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Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains
Published on: January 5, 2016
A human single-chain Fv intrabody blocks aberrant cellular effects of overexpressed alpha-synuclein
Chun Zhou1, Sharareh Emadi, Michael R Sierks
1Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
Abstract:
alpha-Synuclein (alpha-syn) has been identified as the major component of Lewy bodies that characterize neurodegenerative synucleinopathies, including Parkinson's disease. Overexpression of alpha-syn, and prefibrillar alpha-syn oligomers, has been implicated in these pathologies; therefore, prevention of prefibril accumulation, and inhibition of other aberrant effects of overexpressed alpha-syn, could provide novel treatments. Here, we have selected a human single-chan Fv (scFv) antibody, D10, that binds human monomeric wild-type alpha-syn. We demonstrate, by retargeting assays and coimmunoprecipitation, that the D10 scFv is a specific and efficient intracellular antibody (intrabody). By transfecting the D10 scFv gene into an HEK 293 cell line that overexpresses wild-type alpha-syn, we show that the D10 intrabody stabilizes detergent-soluble monomeric alpha-syn and inhibits the formation of detergent-insoluble high-molecular-weight alpha-syn species. In addition, the D10 intrabody ameliorates the decreased cell adhesion that characterizes the alpha-syn-overexpressing cells. Given the important role of alpha-syn pathology, and the facility with which intrabodies can be further engineered in vitro, anti-alpha-syn intrabodies may represent novel molecular therapeutics for synucleinopathies, with implications for other neurodegenerative disorders caused by misfolded accumulated proteins.
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