Abstract
SummaryHere, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.
Highlights
The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells
Single-Molecule Experiments Reveal the Presence of Distinct Oligomeric Species during aS Fibril Formation We have previously demonstrated, using a model amyloidogenic protein, that it is possible to quantify the fraction of oligomers in a sample and estimate their size distribution using the two-color coincidence detection (TCCD) method in diffusion single-molecule fluorescence experiments (Orte et al, 2008)
Cell 149, 1048–1059, May 25, 2012 a2012 Elsevier Inc. 1049 fluorescent probes to investigate the aggregation of aS (Thirunavukkuarasu et al, 2008), we compared the aggregation behavior of aS tagged with the two fluorophores used in the TCCD experiments, Alexa Fluor 488 (AF488) and Alexa Fluor 647 (AF647), to that of the unlabeled protein and established that the presence of the dye molecules does not alter the fundamental features of the aggregation reaction or the appearance of the resulting fibrillar aggregates
Summary
We use single-molecule techniques to study the aggregation of a-synuclein, the protein whose misfolding and deposition is associated with Parkinson’s disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, acting as a source of cytotoxic species
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