Low-resolution structure of a vesicle disrupting α-synuclein oligomer that accumulates during fibrillation

Abstract

One of the major hallmarks of Parkinson disease is aggregation of the protein α-synuclein (αSN). Aggregate cytotoxicity has been linked to an oligomeric species formed at early stages in the aggregation process. Here we follow the fibrillation process of αSN in solution over time using small angle X-ray scattering and resolve four major coexisting species in the fibrillation process, namely monomer, dimer, fibril and an oligomer. By ab initio modeling to fit the data, we obtain a low-resolution structure of a symmetrical and slender αSN fibril in solution, consisting of a repeating unit with a maximal distance of 900 Å and a diameter of ∼180 Å. The same approach shows the oligomer to be shaped like a wreath, with a central channel and with dimensions corresponding to the width of the fibril. The structure, accumulation and decay of this oligomer is consistent with an on-pathway role for the oligomer in the fibrillation process. We propose an oligomer-driven αSN fibril formation mechanism, where the fibril is built from the oligomers. The wreath-shaped structure of the oligomer highlights its potential cytotoxicity by simple membrane permeabilization. This is confirmed by the ability of the purified oligomer to disrupt liposomes. Our results provide the first structural description in solution of a potentially cytotoxic oligomer, which accumulates during the fibrillation of αSN.alpha-synuclein | amyloid | structral nucleus | solution structure P arkinson Disease (PD) is a common neurodegenerative disorder of the brain. Hallmarks of PD are massive death of dopaminergic neurons and formation of intracellular Lewy bodies (LBs). LBs mainly consist of large fibrillar inclusions of α-synuclein (αSN), a 140-residue natively unfolded protein (1). Numerous in vitro studies of αSN fibrillation have shown that fibril formation is a nucleated polymerization and that oligomers form transiently in the lag phase (1-5). Such oligomers, rather than fibrils or monomers, have been suggested to be the neurotoxic species (4, 6), however whether the oligomers are on-or offpathway in fibril formation and whether the cytotoxic species corresponds to the nucleus of fibrillation remain unclear. Neurotoxicity is proposed to arise from a pore-like membrane permeabilization (6) or by destabilization of the membrane allowing nonspecific ion-transport (7). Unambiguous structural information of the cytotoxic species is difficult to obtain due to sample coexistence of different species, sensitivity to sample handling and potential surface-binding artifacts. The morphology of purified oligomers from wildtype and mutant αSN have been studied by atomic force microscopy (AFM) and electron microscopy (EM) (4, 5), but purified oligomers may be structurally and functionally distinct from those in equilibrium with monomers and fibrils. Different conditions can lead to different oligomers (4, 5, 8) with varying ability to disrupt artificial cell membranes (9-12). Ideally, structural studies should be conducted on an unperturbed ensemble of all potential ...

Pressure effects on α-synuclein amyloid fibrils: An experimental investigation on their dissociation and reversible nature

“…Hence, the differences between WT and A30P aSN fibrils detected by Nielsen et al [47], suggesting increased β-sheet pairing distances in A30P fibrils and their more compact interfibrillar arrangement, cannot be evidenced in our experimental set-up, neither the use of a single helix form factor newly adopted to analyse lysozyme amyloid aggregates [48] is suitable for our SAXS curves. In agreement with Atomic Force Microscopy results [49], we have considered that 5 the cylinder radius could be polydisperse, and fibril electron density could be different from the one of globular protein, due to the hypothesis of the presence of internal cavities [50,51]. Due to the moderately low sample concentration, to the high aggregation number of amyloid fibrils that determines a low number density of scattering particles in solution, to the lowest available Q value and to the absence of interaction peaks in our SAXS curves, the interactions between fibrils have been neglected.…”

Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation

“…It has been proposed that amyloid fibrils of different proteins, including α-synuclein (23,24), can grow most efficiently by the addition of oligomers to the fibril ends. In our experiments we used α-synuclein isolated by gel filtration whose CD spectrum is completely consistent with an unfolded monomeric protein (SI Appendix, section 3).…”

Distinct higher-order α-synuclein oligomers induce intracellular aggregation

“…Growing evidence suggests that soluble oligomeric forms of α-syn, which precede amyloid formation, are causative triggers for the dopaminergic cell loss occurring in PD [4,5]. The morphology and associated mode of toxicity displayed by alternative types of α-syn oligomers are largely dependent on the environmental conditions under which they have been prepared [6][7][8][9][10][11]. Numerous in vitro and in vivo studies on α-syn oligomers have demonstrated that a toxic gain-offunction occurs in the disease state via two main mechanisms: (i) Ca 2 + imbalances caused by the formation of pore-like complexes within lipid membranes, and (ii) transmembrane seeding that then results in intracellular aggregation [1].…”