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Peroxiredoxin 5 is a distinct isoform of the peroxiredoxin gene family. The antioxidative and antiapoptotic functions of peroxiredoxin 5 have been extensively demonstrated in cell culture experiments. In the present paper, we provide the first functional analysis of peroxiredoxin 5 in a multicellular organism, Drosophila melanogaster. Similar to its mammalian, yeast or human counterparts, dPrx5 (Drosophila peroxiredoxin 5) is expressed in several cellular compartments, including the cytosol, nucleus and the mitochondrion. Global overexpression of dPrx5 in flies increased resistance to oxidative stress and extended their life span by up to 30% under normal conditions. The dprx5 −/− null flies were comparatively more susceptible to oxidative stress, had higher incidence of apoptosis, and a shortened life span. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis revealed that the dprx5 −/− null mutant had discernible tissue-specific apoptotic patterns, similar to those observed in control flies exposed to paraquat. In addition, apoptosis was particularly notable in oenocytes. During development the dPrx5 levels co-varied with ecdysone pulses, suggesting inter-relationship between ecdystreroids and dPrx5 expression. The importance of dPrx5 for development was further underscored by the embryonic lethal phenotype of progeny derived from the dprx5 −/− null mutant. Results from the present study suggest that the antioxidant and anti-apoptotic activities of dPrx5 play a critical role in development and aging of the fly.

The redox state of tissues tends to become progressively more prooxidizing during the aging process. The hypothesis tested in this study was that enhancement of reductive capacity by overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme for NADPH biosynthesis, could protect against oxidative stress and extend the life span of transgenic Drosophila melanogaster. Overexpression of G6PD was achieved by combining a UAS-G6PD responder transgene at one of four independent loci with either a broad expression (armadillo-GAL4, Tubulin-GAL4, C23-GAL4, and da-GAL4) or a neuronal driver (D42-GAL4 and Appl-GAL4). The mean life spans of G6PD overexpressor flies were extended, in comparison with driver and responder controls, as follows: armadillo-GAL4 (up to 38%), Tubulin-GAL4 (up to 29%), C23-GAL4 (up to 27%), da-GAL4 (up to 24%), D42-GAL4 (up to 18%), and Appl-GAL4 (up to 16%). The G6PD enzymatic activity was increased, as were the levels of NADPH, NADH, and the GSH/GSSG ratio. Resistance to experimental oxidative stress was enhanced. Furthermore, metabolic rates and fertility were essentially the same in G6PD overexpressors and control flies. Collectively, the results demonstrate that enhancement of the NADPH biosynthetic capability can extend the life span of a relatively long-lived strain of flies, which supports the oxidative stress hypothesis of aging.Glucose-6-phosphate dehydrogenase catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate and the reduction of NADP ϩ to NADPH, which is the rate-limiting step in the pentose phosphate pathway. This metabolic pathway serves multiple cellular functions, such as the supply of pentose intermediates for nucleotide synthesis, interconversion of 3-7 carbon sugars for various metabolic purposes, and the generation of reducing power in the form of NADPH. The latter metabolite plays an integral role in a broad range of cellular functions, such as (i) acting as a cofactor in reductive biosynthesis, (ii) detoxification of xenobiotics, and (iii) maintenance of the cellular redox state by providing reducing equivalents to antioxidative systems, among others (1).The concept that maintenance of an optimal redox state is essential to cellular survival is now firmly established. Significant amounts of superoxide anion radical and its stoichiometric product, hydrogen peroxide, are generated in mitochondria and peroxisomes under normal physiological conditions (2). Hydrogen peroxide is detoxified by catalase and peroxidases. The activity of the peroxidases is dependent upon the availability of reduced forms of glutathione (GSH) or thioredoxin, and it results in the oxidation of GSH to GSSG and of reduced thioredoxin red (Trx-(SH) 2 ) 2 to oxidized thioredoxin ox (Trx-(S) 2 ). The reducing equivalents for the reductions of GSSG by glutathione reductase and thioredoxin ox by thioredoxin reductase are supplied by NADPH (3). Thus, the regeneration of two major cellular antioxidants, GSH and thioredoxin, is dependent upon the supply of NADPH. In insects, glutathio...

