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Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay

View ORCID ProfileDerrick Lau, Chloé Magnan, Kathryn Hill, View ORCID ProfileAntony Cooper, View ORCID ProfileYann Gambin, View ORCID ProfileEmma Sierecki
doi: https://doi.org/10.1101/2021.08.09.455607
Derrick Lau
1EMBL Australia Node for Single Molecule Sciences and School of Medical Sciences, Faculty of Medicine, the University of New South Wales, Sydney NSW 2052 Australia
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Chloé Magnan
1EMBL Australia Node for Single Molecule Sciences and School of Medical Sciences, Faculty of Medicine, the University of New South Wales, Sydney NSW 2052 Australia
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Kathryn Hill
2The Australian Parkinson’s Mission, The Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia
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Antony Cooper
2The Australian Parkinson’s Mission, The Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia
3St Vincent’s Clinical School, UNSW Sydney, Sydney, New South Wales, Australia
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  • For correspondence: e.sierecki@unsw.edu.au y.gambin@unsw.edu.au a.cooper@garvan.org.au
Yann Gambin
1EMBL Australia Node for Single Molecule Sciences and School of Medical Sciences, Faculty of Medicine, the University of New South Wales, Sydney NSW 2052 Australia
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  • For correspondence: e.sierecki@unsw.edu.au y.gambin@unsw.edu.au a.cooper@garvan.org.au
Emma Sierecki
1EMBL Australia Node for Single Molecule Sciences and School of Medical Sciences, Faculty of Medicine, the University of New South Wales, Sydney NSW 2052 Australia
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  • For correspondence: e.sierecki@unsw.edu.au y.gambin@unsw.edu.au a.cooper@garvan.org.au
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Abstract

The quantification of α-synuclein (α-syn) aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinson’s disease. However, multiple species such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein and these species are likely to co-exist in biological samples and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify which species can be detected in seeding assays, recombinant oligomers and preformed fibrils were produced and purified to characterise their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent but our results reveal that their seeding behaviour is very different compared to preformed fibrils in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples are likely to dominate the response in seeding assays.

Competing Interest Statement

YG and ES are founders of AttoQuest and inventors of the AttoBright instrument (PCT AU2019/050188)

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted August 09, 2021.
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Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay
Derrick Lau, Chloé Magnan, Kathryn Hill, Antony Cooper, Yann Gambin, Emma Sierecki
bioRxiv 2021.08.09.455607; doi: https://doi.org/10.1101/2021.08.09.455607
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Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay
Derrick Lau, Chloé Magnan, Kathryn Hill, Antony Cooper, Yann Gambin, Emma Sierecki
bioRxiv 2021.08.09.455607; doi: https://doi.org/10.1101/2021.08.09.455607

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