Mutations in chemosensory cilia cause resistance to paraquat in nematode Caenorhabditis elegans

The relationship between oxidative stress and longevity is a matter of concern in various organisms. We isolated mutants resistant to paraquat from nematode Caenorhabditis elegans. One mutant named mev-4 was long-lived and showed cross-resistance to heat and Dyf phenotype (defective in dye filling). Genetic and sequence analysis revealed that mev-4 had a nonsense mutation on the che-11 gene, homologues of which are involved in formation of cilia and flagella in other organisms. The paraquat resistance was commonly observed in various Dyf mutants and did not depend on the daf-16 gene, whereas the extension of life span did depend on it. Expression of antioxidant enzyme genes seemed normal. These results suggest that chemosensory neurons are a target of oxidative stress and influence longevity dependent on the daf-16 signaling in C. elegans.     

Ginkgo biloba extract EGb 761 increases stress resistance and extends life span of Caenorhabditis elegans.

EGb 761, a standardized extract of Ginkgo biloba leaves, has been used in clinical trials for its beneficial effects on brain functions. In mammals, EGb 761 has been shown to enhance cognition, stress resistance, and longevity, but its molecular and cellular mechanisms are not known. In the present investigation, we used the model organism Caenorhabditis elegans to evaluate pharmacological effects of EGb 761 on aging. We tested the theory that EGb 761 augments the natural antioxidant system of C elegans, and thus increases stress resistance and longevity. We found that treatment of the wild-type worms with EGb 761 extended their median life span by 8%. Amongst several purified components of EGb 761, the flavonoid tamarixetin showed the most dramatic effect: it extended the median life span by 25%. Furthermore, EGb 761 increased the wild type's resistance to acute oxidative and thermal stress by 33% and 25%, respectively. Treatment of the prematurely aging mutant worms mev-1 with EGb 761 increased their resistance to acute oxidative and thermal stress by 33% and 11%, respectively. It appears that oxidative stress, a major determinant of life span, as well as other types of stress, can be successfully counteracted by the Ginlkgo biloba extract EGb 761.     

Elevated paraquat resistance can be used as a bioassay for longevity in a genetically based long-lived strain of Drosophila

A long-lived (L) strain of Drosophila melanogaster, derived from a normal-lived (R) strain by artificial selection, has a significantly different adult longevity. Previous work has shown that (1) the two strains age in the same manner, (2) the major genes responsible for much of the L strain's extended longevity are located on the 3rd chromosome, and (3) the extended longevity phenotype is significantly modulated by the larval environment. In this report, we investigate the resistance of the L and R strains to the lethal effects of dietary paraquat. We show that, within the limitations of our described chromosomal and environmental manipulations, the extended longevity phenotype always accompanies the phenotype of elevated paraquat resistance. In addition, reversed selection applied to the L strain results in the simultaneous decrease of both life span and paraquat resistance. Thus, the presence or absence of the latter phenotype may be used as a bioassay for the presence or absence of the extended longevity phenotype, without any necessary implication of causality. Use of this bioassay should greatly speed up the genetic analysis of this system by allowing us to identify long-lived animals at a young age. Finally, we show that the age-related loss of elevated paraquat resistance in both strains precedes all the other age-related functional decrements which we have previously noted in this system.     

Elevated paraquat resistance can be used as a bioassay for longevity in a genetically based long-lived strain of Drosophila.

A long-lived (L) strain of Drosophila melanogaster, derived from a normal-lived (R) strain by artificial selection, has a significantly different adult longevity. Previous work has shown that 1) the two strains age in the same manner, 2) the major genes responsible for much of the L strain's extended longevity are located on the 3rd chromosome, and 3) the extended longevity phenotype is significantly modulated by the larval environment. In this report, we investigate the resistance of the L and R strains to the lethal effects of dietary paraquat. We show that, within the limitations of our described chromosomal and environmental manipulations, the extended longevity phenotype always accompanies the phenotype of elevated paraquat resistance. In addition, reversed selection applied to the L strain results in the simultaneous decrease of both life span and paraquat resistance. Thus, the presence or absence of the latter phenotype may be used as a bioassay for the presence or absence of the extended longevity phenotype, without any necessary implication of causality. Use of this bioassay should greatly speed up the genetic analysis of this system by allowing us to identify long-lived animals at a young age. Finally, we show that the age-related loss of elevated paraquat resistance in both strains precedes all the other age-related functional decrements which we have previously noted in this system.     

Common mechanisms for calorie restriction and adenylyl cyclase type 5 knockout models of longevity

Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared with WT mice. CR applied to AC5 KO results in rapid decrease in body weight, metabolic deterioration, and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome‐wide for brain, heart, skeletal muscle, and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene ontology analysis identified many significantly regulated, tissue‐specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD, and olfactory receptors in both young‐ and intermediate‐age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR mice, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance.     

