The Genetics of Catalase in Drosophila Melanogaster: Isolation and Characterization of Acatalasemic Mutants

Activated oxygen species have been demonstrated to be the important agents in oxygen toxicity by disrupting the structural and functional integrity of cells through lipid peroxidation events, DNA damage and protein inactivation. The biological consequences of free radical damage have long been hypothesized to be a causal agent in many aging-related diseases. Catalase (H2O2:H2O2 oxidoreductase; EC 1.15.1.1) is one of several enzymes involved in the scavenging of oxygen free radicals and free radical derivatives. The structural gene for catalase in Drosophila melanogaster has been localized to region 75D1-76A on chromosome 3L by dosage responses to segmental aneuploidy. This study reports the isolation of a stable deficiency, Df(3L)CatDH104(75C1-2;75F1), that uncovers the catalase locus and the subsequent isolation of six acatalasemic mutants. All catalase mutants are viable under standard culture conditions and recessive lethal mutations within the 75Cl-F1 interval have been shown not to affect catalase activity. Two catalase mutations are amorphic while four are hypomorphic alleles of the Cat+ locus. The lack of intergenic complementation between the six catalase mutations strongly suggests that there is only one functional gene in Drosophila. One acatalesemic mutation was mapped to position 3-47.0 which resides within the catalase dosage sensitive region. While complete loss of catalase activity confers a severe viability effect, residual levels are sufficient to restore viability to wild type levels. These results suggest a threshold effect for viability and offer an explanation for the general lack of phenotypic effects associated with the known mammalian acatalasemics.     

Cooperative action of antioxidant defense systems in Drosophila

Molecular oxygen is key to aerobic life but is also converted into cytotoxic byproducts referred to as reactive oxygen species (ROS). Intracellular defense systems that protect cells from ROS-induced damage include glutathione reductase (GR), thioredoxin reductase (TrxR), superoxide dismutase (Sod), and catalase (Cat). Sod and Cat constitute an evolutionary conserved ROS defense system against superoxide; Sod converts superoxide anions to H(2)O(2), and Cat prevents free hydroxyl radical formation by breaking down H(2)O(2) into oxygen and water. As a consequence, they are important effectors in the life span determination of the fly Drosophila. ROS defense by TrxR and GR is more indirect. They transfer reducing equivalents from NADPH to thioredoxin (Trx) and glutathione disulfide (GSSG), respectively, resulting in Trx(SH)(2) and glutathione (GSH), which act as effective intracellular antioxidants. TrxR and GR were found to be molecularly conserved. However, the single GR homolog of Drosophila specifies TrxR activity, which compensates for the absence of a true GR system for recycling GSH. We show that TrxR null mutations reduce the capacity to adequately protect cells from cytotoxic damage, resulting in larval death, whereas mutations causing reduced TrxR activity affect pupal eclosion and cause a severe reduction of the adult life span. We also provide genetic evidence for a functional interaction between TrxR, Sod1, and Cat, indicating that the burden of ROS metabolism in Drosophila is shared by the two defense systems.     

Age-specific decrease in aerobic efficiency associated with increase in oxygen free radical production in Drosophila melanogaster

This study was designed to test the free radical theory of aging by using Drosophila melanogaster as a model system. Oxygen free radicals are generated by mitochondria during the process of normal oxidative metabolism. Age-specific measurements of oxygen consumption, heat production and anti-oxidant enzyme activity were obtained from two inbred lines of male flies, one selected for longevity and one normal-lived. The findings of this study demonstrate that although oxygen consumption remains relatively constant over the majority of the life span of each line of flies, aerobic efficiency declines with advancing age. This loss of aerobic efficiency manifests itself as a decline in total body metabolism as measured by heat production, and appears to be associated with an age-specific increase in damage inflicted upon mitochondria by oxygen free radicals.     

