Direct Observation of the Interconversion of Normal and Toxic Forms of α-Synuclein

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.

Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates

“…We incubated α‐synuclein, the 140 residue natively unstructured protein whose aggregation is associated with PD, under conditions where it has previously been found to form oligomeric species, and observed a change in the fluorescence intensity (Figure S12) of individual vesicles similar to that described above for Aβ42 18, 32…”

“…Some of these mechanisms are thought to involve specific binding to receptors on the cell membrane1, 5, 9 while others appear to be the consequence of non‐specific membrane disruption 1, 3, 5, 9, 16. A body of data suggests that small soluble aggregates, often called oligomers, rather than mature fibrils, are able to cause the membrane to become permeable to Ca 2+ resulting in Ca 2+ influx and the disruption of Ca 2+ homeostasis 2, 3, 5, 9, 17, 18. Therefore, it is important to quantify and characterize these species within aggregation mixtures in vitro as well as to be able to perform measurements in biological samples, such as cerebrospinal fluid (CSF) 19.…”

Kinetics of the Interactions between Copper and Amyloid‐β with FAD Mutations and Phosphorylation at the N terminus

“…In order to unequivocally establish the oligomerisation mechanism in the presence of metal ions, Aβ peptides labelled with pairs of fluorophores would be necessary for the identification of co‐existing Aβ species. FRET or two‐colour coincidence detection at the single‐molecule level would allow direct measurement of time profiles for both copper‐bound Aβ monomer and copper‐bridged Aβ dimer 45, 46. However, mutations or post‐translational modifications on the N terminus of Aβ would not necessarily increase the reactivity of Aβ.…”

Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells

“…Soluble nanometre‐sized protein oligomers have been identified as the major cytotoxic species in AD and PD,7, 8, 9, 10 but the study of such species has remained challenging, as they tend to be low in abundance and adopt a wide range of heterogeneous structures. To overcome this problem, we have developed an array of single‐molecule techniques11, 12, 13, 14 to observe oligomeric species individually, and have applied them to characterise the aggregation pathway of several disease‐related proteins in vitro.…”