Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Aug 1;289(31):21490-507.
doi: 10.1074/jbc.M113.545749. Epub 2014 Jun 18.

Soluble, prefibrillar α-synuclein oligomers promote complex I-dependent, Ca2+-induced mitochondrial dysfunction

Eric S Luth  1 , Irina G Stavrovskaya  2 , Tim Bartels  1 , Bruce S Kristal  2 , Dennis J Selkoe  3
Affiliations

Soluble, prefibrillar α-synuclein oligomers promote complex I-dependent, Ca2+-induced mitochondrial dysfunction

Eric S Luth et al. J Biol Chem. .

Abstract

α-Synuclein (αSyn) aggregation and mitochondrial dysfunction both contribute to the pathogenesis of Parkinson disease (PD). Although recent studies have suggested that mitochondrial association of αSyn may disrupt mitochondrial function, it is unclear what aggregation state of αSyn is most damaging to mitochondria and what conditions promote or inhibit the effect of toxic αSyn species. Because the neuronal populations most vulnerable in PD are characterized by large cytosolic Ca(2+) oscillations that burden mitochondria, we examined mitochondrial Ca(2+) stress in an in vitro system comprising isolated mitochondria and purified recombinant human αSyn in various aggregation states. Using fluorimetry to simultaneously measure four mitochondrial parameters, we observed that soluble, prefibrillar αSyn oligomers, but not monomeric or fibrillar αSyn, decreased the retention time of exogenously added Ca(2+), promoted Ca(2+)-induced mitochondrial swelling and depolarization, and accelerated cytochrome c release. Inhibition of the permeability transition pore rescued these αSyn-induced changes in mitochondrial parameters. Interestingly, the mitotoxic effects of αSyn were specifically dependent upon both electron flow through complex I and mitochondrial uptake of exogenous Ca(2+). Our results suggest that soluble prefibrillar αSyn oligomers recapitulate several mitochondrial phenotypes previously observed in animal and cell models of PD: complex I dysfunction, altered membrane potential, disrupted Ca(2+) homeostasis, and enhanced cytochrome c release. These data reveal how the association of oligomeric αSyn with mitochondria can be detrimental to the function of cells with high Ca(2+)-handling requirements.

