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α-Synuclein aggregation nucleates through liquid–liquid phase separation

Abstract

α-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson’s disease pathogenesis. However, the early events involved in this process remain unclear. Here, using the in vitro reconstitution and cellular model, we show that liquid–liquid phase separation of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form an amyloid hydrogel that contains oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation, such as low pH, phosphomimetic substitution and familial Parkinson’s disease mutations, also promote α-Syn liquid–liquid phase separation and its subsequent maturation. We further demonstrate α-Syn liquid-droplet formation in cells. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. This work provides detailed insights into the phase-separation behaviour of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in Parkinson’s disease pathogenesis.

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Fig. 1: α-Syn undergoes LLPS in vitro.
Fig. 2: The dynamics of α-Syn in LLPS slows down with time.
Fig. 3: PD-associated aggregation factors promote α-Syn LLPS.
Fig. 4: α-Syn phase-separated droplets mature and age into fibrillar aggregates.
Fig. 5: Site-specific conformational changes and dynamics of α-Syn during LLPS.
Fig. 6: Liquid-like condensates of α-Syn in cells.

Data availability

The authors declare that all the data supporting the findings of this study are available within the article, in the source data files and in the Supplementary Information files. All the data analysis was performed using published tools and packages and has been provided with the paper.

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Acknowledgements

We thank R. Mallik, S. Sen and M. Rao for critical inputs on the manuscript. We also acknowledge IIT Bombay Central Facilities for TEM, FACS and confocal microscopy to perform the experiments. We thank J. B. Udgaonkar for providing the TCSPC facility. We thank R. Reddy and S. Sen for their help in TCSPC microscopy experiments. We acknowledge C. Glabe for the kind gift of the OC antibody. The authors acknowledge DBT (BT/PR22749/BRB/10/1576/2016), Government of India for financial support, and N.S. acknowledges DST SERB (PDF/2016/003736) for funding.

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S.R., R.K., K.P., J.M., R. Panigrahi, S. Mehra, L.G., D.C., A.S.S., S. Maiti and S.B. performed the in vitro and in silico experiments. N.S., S.P., D.D., A.N. and J.G. performed the in cell experiments. All authors participated in analysing the data. S.R. and N.S. contributed equally to this work. The study was conceived by S.K.M. and designed by S.K.M., G.K., R. Padinhateeri, A.K., A.C. and R.R. All authors participated in manuscript writing and approved the manuscript.

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Correspondence to Samir K. Maji.

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Extended data

Extended Data Fig. 1 PD-associated conditions promote α-Syn LLPS.

