An enriched polyphenolic extract obtained from the by-product of Rosa damascena hydrodistillation activates antioxidant and proteostatic modules

BackgroundProlonged maintenance of proteome stability and functionality (proteostasis) is of emerging significance in aging retardation and healthspan.PurposeAn enriched polyphenolic extract obtained from the hydrodistillation of rose petals was tested for its capacity to activate the proteostasis network modules, and thus modulate health- and/or lifespan at the cellular and whole organism level.MethodsThe aqueous extract that remained after the hydrodistillation of Rosa damascena petals, was processed with a polystyrene-FPX66 adsorption resin and sequentially fractionated by FCPC. NMR and UHPLC-HRMS analyses revealed the presence of 28 metabolites, mainly glycosides of kaempferol and quercetin.ResultsThe extract showed high in vitro antioxidant activity and was not toxic in normal human skin fibroblasts, while it promoted the upregulation of NRF2-induced antioxidant genes and main proteostatic modules. Consistently, supplementation of this extract in Drosophila flies’ culture medium induced a cncC/NRF2-mediated upregulation of antioxidant and proteostatic modules. Prolonged administration of the extract in flies’ culture medium was not toxic and did not affect food intake rate or fecundity; also, it delayed the age-related decline of stress tolerance and locomotion performance (neuromuscular functionality) and dose-dependently extended flies’ lifespan.ConclusionOur findings indicate that the enriched polyphenolic extract obtained from the residue of R. damascena hydrodistillation activates cytoprotective cellular modules that, likely, contribute to its potential anti-aging properties.     

A call to fins! Zebrafish as a gerontological model

Among the wide variety of model organisms commonly used for studies on aging, such as worms, flies and rodents, a wide research gap exists between the invertebrate and vertebrate model systems. In developmental biology, a similar gap has been filled by the zebrafish (Danio rerio). We propose that the zebrafish is uniquely suited to serve as a bridge model for gerontology. With high fecundity and economical husbandry requirements, large populations of zebrafish may be generated quickly and cheaply, facilitating large-scale approaches including demographic studies and mutagenesis screens. A variety of mutants identified in such screens have led to modelling of human disease, including cardiac disorders and cancer. While zebrafish longevity is at least 50% longer than in commonly used mouse strains, as an ectothermic fish species, its life span may be readily modulated by caloric intake, ambient temperature and reproductive activity. These features, coupled with a growing abundance of biological resources, including an ongoing genome sequencing project, make the zebrafish a compelling model organism for studies on aging.     

Interactions among morphotype,nutrition, and temperature impact fitness of an invasive fly

Invasive animals depend on finding a balanced nutritional intake to colonize, survive, and reproduce in new environments. This can be especially challenging during situations of fluctuating cold temperatures and food scarcity, but phenotypic plasticity may offer an adaptive advantage during these periods. We examined how lifespan, fecundity, pre‐oviposition periods, and body nutrient contents were affected by dietary protein and carbohydrate (P:C) ratios at variable low temperatures in two morphs (winter morphs WM and summer morphs SM) of an invasive fly, Drosophila suzukii. The experimental conditions simulated early spring after overwintering and autumn, crucial periods for survival. At lower temperatures, post‐overwintering WM lived longer on carbohydrate‐only diets and had higher fecundity on low‐protein diets, but there was no difference in lifespan or fecundity among diets for SM. As temperatures increased, low‐protein diets resulted in higher fecundity without compromising lifespan, while high‐protein diets reduced lifespan and fecundity for both WM and SM. Both SM and WM receiving high‐protein diets had lower sugar, lipid, and glycogen (but similar protein) body contents compared to flies receiving low‐protein and carbohydrate‐only diets. This suggests that flies spend energy excreting excess dietary protein, thereby affecting lifespan and fecundity. Despite having to recover from nutrient depletion after an overwintering period, WM exhibited longer lifespan and higher fecundity than SM in favorable diets and temperatures. WM exposed to favorable low‐protein diet had higher body sugar, lipid, and protein body contents than SM, which is possibly linked to better performance. Although protein is essential for oogenesis, WM and SM flies receiving low‐protein diets did not have shorter pre‐oviposition periods compared to flies on carbohydrate‐only diets. Finding adequate carbohydrate sources to compensate protein intake is essential for the successful persistence of D. suzukii WM and SM populations during suboptimal temperatures.     

