ABSTRACT Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB) are neurodegenerative disorders characterized by the accumulation of α-synuclein aggregates. α-synuclein forms droplets

via liquid-liquid phase separation (LLPS), followed by liquid-solid phase separation (LSPS) to form amyloids, how this process is physiologically-regulated remains unclear. β-synuclein

colocalizes with α-synuclein in presynaptic terminals. Here, we report that β-synuclein partitions into α-synuclein condensates promotes the LLPS, and slows down LSPS of α-synuclein, while

disease-associated β-synuclein mutations lose these capacities. Exogenous β-synuclein improves the movement defects and prolongs the lifespan of an α-synuclein-expressing NL5901

underlying the normal and disease states of PD and DLB with therapeutical potentials. SIMILAR CONTENT BEING VIEWED BY OTHERS ENHANCING MITOCHONDRIAL PROTEOLYSIS ALLEVIATES

ALPHA-SYNUCLEIN-MEDIATED CELLULAR TOXICITY Article Open access 21 June 2024 DISRUPTING THE Α-SYNUCLEIN-ESCRT INTERACTION WITH A PEPTIDE INHIBITOR MITIGATES NEURODEGENERATION IN PRECLINICAL

MODELS OF PARKINSON’S DISEASE Article Open access 19 April 2023 CRBN MODULATES SYNUCLEIN FIBRILLATION _VIA_ DEGRADATION OF DNAJB1 IN MOUSE MODEL OF PARKINSON DISEASE Article Open access 23

October 2024 INTRODUCTION Liquid-liquid phase separation (LLPS) has emerged as a key physiological process in understanding the complex behavior of biomolecules within cellular environments.

Biomolecular condensates formed by phase separated protein and RNA, such as membrane-less organelles, participate in multiple biological processes, including transcription, chromatin

organization, response to stress, and density signaling assembly1. Proteins undergo phase separation through forces including electrostatic interaction, hydrophobic interaction, hydrogen

bonding, dipole-dipole, π-π, and cation-π interaction2. Aberrant phase separations have been reported in many neurodegenerative diseases and cancers3. Parkinson’s disease (PD) and Dementia

with Lewy Bodies (DLB) are common neurodegenerative disorders, characterized by the formation of abnormal aggregates called Lewy bodies that are primarily composed of misfolded α-synuclein

(α-Syn)4,5. Aggregation of α-Syn is one of the major causes of neuronal cell death and is the leading pathogenic factor involved in the progression of PD and DLB. Recent studies suggest that

α-Syn forms liquid droplets via LLPS before converting into hydrogels and aggregates through a non-canonical condensation pathway. Factors exacerbating α-Syn aggregation, such as familial

or sporadic mutations, can promote LLPS of α-Syn and its subsequent maturation into amyloids through liquid-solid phase separation (LSPS)6. The synuclein family has three closely related

proteins: α-synuclein (α-Syn), β-synuclein (β-Syn), and γ-synuclein (γ-Syn)7. With an estimated 50 µM physiological concentration, α-Syn is highly abundant in neuronal synapses8. At

physiological pH, α-Syn forms phase separated condensates in the presence of the crowding agent polyethylene glycol (PEG)9. β-Syn co-localizes and is co-expressed at similar level, with

α-Syn at the presynaptic terminals, but it does not form aggregates under physiological conditions10,11. α- and β-Syn share high similarity (78%) and similar structural components,

encompassing a positively charged N-terminal, a hydrophobic NAC domain, and a negatively charged C-terminal (Fig. 1a and Supplementary Fig. 1a, b), although β-Syn lacks most of the NAC

region. Point mutations in α-Syn associated with familial Parkinson’s disease occur predominantly in the N-terminal region, e.g., A30P, E46K, H50Q, G51D, and A53T while mutations A18T and

A29S lack family history12,13,14,15. Previously, we showed that C-terminal truncation exacerbates the aggregation and cytotoxicity of α-Syn, and some natural compounds inhibit the

aggregation of α-Syn16,17,18. β-Syn is generally considered playing a neuroprotective role and improves PD19, it has been shown to inhibit the aggregation and toxicity of α-Syn by preventing

the formation of toxic oligomers and fibrils20,21. On the other hand, the V70M and P123H mutations in β-Syn seem to be associated with a predisposition to sporadic and familial DLB cases,

respectively22. Brain-specific β-Syn isoforms β-Syn120 and β-Syn104, are detected in the caudate nucleus of all DLB samples23. Interaction between α- and β-Syn have been suggested24,

however, the molecular basis underlying their interplay remain poorly understood. In this study, we investigated the effects of wildtype and pathogenic β-Syn mutants on α-Syn phase

separation and α-Syn-induced Parkinson’s disease, in vitro and in _C. elegans_ models. A set of therapeutic peptides designed based on the α- and β-Syn binding site were validated and found

to inhibit the phase transition of α-Syn, rescued the movement defects, and prolonged the lifespan in a _C. elegans_ PD model. RESULTS CHARACTERIZATION OF IN VITRO LLPS OF Α-SYN AND Β-SYN

Phase separation behaviors are intrinsically determined by protein sequences including intrinsically disordered regions (IDRs), low-complexity domains (LCDs), amino acid charges, and

hydrophobicity25,26. Charge distribution analysis suggested that the C-terminal regions of α-Syn and β-Syn are negatively charged (Supplementary Fig. 1b); FuzDrop predicts that the

C-terminal has high propensity to undergo phase separation serving as a driver for droplet formation (Supplementary Fig. 1c) because PDP ≥ 0.60 are likely to spontaneously phase separate27.

In vitro, using purified α-Syn and β-Syn (Supplementary Fig. 1d), α-Syn formed LLPS condensates, while β-Syn did not undergo spontaneous phase separation and generated no droplet under the

tested condition (10 mM HEPES, 50 mM NaCl, 20% PEG, pH 7.4; 100 μM) (Fig. 1b). The complete fusion of 5(6)-FAM-labeled α-Syn (FAM-α-Syn) and Rhodamine B-labeled α-Syn (Rho-α-Syn)

demonstrated the liquid properties of α-Syn (Supplementary Fig. 1e). To investigate the liquid-like behavior of the condensate, we conducted a fluorescence recovery after photobleaching

(FRAP) assay. Fast fluorescence recovery of the α-Syn droplets was observed within two minutes after bleaching (Supplementary Fig. 1f). We observed under confocal microscopy for 60 min and

found no significant difference in condensates formation (Supplementary Fig. 1g), so the following images were taken within 1 h. These results indicated that α-Syn has a high propensity to

form phase separated condensates. Β-SYN PARTITIONS INTO Α-SYN CONDENSATES AND CO-LOCALIZES WITH Α-SYN We investigated whether and how β-Syn affects α-Syn condensates. When FAM-β-Syn was

added to preformed α-Syn condensates (containing 10% Rho-α-Syn), the green FAM-β-Syn initially formed circles outside of the red α-Syn condensates, then gradually entered the droplet and

were completely partitioned inside α-Syn condensates within 60 min as seen using confocal microscopy (Fig. 1c). Lysozyme was used as a negative control, because FAM-labeled lysozyme was

unable to partition into α-Syn condensates, even after prolonged co-incubation (Supplementary Fig. 1h). Single-droplet fluorescence imaging of droplets was taken with FITC (donor) and

