Curcumin Interacts with α-Synuclein Condensates To Inhibit Amyloid Aggregation under Phase Separation.

The amyloid aggregation of α-synuclein (α-Syn) is highly associated with Parkinson's disease (PD). Discovering α-Syn amyloid inhibitors is one of the strategies for PD therapies. Recent studies suggested that α-Syn undergoes phase separation to accelerate amyloid aggregation. Molecules modulating α-Syn phase separation or transition have the potential to regulate amyloid aggregation. Here, we discovered that curcumin, a small natural molecule, interacts with α-Syn during phase separation. Our study showed that curcumin neither affects the formation of α-Syn condensates nor influences the initial morphology of α-Syn condensates. However, curcumin decreases the fluidity of α-Syn inside the condensates and efficiently inhibits α-Syn from turning into an amyloid. It also inhibits the amyloid aggregations of PD disease-related α-Syn E46K and H50Q mutants under phase separation. Furthermore, curcumin can destabilize preformed α-Syn amyloid aggregates in the condensates. Together, our findings demonstrate that curcumin regulates α-Syn amyloid formation during protein phase separation and reveal that α-Syn amyloid aggregation under phase separation can be modulated by small molecules.

Introduction

The deposition of Lewy bodies and Lewy neurites in the cytoplasm of neurons is a key pathological hallmark of Parkinson’s disease (PD).[1,2] α-Synuclein (α-Syn), a small soluble protein mainly localized at the presynaptic nerve terminal, is the major component of Lewy bodies and Lewy neurites.[3−6] α-Syn is composed of 140 amino acids which are divided into three domains, including the amphipathic N-terminal domain (residues 1–60), a highly hydrophobic NAC domain (residues 60–95), and a proline-rich C-terminal region (residues 95–140).[7,8] It has been accepted that α-Syn is involved in PD pathology by forming toxic amyloid aggregates, although its physiological function has not been fully understood.[9]

Depending on the local environment, the monomeric α-Syn is either disordered or in physiological α-helical conformation.[10−14] α-Syn forms oligomers through intermolecular assemblies and further accumulates to mature amyloid fibrils, which is considered the pathogenic mechanism in PD.[15−17] Previous studies suggested that the hydrophobic NAC domain is the core domain for α-Syn aggregation.[18,19] Recent SSNMR and Cryo-EM studies showed two kinds of polymorphs in α-Syn fibrils: rod and twister polymorphs. The core of rod polymorph fibrils is composed of residues 37–99, while the core of twister polymorph fibrils is composed of residues 43–83.[20,21] Another recent study suggested that α-Syn segment 44–47 is required for the amyloid fibril elongation.[22] Hence, in addition to the NAC domain, residues 37–59 are also critical in forming amyloid aggregates. Furthermore, multiple familial PD disease-related mutants of α-Syn have been identified, including E46K and H50Q. Most of the α-Syn mutants accelerate α-Syn fibrillar formation, confirming the central roles of α-Syn in PD pathology.[23−25] Therefore, the amyloid aggregation has become a critical target for PD therapy.[26−30]

Proteins can condensate into liquid droplets through protein–protein weak multivalent interactions, called liquid–liquid phase separation (LLPS). The protein is highly concentrated in the phase-separated droplets, dozens or hundreds of times higher than that in the dilute phase. Under physiological conditions, the phase-separated droplets are highly regulated and reversible.[31] However, the formation of droplets may be disturbed in some pathological conditions such as gene mutation, abnormal post-translational modification, or cellular stress.[32] Many amyloid aggregation-prone proteins, including α-Syn, undergo LLPS.[33−40] Recent studies suggested that α-Syn initiates the conformation change and amyloid formation from LLPS.[36−42] The phase-separated α-Syn facilitates the liquid-to-solid transition to form mature amyloid fibrils.[39]