Keywords: Complex I; Electron Transport System (ETS); Mitochondria; Mitochondrial Permeability Transition (MPT); Parkinson Disease; Protein Aggregation; alpha-Synuclein.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Prefibrillar, ThTneg 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 (ThTneg) aged protein was sampled from the aggregation lag phase, and ThT-positive (ThTpos) 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 ThTneg αSyn (red traces); and a single 20 μm Ca2+ addition. Mitochondrial membrane potential (ΔΨm) 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, ThTneg 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 ThTpos αSyn taken from the end-stage plateau of fluorescence was compared with vehicle control. Error bars, S.D. of four independent experiments.
FIGURE 2.
FIGURE 2.
Variation in αSyn aggregation kinetics. Aggregation states of five different 0.6 mg/ml αSyn samples incubated on different days under nutation at 37 °C were monitored over time by ThT fluorescence. Note the broken abscissa and variation in duration of the aggregation lag phase prior to ThT positivity. Raw fluorescence values were normalized to the maximal fluorescence signal for each sample. Error bars were omitted to highlight differences among rather than within samples.
FIGURE 3.
FIGURE 3.
Characterization of sonicated and non-sonicated αSyn fibrils. A and B, representative electron micrographs of αSyn aggregated at 2 mg/ml for 5 days under nutation before (A) and after (B) sonication. Sonicated fibrils contained a heterogeneous mixture of species including spherical oligomers and short fibril fragments. Scale bars, 100 nm. C, hydrodynamic radii (Rh) of fibrils and sonicated fibrils as measured by dynamic light scattering. Error bars, S.E. of three and seven replicates for fibrils and sonicated fibrils, respectively. D, background-subtracted fluorescence values of 2.5 μm (based on starting monomer concentration) fibrillar and sonicated αSyn fibrils in the presence of 10 μm ThT expressed as a percentage of signal obtained for fibrillar αSyn. Sonicated fibrils fluoresced with ∼10% of the intensity of fibrils. Error bars, S.D. of three independent experiments. E, an example aggregation time course of 7 μm monomeric αSyn seeded with 1 mol % of fibrillar, sonicated, or additional monomeric αSyn is shown. Sonicated and non-sonicated fibrils accelerated the aggregation of monomeric αSyn compared with additional monomeric αSyn. In this experiment, monomer-seeded αSyn was observed to acquire ThT positivity after 40 h of aging. 100,000 AFU represents the upper limit of detection of our instrument. Error bars, S.D. of 3–4 replicates. Similar data were obtained in three other independent experiments.
FIGURE 4.
FIGURE 4.
Sonicated αSyn fibrils promote Ca2+-mediated mitochondrial dysfunction in a substrate-dependent manner. A, representative traces of basic parameters of isolated liver mitochondria (ΔΨm, extramitochondrial Ca2+ fluorescence, NAD(P)H autofluorescence, and mitochondrial swelling) simultaneously measured by a multichannel fluorimeter and recorded in the presence of a single 20 μm Ca2+ addition, 5 mm glutamate and 5 mm malate as substrates, and either PBS vehicle (black traces) or 1 μm (red traces) or 2 μm (pink traces) sonicated αSyn fibrils. Spikes at 60 s result from the addition of mitochondria; arrows are used to indicate the time of the Ca2+ addition. B, the CRT of mitochondria treated with 1 and 2 μm sonicated αSyn fibrils under complex I conditions (glutamate/malate-dependent respiration) were normalized to vehicle-treated mitochondria. Sonicated αSyn dose-dependently reduced CRT under these conditions. Error bars, S.D. from at least nine independent experiments. *, p < 0.05, ANOVA followed by Tukey's multiple-comparison test. C, the relative CRT of isolated mitochondria respiring under complex I conditions and treated with non-sonicated αSyn fibrils was determined. Error bars, S.D. from five independent experiments. D, representative traces of ΔΨm (left) and extramitochondrial Ca2+ fluorescence (right) of mitochondria treated with a single aliquot of Ca2+ in the presence of succinate/rotenone (complex II-dependent respiration) and either vehicle (black traces) or 2 μm sonicated αSyn fibrils (pink traces). E, the CRT of mitochondria respiring under complex II conditions (succinate/rotenone-dependent respiration) and treated with 1 and 2 μm sonicated αSyn was compared with vehicle-treated mitochondria under identical conditions. Error bars, S.D. from at least four independent experiments.
FIGURE 5.
FIGURE 5.
