and red, respectively. Various concentrations of lipids in Mimic and DOPC membranes are guided by distinct colors: black (0 mM), light blue (0.5 mM), blue (1 mM), green (2 mM), yellow (3 mM), pink (4 mM), and red (5 mM). (D,H,L) AFM images were taken for the samples of αSN₁₂₉ (D), αSN_130CF (H), and αSN_A53T (L) incubated with 5 mM of Mimic (left) or DOPC (right) lipids. The white scale bars indicate 500 nm.

FIGURE 2

Calorimetry-based characterization of interactions between αSNs and Mimic membranes. (A–E) ITC thermograms (upper) and binding isotherms (lower) obtained by titrating αSN_WT (A), αSN₁₂₉ (B), αSN_130CF (C), and αSN_A53T (D) to Mimic membranes are shown. Solid lines in binding isotherms indicate the fit curves based on a one-set of sites binding model. (E) Thermodynamic parameters for the binding of αSNs to Mimic membranes.

FIGURE 3

Kinetic analysis of amyloid formation of αSNs in model membranes. (A–I) Maximum ThT fluorescence intensities (A,D,G), lag times (B,E,H), and elongation rate constants (C,F,I) of amyloidogenesis of αSN₁₂₉ (A–C), αSN_130CF (D–F), and αSN_A53T (G–I) in the absence and presence of Mimic (left) or DOPC membranes (right). The average values calculated from three wells in a microplate are shown with error bars reporting the standard deviation. “n.d.” denotes the concentration of lipids at which no significant increase in the ThT fluorescence intensity throughout the incubation period was observed. Various concentrations of lipids in Mimic and DOPC membranes are guided by distinct colors: black (0 mM), light blue (0.5 mM), blue (1 mM), green (2 mM), yellow (3 mM), pink (4 mM), and red (5 mM).

FIGURE 4

Schematic models for the dual effect of Mimic membranes on αSN amyloidogenesis. (A,B) Two models, the amyloidogenic structure model (A) and the condensation-dilution model (B) are schematically shown. Free monomers, partially- and highly-helical monomers in the membrane-bound forms, and amyloid fibrils are illustrated. The N-terminal region, the non-amyloid component region, and the C-terminal region of αSN are represented in blue, grey, and red, respectively. Increases in the concentration of lipids are indicated by the purple triangle.