Comparison of Various Fragmentation Methods of Alpha-Synuclein Fibrils for Establishing a Parkinson's Disease-Like Model in Cell Culture

Abstract

Objectives Alpha-synuclein (alpha-syn) fibrils, which are associated with Lewy-like inclusions, are currently utilized in the modeling of Parkinson's disease (PD). The fibrils require a length shorter than 100 nm to uptake intracellularly. Our study aims to determine the optimal probe sonication parameters that can be applied in diverse laboratory settings for the fragmentation of fibrils to lengths of <= 100 nm.Methods In our study, human wild-type alpha-syn fibrils were sonicated with 1, 3, and 5 s-on/off pulse applications at varying ambient temperatures (room temperature, ice, and ice-surrounded by dry ice) for a duration of 1 min, with a total of 40 and 60 cycles, respectively. The length of the fibrils was quantified by transmission electron microscopy imaging. The total alpha-syn in SH-SY5Y cells was detected by immunofluorescence staining following the administration of fragmented fibrils after 2-, 4-, and 6-day incubations.Results Although fragmentation was observed under all conditions, fibrils of <= 100 nm were obtained with a 5 s-on/off pulse in ice and ice-surrounded by dry ice ambientes. The fibril length of <= 100 nm was observed on days 2 and 4; however, the highest level of observed intensity was detected on day 6.Conclusions The application of 5 s-on/off consecutive pulses in ice, and ice-surrounded by dry ice ambientes are more effective methods for fragmenting and internalizing fibrils, thereby forming a reproducible cellular PD-like model. In summary, our study provides a significant contribution to the development of an economical and effective sonication methodology for the fragmentation of fibrils in a diverse of laboratory settings.