Exploring the Release of Toxic Oligomers from α-Synuclein Fibrils with Antibodies and STED Microscopy

Abstract

α-Synuclein (αS) is an intrinsically disordered and highly dynamic protein involved in dopamine release at presynaptic terminals. The abnormal aggregation of αS as mature fibrils into intraneuronal inclusion bodies is directly linked to Parkinson's disease. Increasing experimental evidence suggests that soluble oligomers formed early during the aggregation process are the most cytotoxic forms of αS. This study investigated the uptake by neuronal cells of pathologically relevant αS oligomers and fibrils exploiting a range of conformation-sensitive antibodies, and the super-resolution stimulated emission depletion (STED) microscopy. We found that prefibrillar oligomers promptly penetrate neuronal membranes, thus resulting in cell dysfunction. By contrast, fibril docking to the phospholipid bilayer is accompanied by αS conformational changes with a progressive release of A11-reactive oligomers, which can enter into the neurons and trigger cell impairment. Our data provide important evidence on the role of αS fibrils as a source of harmful oligomers, which resemble the intermediate conformers formed de novo during aggregation, underling the dynamic and reversible nature of protein aggregates responsible for α-synucleinopathies.

Keywords: Lewy bodies; PD; amyloid; neurodegeneration; protein aggregation; protein misfolding; synucleinopathies; toxic oligomers.

Figure 1

(A) Dot-blot analysis of αS conformers labelled with conformation-sensitive antibodies A11, OC, and 5G4, and conformation-insensitive 211 antibodies specific for human αS. (B) Representative confocal images of SH-SY5Y cells exposed to αS species at 0.3 μM for 1 h. Red fluorescence shows the cell membranes revealed with wheat germ agglutinin. Green fluorescence indicates the αS species labelled with the indicated antibodies. (C,D) Histograms reporting the semi-quantitative analysis of intracellular (C) and extracellular (D) αS fluorescence expressed as the percentage observed in untreated cells, taken as 100%. (E) MTT reduction in SH-SY5Y cells exposed to αS species at 0.3 µM for 24 h. Error bars indicate S.E.M. The statistical analysis was made by one-way ANOVA followed by Bonferroni’s multiple comparison test relative to untreated cells (* p  <  0.05, ** p  <  0.01, *** p  <  0.001).

Figure 2

Representative confocal images of caspase-3 activity in SH-SY5Y cells treated for different lengths of time (1, 3, 5 and 24 h) with M, OB* and SF at 0.3 µM. Untreated cells are also shown. The fluorescence signals are expressed as the percentage of the values for untreated cells, taken as 100%. The kinetic plots report the caspase-3 derived fluorescence versus time following αS addition to the cell medium. The continuous lines through the data represent the best fits to a single-exponential function for OB* (green line), and a sigmoidal function for SF (blue line). Error bars indicate S.E.M. The statistical analysis was made by one-way ANOVA followed by Bonferroni’s multiple comparison test relative to untreated cells (* p < 0.05, *** p < 0.001).

Figure 3

(A) Representative confocal images showing caspase-3 activity in SH-SY5Y cells treated with αS aggregates at 0.3 µM and A11, OC, 5G4 and 211 antibodies (in a 1:2.5 molar ratio) for 24 h. (B) The histogram reports a semi-quantitative analysis of caspase-3 activity expressed as the percentage of untreated cells represented in Figure 2, taken as 100%. (C) MTT reduction in SH-SY5Y cells treated with αS aggregates (0.3 µM) and with A11, OC, 5G4 and 211 antibodies (in a 1:2.5 molar ratio) for 24 h. Error bars indicate S.E.M. The statistical analysis was made by one-way ANOVA followed by Bonferroni’s multiple comparison test relative to untreated cells (* p < 0.05, ** p < 0.01, *** p < 0.001), or to cells treated with the same αS species without antibodies (° p < 0.05, °° p < 0.01, and °°° p < 0.001).

Figure 4

(A,B) Representative confocal images showing OB* (A) and SF (B) incubated in a glass coverslip at 0.3 μM in SH-SY5Y culture medium without cells for 0 and 24 h at 37 °C. The green-fluorescent signals arise from the staining with the A11 and 211 antibodies, respectively. The pink and yellow boxed areas show higher magnifications of the αS species. (C) Semi-quantitative analysis of the oligomeric αS-derived green fluorescent signal showed in panels A and B, and expressed as number of puncta per µm². Error bars indicate S.E.M. The statistical analysis was made by Student t-test relative to samples of SF at time 0 h (°°° p < 0.001). (D) Western Blotting of the cytosolic (cyto) and membrane (mem) fractions purified from SH-SY5Y cells treated for 24 h with SF or OB* at 0.3 μM. αS species were then probed with conformation-insensitive anti-αS 211 antibodies (Original Western Blot Figure see Figure S1).

Figure 5

Representative STED images of primary rat cortical neurons (A) and human iPSC-derived dopaminergic neurons (B) exposed to OB* and SF at 0.3 µM for 24 h. Red and green fluorescence indicates the cell membranes and the αS species labelled with wheat germ agglutinin and the conformation-insensitive human-specific anti-αS 211 antibodies, respectively, in (A), or by MAP-2 and the anti-oligomer A11 antibodies, respectively, in (B). The pink boxed areas show higher magnifications of the αS species. (C) 3D reconstructions of the z-stack analysis of the cells shown in panels A and B. Neurons were virtually dissected on the zy plane to show more clearly the extracellular (top) and intracellular (middle) αS species labelled with 211 (left images) and A11 antibodies (right images).