Overexpression of a Drosophila homolog of apolipoprotein D leads to increased stress resistance and extended lifespan

Increased Apolipoprotein D (ApoD) expression has been reported in various neurological disorders, including Alzheimer's disease, schizophrenia, and stroke, and in the aging brain . However, whether ApoD is toxic or a defense is unknown. In a screen to identify genes that protect Drosophila against acute oxidative stress, we isolated a fly homolog of ApoD, Glial Lazarillo (GLaz). In independent transgenic lines, overexpression of GLaz resulted in increased resistance to hyperoxia (100% O(2)) as well as a 29% extension of lifespan under normoxia. These flies also displayed marked improvements in climbing and walking ability after sublethal exposure to hyperoxia. Overexpression of Glaz also increased resistance to starvation without altering lipid or protein content. To determine whether GLaz might be important in protection against reperfusion injury, we subjected the flies to hypoxia, followed by recovery under normoxia. Overexpression of GLaz was protective against behavioral deficits caused in normal flies by this ischemia/reperfusion paradigm. This and the accompanying paper by Sanchez et al. (in this issue of Current Biology) are the first to manipulate the levels of an ApoD homolog in a model organism. Our data suggest that human ApoD may play a protective role and thus may constitute a therapeutic target to counteract certain neurological diseases.     

Antioxidant activities of recombinant amphioxus (<Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>) apolipoprotein D

Apolipoprotein D (ApoD), a member of lipocalin, has been recently shown to be involved in regulating protection from oxidative stress. The absence of ApoD in mouse and Drosophila can reduce the resistance to oxidative stress and shorten lifespan. However, little information is available regarding the expression in vitro of ApoD and its biochemical properties. Amphioxus (Branchiostoma belcheri) ApoD, BbApoD, is an archetype of vertebrate ApoD proteins. In this study, the prokaryotic expression plasmid pET32a–BbApoD was constructed and recombinant BbApoD expressed in Escherichia coli BL21 and purified. Antioxidation assays showed that the recombinant BbApoD protein had the capacities to scavenge hydroxyl radicals (≥240 μg/ml) and to prevent nicking of the supercoiled DNA (≥100 μg/ml) in vitro, providing a biochemical evidence for antioxidant role of ApoD. This supports the notion that ApoD is part of the mechanisms regulating protection from oxidative stresses.     

Grasshopper Lazarillo, a GPI-anchored Lipocalin, increases Drosophila longevity and stress resistance, and functionally replaces its secreted homolog NLaz

Lazarillo (Laz) is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein first characterized in the developing nervous system of the grasshopper Schistocerca americana. It belongs to the Lipocalins, a functionally diverse family of mostly secreted proteins. In this work we test whether the protective capacity known for Laz homologs in flies and vertebrates (NLaz, GLaz and ApoD) is evolutionarily conserved in grasshopper Laz, and can be exerted from the plasma membrane in a cell-autonomous manner. First we demonstrate that extracellular forms of Laz have autocrine and paracrine protecting effects for oxidative stress-challenged Drosophila S2 cells. Then we assay the effects of overexpressing GPI-linked Laz in adult Drosophila and whether it rescues both known and novel phenotypes of NLaz null mutants. Local effects of GPI-linked Laz inside and outside the nervous system promote survival upon different stress forms, and extend lifespan and healthspan of the flies in a cell-type dependent manner. Outside the nervous system, expression in fat body cells but not in hemocytes results in protection. Within the nervous system, glial cell expression is more effective than neuronal expression. Laz actions are sexually dimorphic in some expression domains. Fat storage promotion and not modifications in hydrocarbon profiles or quantities explain the starvation-desiccation resistance caused by Laz overexpression. This effect is exerted when Laz is expressed ubiquitously or in dopaminergic cells, but not in hemocytes. Grasshopper Laz functionally restores the loss of NLaz, rescuing stress-sensitivity as well as premature accumulation of aging-related damage, monitored by advanced glycation end products (AGEs). However Laz does not rescue NLaz courtship behavioral defects. Finally, the presence of two new Lipocalins with predicted GPI-anchors in mosquitoes shows that the functional advantages of GPI-linkage have been commonly exploited by Lipocalins in the arthropodan lineage.     

