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Find video protocols related to scientific articles indexed in Pubmed.
Interplay of dFOXO and two ETS-family transcription factors determines lifespan in Drosophila melanogaster.
PLoS Genet.
PUBLISHED: 09-01-2014
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Forkhead box O (FoxO) transcription factors (TFs) are key drivers of complex transcriptional programmes that determine animal lifespan. FoxOs regulate a number of other TFs, but how these TFs in turn might mediate the anti-ageing programmes orchestrated by FoxOs in vivo is unclear. Here, we identify an E-twenty six (ETS)-family transcriptional repressor, Anterior open (Aop), as regulated by the single Drosophila melanogaster FoxO (dFOXO) in the adult gut. AOP, the functional orthologue of the human Etv6/Tel protein, binds numerous genomic sites also occupied by dFOXO and counteracts the activity of an ETS activator, Pointed (Pnt), to prevent the lifespan-shortening effects of co-activation of dFOXO and PNT. This detrimental synergistic effect of dFOXO and PNT appears to stem from a mis-regulation of lipid metabolism. At the same time, AOP activity in another fly organ, the fat body, has further beneficial roles, regulating genes in common with dfoxo, such as the secreted, non-sensory, odorant binding protein (Obp99b), and robustly extending lifespan. Our study reveals a complex interplay between evolutionarily conserved ETS factors and dFOXO, the functional significance of which may extend well beyond animal lifespan.
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Intervening in ageing to prevent the diseases of ageing.
Trends Endocrinol. Metab.
PUBLISHED: 08-28-2014
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Increases in human lifespan worldwide have revealed that advancing age is the predominant risk factor for major life-threatening diseases. Recent work has shown that ageing in diverse animals, including humans, is malleable to specific types of genetic mutation, diet, and drugs that can extend lifespan and improve health during ageing. These findings point to the prospect of broad-spectrum preventive medicine for the diseases of ageing based on intervention in relevant aspects of the ageing process itself.
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C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins.
Science
PUBLISHED: 08-07-2014
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An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question, we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats, but not stop codon-interrupted "RNA-only" repeats in Drosophila caused adult-onset neurodegeneration. Thus, expanded repeats promoted neurodegeneration through dipeptide repeat proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence revealed that both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration.
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Lithium suppresses A? pathology by inhibiting translation in an adult Drosophila model of Alzheimer's disease.
Front Aging Neurosci
PUBLISHED: 07-30-2014
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The greatest risk factor for Alzheimer's disease (AD) is age, and changes in the ageing nervous system are likely contributors to AD pathology. Amyloid beta (A?) accumulation, which occurs as a result of the amyloidogenic processing of amyloid precursor protein (APP), is thought to initiate the pathogenesis of AD, eventually leading to neuronal cell death. Previously, we developed an adult-onset Drosophila model of AD. Mutant A?42 accumulation led to increased mortality and neuronal dysfunction in the adult flies. Furthermore, we showed that lithium reduced A?42 protein, but not mRNA, and was able to rescue A?42-induced toxicity. In the current study, we investigated the mechanism/s by which lithium modulates A?42 protein levels and A?42 induced toxicity in the fly model. We found that lithium caused a reduction in protein synthesis in Drosophila and hence the level of A?42. At both the low and high doses tested, lithium rescued the locomotory defects induced by A?42, but it rescued lifespan only at lower doses, suggesting that long-term, high-dose lithium treatment may have induced toxicity. Lithium also down-regulated translation in the fission yeast Schizosaccharomyces pombe associated with increased chronological lifespan. Our data highlight a role for lithium and reduced protein synthesis as potential therapeutic targets for AD pathogenesis.
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Using doubly-labeled water to measure energy expenditure in an important small ectotherm Drosophila melanogaster.
J Genet Genomics
PUBLISHED: 04-23-2014
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Energy expenditure is a key variable in the study of ageing, and the fruit fly Drosophila melanogaster is a model organism that has been used to make step changes in our understanding of the ageing process. Standard methods for measurement of energy expenditure involve placing individuals in metabolic chambers where their oxygen consumption and CO2 production can be quantified. These measurements require separating individuals from any social context, and may only poorly reflect the environment in which the animals normally live. The doubly-labeled water (DLW) method is an isotope-based technique for measuring energy expenditure which overcomes these problems. However, technical challenges mean that the smallest animals this method has been previously applied to weighed 50-200 mg. We overcame these technical challenges to measure energy demands in Drosophila weighing 0.78 mg. Mass-specific energy expenditure varied between 43 and 65 mW·g(-1). These estimates are considerably higher than estimates using indirect calorimetry of Drosophila in small metabolic chambers (around 18 mW·g(-1)). The methodology we have established extends downwards by three orders of magnitude the size of animals that can be measured using DLW. This approach may be of considerable value in future ageing research attempting to understand the genetic and genomic basis of ageing.
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Lowered insulin signalling ameliorates age-related sleep fragmentation in Drosophila.
PLoS Biol.
PUBLISHED: 04-01-2014
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Sleep fragmentation, particularly reduced and interrupted night sleep, impairs the quality of life of older people. Strikingly similar declines in sleep quality are seen during ageing in laboratory animals, including the fruit fly Drosophila. We investigated whether reduced activity of the nutrient- and stress-sensing insulin/insulin-like growth factor (IIS)/TOR signalling network, which ameliorates ageing in diverse organisms, could rescue the sleep fragmentation of ageing Drosophila. Lowered IIS/TOR network activity improved sleep quality, with increased night sleep and day activity and reduced sleep fragmentation. Reduced TOR activity, even when started for the first time late in life, improved sleep quality. The effects of reduced IIS/TOR network activity on day and night phenotypes were mediated through distinct mechanisms: Day activity was induced by adipokinetic hormone, dFOXO, and enhanced octopaminergic signalling. In contrast, night sleep duration and consolidation were dependent on reduced S6K and dopaminergic signalling. Our findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans.
