Fig 1: aS fibrils gradually release oligomers that are ultimately responsible for their toxicity.a Dot-blot analysis of aS species probed with conformational specific antibody A11 (AHB0052, Thermo Fisher Scientific), OC (AB2286, Sigma-Aldrich), conformation-insensitive polyclonal anti-aS antibody (ab52168, Abcam) and conformation-insensitive monoclonal 211 antibody specific for human aS (sc12767, Santa Cruz Biotechnology). b Representative confocal microscope images showing SF (at 0.3 µM) incubated in CM without cells in wells containing a glass coverslip for 0–24 h at 37 °C. Representative images of OB* incubated for 24 h were also shown as positive control. The green-fluorescent signals derive from the staining with mouse monoclonal 211 anti-aS antibodies and rabbit anti-oligomer A11 polyclonal antibodies, in the first and second rows, respectively, and then Alexa-Fluor 514-conjugated anti-mouse or anti-rabbit secondary antibodies (three independent experiments with one internal replicate). c Representative confocal scanning microscope images showing human iPSC-derived dopaminergic neurons treated for 24 h with OB*/SF/LF at 0.3 µM. Red and green fluorescence indicates mouse anti-MAP-2 antibodies (ab11267, Abcam) and the A11-positive prefibrillar oligomers, respectively. d Representative confocal scanning microscope images showing the median sections of SH-SY5Y cells treated for the lengths of time indicated with OB*/SF/LF at 0.3 µM. Red and green fluorescence indicates the cell membranes labeled with WGA and the A11-positive prefibrillar oligomers, respectively (three independent experiments with four internal replicates). e Kinetic plots reporting A11-intracellular fluorescence following the addition of 0.3 µM of the indicated aS species to SH-SY5Y cells. The continuous lines through the data represent the best fits to exponential and sigmoidal functions (see “Methods”), for OB*, SF, and LF, respectively. f Kinetic plots reporting the MTT reduction versus time elapsed following addition of 0.3 µM of the indicated aS species to SH-SY5Y cells. g Dependence of MTT reduction on the penetration of A11-positive aS in SH-SY5Y cells treated with aS species. MTT reduction values reported in (f) plotted against the aS-derived intracellular fluorescence values reported in (e) of cells treated with OB*/SF/LF at the corresponding times. Experimental errors are S.E.M. (n = 3 with four internal replicates in panel (e); n = 4 with three internal replicates in panel (f); n = 3 with four internal replicates and n = 4 with three internal replicates for MTT reduction and intracellular A11-positive aS, respectively, in panel (g). Samples were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test relative to untreated cells (in panels e and f, *P < 0.05, **P < 0.01, ***P < 0.001; in panel g, P < 0.001). A total of 200–250 cells (e) and 150,000–200,000 cells (f) were analyzed per condition.
Fig 2: aS fibrils are largely localized at the surface of the cellular membrane but ROS generation correlates with the intracellular aS pool.a Representative confocal scanning microscope images showing the basal, median, and apical sections of SH-SY5Y cells treated for 1 h with the indicated aS species at 0.3 µM and the median sections of untreated cells. Red and green fluorescence indicates the cell membranes and the aS species revealed with wheat germ agglutinin (WGA) and polyclonal anti-aS antibodies (Ab52168, Abcam), respectively. The histogram on the right reports a semi-quantitative analysis of the intracellular and extracellular aS-derived fluorescence data expressed as the percentage of endogenous aS fluorescence. b Dependence of ROS production on the intracellular aS-derived fluorescences in SH-SY5Y cells treated with aS species. ROS values reported in Fig. S5 were plotted against the aS-derived fluorescence values reported in Fig. S4d of cells treated with OB* and SF at the corresponding concentrations. c Representative confocal scanning microscope images showing mitochondrial superoxide production detected with MitoSOX probe in living SH-SY5Y cells. Red and green fluorescence indicates MitoSOX staining and aS labeled with AF488 dye, respectively (six independent experiments with one internal replicate). d Dependence of mitochondrial superoxide production on the intracellular AF488-derived fluorescence signal in SH-SY5Y cells treated with aS species. Experimental errors are S.E.M. (n = 4 with three internal replicates in panels (a), (b); n = 6 in panel (d) with one internal replicate). Samples were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test relative to untreated cells (in panel a, *P < 0.05, **P < 0.01, ***P < 0.001; in panel b, P < 0.001; in panel d, P < 0.001). A total of 200–250 cells were analyzed per condition.
Fig 3: Inhibition of oligomer release from fibrils prevents their toxicity.a Representative confocal scanning microscope images showing the median sections of SH-SY5Y cells treated for 24 h with the indicated aS species at 0.3 µM, in the absence or presence of A11 (AHB0052, Thermo Fisher Scientific) and OC (AB2286, Sigma-Aldrich) antibodies at 1:2.5 molar ratio. Red and green fluorescence indicates the cell membranes and the aS species revealed with WGA and polyclonal anti-aS antibodies (ab52168, Abcam), respectively. The arrows in the images show the intracellular green-fluorescent punctae. b Semi-quantitative analysis of the green fluorescence signal referring to panel (a) and derived from intracellular aS species expressed as the percentage of untreated cells. c MTT reduction in SH-SY5Y cells treated for 24 h with the indicated aS species at 0.3 µM in the absence or presence of A11 and OC antibodies (1:2.5 molar ratio). d Dependence of MTT reduction (values reported in panel c) on the aS-derived fluorescence values in cells treated with OB*/SF/LF (values from panel b) in the absence or presence of A11 and OC antibodies (1:2.5 molar ratio). In all panels, experimental errors are S.E.M. (n = 4 with three internal replicates). Samples were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test relative to untreated cells (in panels b and c, *P < 0.05, **P < 0.01, ***P < 0.001) and cells treated with the aS species (in panels b and c, §P < 0.05, §§P < 0.01, §§§P < 0.001). In panel d, P < 0.001. A total of 200–250 cells (a, b), and 150,000–200,000 cells (c) were analyzed per condition.
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