Transcriptional regulation of the Drosophila catalase gene by the DRE/DREF system

Reactive oxygen species (ROS) cause oxidative stress and aging. The catalase gene is a key component of the cellular antioxidant defense network. However, the molecular mechanisms that regulate catalase gene expression are poorly understood. In this study, we have identified a DNA replication-related element (DRE; 5'-TATCGATA) in the 5'-flanking region of the Drosophila catalase gene. Gel mobility shift assays revealed that a previously identified factor called DREF (DRE- binding factor) binds to the DRE sequence in the Drosophila catalase gene. We used site-directed mutagenesis and in vitro transient transfection assays to establish that expression of the catalase gene is regulated by DREF through the DRE site. To explore the role of DRE/DREF in vivo, we established transgenic flies carrying a catalase-lacZ fusion gene with or without mutation in the DRE. The beta-galactosidase expression patterns of these reporter transgenic lines demonstrated that the catalase gene is upregulated by DREF through the DRE sequence. In addition, we observed suppression of the ectopic DREF-induced rough eye phenotype by a catalase amorphic Cat(n1) allele, indicating that DREF activity is modulated by the intracellular redox state. These results indicate that the DRE/DREF system is a key regulator of catalase gene expression and provide evidence of cross-talk between the DRE/DREF system and the antioxidant defense system.     

Increased internal and external bacterial load during Drosophila aging without life-span trade-off

The role of microbial load during aging of the adult fruit fly Drosophila melanogaster is incompletely understood. Here we show dramatic increases in aerobic and anaerobic bacterial load during aging, both inside the body and on the surface. Scanning electron microscopy and cell staining analyses of the surface of aged flies detected structures resembling abundant small bacteria and bacterial biofilms. Bacteria cultured from laboratory flies included aerobic species Acetobacter aceti, Acetobacter tropicalis, and Acetobacter pasteurianus and anaerobic species Lactobacillus plantarum and Lactobacillus sp. MR-2; Lactobacillus homohiochii, Lactobacillus fructivorans, and Lactobacillus brevis were identified by DNA sequencing. Reducing bacterial load and antimicrobial peptide gene expression by axenic culture or antibiotics had no effect on life span. We conclude that Drosophila can tolerate a significant bacterial load and mount a large innate immune response without a detectable trade-off with life span; furthermore, microbes do not seem to limit life span under optimized laboratory conditions.     

Effects of overexpression of copper-zinc and manganese superoxide dismutases,catalase, and thioredoxin reductase genes on longevity in Drosophila melanogaster

The overexpression of antioxidative enzymes such as CuZn-superoxide dismutase (SOD), Mn-SOD, and catalase has previously been reported to extend life span in transgenic flies (Drosophila melanogaster). The purpose of this study was to determine whether life-extending effects persist if the recipient control strains of flies are relatively long-lived. Accordingly, the life spans of large numbers of replicate control and overexpressor lines were determined in two long-lived genetic backgrounds involving a combined total of >90,000 flies. Significant increases in the activities of both CuZn-SOD and catalase had no beneficial effect on survivorship in relatively long-lived y w mutant flies and were associated with slightly decreased life spans in wild type flies of the Oregon-R strain. The introduction of additional transgenes encoding Mn-SOD or thioredoxin reductase in the same genetic background also failed to cause life span extension. In conjunction with data from earlier studies, the results show that increasing the activities of these major antioxidative enzymes above wild type levels does not decrease the rate of aging in long-lived strains of Drosophila, although there may be some effect in relatively short-lived strains.     

The effects of dietary coenzyme Q on Drosophila life span

Reactive oxygen species (ROS), generated as by-products of aerobic metabolism, cause damage to proteins and cellular membranes, and are thus thought to influence senescence. Caenorhabditis elegans fed on diets lacking in ubiquinone coenzyme Q (CoQ), a coenzyme in the oxidative phosphorylation pathway, show increased longevity, possibly because of reduced ROS generation. We test the role of dietary CoQ in determining Drosophila melanogaster longevity by measuring survival and cytochrome c-oxidase activity (a proxy for aerobic metabolic performance) in flies fed wild-type yeast, CoQ-less yeast, or respiratory control (RC) yeast replete with CoQ but independently deficient in mitochondrial respiration. We find no evidence that dietary manipulation of CoQ in D. melanogaster increases life span or decreases age-dependent decline in cytochrome c oxidase activity. Instead, we find evidence that flies fed a diet of respiratory-deficient yeast (CoQ-less or RC) tend to have decreased longevity and increased rates of decline in cytochrome c-oxidase activity [corrected]     

