Bifunctional Anti-Non-Amyloid Component α-Synuclein Nanobodies Are Protective In Situ.
Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn) are closely associated with synucleinopathies, including Parkinson's disease (PD). VH14 is a human single domain intrabody selected against the non-amyloid component (NAC) hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts. We compared this anti-NAC candidate to NbSyn87, which binds to the C-terminus of α-Syn. NbSyn87PEST degrades α-Syn as well or better than VH14PEST. However, while both candidates reduced toxicity, VH14PEST appears more effective in both proteostatic stress and toxicity assays. These results show that the approach of reducing intracellular monomeric targets with novel antibody engineering technology should allow in vivo modulation of proteostatic pathologies.
Neural Stem Cell Institute, Rensselaer, NY, 12144, United States of America; and Department of Biomedical Sciences; University at Albany, Albany, NY, 12208, United States of America.|Wadsworth Center, New York State Department of Health, Albany, NY, 12208, United States of America.
David C
DC
Butler
Wadsworth Center, New York State Department of Health, Albany, NY, 12208, United States of America.
Shubhada N
SN
Joshi
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.
Erwin De
E
Genst
Wadsworth Center, New York State Department of Health, Albany, NY, 12208, United States of America.
Ankit S
AS
Baghel
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.
Christopher M
CM
Dobson
Neural Stem Cell Institute, Rensselaer, NY, 12144, United States of America; and Department of Biomedical Sciences; University at Albany, Albany, NY, 12208, United States of America.|Wadsworth Center, New York State Department of Health, Albany, NY, 12208, United States of America.
Anne
A
Messer
D058227:Amyloidogenic Proteins / Q000276:immunology*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D009474:Neurons / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D051381:Rats; D061905:Single-Domain Antibodies; D013234:Stem Cells; D014162:Transfection; D051844:alpha-Synuclein / Q000737:chemistry* / Q000276:immunology / Q000502:physiology
D058227:Amyloidogenic Proteins; D061905:Single-Domain Antibodies; D051844:alpha-Synuclein; D046988:Proteasome Endopeptidase Complex