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Cell-nonautonomous effects of dFOXO/DAF-16 in aging.
Cell Rep
PUBLISHED: 01-14-2014
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Drosophila melanogaster and Caenorhabditis elegans each carry a single representative of the Forkhead box O (FoxO) family of transcription factors, dFOXO and DAF-16, respectively. Both are required for lifespan extension by reduced insulin/Igf signaling, and their activation in key tissues can extend lifespan. Aging of these tissues may limit lifespan. Alternatively, FoxOs may promote longevity cell nonautonomously by signaling to themselves (FoxO to FoxO) or other factors (FoxO to other) in distal tissues. Here, we show that activation of dFOXO and DAF-16 in the gut/fat body does not require dfoxo/daf-16 elsewhere to extend lifespan. Rather, in Drosophila, activation of dFOXO in the gut/fat body or in neuroendocrine cells acts on other organs to promote healthy aging by signaling to other, as-yet-unidentified factors. Whereas FoxO-to-FoxO signaling appears to be required for metabolic homeostasis, our results pinpoint FoxO-to-other signaling as an important mechanism through which localized FoxO activity ameliorates aging.
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Signaling by IL-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin.
Nat. Immunol.
PUBLISHED: 01-08-2014
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Obesity and resistance to insulin are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation; however, its role in this context remains controversial. Here we found that mice with an inactivated gene encoding the IL-6R? chain of the receptor for IL-6 in myeloid cells (Il6ra(?myel) mice) developed exaggerated deterioration of glucose homeostasis during diet-induced obesity, due to enhanced resistance to insulin. Tissues targeted by insulin showed increased inflammation and a shift in macrophage polarization. IL-6 induced expression of the receptor for IL-4 and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6ra(?myel) mice were resistant to IL-4-mediated alternative polarization of macrophages and exhibited enhanced susceptibility to lipopolysaccharide (LPS)-induced endotoxemia. Our results identify signaling via IL-6 as an important determinant of the alternative activation of macrophages and assign an unexpected homeostatic role to IL-6 in limiting inflammation.
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Gender and longevity: why do men die earlier than women? Comparative and experimental evidence.
Best Pract. Res. Clin. Endocrinol. Metab.
PUBLISHED: 06-22-2013
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Sex differences in lifespan exist world-wide, with women outliving men by more than a decade in some countries. The gender gap is not a uniquely human phenomenon; most sexually reproducing species examined show sex differences in patterns of ageing, yet a comprehensive explanation does not exist. Here, we discuss how ageing responds to natural selection on traits that arise as a consequence of sexuality. Sexual dimorphisms in vertebrates are mediated by sex-steroids, such as androgens and oestrogens, and we examine their regulation of biological processes that can affect ageing and lifespan. The sexes can respond differently to dietary restriction and altered activity of nutrient-sensing pathways, with females showing a greater plasticity for life extension. We suggest that the cross-regulation of steroid hormone and nutrient-sensing signalling pathways is a promising process for further study in understanding the biological basis for the gender gap.
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The hallmarks of aging.
Cell
PUBLISHED: 06-11-2013
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Aging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death. This deterioration is the primary risk factor for major human pathologies, including cancer, diabetes, cardiovascular disorders, and neurodegenerative diseases. Aging research has experienced an unprecedented advance over recent years, particularly with the discovery that the rate of aging is controlled, at least to some extent, by genetic pathways and biochemical processes conserved in evolution. This Review enumerates nine tentative hallmarks that represent common denominators of aging in different organisms, with special emphasis on mammalian aging. These hallmarks are: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. A major challenge is to dissect the interconnectedness between the candidate hallmarks and their relative contributions to aging, with the final goal of identifying pharmaceutical targets to improve human health during aging, with minimal side effects.
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A holidic medium for Drosophila melanogaster.
Nat. Methods
PUBLISHED: 04-28-2013
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A critical requirement for research using model organisms is a well-defined and consistent diet. There is currently no complete chemically defined (holidic) diet available for Drosophila melanogaster. We describe a holidic medium that is equal in performance to an oligidic diet optimized for adult fecundity and lifespan. This holidic diet supports development over multiple generations but at a reduced rate. Over 7 years of experiments, the holidic diet yielded more consistent experimental outcomes than did oligidic food for egg laying by females. Nutrients and drugs were more available to flies in holidic medium and, similar to dietary restriction on oligidic food, amino acid dilution increased fly lifespan. We used this holidic medium to investigate amino acid-specific effects on food-choice behavior and report that folic acid from the microbiota is sufficient for Drosophila development.
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Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I.
Nat. Med.
PUBLISHED: 04-15-2013
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Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.
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Polymorphism in the neurofibromin gene, Nf1, is associated with antagonistic selection on wing size and development time in Drosophila melanogaster.
Mol. Ecol.
PUBLISHED: 02-10-2013
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In many invertebrates, body size shows genetically based clines, with size increasing in colder climates. Large body size is typically associated with prolonged development times. We consider variation in the CNS-specific gene neurofibromin 1 (Nf1) and its association with body size and development time. We identified two major Nf1 haplotypes in natural populations, Nf1-insertion-A and Nf1-deletion-G. These haplotypes are characterized by a 45-base insertion/deletion (INDEL) in Nf1 intron 2 and an A/G synonymous substitution (locus L17277). Linkage disequilibrium (LD) between the INDEL and adjacent sites is high but appears to be restricted within the Nf1 gene interval. In Australia, the frequency of the Nf1-insertion-A haplotype increases with latitude where wing size is larger, independent of the chromosomal inversion In(3R)Payne. Unexpectedly, the Nf1-insertion-A haplotype is negatively associated with wing size. We found that the Nf1-insertion-A haplotype is enriched in females with shorter development time. This suggests that the Nf1 haplotype cline may be driven by selection for development time rather than size; females from southern (higher latitude) D. melanogaster populations maintain a rapid development time despite being relatively larger, and the higher incidence of Nf1-insertion-A in Southern Australia may contribute to this pattern, whereas the effects of the Nf1 haplotypes on size may be countered by other loci with antagonistic effects on size and development time. Our results point to the potential complexity involved in identifying selection on genetic variants exhibiting pleiotropic effects when studies are based on spatial patterns or association studies.
