Fig. 6: A53T α-Syn induces mPTP opening, and mROS accelerates oligomerization and cell death.
From: Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity
ai, Representative time course images showing that Δψm reduction (TMRM) is followed by an increase of cytoplasmic calcium level (Fluo-4) at the point of mPTP opening. aii,aiii, Representative traces from the cells treated with 500 nM of WT or A53T α-Syn, respectively. aiv, A53T-treated cells require lower concentrations of ferutinin to open the mPTP than WT-treated cells (n = 4 or 6 independent experiments). bi, Representative time course images showing that apoptosis (NucView) is induced after a substantial loss of Δψm after ferutinin-induced PTP opening. bii,biii, Representative traces and WT-treated or A53T-treated cells. biv, A53T α-Syn treatment induces earlier PTP opening than WT α-Syn (n = 9 or 19 cells over two independent experiments). c, mPTP opening in isolated mitochondria from permeabilized cells. ci, Representative time course images of mPTP opening after applying AF-488-A53T α-Syn. cii,ciii, The mitochondrial area (ROI 1 area) exhibited a rapid loss of Δψm, whereas the extra-mitochondrial area (ROI 2) exhibited increased intensity of Rhod-5N after mPTP opening. civ, Quantitative histogram showing that PTP opening occurs earlier in A53T-treated than WT-treated mitochondria (n = 10 or 13 cells over two independent experiments). d, FRET intensity and FRET efficiency of A53T are reduced by treatment with mito-TEMPO. di, The representative images. dii,diii, Mito-TEMPO-treated cells show reduced A53T FRET intensity (dii; n = 3 or 4 independent experiments) and efficiency (diii; n = 20 or 15 cells over three independent experiments, and error bars represent 95% CIs). div, Application of Trolox to cells reduced FRET intensity signal by reducing uptake of donor (dii–div; n = 3 or 4 independent experiments). ei,eii, Cell death was induced by 48-hour incubation of A53T but not by WT or A30P/E46K (n = 3 independent experiments). fi,fii, A53T-induced cell death was rescued by treatment with mito-TEMPO (n = 4 or 5 independent experiments). Note: 100 μM Trolox and 0.5 μM mito-TEMPO (MitoT) were pre-treated 30 minutes before α-Syn application. Note: Data are represented as data ± s.e.m. (box). *P < 0.05, **P < 0.005 and ***P < 0.0005. Detailed statistical information is shown in Supplementary Table 1. a.u., arbitrary units.