Circadian regulation of response to oxidative stress in Drosophila melanogaster

Circadian rhythms are fundamental biological phenomena generated by molecular genetic mechanisms known as circadian clocks. There is increasing evidence that circadian synchronization of physiological and cellular processes contribute to the wellness of organisms, curbing pathologies such as cancer and premature aging. Therefore, there is a need to understand how circadian clocks orchestrate interactions between the organism’s internal processes and the environment. Here, we explore the nexus between the clock and oxidative stress susceptibility in Drosophila melanogaster. We exposed flies to acute oxidative stress induced by hydrogen peroxide (H₂O₂), and determined that mortality rates were dependent on time at which exposure occurred during the day/night cycle. The daily susceptibility rhythm was abolished in flies with a null mutation in the core clock gene period (per) abrogating clock function. Furthermore, lack of per increased susceptibility to H₂O₂ compared to wild-type flies, coinciding with enhanced generation of mitochondrial H₂O₂ and decreased catalase activity due to oxidative damage. Taken together, our data suggest that the circadian clock gene period is essential for maintaining a robust anti-oxidative defense.     

Trends in oxidative aging theories

The early observations on the rate-of-living theory by Max Rubner and the report by Gershman that oxygen free radicals exist in vivo culminated in the seminal proposal in the 1950s by Denham Harman that reactive oxygen species are a cause of aging (free radical theory of aging). The goal of this review is to analyze recent findings relevant in evaluating Harman's theory using experimental results as grouped by model organisms (i.e., invertebrate models and mice). In this regard, we have focused primarily on recent work involving genetic manipulations. Because the free radical theory of aging is not the only theorem proposed to explain the mechanism(s) involved in aging at the molecular level, we also discuss how this theory is related to other areas of research in biogerontology, specifically, telomere/cell senescence, genomic instability, and the mitochondrial hypothesis of aging. We also discuss where we think the free radical theory is headed. It is now possible to give at least a partial answer to the question whether oxidative stress determines life span as Harman posed so long ago. Based on studies to date, we argue that a tentative case for oxidative stress as a life-span determinant can be made in Drosophila melanogaster. Studies in mice argue for a role of oxidative stress in age-related disease, especially cancer; however, with regard to aging per se, the data either do not support or remain inconclusive on whether oxidative stress determines life span.     

Expression of bovine superoxide dismutase in Drosophila melanogaster augments resistance of oxidative stress.

Superoxide dismutases (SOD) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eucaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (O2-) to H2O2 and O2. Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of O2- and oxidative injury to biological structures, we generated transgenic strains of Drosophila melanogaster overproducing CuZn SOD. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin 5c gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Our data show that expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an O2(-)-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines. The highest level of expression of the active enzyme in adults was 1.60 times the normal value. Higher levels may have led to the formation of toxic levels of H2O2 during development, since flies that died during the process of eclosion showed an unusual accumulation of lipofuscin (age pigment) in some of their cells. In conclusion, our data show that free-radical detoxification has a minor by positive effect on mean longevity for several strains.     

The effects of age on radiation resistance and oxidative stress in adult Drosophila melanogaster

Drosophila melanogaster (fruit fly) is a well-established model organism for genetic studies of development and aging. We examined the effects of lethal ionizing radiation on male and female adult Drosophila of different ages, using doses of radiation from 200 to 1500 Gy. Fifty percent lethality 2 days postirradiation (LD(50/2)) in wild-type 1-day-old adult fruit flies was approximately 1238 Gy for males and 1339 Gy for females. We observed a significant age-dependent decline in the radiation resistance of both males and females. Radiation damage is postulated to occur by the generation of oxygen radicals. An age-related decline in the ability of flies to resist an agent that induces oxygen radicals, paraquat, was observed when comparing 10- and 20-day adults. Female flies are more resistant to paraquat than male flies. Oxidative stress mediated by paraquat was additive with sublethal exposures to radiation in young adults. Therefore, the ability to repair the damage caused by oxygen radicals seems to decline with the age of the flies. Because Drosophila adults are largely post-mitotic, our data suggest that adult Drosophila melanogaster can serve as an excellent model to study the factors responsible for radiation resistance in post-mitotic tissue and age-dependent changes in this resistance.     

Role of oxidative stress in Drosophila aging.