a, The aggregation growth curves are used for lag time (tlag) calculation using the equation (9) given in the Methods section of the supplementary information. The lag time for aggregation is decreased significantly in the presence of the PD-factors (tlag for Cu+2, Fe+3, liposome, S129E, A53T and E46K are 3.9, 3.2, 5.6, 5.2, 3.1 and 2.9 days, respectively) compared to the WT α-Syn in presence of 10% PEG (tlag of 10.5 days). Data represents mean ± SEM for n=3 independent experiments. p-values are denoted with ***p≤0.001. p-values are calculated for WT+Cu+2 (***p=0.001), WT+Fe+3 (***p=9e-04), WT+liposome (***p=3e-04), S129E+PEG (***p=1.8e-04), A53T+PEG (***p=8.9e-05) and E46K+PEG (***p=7.8e-05), with respect to WT+PEG. b, TEM images showing the morphology of the aggregates formed in various conditions post 30 days of incubation. α-Syn formed fibrils in the presence of each of the additives (50 μM Cu+2, 50 μM Fe+3, 1 mM liposome, and 200 μM S129E phosphomimetic). Fibril formation by A53T and E46K α-Syn (+10% PEG-8000) under LLPS conditions are also shown. c, Left panel: Representative DIC images of 200 μM α-Syn showing liquid droplet formation in presence of different concentrations of the additives. It is important to note that in presence of 100 μM Cu+2, Fe+3 and >1 mM liposome, α-Syn shows aggregated structures under the microscope. S129E phosphomimetic α-Syn shows droplet formation at a faster rate than WT (within 24 h) in the presence of 1, 5 and 10% PEG. Right panel shows LLPS with 5 μM of α-Syn in the presence of indicated additives (100 μM Cu+2, 1 mM liposome; except for Fe+3 for ≤100 μM concentration). S129E α-Syn also shows droplet formation at 5 μM concentration in presence of 15% PEG (~24 h). d, DIC and TEM images of 200 μM α-Syn +10% PEG in presence of 200 μM dopamine showing appearance of droplets only after 20 days of incubation at 37 °C. To note, the droplets do not grow in size even after 30 days. e, The % fluorescence recovery (left) and apparent diffusion coefficient (right) of liquid droplets formed at d2 in presence of various additives after photobleaching. The % fluorescence recovery (left) is analyzed from n=3 independent experiments. p-values are denoted with ***p≤0.001, * p≤0.05, NS (non-significant) p>0.05. p-values for WT+Cu+2 (*p=0.04), WT+Fe+3 (***p=0.0006), WT+liposome (NS, p>0.99), S129E+PEG (NS, p>0.99), A53T+PEG (NS, p>0.99) and E46K+PEG (NS, p>0.99) are calculated with respect to WT+PEG. The apparent diffusion coefficients (right) at d2 are calculated to be 0.42 μm2s-1 for droplets formed in presence of 10% PEG and 0.14, 0.20, 0.34, 0.30, 0.189, 0.08 μm2s-1 for droplets formed in presence of Cu+2, Fe+3, liposome, S129E, A53T(+10% PEG) and E46K(+10% PEG), respectively. ω (radius of the bleached region) = 2 μm. Data represents mean ± SEM for n=3 independent experiments. p-values are denoted with ***p≤0.001. p-values for WT+Cu+2 (***p=2.12e-08), WT+Fe+3 (***p=1.5e-07), WT+liposome (***p=9e-04), S129E+PEG (***p=5.5e-04), A53T+PEG (***p=1.3e-07) and E46K+PEG (***p=1.9e-08), with respect to WT+PEG. The p-values are calculated with the help of one way ANOVA followed by Student-Newman-Keuls (SNK) post hoc test with a 95% confidence interval (a, e), All the experiments are performed three times with similar observations (b-d). Source Data

Extended Data Fig. 2 Effect of familial mutations of α-Syn on LLPS, liquid-to-solid transition and aggregation.

a, α-Syn and its two familial mutants A53T and E46K are incubated in presence of 10% PEG at 37 °C for LLPS. At different time intervals, microscopy images are taken and the number and size of the liquid droplets is quantified. The number of droplets (n=200) are counted from five different microscopic fields (63X magnification) and data represents mean ± standard deviation (SD) for n=3 independent experiments. The data shows more in number and larger size droplets formed by both familial mutants compared to WT type α-Syn. For number distribution (left), the p-values are NS p>0.99 (A53T, d2), ***p=4.4e-05 (E46K, d2), NS, p=0.227 (A53T, d3), ***p=7.6e-09 (E46K, d3), ***p=9e-08 (A53T, d5), ***p=7.2e-08 (E46K, d5), ***p=7e-04 (A53T, d7), ***p=3.3e-05 (E46K, d7), NS p=0.0537 (A53T, d15) and NS p=0.05 (E46K, d15) with respect to WT. For size distribution (right) the p values are NS, p=0.05 (A53T, d2), ***p<1e-12 (E46K, d2), **p=0.00510 (A53T, d3), *p=0.047 (E46K, d3), ***p<1e-12 (A53T, E46K d5), NS p=0.32 (A53T, d7), ***p<1e-12 (E46K, d7) and ***p<1e-12 (A53T, E46K d15) with respect to WT. The range of p values is taken as (*p≤0.05; **p≤0.005; ***p≤0.001; NS, p>0.05, NS: non-significant). The significance of the data is analyzed with the help of one way ANOVA followed by Student-Newman-Keuls (SNK) post hoc test with a 95% confidence interval. The significance is calculated with respect to WT α-Syn. b, Light scattering measurements at 350 nm for 200 μM WT, A53T and E46K α-Syn in presence of 10% PEG. The plot shows greater increase in light scattering for E46K followed by A53T and WT protein, suggesting higher extent of phase separation and liquid droplet formation by mutants compared to the WT protein. n=2 independent experiments. c, Comparison of aggregation kinetics by WT, A53T and E46K α-Syn under LLPS condition (200 μM α-Syn + 10% PEG, no agitation, incubated at 37°C) monitored by ThT fluorescence over the course of 30 days. n=3 independent experiments. Values represent mean ± SEM. The data shows faster aggregation kinetics by mutants over WT protein. d and e, Fluorescence images of A53T and E46K α-Syn droplets showing positive ThioS co-partitioning after d2 of incubation. TEM image reveals the presence of droplets at the early days of incubation and formation of amyloid fibrils at the later stage (d30). f, The gel inversion test for A53T and E46K α-Syn solution in presence of 10% PEG after 30 days of incubation (under LLPS conditions) depicting hydrogel formation (left) and the corresponding scanning electron microscope (SEM) showing hydrogel networks (right). g, Bulk rheology of A53T and E46K α-Syn hydrogels showing higher storage modulus (G’) than loss modulus (G’’) for both proteins confirming their gel-state. n=2 independent experiments. All the experiments are performed three times with similar observations (d-f). Source Data