A number of small natural molecules, including polyphenols, have been discovered to inhibit the aggregation of amyloid proteins and alleviate the cellular toxicity caused by amyloid proteins.[43−49] For example, the green tea polyphenol epigallocatechin-3-gallate (EGCG) prevents the tau protein from forming toxic oligomers.[50] Another polyphenol resveratrol could convert soluble oligomers and fibrils of amyloid Aβ into non-toxic aggregated species.[51] Multiple polyphenols have been discovered to inhibit α-Syn aggregation, including curcumin, myricetin, EGCG, hydroxycinnamic acids, rosmarinic acid, and ferulic acid.[47,52−59] Furthermore, some small natural polyphenols can destabilize the preformed α-Syn aggregates in vitro.[47]

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6,-heptadiene-3,5-dione) is a non-flavonoid biphenolic compound extracted from the root of the Curcuma longa plant. It has anti-inflammatory, anticarcinogenic, and anti-oxidant abilities.[60] In addition, curcumin is able to slow down the progression of neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and PD.[61−63] In the past decades, the effects of curcumin on α-Syn aggregation have been widely studied. Previous biochemical studies found that curcumin efficiently blocked α-Syn aggregation in vitro.[54,64] Several modified analogues of curcumin with improved stability have also been proven effective in inhibiting α-Syn amyloid aggregation and depolymerizing α-Syn fibrils.[65] Moreover, the combination of curcumin and β-cyclodextrin could inhibit α-Syn aggregation and break up the preformed aggregates almost completely at appreciably low concentrations.[66,67] Interestingly, other studies reported that curcumin may bind to oligomers and fibrils that accelerate α-Syn fibrillation, producing less-toxic α-Syn aggregates.[68,69] Cellular studies showed that curcumin could reduce the toxicity of SH-SY5Y cells treated with α-Syn oligomers.[70] It was reported that curcumin could prevent HKI release and ROS enhancement triggered by α-Syn fibrils in mitochondria.[71]

In this work, we examined whether curcumin plays a role in the phase separation and amyloid formation during α-Syn LLPS. We discovered that curcumin binds to α-Syn condensates directly and reduces protein fluidity inside the condensates. However, it does not affect the formation and initial morphology of α-Syn condensates. While the phase-separated α-Syn gradually turned into an amyloid with the extension of incubation time, curcumin efficiently inhibited the formation of amyloid aggregates in a dose-dependent manner. Interestingly, curcumin could also impede the amyloid aggregation of α-Syn E46K and H50Q mutants during phase separation. Moreover, we found that curcumin dose-dependently disassembles the preformed amyloid aggregates in condensates. Altogether, our findings demonstrate that curcumin can target α-Syn during phase separation and prevent the phase transition of α-Syn to form amyloid aggregates.

Results

Curcumin Interacts with α-Syn Condensates but Does Not Affect the Morphology

α-Syn undergoes LLPS in vitro in the presence of a crowding agent, which mimics the cellular physiological conditions.[39,40] We labeled α-Syn with the fluorescence dye rhodamine (Rhod) and mixed it with the unlabeled α-Syn at a molar ratio of 1:9. Using a confocal microscope, we observed α-Syn formed liquid droplets in the presence of 20% PEG-10000 as we previously observed (Figure A).[40] To test whether curcumin regulates α-Syn LLPS, we mixed curcumin with α-Syn in the presence of PEG, and no noticeable morphology change was observed (Figure A). We further analyzed the condensates by quantifying the size and numbers. Curcumin affected neither the size nor the numbers of α-Syn condensates (Figure S1).

Figure 1

Curcumin interacts with α-Syn in the condensates. (A) Fluorescence and differential interference contrast (DIC) images showing the formation of Rhod-labeled α-Syn phase-separated condensates in the absence or presence of 25 μM curcumin. The total α-Syn concentration is 200 μM. Scale bar, 5 μm. (B) Fluorescence emission spectra of curcumin with or without α-Syn. α-Syn formed condensates in the presence of PEG. Curcumin fluorescence was increased with a blue shift in the presence of α-Syn condensates. The final concentrations of α-Syn and curcumin are 200 and 25 μM, respectively.

