FIGURE 1.

Prefibrillar, ThT^neg αSyn sensitizes mitochondria to Ca²⁺-induced dysfunction in a substrate-dependent manner. A, sample aggregation time course of recombinant αSyn as measured by the fluorescence of the β-sheet-binding dye ThT. 0.6 mg/ml αSyn was aged at 37 °C under nutation, and ThT fluorescence was monitored periodically. αSyn was sampled at three different stages of aggregation for application to mitochondria. Unaged monomeric protein was obtained prior to 37 °C incubation, ThT-negative (ThT^neg) aged protein was sampled from the aggregation lag phase, and ThT-positive (ThT^pos) aged protein was collected at the end stage of aggregation once ThT fluorescence had plateaued. B, representative traces of basic parameters of isolated liver mitochondria simultaneously measured by a multichannel fluorimeter and recorded in the presence of 5 mm glutamate and 5 mm malate as substrates; either PBS vehicle (black traces), 1 μm monomeric αSyn (blue traces), or 1 μm ThT^neg αSyn (red traces); and a single 20 μm Ca²⁺ addition. Mitochondrial membrane potential (ΔΨ_m) was measured as changes in TMRM fluorescence. Higher fluorescence intensity corresponds to depolarized mitochondria. Ca²⁺ concentration in the mitochondrial suspension was measured as Ca-Green 5N fluorescence signal. The redox status of pyridine nucleotides was measured by NAD(P)H autofluorescence. Lower intensity corresponds to a more oxidized state of NAD(P)H. Mitochondrial swelling was measured by the change in light scattering of the mitochondrial suspension. A decrease in scattering indicates a dilution of mitochondrial solutes due to swelling. Spikes result from the additions of Ca²⁺, αSyn, or vehicle and alamethicin (Ala), which was used at the conclusion of the measurement to induce maximal swelling. C, quantification of the ability of aged, ThT^neg but not monomeric αSyn to reduce the mitochondrial retention time of 20 μm Ca²⁺ under complex I (glutamate and malate as substrates) but not complex II conditions (succinate as a substrate plus rotenone to inhibit complex I). CRT was defined as time from Ca²⁺ addition to the ultimate plateau of Ca-Green 5N fluorescence. Error bars, S.D. from at least five independent experiments. *, p < 0.05, ANOVA followed by Tukey's multiple-comparison test. D, the relative CRT of ThT^pos αSyn taken from the end-stage plateau of fluorescence was compared with vehicle control. Error bars, S.D. of four independent experiments.