Downstream targets and compartment boundaries of oxidative stress revealed by RNAi-mediated silencing of Sod1 and Sod2. J. Hu 1, F. Missirlis 2, K. Kirby 1, A.J. Hilliker 3, J.P. Phillips 1. 1) Dept of Mol Biol and Genetics, University of Guelph, Canada; 2) NICHD, NIH, Bethesda, MD; 3) Dept Biol., York University, Canada.
Mutational and transgenic studies have established a fundamental role for reactive oxygen (RO) metabolism in life history traits in Drosophila. To circumvent the absence of germline mutations, we recently reported that ablation of mitochondrial SOD2 through expression of a GAL4-regulated, inverted-repeat Sod2 RNAi transgene leads to RO-mediated 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. (Kirby, et al. Proc Nat Acad Sci USA in press). Here we report that expression of a GAL4-regulated, inverted-repeat Sod1 RNAi transgene phenocopies SOD1-null mutants through specific loss of SOD1, causing hypersensitivity to applied oxidative stress and an 80 % reduction in adult lifespan. Using the selective sensitivity of cytoplasmic and mitochondrial aconitases to RO-mediated [4Fe-4S] cluster inactiviation, we monitored the compartment-specific, downstream effects of oxidative stress conferred by RNAi-mediated loss of SOD1 and SOD2. Loss of SOD2 confers reversible inactivation of mitochondrial aconitase while loss of SOD1 confers conversion of the bi-functional cytoplasmic aconitase to iron regulatory protein-1 (IRP-1). IREs have been identified in Drosophila mRNAs specifying ferritin and the Ip subunit of SDH, making these genes potential downstream targets of SOD1 deficiency. The linkage between oxidative stress and iron metabolism revealed by these studies suggests that perturbations in iron metabolism may underwrite at least some of the phenotypic symptoms of SOD1 deficiency and may be an important factor in normal aging. The results also challenge the view that superoxide emanating from the respiratory chain is an important source of oxidative stress in the cytoplasmic compartment.