Primary and Secondary Drug Screening Assays for Friedreich Ataxia

Abstract

Friedreich ataxia (FRDA) is an autosomal recessive neuro-and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix for the assembly of iron-sulfur clusters (ISCs), which are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain (ETC). Decreased expression of frataxin or the yeast frataxin orthologue, Yfh1p, is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. Using yeast depleted of Yfh1p, a high-throughput screening (HTS) assay was developed in which mitochondrial function was monitored by reduction of the tetrazolium dye WST-1 in a growth medium with a respiration-only carbon source. Of 101 200 compounds screened, 302 were identified that effectively rescue mitochondrial function. To confirm activities in mammalian cells and begin understanding mechanisms of action, secondary screening assays were developed using murine C2C12 cells and yeast mutants lacking specific complexes of the ETC, respectively. The compounds identified in this study have potential relevance for other neurodegenerative disorders associated with mitochondrial dysfunction, such as Parkinson disease.

Ferroptosis as a Novel Therapeutic Target for Friedreich’s Ataxia

“…1) at concentrations ranging from 50 mM to 5 nM in the murine I154F FRDA fibroblasts. These cells are sensitive to combined treatment with iron, in the soluble form of FAC, and BSO (which inhibits glutathione synthesis) at concentrations that by themselves are nontoxic but together cause a synergistic loss of viability (Cotticelli et al, 2012). Under the assay conditions, 75% of the cells died 48 hours following treatment ( Fig.…”

“…Patient-derived primary fibroblasts, although a clinically unaffected cell type, are sensitive to treatment with iron (Wong et al, 2000) and BSO (Jauslin et al, 2002), which inhibits glutathione synthesis. Based on these data, we developed a cellular model of FRDA in which primary FRDA fibroblasts were treated with a synergistic combination of FAC and BSO, which we have used successfully to screen for potential therapeutics for FRDA (Cotticelli et al, 2012). We demonstrate herein that the ferroptosis inhibitors Fer-1, SRS11-92, and XJB-5-131 are efficacious in protecting FRDA cells in this model, whereas caspase inhibitors are not.…”

“…Both murine and human FRDA cells show increased sensitivity to treatment with ferric ammonium citrate (FAC), iron content 16.5%-18.5% (Sigma, St. Louis, MO), and L-buthionine (S,R)sulfoximine (BSO), 99% purity (Acros Organics, West Chester, PA), which inhibits the rate-limiting step of glutathione synthesis. Under the assay conditions we use, concentrations of iron and BSO that by themselves are nontoxic together cause a synergistic loss of viability (Cotticelli et al, 2012). Drugs are typically added 2 hours after BSO treatment.…”

“…Drugs are typically added 2 hours after BSO treatment. The assay has been described in detail and validated multiple times (Cotticelli et al, 2012(Cotticelli et al, , 2013. Idebenone (purity $98%) was obtained from Spectrum Chemical (New Brunswick, NJ); carbobenzoxy-valylalanyl-aspartyl-[O-methyl]-fluoromethylketone (purity $95%) was obtained from Adipogen (San Diego, CA); staurosporine (purity $98%) and Z-DEVD-FMK (purity $95%) (carbobenzoxy-aspartyl-glutamylvalyl-aspartyl-[O-methyl]-fluoromethylketone) were obtained from Cayman Chemicals (Ann Arbor, MI).…”

Contribution of Yeast Models to Neurodegeneration Research

“…Absence of Yfh1p, likewise of its human orthologue, results in mitochondrial iron accumulation, mitochondrial dysfunction, and oxidative stress [12, 13]. Recently, the yeast model was applied to the high-throughput screening (HTS) of compounds able to rescue mitochondrial function [14]. This was possible because yeast is one of the rare eukaryotes with a good fermenting capacity allowing the analysis of commonly disease-associated mitochondrial defects that would be lethal in other systems [15].…”