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MTERF3 regulates mitochondrial ribosome biogenesis in invertebrates and mammals.
PLoS Genet.
PUBLISHED: 01-03-2013
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Regulation of mitochondrial DNA (mtDNA) expression is critical for the control of oxidative phosphorylation in response to physiological demand, and this regulation is often impaired in disease and aging. We have previously shown that mitochondrial transcription termination factor 3 (MTERF3) is a key regulator that represses mtDNA transcription in the mouse, but its molecular mode of action has remained elusive. Based on the hypothesis that key regulatory mechanisms for mtDNA expression are conserved in metazoans, we analyzed Mterf3 knockout and knockdown flies. We demonstrate here that decreased expression of MTERF3 not only leads to activation of mtDNA transcription, but also impairs assembly of the large mitochondrial ribosomal subunit. This novel function of MTERF3 in mitochondrial ribosomal biogenesis is conserved in the mouse, thus we identify a novel and unexpected role for MTERF3 in coordinating the crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression.
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Dietary restriction extends lifespan in wild-derived populations of Drosophila melanogaster.
PLoS ONE
PUBLISHED: 01-01-2013
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Dietary restriction (DR) can result in lifespan-extension and improved function and health during ageing. Although the impact of DR on lifespan and health has been established in a variety of organisms, most DR experiments are carried out on laboratory strains that have often undergone adaptation to laboratory conditions. The effect of DR on animals recently derived from wild populations is rarely assessed. We measured the DR response of four populations of Drosophila melanogaster within two generations of collection from the wild. All populations responded to DR with an increase in lifespan and a decrease in female fecundity, similarly to a control, laboratory-adapted strain. These effects of DR are thus not a result of adaptation to laboratory conditions, and reflect the characteristics of natural populations.
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Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor.
Aging Cell
PUBLISHED: 07-12-2011
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Mammals possess multiple insulin-like growth factor (IGF) binding proteins (IGFBPs), and related proteins, that modulate the activity of insulin/IGF signalling (IIS), a conserved neuroendocrine signalling pathway that affects animal lifespan. Here, we examine if increased levels of an IGFBP-like protein can extend lifespan, using Drosophila as the model organism. We demonstrate that Imaginal morphogenesis protein-Late 2 (IMP-L2), a secreted protein and the fly homologue of the human IGFBP7 tumour suppressor, is capable of binding at least two of the seven Drosophila insulin-like peptides (DILPs), namely native DILP2 and DILP5 as present in the adult fly. Increased expression of Imp-L2 results in phenotypic changes in the adult consistent with down-regulation of IIS, including accumulation of eIF-4E binding protein mRNA, increase in storage lipids, reduced fecundity and enhanced oxidative stress resistance. Increased Imp-L2 results in up-regulation of dilp2, dilp3 and dilp5 mRNA, revealing a feedback circuit that is mediated via the fly gut and/or fat body. Importantly, over-expression of Imp-L2, ubiquitous or restricted to DILP-producing cells or gut and fat body, extends lifespan. This enhanced longevity can also be observed upon adult-onset induction of Imp-L2, indicating it is not attributable to developmental changes. Our findings point to the possibility that an IGFBP or a related protein, such as IGFBP7, plays a role in mammalian aging.
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Death and dessert: nutrient signalling pathways and ageing.
Curr. Opin. Cell Biol.
PUBLISHED: 07-04-2011
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Reduction in nutrient intake without malnutrition can delay ageing and extend healthy life in diverse organisms from yeast to primates. This effect can be recapitulated by genetic or pharmacological dampening of the signal through nutrient signalling pathways, making them a promising target for intervention into human ageing and age-related diseases. Here we review the current knowledge of the interactions between nutrient signalling pathways and ageing, focusing on the findings emerged in the past few years.
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Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila.
Nature
PUBLISHED: 06-10-2011
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Overexpression of sirtuins (NAD(+)-dependent protein deacetylases) has been reported to increase lifespan in budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans and Drosophila melanogaster. Studies of the effects of genes on ageing are vulnerable to confounding effects of genetic background. Here we re-examined the reported effects of sirtuin overexpression on ageing and found that standardization of genetic background and the use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high-level sir-2.1 overexpression abrogated the longevity increase, but did not abrogate sir-2.1 overexpression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low-copy-number sir-2.1 overexpression also abrogated longevity. A Drosophila strain with ubiquitous overexpression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported, but was not long-lived relative to the appropriate transgenic controls, and nor was a new line with stronger overexpression of dSir2. These findings underscore the importance of controlling for genetic background and for the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life-extending effect of dietary restriction on ageing in Drosophila has also been reported to be dSir2 dependent. We found that dietary restriction increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects of sirtuins on lifespan in C. elegans and Drosophila.
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dFOXO-independent effects of reduced insulin-like signaling in Drosophila.
