Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia

PLoS One. 2013;8(2):e55940. doi: 10.1371/journal.pone.0055940. Epub 2013 Feb 13.

Abstract

Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Drug Evaluation, Preclinical / methods*
  • Frataxin
  • Friedreich Ataxia / drug therapy*
  • Friedreich Ataxia / genetics
  • Gene Library
  • Genes, Reporter
  • Genomics
  • HeLa Cells
  • Humans
  • Iron-Binding Proteins / genetics*
  • Trinucleotide Repeat Expansion
  • Up-Regulation

Substances

  • Iron-Binding Proteins

Grants and funding

This work was supported by the Muscular Dystrophy Association (USA) (grants 3657, 3725, 69033), the National Health and Medical Research Council (Australia) (grant 491234), the Friedreich’s Ataxia Research Alliance (USA), the Brockhoff Foundation (Australia), the Friedreich Ataxia Research Association (Australasia), Seek A Miracle (USA) and the Victorian Government’s Operational Infrastructure Support Program. MBD is a National Health and Medical Research Council Practitioner Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.