Rhodamine B (Rho) (acceptor) as the intermolecular Förster resonance energy transfer (FRET) pair. Upon excitation of the donor FITC, enhanced emission fluorescence of Rhodamine B indicated

intermolecular FRET, suggesting interaction between α-Syn and β-Syn within the condensates (Fig. 1d). Moreover, by bleaching the Rho-channel and detecting the FITC channel, we found

decreased red fluorescence and increased green fluorescence, which also indicated the interaction between the α-Syn and β-Syn (Fig. 1e, f). Β-SYN PROMOTES LIQUID-LIQUID PHASE SEPARATION OF

Α-SYN THROUGH ELECTROSTATIC INTERACTIONS To examine whether and how β-Syn affects α-Syn LLPS, we compared the morphological characteristics of the condensates. We quantified the number and

area of condensates, and found increased number and mean area of α-Syn/β-Syn (α/β-Syn) complex condensates compared with those of the α-Syn alone (Fig. 2a, b), indicating that β-Syn may

enhance the LLPS propensity of α-Syn. A turbidity assay indicated markedly increased turbidity of α/β-Syn condensates compared to that of α-Syn alone (Fig. 2c). Sedimentation assay can

quantitatively examine phase-separated proteins28, it has been suggested that the precipitate mainly contains phase-separated or aggregated proteins, while the supernatant contains soluble

proteins29. The sedimentation assay indicated that in the mixture of α-Syn and β-Syn, most of the proteins (~86%) were found in the precipitate; while most of the β-Syn was in the

supernatant (~87%) in the control β-Syn alone (Fig. 2d, e), suggesting that β-Syn promotes α-Syn phase separation. To identify the components within the LLPS precipitates, we re-suspended

the precipitate from the sedimentation assay with LLPS buffer, and observed condensates (Supplementary Fig. 2a). FRAP assay on the condensates of re-suspended precipitate indicated that the

re-suspended precipitate retained the condensate morphology and FRAP behaviors, albeit at reduced level (Supplementary Fig. 2b). Moreover, no obvious ThT fluorescence was detected from the

precipitate and supernatant (Supplementary Fig. 2c), indicating no formation of amyloids. These results suggest that the precipitates were mainly condensates but not amyloid fibrils. In

addition, FRAP data demonstrated the enhanced mobility of α-Syn within the α/β-Syn condensates compared with that of α-Syn alone (~50% recovery for α-Syn alone, ~72% recovery for α/β-Syn)

(Fig. 2f, g). α-Syn condensates with different sizes gave no significant difference/association in the bleaching recovery ability with the size/volume of the condensates (Supplementary Fig. 

3a, b). We also investigated whether molecular crowding affects the LLPS behaviors of α-Syn in response to equimolar β-Syn, and increasing concentrations of α-Syn and β-Syn resulted in

increasing numbers of co-condensates (Supplementary Fig. 3c). Next, we determined the primary interactions driving the formation of α/β-Syn condensates. Increasing the concentration of NaCl

(100–500 mM) gradually shrank the size and eventually abolished the formation of α/β-Syn condensates, while the addition of 1,6-hexanediol (1,6-HD; 5–20%) did not disrupt α/β-Syn condensates

formation but led to clustered morphology (Fig. 2h-j). We further assessed the 1,6-HD effects by sedimentation assay and FRAP assay. In the sedimentation assay, the presence of 1,6-HD

(0–20%) did not obviously affect protein levels in the supernatant (Supplementary Fig. 4a, b). Furthermore, the presence of 1,6-HD (0–20%) caused increased condensates clustering

(Supplementary Fig. 4c) and affected the fluidity of α-Syn condensates and α-Syn/β-Syn co-condensates (Supplementary Fig. 4d, e). In the presence of increasing concentration of salt, the

α/β-Syn condensates decreased sharply with a complete dissolution of droplets at 500 mM NaCl indicating that intermolecular electrostatic interactions are the driving force within the

condensates (Supplementary Fig. 4f). The addition of negatively charged heparin (1 or 10 mg/mL), which affects the ionic charge-charge interactions30, abolished the formation of α/β-Syn

condensates (Supplementary Fig. 4g). The control α-Syn condensates also disappeared when treated with 500 mM NaCl or 10 mg/mL heparin (Supplementary Fig. 4h); turbidity measurements

suggested that compared with α-Syn alone, disrupting α-Syn + β-Syn condensate formation required higher ionic strength (Supplementary Fig. 4i, j). These results collectively suggested that

β-Syn promotes the LLPS of α-Syn with electrostatic interactions as the primary driving force. Β-SYN DELAYS THE LIQUID-SOLID PHASE SEPARATION OF Α-SYN Wetting behaviors indicate the fluid

property of liquid droplets when spreading on solid surface, phase separated droplets dynamically fuse with each other to generate larger droplets with irregular shapes termed as protein

rafts31,32; while hydrogels and aggregates lose fluidity property and do not wet or change shape33 (Supplementary Fig. 5 and Supplementary Movie 1). We monitored the behavior of the

condensates over time to detect liquid-solid phase separation (LSPS) using DIC microscopy and observed that the α/β-Syn condensates fused into protein rafts on the glass surface and lasted

for 3 h (Fig. 3a), indicating liquid properties. In contrast, α-Syn alone showed no wetting behavior throughout the experiment, indicating amyloid formation or gel-like transition within the

condensates (Fig. 3a). α-Syn has been reported to nucleate through LLPS6, then aggregating into fibrils with prion-like characteristics34. Next, we investigated whether β-Syn affects the

amyloid transition of α-Syn using a Thioflavin T (ThT) assay, which detects protein aggregates and amyloid-like assemblies by exhibiting a characteristic emission band at 482 nm. The ThT

fluorescence of α-Syn alone exhibited a significant increase at day 2 (Fig. 3b, left), while the presence of equimolar β-Syn decreased the maximum fluorescence intensity of α-Syn (Fig. 3b,

left). And β-Syn alone showed no tendency to form fibrils even after incubation for 7 days (Fig. 3b, right). Morphologically, fibril-like structures started to form around α-Syn condensates

at day 3 as observed by transmission electron microscopy (TEM) and there were more fibril-like structures surrounding the droplets at day 7. In contrast, there were no noticeable fibril-like

structures formed in β-Syn alone or α/β-Syn condensates (Fig. 3c). We further used Raman spectroscopy, which determines the secondary structural characteristics of proteins, on single

droplets at day 7 (Fig. 3d–f). To identify changes in secondary structures, we deconvoluted the 1600–1700 cm−1 amide-I region, which represents secondary structural elements (Fig. 3f). The

results suggested that α-Syn condensates are mainly composed of β-sheet structures (60.56%), while the addition of equimolar β-Syn dramatically decreased the β-sheet content to 24.62% (Fig. 

