A human single-chain Fv intrabody blocks aberrant cellular effects of overexpressed alpha-synuclein.
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.
Zhou
Chun
C
Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
Emadi
Sharareh
S
Sierks
Michael R
MR
Messer
Anne
A
D000818:Animals; D000918:Antibody Specificity; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002522:Chlorocebus aethiops; D003902:Detergents / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D012995:Solubility / Q000187:drug effects; D051843:Synucleins; D051844:alpha-Synuclein
D003902:Detergents; D007128:Immunoglobulin Fragments; D009419:Nerve Tissue Proteins; C497604:SNCA protein, human; D051843:Synucleins; D051844:alpha-Synuclein