Silencing of human alpha-synuclein in vitro and in rat brain using lentiviral-mediated RNAi

Exp Neurol. 2006 Apr;198(2):382-90. doi: 10.1016/j.expneurol.2005.12.024. Epub 2006 Feb 7.


Human alpha-synuclein overexpression and its toxic accumulation in neurons or glia are known to play key roles in the pathogenesis of Parkinson's disease and other related neurodegenerative synucleinopathies. Several single point mutations in the alpha-synuclein gene, as well as gene duplication and triplication, have been linked to familial Parkinson's disease. Moreover, genetic variability of the alpha-synuclein gene promoter is associated with idiopathic Parkinson's disease. Silencing of the human alpha-synuclein gene by vector-based RNA interference (RNAi) is a promising therapeutic approach for synucleinopathies. Here, we report identification of a 21-nucleotide sequence in the coding region of human alpha-synuclein that constitutes an effective target for robust silencing by RNAi and demonstrate allele-specific silencing of the A53T mutant of human alpha-synuclein. Furthermore, we have developed a plasmid vector-based RNAi for silencing of human alpha-synuclein in vitro. Lastly, using a dual cassette lentivirus that co-expresses an alpha-synuclein-targeting small hairpin RNA (shRNA) and enhanced green fluorescent protein (EGFP) as a marker gene, we demonstrate effective silencing of endogenous human alpha-synuclein in vitro in the human dopaminergic cell line SH-SY5Y and also of experimentally expressed human alpha-synuclein in vivo in rat brain. Our results demonstrate potent silencing of human alpha-synuclein expression in vitro and in vivo by viral vector-based RNAi and provide the tools for developing effective gene silencing therapeutics for synucleinopathies, including Parkinson's disease.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • Brain / drug effects
  • Brain / metabolism*
  • Brain / virology
  • Dose-Response Relationship, Drug
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / physiology*
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Lentivirus / physiology*
  • Male
  • Mutant Proteins / physiology
  • Phosphopyruvate Hydratase / metabolism
  • RNA Interference / drug effects
  • RNA Interference / physiology*
  • RNA, Small Interfering / pharmacology
  • Rats
  • Rats, Wistar
  • Transfection / methods
  • alpha-Synuclein / genetics
  • alpha-Synuclein / metabolism*


  • Mutant Proteins
  • RNA, Small Interfering
  • alpha-Synuclein
  • Green Fluorescent Proteins
  • Phosphopyruvate Hydratase