3g). PEG is a commonly used molecular crowder that promotes phase separation (i.e., addition of PEG creates LLPS conditions). We also monitored the fibrillation kinetics of α/β-Syn complex

in the absence of PEG, where fibrillation was induced by continuous shaking (i.e., without PEG creates non-LLPS conditions). We applied ThT-based kinetic assay to detect amyloid fibril

formation (Supplementary Fig. 6a), dynamic light scattering (DLS) to measure the particle size during aggregation (Supplementary Fig. 6b), TEM to characterize amyloid morphology

(Supplementary Fig. 6c), far-UV circular dichroism (CD) spectroscopy to analyze secondary structure components (Supplementary Fig. 6d), and immuno-dot blots to detect the formation of

oligomers and fibrils during incubation (Supplementary Fig. 6e, f). Together, these results also indicated that β-Syn inhibits the liquid-solid transition of α-Syn and prevents

transformation of α-Syn into highly ordered amyloid fibrils (Supplementary Fig. 6). DISTINCT EFFECTS OF Β-SYN ON PHASE SEPARATION OF PATHOGENIC Α-SYN MUTATIONS Approximately 10% of PD

patients have familial/sporadic single-point mutations, including A18T, A29S, A30P, E46K, H50Q, G51D, and A53T35. We expressed and purified these mutants (Supplementary Fig. 7a), and the

LLPS of wildtype and mutant α-Syn were imaged using confocal microscopy. E46K formed more and larger condensates than wildtype α-Syn, while no noticeable difference in size was observed for

A18T, A30P, and A53T (Supplementary Fig. 7b–d). The FRAP experiments suggested reduced FRAP recovery of these α-Syn mutants except for A30P (Supplementary Fig. 7e). A30P alone formed less

number of condensates than wildtype α-Syn, but abundant co-condensates with β-syn; moreover, A30P and E46K formed β-syn co-condensates with sizes larger than that of α-Syn/β-Syn

co-condensates (Supplementary Fig. 8a–c). Interestingly, the FRAP results showed that the presence of equimolar β-Syn increased the protein mobility within α-Syn mutants condensates,

including A18T, A29S, A30P and G51D condensates (~17% recovery for A18T alone, ~45% recovery for A18T with β-Syn; ~19% recovery for A29S alone, ~27% recovery for A29S with β-Syn; ~34%

recovery for A30P alone, ~46% recovery for A30P with β-Syn; ~7% recovery for G51D alone, ~33% recovery for G51D with β-Syn) (Supplementary Fig. 8d); while the presence of equimolar β-Syn did

not alter the mobility of E46K or H50Q condensates (~15% recovery for E46K alone, ~12% recovery for E46K with β-Syn; ~21% recovery for H50Q alone, ~22% recovery for H50Q with β-Syn)

(Supplementary Fig. 8d), and caused an apparent decrease in fluidity of A53T condensates (~43% recovery for A53T alone, ~33% recovery for A53T with β-Syn) (Supplementary Fig. 8d). We

performed FRET assay for these α-Syn mutants with β-Syn, and found that both WT and mutant α-Syn exhibited FRET effect with β-Syn (Supplementary Fig. 9a, b). Specifically, the A18T, A30P,

and E46K α-Syn mutants showed enhanced FRET with β-Syn, while the A29S and A53T α-Syn mutants showed decreased FRET with β-Syn compared to the α-Syn WT; the FRET of H50Q and G51D α-Syn

mutants with β-Syn were similar to that of the α-Syn WT (Supplementary Fig. 9b). DLB ASSOCIATED Β-SYN MUTANTS PROMOTE THE LIQUID-SOLID PHASE SEPARATION OF Α-SYN Wildtype β-Syn is considered

to inhibit abnormal α-Syn aggregation, however, two β-Syn mutants, V70M and P123H, have been shown to exacerbate the potential neurotoxicity in DLB patients, and splicing isoforms β-Syn120

and β-Syn104 are detected brain-specifically in patients with Lewy body disease23,36. We analyzed the distribution of charges and phase separation propensities of β-Syn mutants (Fig. 4a and

Supplementary Fig. 10a–h) and expressed and purified these β-Syn mutants or isoforms (Supplementary Fig. 10i). Consistent with wildtype β-Syn, none of the β-Syn mutants exhibited phase

separation or formation of obvious droplets or aggregates under tested conditions (Supplementary Fig. 10j). After mixing V70M, P123H or β-Syn120 with equimolar α-Syn, mesh-like structures

were observed (Fig. 4b). Consistent with these data, turbidity assays indicated a sharp turbidity increase when mixing wildtype α-Syn with mutant β-Syn (Fig. 4c). The FRAP analysis

demonstrated mildly decreased internal mobility of α-Syn within the condensates in the presence of β-Syn120 and β-Syn104, compared to that of α-Syn alone (~40% recovery for α-Syn alone, ~30%

recovery for α-Syn with β-Syn120, ~28% recovery for α-Syn with β-Syn104), whereas the condensates formed in the presence of equimolar V70M or P123H with α-Syn undergo a faster transition

into a solid-like state (~12% recovery for α-Syn with V70M and ~11% recovery for P123H) (Fig. 4d). After 7 days incubation, α-Syn formed amyloid fibrils or gel-like structures surrounding

the condensates in the presence of β-Syn mutants as observed by TEM (Fig. 4e). To investigate the partitioning behavior of β-Syn mutants, β-Syn mutants or isoforms were added to preformed

α-Syn condensates. All mutants or isoforms fully partitioned into α-Syn condensates within 60 min (Supplementary Fig. 11a), except for P123H which took 180 min (Supplementary Fig. 11b). We

further monitored the LSPS under PEG-free conditions. ThT-based kinetic assays showed that wildtype and mutants/isoforms of β-Syn, when incubated alone, did not form fibrils within 96 h

(Supplementary Fig. 12a). α-Syn reached the plateau stage after 60 h incubation with a lag time of 29.37 ± 0.62 h. The presence of equimolar β-Syn significantly decreased the maximum

fluorescence intensity and prolonged the lag time to 33.84 ± 0.45 h, whereas the presence of equimolar V70M or P123H β-Syn caused a rapid transformation of α-Syn into amyloid fibrils, with

decreased lag time of 28.62 ± 0.89 h and 28.59 ± 0.61 h, and P123H also increased the maximum fluorescence intensity (Supplementary Fig. 12b). Notably, isoforms β-Syn104 or β-Syn120

significantly decreased the lag time of α-Syn to 7.40 ± 0.84 h or 9.58 ± 0.93 h, respectively (Supplementary Fig. 12b). After 96 h incubation, α-Syn alone or with equimolar V70M or P123H

formed typical linear ribbon-like fibrils, while the presence of equimolar wildtype β-Syn, β-Syn104 or β-Syn120 resulted in the formation of amorphous aggregates as observed by TEM

(Supplementary Fig. 12c). WILDTYPE Β-SYN ALLEVIATES WHILE V70M/P123H MUTATIONS EXACERBATE Α-SYN-INDUCED NEURODEGENERATION To study the effects of wildtype and mutant β-Syn on α-Syn in vivo,

we established a _C. elegans_ model by co-expressing α-Syn with wildtype or mutant β-Syn37. Exogenous DNA was introduced into the developing oocytes within the reproductive system of adult

hermaphrodites to produce transgenic progeny worms (Fig. 5a; Supplementary Table 1). In wildtype N2 strains and β-Syn overexpressing strains, aggregates were barely observed in the head of

the worms (Fig. 5b). Next, we crossed wildtype and mutant β-Syn overexpressing worm strains with the NL5901 strain (a PD model that overexpresses α-Syn). The wildtype β-Syn, V70M, and P123H

mutants were partially co-localized with wildtype α-Syn in _C. elegans_ (Fig. 5b). Furthermore, FRAP analysis demonstrated that co-expression of wildtype β-Syn enhanced α-Syn mobility within

inclusions in NL5901 worms, while the V70M and P123H β-Syn mutants decreased α-Syn mobility within inclusions in NL5901 worms (~34% recovery for NL5901 strains, ~61% recovery for

