Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids

Abstract

The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson's disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.

Keywords: CSF; Parkinson’s; biomarkers; single-molecule.

Figure 1

(A) Molecular structure of ThT. In the unbound state, ThT has a low fluorescence intensity; however, when bound to extended β-sheet containing oligomers or fibrils, the fluorescence quantum yield increases significantly (more then 4000 fold). (B) Fluorescence intensity of an individual αS aggregate. The red trace shows the AF647 fluorescence, which decreases over time in stepwise drops, due to photobleaching of each dye molecule. The green trace shows the intensity of ThT, which remains constant over time, since exchange of ThT occurs at a much higher rate than photobleaching of the individual ThT molecules. (C) AF647 fluorescence image of monomeric αS. (D) SAVE image of monomeric αS, there are no visible puncta, since ThT does not bind with an increase in fluorescence to monomeric protein. (E) AF647 image of aggregated αS. The brighter puncta correspond to aggregates, and the dimmer puncta monomeric protein. (F) SAVE images of the same regions as in (E). Only the aggregates (bottom) give rise to fluorescent signal, and these correlate well with the brighter puncta in (E). The scale bars in (C)–(F) are 5 μm and in the zoom are 500 nm in length. AF647 intensity histograms of labeled monomeric and aggregated αS are shown in (G) and (H), respectively. As expected, the aggregated αS has an intensity distribution shifted to higher values than for monomeric αS.

Figure 2

SAVE images of αS species incubated under aggregating conditions. At 2 h, no fluorescent puncta were visible, since only monomeric protein is present. However, after 4 h, diffraction-limited puncta corresponding to oligomers or small fibrils (<250 nm) become visible. Eventually, after 21 h, larger fibrillar species are present. Scale bars are 5 μm and 500 nm in the zoomed insets.

Figure 3

SAVE images of AF647 labeled Aβ and tau aggregates. Aβ was imaged via its AF647 fluorescence (A) and using SAVE (B) after 3 h of aggregation at a concentration of 100 pM, and Tau via AF647 fluorescence (C) and SAVE (D) after 45 minutes at a concentration of 37.5 pM. The brighter puncta in (A) and (C) are coincident with the puncta in (B) and (D), respectively. Scale bars are 10 μm and 1 μm in the zoomed insets. The green circles highlight example aggregated species.

Figure 4

(A) AFM image of enriched oligomers. (B) DLS peak for monomer (blue), enriched oligomers (red), and fibrils (green) (mean from three measurements, error bars are from the standard deviation). (C) CD spectra of monomer, enriched oligomers, and fibrils.

Figure 5

(A) SAVE images of enriched oligomers at a range of concentrations in 5 μM ThT. Scale bars are 5 μm B. Number of events detected when a range of concentrations of enriched oligomers (mean ± SD, n = 30 images). There is a linear detection regime between 0.1 and 10 nM. At lower concentrations, the number of oligomers detected is similar to the number of events detected from buffer alone, while at higher concentrations, the field-of-view is saturated, and counting individual puncta becomes problematic.

Figure 6

(A) Photon count intensity distribution histograms from two representative samples of CSF (PD (red), sample 10; controls (blue), sample 23); these cases give rise to counts closest to mean for the PD and HC samples, respectively. (B) Representative TIRF images from sample 10 (red) and sample 23 (blue). For each sample, 27 images were taken (three 3 × 3 grid scans), and the number of puncta counted. For sample 10, 1201 oligomers were detected, whereas for the HC, only 348 oligomers were observed. Green circles show detected oligomers. Scale bar is 5 μm in the main images, and 500 nm in the zoomed image. (C) Histogram of mean number of oligomers for each sample, red bars are from PD CSF samples, and blue from HC samples. (D) Box plots of the same data for the number of oligomers detected. Horizontal lines show the mean counts for PD and HC samples. (E) Box plots of total αS concentrations from ELISA measurement.