Fig. 4

Cytotoxicity and death of dopaminergic neurons induced by α-synuclein phosphorylation at T64. (A) RNA sequence analysis of the SH-SY5Y cells transfected with WT, T64A, T64D, or T64E α-synuclein. RNA was isolated 7 d after electroporation. The mRNAs with a more than twofold increase (red) or decrease (blue) in both the T64D and T64E samples are listed. (B) Mitochondrial membrane potential in the SH-SY5Y cells transfected with WT or T64D α-synuclein. Mitochondrial membrane potential was measured 5 d after electroporation using a JC-1 MitoMP Detection Kit. The numbers on the bar graph indicate the total cell number analyzed. *P = 0.0150, ***P < 0.0001. (C) Lysosomal pH in the SH-SY5Y cells transfected with WT or T64D α-synuclein. Lysosomal pH was evaluated 5 d after electroporation using LysoSensor Yellow/Blue DND-160 (PDMPO). The numbers on the bar graph indicate the total cell number analyzed. ***P < 0.0001. (D) Cell death or viability in the HEK293T cells transfected with WT, T64D, T64A, A53T, or A30P α-synuclein. Cell death was measured by LDH Cytotoxicity Detection Kit 4 d after plasmid transfection using Lipofectamine 3000 reagent. Cell viability was measured by Cell Counting Kit-8 4 d after plasmid transfection using Lipofectamine 3000 reagent. N = 10 samples/group. Cell death: WT vs. T64D: P < 0.0001, T64A vs. T64D: P = 0.0002, WT vs. A30P: P = 0.0012, WT vs. A53T: P = 0.0016. Cell viability: WT vs. T64D: P < 0.0001, T64A vs. T64D: P < 0.0001. N.S.: not significant. (E) Western blotting of cleaved Caspase-3 in the HEK293T or SH-SY5Y cells transfected with WT, T64D, T64A, A53T or A30P α-synuclein. HEK293T cells were collected 4 d after plasmid transfection using Lipofectamine 3000 and SH-SY5Y cells were collected 5 d after electroporation. The graphs show quantitative analysis of cleaved Caspase-3 using N = 6 (HEK293T) or 4 (SH-SY5Y) samples/group. HEK293T: WT vs. T64D: P = 0.0015, T64A vs. T64D: P = 0.003, WT vs. A30P: P = 0.0052, WT vs. A53T: P = 0.0038. For the ANOVA comparison among T64D, A53T and A30P, the F value = 0.0508 and P = 0.9506 (N.S.). SH-SY5Y: WT vs. T64D: P = 0.0145, T64A vs. T64D: P = 0.0099, WT vs. A30P: P = 0.0057, WT vs. A53T: P = 0.0039. For the ANOVA comparison among T64D, A53T and A30P, the F value = 0.0364 and P = 0.9644 (N.S.). N.S.: not significant. (F) Caspase-3-positive SH-SY5Y cells (white arrow) transfected with T64D α-synuclein. SH-SY5Y cells were subjected to immunofluorescence 5 d after electroporation. The graph shows the percentage of caspase-3-positive SH-SY5Y cells transfected with WT, T64A or T64D α-synuclein. The numbers on the bar graph indicate the total cell number counted. *P = 0.0043, **P = 0.0027. (G) Images of Zebrafish: Control fish, Tg(NBT:human α-synuclein WT IRES GFP)Tg(NBT:human α-synuclein T64D IRES GFP) and Tg(NBT:human α-synuclein T64A IRES GFP) at 5 dpf. (H) Western blotting of TH, Caspase-3 and α-synuclein in zebrafish: Control fish, Tg(NBT:human α-synuclein WT IRES GFP)Tg(NBT:human α-synuclein T64D IRES GFP) and Tg(NBT:human α-synuclein T64A IRES GFP) at 5 dpf. The graphs show quantitative analysis of TH and Caspase-3 using N = 6 fishes/group. Dunnett's test was applied (Control = Control fish). **P = 0.0038 (TH), **P = 0.0067 (cleaved Caspase-3). (I) Dopaminergic neurons in zebrafish: Control fish, Tg(NBT:human α-synuclein WT IRES GFP)Tg(NBT:human α-synuclein T64D IRES GFP) and Tg(NBT:human α-synuclein T64A IRES GFP) at 5 dpf. The graph shows the number of DC2 and DC4 dopaminergic neurons of the posterior tuberculum. N = 10 fishes/group. Dunnett's test was applied (Control = Control fish). ***P < 0.0001. (J) Cell death in zebrafish brain: Control fish, Tg(NBT:human α-synuclein WT IRES GFP)Tg(NBT:human α-synuclein T64D IRES GFP) and Tg(NBT:human α-synuclein T64A IRES GFP) at 5 dpf. Cell death was analysed using Acridine Orange Staining. N = 8 fishes/group. Dunnett's test was applied (Control = Control fish). ***P = 0.0002. (K) Spontaneous swimming movement in zebrafish. Control fish and Tg(NBT:human α-synuclein T64D IRES GFP) at 5 dpf. N = 12 fishes/group. **P = 0.0071 (Distance), P = 0.0070 (Velocity). Control fish and Tg(NBT:human α-synuclein T64A IRES GFP) at 5 dpf. N = 12 fishes/group. N.S.: not significant. Tracking shows a representative trace of the zebrafish movements for 1 min.