RNAi-mediated suppression of the mitochondrial iron chaperone, frataxin, in Drosophila

Abstract

The mitochondrial iron chaperone, frataxin, plays a critical role in cellular iron homeostasis and the synthesis and regeneration of Fe-S centers. Genetic insufficiency for frataxin is associated with Friedreich's Ataxia in humans and confers loss of function of Fe-containing proteins including components of the respiratory chain and mitochondrial and cytosolic aconitases. Here, we report the use of RNA-interference (RNAi) to suppress frataxin in the multicellular eukaryote, Drosophila. Phenotypically, suppression of the Drosophila frataxin homologue (dfh) confers distinct phenotypes in larvae and adults, leading to giant long-lived larvae and to conditional short-lived adults. Deficiency of the DFH protein results in diminished activities of numerous heme- and iron-sulfur-containing enzymes, loss of intracellular iron homeostasis and increased susceptibility to iron toxicity. In parallel with the differential larval and adult phenotypes, our results indicate that dfh silencing differentially dysregulates ferritin expression in adults but not in larvae. Moreover, silencing of dfh in the peripheral nervous system, a specific focus of Friedreich's pathology, permits normal larval development but imposes a marked reduction in adult lifespan. In contrast, dfh silencing in motorneurons has no deleterious effect in either larvae or adults. Finally, overexpression of Sod1, Sod2 or Cat does not suppress the failure of DFH-deficient animals to successfully complete eclosion, suggesting a minimal role of oxidative stress in this phenotype. The robust developmental, biochemical and tissue-specific phenotypes conferred by DFH deficiency in Drosophila provide a platform for identifying genetic, nutritional and environmental factors, which ameliorate the symptoms arising from frataxin deficiency.

Hydrogen peroxide scavenging rescues frataxin deficiency in a Drosophila model of Friedreich's ataxia

“…As reported earlier, DFH deficiency imposed by expression of UAS-DfhIR transgenes controlled by the C96-Gal4 driver confers marked early-onset adult mortality that is reflected as a 40% reduction in media adult life span (17). The C96-Gal4 driver promotes robust expression in the PNS and oenocytes with low-level expression in many other tissues (A.J.H., unpublished data).…”

“…The Drosophila FRDA model used in this work employs the C96-Gal4 driver to reduce endogenous DFH via RNAi and to coordinately augment native levels of antioxidant enzymes (17). As reported earlier, DFH deficiency imposed by expression of UAS-DfhIR transgenes controlled by the C96-Gal4 driver confers marked early-onset adult mortality that is reflected as a 40% reduction in media adult life span (17).…”

“…Yeast, Drosophila, mouse, and cell culture models of frataxin deficiency have revealed important roles for frataxin and its homologs in mitochondrial iron storage (7)(8)(9)(10)(11)(12)(13)(14)(15), regulation of intracellular iron trafficking (7,8,10,16,17), iron-sulfur (Fe-S) cluster (16)(17)(18)(19)(20)(21) and heme biogenesis (14,22), and reactivation of the labile Fe-S cluster of mitochondrial aconitase (mACON) (22,23). A role for frataxin in preventing formation of deleterious reactive oxygen species (ROS) has been well established (12,15,24), invoking a paradigm of FRDA pathology in which ROS toxicity leads to mitochondrial dysfunction with subsequent cell death (for review, see refs.…”

“…We recently reported the development of a Drosophila model of FRDA (17) that takes advantage of Gal4/UAS transgenebased RNAi-methodology to impose down-regulation of the Drosophila fraxatin homolog (Dfh) (30). In this model, Dfh suppression recapitulates the principal biochemical hallmarks of FRDA, including diminished activity of Fe-S-containing enzymes, susceptibility to iron toxicity, loss of intracellular iron homeostasis, and early-onset adult mortality.…”

Characterization of mitochondrial ferritin in Drosophila

“…Collectively, these results suggest that mitochondrial ferritin overexpression may have subtle effects on global iron metabolism. Given the recent development of a Drosophila model for suppression of frataxin expression it would be interesting to test whether Fer3HCH can function protectively under pathological conditions (31). Because mitochondrial ferritin is a nuclear-encoded protein and its levels can change dramatically (as shown in sideroblastic anemia patients), it is likely that a complex nuclear-mitochondrial communication system exists within the cell to protect mitochondria from iron overload (32).…”