α-Syn;β-Syn strains, ~10% recovery for α-Syn;V70M strains, ~20% recovery for α-Syn;P123H strains) (Fig. 5c). The thrashing assay, as a behavioral indicator for the movement and behavior of

_C. elegans_, assesses nervous system function38. When we overexpressed β-Syn in NL5901 strains, an improvement in movement was observed in α-Syn;β-Syn strains (Fig. 5d); while significantly

impaired movements were observed in α-Syn;V70M and α-Syn;P123H strains, as compared with that of the NL5901 strains (Fig. 5d; Supplementary Movies 2–9). Additionally, the impact of wildtype

and mutant β-Syn on the lifespan of N2 and NL5901 strains were assessed. Notably, decreased lifespans were observed in N2 strains that overexpress wildtype or mutant β-Syn (median lifespan

9.63 ± 1.80 days for wildtype β-Syn group, 8.67 ± 3.06 days for V70M group, and 6.67 ± 1.15 days for P123H group _vs_. 13.67 ± 0.58 days for N2 controls) (Fig. 5e). On the other hand,

wildtype β-Syn could extend the lifespan of NL5901 strains (median lifespan 14.33 ± 2.08 days for α-Syn;β-Syn group _vs_. 10.25 ± 0.50 days for NL5901 control group), while co-expression of

V70M or P123H mutants markedly decreased the median lifespan of NL5901 (median lifespan 5.67 ± 0.58 days for α-Syn;V70M group and 7.00 ± 1.00 days for α-Syn;P123H group _vs_. 10.25 ± 0.50

days for the NL5901 control group) (Fig. 5f). Taken together, consistent with the in vitro results (Fig. 4), the _C_. _elegans_ experiments indicated that wildtype β-Syn maintains the LLPS

properties of α-Syn condensates, while the DLB associated β-Syn mutants V70M and P123H exacerbated the toxic LSPS of α-Syn and increased the PD-like symptoms in _C_. _elegans_. RATIONALLY

DESIGNED Β-SYN DERIVED PEPTIDES INHIBIT THE LLPS AND LSPS OF Α-SYN AND PROTECT AGAINST Α-SYN-INDUCED NEURODEGENERATION The inhibitory effects of β-Syn on α-Syn LSPS inspired us to identify

peptide fragments that inhibit α-Syn phase transitions. AlphaFold 2 Colab identified the key residues for the interactions in the N-terminal region of α-Syn and β-Syn, with several hot spots

between residues 33–48 of β-Syn (Fig. 6a). The workflow for screening and evaluating peptides for the inhibition of α-Syn phase separation was shown in Fig. 6b. A virtual step-by-step

screening protocol overlapping decapeptide within the β-Syn 33-48 region was performed (Supplementary Table 2). Two decapeptides, 36–45 and 37–46, showed the highest binding affinity to

α-Syn, and the binding energy of their _retro-inverso_ peptides was further determined (Supplementary Table 2). Based on virtual calculations, peptides corresponding to β-Syn45-36 and

β-Syn37-46 that have the highest binding affinity for α-Syn were chosen, and further FRAP analysis showed that only the presence of 5-fold molar β-Syn45-36 increased the protein mobility

within α-Syn condensates (~26% recovery for α-Syn alone, ~41% recovery for α-Syn with β-Syn45-36) (Supplementary Fig. 13a). To enhance the binding affinity with α-Syn, we chose to modify

residues β-Syn45-36 (called P0) with alanine scanning to identify the residues on P0 that affect binding affinity to α-Syn and found that the lysine at position 3 can be substituted

(Supplementary Table 3). We first attempted to substitute this lysine with hydrophobic amino acids and found decreased binding energy with α-Syn (Supplementary Table 4). Based on the

importance of electrostatic interaction between α-Syn and β-Syn for LLPS (Fig. 2h and Supplementary Fig. 4) and the positive charge on α-Syn N-terminus (Supplementary Fig. 1b), we next

systematically substituted amino acids that would increase the net negative charge, and five additional peptides with the highest binding energy rank were chosen for further experiments

(P1–P5; Supplementary Table 4). We first examined in vitro phase separation. FRET microscopic imaging was used to study the interaction between the peptides and α-Syn. The results suggested

colocalization and interaction between the donor-peptides and acceptor-α-Syn (Supplementary Fig. 13b). And we found that all six peptides were able to partition into preformed α-Syn

condensates (Supplementary Fig. 13c). FRAP analysis demonstrated that the addition of equimolar peptides showed no obvious effect on the fluorescence recovery of α-Syn (Supplementary Fig. 

13d, e). At a 5:1 molar ratio of peptide to α-Syn, P4, and P5 completely inhibited the phase separation of α-Syn and eliminated droplet formation (Fig. 6c). Peptides P1–P3 showed no obvious

effect on the fluorescence recovery of α-Syn condensates within 120 s after bleaching (Fig. 6d). We determined the effect of these peptides on α-Syn fibril formation in PEG-free conditions.

In a ThT-based kinetic assay, the presence of equimolar peptides significantly decreased the maximum fluorescence intensity of α-Syn (Supplementary Fig. 14a). Morphologically, after 96 h

incubation, α-Syn alone formed typical linear ribbon-like fibrils, while co-incubation with equimolar peptides resulted in short linear fibrils or amorphous aggregates (Supplementary Fig. 

14b). Further, an immuno-dot blot assay showed that peptides P3, P4 and P5 effectively inhibited the production of oligomers and fibrils during a 96 h incubation (Supplementary Fig. 14c, d).

The CD spectroscopy results showed that after 96 h incubation, compared to α-Syn alone, the presence of peptides significantly reduced the negative peak at 218 nm indicative of β-sheet

structures, suggesting their inhibitory effects on α-Syn aggregation (Supplementary Fig. 14e). However, these peptides were unable to disassemble preformed α-Syn fibrils (Supplementary Fig. 

