α-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson's disease

Abstract

Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson's disease.

Fig. 1

Characterisation of oligomers and their effect on mitochondria. a Representative SAVE images of early oligomers (4 h), late oligomers (8 h), and fibrils (24 h). Zoom and representative two-dimensional Gaussian distribution fits are shown in the insets. The scale bar is 5 µm and 1 µm in the zoom, and the colour bar shows the Gaussian amplitude (×10⁴ photons). b Quantification of aggregation. Each detected species was fitted to a two-dimensional Gaussian distribution, and histograms of the widths (FWHM) along the longest axis, and the total integrated intensities are shown for each time-point. c Representative traces from single experiments, of NADH autofluorescence in WT neurons exposed to either monomers (n = 15 cells), oligomers (n = 32 cells), or oligomers ± CsA (n = 9 cells and n = 6 cells, respectively). d Representative Rh123 traces of cells challenged with oligomers. e Quantification of mitochondrial depolarisation upon either monomeric or oligomeric application. N = 3 experiments; Oligomers: n ≥ 60 neurons/astrocytes; Monomers: n ≥ 50 neurons/astrocytes. f Representative Rh123 traces of cells challenged with oligomers. g Representative Rh123 traces of cells challenged with oligomers ± CsA. h Representative traces of Rh123 and fura-2 in WT neurons exposed to glutamate in the presence of monomers or oligomers. i Representative images of WT rat neurons labelled with fluo-4 (cytosolic calcium) and TMRM (ΔΨm) before (0 min) and after (4 min) high laser exposure which represents PTP opening. A representative trace of TMRM and Fluo-4 fluorescence where the drop in TMRM fluorescence precedes increase in Fluo-4 fluorescence. Quantification of the time until PTP opening in WT cells pre-exposed to oligomeric α-synuclein. N = 3 experiments; n = 43 cells for control and n = 20 cells for oligomers. Two-tailed Student’s t-test for e and i. Scatter points represent individual cells for e and i. Scale bar = 10 μm. Data represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2

Oligomers but not monomers induce PTP opening. a Mitochondrial swelling was measured by changes in optical density (D) in the presence of buffer, monomers, and oligomers. Complete mitochondrial swelling was achieved by the addition of 5 μM alamethicin. b Schematic diagram of mitoplast preparation and patch-clamp experiment. c Patch clamp recording of the inner membrane from control mitoplasts prepared in calcium-free media in the absence of mitochondrial energy substrates. d Patch clamp recording of the inner membrane from control mitoplasts prepared in calcium media in the presence of succinate showing activation of PTP. e Typical multi-conductance state of single PTP channel, induced by calcium (positive control). f Novel channel activity detected in the presence of α-synuclein oligomers in the pipette solution, other conditions the same as in a. g Oligomer-induced gradual development of channel activity (i and ii), which later transformed into full conductance channel resembling PTP (iii) which was inactivated/closed by the application of high negative potential (iv). h Current amplitude histogram of the channel shown in e. i Detection frequency of the PTP channel in different experimental conditions. j Effects of PTP inhibitors CSA (1 µM) and ADP (1 mM) on channel activity. k, l Current amplitude histogram of the channel shown in j

Fig. 3

Oligomers co-localise with ATP synthase. a Representative images of rat neuronal co-cultures treated with either monomers or oligomers over 7 days and probed with a filament specific antibody. The nucleus can be seen in blue (DAPI). b Representative immunocytochemistry images of rat neuronal co-cultures treated with oligomers. The cells were probed for ATP synthase subunit-α and filament α-synuclein. The nucleus can be seen in blue (DAPI). Scale bar = 10 μm. c Left: diffraction limited image of α-synuclein (scale bar = 5 µm). Right: super-resolved images of α-synuclein and ATP synthase and merged imaged (scale bars are 500 nm). Schematic diagram of DNA-PAINT: The secondary antibody conjugated with a docking strand binds the primary antibody. A fluorophore labelled complementary imaging DNA strand binds the docking strand on the secondary antibody allowing super-resolution imaging. d Representative PLA images of cells treated with either monomers, oligomers or no synuclein probing for ATP synthase subunit α and filament α-synuclein. Cultures were counterstained with the neuronal marker, MAP2. Scale bar = 10 μm. e Quantification of PLA signals per cell. N = 3 experiments. Oligomers: n = 182 cells; Monomers: n = 117; Control: n = 134 cells. f Representative PLA images of cells treated with oligomers probing for ATP synthase subunit α and filament α-synuclein. TOMM20 was probed to validate the mitochondrial localisation of the PLA signal. Two-tailed Student’s t-test for e. Scatter points represent number of puncta per cell. Data represented as mean ± SEM. ***p < 0.001