We performed fluorescence measurements to examine whether curcumin directly interacts with α-Syn condensates. Curcumin was excited at 426 nm, and it showed a weak fluorescence at approximately 540 nm. When we mixed curcumin with α-Syn condensates, a dramatic increase in fluorescence intensity with a blue shift to 510 nm was observed (Figure B). In contrast, α-Syn, mostly in monomers, does not induce noticeable fluorescence change in the solution without PEG (Figure B). These data suggested that curcumin efficiently interacts with the hydrophobic regions of the α-Syn protein in the condensates.[68] However, whether curcumin directs α-Syn in monomers or oligomers needs to be further examined.

Curcumin Reduces α-Syn Protein Fluidity inside the Condensates

We then examined how curcumin affects the formation of α-Syn condensates. We employed sedimentation and turbidity assays to quantify the phase-separated α-Syn protein.[72,73] In a sedimentation-based assay, the fraction of phase-separated α-Syn, indicated by protein recovered from the condensed phase in pellets, was not affected by curcumin (Figure A). Protein phase separation leads to the increase in turbidity, which can be monitored by measuring the absorbance at 600 nm.[72] We found that α-Syn liquid droplets caused high turbidity in the presence of PEG and curcumin has less effect, confirming that the total amounts of phase-separated α-Syn were not affected. (Figure B).[40,72] These studies demonstrated that curcumin affects neither the initial morphology of α-Syn condensates nor the total amounts of phase-separated α-Syn.

Figure 2

Curcumin decreases the fluidity of the α-Syn protein inside the condensates. (A) Sedimentation-based assays showing the distributions of α-Syn in the supernatant (S) and pellet (P) in the absence or presence of curcumin. (B) Turbidity assays showing the effect of curcumin on the formation of phase-separated α-Syn. Turbidity was evaluated by monitoring the absorbance at 600 nm. Data are presented as mean ± SD (n = 3 independent replicates). P values were calculated using Student’s t test. n.s., P > 0.05. (C) Representative FRAP images of α-Syn condensates in the absence (top) or presence (bottom) of curcumin. The fluorescence images of prebleached, bleached (0 s), and bleached after 120 s recovery are shown. Scale bar, 2 μm. (D) The normalized FRAP curves of α-Syn condensates in the absence (black) or presence (red) of curcumin shown in (C). Data are presented as mean ± SD (n = 3 independent replicates). The concentrations of α-Syn and curcumin are 200 and 25 μM, respectively. All the experiments were carried out in the presence of 20% PEG-10,000.

The protein inside the liquid droplets has high fluidity, which can be measured by fluorescence recovery after photobleaching (FRAP) experiments.[40] In our conditions, the fluorescence intensity of α-Syn droplets could fully reach the prebleaching state at 120 s after fluorescence bleaching, confirming the phase state of liquid droplets (Figure C,D). Unexpectedly, an apparent decrease in FRAP was observed in the α-Syn condensates with curcumin, primarily caused by α-Syn–curcumin interactions (Figure C,D). Our data suggested that curcumin interacts with α-Syn and hinders the protein fluidity inside the condensates, although it does not influence the initial formation of α-Syn condensates.

Curcumin Inhibits the Amyloid Formation of Phase-Separated α-Syn

Protein phase separation can promote α-Syn amyloid aggregation by facilitating the rate-limiting protein nucleation. Next, we studied how curcumin regulates α-Syn amyloid aggregation through phase separation. We used Thioflavin T (ThT), an amyloid-specific dye, to monitor α-Syn amyloid formation during phase separation.[74] While curcumin slightly affected ThT fluorescence in the absence of α-Syn, the wholly matured α-Syn fibrils dramatically increased the ThT fluorescence intensity, which was decreased by adding the compound (Figure S2). A time-dependent increase in ThT fluorescence was observed after the incubation of α-Syn condensates at 37 °C, indicating that α-Syn gradually turned into an amyloid in the condensates (Figure A). We did not observe the apparent lag phase in the ThT fluorescence experiments during incubation. We speculate that LLPS gives an extremely high protein concentration of α-Syn in the condensates, leading to rapid protein self-assembly and a high aggregation rate. Interestingly, curcumin reduced the ThT fluorescence in a concentration-dependent manner (Figure A). Furthermore, it slightly decreased the initial aggregation rates of α-Syn. The corresponding time-dependent fluorescence images showed that curcumin had less effect on the morphology of α-Syn condensates (Figure S3). Based on the ThT fluorescence intensity of the wholly matured α-Syn fibrils, we estimated that nearly 50% of α-Syn formed complete fibrillization in the condensates after incubation at 48 h. The uncomplete fibrillization of α-Syn in the reactions may be due to the incubation conditions without shaking. The solidified α-Syn condensates could not further recruit free α-Syn monomers in the solution.