Insects as a model system for aging studies

As the human lifespan has increased dramatically in recent decades, the amount of aging research has correspondingly increased. To investigate mechanisms of aging, an efficient model system is required. Although mammalian animal models are essential for aging studies, they are sometimes inappropriate due to their long lifespans and high maintenance costs. In this regard, insects can be effective alternative model systems for aging studies, as insects have a relatively short lifespan and cost less to maintain. Many species of insects have been used as model systems for aging studies, especially fruit flies, silkworm moths and several social insects. Fruit flies are most commonly used for aging studies due to the wide availability of abundant resources such as mutant stocks, databases and genetic tools. Silkworm moths are also good tools for studying aging at the tissue level due to their relatively large size. Last, social insects such as ants and bees are good for investigating lifespan determinants, as their lifespans significantly differ according to caste despite a constant genotype among the population. In this review, we discuss the current status and future prospects of aging research using insect model systems.     

Rapamycin increases lifespan and inhibits spontaneous tumorigenesis in inbred female mice

The nutrient-sensing TO R (target of rapamycin) pathway is involved in cellular and organismal aging. Rapamycin, an inhibitor of TO R, extends lifespan in yeast, fruit flies and genetically heterogeneous mice. Here, we demonstrate that lifelong administration of rapamycin extends lifespan in female 129/Sv mice characterized by normal mean lifespan of 2 y. Importantly, rapamycin was administrated intermittently (2 weeks per month) starting from the age of 2 mo. Rapamycin inhibited age-related weight gain, decreased aging rate, increased lifespan (especially in the last survivors) and delayed spontaneous cancer. 22.9% of rapamycin-treated mice survived the age of death of the last mouse in control group. Thus we demonstrated for the first time in normal inbred mice that lifespan can be extended by rapamycin. This opens an avenue to develop optimal doses and schedules of rapamycin as an anti-aging modality.     

Adaptation of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> to unfavorable growth medium affects lifespan and age-related fecundity

Experimental adaptation of Drosophila melanogaster to nutrient-deficient starch-based (S) medium resulted in lifespan shortening, increased early-life fecundity, accelerated reproductive aging, and sexually dimorphic survival curves. The direction of all these evolutionary changes coincides with the direction of phenotypic plasticity observed in non-adapted flies cultured on S medium. High adult mortality rate caused by unfavorable growth medium apparently was the main factor of selection during the evolutionary experiment. The results are partially compatible with Williams’ hypothesis, which states that increased mortality rate should result in relaxed selection against mutations that decrease fitness late in life, and thus promote the evolution of shorter lifespan and earlier reproduction. However, our results do not confirm Williams’ prediction that the sex with higher mortality rate should undergo more rapid aging: lifespan shortening by S medium is more pronounced in naive males than females, but it was female lifespan that decreased more in the course of adaptation. These data, as well as the results of testing of F₁ hybrids between adapted and control lineages, are compatible with the idea that the genetic basis of longevity is different in the two sexes, and that evolutionary response to increased mortality rate depends on the degree to which the mortality is selective. Selective mortality can result in the development of longer (rather than shorter) lifespan in the course of evolution. The results also imply that antagonistic pleiotropy of alleles, which increase early-life fecundity at the cost of accelerated aging, played an important role in the evolutionary changes of females in the experimental lineage, while accumulation of deleterious mutations with late-life effects due to drift was more important in the evolution of male traits.     

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.     

Pharmacology,genomics, and the evolutionary biology of ageing

Aging is a multifold process affected by many genes and thus many biochemical pathways. This conclusion is underscored by the failure to find simple central controls for the aging process during the 20th Century. This situation poses a fundamental challenge to anti-aging medicine: how to develop effective therapies for a genomically complex pathology. We propose such a strategy. As a first step, we recommend the use of model systems in which significant genetic intervention is not proscribed or impractical. Second, we propose that work with such model systems begin with selected lines that have genetic enhancements that allow increased lifespan. Third, genomic methods should be used to identify a number of biochemical pathways for increasing lifespan. Fourth, biochemical pathways that have been identified in model systems would then be available for pharmaceutical development, first in rodents, eventually in a clinical human population. This may seem to be a cumbersome R&D strategy, but starting with human populations or inadequately pre-screened compounds would be unlikely to succeed because of the complexity of the aging problem.     