14f). We further assessed the effects of these peptides in vivo using _C. elegans_ strains N2 and NL5901. Determination of lifespan in N2 strains suggested that none of the six peptides

showed obvious toxicity (Supplementary Fig. 15), and the α-Syn inclusions found in NL5901 after 3 days of treatment showed no obvious morphological differences compared with those of the

untreated group (Fig. 6e). To investigate α-Syn mobility within inclusions, we conducted FRAP experiments. Interestingly, treating NL5901 worms with peptides P0, P1, P3, P4 and P5 mildly

improved the fluorescence intensity recovery after bleaching (~24% recovery for NL5901, ~30% recovery for NL5901 treated with P0, ~26% recovery for NL5901 treated with P1, ~28% recovery for

NL5901 treated with P3, ~32% recovery for NL5901 treated with P4, ~35% recovery for NL5901 treated with P5), while P2 showed no obvious effects (Fig. 6f). Except for P2, the rest of peptides

significantly increased the movement of NL5901 worms in the thrashing assay, suggesting that these peptides can reduce PD-like movement disorders (Fig. 6g; the locomotion of _C. elegans_

are shown in Supplementary movies 10-16). Consistently, determination of lifespan showed that, except for P2, these peptides significantly extended the lifespan of NL5901 that expresses

human α-Syn (median lifespan 12.67 ± 1.15 days for P0 treatment, 12.25 ± 0.50 days for P1 treatment, 10.25 ± 0.50 days for P2 treatment, 12.67 ± 0.58 days for P3 treatment, 14.40 ± 0.89 days

for P4 treatment, and 13.67 ± 0.58 days for P5 treatment _vs_. 9.67 ± 0.58 days for the untreated NL5901 controls) (Fig. 6h, i). DISCUSSION Both α-Syn and β-Syn show similar expression

levels in the brain and colocalize in presynaptic terminals39,40. α-Syn has been associated with formation of pathological aggregates named Lewy bodies41. Under physiological conditions,

β-Syn does not undergo spontaneous aggregation42, suggesting that these molecules have opposing roles. β-Syn expression is drastically diminished in the brains from patients that presented

clinically as DLB with short disease duration43. The levels of β-Syn are elevated in PD patients, while the levels of α-Syn remain unchanged, suggesting that β-Syn plays a chaperone role for

α-Syn aggregation; the increased levels of β-Syn in PD patients may serve to prevent further aggregation of α-Syn and maintain the balance44,45,46,47. Recent studies suggested aberrant

phase separation of α-Syn precedes pathological protein aggregation3. Over time, α-Syn moves from liquid-liquid phase separation to liquid-solid phase separation forming irreversible

amyloids leading to disease48. However, the role and stage in which β-Syn affects α-Syn phase separation, as well as the mechanistic basis, remain unclear. LLPS reflects normal protein

behavior in vivo, which maintains proper physiological functions and inhibits pathological aggregation49,50. Here, we studied the effect of β-Syn on the phase separation of α-Syn (Fig. 6j)

and found that wildtype β-Syn facilitates LLPS of α-Syn and delays the progression to LSPS of α-Syn through interactions at their N-terminal domains (Figs. 2, 3 and 6a). Familial/sporadic

mutations account for approximately 10% of total PD cases51. Our study suggests that β-Syn not only inhibits amyloid formation by wildtype α-Syn, it also affects the phase transition of most

of the PD-related α-Syn mutations we studied, suggesting a potential intervention strategy for patients carrying these mutations. β-Syn mutants (V70M and P123H) and the brain-specific

splicing isoforms (β-Syn120 and β-Syn104) are detected in Lewy body diseases22,23. V70M is associated with sporadic cases of DLB while P123H is associated with familial DLB52.

Histopathological and immunohistochemical analysis of brain sections from DLB patients with the P123H mutation revealed widespread Lewy body pathology and α-Syn aggregation, without evidence

of β-Syn aggregation22. In DLB patients, the gene expression ratios of the β-Syn120 and β-Syn104 in the caudate nucleus are significantly increased, whereas no β-Syn104 is found in normal

brains23. The two isoforms resulting from alternative splicing, exclude exon 4 or exon 6 resulting in the N-terminal or C-terminal shortening of the protein23. V70M and P123H have been

suggested to worsen the neuropathological effects in transgenic mice36,53, while the pathological effects of β-Syn120 and β-Syn104 isoforms have not been reported. Our results suggest that

β-Syn or its mutants have no propensity to undergo LLPS or form fibrils in vitro (Supplementary Figs. 10j and 12a). Notably, these β-Syn mutants and isoforms lose the ability to regulate the

LLPS of α-Syn, and even promote the LSPS of α-Syn (Fig. 4 and Supplementary Fig. 12). Our studies using a _C. elegans_ model also suggested that V70M and P123H mutations induce

neurotoxicity and exacerbate α-Syn-induced neurodegeneration (Fig. 5). It has been noted that for TDP-43 pathology observed in the familial and sporadic amyotrophic lateral sclerosis (ALS)

and frontotemporal lobar degeneration (FTLD) cases54, shortened TDP-43 isoforms with splicing sites in exon 6, exhibit aberrant cytoplasmic aggregation, contributes to pathology in ALS55,56,

implicating that the flawed splicing mechanism may underlying the generation of pathogenic β-Syn isoforms and TDP-43 splice isoforms. This interesting finding also suggests that the

physiological balance between α-Syn and β-Syn is finely tuned. Currently, therapies for PD or DLB are primarily designed to alleviate clinical symptoms, there are still no proven safe and

effective treatments53. Levodopa, which supplements the levels of dopamine in the brain, is primarily used to treat PD symptoms57, however, its long-term use leads to issues such as

dyskinesias, non-motor side effects, and levodopa resistance58. Monoclonal antibodies against α-Syn, prasinezumab, and cinpanemab, failed in clinical trials for early Parkinson’s disease

patients59,60, which may be difficult to cross the blood-brain barrier due to its large molecular size61. Compared to monoclonal antibodies, peptides are much smaller facilitating passage

through the blood-brain barrier62 and demonstrate higher target specificity compared with small molecules. Two peptides (P4 and P5) used in this study showed good inhibitory effects on the

formation of phase separated condensates and fibrils in vitro and in a _C. elegans_ model (Fig. 6 and Supplementary Fig. 14). The peptides may disrupt multivalent interactions of α-Syn that

are necessary for liquid-liquid phase separation63,64. Although they did not disaggregate preformed α-Syn fibrils in our study (Supplementary Fig. 14f), they may still be further explored to

attenuate the progression of the disease. In summary, we have discovered a Yin-Yang balance between α-Syn and β-Syn in health and disease. This delicate balance is fine-tuned by the

interaction and the resulting phase separation and may be disrupted by factors like familial/sporadic mutations. This finding inspired us to design peptides derived from β-Syn that could

inhibit the amyloid transition of α-Syn. As a proof of concept, the rationally designed peptides improved the PD-like symptoms and increased the lifespan of _C. elegans_ PD model. METHODS

_C. ELEGANS_ STRAINS All strains were cultured on standard nematode growth media (NGM) plates seeded with _Escherichia coli_ OP5065. N2 (wildtype), NL5901

(_pkIs2386_[_unc-54p_::α-synuclein::YFP]), and the transgenic strains were used in this study (Supplementary Table 1). N2 is the wildtype strains, NL5901 is the α-Syn overexpressing strains,

and the transgenic strains were generated in house by microinjection and standard genetic crosses as follows66,67. Transgenic worms were generated by injecting 20 ng/μL plasmid together

with 20 ng/μL _lin-44p_::GFP (as an injection marker) into N2 strains using FemtoJet 4i (Eppendorf, Hamburg, Germany). After microinjection, progeny carrying both the transgene (red

fluorescence) and the injection marker (green fluorescence) were isolated; individuals with stable transgene expression were selected and validated as new strains and crossed with male

NL5901. Age-synchronized worms were generated by transferring reproductive adults onto fresh NGM plates for a period of 3 h to facilitate egg laying. MATERIALS

2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (catalog number: H109406) and Rhodamine B isothiocyanate (catalog number: R105502) were obtained from Aladdin (Shanghai,

