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

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.


Atomic force microscopy
After incubating αSN monomers with Mimic and DOPC membranes at 5 mM lipids, sample drops of 50 µM αSNs were deposited on freshly cleaved mica plates. Following 1 min, the remaining solution was blown off with compressed air and further air-dry.
Atomic force microscopy images were acquired using a Digital Instruments Nanoscope IIIa scanning microscope (Veeco, Santa. Barbara, CA) with a Si microcantilever.

Circular dichroism spectroscopy
Circular dichroism (CD) experiments were carried out on a JASCO J820 spectrophotometer (Tokyo, Japan) at 37 o C. The far-UV CD spectra of αSNs in 20 mM sodium phosphate buffer (pH 7.4) containing 100 mM NaCl and various concentrations of lipids of DOPC and Mimic membranes were recorded using a quartz cuvette with a 0.1-mm path length. After subtracting the solvent background, CD signals were presented as the mean residue ellipticity (deg•cm 2 • dmol −1 ). The content of secondary structures was S4 predicted using the BeStSel algorithm (Micsonai et al., 2015).

Determination of the population of membrane-bound αSNs.
To calculate the population of membrane-bound αSNs, free αSN (FαSN) is assumed to bind to multiple lipid molecules (LipidN) with a Kd based on the following scheme (Galvagnion et al., 2015;Terakawa, Lee, and Kinoshita et al., 2018): where N represents the number of lipids interacting with one αSN.
Thus, Kd is defined by the following equation: where [FαSN], [LipidN], and [BαSN(Lipid)N] represent the concentration of free αSN, lipid molecules responsible for binding to one αSN, and αSN:lipid complex, respectively.

Equation S3
Equation S1 was substituted with equations S2 and S3 to yield the following equation: The population of membrane-bound αSNs (Pb) is defined as follows:

Equation S5
Equation S4 was substituted into S5 to give the following equation: where [αSN]t was 50 µM. The values of Kd and N were obtained from the ITC measurements. The N values of αSNWT, αSN129, αSN130CF, and αSNA53T were 47.6, 50, 55.6, and 52.6, respectively.

Determination of the helix content per percentage of bound αSN
The helix content per percentage of bound αSN (Hb) was calculated using the following equation: where Hi is the helix content of αSN in the absence of Mimic lipids. Ht and Pb are the total helix content and the population of bound αSN at the desired concentration of Mimic lipids, respectively. Ht and Hi were obtained by analyzing the CD spectra using the BeStSel algorithm (Micsonai et al., 2015). yellow (3 mM), pink (4 mM), and red (5 mM). Results were reproduced with modifications from our previous study (Terakawa, Lee, and Kinoshita et al., 2018).