Pharmacology,Genomics, and the Evolutionary Biology of Ageing

Aging is a multifold process affected by many genes and thus many biochemical pathways. This conclusion is underscored by the failure to find simple central controls for the aging process during the 20th Century. This situation poses a fundamental challenge to anti-aging medicine: how to develop effective therapies for a genomically complex pathology. We propose such a strategy. As a first step, we recommend the use of model systems in which significant genetic intervention is not proscribed or impractical. Second, we propose that work with such model systems begin with selected lines that have genetic enhancements that allow increased lifespan. Third, genomic methods should be used to identify a number of biochemical pathways for increasing lifespan. Fourth, biochemical pathways that have been identified in model systems would then be available for pharmaceutical development, first in rodents, eventually in a clinical human population. This may seem to be a cumbersome R&D strategy, but starting with human populations or inadequately pre-screened compounds would be unlikely to succeed because of the complexity of the aging problem.     

Strain‐specific effects of parental age on offspring in Drosophila melanogaster

Maternal age is generally known to be negatively correlated with the lifespan of offspring in several animal models including yeast, rotifers, flies, and possibly in humans. However, several reports have shown positive effects of parental age on offspring lifespan. Thus, there was a need to investigate further the inconsistent results on the effect of parental age on lifespan. In this study, the effects of parental age on offspring fitness and lifespan were examined by using Drosophila melanogaster. The lifespan of offspring from old parents was significantly increased compared with that of the young counterparts in the Canton‐S (CS) strain but not in other D. melanogaster strains, such as Oregon‐R (OR) and w¹¹¹⁸. To find out why the lifespan is increased in the offspring from old parents in CS flies, fitness components that could modulate lifespan were examined in CS flies. Egg weight and body weight were reduced by parental aging and the offspring of old fathers or old mothers developed faster than that of the young. In addition, the offspring of old parents had increased resistance to oxidative and heat shock stresses. However, reproductive capacity, mating preference, and food intake were unaffected by parental aging. These results indicate that parental aging in CS strain D. melanogaster has beneficial effects on the lifespan and fitness of offspring. The presence of strain‐specific manner effects suggests that genetic background might be a significant factor in the parental age effect.     

EFFECTS ON FITNESS COMPONENTS OF P-ELEMENT INSERTS IN DROSOPHILA MELANOGASTER: ANALYSIS OF TRADE-OFFS

We analyzed the trade-offs between fitness components detected in four experiments in which traits were manipulated by inserting small (control) and large (treatment) P-elements into the Drosophila melanogaster genome. Treatment effects and the interactions of treatment with temperature, experiment, and line were caused by the greater length and different positions of the treatment insert. In inbred flies, the treatment decreased early and total fecundity. Whether it increased the lifespan of mated females depended upon adult density. Analysis of line-by-treatment-by-temperature interactions revealed hidden trade-offs that would have been missed by other methods. They included a significant trade-off between lifespan and early fecundity. At 25°C high early fecundity was associated with decreased reproductive rates and increased mortality rates 10–15 days later and persisting throughout life, but not at 29.5°C. Correlations with Gompertz coefficients suggested that flies that were heavier at eclosion also aged more slowly and that flies that aged more slowly had higher fecundity late in life at 25°C. The results support the view that lifespan trades off with fecundity and that late fecundity trades off with rate of aging in fruitflies. Genetic engineering is an independent method for the analysis of trade-offs that complements selection experiments.     

Repeated stress exposure results in a survival–reproduction trade-off in Drosophila melanogaster

While insect cold tolerance has been well studied, the vast majority of work has focused on the effects of a single cold exposure. However, many abiotic environmental stresses, including temperature, fluctuate within an organism''s lifespan. Given that organisms may trade-off survival at the cost of future reproduction, we investigated the effects of multiple cold exposures on survival and fertility in the model organism Drosophila melanogaster. We found that multiple cold exposures significantly decreased mortality compared with the same length of exposure in a single sustained bout, but significantly decreased fecundity (as measured by r, the intrinsic rate of increase) as well, owing to a shift in sex ratio. This change was reflected in a long-term decrease in glycogen stores in multiply exposed flies, while a brief effect on triglyceride stores was observed, suggesting flies are reallocating energy stores. Given that many environments are not static, this trade-off indicates that investigating the effects of repeated stress exposure is important for understanding and predicting physiological responses in the wild.     

