Effect of freeze-thaw cycle induced oligomers on alpha-synuclein liquid-liquid phase separation and aggregation.

Protein aggregation in the form of amyloid fibrils is linked with the onset and progression of several widespread and incurable disorders, including neurodegenerative Alzheimer's and Parkinson's diseases. Despite decades of research and countless attempts to discover potential aggregation-altering treatment modalities, there are still no effective cures for the majority of amyloid-related disorders. This is, in part, due to our incomplete understanding of the underlying factors that influence the phase separation and aggregation of these various disease-related proteins. Over the last few years, it has been shown that even factors such as storage conditions or sample preincubation times can affect the formation kinetics of amyloid fibrils. In the case of alpha-synuclein, even the protein purification procedure yielded different concentrations of oligomeric species, which altered the aggregation mechanism. In this work, we further explore the role of alpha-synuclein oligomers by inducing their formation via freeze-thaw cycles and analyzing their effect on liquid-liquid and liquid-solid phase transitions. Using real-time kinetic monitoring, brightfield microscopy, Fourier-transform infrared spectroscopy and atomic force microscopy, we show that the presence of freeze-thaw cycle induced alpha-synuclein oligomers has significant effects on its condensate assembly and subsequent aggregation, as well as the final structure of the formed aggregates.