Spontaneous nucleation and fast aggregate-dependent proliferation of α-synuclein aggregates within liquid condensates at neutral pH

Abstract

The aggregation of α-synuclein into amyloid fibrils has been under scrutiny in recent years because of its association with Parkinson’s disease. This process can be triggered by a lipid-dependent nucleation process, and the resulting aggregates can proliferate through secondary nucleation under acidic pH conditions. It has also been recently reported that the aggregation of α-synuclein may follow an alternative pathway, which takes place within dense liquid condensates formed through phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we used fluorescence-based assays to enable a kinetic analysis of the microscopic steps underlying the aggregation process of α-synuclein within liquid condensates. Our analysis shows that at pH 7.4, this process starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Our results thus reveal the microscopic mechanism of α-synuclein aggregation within condensates through the accurate quantification of the kinetic rate constants for the appearance and proliferation of α-synuclein aggregates at physiological pH.

A scale-invariant log-normal droplet size distribution below the critical concentration for protein phase separation

“…Human wild-type α-synuclein and A90C cysteine variants were purified from Escherichia coli BL21 (DE3)-gold (Agilent Technologies) expressing plasmid pT7-7 encoding for α-synuclein as previously described 35,42,43 . Following purification in 50 mM trisaminomethane-hydrochloride (Tris-HCL) at pH 7.4, α-synuclein was concentrated using Amicon Ultra-15 centrifugal filter units (Merck Millipore).…”

Macromolecular Crowding Promotes Re-entrant Liquid–Liquid Phase Separation of Human Serum Transferrin and Prevents Surface-Induced Fibrillation

“…Recently, Vendruscolo and co-workers studied the aggregation kinetics of α-synuclein within liquid condensates and showed that α-synuclein can undergo spontaneous homogeneous primary nucleation and fast aggregate-dependent proliferation within condensates at physiological pH. 43 Similarly, using a family of chimeric peptides (ACC 1−13 K n ), Winter and co-workers demonstrated that the early fibrils originate at the droplet/bulk interface within single liquid droplets in the ATP-triggered amyloidogenic pathway. 38 While the interior of these biomolecular condensates is highly dynamic and heterogeneous in nature, their solid-like fibrillar aggregates have relatively defined and homogeneous structural composition.…”

“…Over the past decade, considerable efforts have been made to understand the fundamental mechanism behind the LLPS of various proteins, particularly intrinsically disordered proteins/regions (IDPs/IDRs) containing low-complexity domains/regions (LCDs/LCRs) in their polypeptide sequence. − LLPS of biomolecules is a spontaneous and thermodynamically favorable liquid–liquid demixing phenomenon where the enthalpy change associated with the multivalent attractive intermolecular interactions and the increase in entropy associated with the release of water molecules from the surfaces of biomolecules compensate the entropy loss due to the association of biomolecules. Due to these enthalpic and entropic contributions, the LLPS of biomolecules is highly sensitive on various internal and external factors including composition, concentration, temperature, pressure, pH, ionic strength, and crowding. , The phase-separated biomolecule-rich phase contains highly dynamic liquid-like membraneless condensates which are stabilized by various multivalent intermolecular forces including electrostatic, hydrophobic, hydrogen bonding, dipole–dipole, π–π, and/or cation−π interactions. ,, Recent studies have demonstrated that these membraneless condensates act as dynamic intermediates during the formation of amyloid aggregates of various disease-associated proteins or polypeptides. − ,,,− Earlier, Maji and co-workers quantified the conformational heterogeneity during liquid-to-solid phase transition of α-synuclein and showed that the interior of phase-separated droplets is heterogeneous with the presence of monomers, oligomers, and fibrils. , More importantly, it has been observed that the phase-separated droplets act as nucleation scaffolds for protein aggregation and liquid-to-solid phase transition. Recently, Vendruscolo and co-workers studied the aggregation kinetics of α-synuclein within liquid condensates and showed that α-synuclein can undergo spontaneous homogeneous primary nucleation and fast aggregate-dependent proliferation within condensates at physiological pH .…”

“…Due to these enthalpic and entropic contributions, the LLPS of biomolecules is highly sensitive on various internal and external factors including composition, concentration, temperature, pressure, pH, ionic strength, and crowding. , The phase-separated biomolecule-rich phase contains highly dynamic liquid-like membraneless condensates which are stabilized by various multivalent intermolecular forces including electrostatic, hydrophobic, hydrogen bonding, dipole–dipole, π–π, and/or cation−π interactions. ,, Recent studies have demonstrated that these membraneless condensates act as dynamic intermediates during the formation of amyloid aggregates of various disease-associated proteins or polypeptides. − ,,,− Earlier, Maji and co-workers quantified the conformational heterogeneity during liquid-to-solid phase transition of α-synuclein and showed that the interior of phase-separated droplets is heterogeneous with the presence of monomers, oligomers, and fibrils. , More importantly, it has been observed that the phase-separated droplets act as nucleation scaffolds for protein aggregation and liquid-to-solid phase transition. Recently, Vendruscolo and co-workers studied the aggregation kinetics of α-synuclein within liquid condensates and showed that α-synuclein can undergo spontaneous homogeneous primary nucleation and fast aggregate-dependent proliferation within condensates at physiological pH . Similarly, using a family of chimeric peptides (ACC 1–13 K n ), Winter and co-workers demonstrated that the early fibrils originate at the droplet/bulk interface within single liquid droplets in the ATP-triggered amyloidogenic pathway .…”

Secondary Processes Dominate the Quiescent, Spontaneous Aggregation of α-Synuclein at Physiological pH with Sodium Salts

“…αS aggregation can be promoted by lipid membranes − or by shaking, which introduces air–water interfaces, , and is used in diagnostic assays based on biosamples . The spontaneous aggregation of αS has been recently reported within liquid condensates formed by liquid–liquid phase separation. , In these condensates, the concentration of αS reaches millimolar levels, thereby dramatically enhancing the speed of the aggregation process. However, the quest remains open for assays under conditions where spontaneous αS aggregation can be observed in the absence of condensation.…”