Transgene-mediated RNAi and reverse genetic analysis of the aging-related gene, Sod1 in Drosophila melanogaster

Drosophila melanogaster is a useful model organism to study the fundamental role of reactive oxygen (RO) metabolism in complex life history traits, including aging. In this study, a novel transgene-based RNA interference (RNAi) system was developed to differentially suppress the expression of the endogenous Cu/Zn superoxide dismutase (Sod1) gene via the action of in vivo-synthesized Sod1 double-stranded RNAs. This potent reverse genetic tool facilitates further investigation of aging-associated gene function at subcellular and organismal levels under various spatial and temporal expression patterns.; SOD1 RNAi adults bearing the UAS-Sod1 inverted repeat (IR) and daG32-GAL4 transgenes show a specific reduction of the endogenous Sod1 transcript and the SOD1 protein. Such flies phenocopy mutants homozygous for null alleles of Sod1. Forty different GAL4 drivers were used to suppress SOD1 in specific targeted tissues. SOD1 RNAi induced by PNS-GAL4 in the peripheral nervous system (PNS) gave the most striking effect on adult lifespan, suggesting that PNS may be a crucial tissue for maintaining normal lifespan. Co-suppression of SOD1 and SOD2 by RNAi shows no additive effect on preadult development or the reduction of adult lifespan. Overexpression of SOD1 does not rescue the early onset mortality in SOD2 RNAi flies. This suggests that SOD1 and SOD2 act independently of one another in cytosol and mitochondria. The subcellular-compartmentalized nature of oxidative stress defense was further supported by the protection of the integrity of cytosolic aconitase from superoxide attack by SOD1, but not by SOD2. Finally, the fact that dietary restriction (DR) mitigates RO-mediated premature aging in SOD1 deficient flies suggests that RO metabolism plays an important role in DR.; This successfully established RNAi system is the first application in Drosophila aging studies. The significance of this research is to gain more insight into RO-mediated aging in Drosophila.