We review the role that oxidative damage plays in regulating the lifespan of the fruit fly, Drosophila melanogaster. Results from our laboratory show that the lifespan of Drosophila is inversely correlated to its metabolic rate. The consumption of oxygen by adult insects is related to the rate of damage induced by oxygen radicals, which are purported to be generated as by-products of respiration. Moreover, products of activated oxygen species such as hydrogen peroxide and lipofuscin are higher in animals kept under conditions of increased metabolic rate. In order to understand the in vivo relationship between oxidative damage and the production of the superoxide radical, we generated transgenic strains of Drosophila melanogaster that synthesize excess levels of enzymatically active superoxide dismutase. This was accomplished by P-element transformation of Drosophila melanogaster with the bovine cDNA for CuZn superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide radical to hydrogen peroxide and water. Adult flies that express the bovine SOD in addition to native Drosophila SOD are more resistant to oxidative stresses and have a slight but significant increase in their mean lifespan. Thus, resistance to oxidative stress and lifespan of Drosophila can be manipulated by molecular genetic intervention. In addition, we have examined the ability of adult flies to respond to various environmental stresses during senescence. Resistance to oxidative stress, such as that induced by heat shock, is drastically reduced in senescent flies. This loss of resistance is correlated with the increase in protein damage generated in old flies by thermal stress and by the insufficient protection from cellular defense systems which includes the heat shock proteins as well as the oxygen radical scavenging enzymes. Collectively, results from our laboratory demonstrate that oxidative damage plays a role in governing the lifespan of Drosophila during normal metabolism and under conditions of environmental stress.     

The effect of ablation of the gene for H<Superscript>+</Superscript>-transporting NAD/NADP transhydrogenase on the life spans of nematodes and mammals

Mitochondrial transhydrogenase catalyzes the reaction; H_out⁺ + NADP⁺ + NADH = NAD⁺ + NADPH + H_in⁺. The maintenance of the NADPH pool increases the mitochondrial antioxidant potential. Therefore, according to the commonly adopted free radical theory of aging, ablation of the transhydrogenase gene should reduce the life span. However, contrary to this reasoning, the life span of Caenorhabditis elegans nematodes with null mutations in the gene does not differ from that in wild-type worms. This fact indicates that free radical damage of mitochondria is not associated with aging. Meta analysis of data on the life span in mice possessing a spontaneous mutation in the transhydrogenase gene shows that a lack of this enzyme does not accelerate aging in mammals either. The heart is the tissue with the highest transhydrogenase production rate, and it is likely that this enzyme contributes to the protection of cardiac myocytes from oxidative stress.     

Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly

The oxidative stress hypothesis of aging predicts that a reduction in the generation of mitochondrial reactive oxygen species (ROS) will decrease oxidative damage and extend life span. Increasing mitochondrial proton leak-dependent state 4 respiration by increasing mitochondrial uncoupling is an intervention postulated to decrease mitochondrial ROS production. When human UCP2 (hUCP2) is targeted to the mitochondria of adult fly neurons, we find an increase in state 4 respiration, a decrease in ROS production, a decrease in oxidative damage, heightened resistance to the free radical generator paraquat, and an extension in life span without compromising fertility or physical activity. Our results demonstrate that neuronal-specific expression of hUCP2 in adult flies decreases cellular oxidative damage and is sufficient to extend life span.     

Mice lacking catalase develop normally but show differential sensitivity to oxidant tissue injury

Catalase plays a major role in cellular antioxidant defense by decomposing hydrogen peroxide, thereby preventing the generation of hydroxyl radical by the Fenton reaction. The degree of catalase deficiency in acatalasemic and hypocatalasemic mice varies from tissue to tissue. They therefore may not be suitable for studying the function of this enzyme in certain models of oxidant-mediated tissue injury. We sought to generate a new line of catalase null mice by the gene targeting technique. The mouse catalase (Cat or Cas1) gene was disrupted by replacing parts of intron 4 and exon 5 with a neomycin resistance cassette. Homozygous Cat knockout mice, which are completely deficient in catalase expression, develop normally and show no gross abnormalities. Slices of liver and lung and lenses from the knockout mice exhibited a retarded rate in decomposing extracellular hydrogen peroxide compared with those of wild-type mice. However, mice deficient in catalase were not more vulnerable to hyperoxia-induced lung injury; nor did their lenses show any increased susceptibility to oxidative stress generated by photochemical reaction, suggesting that the antioxidant function of catalase in these two models of oxidant injury is negligible. Further studies showed that cortical injury from physical impact caused a significant decrease in NAD-linked electron transfer activities and energy coupling capacities in brain mitochondria of Cat knockout mice but not wild-type mice. The observed decrease in efficiency of mitochondrial respiration may be a direct result of an increase in mitochondrion-associated calcium, which is secondary to the increased oxidative stress. These studies suggest that the role of catalase in antioxidant defense is dependent on the type of tissue and the model of oxidant-mediated tissue injury.     