THE SYMMETRY OF CORRELATED SELECTION RESPONSES IN ADAPTIVE EVOLUTION: AN EXPERIMENTAL STUDY USING DROSOPHILA

The relationship between the processes of density-dependent and age-specific selection has been investigated by examining a common phenotype, urea resistance, which has apparently evolved in response to each of these selection mechanisms. Twenty populations that have experienced differing levels of age-specific selection show differences in egg-to-adult viability in environments with high levels of urea. Among this group of populations, it appears that resistance to urea is correlated with longevity, but not development time. Ten populations kept at extreme larval densities for many generations also show responses to urea: those kept at high larval densities appear to be most resistant to urea. However, these populations show no differences in adult longevity. An additional five populations were selected directly for urea resistance by adding this compound to the larval food environment. Again, there was a strong response to this artificial selection, with urea resistance increasing dramatically, but these populations showed no response in adult longevity or resistance to crowding when compared to five control populations. There is clearly no simple relationship between longevity and larval urea resistance. It may be that age-specific and density-dependent selection induce similar changes in this phenotype, but do so through different genetic and physiological pathways. We suggest that these data are not consistent with the view of constant and symmetric genetic variance-covariance matrices. These data support a more prominent role for observations of evolutionary trajectories rather than static measurements of genetic components of variance.     

The Role of Oxidative Stress in the Longevity and Insecticide Resistance Phenotype of the Major Malaria Vectors Anopheles arabiensis and Anopheles funestus

Oxidative stress plays numerous biological roles, both functional and pathological. The role of oxidative stress in various epidemiologically relevant biological traits in Anopheles mosquitoes is not well established. In this study, the effects of oxidative stress on the longevity and insecticide resistance phenotype in the major malaria vector species An. arabiensis and An. funestus were examined. Responses to dietary copper sulphate and hydrogen peroxide were used as proxies for the oxidative stress phenotype by determining the effect of copper on longevity and hydrogen peroxide lethal dose. Glutathione peroxidase and catalase activities were determined colorimetrically. Oxidative burden was quantified as protein carbonyl content. Changes in insecticide resistance phenotype were monitored by WHO bioassay. Insecticide resistant individuals showed an increased capacity for coping with oxidative stress, mediated by increased glutathione peroxidase and catalase activity. This effect was observed in both species, as well as in laboratory strains and F₁ individuals derived from wild-caught An. funestus mothers. Phenotypic capacity for coping with oxidative stress was greatest in strains with elevated Cytochrome P450 activity. Synergism of oxidative stress defence enzymes by dietary supplementation with haematin, 3-Amino-1, 2, 4-triazole and Sodium diethyldithiocarbamate significantly increased pyrethroid-induced mortality in An. arabiensis and An. funestus. It is therefore concluded that defence against oxidative stress underlies the augmentation of the insecticide resistance phenotype associated with multiple blood-feeding. This is because multiple blood-feeding ultimately leads to a reduction of oxidative stress in insecticide resistant females, and also reduces the oxidative burden induced by DDT and pyrethroids, by inducing increased glutathione peroxidase activity. This study highlights the importance of oxidative stress in the longevity and insecticide resistance phenotype in malaria vectors.     

Modulation of methuselah expression targeted to Drosophila insulin‐producing cells extends life and enhances oxidative stress resistance

Ubiquitously reduced signaling via Methuselah (MTH), a G‐protein‐coupled receptor (GPCR) required for neurosecretion, has previously been reported to extend life and enhance stress resistance in flies. Whether these effects are due to reduced MTH signalling in specific tissues remains unknown. We determined that reduced expression of mth targeted to the insulin‐producing cells (IPCs) of the fly brain was sufficient to extend life and enhance oxidative stress resistance. Paradoxically, we discovered that overexpression of mth targeted to the same cells has similar phenotypic effects to reduced expression due to MTH's interaction with β‐arrestin, which uncouples GPCRs from their G‐proteins. We confirmed the functional relationship between MTH and β‐arrestin by finding that IPC‐targeted overexpression of β‐arrestin alone mimics the longevity phenotype of reduced MTH signaling. As reduced MTH signaling also inhibits insulin secretion from the IPCs, the most parsimonious mechanistic explanation of its longevity and stress‐resistance enhancement might be through reduced insulin/IGF signaling (IIS). However, examination of phenotypic features of long‐lived IPC‐mth modulated flies as well as several downstream IIS targets implicates enhanced activity of the JNK stress‐resistance pathway more directly than insulin signaling in the longevity and stress‐resistance phenotypes.     