Aging Cell
PUBLISHED: 05-06-2011
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The insulin/insulin-like growth factor-like signaling (IIS) pathway in metazoans has evolutionarily conserved roles in growth control, metabolic homeostasis, stress responses, reproduction, and lifespan. Genetic manipulations that reduce IIS in the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the mouse have been shown not only to produce substantial increases in lifespan but also to ameliorate several age-related diseases. In C. elegans, the multitude of phenotypes produced by the reduction in IIS are all suppressed in the absence of the worm FOXO transcription factor, DAF-16, suggesting that they are all under common regulation. It is not yet clear in other animal models whether the activity of FOXOs mediate all of the physiological effects of reduced IIS, especially increased lifespan. We have addressed this issue by examining the effects of reduced IIS in the absence of dFOXO in Drosophila, using a newly generated null allele of dfoxo. We found that the removal of dFOXO almost completely blocks IIS-dependent lifespan extension. However, unlike in C. elegans, removal of dFOXO does not suppress the body size, fecundity, or oxidative stress resistance phenotypes of IIS-compromised flies. In contrast, IIS-dependent xenobiotic resistance is fully dependent on dFOXO activity. Our results therefore suggest that there is evolutionary divergence in the downstream mechanisms that mediate the effects of IIS. They also imply that in Drosophila, additional factors act alongside dFOXO to produce IIS-dependent responses in body size, fecundity, and oxidative stress resistance and that these phenotypes are not causal in IIS-mediated extension of lifespan.
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The bicoid stability factor controls polyadenylation and expression of specific mitochondrial mRNAs in Drosophila melanogaster.
PLoS Genet.
PUBLISHED: 04-14-2011
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The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation.
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Dietary restriction and aging: a unifying perspective.
Cell Metab.
PUBLISHED: 04-11-2011
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Dietary restriction (DR) and mutations in nutrient signaling pathways can extend healthy life span in diverse organisms. Studying the interaction between these interventions should reveal mechanisms of aging, but has yielded some apparently contradictory results. A multidimensional representation of nutrition, called the geometric framework, can better describe the responses of life span and other traits, including metabolism, and can reconcile these apparent contradictions. We provide examples showing that it is more informative to analyze DR in terms of dietary balance and that dietary optimization for life span is critical for studies examining the biology of aging and other traits.
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Unraveling the biological roles of reactive oxygen species.
Cell Metab.
PUBLISHED: 04-05-2011
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Reactive oxygen species are not only harmful agents that cause oxidative damage in pathologies, they also have important roles as regulatory agents in a range of biological phenomena. The relatively recent development of this more nuanced view presents a challenge to the biomedical research community on how best to assess the significance of reactive oxygen species and oxidative damage in biological systems. Considerable progress is being made in addressing these issues, and here we survey some recent developments for those contemplating research in this area.
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Molecular basis of adaptive shift in body size in Drosophila melanogaster: functional and sequence analyses of the Dca gene.
Mol. Biol. Evol.
PUBLISHED: 04-04-2011
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Latitudinal body size clines in animals conforming to Bergmanns rule occur on many continents but isolating their underlying genetic basis remains a challenge. In Drosophila melanogaster, the gene Dca accounts for approximately 5-10% of the natural wing size variation (McKechnie SW, Blacket MJ, Song SV, Rako L, Carroll X, Johnson TK, Jensen LT, Lee SF, Wee CW, Hoffmann AA. 2010. A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila. Mol Ecol. 19:775-784). We present here functional evidence that Dca is a negative regulator of wing size. A significant negative latitudinal cline of Dca gene expression was detected in synchronized third instar larvae. In addition, we clarified the evolutionary history of the three most common Dca promoter alleles (Dca237-1, Dca237-2, and Dca247) and showed that the insertion allele (Dca247), whose frequency increases with latitude, is associated with larger wing centroid size and higher average cell number in male flies. Finally, we showed that the overall linkage disequilibrium (LD) was low in the Dca promoter and that the insertion/deletion polymorphism that defines the Dca alleles was in strong LD with two other upstream sites. Our results provide strong support that Dca is a candidate for climatic adaptation in D. melanogaster.
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A longer and healthier life with TOR down-regulation: genetics and drugs.
Biochem. Soc. Trans.
PUBLISHED: 03-25-2011
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Genetic down-regulation of a major nutrient-sensing pathway, TOR (target of rapamycin) signalling, can improve health and extend lifespan in evolutionarily distant organisms such as yeast and mammals. Recently, it has been demonstrated that treatment with a pharmacological inhibitor of the TOR pathway, rapamycin, can replicate those findings and improve aging in a variety of model organisms. The proposed underlying anti-aging mechanisms are down-regulated translation, increased autophagy, altered metabolism and increased stress resistance.
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Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signalling.
Mol. Syst. Biol.
PUBLISHED: 03-10-2011
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FoxO transcription factors, inhibited by insulin/insulin-like growth factor signalling (IIS), are crucial players in numerous organismal processes including lifespan. Using genomic tools, we uncover over 700 direct dFOXO targets in adult female Drosophila. dFOXO is directly required for transcription of several IIS components and interacting pathways, such as TOR, in the wild-type fly. The genomic locations occupied by dFOXO in adults are different from those observed in larvae or cultured cells. These locations remain unchanged upon activation by stresses or reduced IIS, but the binding is increased and additional targets activated upon genetic reduction in IIS. We identify the part of the IIS transcriptional response directly controlled by dFOXO and the indirect effects and show that parts of the transcriptional response to IIS reduction do not require dfoxo. Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the indirect and dfoxo-independent effects. We demonstrate genome-wide evolutionary conservation of dFOXO targets between the fly and the worm Caenorhabditis elegans, enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.
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Electrophysiological recordings from the giant fiber pathway of D. melanogaster.
J Vis Exp
PUBLISHED: 02-10-2011
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When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.
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Measurement of H2O2 within living Drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix.
Cell Metab.
PUBLISHED: 02-03-2011
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Hydrogen peroxide (H(2)O(2)) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H(2)O(2) in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H(2)O(2) levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H(2)O(2) to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H(2)O(2) that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H(2)O(2) with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H(2)O(2) correlates with aging, it may not be causative.
