Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells.

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Whiten, Daniel R 
Zuo, Yukun 
Calo, Laura 
Choi, Minee-Liane 
De, Suman 

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid-specific aptamer, we demonstrate that this technique is able to detect and super-resolve a range of aggregated species, including those formed by α-synuclein and amyloid-β. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.

DNA PAINT, alpha-synuclein, amyloid formation, aptamers, induced pluripotent stem cells, neurodegenerative disorders, Alzheimer Disease, Amyloid beta-Peptides, Aptamers, Peptide, Humans, Neurons, Parkinson Disease, Protein Aggregates, Protein Aggregation, Pathological, alpha-Synuclein
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Royal Society (RP150066)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (701013)
The Royal Society (uf120277)
Royal Society (URF\R\180029)
Royal Society (RGF\EA\181021)
Medical Research Council (MR/K015850/1)
European Research Council (669237)
M.H.H. was supported by a Junior Research Fellowship at Christ’s College, University of Cambridge, and the Herchel Smith Foundation. Y.Z. was supported by Cambridge Trust and Chinese Scholarship Council. PF was supported by Boehringer Ingelheim Fonds, and the German National Merit Foundation. S.D. is funded by a Marie-Curie Individual Fellowship. C.M.D. is supported by the UK Biotechnology and Biochemical Sciences Research Council and the Wellcome Trust. This work was also supported by the Cambridge Centre for Misfolding Diseases (P.F., and C.M.D.), the Royal Society (D.K.), the European Research Council with an ERC Advanced Grant (669237) (D.R.W and D.K.), and the Allen Distinguished Investigator Program, through The Paul G. Allen Frontiers Group (M.H.H.). M.G.S. and L.C. were supported by the Cambridge Biomedical Research Centre at Addenbrooke’s hospital, Cambridge and the Allen Foundation.