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

Abstract

The formation of amyloid fibrils by the intrinsically disordered protein α-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that α-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of α-synuclein aggregation and aggregate spreading in the context of Parkinson disease.seeding | prion-like behavior | neurodegenerative disease | kinetic analysis | electrostatic interactions T he conversion of soluble peptide and protein molecules into insoluble amyloid fibrils is of great interest in fields of science ranging from molecular medicine to nanotechnology (1). The formation of amyloid fibrils is a characteristic feature of a substantial number of increasingly common medical disorders, including neurodegenerative conditions such as Alzheimer's and Parkinson diseases (2). Elucidating the fundamental mechanistic steps involved in the conversion from the soluble to the fibrillar forms of the peptides and proteins involved in such disorders is crucial for understanding their origin and proliferation, and hence for exploring in a rational manner new and effective therapeutic strategies through which to combat their onset or progression (3).One general aspect of amyloid diseases is that once the first aggregates are formed it is very difficult to stop or reverse the aggregation process. This implies that aggregation needs to be studied in both the absence and presence of preformed aggregates, commonly known as seeds, to deepen our understanding of the mechanism of the self-assembly process in vivo. It is possible to define from in vitro studies the rate constants for the multiplicity of microscopic steps that increase the number and total mass of the different types of aggregates that are populated during this process. Such an analysis has recently been carried out for the Aβ42 peptide (4) associated with Alzheimer's disease by combining experimental and theoretical methodolo...

SOD1 aggregation in ALS mice shows simplistic test tube behavior

“…A general concern in studies of protein aggregation is that the data show high intrinsic variability and require good statistics to pin down (2,21). The phenomenon is here illustrated by the aggregation of the SOD1 barrel (SOD1 barrel ) (22,23) in pure buffer and 5 M urea (Fig.…”

“…Involvement of D. Unlike other precursors of neurodegenerative disease (21,29,30), the SOD1 monomer is not at all times disordered but can hide its sticky sequence material by folding up into a well-ordered and perfectly soluble β-barrel structure (7) (Fig. 1C).…”

Mécanismes fondamentaux de formation de fibres amyloïdes par la protéine α-synucléine dans la maladie de Parkinson

“…Nos deux études expérimentales systématiques [9,10] nous ont permis de mettre en évidence les deux étapes fondamentales du mécanisme de formation de fibres amyloïdes par la protéine -syn, processus lié à la maladie de Parkinson. Tout d'abord, nous avons montré que les vésicules lipidiques induisent l'étape d'agrégation d'-syn en stimulant le taux de nucléation par au moins 3 ordres de grandeur.…”

“…Dans le but de répondre à cette question, nous avons utilisé des systèmes minimalistes pour étudier le comportement de la protéine in vitro en présence de petites vésicules lipidiques ou de faibles quantités de fibres. Nous avons combiné deux approches : une approche expérimentale, dans laquelle l'influence des différents réactifs (-syn, lipides et fibres) sur la cinétique de formation de fibres amyloïdes était systématique-ment étudiée, et une approche théo-rique, fondée sur l'analyse cinétique de nos données expérimentales, ce qui a l'ordre du millimolaire à température ambiante, bien que des fibres déjà formées croissent dans ces conditions [10]. Dans la majorité des études publiées, l'agrégation d'-syn est étudiée sous agitation et il est maintenant établi que l'interface air-eau [11] ou les parois du récipient de réaction jouent le rôle de la surface où se passe la nucléation.…”

“…De plus, l'observation de corps de Lewy 2 dans des précurseurs neuronaux issus de cellules souches embryonnaires dix ans après leur transplantation chez des patients atteints de la maladie de Parkinson renforce l'hypothèse de la propagation d'agrégats d'une cellule neuronale à une autre [13]. Pour étu-dier les phénomènes de propagation et multiplication de fibres in vitro, nous avons incubé de très faibles quantités de fibres (nanomolaire) en présence d'un large excès d'-syn monomérique (micromolaire) [10]. À pH neutre et en l'absence d'agitation, le principal phénomène observé est la croissante lente de fibres via l'addition de protéine monomérique une à une.…”