Figure 3

Curcumin inhibits α-Syn amyloid aggregation in the condensates. (A) Kinetics of α-Syn amyloid aggregation in the absence (black) or presence of the indicated molar ratio of curcumin. The enhanced ThT fluorescence indicates the formation of α-Syn amyloid aggregates at 485 nm. Data were normalized with the ThT fluorescence intensity of completed matured fibrils and presented as mean ± SD (n = 3 independent replicates). The total α-Syn concentration is 200 μM. Curcumin concentrations are 10 μM (red), 25 μM (blue), and 50 μM (green), respectively. (B) Transmission electron micrographs showing the fibrillar formation of α-Syn condensates in the absence or presence of 50 μM curcumin after incubation at 37 °C for 48 h. The α-Syn concentration is 200 μM. All the experiments were carried out in the presence of 20% PEG-10,000.

Then, we used transmission electron microscopy (TEM) to observe the detailed morphology of α-Syn condensates. We found that α-Syn formed amyloid fibrils matured from the condensates after 48 h incubation (Figure B). However, the fibrils were largely reduced by curcumin, demonstrating that curcumin blocks α-Syn amyloid aggregation in the condensates (Figure B). Interestingly, FRAP experiments showed that the fluorescence intensity of α-Syn condensates could not be recovered after 48 h incubation, confirming that α-Syn aggregated into a solid-like state after incubation (Figure A–C). An increased FRAP was observed with α-Syn condensates in the presence of curcumin, indicating that the α-Syn/curcumin condensates are partially solid (Figure A–C). However, we were not able to verify the formation of amyloid aggregation by circular dichroism (CD) spectroscopy (Chapter S1). Therefore, the exact secondary structure of the α-Syn condensates in the presence of curcumin needs to be further studied.

Figure 4

The morphology of α-Syn condensates is not altered by curcumin after incubation. (A) Fluorescence and DIC images showing the morphology of α-Syn condensates in the absence or presence of 25 μM curcumin after incubation at 37 °C for 48 h. Scale bar, 5 μm. (B) Representative FRAP images of α-Syn condensates in the absence (top) or presence (bottom) of curcumin after incubation for 48 h. The fluorescence images of prebleached, bleached (0 s), and bleached after 120 s recovery are shown. Scale bar, 2 μm. (C) The normalized FRAP curves of α-Syn condensates in the absence (black) or presence (red) of curcumin shown in (B). Data are presented as mean ± SD (n = 3 independent replicates). The total α-Syn concentration is 200 μM. All the experiments were carried out in the presence of 20% PEG-10,000.

Amyloid Aggregations of α-Syn E46K and H50Q Mutant Condensates Were Inhibited by Curcumin

Multiple familial PD disease-related mutants have been identified. Most of the mutations accelerate the amyloid aggregation and increase cytotoxicity, supporting the importance of α-Syn aggregation in PD pathogenesis. A recent study suggested that E46K, A53T, or phosphomimetic S129E mutation could promote α-Syn LLPS, accelerating amyloid aggregation of α-Syn in condensates.[39] We chose the E46K mutant, which underwent LLPS dramatically in the previous study, to examine curcumin’s effect. Moreover, we tested a new mutant H50Q which has not been examined in the phase separation. Confocal microscopy studies showed that α-Syn E46K and H50Q mutants underwent LLPS, and curcumin did not change the morphology of the condensates (Figure A,B). We then examined if curcumin inhibits the aggregation of E46K and H50Q mutants during phase separation. As shown in Figure C,D, α-Syn E46K and H50Q mutants form α-Syn amyloid aggregation in the condensates. Curcumin inhibited the amyloid aggregation of E46K and H50Q mutants efficiently (Figure C,D). Together, these data suggested that curcumin could block both wide-type and PD disease-related α-Syn aggregation during α-Syn phase transition.

