Abstract
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.
Highlights
Cell-to-cell transmission of proteopathic aggregates is increasingly understood to represent the underlying cause of the stereotypical spread of α-synuclein pathology observed in Parkinson’s disease [1]
A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils was removed and replaced with 50 μl of fresh Neuro2A medium
A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils control) were added to each well, and a single 3/32inch polytetrafluoroethylene bead (McMaster-Carr, Atlanta, GA) was added to each well and the plate sealed with foil
Summary
Cell-to-cell transmission of proteopathic aggregates is increasingly understood to represent the underlying cause of the stereotypical spread of α-synuclein pathology observed in Parkinson’s disease [1]. A variety of model systems have been developed to replicate this process in vivo [2,3,4,5,6,7] as well as in primary and immortalized cell culture [8,9,10]. Taken together, these experimental models have identified specific cellular components responsible for the internalization and transmission of α-synuclein. It has been difficult to develop experimental methods that permit the biochemical discrimination of intracellular PFFs from those remaining extracellularly, for example PFFs coating the cell surface.
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