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Replication of extended lifespan phenotype in mice with deletion of insulin receptor substrate 1.
PLoS ONE
PUBLISHED: 01-25-2011
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We previously reported that global deletion of insulin receptor substrate protein 1 (Irs1) extends lifespan and increases resistance to several age-related pathologies in female mice. However, no effect on lifespan was observed in male Irs1 null mice. We suggested at the time that the lack of any effect in males might have been due to a sample size issue. While such lifespan studies are essential to our understanding of the aging process, they are generally based on survival curves derived from single experiments, primarily due to time and economic constraints. Consequently, the robustness of such findings as a basis for further investigation has been questioned. We have therefore measured lifespan in a second, separate cohort of Irs1 null female mice, and show that, consistent with our previous finding, global deletion of Irs1 significantly extends lifespan in female mice. In addition, an augmented and completed study demonstrates lifespan extension in male Irs1 null mice. Therefore, we show that reduced IRS1-dependent signalling is a robust mechanism through which mammalian lifespan can be modulated.
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Some highlights of research on aging with invertebrates, 2010.
Aging Cell
PUBLISHED: 12-07-2010
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This annual review focuses on invertebrate model organisms, which continue to yield fundamental new insights into mechanisms of aging. This year, the budding yeast has been used to understand how asymmetrical partitioning of cellular constituents at cell division can produce a rejuvenated offspring from an aging parent. Blocking of sensation of carbon dioxide is shown to extend fly lifespan and to mediate the lifespan-shortening effect of sensory exposure to fermenting yeast. A new study of daf-16, the key forkhead transcription factor that mediates extension of lifespan by mutants in the insulin-signalling pathway in Caenorhabditis elegans, demonstrates that expression of tissue-specific isoforms with different patterns of response to upstream signalling mediates the highly pleiotropic effects of the pathway on lifespan and other traits. A new approach to manipulating mitochondrial activity in Drosophila, by introducing the yeast NADH-ubiquinone oxidoreductase, shows promise for understanding the role of mitochondrial reactive oxygen species in aging. An exciting new study of yeast and mammalian cells implicates deterioration of the nuclear pore, and consequent leakage of cytoplasmic components into the nucleus, as an important cause of aging in postmitotic tissues. Loss of, or damage to, chromosome-associated histones is also implicated in the determination of lifespan in yeast, worms and fruit flies. The relationship between functional aging, susceptibility to aging-related disease and lifespan itself are explored in two studies in C. elegans, the first examining the role of dietary restriction and reduced insulin signalling in cognitive decline and the second profiling aggregation of the proteome during aging. The invertebrates continue to be a power house of discovery for future work in mammals.
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Insulin signalling regulates remating in female Drosophila.
Proc. Biol. Sci.
PUBLISHED: 08-25-2010
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Mating rate is a major determinant of female lifespan and fitness, and is predicted to optimize at an intermediate level, beyond which superfluous matings are costly. In female Drosophila melanogaster, nutrition is a key regulator of mating rate but the underlying mechanism is unknown. The evolutionarily conserved insulin/insulin-like growth factor-like signalling (IIS) pathway is responsive to nutrition, and regulates development, metabolism, stress resistance, fecundity and lifespan. Here we show that inhibition of IIS, by ablation of Drosophila insulin-like peptide (DILP)-producing median neurosecretory cells, knockout of dilp2, dilp3 or dilp5 genes, expression of a dominant-negative DILP-receptor (InR) transgene or knockout of Lnk, results in reduced female remating rates. IIS-mediated regulation of female remating can occur independent of virgin receptivity, developmental defects, reduced body size or fecundity, and the receipt of the female receptivity-inhibiting male sex peptide. Our results provide a likely mechanism by which females match remating rates to the perceived nutritional environment. The findings suggest that longevity-mediating genes could often have pleiotropic effects on remating rate. However, overexpression of the IIS-regulated transcription factor dFOXO in the fat body-which extends lifespan-does not affect remating rate. Thus, long life and reduced remating are not obligatorily coupled.
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Inhibition of GSK-3 ameliorates Abeta pathology in an adult-onset Drosophila model of Alzheimers disease.
PLoS Genet.
PUBLISHED: 07-23-2010
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Abeta peptide accumulation is thought to be the primary event in the pathogenesis of Alzheimers disease (AD), with downstream neurotoxic effects including the hyperphosphorylation of tau protein. Glycogen synthase kinase-3 (GSK-3) is increasingly implicated as playing a pivotal role in this amyloid cascade. We have developed an adult-onset Drosophila model of AD, using an inducible gene expression system to express Arctic mutant Abeta42 specifically in adult neurons, to avoid developmental effects. Abeta42 accumulated with age in these flies and they displayed increased mortality together with progressive neuronal dysfunction, but in the apparent absence of neuronal loss. This fly model can thus be used to examine the role of events during adulthood and early AD aetiology. Expression of Abeta42 in adult neurons increased GSK-3 activity, and inhibition of GSK-3 (either genetically or pharmacologically by lithium treatment) rescued Abeta42 toxicity. Abeta42 pathogenesis was also reduced by removal of endogenous fly tau; but, within the limits of detection of available methods, tau phosphorylation did not appear to be altered in flies expressing Abeta42. The GSK-3-mediated effects on Abeta42 toxicity appear to be at least in part mediated by tau-independent mechanisms, because the protective effect of lithium alone was greater than that of the removal of tau alone. Finally, Abeta42 levels were reduced upon GSK-3 inhibition, pointing to a direct role of GSK-3 in the regulation of Abeta42 peptide level, in the absence of APP processing. Our study points to the need both to identify the mechanisms by which GSK-3 modulates Abeta42 levels in the fly and to determine if similar mechanisms are present in mammals, and it supports the potential therapeutic use of GSK-3 inhibitors in AD.
