Fig 1: aS fibrils gradually destabilize membrane integrity resulting in neuronal dysfunction.a Representative confocal microscope images showing primary rat cortical neurons loaded with the calcein-AM probe for 10 min and then treated for 1 h with the indicated 0.3 µM aS species. Semi-quantitative analyses of the calcein-derived fluorescence signal in primary rat cortical neurons and SH-SY5Y cells. b Representative confocal microscope images showing the Ca2+-derived fluorescence in primary rat cortical neurons treated for 15 min with the indicated 0.3 µM aS species and then loaded with the Fluo-4 AM probe. Semi-quantitative analysis of the intracellular Ca2+-derived fluorescence in primary rat cortical neurons and SH-SY5Y cells. c Time-course analysis of the intracellular Ca2+-derived fluorescence in SH-SY5Y cells treated for the lengths of time indicated with OB*/SF/LF at 0.3 µM. d Representative confocal microscope images of human iPSC-derived dopaminergic neurons expressing MAP-2 (ab32454, Abcam) and TH (sc-25269, Santa Cruz Biotechnology) markers at 14–18 days of maturation (three independent experiments with one internal replicate). Approximately 75% of the cells are TH positive (estimated by immunostaining). Nuclei were stained with DAPI. e Representative confocal microscope images showing caspase-3-derived fluorescence in human iPSC-derived dopaminergic neurons treated for 24 h with the indicated aS species at 0.3 µM. f Semi-quantitative analysis of the caspase-3-derived fluorescence in human iPSC-derived dopaminergic neurons and SH-SY5Y cells treated for 24 h with the indicated aS species at 0.3 µM. f MTT reduction in primary rat cortical neurons and SH-SY5Y cells treated for 24 h with the indicated 0.3 µM aS species. In all panels data are expressed as the percentage of the value for untreated cells. Experimental errors are S.E.M. (n = 3 with two internal replicates and n = 4 with three internal replicates for cortical neurons and SH-SY5Y cells, respectively, in panels (a), (b); n = 4 in panel c with one internal replicate; n = 3 with two internal replicates and n = 4 with three internal replicates for iPSC-derived dopaminergic neurons and SH-SY5Y cells, respectively, in panel f; n = 6 with three internal replicates in panel (g)). Samples were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test relative to untreated cells (in panels a, b, f, and g, *P < 0.05, **P < 0.01, ***P < 0.001). A total of 200–250 cells (a–f) and 150,000–200,000 cells (g) were analyzed per condition.