Allerton, S. C. | Search bioRxiv

bioRxiv2024

Molecular rotors provide insight into the mechanism of formation and conversion of α-synuclein aggregates

-Synuclein is an intrinsically disordered protein that forms amyloids in Parkinsons disease. Currently, detection methods predominantly report on the formation of mature amyloids but have poor sensitivity to the early-stage, toxic oligomers. Molecular rotors are fluorophores that sense changes in the viscosity of their local environment. Here, we monitor -synuclein oligomer formation using the fluorescence lifetime of molecular rotors. We detect oligomer formation and conversion into amyloids for wild type and two -synuclein variants; the pathological mutant A30P and {Delta}P -synuclein, which lacks a master regulator region of aggregation (residues 36-42). We report that A30P -synuclein shows a similar rate of oligomer formation compared to wild type -synuclein, whereas {Delta}P -synuclein shows delayed oligomer formation. Additionally, both variants demonstrate a slower conversion of oligomers to amyloids. Our method provides a quantitative approach to unveiling the complex mechanism of -synuclein aggregation which is key to understanding the pathology of Parkinsons disease.

biochemistry↗

bioRxiv2024

The N-terminal Region of alpha-Synuclein Controls Amyloid Nucleation at the Condensate Interface

-Synuclein self-assembles into amyloid fibrils during neurodegeneration. The protein can also self-assemble via liquid-liquid phase separation to form biomolecular condensates. The link between these processes is evident, as -synuclein condensates can mature into amyloids. However, the mechanisms driving this maturation remain largely unknown, particularly when incorporating pathological post-translational modifications known to affect -synuclein self-assembly in the absence of LLPS, such as N-terminal truncation. Moreover, condensates are primarily studied as isolated entities; however, it is increasingly evident that they interact with various cellular components and surfaces. Here, we developed a microscopy-based quantitative real-time imaging protocol to investigate how N-terminal truncation influences -synuclein condensate formation, well surface wetting, and maturation. We found that increasing -synuclein truncation, which reduces N-terminal hydrophobicity, inhibits condensate sedimentation, enhances surface wettability, and accelerates maturation. Additionally, by increasing well surface hydrophobicity we decreased -synuclein condensate wettability, delaying their maturation. Thus, we propose that enhanced wettability, which increases the condensate surface-to-volume ratio, promotes -synuclein nucleation at the condensate-bulk solution interface, thereby accelerating maturation. Our results reveal distinct mechanistic roles for -synuclein N-terminal residues and indicate that condensate wetting on cellular surfaces, such as synaptic vesicles, may drive toxic aggregate formation during neurodegeneration.

biochemistry↗