Mutations associated with familial Parkinson's disease alter the initiation and amplification steps of α-synuclein aggregation

Abstract

Parkinson's disease is a highly debilitating neurodegenerative condition whose pathological hallmark is the presence in nerve cells of proteinacious deposits, known as Lewy bodies, composed primarily of amyloid fibrils of α-synuclein. Several missense mutations in the gene encoding α-synuclein have been associated with familial variants of Parkinson's disease and have been shown to affect the kinetics of the aggregation of the protein. Using a combination of experimental and theoretical approaches, we present a systematic in vitro study of the influence of disease-associated single-point mutations on the individual processes involved in α-synuclein aggregation into amyloid fibrils. We find that lipid-induced fibril production and surface catalyzed fibril amplification are the processes most strongly affected by these mutations and show that familial mutations can induce dramatic changes in the crucial processes thought to be associated with the initiation and spreading of the aggregation of α-synuclein.

Keywords: familial Parkinson’s disease; kinetic analysis; lipid-induced aggregation; neurodegenerative disease; seeded aggregation.

Fig. 1.

Disease-associated mutations of α-synuclein and specific steps in the mechanism of aggregation. (Upper) Schematic illustration of the mutational variants of α-synuclein associated with familial Parkinson’s disease. The amino acid substitution and the range of the age of onset reported in patients are indicated in each case (19). (Lower) Schematic summary of individual processes involved in the aggregation of WT α-synuclein (20, 21).

Fig. 2.

The rate of lipid-induced α-synuclein aggregation is dramatically altered by the disease-related mutations. (A–F) Change in ThT fluorescence intensity when monomeric α-synuclein [WT (A), A30P (B), E46K (C), H50Q (D), G51D (E), and A53T (F)] was incubated in the absence (black) and in the presence of 100 μM DMPS under quiescent conditions at pH 6.5 and 30 °C. The protein concentrations used in this study were: 20 μM (dark blue), 40 μM (light blue), 60 μM (green), 80 μM (light green), and 100 μM (yellow). The Insets in A, B, and F show the AFM images of the fibrils formed under these conditions. (Scale bars: 1 μm.) a.u., arbitrary units. (G) Schematic illustration of α-synuclein interacting with a lipid bilayer highlighting the positions of the mutations in the membrane sensor region which has been reported to modulate the membrane binding affinity. Adapted from ref. with permission from Macmillan Publishers Ltd: Nature Communications, copyright (2014). (H) Relative rate of lipid-induced α-synuclein aggregation, determined by fitting the early times of the aggregation curves of each mutant to a one-step nucleation model (SI Appendix, Fig. S3).

Fig. 3.

The elongation rate of α-synuclein is only mildly affected by the disease-associated mutations. (A) Change in ThT fluorescence when monomeric α-synuclein at different concentrations [10 μM (black), 30 μM (blue), 50 μM (light blue), 70 μM (green), and 100 μM (yellow)] was incubated in the presence of 5 μM preformed fibrils of the WT protein under quiescent conditions at pH 6.5 and 37 °C. a.u., arbitrary units. (B) Normalized ratios of the elongation rates of the disease-associated variants relative to that of each variant monomer elongating its own preformed fibrils. Note that the elongation of seeds by monomers of the same type (marked by *) is always among the fastest (except for A30P).

Fig. 4.

The rates of fibril amplification of WT α-synuclein, A30P, E46K, and A53T, but not that of H50Q and G51D, are enhanced at mildly acidic pH. (A) Change in ThT fluorescence intensity when monomeric α-synuclein (WT, A30P, E46K, H50Q, G51D, and A53T) was incubated in the presence of 35 nM preformed fibrils of the WT protein under quiescent conditions at pH 4.8 and 37 °C. The protein concentrations used in this study were 20 μM (black), 30 μM (dark blue), 40 μM (light blue), 50 μM (green), 70 μM (red), and 100 μM (yellow). (B) The relative rates of fibril amplification of the α-synuclein variants were averaged over the concentrations studied here and normalized relative to the rate of the fastest aggregating monomeric variant (i.e., A53T α-synuclein).

Fig. 5.

Mutations associated with familial Parkinson’s disease alter the lipid-induced aggregation and fibril amplification steps of α-synuclein amyloid formation. The plot summarizes the effects of the five single-amino acid mutations studied in this work on the rates of lipid-induced aggregation and fibril amplification at mildly acidic pH. The data were normalized relative to the rates of WT (black circle) α-synuclein.