Figure 5

Curcumin inhibits the amyloid aggregation of α-Syn E46K and H50Q mutants in the condensates. Fluorescence and DIC images showing the formation of Rhod-labeled α-Syn E46K (A) and H50Q (B) phase-separated condensates in the absence or presence of curcumin. The kinetics of amyloid aggregation for α-Syn E46K (C) and H50Q (D) mutants in the absence (black) or presence of curcumin (red) were indicated by the enhanced ThT fluorescence. Data were normalized with the ThT fluorescence intensity of completed matured fibrils and presented as mean ± SD (n = 3 independent replicates). The concentrations of α-Syn and curcumin are 200 and 25 μM, respectively. All the experiments were carried out in the presence of 20% PEG-10,000.

Curcumin Destabilizes the Amyloid Aggregates Matured from α-Syn Phase Separation

Then we tested whether curcumin could disassemble preformed α-Syn amyloid aggregates in the condensates. We prepared the phase-separated α-Syn aggregates by continuously incubating α-Syn condensates at 37 °C. Curcumin was then added to these preformed α-Syn aggregates in a different concentration. We found that curcumin reduced the ThT fluorescence intensity of α-Syn aggregates in a dose-dependent manner (Figure A). TEM images showed that the number of visible fibrils decreased in the presence of curcumin. However, it does not allow precisely quantifying the amount of the aggregated protein in the whole sample (Figure B). Curcumin is active at concentrations lower than one molecule per α-Syn molecule. Therefore, it is unlikely that the fibrils were dissociated into monomers. Due to the lack of curcumin-α-Syn structural information, the mechanism of aggregation destabilization by curcumin also needs further investigation. Together, our findings reveal that curcumin not only inhibits the formation of α-Syn amyloid aggregates but also disassembles the preformed amyloid aggregates during the phase separation process.

Figure 6

Curcumin destabilizes the preformed α-Syn amyloid aggregates matured from phase transition. (A) The preformed α-Syn amyloid aggregates (200 μM) were mixed with 10, 25, and 50 μM curcumin, respectively. ThT fluorescence was measured to indicate the destabilization of α-Syn amyloid aggregates by curcumin. Data are presented as mean ± SD (n = 3 independent replicates). P values were calculated using Student’s t test. **P < 0.01. ***P < 0.001. ****P < 0.0001. The preformed α-Syn amyloid aggregates were prepared by incubation of α-Syn condensates at 37 °C for 48 h. (B) Transmission electron micrographs showing the preformed α-Syn amyloid aggregates (200 μM) were reduced after incubation with 50 μM curcumin for 12 h. All the experiments were carried out in the presence of 20% PEG-10,000.

Discussion

The aggregation of α-Syn is a critical target for PD treatment. In the past decades, many efforts have been performed on the screen or rational design of reagents targeting α-Syn aggregations.[28,29,75] Multiple studies demonstrated that α-Syn aggregation was accelerated by protein phase separation, making phase-separated α-Syn a new drug target for PD treatment.[39] Recent studies reported that small molecules could modulate protein LLPS by promoting or preventing the formation of liquid droplets.[76,77] It is a promising strategy to target α-Syn aggregation by interruption of phase separation or transition, both of which affect α-Syn turning into an amyloid.