A complex II defect affects mitochondrial structure,leading to ced-3- and ced-4-dependent apoptosis and aging

The mev-1(kn1) mutation of Caenorhabditis elegans is in Cyt-1, which encodes a subunit of succinate-coenzyme Q oxidoreductase in the mitochondrial electron transport chain. Mutants are hypersensitive to oxidative stress and age precociously in part because of increased superoxide anion production. Here, we show that mev-1 mutants are defective in succinate-coenzyme Q oxidoreductase, possess ultrastructural mitochondrial abnormalities (especially in muscle cells), show a loss of membrane potential, have altered CED-9 and Cyt-1 protein levels under hyperoxia, and contain ced-3-and ced-4-dependent supernumerary apoptotic cells. These defects likely explain the failure of mev-1 to complete embryonic development under hyperoxia as well as its reduced life span.     

A defect in the cytochrome b large subunit in complex II causes both superoxide anion overproduction and abnormal energy metabolism in Caenorhabditis elegans

A mev-1(kn1) mutant of the nematode Caenorhabditis elegans is defective in the cytochrome b large subunit (Cyt-1/ceSDHC) in complex II of the mitochondrial electron transport chain. We have previously shown that a mutation in mev-1 causes shortened life span and rapid accumulation of aging markers such as fluorescent materials and protein carbonyls in an oxygen-dependent fashion. However, it remains unclear as to whether this hypersensitivity is caused by direct toxicity of the exogenous oxygen or by the damage of endogenous reactive oxygen species derived from mitochondria. Here we report important biochemical changes in mev-1 animals that serve to explain their abnormalities under normoxic conditions: (i) an overproduction of superoxide anion from mitochondria; and (ii) a reciprocal reduction in glutathione content even under atmospheric oxygen. In addition, unlike wild type, the levels of superoxide anion production from mev-1 mitochondria were significantly elevated under hyperoxia. Under normal circumstances, it is well known that superoxide anion is produced at complexes I and III in the electron transport system. Our data suggest that the mev-1(kn1) mutation increases superoxide anion production at complex II itself rather than at complexes I and III. The mev-1 mutant also had a lactate level 2-fold higher than wild type, indicative of lactic acidosis, a hallmark of human mitochondrial diseases. These data indicate that Cyt-1/ceSDHC plays an important role not only in energy metabolism but also in superoxide anion production that is critically involved in sensitivity to atmospheric oxygen.     

No evidence of elevated germline mutation accumulation under oxidative stress in Caenorhabditis elegans

Variation in rates of molecular evolution has been attributed to numerous, interrelated causes, including metabolic rate, body size, and generation time. Speculation concerning the influence of metabolic rate on rates of evolution often invokes the putative mutagenic effects of oxidative stress. To isolate the effects of oxidative stress on the germline from the effects of metabolic rate, generation time, and other factors, we allowed mutations to accumulate under relaxed selection for 125 generations in two strains of the nematode Caenorhabditis elegans, the canonical wild-type strain (N2) and a mutant strain with elevated steady-state oxidative stress (mev-1). Contrary to our expectation, the mutational decline in fitness did not differ between N2 and mev-1. This result suggests that the mutagenic effects of oxidative stress in C. elegans are minor relative to the effects of other types of mutations, such as errors during DNA replication. However, mev-1 MA lines did go extinct more frequently than wild-type lines; some possible explanations for the difference in extinction rate are discussed.     

Acacetin promotes healthy aging by altering stress response in Caenorhabditis elegans

The progression in lifespan has been associated with elevated intracellular reactive oxygen species (ROS) and oxidative stress level which contributes to development of age related disorders. The discovery of lifespan modulating phytomolecules may promote development of natural therapies against age related afflictions. Acacetin (5,7-dihydroxy-4-methoxyflavone), is a naturally occurring flavonoid known to possess therapeutic properties. To this end, the present study evaluates effect of acacetin (AC) on lifespan, stress and neurotoxicity for the first time by using well-established free living, multicellular Caenorhabditis elegans model system. The 25?μM dose of AC significantly prolonged the mean lifespan of worms by 27.31% in comparison to untreated control and other tested doses of AC. Additionally, AC enhanced stress resistance against oxidative and thermal stress in worms. Furthermore, AC attenuated age related intracellular ROS level, aggregation of age pigment lipofuscin and increased the mean survival in stress hypersensitive mev-1 mutant by 40.5%. AC supplementation also reduced the alpha synuclein aggregation in transgenic worm model of Parkinson’s disease. The enhanced stress resistance, lifespan and alleviation of age related pathology can be attributed to increment in stress modulatory enzymes like superoxide dismutase (SOD) and catalase (CAT) level. Altogether the results suggest AC exposure maintains stress level, health span and extends mean lifespan of C. elegans. The longevity promoting and neuromodulatory effects of AC are mediated by up regulation of the stress response genes sod-3 and gst-4. The present finding gives new insights of natural remedies and their future prospects in developing therapeutic interventions for managing age related diseases.     