Fig. 4

Oligomeric α-synuclein generates ROS and induces lipid peroxidation. a Representative trace of ROS generation by xanthine oxidase/xanthine (N = 3 experiments; n = 14 regions), monomers (N = 3 experiments; n = 10 regions), and oligomers (N = 6 experiments; n = 24 regions) in a cell-free system. b Quantitative analysis of the cell-free DHE oxidation. c Representative images of C11-BODIPY 581/591 labelled cells and areas analysed: (n) neurons, (a) astrocytes, and (p) processes before and after permeabilization with 30 µM Digitonin. d Averaged traces of oligomer-induced lipid peroxidation of neurons and e astrocytes before permeabilization. f Averaged traces of lipid peroxidation after permeabilisation of mitochondria in neurons (f), astrocytes (g), and mixed processes (h). i Quantitative analysis of the data of neurons (N = 3 experiments; n = 26 cells), astrocytes (N = 3 experiments; n = 16 cells), neuronal mitochondria (N = 3 experiments; n = 21 regions), astrocytic mitochondria (N = 3 experiments; n = 20 regions), and mixed processes mitochondria (N = 3 experiments; n = 28 regions). j Representative images and k cell death quantification of cells treated with either control buffer, oligomers (n = 264 cells), oligomers + CsA (n = 189 cells), or oligomers + MitoQ (n = 258 cells). N = 3 experiments. Two-tailed Student’s t-test (b, i). One-way ANOVA with Bonferroni correction (f) and scatter points represent sampled fields of view (k). Data represented as mean ± SEM. Scale bar = 10μm. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

Impaired mitochondrial function in SNCA triplication iPS derived neurons. a Representative ICC images of control, isogenic control, and triplication cells probed for filament α-synuclein. The nucleus can be seen in blue (DAPI). b Representative PLA images and quantification of control and triplication iPS derived neurons showing a close proximity between filament α-synuclein and ATP synthase with the nucleus in blue (DAPI). TOMM20 was probed for to validate the mitochondrial localisation of the PLA signal. N = 3 experiments; control n = 155 cells, and triplication n = 115 cells. c Representative images of control and triplication iPS derived neurons loaded with TMRM and d ΔΨm quantification. N = 3 experiments, n ≥ 32 cells. e Representative traces of TMRM fluorescence in control and SNCA triplication neurons after addition of oligomycin (2 μg/ml), rotenone (1 μM), and FCCP (1 μM). f Representative trace of NADH in control and triplication iPS derived neurons. FCCP is applied to maximise respiration and therefore minimise the NADH pool and NaCN is added to block the mitochondrial respiration and therefore maximise the NADH pool. g Quantification of the redox index in control (N = 3 experiments, n = 112 cells) and triplication iPS derived neurons (N = 3 experiments, n = 148 cells). Two-tailed Student’s t-test for b, d, g. b Scatter points on scatter column represent number of puncta per cell. d, g Scatter points represent individual cells/areas. Data represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 6

Lowered PTP opening threshold and increased cell death in SNCA triplication iPS derived neurons. a Representative images of mitochondria labelled with TMRM before ferutinin was added (0 min), immediately before mPTP opening (12 min), and after mPTP opening (20 min). b Representative traces of PTP opening in control and triplications in response to ferutinin. c Proportion of cells (%) that fully opened PTP in response to 25 μM ferutinin. N = 3 experiments, control n = 125 cells, and triplication n = 82 cells. d Representative traces of PTP opening in SNCA triplication neurons ± CsA in response to high laser. e Quantification of the time until laser-induced PTP opening in control (N = 3 experiments; n = 77 areas), SNCA triplication neurons (N = 3 experiments; n = 82 areas), and SNCA triplication neurons + CsA (N = 3 experiments; n = 49 areas). f Representative images and g quantification of cell death in control, isogenic control, and triplication neurons challenged with control buffer or oligomers ± CsA. N = 3 experiments, n ≥ 763 cells. g Scatter points represent individual fields of view. Two-tailed Student’s t-test for c. One-way ANOVA with Bonferroni correction (e, g). Scatter points represent sampled fields of view. Data represented as mean ± SEM. Scale bar = 10 μm. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 7

Schematic diagram of oligomeric α-synuclein effects on mitochondria. Monomeric α-synuclein interacts with ATP synthase and improves the efficiency of ATP synthesis. Oligomeric α-synuclein also interacts with ATP synthase but conversely, impairs respiration, and depolarises the mitochondria. Oligomers induce ROS production, leading to lipid peroxidation and oxidation of key mitochondrial proteins. Together, these oligomer-induced events open the mitochondrial permeability transition pore