The release of toxic oligomers from α-synuclein fibrils induces dysfunction in neuronal cells

Abstract

The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson's disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species.

Fig. 1. αS fibrils interact weakly with the surface of the lipid bilayer of synthetic membranes.

a PRE effects measured using MAS ssNMR for OB*/SF/LF using SUVs with DOPE:DOPS:DOPC lipid composition (molar ratio of 5:3:2) with a paramagnetic center (PC) on the bilayer surface on the hydrophilic head group (left) or in the membrane interior at carbon 16 of the lipid chain (right). ¹³C–¹³C DARR spectra measured in the presence and absence of the PC-labeled lipids are shown in blue and red, respectively. For comparison, the plots of OB* are drawn using OB* data from our previous investigation. b Changes in the far-UV CD spectrum of 10 μM OB*/SF/LF and increasing concentrations of SUVs: 1 mM (red), 2 mM (blue), and 8 mM (yellow). The spectra in the absence of SUVs was subtracted in each case from that acquired in their presence. c Calcein release from SUVs (% of total intravescicular calcein—signal normalized with respect to the treatment with 1% v/v Triton X-100, see Supplementary Information for more details) upon incubation of the vesicles with the indicated αS species.

Fig. 2. αS fibrils are largely localized at the surface of the cellular membrane but ROS generation correlates with the intracellular αS 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 αS species at 0.3 μM and the median sections of untreated cells. Red and green fluorescence indicates the cell membranes and the αS species revealed with wheat germ agglutinin (WGA) and polyclonal anti-αS antibodies (Ab52168, Abcam), respectively. The histogram on the right reports a semi-quantitative analysis of the intracellular and extracellular αS-derived fluorescence data expressed as the percentage of endogenous αS fluorescence. b Dependence of ROS production on the intracellular αS-derived fluorescences in SH-SY5Y cells treated with αS species. ROS values reported in Fig. S5 were plotted against the αS-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 αS 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 αS 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. αS 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 αS 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 αS 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 αS 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 αS 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 αS 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.

Fig. 4. αS fibrils gradually release oligomers that are ultimately responsible for their toxicity.

a Dot-blot analysis of αS species probed with conformational specific antibody A11 (AHB0052, Thermo Fisher Scientific), OC (AB2286, Sigma-Aldrich), conformation-insensitive polyclonal anti-αS antibody (ab52168, Abcam) and conformation-insensitive monoclonal 211 antibody specific for human αS (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-αS 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 αS 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 αS species to SH-SY5Y cells. g Dependence of MTT reduction on the penetration of A11-positive αS in SH-SY5Y cells treated with αS species. MTT reduction values reported in (f) plotted against the αS-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. 5. Visualization of αS fibrils outside and oligomers inside cells at high resolution.

a, b Representative STED images of primary rat cortical neurons that were untreated (a) or treated (b) with OB* (left) and SF (right) for 14 and 24 h. Red and green fluorescence indicates the cell membranes and the αS species revealed by WGA and the conformation-insensitive and human αS-specific 211 antibodies (sc12767, Santa Cruz Biotechnology), respectively. Higher magnifications of the αS species are shown in the boxed areas (three independent experiments with two internal replicates). c Semi-quantitative analysis of the intracellular and extracellular 211-derived fluorescence data referring to panel (b). Experimental errors are S.E.M. (n = 3 with two internal replicates). Samples were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test relative to cells treated with OB* for 14 and 24 h (in panel c, ^§§P < 0.01, ^§§§P < 0.001). d 3D reconstruction of the z-stack analysis (5-μm-thick slices) of the specimens shown in panel (b). A primary neuron was virtually dissected on the zy plane to show more clearly the extracellular (top) and intracellular (middle) αS species. A total of 40–60 cells were analyzed per condition (three independent experiments with four internal replicates). e Representative STED images of SH-SY5Y cells that were treated with AF488-OB* (left) and AF488-SF (right) for 24 h. Red and green fluorescence indicates the cell membranes labeled with WGA and αS labeled with AF488 dye, respectively. Higher magnifications of the αS species are shown in the boxed areas (three independent experiments with one internal replicate). f 3D reconstruction of the z-stack analysis (5-μm-thick slices) of the specimens shown in panel (e). Other details as in panel d (three independent experiments with one internal replicate). In all panels blue arrows indicate either fibrillar or oligomeric αS species.

Fig. 6. 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 αS 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 αS species revealed with WGA and polyclonal anti-αS 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 αS species expressed as the percentage of untreated cells. c MTT reduction in SH-SY5Y cells treated for 24 h with the indicated αS 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 αS-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 αS 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.