100,000 × g soluble fraction of sonicated αSyn fibrils contains bioactive oligomers. A and B, representative electron micrographs of sonicated αSyn fibrils fractionated into a 100,000 × g supernatant (A) and pellet (B). Large fibril fragments pellet at this speed, whereas smaller, rounded oligomers remain soluble. Scale bars, 100 nm. C, comparison of the hydrodynamic radii (Rh) of αSyn monomer and the 100,000 × g supernatant and pellet of sonicated αSyn fibrils as measured by dynamic light scattering. Error bars, S.E. of 3, 5, and 3 replicates for fibrils, 100,000 × g supernatant, and 100,000 × g pellet, respectively. D, soluble oligomers present in the 100,000 × g supernatant can seed the aggregation of monomeric αSyn. Monomeric αSyn at 20 μm was seeded with 1 mol % of additional monomeric αSyn or the 100,000 × g supernatant of sonicated fibrils. An aggregation time course representative of three independent experiments is shown. Error bars, S.D. of 3–4 replicates. E, CRT reductions upon treatment with the 100,000 × g supernatant and pellet of the total sonicated material were compared. Data are represented as the percentage of CRT reduction compared with total sonicated material from the same αSyn preparation. Error bars, S.D. from three independent experiments. *, p < 0.05 using student's t test.
FIGURE 6.
FIGURE 6.
αSyn-induced changes in Ca2+ flux and swelling are due to mPTP activity and are accompanied by cytochrome c release. A, comparison of extramitochondrial Ca2+ fluorescence (left) and swelling (right) of mitochondria incubated with vehicle control (black), 1 μm sonicated αSyn fibrils (red), and 1 μm sonicated fibrils plus 1 μm CsA (purple). All experiments were conducted with the addition of 20 μm Ca2+ under complex I conditions. 30-μl aliquots were removed at the indicated times (1–4 and Ala) and were further processed for analysis in B. The addition of alamethicin is indicated by asterisks. Traces are representative of four independent experiments. B, aliquots removed from the mitochondrial suspensions at times noted in A were centrifuged at 14,000 × g for 5 min. The supernatants were run on SDS-PAGE, Western blotted, and probed with an antibody against cytochrome c to detect cytochrome c released from damaged mitochondria. Western blots are representative of four independent experiments.
FIGURE 7.
FIGURE 7.
Electron microscopy confirms that CsA prevents αSyn-induced changes in mitochondrial swelling. A–D, electron micrographs of fixed and sectioned mitochondrial pellets sampled from different stages of the fluorimetric Ca2+ retention assay. A, mitochondria at baseline (prior to the Ca2+ addition). B, vehicle-treated mitochondria fixed after complete Ca2+-induced swelling as measured by plateau in absorbance at 587 nm. C, sonicated αSyn-treated mitochondria collected and fixed following complete Ca2+-induced swelling. D, CsA-pretreated mitochondria incubated with sonicated αSyn, treated with Ca2+, and fixed at the same time point as in C. Scale bars, 500 nm. E, mean mitochondrial diameter from the sample categories depicted in A–D. Error bars, S.D. from three independent experiments. *, p < 0.05, ANOVA followed by Tukey's multiple-comparison test. ns, not significant.
FIGURE 8.
FIGURE 8.
αSyn does not affect mitochondrial parameters in the absence of exogenous Ca2+. A, comparison of mitochondrial suspensions incubated with vehicle control (black) or 1 μm sonicated αSyn fibrils (red) under complex I conditions in the absence of exogenous Ca2+. The addition of sonicated αSyn resulted in no alteration of steady state ΔΨm, Ca2+ flux, oxidation state of pyridine nucleotides, or membrane swelling. Alamethicin (Ala) was added to induce complete swelling after 1 h. Arrows, the addition of mitochondria. Traces are representative of four independent experiments. B and C, representative swelling traces of mitochondria incubated with either vehicle (black) or 2 μm sonicated fibrils (red) under complex I conditions and treated with 1 μm (B) or 10 μm (C) phenylarsine oxide (PhAsO). αSyn incubation did not sensitize mitochondria to undergo mPTP-related swelling in the presence of phenylarsine oxide.
FIGURE 9.
FIGURE 9.