Multiple recent studies suggested that α-Syn might take two pathways to form amyloid aggregates.[38,39] In the traditional deposition pathway, α-Syn forms oligomers and grows into matured fibrils. In the condensation pathway, α-Syn undergoes LLPS, facilitating the amyloid aggregation in the condensates. Some studies showed that curcumin inhibits α-Syn fibrillization and destabilizes preformed aggregates in solution.[54,64−67,71] However, other reports suggested that curcumin binds to oligomers and fibrils that accelerate α-Syn fibrillation to produce morphologically different amyloid fibrils.[68,69] The observation of distinct roles might be due to the different experimental conditions, such as the protein purification method and the pH conditions.[69] Molecules bound to α-Syn may regulate its phase separation or transition.[39] For example, we recently reported that Mn2+ induces α-Syn to form irregular solid-like condensates.[40] Curcumin is a vital lead compound that regulates α-Syn amyloid aggregation in the deposition pathway and releases cellular toxicity. However, how curcumin regulates α-Syn amyloid aggregation in the condensation pathway remains unknown. Here, we studied the roles of curcumin on α-Syn amyloid aggregation under LLPS conditions. Interestingly, our findings demonstrate that curcumin inhibits α-Syn amyloid aggregation and destabilizes the preformed aggregates in the condensates (Figure ).[54,64−67,71]

Figure 7

Schematic illustration showing that curcumin inhibits α-synuclein amyloid aggregation under phase separation.

Different from the recent studies showing that small molecules modulate liquid droplet formation, curcumin has less effect on the initial morphology and total amounts of α-Syn condensates.[76,77] However, curcumin interacts with α-Syn condensates directly and decreases the molecular diffusion of the α-Syn protein inside the condensates, as indicated by fluorescence and FRAP experiments. Previous studies suggested that curcumin binds to the hydrophobic surfaces of α-Syn oligomers and fibrils.[68] The concentrated α-Syn forms liquid droplets with the requirement of the central hydrophobic region, which might be more exposed during LLPS.[39] Our study indicated that phase separation facilitates the hydrophobic interactions between curcumin and α-Syn, hindering protein diffusion. Since the hydrophobic region is the primary domain for α-Syn intermolecular assembly, the curcumin−α-Syn interactions could further inhibit α-Syn amyloid aggregation in the condensates.[78] It should be noted that the condensates are apparently different from the oligomers. α-Syn oligomers are solidified substances, but the condensates are typical spherical droplets with liquid properties, although they are both formed by the intermolecular assembly. Furthermore, the size of the prepared α-Syn oligomers is usually at the level of nanometers, but the diameters of condensates can reach micrometers. Indeed, recent studies reported that α-Syn could further form oligomers and fibrils in the condensates by a liquid-to-solid transition.[38,39] Interestingly, we discovered that curcumin also inhibited the amyloid aggregations of two PD disease-related E46K and H50Q mutants in the condensates. Besides the inhibition of amyloid aggregation, curcumin can destabilize the preformed amyloid aggregates matured in the condensates, confirming our conclusion that curcumin interacts with α-Syn through the hydrophobic interaction.

Together, we discovered that curcumin interacts with α-Syn in the condensates during phase separation. Curcumin disturbs the α-Syn protein fluidity in the condensates and inhibits the α-Syn protein from turning into an amyloid, although it has less effect on the initial formation of α-Syn condensates. These findings demonstrate that curcumin efficiently inhibits α-Syn amyloid aggregation and destabilizes preformed amyloid aggregation during phase separation. Our study reveals that α-Syn amyloid aggregation under the condition of phase separation can be targeted by small molecules like curcumin.

Materials and Methods

Protein Expression and Purification

Recombinant α-synuclein (α-Syn) was expressed in E. coli as previously described.[40,79−82] Briefly, the cDNA encoded human α-Syn gene was cloned into a pETSUMO vector. The recombinant plasmid was transformed into a BL21 (DE3) Competent Cell. The protein expression was induced by adding 1 mM isopropyl β-d-galactopyranoside (IPTG) when the OD of bacteria reached 0.6–0.8. The culture continued to grow for 2 h at 37 °C, and the pellets were collected by centrifugation. After lysis, the His6-SUMO-α-Syn fusion protein was purified by nickel affinity chromatography. The extra His6 tags were removed by digestion with SUMO protease. The purified untagged protein was subsequently dialyzed overnight against a protein storage buffer (25 mM Tris–HCl [pH 7.4], 50 mM NaCl).[40]

Preparation of Curcumin Stock Solution

The stock solution (10 mM) of curcumin was prepared in DMSO. For each experiment, curcumin was diluted by protein storage buffer to the required concentration.