Calorie restriction delays lipid oxidative damage in Drosophila melanogaster

The oxidative stress hypothesis predicts that the accumulation of oxidative damage to a variety of macromolecules is the molecular trigger driving the process of aging. Although an inverse relationship between oxidative damage and lifespan has been established in several different species, the precise relationship between oxidative damage and aging is not fully understood. Drosophila melanogaster is a favored model organism for aging research. Environmental interventions such as ambient temperature and calorie restriction can alter adult lifespan to provide an excellent system to examine the relationship between oxidative damage, aging and lifespan. We have developed an enzyme-linked immunosorbent assay (ELISA) using commercially available reagents for measuring 4-hydroxy-2-nonenal (HNE) in proteins, a marker for oxidative damage to lipids, and present data in flies to show that HNE adducts accumulate in an age-dependent manner. With immunohistology, we also find the primary site of HNE accumulation is the pericerebral fat body, where induction of dFOXO was recently shown to retard aging. When subjected to environmental interventions that shorten lifespan, such as elevated ambient temperature, the chronological accumulation of HNE adduct is accelerated. Conversely, interventions that extend lifespan, such as lower ambient temperature or low calorie diets, slow the accumulation of HNE adduct. These studies associate damage from lipid peroxidation with aging and lifespan in Drosophila and show that calorie restriction in flies, as in mammals, slows the accumulation of lipid related oxidative damage.     

Analyses of fruit flies that do not express selenoproteins or express the mouse selenoprotein, methionine sulfoxide reductase B1, reveal a role of selenoproteins in stress resistance

Selenoproteins are essential in vertebrates because of their crucial role in cellular redox homeostasis, but some invertebrates that lack selenoproteins have recently been identified. Genetic disruption of selenoprotein biosynthesis had no effect on lifespan and oxidative stress resistance of Drosophila melanogaster. In the current study, fruit flies with knock-out of the selenocysteine-specific elongation factor were metabolically labeled with (75)Se; they did not incorporate selenium into proteins and had the same lifespan on a chemically defined diet with or without selenium supplementation. These flies were, however, more susceptible to starvation than controls, and this effect could be ascribed to the function of selenoprotein K. We further expressed mouse methionine sulfoxide reductase B1 (MsrB1), a selenoenzyme that catalyzes the reduction of oxidized methionine residues and has protein repair function, in the whole body or the nervous system of fruit flies. This exogenous selenoprotein could only be expressed when the Drosophila selenocysteine insertion sequence element was used, whereas the corresponding mouse element did not support selenoprotein synthesis. Ectopic expression of MsrB1 in the nervous system led to an increase in the resistance against oxidative stress and starvation, but did not affect lifespan and reproduction, whereas ubiquitous MsrB1 expression had no effect. Dietary selenium did not influence lifespan of MsrB1-expressing flies. Thus, in contrast to vertebrates, fruit flies preserve only three selenoproteins, which are not essential and play a role only under certain stress conditions, thereby limiting the use of the micronutrient selenium by these organisms.     

Regulation of longevity by regulator of G-protein signaling protein, Loco

Regulator of G-protein signaling (RGS) proteins contribute to G-protein signaling pathways as activators or repressors with GTPase-activating protein (GAP) activity. To characterize whether regulation of RGS proteins influences longevity in several species, we measured stress responses and lifespan of RGS-overexpressing and RGS-lacking mutants. Reduced expression of Loco, a RGS protein of Drosophila melanogaster, resulted in a longer lifespan for both male and female flies, also exhibiting stronger resistance to three different stressors (starvation, oxidation, and heat) and higher manganese-containing superoxide dismutase (MnSOD) activity. In addition, this reduction in Loco expression increased fat content and diminished cAMP levels. In contrast, overexpression of both genomic and cDNA loco gene significantly shortened the lifespan with weaker stress resistance and lower fat content. Deletion analysis of the Loco demonstrated that its RGS domain is required for the regulation of longevity. Consistently, when expression of RGS14, mammalian homologue of Loco, was reduced in rat fibroblast cells, the resistance to oxidative stress increased with higher MnSOD expression. The changes of yeast Rgs2 expression, which shares a conserved RGS domain with the fly Loco protein, also altered lifespan and stress resistance in Saccharomyces cerevisiae. Here, we provide the first evidence that RGS proteins with GAP activity affect both stress resistance and longevity in several species.     

A model of oxidative stress management: moderation of carbohydrate metabolizing enzymes in SOD1-null Drosophila melanogaster