China); Thioflavin-T (ThT) (catalog number: T3516), Fluorescein 5-isothiocyanate (FITC) (catalog number: F7250) and 1,6-hexanediol (catalog number: 240117) were obtained from Sigma Aldrich

(St. Louis, MO); 5(6)-carboxyfluorescein (5(6)-FAM SE) (catalog number: HY15937) was obtained from MCE (New Jersey, NY); isopropyl β-D-1-thiogalactopyranoside (IPTG) (catalog number: 1122GR)

was obtained from Biofroxx (Guangzhou, China); heparin sodium (catalog number: S12004) was obtained from Yuanye Biotech (Shanghai, China); A11 (oligomer-specific antibody; 1:2000; catalog

number: ab126892) and OC antibody (fibril-specific antibody; 1:2000; catalog number: ab201062) were obtained from Abcam (Cambridge, UK); streptomycin sulfate (catalog number: BS142) and PEG

4000 (catalog number: BS174) were obtained from Biosharp (Beijing, China); glacial acetic acid (catalog number: 10000208) and ammonium sulfate (catalog number: 10002918) were obtained from

Sinopharm (Shanghai, China); HPLC grade acetonitrile (catalog number: A998-4) was obtained from Fisher Chemical (Geel, Belgium); uranyl acetate (catalog number: 02624-AB) was obtained from

SPI Supplies (West Chester, PA). BIOINFORMATIC ANALYSES Phase separation propensities were analyzed using FuzDrop version 1.0 with default parameters

(https://fuzdrop.bio.unipd.it/predictor), the charge distribution was analyzed using the Classification of Intrinsically Disordered Ensemble Regions (CIDER) version 1.7 with default

parameters (https://pappulab.wustl.edu/CIDER/analysis/)32, the interactions between α-Syn and peptides derived from β-Syn were predicted using AlphaFold 2 (Version 1.5.5;

https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb)68 on the ColabFold with specific adjustments to the default parameters, the “alphafold2_multimer_V1”

model type was used to process multimeric protein complexes, with pair_mode set to “unpaired_paired” and num_recycles set to 3, and the max_msa was set to 512:1024, while all other settings

were kept at their default values. The predictions were included an automated energy-minimization step. Following the multisequence alignment, three-dimensional models were generated. The

predictions were executed on the Google Colab platform. RECOMBINANT PROTEIN EXPRESSION AND PURIFICATION Expression and purification of wildtype and mutant α-Syn (A18T, A29S, A30P, E46K,

H50Q, G51D, A53T), wildtype and mutant β-Syn (V70M, P123H, β-Syn120 and β-Syn104) were performed as follows18. Briefly, plasmids expressing these proteins were transformed into _E.coli_ BL21

(DE3), and expression was induced by adding 1 mM IPTG. Collected cells were boiled for 10 min to lyse cells and denature the protein, after centrifugation, the supernatant was collected,

and 10% (w/v) streptomycin sulfate (136 μL/mL of supernatant) and glacial acetic acid (222 μL/mL of supernatant) were added to precipitate nucleic acids. Then, an equal volume of saturated

ammonium sulfate was added to the supernatant to precipitate protein. The precipitate was washed 3 times with 50% saturated ammonium sulfate and 50% ethanol containing 100 mM ammonium

acetate. After lyophilization, proteins were dissolved by 6 M guanidine hydrochloride, then purification was performed using a semi-preparative Welch Ultimate XB-C18 column (Shanghai, China)

through a Hitachi L-2000 HPLC system (Tokyo, Japan), and detected at 280 nm69. Following gradient was used with a constant flow rate of 3 mL/min. The mobile phase contained 0.1% TFA. After

injection of 400 μL sample, the gradient was run with 30% acetonitrile for 2 min, followed by an isocratic elution to 50% acetonitrile for 8 min; the gradient was subsequently increased to

70% acetonitrile within 2 min and then decreased to 30% within 2 min. The purity of the preparations was >95% as demonstrated by SDS-PAGE with Coomassie blue staining. PROTEIN LABELING

Labeling α-Syn with Rhodamine B, and β-Syn with 5(6)-FAM and FITC was performed as follows17,70. Briefly, the indicated protein was incubated with a 10-fold molar excess of fluorescent dyes

at room temperature for 4 h; then, NH4Cl was added to quench the excess dye, and then dialyzed against pure water for at least 4 times to remove excess free dye, and finally lyophilized at 4

 °C. The proteins labeled are designated as Rho-α-Syn, FAM-β-Syn, and FITC-β-Syn, respectively. The labeling degree of protein with dye was calculated by following formula71. Firstly, we

calculated the protein concentration in the samples using the following formula: $${{{\rm{Protein}}}}\; {{{\rm{concentration}}}} \left(M\right)=\frac{{A}_{280}-({A}_{\max }\times

{{{\rm{CF}}}})}{\varepsilon }$$ (1) Where _ε_ = protein molar extinction coefficient. _A_max = Absorbance value of the protein-dye conjugate measured at the wavelength maximum of dye. _A_280

 = Absorbance value of protein-dye conjugate measured at 280 nm, and CF = Correction factor, calculated using the following formula: $${{{\rm{CF}}}}=\frac{{A}_{280}}{{A}_{\max }}$$ (2) Where

_A_280 = Absorbance value of dye measured at 280 nm and _A_max = Absorbance value of dye measured at the wavelength maximum of dye. We calculated the CF for Rhodamine B, 5(6)-FAM, and FITC

to be 0.33, 0.22, and 0.32, respectively. Subsequently, the degree of labeling was calculated using the following formula: $${{{\rm{Moles}}}}\; {{{\rm{dye}}}}\; {{{\rm{per}}}}\;

{{{\rm{mole}}}}\; {{{\rm{protein}}}}=\frac{{A}_{\max }{{{\rm{of}}}}\; {{{\rm{the}}}}\; {{{\rm{labeled}}}}\; {{{\rm{protein}}}}}{{\varepsilon }^{{\prime} }\times {{{\rm{protein}}}}\;

{{{\rm{concentration}}}} \, (M)}$$ (3) Where _ε_’ = molar extinction coefficient of the fluorescent dye. We determined a labeling ratio of 0.64–1.00 moles of dye per mole of protein in our

protein labeling experiment. It indicated a successful protein-fluorophore conjugation72. IN VITRO LIQUID-LIQUID PHASE SEPARATION (LLPS) To study LLPS, purified proteins were freshly

dissolved in buffer containing 10 mM HEPES, 50 mM NaCl and 20% (w/v) PEG 4000 (pH 7.4) at 25 °C73. All the LLPS samples were mixed by 10% labeled protein and 90% unlabeled protein to

minimize the effect of protein labeling on LLPS, and the final protein concentration of each sample was 100 μM. To assess the intermolecular forces between droplets, 500 mM NaCl or 10%

1,6-HD were added. These LLPS samples were dropped on the glass slides then covered with a 24 mm coverslip and sealed with nail polish around the edges. The liquid droplet formation in vitro

was observed and imaged using a Nikon AX 100×/1.49 NA oil immersion objective (Tokyo, Japan) under DIC (Differential Interference Contrast) and appropriate fluorescence channels (488 nm for

FITC and 5(6)-FAM, 561 nm for Rhodamine B). Confocal imaging was performed using NIS Viewer AX software, with laser powers set to under 5%, and the pinhole size set to 1 AU. FLUORESCENCE

RECOVERY AFTER PHOTOBLEACHING (FRAP) For in vitro FRAP study, LLPS sample preparation was as described above. Bleaching was done using a 561 nm laser at 100% intensity for selected region of

interests (ROI). After acquiring three images before bleaching, the recovery was monitored for 120 s. FRAP experiments were performed using a Nikon AX with a 100X oil immersion objective26.