Nectarine promotes longevity in Drosophila melanogaster

Fruits containing high antioxidant capacities and other bioactivities are ideal for promoting longevity and health span. However, few fruits are known to improve the survival and health span in animals, let alone the underlying mechanisms. Here we investigate the effects of nectarine, a globally consumed fruit, on life span and health span in Drosophila melanogaster. Wild-type flies were fed standard, dietary restriction (DR), or high-fat diet supplemented with 0-4% nectarine extract. We measured life span, food intake, locomotor activity, fecundity, gene expression changes, and oxidative damage indicated by the level of 4-hydroxynonenal-protein adduct in these flies. We also measured life span, locomotor activity, and oxidative damage in sod1 mutant flies on the standard diet supplemented with 0-4% nectarine. Supplementation with 4% nectarine extended life span, increased fecundity, and decreased expression of some metabolic genes, including a key gluconeogenesis gene, PEPCK, and oxidative stress-response genes, including peroxiredoxins, in female wild-type flies fed the standard, DR, or high-fat diet. Nectarine reduced oxidative damage in wild-type females fed the high-fat diet. Moreover, nectarine improved the survival of and reduced oxidative damage in female sod1 mutant flies. Together, these findings suggest that nectarine promotes longevity and health span partly by modulating glucose metabolism and reducing oxidative damage.     

Long-lived worms and aging

Several investigators have generated long-lived nematode worms (Caenorhabditis elegans) in the past decade by mutation of genes in the organism in order to study the genetics of aging and longevity. Dozens of longevity assurance genes (LAG) that dramatically increase the longevity of this organism have been identified. All long-lived mutants of C. elegans are also resistant to environmental stress, such as high temperature, reactive oxygen species (ROS), and ultraviolet irradiation. Double mutations of some LAGs further extended life span up to 400%, providing more insight into cellular mechanisms that put limits on the life span of organisms. With the availability of the LAG mutants and the combined DNA microarray and RNAi technology, the understanding of actual biochemical processes that determine life span is within reach: the downstream signal transduction pathway may regulate life span by up-regulating pro-longevity genes such as those that encode antioxidant enzymes and/or stress-response proteins, and down-regulating specific life-shortening genes. Furthermore, longevity could be modified through chemical manipulation. Results from these studies further support the free radical theory of aging, suggest that the molecular mechanism of aging process may be shared in all organisms, and provide insight for therapeutic intervention in age-related diseases.     

Rap-GEF/Rap signaling restricts the formation of supernumerary spermathecae in Drosophila melanogaster

Sperm storage in the female is a key factor for reproductive success in a variety of organisms, including Drosophila melanogaster. The spermathecae (SP) are the Drosophila organs for long-term storage. While wild-type female flies have two SP, occasionally, three or four SP have been observed in mutant flies. However, the molecular mechanism of SP formation is unknown. Here we show that loss of function of a Drosophila Rap-GEF (GEF26) result in an occurrence of the supernumerary SP; females have three SP (varies from 11 to 62% in different allele combinations) instead of the normal two SP. In addition, the Gef26 mutant flies also have ectopic wing veins and extra mechanosensory organs. The supernumerary SP phenotype of the Gef26 mutation can be enhanced by the Drosophila Rap mutations and rescued by overexpressing the cell adhesion molecule DE-cadherin. These data suggest that the Rap-GEF/Rap signaling controls the formation of supernumerary spermathecae through modulating cell adhesion in Drosophila.     

The effects of catalase gene overexpression on life span and resistance to oxidative stress in transgenic Drosophila melanogaster.

Oxygen free radicals and hydroperoxides have been postulated to play a causal role in the aging process, implying that antioxidant enzymes may act as longevity determinants. Catalase (H2O2:H2O2 oxidoreductase; EC1.11.1.6) is the sole enzyme involved in the elimination of H2O2 in Drosophila melanogaster; glutathione peroxidase being absent. A genomic fragment containing the Drosophila catalase gene was used to construct transgenic Drosophila lines by means of P element-mediated transformation. Enhanced levels of catalase (up to 80%) did not prolong the life span of flies, nor did they provide improved protection against oxidative stress induced by hyperoxia or paraquat treatment. However, enhanced resistance to hydrogen peroxide was observed in the overexpressors.