Changes in the expression of four heat shock proteins during the aging process in Brachionus calyciflorus (rotifera)

Heat shock proteins (HSPs) are molecular chaperones and have an important role in the refolding and degradation of misfolded proteins, and these functions are related to aging. Rotifer is a useful model organism in aging research, owing to small body size (0.1–1 mm), short lifespan (6–14 days), and senescence phenotypes that can be measured relatively easily. Therefore, we used rotifer as a model to determine the role of four typical hsp genes on the aging process in order to provide a better understanding of rotifer aging. We cloned cDNA encoding hsp genes (hsp40, hsp60, hsp70, and hsp90) from the rotifer Brachionus calyciflorus Pallas, analyzed their molecular characteristics, determined its modulatory response under different temperatures and H₂O₂ concentrations and investigated the changes in expression of these genes during the aging process. We found that Bchsp70 mRNA expression significantly decreased with aging. In addition, we also studied the effects of dietary restriction (DR) and vitamin E on rotifer lifespan and reproduction and analyzed the changes in expression of these four Bchsp genes in rotifers treated with DR and vitamin E. The results showed that DR extended the lifespan of rotifers and reduced their fecundity, whereas vitamin E had no significant effect on rotifer lifespan or reproduction. Real-time PCR indicated that DR increased the expression of these four Bchsps. However, vitamin E only improved the expression of Bchsp60, and reduced the expression of Bchsp40, Bchsp70, and Bchsp90. DR pretreatment also increased rotifer survival rate under paraquat-induced oxidative stress. These results indicated that hsp genes had an important role in the anti-aging process.     

Delayed behavioural aging and altered mortality in Drosophila beta integrin mutants

The genetic basis for aging is being intensely investigated in a variety of model systems. Much of the focus in Drosophila has been on the molecular-genetic determinants of lifespan, whereas the molecular-genetic basis for age-related functional declines has been less vigorously explored. We evaluated behavioural aging and lifespan in flies harbouring loss-of-function mutations in myospheroid, the gene that encodes betaPS, a beta integrin. Integrins are adhesion molecules that regulate a number of cellular processes and developmental events. Their role in aging, however, has received limited attention. We report here that age-related declines in locomotor activity are ameliorated and that mean lifespan is increased in myospheroid mutants. The delayed functional senescence and altered mortality in myospheroid flies are independent of changes in body size, reproduction or stress resistance. Our data indicate that functional senescence and age-dependent mortality are influenced by beta integrins in Drosophila.     

Accelerating aging by mouse reverse genetics: a rational approach to understanding longevity

Investigating the molecular basis of aging has been difficult, primarily owing to the pleiotropic and segmental nature of the aging phenotype. There are many often interacting symptoms of aging, some of which are obvious and appear to be common to every aged individual, whereas others affect only a subset of the elderly population. Although at first sight this would suggest multiple molecular mechanisms of aging, there now appears to be almost universal consensus that aging is ultimately the result of the accumulation of somatic damage in cellular macromolecules, with reactive oxygen species likely to be the main damage-inducing agent. What remains significant is unravelling how such damage can give rise to the large variety of aging symptoms and how these can be controlled. Although humans, with over a century of clinical observations, remain the obvious target of study, the mouse, with a relatively short lifespan, easy genetic accessibility and close relatedness to humans, is the tool par excellence to model aging-related phenotypes and test strategies of intervention. Here we present the argument that mouse models with engineered defects in genome maintenance systems are especially important because they often exhibit a premature appearance of aging symptoms. Confirming studies on human segmental progeroid syndromes, most of which are based on heritable mutations in genes involved in genome maintenance, the results thus far obtained with mouse models strongly suggest that lifespan and onset of aging are directly related to the quality of DNA metabolism. This may be in keeping with the recent discovery of a possible 'universal survival' pathway that improves antioxidant defence and genome maintenance and simultaneously extends lifespan in the mouse and several invertebrate species.     

Hyaluronan-chondroitin hybrid oligosaccharides as new life science research tools

Mitochondrial trifunctional protein (MTP), which consists of the MTPα and MTPβ subunits, catalyzes long-chain fatty acid β-oxidation. MTP deficiency in humans results in Reye-like syndrome. Here, we generated Drosophila models of MTP deficiency by targeting two genes encoding Drosophila homologs of human MTPα and MTPβ, respectively. Both Mtpα(KO) and Mtpβ(KO) flies were viable, but demonstrated reduced lifespan, defective locomotor activity, and reduced fecundity represented by the number of eggs laid by the females. The phenotypes of Mtpα(KO) flies were generally more striking than those of Mtpβ(KO) flies. Mtpα(KO) flies were hypersensitive to fasting, and retained lipid droplets in their fat body cells as in non-fasting conditions. The amount of triglyceride was also unchanged upon fasting in Mtpα(KO) flies, suggesting that lipid mobilization was disrupted. Finally, we showed that both Mtpα(KO) and Mtpβ(KO) flies accumulated acylcarnitine and hydroxyacylcarnitine, diagnostic markers of MTP deficiencies in humans. Our results indicated that both Mtpα(KO) and Mtpβ(KO) flies were impaired in long-chain fatty acid β-oxidation. These flies should be useful as a model system to investigate the molecular pathogenesis of MTP deficiency.     