Metabolic Signatures of Extreme Longevity in Northern Italian Centenarians Reveal a Complex Remodeling of Lipids,Amino Acids,and Gut Microbiota Metabolism

The aging phenotype in humans has been thoroughly studied but a detailed metabolic profiling capable of shading light on the underpinning biological processes of longevity is still missing. Here using a combined metabonomics approach compromising holistic ¹H-NMR profiling and targeted MS approaches, we report for the first time the metabolic phenotype of longevity in a well characterized human aging cohort compromising mostly female centenarians, elderly, and young individuals. With increasing age, targeted MS profiling of blood serum displayed a marked decrease in tryptophan concentration, while an unique alteration of specific glycerophospholipids and sphingolipids are seen in the longevity phenotype. We hypothesized that the overall lipidome changes specific to longevity putatively reflect centenarians'' unique capacity to adapt/respond to the accumulating oxidative and chronic inflammatory conditions characteristic of their extreme aging phenotype. Our data in centenarians support promotion of cellular detoxification mechanisms through specific modulation of the arachidonic acid metabolic cascade as we underpinned increased concentration of 8,9-EpETrE, suggesting enhanced cytochrome P450 (CYP) enzyme activity. Such effective mechanism might result in the activation of an anti-oxidative response, as displayed by decreased circulating levels of 9-HODE and 9-oxoODE, markers of lipid peroxidation and oxidative products of linoleic acid. Lastly, we also revealed that the longevity process deeply affects the structure and composition of the human gut microbiota as shown by the increased extrection of phenylacetylglutamine (PAG) and p-cresol sulfate (PCS) in urine of centenarians. Together, our novel approach in this representative Italian longevity cohort support the hypothesis that a complex remodeling of lipid, amino acid metabolism, and of gut microbiota functionality are key regulatory processes marking exceptional longevity in humans.     

Lipid metabolism and diet: possible mechanisms of slow aging

The ability to survive to an extremely old age is a consequence of complex interactions among genes, environment, lifestyle and luck. In the last two centuries, life expectancy in western countries has doubled, increasing from 40 to 81 years (79 for males and 82 for females). The candidate factors to determine such mortality reduction are reduced exposure to infections and the subsequent reduction in inflammatory responses, and to some extent, improvement in diet and nutrition. Among the people born at the beginning of the previous century, a small portion of individuals (1 in 10,000 born) have reached 100 years, surviving approximately 20 years more than the general population. The successful longevity of these individuals shows a familial component, possibly genetic, as underlined by the centenarian sibling's increased chance of reaching 100 years of age compared to the general population. Genetic studies on long living individuals have led to the discovery of potential genetic causes of extreme longevity. These discoveries have highlighted the role of lipid metabolism as a potential key player in the ability to survive to extreme old age. Additional studies on the longevity phenotype have confirmed the role of lipids and lipid-associated cell activities in the predisposition to longevity, from lower eukaryotes to humans. The main focus of this review is the appreciation of demographic survival data and changes in recent diet with the above mentioned genetic and phenotypic biomarkers of longevity, in order to elucidate hypotheses on mechanisms of slow aging and disease resistance.     

Comparative biochemical and stress analysis of genetically selected drosophila strains with different longevities

We have performed a comparative analysis of the effects of age of reproduction on the biochemical (protein, lipid, and glycogen content) and stress resistance (ability to survive starvation, desiccation, and exogenous paraquat) parameters on 10 sister lines of five different Drosophila strains. Four pairs of these sister lines were selected under different regimens for either early or delayed reproduction; the fifth pair was maintained in a nonselected state and served as the baseline strain to which all others were compared. It is generally accepted that the early regimens give rise to short-lived phenotypes, whereas the delayed regimens give rise to long-lived phenotypes. Our results suggest that a mechanism involving lipid and starvation resistance is not operative in our long-lived strains. In addition, a mechanism involving glycogen content and desiccation resistance is only weakly supported. Finally, there is strong support for a mechanism that gives rise to enhanced paraquat resistance and therefore may involve regulatory changes in the pattern of ADS gene expression. In addition, the 15-day early age at reproduction regimen (M type) shows qualitatively similar responses to that of the late age at reproduction regimen (L type). These results suggest that correlations between biochemical traits and longevity must be interpreted with caution. We discuss possible reasons for these results, including the possibility of multiple mechanisms, each leading to a different extended longevity phenotype.