The recovery of the bleached spots was recorded and analyzed using NIS Viewer AX software provided with the instrument. FRAP recovery data was recorded for at least 6 repeats for each

sample. PARTITIONING BEHAVIOR OF WILDTYPE AND MUTANT Β-SYN IN Α-SYN CONDENSATES 150 μM of α-Syn (containing 10% Rho-α-Syn) was prepared in a 50 mM NaCl buffer containing 20% PEG and

incubated for 30 min to form α-Syn condensates. Subsequently, 30 μM FAM labeled wildtype or mutant β-Syn was added into preformed α-Syn condensates. The partition of the wildtype or mutant

β-Syn into α-Syn condensates was observed using a Nikon AX with a 100X oil immersion objective at regular intervals74. Image Pro software was used for analyzing images. FÖRSTER RESONANCE

ENERGY TRANSFER (FRET) EXPERIMENT Fluorescence pairs, FITC-β-Syn (donor) and Rho-α-Syn (acceptor), FITC-peptides (donor) and Rho-α-Syn (acceptor), were used in FRET75. Complex condensates

were formed by equimolar ratio of 100 μM α-Syn and 100 μM β-Syn/peptides. Samples for the imaging were prepared as described above. FRET experiment was performed with a Nikon AX with a 100X

oil immersion objective (_λ_ex = 488 nm and _λ_em = 590 nm). For FRET acceptor photobleaching experiments76, 100% laser power (at excitation wavelength of 561 nm) was applied to bleach the

red signal in the region of interest (ROI), and record green and red fluorescence intensities change before and after bleaching. The FRET efficiency was calculated by the FRET acceptor

photobleaching experiments77. FRET efficiencies for each bleaching step were calculated by the following formula: $${{{\rm{FRET\;

efficiency}}}}=1-\frac{{I}_{{{{\rm{pre}}}}}}{{I}_{{{{\rm{post}}}}}}$$ (4) Where _I_pre is the fluorescence intensity of the FRET donor before photobleaching and _I_post is postbleaching.

WETTING ASSAY Wetting describes the ability of a liquid to spread over a solid surface. Liquid droplets fuse and generate larger puncta (protein rafts) at the bottom whereas hydrogels and

protein aggregates do not wet or change shape78. LLPS samples of 100 μM α-Syn, 100 μM β-Syn, or 100 μM α/β-Syn were prepared as described above, then immediately transferred to confocal dish

for confocal imaging. Samples were imaged after 5 min, 30 min, 1 h, 3 h and 19 h using a Nikon AX with a 100X oil immersion objective. FAR-UV CIRCULAR DICHROISM (CD) Far-UV CD was performed

to study the solution secondary structure17,79,80. Sample concentration was 10 μM and the CD spectra were obtained from 260 nm to 200 nm range with a 1 nm bandwidth, 100 nm/min scanning

speed. Measurements were analyzed using a JASCO-810 circular dichroism spectropolarimeter (JASCO, Japan) under continuous N2 flow. All spectra were blank subtracted. The data obtained were

converted into the mean residue ellipticity [θ]. All experiments were repeated at least three times. TURBIDITY ASSAY The phase-separated samples were immediately transferred to a transparent

96-well plate and the absorbance was measured at 405 nm81. Turbidity measurements were conducted using a Tecan microplate reader (NSW, Australia). All experiments were repeated at least

three times. SEDIMENTATION ASSAY Phase-separated proteins were quantified using the sedimentation assay78. Briefly, a 100 μL mixed solution was prepared for phase separation, the mixture was

centrifuged at 16,200 × _g_ for 10 min to separate the supernatant and precipitate; the pellet fraction was re-suspended with the same buffer to the equal volume as supernatant fraction.

SDS loading buffer was added to the precipitate and supernatant, boiled for 15 min, then resolved by 13.5% SDS-PAGE and stained with Coomassie Brilliant Blue. The protein loading amount was

5 μg for each group. Determine the proportion of total protein in the phase-separated droplets was quantified by Image J. TRANSMISSION ELECTRON MICROSCOPY (TEM) Samples for TEM were prepared

as follows82,83. Briefly, 5 μL samples were deposited on a carbon film of 400 mesh copper grid (Zhongjingkeyi Tech., Beijing, China) for 3 min. The grids were washed once with ddH2O,

stained with 1% (w/v) uranyl acetate for 90 s, washed twice with ddH2O then air-dried. Samples were imaged with a H-8100 transmission electron microscope operated at 120 kV (Hitachi, Tokyo,

Japan). IMMUNO-DOT-BLOT ASSAYS Dot blot assay was performed as follows84, 2 μL samples were spotted onto a nitrocellulose filter membrane (Bio-Rad, Hercules, CA), air-dried. 5% non-fat milk

was used to block the membrane for 1 h at room temperature and then incubated with OC antibody (fibril-specific antibody; 1:2000) or A11 antibody (oligomer-specific antibody; 1:2000) at 4 °C

overnight, followed by incubating with corresponding secondary antibody (1:2000) for 3 h at room temperature. An ECL chemiluminescence kit (Advansta, San Jose, CA) was used for blot

development. DYNAMIC LIGHT SCATTERING (DLS) ANALYSIS DLS was performed using a zeta pals potential analyzer (Brookhaven Instruments, Holtsville, NY)18,85. Briefly, 100 μM α-Syn, β-Syn, or

α/β-Syn samples in the absence of 20% PEG were measured with a scattering angle of 90°. A sample volume of 300 μL was used for all the reactions. Each sample was scanned three times (30 s

per scan) and the mean particle size was recorded. MICRO-RAMAN SPECTROSCOPY Micro-Raman spectra were measured using a micro-Raman spectrometer (LabRAM HR800, Horiba, Japan). For

single-droplet Raman measurement, 5 μL samples were placed on a glass slide covered with a coverslip and droplets were focused using a ×50 long working distance objective lens. An NIR laser

(532 nm) with an exposure time of 120 s and 600 mW (100%) laser power was used to excite the samples86. All experiments were repeated at least three times. The collected Raman spectra were

baseline corrected and plotted using Origin 2021. RATIONALLY DESIGNED Β-SYN DERIVED PEPTIDES INHIBITS THE PHASE TRANSITION OF Α-SYN Using Alphafold 2 Colab and alanine scanning