The response to oxidative stress involves numerous genes and mutations in these genes often manifest in pleiotropic ways that presumably reflect perturbations in ROS-mediated physiology. The Drosophila melanogaster SOD1-null allele (cSODn108) is proposed to result in oxidative stress by preventing superoxide breakdown. In SOD1-null flies, oxidative stress management is thought to be reliant on the glutathione-dependent antioxidants that utilize NADPH to cycle between reduced and oxidized form. Previous studies suggest that SOD1-null Drosophila rely on lipid catabolism for energy rather than carbohydrate metabolism. We tested these connections by comparing the activity of carbohydrate metabolizing enzymes, lipid and triglyceride concentration, and steady state NADPH:NADP(+) in SOD1-null and control transgenic rescue flies. We find a negative shift in the activity of carbohydrate metabolizing enzymes in SOD1-nulls and the NADP(+)-reducing enzymes were found to have significantly lower activity than the other enzymes assayed. Little evidence for the catabolism of lipids as preferential energy source was found, as the concentration of lipids and triglycerides were not significantly lower in SOD1-nulls compared with controls. Using a starvation assay to impact lipids and triglycerides, we found that lipids were indeed depleted in both genotypes when under starvation stress, suggesting that oxidative damage was not preventing the catabolism of lipids in SOD1-null flies. Remarkably, SOD1-nulls were also found to be relatively resistant to starvation. Age profiles of enzyme activity, triglyceride and lipid concentration indicates that the trends observed are consistent over the average lifespan of the SOD1-nulls. Based on our results, we propose a model of physiological response in which organisms under oxidative stress limit the production of ROS through the down-regulation of carbohydrate metabolism in order to moderate the products exiting the electron transport chain.     

Thioredoxin-2 affects lifespan and oxidative stress in Drosophila

Thioredoxins are proteins that have thiol-reducing activity and a characteristic conserved active site (WCGPC). They have several documented functions, e.g. roles in defences against oxidative stress and as electron donors for ribonucleotide-reductase. In Drosophila melanogaster there are three "classical" thioredoxins with the conserved active site: deadhead, ThioredoxinT and Thioredoxin-2. Here, we report the creation of null-mutations in the Thioredoxin-2 (Trx-2) gene. Characterization of two Trx-2 mutants indicated that Trx-2 affects the lifespan of D. melanogaster, and is involved in the organism's oxidative stress protection system. We found that the mutants have a shorter lifespan than wild-type flies, and thioredoxin double mutant flies showed lower tolerance to oxidative stress than wild-type flies, while flies carrying multiple copies of a Trx-2 rescue construct showed higher tolerance. These findings suggest that Trx-2 has modest or redundant functions in Drosophila physiology under unstressed conditions, but could be important during times of environmental stress.     

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease

ApoD (apolipoprotein D) is up-regulated in AD (Alzheimer's disease) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo, but the mechanism is unknown. Specific methionine residues may inhibit lipid peroxidation by reducing radical-propagating L-OOHs (lipid hydroperoxides) to non-reactive hydroxides via a reaction that generates MetSO (methionine sulfoxide). Since apoD has three conserved methionine residues (Met(49), Met(93) and Met(157)), we generated recombinant proteins with either one or all methionine residues replaced by alanine and assessed their capacity to reduce HpETEs (hydroperoxyeicosatetraenoic acids) to their HETE (hydroxyeicosatetraenoic acid) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the methionine residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated that Met(93) was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD methionine oxidation was associated with formation of apoD aggregates that were also detected in the hippocampus of AD patients. In conclusion, conversion of HpETE into HETE is mediated by apoD Met(93), a process that may contribute to apoD antioxidant function.     

Mating increases starvation resistance and decreases oxidative stress resistance in Drosophila melanogaster females

Mating stimulates complex physiological changes in females of Drosophila melanogaster. Long-term effects of mating are manifested in increased fecundity and shortened lifespan. It is not clear how mating affects stress resistance in fly females. We addressed this question here and found that mated and highly fecund wild-type D. melanogaster females have significantly higher resistance to starvation throughout their lifetime than age-matched virgin females. Mean survival time under starvation was age dependent with maximum survival time observed in 15-day-old mated females. Mating-induced increase in starvation resistance was associated with significantly higher fat reserves stored as triacylglycerols. While mated females had higher resistance to starvation, their resistance to oxidative stress was significantly lower than in age-matched virgins. Our study revealed that mating leads to an opposing relationship between resistance to starvation and resistance to oxidative stress in Drosophila females. Thus, shortened lifespan of mated females is associated with their high-fat content and greater susceptibility to oxidative stress.     

Expression of EphA receptors and ligands during chick cerebellar development

Apolipoprotein D (ApoD) is a secreted protein that belongs to the lipocalin family. We describe the expression pattern of ApoD during mouse embryogenesis by in situ hybridization using RNA probes. ApoD is expressed at E9 in mesenchymal cells in the rombencephalic–mesencephalic region. At E9.5 the cephalic ApoD-positive cells appear in the mesenchyme, and at later stages (starting at E10.5) ApoD expression is seen in meninges. Within the neuroepithelium, ApoD is expressed in pericytes surrounding brain and spinal cord capillaries from E10.5 to birth. Other places of expression of ApoD are the mesenchyme surrounding the olfactory epithelium and semicircular canals, as well as chondroblasts of skull and vertebrae.     

