In situ kinetic measurements of α-synuclein aggregation reveal large population of short-lived oligomers

Abstract

Knowledge of the mechanisms of assembly of amyloid proteins into aggregates is of central importance in building an understanding of neurodegenerative disease. Given that oligomeric intermediates formed during the aggregation reaction are believed to be the major toxic species, methods to track such intermediates are clearly needed. Here we present a method, electron paramagnetic resonance (EPR), by which the amount of intermediates can be measured over the course of the aggregation, directly in the reacting solution, without the need for separation. We use this approach to investigate the aggregation of α-synuclein (αS), a synaptic protein implicated in Parkinson's disease and find a large population of oligomeric species. Our results show that these are primary oligomers, formed directly from monomeric species, rather than oligomers formed by secondary nucleation processes, and that they are short-lived, the majority of them dissociates rather than converts to fibrils. As demonstrated here, EPR offers the means to detect such short-lived intermediate species directly in situ. As it relies only on the change in size of the detected species, it will be applicable to a wide range of self-assembling systems, making accessible the kinetics of intermediates and thus allowing the determination of their rates of formation and conversion, key processes in the self-assembly reaction.

Fig 1

a) Overall reaction pathways and intermediates of α-synuclein aggregation shown schematically. Blue spheres: monomers within oligomers (shapes of oligomers are arbitrary). Red stars: R1 nitroxide spin label (see b). b) Molecular structure of the R1 spin label attached to the protein. No specific kinetic pathways are shown.

Fig 2. Room temperature EPR spectra of α-synuclein (R1-αS) at different time points of aggregation.

Full spectra: Inset, the box shows the region zoomed into. Zoomed-in spectra: Amplitude expanded four-fold with respect to inset. a) Start of aggregation (t = 0). b) 9 hours of aggregation. c) 24 hours of aggregation. d) 42 hours of aggregation. Black: Experimental spectra. Red: Simulated spectra. Arrow: feature of broad spectral component (see text).

Fig 3. Aggregation of α-synuclein as a function of time derived from EPR.

Amount of fast fraction (green dots) caused by monomers. Amount of medium fraction (blue dots) assigned to oligomers. Amount of slow fraction (red dots) assigned to fibrils. The solid lines are the fractions of monomer, oligomer and fibrils predicted from the best fit of the model, with a reaction order of n = 7.

Fig 4. Schematic of the kinetic model used to fit the experimental data, showing species, rate constants and processes considered in the kinetic model.

Because monomers and oligomers reach pre-equilibrium on a timescale significantly faster than that of the measurement intervals, altering this model to make oligomers off-pathway does not affect the fit quality. As such, the oligomers cannot be resolved as on- or off-pathway and must instead be considered part of the reactant ensemble at this experimental time resolution [42]. Left: Oligomer formation (k_o) and dissociation (k_d) interconvert monomers (m) and oligomers (O). Conversion (k_c) or nucleation (k_n), green arrow, leads to fibrils (right). Elongation, (k₊) grows existing fibrils, M denotes the monomer equivalent fibril concentration and P the number concentration of fibrils.