Invertebrates as model organisms for research on aging biology

Invertebrate model systems, such as nematodes and fruit flies, have provided valuable information about the genetics and cellular biology involved in aging. However, limitations of these simple, genetically tractable organisms suggest the need for other model systems, some of them invertebrate, to facilitate further advances in the understanding of mechanisms of aging and longevity in mammals, including humans. This paper introduces 10 review articles about the use of invertebrate model systems for the study of aging by authors who participated in an ‘NIA-NIH symposium on aging in invertebrate model systems’ at the 2013 International Congress for Invertebrate Reproduction and Development. In contrast to the highly derived characteristics of nematodes and fruit flies as members of the superphylum Ecdysozoa, cnidarians, such as Hydra, are more ‘basal’ organisms that have a greater number of genetic orthologs in common with humans. Moreover, some other new model systems, such as the urochordate Botryllus schlosseri, the tunicate Ciona, and the sea urchins (Echinodermata) are members of the Deuterostomia, the same superphylum that includes all vertebrates, and thus have mechanisms that are likely to be more closely related to those occurring in humans. Additional characteristics of these new model systems, such as the recent development of new molecular and genetic tools and a more similar pattern to humans of regeneration and stem cell function suggest that these new model systems may have unique advantages for the study of mechanisms of aging and longevity.     

Activation of AMPK by the Putative Dietary Restriction Mimetic Metformin Is Insufficient to Extend Lifespan in Drosophila

The biguanide drug, metformin, commonly used to treat type-2 diabetes, has been shown to extend lifespan and reduce fecundity in C. elegans through a dietary restriction-like mechanism via the AMP-activated protein kinase (AMPK) and the AMPK-activating kinase, LKB1. We have investigated whether the longevity-promoting effects of metformin are evolutionarily conserved using the fruit fly, Drosophila melanogaster. We show here that while feeding metformin to adult Drosophila resulted in a robust activation of AMPK and reduced lipid stores, it did not increase lifespan in either male or female flies. In fact, we found that when administered at high concentrations, metformin is toxic to flies. Furthermore, no decreases in female fecundity were observed except at the most toxic dose. Analysis of intestinal physiology after metformin treatment suggests that these deleterious effects may result from disruptions to intestinal fluid homeostasis. Thus, metformin appears to have evolutionarily conserved effects on metabolism but not on fecundity or lifespan.     

Acyl coenzyme A binding protein (ACBP): An aging- and disease-relevant “autophagy checkpoint”

Acyl coenzyme A binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is a phylogenetically ancient protein present in some eubacteria and the entire eukaryotic radiation. In several eukaryotic phyla, ACBP/DBI transcends its intracellular function in fatty acid metabolism because it can be released into the extracellular space. This ACBP/DBI secretion usually occurs in response to nutrient scarcity through an autophagy-dependent pathway. ACBP/DBI and its peptide fragments then act on a range of distinct receptors that diverge among phyla, namely metabotropic G protein-coupled receptor in yeast (and likely in the mammalian central nervous system), a histidine receptor kinase in slime molds, and ionotropic gamma-aminobutyric acid (GABA)_A receptors in mammals. Genetic or antibody-mediated inhibition of ACBP/DBI orthologs interferes with nutrient stress-induced adaptations such as sporulation or increased food intake in multiple species, as it enhances lifespan or healthspan in yeast, plant leaves, nematodes, and multiple mouse models. These lifespan and healthspan-extending effects of ACBP/DBI suppression are coupled to the induction of autophagy. Altogether, it appears that neutralization of extracellular ACBP/DBI results in “autophagy checkpoint inhibition” to unleash the anti-aging potential of autophagy. Of note, in humans, ACBP/DBI levels increase in various tissues, as well as in the plasma, in the context of aging, obesity, uncontrolled infection or cardiovascular, inflammatory, neurodegenerative, and malignant diseases.