Article Figures & Data
Figures
- Figure 1.
Mitochondrial membrane potential is lower in synuclein-deficient cells and can be rescued by exogenous monomeric α-synuclein. A, Basal Δψm was quantified in WT, AKO, and TKO cells. B, Representative traces of TMRM fluorescence in WT and TKO primary cells after addition of oligomycin (2 μg/ml), rotenone (1 μm), and FCCP (1 μm). C, Representative images of cells loaded with TMRM before and after α-synuclein addition. *p < 0.05; ***p < 0.001; n = 3 independent experiments. Scale bar, 20 μm.
- Figure 2.
NADH autofluorescence measurements reveal increased respiration in TKO cells. A, Representative traces of NADH in healthy cells. FCCP (1 μm) is applied to maximize respiration and therefore minimize the NADH pool; NaCN (1 mm) is added to block the mitochondrial respiration and therefore maximize the NADH pool. B, Redox index is significantly lower in TKO compared with WT cells. C, Mitochondrial pool of NADH is significantly larger in TKO compared with WT cells, indicating normal substrate supply. *p < 0.05; ***p < 0.001; n = 3 experiments.
- Figure 3.
TKO mitochondria are uncoupled, respire faster, and have a lower ATP synthase efficiency. A, Representative traces of oxygen consumption in the presence of mitochondrial substrates (V2; 5 mm glutamate/malate). B, Quantification of basal respiration (V2) revealed a significantly faster respiration in synuclein-deficient mitochondria compared with WT mitochondria (n ≥ 3 experiments). C, Application of the alternate TCA substrates malate and sodium pyruvate mirrored the higher V2 observed in B (n = 3 experiments). D, Quantification of RCR showed that TKO mitochondria are uncoupled and that α-, β-, or γ-synuclein lowers the RCR. E, Quantification of V3 and V4 of WT and TKO mitochondria with and without α-synuclein. F, Representative traces of oxygen consumption (V3) in the absence and presence of monomeric α-synuclein. G, Quantification of ADP:O in WT and TKO mitochondria in the presence and absence of α-, β-, or γ-synuclein. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 4.
ATP synthase efficiency can be rescued by application of monomeric synuclein. A, Quantification of the effects on ADP:O when 35 or 100 nm monomeric α-synuclein is applied to WT or TKO mitochondria (n = 3 experiments). B, Quantification of AKO ADP:O in the presence or absence of α-synuclein. C, Quantification of the effects on ADP:O when monomeric mutant A30P α-synuclein is applied to TKO mitochondria. D, Quantification of ADP:O in the presence of GDP (a UCP inhibitor) and/or α-synuclein. E, Representative traces of V2 activation by α-, β-, or γ-synuclein. E, Representative traces of oxygen consumption upon oligomycin, α-, β-, or γ-synuclein and FCCP application. G, Quantification of FCCP induced maximum respiration in the presence of α, β, or γ-synuclein. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 5.
ATP levels in TKO brain tissue are significantly lower compared with WT. A, Quantification of ATP levels in WT and TKO cortex and midbrain tissue by colorimetric measurements (n = 3). B, Quantification of basal ATP levels in WT and TKO neuronal cocultures using a FRET-based mitochondrial ATP probe (n = 5). C, Representative images of cells transfected with the mitochondrial ATP probe before and after permeabilization. D, E, Representative trace of kinetic changes in mitochondrial ATP of WT cells in the presence of mitochondrial substrates (malate/glutamate), ADP, and oligomycin in the absence (D) or presence (E) of monomeric α-synuclein. F, ATP amplitude quantification upon ADP application in the presence or absence of monomeric α-synuclein in WT or TKO (n ≥ 4). *p < 0.05; ***p < 0.00. Scale bar, 10 μm.
- Figure 6.
ATP synthase is more efficient in the presence of monomeric α-synuclein. A, Diagram displaying inhibitory effects of oligomycin, iodoacetic acid (IAA), and NaCN. B, Representative Mag-Fura traces of WT and TKO cocultures after treatment with oligomycin and IAA, which block oxidative phosphorylation and glycolysis. These inhibitors allow assessment of the total ATP pool. C, Representative Mag-Fura traces of WT and TKO cocultures after treatment with NaCN and IAA, which blocks mitochondrial respiration and glycolysis. D, Representative Mag-Fura traces of TKO cocultures after treatment with NaCN and IAA in the presence of 100 nm monomeric α-synuclein. E, Quantification of the time until collapse in cells exposed to oligomycin and IAA, NaCN and IAA, NaCN, or IAA in the presence of monomeric α-synuclein. n = 3 experiments; ***p < 0.001.
- Figure 7.
Monomeric α-synuclein binds and improves ATP synthase efficiency. A, Quantification of ATP synthase activity in the presence and absence of 100 nm monomeric α-synuclein. Bi, Representative images of PLA showing α-synuclein and ATP synthase subunit α interaction in SH-SY5Y and rat neuronal cocultures. The nucleus is stained in blue (DAPI). Note that the α-synuclein antibody does not detect endogenous rat α-synuclein. Bii, Representative image of a technical control in which one primary antibody was omitted. C, Western blot showing an interaction between α-synuclein and the ATP synthase α-subunit (ATP5A) in the eluent that was exposed to exogenous α-synuclein. The membrane was reprobed with the antibody specific for α-synuclein to visualize the bait used for the co-IP. D, Schematic diagram illustrating the effects of α-synuclein deficiency and rescue within the mitochondria. *p < 0.05; n = 4 experiments. Scale bar, 10 μm.
In this issue
