A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils

• Published in: PloS one 2021-01, Vol.16 (1), p.e0241161
• Main Authors: Jarvela, Timothy S, Chaplot, Kriti, Lindberg, Iris
• Format: Article
• Language: English
• Subjects: alpha-Synuclein - genetics; alpha-Synuclein - metabolism; Anatomy; Animals; Biological Assay; Biology and Life Sciences; Cell culture; Cell Line; Chromatography; Development and progression; Endopeptidases - genetics; Endopeptidases - metabolism; Engineering and Technology; Ethylenediaminetetraacetic acids; Experiments; Fibrils; Glycerol; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Health aspects; Identification and classification; Internalization; Medical treatment; Methods; Mice; Movement disorders; Nerve proteins; Nervous system; Neurobiology; Neurodegenerative diseases; Neurons - metabolism; Neurosciences; Osmotic shock; Parkinson's disease; Physical Sciences; Physiological aspects; Plasmids; Protease; Proteases; Protein Aggregation, Pathological - genetics; Protein Aggregation, Pathological - metabolism; Protein Aggregation, Pathological - pathology; Proteinase; Proteins; Proteomics; Research and Analysis Methods; Room temperature; Seeds; Sucrose; Sugar; Synuclein; Toolkits
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0241161

Alpha-synuclein pre-formed fibrils (PFFs) represent a promising model system for the study of cellular processes underlying cell-to-cell transmission of alpha-synuclein proteopathic aggregates. However, the ability to differentiate the fate of internalized PFFs from those which remain in the extracellular environment remains limited due to the propensity for PFFs to adhere to the cell surface. Removal of PFFs requires repeated washing and/or specific quenching of extracellular fluorescent PFF signals. In this paper we present a new method for analyzing the fate of internalized alpha-synuclein. We inserted a tobacco etch virus (TEV) protease cleavage site between alpha-synuclein and green fluorescent protein and subjected cells to brief treatment with TEV protease after incubation with tagged PFFs. As the TEV protease is highly specific, non-toxic, and active under physiological conditions, protection from TEV cleavage can be used to distinguish internalized PFFs from those which remain attached to the cell surface. Using this experimental paradigm, downstream intracellular events can be analyzed via live or fixed cell microscopy as well as by Western blotting. We suggest that this method will be useful for understanding the fate of PFFs after endocytosis under various experimental manipulations.