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Ageing in Drosophila: the role of the insulin/Igf and TOR signalling network.
Exp. Gerontol.
PUBLISHED: 06-29-2010
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A remarkable discovery of recent years is that, despite the complexity of ageing, simple genetic interventions can increase lifespan and improve health during ageing in laboratory animals. The pathways involved have often proved to sense nutrients and to match costly activities of organisms, such as growth, metabolism and reproduction, to nutrient status. For instance, the insulin/insulin-like growth factor and Target of Rapamycin signalling network has proved to play a function in ageing, from yeast to mammals, seemingly including humans. In the fruit fly Drosophila, altered activity of several components of this network can increase lifespan and improve locomotor and cardiac function during ageing. The fly brain, fat body (equivalent of mammalian liver and white adipose tissue) and the germ line are important in determination of lifespan, with considerable communication between different tissues. Cellular detoxification pathways, increased autophagy and altered protein synthesis have all been implicated in increased lifespan from reduced IIS/TOR activity, with the role of defence against oxidative stress unresolved. Reduced IIS/TOR signalling can alter or block the response of lifespan to dietary restriction. Reduced IIS can act acutely to lower death rate, implying that it may ameliorate the effects of ageing-related damage, rather than preventing it.
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Extending healthy life span--from yeast to humans.
Science
PUBLISHED: 04-17-2010
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When the food intake of organisms such as yeast and rodents is reduced (dietary restriction), they live longer than organisms fed a normal diet. A similar effect is seen when the activity of nutrient-sensing pathways is reduced by mutations or chemical inhibitors. In rodents, both dietary restriction and decreased nutrient-sensing pathway activity can lower the incidence of age-related loss of function and disease, including tumors and neurodegeneration. Dietary restriction also increases life span and protects against diabetes, cancer, and cardiovascular disease in rhesus monkeys, and in humans it causes changes that protect against these age-related pathologies. Tumors and diabetes are also uncommon in humans with mutations in the growth hormone receptor, and natural genetic variants in nutrient-sensing pathways are associated with increased human life span. Dietary restriction and reduced activity of nutrient-sensing pathways may thus slow aging by similar mechanisms, which have been conserved during evolution. We discuss these findings and their potential application to prevention of age-related disease and promotion of healthy aging in humans, and the challenge of possible negative side effects.
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Biomarkers of aging in Drosophila.
Aging Cell
PUBLISHED: 03-29-2010
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Low environmental temperature and dietary restriction (DR) extend lifespan in diverse organisms. In the fruit fly Drosophila, switching flies between temperatures alters the rate at which mortality subsequently increases with age but does not reverse mortality rate. In contrast, DR acts acutely to lower mortality risk; flies switched between control feeding and DR show a rapid reversal of mortality rate. Dietary restriction thus does not slow accumulation of aging-related damage. Molecular species that track the effects of temperatures on mortality but are unaltered with switches in diet are therefore potential biomarkers of aging-related damage. However, molecular species that switch upon instigation or withdrawal of DR are thus potential biomarkers of mechanisms underlying risk of mortality, but not of aging-related damage. Using this approach, we assessed several commonly used biomarkers of aging-related damage. Accumulation of fluorescent advanced glycation end products (AGEs) correlated strongly with mortality rate of flies at different temperatures but was independent of diet. Hence, fluorescent AGEs are biomarkers of aging-related damage in flies. In contrast, five oxidized and glycated protein adducts accumulated with age, but were reversible with both temperature and diet, and are therefore not markers either of acute risk of dying or of aging-related damage. Our approach provides a powerful method for identification of biomarkers of aging.
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Regulation of lifespan, metabolism, and stress responses by the Drosophila SH2B protein, Lnk.
PLoS Genet.
PUBLISHED: 02-12-2010
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Drosophila Lnk is the single ancestral orthologue of a highly conserved family of structurally-related intracellular adaptor proteins, the SH2B proteins. As adaptors, they lack catalytic activity but contain several protein-protein interaction domains, thus playing a critical role in signal transduction from receptor tyrosine kinases to form protein networks. Physiological studies of SH2B function in mammals have produced conflicting data. However, a recent study in Drosophila has shown that Lnk is an important regulator of the insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway during growth, functioning in parallel to the insulin receptor substrate, Chico. As this pathway also has an evolutionary conserved role in the determination of organism lifespan, we investigated whether Lnk is required for normal lifespan in Drosophila. Phenotypic analysis of mutants for Lnk revealed that loss of Lnk function results in increased lifespan and improved survival under conditions of oxidative stress and starvation. Starvation resistance was found to be associated with increased metabolic stores of carbohydrates and lipids indicative of impaired metabolism. Biochemical and genetic data suggest that Lnk functions in both the IIS and Ras/Mitogen activated protein Kinase (MapK) signaling pathways. Microarray studies support this model, showing transcriptional feedback onto genes in both pathways as well as indicating global changes in both lipid and carbohydrate metabolism. Finally, our data also suggest that Lnk itself may be a direct target of the IIS responsive transcription factor, dFoxo, and that dFoxo may repress Lnk expression. We therefore describe novel functions for a member of the SH2B protein family and provide the first evidence for potential mechanisms of SH2B regulation. Our findings suggest that IIS signaling in Drosophila may require the activity of a second intracellular adaptor, thereby yielding fundamental new insights into the functioning and role of the IIS pathway in ageing and metabolism.
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DILP-producing median neurosecretory cells in the Drosophila brain mediate the response of lifespan to nutrition.
