A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils.
PublisherPublic Library of Science
MetadataShow full item record
AbstractAlpha-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.
Tobacco etch virus
Green Fluorescent Proteins
Identifier to cite or link to this itemhttp://hdl.handle.net/10713/14734
- Selective imaging of internalized proteopathic α-synuclein seeds in primary neurons reveals mechanistic insight into transmission of synucleinopathies.
- Authors: Karpowicz RJ Jr, Haney CM, Mihaila TS, Sandler RM, Petersson EJ, Lee VM
- Issue date: 2017 Aug 11
- Trehalose does not improve neuronal survival on exposure to alpha-synuclein pre-formed fibrils.
- Authors: Redmann M, Wani WY, Volpicelli-Daley L, Darley-Usmar V, Zhang J
- Issue date: 2017 Apr
- α-Synuclein (αSyn) Preformed Fibrils Induce Endogenous αSyn Aggregation, Compromise Synaptic Activity and Enhance Synapse Loss in Cultured Excitatory Hippocampal Neurons.
- Authors: Wu Q, Takano H, Riddle DM, Trojanowski JQ, Coulter DA, Lee VM
- Issue date: 2019 Jun 26
- Synthetic alpha-synuclein fibrils cause mitochondrial impairment and selective dopamine neurodegeneration in part via iNOS-mediated nitric oxide production.
- Authors: Tapias V, Hu X, Luk KC, Sanders LH, Lee VM, Greenamyre JT
- Issue date: 2017 Aug
- Following the fate of endocytosed fibrils.
- Authors: Hasegawa M, Suzuki G
- Issue date: 2017 Aug 11