(https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb), we simulated the interaction between α-Syn and β-Syn to identify the hot spot residues. Next, we

designed and screened a series of peptides derived from the hot spot regions. Binding affinities were analyzed using PDBePISA (https:/www.ebi.ac.uk/msd-srv/prot_int/pistart.html), and

visualization was carried out using PyMOL 2.6.087. By comparing predicted binding energies (ΔiG) of β-Syn-derived modified peptides with α-Syn, we designed peptides with the potential to

inhibit α-Syn aggregation. All peptides were chemically synthesized by Synpeptide Inc. (Nanjing, China). THIOFLAVIN T (THT) EXPERIMENT ThT kinetic experiments were performed as

follows17,83,88. Briefly, the protein was dissolved in a 50 mM PBS buffer containing 100 mM NaCl at pH 7.4 to a final concentration of 100 μM. For antagonistic peptides assay, peptides were

dissolved in the same buffer equimolar to α-Syn. Subsequently, the protein solution was centrifuged at 15,000 × _g_ for 30 min to remove any insoluble aggregates. Following this, 500 μL of

indicated protein solution was incubated on a rotating shaker at 37 °C and 220 rpm for a total of 96 h. 10 μL aliquots were mixed with ThT (final concentration of 20 μM) and detected every

12 h using an FL-2700 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The excitation wavelength was set at 450 nm, while the emission wavelength was set at 482 nm. For recording ThT

fluorescence spectrum, LLPS samples of 300 μL (containing 20 μM ThT) were prepared as described above at 37 °C26. The samples were excited at 450 nm, and emission spectra were recorded from

465 to 600 nm, with excitation and emission slits set to 5 nm. The ThT fluorescence assay was conducted using a Hitachi FL-2700 fluorometer. Each experiment was performed in triplicate to

ensure reproducibility. THRASHING ASSAY Synchronized _C_. _elegans_ were cultured on NGM plates at 20 °C until they reached the young adult stage. To assess thrashing behaviors, worms were

placed in 20 μL of M9 buffer and allowed to recover for 30 s before counting the number of body bends within 30 s38. For peptide treatments, the synchronized worms at L4 stage were fed the

indicated peptides (stock solutions were mixed with _E. coli_ OP50 to a final concentration of 100 μM and spread on NGM plates) for another three days. The thrashing assay was repeated 3

times and 10 worms were used for each condition. Videos on worm movements were taken by a stereo microscope (Mshot MZ62, Guangzhou, China) equipped with a camera (Sony Exmor CMOS, Japan).

LIFESPAN ASSAY All strains were cultured on NGM plates for 2 generations without starvation at 20 °C. For the assay, 20 synchronized L4 stage worms were cultivated on NGM plates containing

fresh OP50. Worms were transferred to new plates every other day. Worms that do not respond to mechanical stimulation from a platinum wire were recorded as dead89. N2 strain was used as a

control. The lifespan assay was repeated 3 times and 20 worms were used each time for each condition. STATISTICS AND REPRODUCIBILITY The data are presented as means ± SD. Statistical

analyses were performed using GraphPad Prism 8 unless indicated otherwise. The statistical significance was calculated using one-way analysis of variance (ANOVA) followed by a Dunnett’s

multiple comparisons test with a 95% confidence interval. At least three independent experiments were performed with similar results. Differences were considered statistically significant at

_P_ < 0.05. REPORTING SUMMARY Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. DATA AVAILABILITY All data are

available in the main text or the Supplementary information. The net charge per residue (NCPR) diagram are available in the Classification of Intrinsically Disordered Ensemble Regions

(CIDER) (https://pappulab.wustl.edu/CIDER/analysis/). The residue-based droplet-promoting probabilities (PDP) results are available in FuzDrop (https://fuzdrop.bio.unipd.it/predictor). The

analysis results of AlphaFold 2 predictions generated in this study are deposited in the GitHub repository (https://github.com/HK-Lab-YLW/HK_natcomms_24-28818_data-AF2-data)90. Source data

are provided with this paper. CODE AVAILABILITY AlphaFold 2 Complex is open source and available at Github (https://github.com/HK-Lab-YLW/HK_natcomms_24-28818_data-AF2-data)90. REFERENCES *

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references ACKNOWLEDGEMENTS This work is supported by the National Key R&D Program of China (2023YFC2507900 to K.H., 2023YFC2307000 to H.C., and 2022YFA0806100 to K.H.), the Natural

Science Foundation of China (82273838 and 31971066 to K.H., 32371171 to H.C.), and the Natural Science Foundation of Hubei Province (2021CFA004 to K.H.). The work was technically supported

by the Analytical and Testing Center of Huazhong University of Science and Technology and the Medical Subcenter. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * School of Pharmacy, Tongji

Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology, Wuhan, China Xi Li, Linwei Yu, 

Tianyi Shi, Yu Zhang, Yushuo Xiao, Chen Wang, Liangliang Song, Ning Li, Xinran Liu, Yuchen Chen, Li Xu, Hong Chen & Kun Huang * Hubei Key Laboratory of Cell Homeostasis, Frontier Science

Center for Immunology and Metabolism, College of Life Sciences, Wuhan University, Wuhan, China Xikai Liu & Ling Zheng * Foundational Sciences, Central Michigan University College of

Medicine, Mt. Pleasant, MI, USA Robert B. Petersen * Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Xiang Cheng

* Department of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China Weikang Xue & Yanxun V. Yu * Tongji-Rong Cheng Biomedical

Center, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Kun Huang Authors * Xi Li View author publications You can also search for this author inPubMed 

Google Scholar * Linwei Yu View author publications You can also search for this author inPubMed Google Scholar * Xikai Liu View author publications You can also search for this author

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author inPubMed Google Scholar * Yushuo Xiao View author publications You can also search for this author inPubMed Google Scholar * Chen Wang View author publications You can also search for

this author inPubMed Google Scholar * Liangliang Song View author publications You can also search for this author inPubMed Google Scholar * Ning Li View author publications You can also

search for this author inPubMed Google Scholar * Xinran Liu View author publications You can also search for this author inPubMed Google Scholar * Yuchen Chen View author publications You

can also search for this author inPubMed Google Scholar * Robert B. Petersen View author publications You can also search for this author inPubMed Google Scholar * Xiang Cheng View author

publications You can also search for this author inPubMed Google Scholar * Weikang Xue View author publications You can also search for this author inPubMed Google Scholar * Yanxun V. Yu

View author publications You can also search for this author inPubMed Google Scholar * Li Xu View author publications You can also search for this author inPubMed Google Scholar * Ling Zheng

View author publications You can also search for this author inPubMed Google Scholar * Hong Chen View author publications You can also search for this author inPubMed Google Scholar * Kun

Huang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS K.H., L.Z., and X.L. conceptualized the study. X.L., L.W.Y., X.K.L. performed the

research and analyzed the data with image analyses tools and developed the methodology. Y.S.X., C.W., L.L.S., X.C., N.L. performed in silico calculations and analyzed the data. W.K.X.

performed the _C. elegans_ microinjection. X.L., L.W.Y., Y.C.C., T.Y.S., Y.Z., L.X., X.R.L. assisted in visualization of data generated. K.H., H.C. supervised all aspects of the study. The

original manuscript draft was written by X.L., with review and editing by K.H., R.B.P, Y.X.Y. CORRESPONDING AUTHORS Correspondence to Hong Chen or Kun Huang. ETHICS DECLARATIONS COMPETING

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ARTICLE CITE THIS ARTICLE Li, X., Yu, L., Liu, X. _et al._ β-synuclein regulates the phase transitions and amyloid conversion of α-synuclein. _Nat Commun_ 15, 8748 (2024).

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