On the importance of fatty acid composition of membranes for aging

The membrane pacemaker theory of aging is an extension of the oxidative stress theory of aging. It emphasises variation in the fatty acid composition of membranes as an important influence on lipid peroxidation and consequently on the rate of aging and determination of lifespan. The products of lipid peroxidation are reactive molecules and thus potent damagers of other cellular molecules. It is suggested that the feedback effects of these peroxidation products on the oxidative stress experienced by cells is an important part of the aging process. The large variation in the chemical susceptibility of individual fatty acids to peroxidation coupled with the known differences in membrane composition between species can explain the different lifespans of species, especially the difference between mammals and birds as well as the body-size-related variation in lifespan within mammals and birds. Lifespan extension by calorie-restriction can also be explained by changes in membrane fatty acid composition which result in membranes more resistant to peroxidation. It is suggested that lifespan extension by reduced insulin/IGF signalling may also be mediated by changes in membrane fatty acid composition.     

The identification of zebrafish mutants showing alterations in senescence-associated biomarkers

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated beta-galactosidase (SA-beta-gal) in our current study. We validated the use of embryonic SA-beta-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-beta-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-beta-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-beta-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms.     

Early Life Hormetic Treatments Decrease Irradiation-Induced Oxidative Damage,Increase Longevity,and Enhance Sexual Performance during Old Age in the Caribbean Fruit Fly

Early life events can have dramatic consequences on performance later in life. Exposure to stressors at a young age affects development, the rate of aging, risk of disease, and overall lifespan. In spite of this, mild stress exposure early in life can have beneficial effects on performance later in life. These positive effects of mild stress are referred to as physiological conditioning hormesis. In our current study we used anoxia conditioning hormesis as a pretreatment to reduce oxidative stress and improve organismal performance, lifespan, and healthspan of Caribbean fruit flies. We used gamma irradiation to induce mild oxidative damage in a low-dose experiment, and massive oxidative damage in a separate high-dose experiment, in pharate adult fruit flies just prior to adult emergence. Irradiation-induced oxidative stress leads to reduced adult emergence, flight ability, mating performance, and lifespan. We used a hormetic approach, one hour of exposure to anoxia plus irradiation in anoxia, to lower post-irradiation oxidative damage. We have previously shown that this anoxic-conditioning treatment elevates total antioxidant capacity and lowers post-irradiation oxidative damage to lipids and proteins. In this study, conditioned flies had lower mortality rates and longer lifespan compared to those irradiated without hormetic conditioning. As a metric of healthspan, we tracked mating both at a young age (10 d) and old age (30 d). We found that anoxia-conditioned male flies were more competitive at young ages when compared to unconditioned irradiation stressed male flies, and that the positive effects of anoxic conditioning hormesis on mating success were even more pronounced in older males. Our data shows that physiological conditioning hormesis at a young age, not only improves immediate metrics of organismal performance (emergence, flight, mating), but the beneficial effects also carry into old age by reducing late life oxidative damage and improving lifespan and healthspan.     

Oxidative stress and aberrant signaling in aging and cognitive decline

Brain aging is associated with a progressive imbalance between antioxidant defenses and intracellular concentrations of reactive oxygen species (ROS) as exemplified by increases in products of lipid peroxidation, protein oxidation, and DNA oxidation. Oxidative conditions cause not only structural damage but also changes in the set points of redox-sensitive signaling processes including the insulin receptor signaling pathway. In the absence of insulin, the otherwise low insulin receptor signaling is strongly enhanced by oxidative conditions. Autophagic proteolysis and sirtuin activity, in turn, are downregulated by the insulin signaling pathway, and impaired autophagic activity has been associated with neurodegeneration. In genetic studies, impairment of insulin receptor signaling causes spectacular lifespan extension in nematodes, fruit flies, and mice. The predicted effects of age-related oxidative stress on sirtuins and autophagic activity and the corresponding effects of antioxidants remain to be tested experimentally. However, several correlates of aging have been shown to be ameliorated by antioxidants. Oxidative damage to mitochondrial DNA and the electron transport chain, perturbations in brain iron and calcium homeostasis, and changes in plasma cysteine homeostasis may altogether represent causes and consequences of increased oxidative stress. Aging and cognitive decline thus appear to involve changes at multiple nodes within a complex regulatory network.     

The insulin-regulated CREB coactivator TORC promotes stress resistance in Drosophila

In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein (CREB) coactivator transducer of regulated CREB activity 2 (TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1. Here we show that starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augmented TORC activity in adult flies, our results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance.