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.
Oxidative stress has been widely implicated as an important factor in the aging process. Because mitochondrial respiration is the principal source of reactive oxygen within cells, the mitochondrially localized superoxide dismutase (SOD) 2 is thought to play an important front-line defensive role against aging-related oxidative stress. Although genetic studies with mutants deficient in SOD1, the predominantly cytosolic isoform of SOD, have been instrumental in elucidating the role of reactive oxygen metabolism in aging in Drosophila, the lack of available mutations in the Sod2 gene has hampered an equivalent analysis of the participation of this important antioxidant enzyme in the Drosophila aging model. Here we report that ablation of mitochondrial SOD2 through expression of a GAL4-regulated, inverted-repeat Sod2 RNA-interference transgene in an otherwise normal animal causes increased endogenous oxidative stress, resulting in loss of essential enzymatic components of the mitochondrial respiratory chain and the tricarboxylic acid cycle, enhances sensitivity to applied oxidative stress, and causes early-onset mortality in young adults. In sharp contrast, ablation of SOD2 has no overt effect on the development of larvae and pupae, which may reflect a fundamental transition in oxygen utilization and͞or reactive oxygen metabolism that occurs during metamorphosis from larval to adult life.
Copper,zinc superoxide dismutase (SOD1) in mammals is activated principally via a copper chaperone (CCS) and to a lesser degree by a CCS-independent pathway of unknown nature. In this study, we have characterized the requirement for CCS in activating SOD1 from Drosophila. A CCS-null mutant (Ccs n29E ) of Drosophila was created and found to phenotypically resemble Drosophila SOD1-null mutants in terms of reduced adult life span, hypersensitivity to oxidative stress, and loss of cytosolic aconitase activity. However, the phenotypes of CCS-null flies were less severe, consistent with some CCS-independent activation of Drosophila SOD1 (dSOD1). Yet SOD1 activity was not detectable in Ccs n29E flies, due largely to a striking loss of SOD1 protein. In contrast, human SOD1 expressed in CCS-null flies is robustly active and rescues the deficits in adult life span and sensitivity to oxidative stress. The dependence of dSOD1 on CCS was also observed in a yeast expression system where the dSOD1 polypeptide exhibited unusual instability in CCS-null (ccs1⌬) yeast. The residual dSOD1 polypeptide in ccs1⌬ yeast was nevertheless active, consistent with CCS-independent activation. Stability of dSOD1 in ccs1⌬ cells was readily restored by expression of either yeast or Drosophila CCS, and this required copper insertion into the enzyme. The yeast expression system also revealed some species specificity for CCS. Yeast SOD1 exhibits preference for yeast CCS over Drosophila CCS, whereas dSOD1 is fully activated with either CCS molecule. Such variation in mechanisms of copper activation of SOD1 could reflect evolutionary responses to unique oxygen and/or copper environments faced by divergent species.
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