Supramolecular Non-Amyloid Intermediates in the Early Stages of α-Synuclein Aggregation

Abstract

The aggregation of α-synuclein is associated with progression of Parkinson's disease. We have identified submicrometer supramolecular structures that mediate the early stages of the overall mechanism. The sequence of structural transformations between metastable intermediates were captured and characterized by atomic force microscopy guided by a fluorescent probe sensitive to preamyloid species. A novel ~0.3-0.6 μm molecular assembly, denoted the acuna, nucleates, expands, and liberates fibers with distinctive segmentation and a filamentous fuzzy fringe. These fuzzy fibers serve as precursors of mature amyloid fibrils. Cryo-electron tomography resolved the acuna inner structure as a scaffold of highly condensed colloidal masses interlinked by thin beaded threads, which were perceived as fuzziness by atomic force microscopy. On the basis of the combined data, we propose a sequential mechanism comprising molecular, colloidal, and fibrillar stages linked by reactions with disparate temperature dependencies and distinct supramolecular forms. We anticipate novel diagnostic and therapeutic approaches to Parkinson's and related neurodegenerative diseases based on these new insights into the aggregation mechanism of α-synuclein and intermediates, some of which may act to cause and/or reinforce neurotoxicity.

Differential Effects on Light Chain Amyloid Formation Depend on Mutations and Type of Glycosaminoglycans

“…10 summarizes the high polymorphism observed in the aggregates formed in the presence of chondroitin sulfate A for all proteins. All aggregates observed match with the description of early intermediates in the fibril formation pathway (often referred to as protofibrils, oligomers, or prefibrillar species) of amyloidogenic proteins (47), which have non-fibrillar morphology. For AL-09 and I Y87H, different aggregate populations coexisted in the reactions with chondroitin sulfate A. AL-09 formed a mixture of spherical, protofibrillar aggregates and dense fibrillar material (Fig.…”

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

“…Despite the low level -sometimes complete lack -of ThT binding to non-fibrillar intermediates compared to mature fibrils, these species can influence the ThT fluorescence level both by reducing overall fibril yield and affecting the kinetics of fibril formation. Our description is largely based on the very systematic framework proposed by Fauerbach and colleagues [21] to systematize known oligomeric and fibrillar species.…”

“…Several sheets usually associate with each other, forming large and sometimes twisted structures visible by microscopy techniques [22]. Early electron microscopy studies of amyloid fibrils tended to describe all amyloid using a single set of structural characteristics; however, there are some significant, albeit possibly superficial, differences between reported morphologies [21,[23][24][25]. The average length can vary from quite short (<100 nm) to very long (>1000 nm); in addition, the lateral association can be dramatically different, with some fibril types showing single fibrils whereas changing buffer conditions or protein type can lead to highly associated fibril structures.…”

Biophysical properties and cellular toxicity of covalent crosslinked oligomers of α-synuclein formed by photoinduced side-chain tyrosyl radicals

“…Amyloid fibril formation in vitro has been monitored by several methods [20], the most widely used being the change in thioflavin-T (ThioT) fluorescence upon binding to cross-b-sheet structures [21] and, more recently, by environmental sensitive probes indicating the transient occurrence of early aggregation intermediates [22][23][24]. Complementary AFM and cryo-EM tomography images also provide evidence for supramolecular prefibrillar forms of aS [25].…”