Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells

Abstract

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.

Keywords: DNA PAINT; alpha-synuclein; amyloid formation; aptamers; induced pluripotent stem cells; neurodegenerative disorders.

Figure 1

The concept of ADPAINT. A) Schematic representation of ADPAINT showing an aggregate bound by multiple aptamers. The DNA docking strand on the aptamer is transiently bound by the complementary imaging strand to generate a SR image. B) Example time montage of an oligomer undergoing ADPAINT. Each sub‐image is separated by 0.5 s, moving through time from left to right then top to bottom; scale bar: 1 μm. C) Intensity profile of the oligomer in (B). Each intensity burst represents the binding of the imaging strand to the aptamer. Grey: raw intensity profile, blue: using a Chung–Kennedy filter31 with a window of five frames applied. D) Examples of diffraction‐limited (DL, using thioflavin‐T) and super‐resolved (using ADPAINT) images of an αS and Aβ oligomer and fibril. Scale bar: 500 nm.

Figure 2

ADPAINT enables the imaging of a range of species formed during the aggregation of αS. A) Example aggregates are shown in SR on the left, with their corresponding nearest neighbour (NN) plots shown on the right, highlighting hotspots of localisation density. Scale bar: 200 nm. B) The number of aggregates increases over time, and C) the number of localisations also increases as the species get larger; this is shown by D) the mean length increases. Data shown are mean±SD of three independent aggregation reactions. E) The percentage of liposomes permeabilised upon addition of aggregates from the different time‐points (mean±SD over 16 fields of view (69×69 μm)).

Figure 3

ADPAINT in iPSCs. A) iPSCs from a PD patient with a triplication of the SNCA gene and from a healthy control. Protein aggregates were imaged by using pFTAA (green) or ADPAINT (red). Scale bars: 5 μm (top) and 100 nm (middle). Compared to control cells, SNCA triplication cells show B) significantly more aggregates and increases in C) the number of localisations and D) the average length of the aggregates. The data shown are means±SD of at least 27 fields of view. * p<0.05, ** p<0.001, *** p<0.0001; analysed by t‐test.