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.

Alpha synuclein aggregation drives ferroptosis: an interplay of iron, calcium and lipid peroxidation

“…We investigated how α-synuclein aggregates disrupt membranes and induce ion fluxes. Using a ATTO-425-labeled Aptamer that recognizes oligomeric aggregates of αsynuclein [32], we confirmed the increase in aggregates in the SNCA x3 cells, demonstrated both by intensity, and area of cell occupied by aggregates. (Fig.…”

“…2 a ). This aptamer, using super resolution microscopy (ADPAINT) has demonstrated an increase in larger aggregates in SNCA x3 neurons [ 32 ]. We performed an oligomer ELISA to measure the soluble aggregates in the SNCA x3 and CTRL cell lysates (Supplementary Fig.…”

“…Cells were permeabilized with 0.25% Triton X 100 and blocked with 10% Normal Goat Serum for 20 min followed by another 3 h with 0.1% Triton X—100 and 10% Normal Goat Serum. Then cells were incubated overnight with 0.5 µ m ATTO-425 labeled Aptamer [ 32 ]. Cells were washed three times with PBS and imaged.…”

PAINT using proteins: A new brush for super‐resolution artists

“…Since its initial implementation in 2006, PAINT-based SMLM has seen many innovative applications. 1,2,5,[7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]36 The development of DNA-PAINT in 2010 represented a sea change, by providing a means to incorporate high specificity into the transient interaction. DNA-PAINT has since been optimized and extended in many ways.…”

“…Because these fluorescent bursts are spatially and temporally separated, the center of each can be identified, and a superresolution image of the membrane constructed by summing these individual localizations (Figure 1a-d). PAINT using different small molecule fluorophores has also been used to map the hydrophobic surfaces of different amyloid aggregates, [5][6][7][8] again taking advantage of both the transient interaction and increase in fluorescence upon binding in a hydrophobic environment.…”