Effects of fibril length on α-synuclein cellular uptake efficiency and pathway

Examensarbete för masterexamen
Master Thesis
Biotechnology (MPBIO), MSc
Zhang, Xiaolu
Protein aggregation and amyloid formation are central to the pathology of several neurodegenerative disorders including Parkinson’s disease (PD). The major pathological hallmark of Parkinson’s disease is the appearance of cytosolic e.g. intracellular inclusions, socalled Lewy bodies, consisting of fibrillar forms of the protein α-synuclein. It has been hypothesized that α-synuclein has several different kinds of inter-related toxic consequences, including cell to cell transmission of protein aggragation, but it is still unclear exactly how αsynuclein affects primarily dopaminergic neurons in the PD brain. The detailed mechanisms of how α-synuclein leads to the nerve cells degeneration therefore remain to be found. This thesis detail with how α-synuclein amyloid fibril damage nerve cells and specifically explores how αsynuclein fibrils of different lengths are taken up by cells. This is important for the understanding of α-synuclein toxicity in the general and for the propensity of α-synuclein fibrils to spread between cells in particular. This study explors the mechanism of human neuroblastoma cells SH-SY5Y uptake of αsynuclein fragments with different lengths by confocal fluorescence laser scanning microscopy and quantification of the cell uptake efficiency by flow cytometry. Partially fluorescently labelled α-synuclein fibrils were prepared by co-incubation of wildtype and A90C-mutant αsynuclein labelled with Hilyte Fluor 488 dye via maleimide coupling. Fragments of different lengths were prepared by stirring or probe sonication of the pre-formed fibrils. Atomic force microscopy and fluorescence spectroscopy were used to characterize the α-synuclein fibril fragments. By comparing uptake in cells incubated with fibrils fragments at 4 ℃ VS 37 ℃ I show that SH-SY5Y cells take up α-synuclein fibrils via endocytosis pathways and that shorter fibrils can be taken more readily compared to longer fibrils. Α-synuclein monomers cell uptake is comparing very inefficient. Confocal fluorescence laser scanning microscopy images show that the shorter fibrils accumulated more vesicles around cell nuclei compared to longer fibrils. This project reveal that α-synuclein fibrils cell upake efficiency is related with fibrils’ lengths. This study shows that it is a promising start for future studies on uptake, cell to cell transmission and toxicity of α-synuclein fibrils with different lengths.
Cellbiologi , Cell- och molekylärbiologi , Medicinsk bioteknologi (med inriktning mot cellbiologi) , Livsvetenskaper , Cell Biology , Cell and Molecular Biology , Medical Biotechnology (with a focus on Cell Biology) , Life Science
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