Mitochondrial Ca2+ cycling is necessary for αSyn-induced mPTP induction but not binding. A, extramitochondrial Ca2+ (as determined by Ca-Green 5N fluorescence) of mitochondrial suspensions incubated with vehicle control (black) or sonicated αSyn fibrils (red) and challenged with 20 μm Ca2+ in the absence (top) or presence of 10 μm Ru360, an inhibitor of the mitochondrial Ca2+ uniporter (bottom). Note that Ru360 prevents uptake of exogenous Ca2+. Traces are representative of four independent experiments. B, measurements of mitochondrial swelling (as determined by the decrease in absorbance at 587 nm) obtained from incubations of the same αSyn and mitochondrial preparations shown in A. 20 μm Ca2+ was added to mitochondrial suspensions in the absence (top) or presence of 10 μm Ru360 (bottom). In this example, the initial absorbance of the αSyn-treated mitochondrial suspension is somewhat lower than that of the vehicle-treated suspension (due to small differences in the total mitochondrial protein present); however, in neither case is there a reduction in absorbance over time, indicating that there is no Ca2+-induced swelling in the presence of Ru360. Traces are representative of four independent experiments. C, SDS-PAGE/Western blot of the supernatant (S), washed pellets (W1–W4), and lysed pellets (L) of mitochondrial suspensions after brief incubation with sonicated αSyn in the presence or absence of 20 μm Ca2+. Membranes were probed for αSyn (top) and cytochrome c (bottom). The majority of the incubated αSyn remains in the supernatant, but a fraction resists four washes in assay buffer and is specifically associated with the mitochondrial pellet. As expected, cytochrome c is only detectable in the lysed mitochondrial pellets, suggesting that mitochondria remain intact after these washes. Blots are representative of four independent experiments. D, quantification of total αSyn immunoreactivity of lysed mitochondrial pellets after incubation with sonicated αSyn in the presence or absence of Ca2+ and after extensive washing. Total lane immunoreactivity was normalized to samples in which 20 μm Ca2+ was added to the αSyn/mitochondria suspension. Error bars, S.D. of four independent experiments.
FIGURE 10.
FIGURE 10.
Sonicated αSyn can inhibit complex I activity. A, mechanically disrupted mitochondria were incubated with vehicle (black), monomeric αSyn (blue), sonicated αSyn fibrils (red), or intact fibrils (green), and the oxidation of supplied NADH was monitored by the absorbance at 340 nm before and after the addition of rotenone (indicated by the dashed line). The post-rotenone absorbance slope was subtracted from the pre-rotenone slope to obtain the rotenone-sensitive activity. B, rotenone-sensitive complex I activity in mitochondria incubated with various αSyn forms was normalized to vehicle-treated mitochondria. Error bars, S.D. of three independent experiments. *, p < 0.05 when compared with all other groups, ANOVA followed by Tukey's multiple comparisons test.
FIGURE 11.
FIGURE 11.
Sonicated αSyn reduces the Ca2+ retention of brain mitochondria without measureable enhancement of ROS production. A, representative traces of the Ca-Green 5N fluorescence of vehicle-treated (black) and 2 μm sonicated αSyn-treated brain mitochondria (red) under complex I conditions. αSyn-treated mitochondria were able to buffer fewer Ca2+ additions to the solution. B, quantification of the relative CRC of vehicle- and sonicated αSyn-treated mitochondria. Error bars, S.D. from eight independent experiments. *, p < 0.05 using Student's t test. C, ROS production from vehicle-treated (black) and sonicated αSyn-treated (red) mitochondria (left) was measured using the dye Amplex Red concurrently with CRC (right). D, the mean slopes of the increase in Amplex Red fluorescence during Ca2+ accumulation were quantified. Despite its ability to reduce CRC, sonicated αSyn did not lead to a significant increase in ROS production in this system. Error bars, S.D. of five independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Spillantini M. G., Schmidt M. L., Lee V. M., Trojanowski J. Q., Jakes R., Goedert M. (1997) α-Synuclein in Lewy bodies. Nature 388, 839–840 - PubMed
    1. Chartier-Harlin M.-C., Kachergus J., Roumier C., Mouroux V., Douay X., Lincoln S., Levecque C., Larvor L., Andrieux J., Hulihan M., Waucquier N., Defebvre L., Amouyel P., Farrer M., Destée A. (2004) α-Synuclein locus duplication as a cause of familial Parkinson's disease. Lancet 364, 1167–1169 - PubMed
    1. Singleton A. B., Farrer M., Johnson J., Singleton A., Hague S., Kachergus J., Hulihan M., Peuralinna T., Dutra A., Nussbaum R., Lincoln S., Crawley A., Hanson M., Maraganore D., Adler C., Cookson M. R., Muenter M., Baptista M., Miller D., Blancato J., Hardy J., Gwinn-Hardy K. (2003) α-Synuclein locus triplication causes Parkinson's disease. Science 302, 841. - PubMed
    1. Kara E., Lewis P. A., Ling H., Proukakis C., Houlden H., Hardy J. (2013) α-Synuclein mutations cluster around a putative protein loop. Neurosci. Lett. 546, 67–70 - PMC - PubMed
    1. Kim H.-J. (2013) α-Synuclein expression in patients with Parkinson disease: a clinician's perspective. Exp. Neurobiol. 22, 77–83 - PMC - PubMed

Publication types

LinkOut - more resources

-