Extended Data Fig. 3 Spatially-resolved FRET study within single liquid droplets.

Spatially-resolved fluorescence spectra, which are measured from single phase separated droplets formed at the indicated time points from fluorescein labeled (donor) and rhodamine labeled (acceptor) α-Syn proteins (fluorescein and rhodamine labeled at 74th Cys). The solid lines represent rhodamine emission when excited at 488 nm for five individual droplets (LD 1 to 5); whereas dotted lines represent rhodamine emission under no FRET scenario for those five respective droplets. The thick line is the average of these five spectra when FRET occurs. These spectra are utilized to extract intensity enhancement factor (IEFET) at different days (d2, d7 and d24). The spectral broadening at day 24 (d24) suggests the formation of amyloid aggregates. n=3 independent experiments. Source Data

Extended Data Fig. 4 Comparative aggregation profile and LLPS behavior of synuclein family of proteins.

a, (Top) Domain organization of α-Syn. The N-terminus (blue), non-amyloid-β component (NAC) (red) and C-terminus (grey) are shown. Bottom: Multiple sequence alignment of the members of the synuclein family generated through Clustal-W omega. α-Syn core (30-110 amino acids) is represented with symbol . The glycine residues (67, 84 and 86), and C-terminal tyrosine residues are highlighted in yellow and cyan, respectively. Residues in the β- and γ-synucleins that are different from those in α-Syn are colored. b, Comparative aggregation profile under non-rotating LLPS condition (200 μM protein + 10% PEG, no agitation, incubated at 37 °C) for α, β, γ and core-Syn monitored using ThT fluorescence. The core α-Syn shows faster aggregation kinetics with smaller lag time (~4 d) compared to the full-length α-Syn (lag time ~10.5 d). β-Syn and γ-Syn do not show any ThT positive aggregation during the indicated incubation time. n=2 independent experiments. c, LLPS behavior of β, γ and core-Syn (200 μM protein, 10% PEG, pH 7.4, 37°C, no agitation) showing no LLPS for β or γ-Syn. The core α-Syn (30-110) shows faster LLPS (within 1 day) than full length WT α-Syn (which takes 2 days under identical conditions). At ~d6, core α-Syn (30-110) starts to form aggregate-like structures visible under microscope (white triangular pointer). d, TEM images of α, β, γ and core-Syn after one month of incubation under LLPS condition showing fibril formation for full-length and core α-Syn. However, β or γ-Syn does not show any amyloid formation. All the experiments are performed three times with similar observations (c-d). Source Data

Extended Data Fig. 5 In cell analysis of liquid-like condensates of α-Syn.