The innate immune response tends to become hyperactive and proinflammatory in older organisms. We investigated connections between activity of the immune-related genes and aging using the Drosophila model. A hallmark of Drosophila immunity is the production of antimicrobial peptides (AMP), whose expression is triggered via activation of the Toll and Imd immune pathways and regulated by NF-ĸB-like transcription factors, Dif/Dorsal and Relish. It was previously shown that overexpression of the upstream component of the immune pathways shortens lifespan via activation of the Relish-dependent immune response. Here we show that direct overexpression of the Relish target AMP genes broadly at high levels or in the fat body induced apoptosis, elicited depolarization of the mitochondria and significantly shortened lifespan. Underexpression of Relish in the fat body beginning in the second half of lifespan prevented overactivation of AMPs and extended longevity. Unlike infection-induced responses, the age-related increase in AMPs does not require the upstream recognition/transduction module of the Imd pathway. It does however require downstream elements, including Relish and Ird5, a component of the downstream IKK complex. Together, these results established causal links between high-level production of antimicrobial peptides and longevity.

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.

Drosophila mitochondria contain two peroxidases, peroxiredoxin 3 (dPrx3) and peroxiredoxin 5 (dPrx5), which together constitute the sole known intra-mitochondrial mechanism for the catalytic removal of hydrogen-and organic-peroxides. dPrx3 exists exclusively within mitochondria, whereas dPrx5 is also present in some other intracellular compartments. Levels of these two peroxiredoxins were genetically manipulated, singly and together, in D. melanogaster, for the purpose of understanding their respective functions. Under-expression of dPrx3 by 90–95% had no discernable effect on life span under normal or oxidative stress conditions; the dPrx5 null flies were previously reported to exhibit a 10% shortening of mean life span and an increase in sensitivity to oxidative stress. Flies under-expressing both dPrx3 and dPrx5 showed an 80% decrease in life span, severe disruption in thiol homeostasis and a massive induction of apoptosis in the muscle and digestive system tissues. The early mortality in flies, under-expressing both peroxiredoxins, was partially offset by over-expression of thioredoxin reductase but not mitochondrion-targeted catalase. These results suggest that mitochondrial peroxiredoxins confer specific protection for thioredoxin/glutathione systems, play a critical role in the maintenance of global thiol homeostasis, prevent the age-associated apoptosis and premature death.

Peroxiredoxin 4 (Prx4) has been implicated in a wide variety of biological processes, including development, progression of cancer, inflammation, and antioxidant function. The purpose of this study was to provide further insight into its multiple roles at the whole-animal level, using Drosophila. Reduced expression of dPrx4 (up to 90%) resulted in greater sensitivity to oxidative stress, an elevated H₂O₂ flux, and increases in lipid peroxidation, but no effect on longevity. Overexpression at low levels (<2-fold) gave reduced levels of oxidative damage and tended to show an increase in longevity. Flies expressing dPrx4 globally at high levels (>5-fold) had a dramatically reduced life span (by 20-80%) and increased apoptosis. Analysis of these overexpressors revealed an aberrant redistribution of the dPrx4 protein from the endoplasmic reticulum (ER) to cytosol and hemolymph. In addition to the known proapoptotic effects of the cytosolic form of dPrx4, dPrx4 overexpression triggered an NF-κB-mediated proinflammatory response, similar to that observed in cells under ER stress or when microbially challenged. Finally, we provide the first evidence that dPrx4, on secretion into the hemolymph, elicits a JAK/STAT-mediated response. The effects on fly survival and homeostasis appear to represent a combination of differential effects dictated in large part by dPrx4 subcellular and tissue-specific localization.

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