Lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation

Abstract

α-Synuclein (α-syn) is a 140-residue intrinsically disordered protein that is involved in neuronal and synaptic vesicle plasticity, but its aggregation to form amyloid fibrils is the hallmark of Parkinson's disease (PD). The interaction between α-syn and lipid surfaces is believed to be a key feature for mediation of its normal function, but under other circumstances it is able to modulate amyloid fibril formation. Using a combination of experimental and theoretical approaches, we identify the mechanism through which facile aggregation of α-syn is induced under conditions where it binds a lipid bilayer, and we show that the rate of primary nucleation can be enhanced by three orders of magnitude or more under such conditions. These results reveal the key role that membrane interactions can have in triggering conversion of α-syn from its soluble state to the aggregated state that is associated with neurodegeneration and to its associated disease states.

Figure 1. Modulation of the kinetics of α-synuclein amyloid formation by lipid vesicles.

(a) Change in the CD signal of α-syn (25 μM (blue) and 50 μM (black)) measured at 222 nm as a function of [DMPS]:[ α-syn] (M:M) ratios. The data (blue dots, 25 μM) fit well to a single step binding model (Eq. 6, K_D = 3.8 ± 1.3 • 10^-7 M and L = 28.2 ± 0.8, blue line). L is the stoichiometry of the reaction and corresponds to the average number of lipid molecules bound to each molecule of monomeric α-syn. (b, d) Duplicates of fluorescence measurements monitored as a function of time when α-syn (50 μM) was incubated in the absence (grey) and presence of increasing concentrations of DMPS (100 (purple), 200 (dark blue), 300 (blue), 400 (cyan), 500 (green), 750 (light green), 1000 (yellow), 1500 (orange), 2000 (red) μM). (c) Variation in the maximum rate of aggregation of α-syn with changes in the DMPS: α-syn ratio. For DMPS: α-syn ratios above 40, no stimulation of amyloid formation is observed because effectively all the α-syn monomers required for the growth of the aggregates are bound to the surfaces of the vesicles. The data correspond to mean values ± s.d.

Figure 2. Dissolution of α-syn fibrils in the presence of an excess of DMPS SUVs.

(a) Change in the ThT fluorescence with time when 20 μM pre-formed amyloid fibrils were incubated in the presence of an excess of DMPS SUVs at 37°C (2 mM (grey), 4 mM (blue) and 8 mM (green)). (b) Change in the CD signal of α-syn when 20 μM pre-formed amyloid fibrils were incubated in the presence of 2 mM DMPS SUVs at 37°C (t = 0 (grey), 5 (purple), 17 (dark blue), 39 (blue), 81 (green), 165 (light green), 205 (yellow), 275 min (orange)). Insert: change in the CD signal of α-syn measured at 222 nm as a function of time.

Figure 3. Effect of the presence of salts on the binding of α–syn to DMPS SUVs and on its kinetics of amyloid formation.

(a) Change in the CD signal measured at 222 nm of 50 μM of α-syn monitored in the presence of increasing concentrations of DMPS SUVs in the absence (grey) and the presence of 25 (blue) and 50 mM NaCl (green) at 30°C. (b) Change in the ThT fluorescence when 50 μM α-syn is incubated in the presence of 100 μM DMPS in the absence (grey) and presence of 25 (blue) and 50 mM NaCl (green) at 30°C).

Figure 4. Effect of the variation of the concentration of DMPS SUVs and free monomeric α-syn on the kinetics of α-syn amyloid formation.

(a,b) Duplicates of the change in the fluorescence signal of ThT when increasing concentrations of α-syn (60 (black), 80 (blue), 100 (light blue), 125 (dark green), 175 (green), 200 μM (yellow)) were incubated in the presence of a constant concentration of DMPS (300 μM) (a), and when free monomeric α-syn (140 μM) was incubated in the presence of increasing concentrations of DMPS (60 (black), 120 (purple), 180 (dark blue), 240 (light blue), 300 (dark green), 450 (light green), 600 (yellow), 1200 (orange) μM) (b). (c,d) Change in the concentration of α-syn that is converted into fibrils as a function of the concentration of α-syn free in solution (c), or bound to the SUVs (d). The red lines correspond to the change in the concentration of α-syn that would be converted into fibrils as a function of the concentration of α-syn free in solution (c), or bound to the SUVs (d), if secondary processes were to dominate the kinetics of α-syn amyloid formation ([α-syn_fibrils]=[α-syn_total]_initial).

Figure 5. Differences in the morphology of α-syn aggregates formed in the presence and absence of DMPS SUVs.

(a,b) AFM images of aggregates of α-syn formed after incubation of 200 μM monomeric α-syn in the presence of 600 μM DMPS SUVs ((b) Expanded region of the image in (a)) and (c) in the presence of preformed seed fibrils . (d, e) Changes in the ThT fluorescence signal observed (d) when the solution of the remaining free monomers is incubated in the presence of fresh DMPS SUVs (300 μM), and (e) after sonication of the reaction mixture for 10s at the end of the process of amyloid formation. The concentrations of α-syn converted into fibrils were found to be 20 (d) and 50 μM (e), respectively. The scale bars of the AFM images correspond to 1 μm (a,e), 500 nm (b,d) and 4 μm (c).

Figure 6. Global kinetic analysis of α-syn aggregation data with a two-step nucleation model.

(a,b) Global fits of the early time-points of the α-syn aggregation curves obtained for the different monomer and DMPS concentrations using a two-step nucleation mechanism (see Methods for details) (Eq. 16, k_nk₊ = 1.2 • 10^-5 M^-(n+1) s^-2, K_M = 125 μM , n = 0.2, k_b = 1.9 • 10^-5 s^-1) (see Fig. 4a,b for the complete time curves). The scheme summarises the proposed mechanism of amyloid formation by α-syn in the presence of DMPS SUVs based on the experimental evidence described in this paper.