Chemical Labeling

The α-Syn protein was labeled by a Rhod labeling kit following the manufacturer’s instructions (ThermoFisher Scientific, USA) as we previously described. The protein was dialyzed against a labeling buffer (50 mM sodium borate, pH 8.5) and then mixed with 15-fold molar excess of fluorescence dye. After incubation at room temperature for 1 h, the free dye in the solution was removed by overnight dialysis against the protein storage buffer.[40]

Confocal Imaging of α-Syn Condensates

The α-Syn condensates were prepared in the presence of PEG-10,000. Unlabeled and Rhod-labeled α-Syn were mixed at a molar ratio of 9:1. The α-Syn mixture was incubated with or without curcumin in the presence of 20% PEG-10,000 (w/v) to form condensates. The α-Syn condensates were visualized with a 100× oil immersion objective under a Nikon A1 microscope (Nikon Corporation, Japan).[40]

Fluorescence Measurement

The fluorescence measurements were carried out on BioTek Synergy HT microplate reader. The curcumin fluorescence was monitored with an excitation wavelength of 426 nm and an emission wavelength of 450–650 nm.

FRAP Analysis

The FRAP analysis was carried out as we previously described.[40] The Rhod-labeled α-Syn condensates were loaded onto a glass slide. The sample was then covered with a coverslip, sealed, and dried with nitrogen. FRAP experiments were performed using a Nikon A1 microscope, and the measurements involved 2 prebleaching frames, 1 flash of bleaching (100% of laser power), and 12 postbleaching frames. Fluorescence photobleaching and recovery were conducted using a 561 nm laser. Data were normalized to the maximal prebleach and minimal postbleach fluorescence intensities.

Sedimentation-Based Assay

Sedimentation and electrophoresis assays were performed as we previously described.[40] The 50 μL samples of α-Syn condensates were separated from free protein by centrifugation at 16,000g for 10 min. The supernatant and pellet were collected individually, and the pellet was washed and resuspended with 50 μL of the same buffer. The same volume of samples from the supernatant and pellet was analyzed by SDS-PAGE.

Turbidity Assay

In the turbidity assay, the samples were added to a transparent 96-well plate. The absorbance at 600 nm was measured using a BioTek Synergy HT microplate reader.[80] Full accounting of statistical significance was included for each data based on at least three independent experiments.

ThT Fluorescence

The amyloid aggregation of α-Syn was indicated by ThT fluorescence intensity. The ThT fluorescence assay was carried out as we previously described.[40,74] 200 μM WT α-Syn and the indicated concentrations of curcumin were incubated at 37 °C for 48 h without shaking. The samples were diluted by 40-fold, and the final concentration of ThT is 40 μM. Fluorescence was measured every 12 h on a BioTek Synergy HT microplate reader with an excitation wavelength of 440 nm and an emission wavelength of 485 nm. In the control experiment, the wholly matured α-Syn fibrils were prepared by shaking the α-Syn protein (200 μM) at 37 °C for 7 days without PEG. Curcumin and α-Syn fibrils were diluted to equal concentrations as we used in the kinetic assays. The ThT fluorescence was monitored with excitation at 440 nm and an emission range of 468–600 nm using a BioTek Synergy HT microplate reader.

We prepared the preformed amyloid aggregates by incubating the phase-separated α-Syn at 37 °C for 48 h for the destabilization of preformed aggregates. The α-Syn amyloid aggregates were incubated with the indicated concentration of curcumin. The ThT fluorescence was measured on a BioTek Synergy HT microplate reader after incubation at 37 °C. Data are presented as the percentage of the fluorescence change without curcumin.

Transmission Electronic Microscopy

α-Syn condensate samples were prepared in the absence or presence of curcumin with incubation at 37 °C for 48 h. The samples were loaded onto the carbon-coated copper grids for 1 min and subsequently stained with 2% uranyl acetate for 1 min. The TEM images were obtained on an H-7650 TEM (HITACHI) at an acceleration voltage of 80 kV.[40]