Aging Cell
PUBLISHED: 02-12-2010
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Dietary restriction extends lifespan in diverse organisms, but the gene regulatory mechanisms and tissues mediating the increased survival are still unclear. Studies in worms and flies have revealed a number of candidate mechanisms, including the target of rapamycin and insulin/IGF-like signalling (IIS) pathways and suggested a specific role for the nervous system in mediating the response. A pair of sensory neurons in Caenorhabditis elegans has been found to specifically mediate DR lifespan extension, but a neuronal focus in the Drosophila nervous system has not yet been identified. We have previously shown that reducing IIS via the partial ablation of median neurosecretory cells in the Drosophila adult brain, which produce three of the seven fly insulin-like peptides, extends lifespan. Here, we show that these cells are required to mediate the response of lifespan to full feeding in a yeast dilution DR regime and that they appear to do so by mechanisms that involve both altered IIS and other endocrine effects. We also present evidence of an interaction between these mNSCs, nutrition and sleep, further emphasising the functional homology between the DILP-producing neurosecretory cells in the Drosophila brain and the hypothalamus of mammals in their roles as integration sites of many inputs for the control of lifespan and behaviour.
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Molecular evolution and functional characterization of Drosophila insulin-like peptides.
PLoS Genet.
PUBLISHED: 01-25-2010
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Multicellular animals match costly activities, such as growth and reproduction, to the environment through nutrient-sensing pathways. The insulin/IGF signaling (IIS) pathway plays key roles in growth, metabolism, stress resistance, reproduction, and longevity in diverse organisms including mammals. Invertebrate genomes often contain multiple genes encoding insulin-like ligands, including seven Drosophila insulin-like peptides (DILPs). We investigated the evolution, diversification, redundancy, and functions of the DILPs, combining evolutionary analysis, based on the completed genome sequences of 12 Drosophila species, and functional analysis, based on newly-generated knock-out mutations for all 7 dilp genes in D. melanogaster. Diversification of the 7 DILPs preceded diversification of Drosophila species, with stable gene diversification and family membership, suggesting stabilising selection for gene function. Gene knock-outs demonstrated both synergy and compensation of expression between different DILPs, notably with DILP3 required for normal expression of DILPs 2 and 5 in brain neurosecretory cells and expression of DILP6 in the fat body compensating for loss of brain DILPs. Loss of DILP2 increased lifespan and loss of DILP6 reduced growth, while loss of DILP7 did not affect fertility, contrary to its proposed role as a Drosophila relaxin. Importantly, loss of DILPs produced in the brain greatly extended lifespan but only in the presence of the endosymbiontic bacterium Wolbachia, demonstrating a specific interaction between IIS and Wolbachia in lifespan regulation. Furthermore, loss of brain DILPs blocked the responses of lifespan and fecundity to dietary restriction (DR) and the DR response of these mutants suggests that IIS extends lifespan through mechanisms that both overlap with those of DR and through additional mechanisms that are independent of those at work in DR. Evolutionary conservation has thus been accompanied by synergy, redundancy, and functional differentiation between DILPs, and these features may themselves be of evolutionary advantage.
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Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster.
Cell Metab.
PUBLISHED: 01-16-2010
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The target of rapamycin (TOR) pathway is a major nutrient-sensing pathway that, when genetically downregulated, increases life span in evolutionarily diverse organisms including mammals. The central component of this pathway, TOR kinase, is the target of the inhibitory drug rapamycin, a highly specific and well-described drug approved for human use. We show here that feeding rapamycin to adult Drosophila produces the life span extension seen in some TOR mutants. Increase in life span by rapamycin was associated with increased resistance to both starvation and paraquat. Analysis of the underlying mechanisms revealed that rapamycin increased longevity specifically through the TORC1 branch of the TOR pathway, through alterations to both autophagy and translation. Rapamycin could increase life span of weak insulin/Igf signaling (IIS) pathway mutants and of flies with life span maximized by dietary restriction, indicating additional mechanisms.
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Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.
Science
PUBLISHED: 10-03-2009
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Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.
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Amino-acid imbalance explains extension of lifespan by dietary restriction in Drosophila.
Nature
PUBLISHED: 09-08-2009
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Dietary restriction extends healthy lifespan in diverse organisms and reduces fecundity. It is widely assumed to induce adaptive reallocation of nutrients from reproduction to somatic maintenance, aiding survival of food shortages in nature. If this were the case, long life under dietary restriction and high fecundity under full feeding would be mutually exclusive, through competition for the same limiting nutrients. Here we report a test of this idea in which we identified the nutrients producing the responses of lifespan and fecundity to dietary restriction in Drosophila. Adding essential amino acids to the dietary restriction condition increased fecundity and decreased lifespan, similar to the effects of full feeding, with other nutrients having little or no effect. However, methionine alone was necessary and sufficient to increase fecundity as much as did full feeding, but without reducing lifespan. Reallocation of nutrients therefore does not explain the responses to dietary restriction. Lifespan was decreased by the addition of amino acids, with an interaction between methionine and other essential amino acids having a key role. Hence, an imbalance in dietary amino acids away from the ratio optimal for reproduction shortens lifespan during full feeding and limits fecundity during dietary restriction. Reduced activity of the insulin/insulin-like growth factor signalling pathway extends lifespan in diverse organisms, and we find that it also protects against the shortening of lifespan with full feeding. In other organisms, including mammals, it may be possible to obtain the benefits to lifespan of dietary restriction without incurring a reduction in fecundity, through a suitable balance of nutrients in the diet.
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The endosymbiont Wolbachia increases insulin/IGF-like signalling in Drosophila.
Proc. Biol. Sci.
