Direct membrane association drives mitochondrial fission by the Parkinson disease-associated protein alpha-synuclein - PubMed (original) (raw)

Mitochondrial fragmentation precedes any disturbance in mitochondrial function or toxicity. A and B, HeLa cells were transfected with mitoGFP and either empty vector control (con) or α-synuclein (+syn) and then loaded with the membrane potentially sensitive dye TMRM (1 n

m

) for 1 h before imaging live. Selected on the basis of GFP, TMRM fluorescence was quantified in individual cells. Treatment with the proton ionophore FCCP (2.5 μ

m

) for 4 min before re-imaging causes a marked redistribution of TMRM fluorescence away from mitochondria (arrows). Scale bar indicates 10 μm. Quantitation of TMRM fluorescence (B) shows no effect of synuclein. The values indicate mean ± S.E. n = 12–49 cells per condition from four independent transfections. C, COS cells were transfected with either vector control or α-synuclein. 1–2 days later, oxygen consumption was measured in the basal state (10 m

m

glutamate and 2.5 m

m

malate) and, after addition of the ATP synthase inhibitor oligomycin (oligo) (1 μg/ml), the proton ionophore FCCP (1 μ

m

) and the complex I inhibitor rotenone (rot) (500 n

m

). Synuclein impairs base-line respiration at 48 h but not 24 h, and the effect is greater in mitochondria uncoupled with FCCP. *, p < 0.01; **, p < 0.005; ***, p < 0.001; NS, not significant by one-way analysis of variance and Newman-Keuls post hoc test. n = 4–9 experiments per group. D, COS cells were transfected with vector control, α-synuclein, or azurite (azur), and after 16 h, replated onto 96-well plates. At 24, 48, 72, and 96 h after transfection, the cells were treated with either calcein green-AM (1 μ

m

) to count live cells, ethidium bromide (5 μ

m

) to count dead cells, or after 70% methanol followed by ethidium bromide to count total cells. α-Synuclein also has no effect on cell survival at 24 h, when fragmentation has already occurred in essentially all cells (

supplemental Fig. S8_B_

). By 48 h, however, there is a small increase in the number of dying cells that persists for the duration of the experiment (p < 0.01 versus control and azurite at 48, 72, and 96 h by one-way analysis of variance and Newman-Keuls post hoc test). Data show mean ± S.E., n = 6 wells per group.