The Role of Stable α-Synuclein Oligomers in the Molecular Events Underlying Amyloid Formation

Abstract

Studies of proteins' formation of amyloid fibrils have revealed that potentially cytotoxic oligomers frequently accumulate during fibril formation. An important question in the context of mechanistic studies of this process is whether or not oligomers are intermediates in the process of amyloid fibril formation, either as precursors of fibrils or as species involved in the fibril elongation process or instead if they are associated with an aggregation process that is distinct from that generating mature fibrils. Here we describe and characterize in detail two well-defined oligomeric species formed by the protein α-synuclein (αSN), whose aggregation is strongly implicated in the development of Parkinson's disease (PD). The two types of oligomers are both formed under conditions where amyloid fibril formation is observed but differ in molecular weight by an order of magnitude. Both possess a degree of β-sheet structure that is intermediate between that of the disordered monomer and the fully structured amyloid fibrils, and both have the capacity to permeabilize vesicles in vitro. The smaller oligomers, estimated to contain ∼30 monomers, are more numerous under the conditions used here than the larger ones, and small-angle X-ray scattering data suggest that they are ellipsoidal with a high degree of flexibility at the interface with solvent. This oligomer population is unable to elongate fibrils and indeed results in an inhibition of the kinetics of amyloid formation in a concentration-dependent manner.

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

“…Results show that high pressures available at the SAXS beamline -until 1.6 kbar -can subtly modify fibrils' sizes and density features, maintaining their overall morphological structure, and induce the fragmentation into soluble disordered aSN oligomers. The possible simultaneous presence of different oligomeric species, as recently detected and described in different experimental conditions by SAXS experiments [61,8,46], can be pointed up from our SAXS curves. Both wild type and familial mutants of aSN amyloid fibrils show that at increasing pressures the fraction of soluble disordered states is increasing.…”

Structural Insights into Amyloid Oligomers of the Parkinson Disease-related Protein α-Synuclein

“…This task has been hampered by the transient nature and inherent polydispersity of such species and the low amount they can be produced in vitro. Several methods have been developed to generate stable oligomers within a short time frame at the required yields to allow their characterization (13,14,28,30). These methods could lead to structurally and functionally distinct oligomeric species.…”

“…Despite the experimental difficulties detailed above, some progress has been made on our current knowledge of the structure of oAS. Indeed, it has been demonstrated that oAS contain a substantial amount of non-fibrillar ␤-sheets (13,14) and that they are arranged in an antiparallel orientation (15,30 (17). In this work, we relied on the rational site-specific positioning of fluorescent tags along the protein sequence to obtain a more detailed representation of the internal architecture of oAS.…”

Parkinson's disease in the spotlight : unraveling nanoscale alpha-synuclein oligomers using ultrasensitive single-molecule spectroscopy

“…This result is in good agreement with a recently performed SAXS study on oligomers formed under similar conditions in which the aggregation number was determined to be about 30 monomers per oligomer [81]. Additionally, we showed that for αS aggregation the fluorescent labels used do not have an influence.…”

“…• C yielded a low-resolution structure of ellipsoidal shaped oligomers with a radius of about 4.5 nm and a radius to length ratio of about two [54,81] (see figure 1.5). In addition to these preparation protocols without any additional compounds present during aggregation, it has been shown that the morphology of αS oligomers is affected by molecular crowding [82] or by the addition of lipids [52,[65][66][67][68], or organic solvents [56,69], or metal ions [56,57,70].…”