a, Fluorescence and DIC merged images of iron-treated HeLa cells expressing C4-α-Syn, which is stained with FlAsH-EDT2. The images are captured at 24 h and 48 h post treatment. The white dotted segmentation marks the nuclear boundary. The experiments are repeated independently five times with similar observations b, Quantification of the droplet numbers for the iron-treated cells. Number of cells for droplet count from three independent live experiments are n = 10 for 24 h and n=13 for 48 h. Data is presented as mean ± SEM (Paired two tailed Students-t-test analysis calculated the ***p value to be 0.00032). c, α-Syn liquid droplets do not associate with membranes or lipid. Representative confocal images of Nile red, LysoTracker-red and MitoTracker dye staining of cells showing that the FlAsH stained C4-α-Syn droplets (24 h) do not colocalize with the cellular lipid droplets or membrane-bound organelles, mitochondria and lysosomes, respectively. The images are acquired at 63X magnification. The experiments are repeated independently twice with similar results. Scale bar=10 μm. d, Representative confocal live cell images of 10 µM Cu+2 treated HeLa cells stained with FlAsH-EDT2 at indicated time-points. The cells showing cytoplasmic liquid-like droplet formation that localize to perinuclear region with time (36 h). The experiments are repeated independently three times showing similar observations. e, f, Quantification of the diameter (e) and circularity (f) of the α-Syn droplets in cells formed due to10 µM Cu+2 treatment. The number of images processed from three independent experiments are n=12 (16 h), n= 13 (24 h), n= 10 (36 h); and the total number of droplets accounted is n = 733 (for 16 h), n = 650 (for 24 h) and n = 1190 (for 36 h). Data is presented as a dot plot with the corresponding mean represented as dash lines. g, FRAP recovery of 10 µM Cu+2 induced droplets in cells at given time-points. Left panel: The images correspond to the pre-bleach and post-bleach droplets represented in thermal pseudocolour. Right panel: Normalized fluorescence recovery curves showing slow recovery for 24 h droplets (~18.8 s) compared to 16 h (~4.9 s). Less than 12% recovery is observed for droplets at 36 h. Data are shown as mean ± SEM (n = 3 independent experiments). Source Data

Extended Data Fig. 6 α-Syn LLPS transformed into aggresomes in cells as detected using ProteoStat dye staining.

a, Confocal images of the iron and copper-treated and untreated cells, at the indicated time points, stained with FlAsH-EDT2 (α-Syn) and ProteoStat dye (red). DAPI (blue) is for nuclear staining. The data shows aggresome formation by iron and copper treated cells. Each individual experiment is repeated twice showing similar results. b-d, Aggresome propensity factor (measured by ProteoStat) is determined using FACS analysis (refer to Methods). b, Representative scatter plots for gating of cells (left) and the corresponding histograms for the fluorescence intensity in PE-Texas-Red A channel (right) are shown (n=2 independent experiments). Overlaid staggered plots for comparison of the intensities at different time-points (top) and histogram for aggresome propensity factor (bottom) are given in c and d, The iron and copper treatment data are given in c and d, respectively. The mean fluorescence intensity values from untreated and iron/copper-treated samples are used to compute the aggresome propensity factor using the equation given in the methods section. Proteasomal inhibitor, 5 µM MG132, is used as a positive control for aggresome induction. The data showing considerable increase in aggresome formation at 48 h (iron-treated) and 36 h (copper treated) compared to earlier time-points of treatment. Both the experiments are repeated twice (n=2 independent experiments) and the values are given as mean. Source Data

Supplementary information

Supplementary Information

Supplementary Materials and Methods, Figs. 1–17, Table 1, Video legends 1–5 and references.

Reporting Summary

Supplementary Video 1

Liquid–liquid phase separation, fusion and growth of α-synuclein droplets in vitro.

Supplementary Video 2

Ostwald ripening of liquid droplets.

Supplementary Video 3

α-Synuclein forms liquid droplets in cells.

Supplementary Video 4

α-Synuclein droplets cluster at the perinuclear region upon maturation.

Supplementary Video 5

Disruption of microtubules leading to clumping of α-synuclein droplets in cell.

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Ray, S., Singh, N., Kumar, R. et al. α-Synuclein aggregation nucleates through liquid–liquid phase separation. Nat. Chem. 12, 705–716 (2020). https://doi.org/10.1038/s41557-020-0465-9

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