Objective: (1) To develop an ultra-sensitive method to visualize single amyloid oligomers and (2) to compare the oligomer load in CSF from patients with PD and controls.

Background: The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders. It is increasingly recognized that soluble oligomeric intermediates of these proteins are the main pathogenic agents that trigger neurodegeneration. Previous studies of CSF samples using a dual-ELISA method suggest that aggregated alpha-synuclein is more abundant in CSF from PD patients than in healthy individuals. However the ability to detect specifically the toxic oligomeric species has been limited by their low abundance and heterogeneous nature. Single-molecule fluorescence techniques are extremely sensitive and can characterize the rarest of species, although the application of such techniques to study proteins in biofluids has not been possible.

Methods: We utilized a dye whose fluorescence emission is switched on upon binding to the structural beta sheet motif that exists in amyloid protein aggregates (thioflavin T). Single molecule total internal reflection microscopy enabled us to visualize these aggregates one at a time. We calibrated this method using oligomers formed from recombinant labelled alpha-synuclein. Next, we measured the number and brightness of oligomers in 18 control CSF samples and 18 PD CSF samples.

Results: Our method detected recombinant alpha-synuclein oligomers (with no detection of the monomers) to a concentration of ∼10pM. Interestingly the number of oligomers was significantly increased in the PD CSF samples (0.013 ± 0.005 oligomers/μm2, mean ± S.D.), compared to healthy controls (0.006 ± 0.004 oligomers/μm2, mean ± S.D.); 95% confidence interval: -0.01 to – 0.004, p<0.0001. The brightness of the oligomers was higher for the PD samples, indicating either that the oligomers were larger or more structured.

Conclusions: We have shown that the ultrasensitive detection of aggregated protein in CSF is feasible, and differentiates between disease and non-disease states. This approach measures the entire oligomer load in biofluids, and simultaneously allows the structural characterization of these oligomers. Oligomer detection may be a valuable tool for diagnostics, and biomarker discovery in PD.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/a-novel-single-molecule-imaging-approach-to-detect-individual-protein-oligomers-in-csf-from-patients-with-parkinsons-disease-pd/