Characterising pathological oligomers with single-molecule microscopy

Session

Session 2 - Early Career Researchers Session: the Science of Tomorrow Today

Authors

Rebecca Saleeb (1), Craig Leighton (1), Ji-Eun Lee (1), Mathew Horrocks (1)

Affiliations

1. University of Edinburgh

Keywords

Single-molecule, total internal reflection fluorescence microscopy, neurodegeneration

Abstract text

Neurodegenerative diseases are characterised by the aberrant accumulation of aggregated protein, with disease type determined by the identity and location of proteinopathy as confirmed post-mortem. At present, however, clinical diagnosis is purely symptomatic, based on the motor or cognitive impairment that results from irreversible neurodegeneration. Increasing evidence suggests the early-stage oligomeric intermediates that precede formation of large protein inclusions are the toxic entity, presenting an intriguing candidate biomarker of disease prior to symptom onset. However, oligomers are very small (tens of nanometers) and their extremely low concentration in human biofluids (estimated to be in the picomolar range) is further masked by the presence of healthy monomer, thus eluding ensemble detection approaches. 

To address this, we developed STAPull (Single-molecule Two-colour Aggregate Pulldown)¹. In STAPull, we employ surface passivation and functionalisation to immobilise target proteins on coverglass at single-molecule densities for TIRF microscopy analysis. By combining differently labelled variants of the same monoclonal antibody (schematised in figure 1A), we are able to differentiate oligomer structures from healthy monomer based on the signal colocalization that occurs with the increased epitope availability of higher order structures (figure 1B). 

 

Our results have shown that STAPull is not only capable of detecting the toxic oligomer structures that precede fibril formation, but that it can do so at physiologically-relevant concentrations. By analysing cerebrospinal fluid (CSF) taken from Parkinson’s disease, Alzheimer’s disease or healthy patients, STAPull was able to detect a significantly higher burden of the Parkinson’s-associated alpha-synuclein aggregates in Parkinson’s patients alone (see figure 1C). 

 

We have now combined this technology with exchange-PAINT in a workflow dubbed ‘ConTOR’ (Constituent Topography at Oligomer Resolution). ConTOR allows us to probe not only aggregate number, but also their size, morphology, and surface composition. Indeed, early data demonstrates an elevated fraction of truncated and phosphorylated alpha-synuclein aggregates in Parkinson’s disease patient CSF.

 

This work is a step towards developing early biofluid-based diagnostics for neurodegenerative disease. As current diagnosis is based on symptom manifestation, patients are identified for clinical trial when the disease is already advanced. New biomarker-based technologies are thus urgently needed to identify patients at the earliest stages, when promising drug candidates may be able to take effect.

Figure 1 – (a) Schematic of the STAPull concept: a glass coverslip is silanised and conjugated to PEG, of which 5% is biotinylated, and treated sequentially with streptavidin, biotinylated capture antibody, and the target biomolecule to surface-immobilise. Following incubation with the sample, pulled-down protein is probed using a mixture of AF488- and AF647- labelled monoclonal detection antibody such that (i) no bound protein produces no signal, (ii) monomeric protein produces single-colour signal, and (iii) oligomeric protein produces two-colour signal when imaged via TIRF microscopy. (b) demonstrative composite and single-channel STAPull images for monomeric (top) or oligomeric (bottom) recombinant a-synuclein protein. (c) Representative STAPull images of the a-synuclein aggregate burden in patients diagnosed with Parkinson’s disease (PD), Alzheimer’s disease (AD) or healthy control (HC) individuals. The boxed region indicated is enlarged in the row below. A dot plot quantification by diagnosis (right) demonstrates STAPull’s specific detection of significantly increased a-synuclein in Parkinson’s disease cases. Mean denoted by the horizontal line and standard error by vertical bars, statistical significance between diagnostic groups was confirmed by one-way ANOVA, where PD samples alone differed from other groups based on Tukey means comparison. Scale bars are 5 mm in full-field, 2 mm in enlarged regions. 

References

Saleeb RS, Leighton C, Lee JE, O'Shaughnessy J, Jeacock K, Chappard A, Cumberland R, Zhao T, Ball SR, Sunde M, Clarke DJ, Piché K, McPhail JA, Louwrier A, Angers R, Gandhi S, Downey P, Kunath T, Horrocks MH. Two-color coincidence single-molecule pulldown for the specific detection of disease-associated protein aggregates. Sci Adv. 2023 Nov 17;9(46)