PUBLISHED: 08-19-2009
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Insulin/IGF-like signalling (IIS) is an evolutionarily conserved pathway that has diverse functions in multi-cellular organisms. Mutations that reduce IIS can have pleiotropic effects on growth, development, metabolic homeostasis, fecundity, stress resistance and lifespan. IIS is also modified by extrinsic factors. For instance, in the fruitfly Drosophila melanogaster, both nutrition and stress can alter the activity of the pathway. Here, we test experimentally the hypothesis that a widespread endosymbiont of arthropods, Wolbachia pipientis, can alter the degree to which mutations in genes encoding IIS components affect IIS and its resultant phenotypes. Wolbachia infection, which is widespread in D. melanogaster in nature and has been estimated to infect 30 per cent of strains in the Bloomington stock centre, can affect broad aspects of insect physiology, particularly traits associated with reproduction. We measured a range of IIS-related phenotypes in flies ubiquitously mutant for IIS in the presence and absence of Wolbachia. We show that removal of Wolbachia further reduces IIS and hence enhances the mutant phenotypes, suggesting that Wolbachia normally acts to increase insulin signalling. This effect of Wolbachia infection on IIS could have an evolutionary explanation, and has some implications for studies of IIS in Drosophila and other organisms that harbour endosymbionts.
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Some highlights of research on aging with invertebrates, 2009.
Aging Cell
PUBLISHED: 07-22-2009
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This annual review focuses on invertebrate model organisms, which shed light on new mechanisms in aging and provide excellent systems for both genome-wide and in-depth analysis. This year, protein interaction networks have been used in a new bioinformatic approach to identify novel genes that extend replicative lifespan in yeast. In an extended approach, using a new, human protein interaction network, information from the invertebrates was used to identify new, candidate genes for lifespan extension and their orthologues were validated in the nematode Caenorhabditis elegans. Chemosensation of diffusible substances from bacteria has been shown to limit lifespan in C. elegans, while a systematic study of the different methods used to implement dietary restriction in the worm has shown that they involve mechanisms that are partially distinct and partially overlapping, providing important clarification for addressing whether or not they are conserved in other organisms. A new theoretical model for the evolution of rejuvenating cell division has shown that asymmetrical division for either cell size or for damaged cell constituents results in increased fitness for most realistic levels of cellular protein damage. Work on aging-related disease has both refined our understanding of the mechanisms underlying one route to the development of Parkinsons disease and has revealed that in worms, as in mice, dietary restriction is protective against cellular proteotoxicity. Two systematic studies genetically manipulating the superoxide dismutases of C. elegans support the idea that damage from superoxide plays little or no role in aging in this organism, and have prompted discussion of other kinds of damage and other kinds of mechanisms for producing aging-related decline in function.
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A Drosophila insulin-like peptide promotes growth during nonfeeding states.
Dev. Cell
PUBLISHED: 06-24-2009
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In metazoans, tissue growth relies on the availability of nutrients--stored internally or obtained from the environment--and the resulting activation of insulin/IGF signaling (IIS). In Drosophila, growth is mediated by seven Drosophila insulin-like peptides (Dilps), acting through a canonical IIS pathway. During the larval period, animals feed and Dilps produced by the brain couple nutrient uptake with systemic growth. We show here that, during metamorphosis, when feeding stops, a specific DILP (Dilp6) is produced by the fat body and relays the growth signal. Expression of DILP6 during pupal development is controlled by the steroid hormone ecdysone. Remarkably, DILP6 expression is also induced upon starvation, and both its developmental and environmental expression require the Drosophila FoxO transcription factor. This study reveals a specific class of ILPs induced upon metabolic stress that promotes growth in conditions of nutritional deprivation or following developmentally induced cessation of feeding.
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Quantification of food intake in Drosophila.
PLoS ONE
PUBLISHED: 05-26-2009
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Measurement of food intake in the fruit fly Drosophila melanogaster is often necessary for studies of behaviour, nutrition and drug administration. There is no reliable and agreed method for measuring food intake of flies in undisturbed, steady state, and normal culture conditions. We report such a method, based on measurement of feeding frequency by proboscis-extension, validated by short-term measurements of food dye intake. We used the method to demonstrate that (a) female flies feed more frequently than males, (b) flies feed more often when housed in larger groups and (c) fly feeding varies at different times of the day. We also show that alterations in food intake are not induced by dietary restriction or by a null mutation of the fly insulin receptor substrate chico. In contrast, mutation of takeout increases food intake by increasing feeding frequency while mutation of ovo(D) increases food intake by increasing the volume of food consumed per proboscis-extension. This approach provides a practical and reliable method for quantification of food intake in Drosophila under normal, undisturbed culture conditions.
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Expression of human uncoupling protein-3 in Drosophila insulin-producing cells increases insulin-like peptide (DILP) levels and shortens lifespan.
Exp. Gerontol.
PUBLISHED: 04-23-2009
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Uncoupling proteins (UCPs) can dissipate mitochondrial protonmotive force by increasing the proton conductance of the inner membrane and through this effect could decrease ROS production, ameliorate oxidative stress and extend lifespan. We investigated whether ubiquitous, pan-neuronal or neurosecretory cell-specific expression of human UCP3 (hUCP3) in adult Drosophila melanogaster affected lifespan. Low, ubiquitous expression of hUCP3 at levels found in rodent skeletal muscle mitochondria did not affect proton conductance in mitochondria isolated from whole flies, but high pan-neuronal expression of hUCP3 increased the proton conductance of mitochondria isolated from fly heads. Expression of hUCP3 at moderate levels in adult neurons led to a marginal lifespan-extension in males. However, high expression of hUCP3 in neuronal tissue shortened lifespan. The life-shortening effect was replicated when hUCP3 was expressed specifically in median neurosecretory cells (mNSC), which express three of the Drosophila insulin-like peptides (DILPs). Expression of hUCP3 in the mNSC did not alter expression of dilp2, dilp3 or dilp5 mRNA, but led to increased amounts of DILP2 in fly heads. These data suggest that lowering mitochondrial coupling by high expression of hUCP3 alters mNSC function in a way that appears to increase DILP-levels in fly heads and lead to a concomitant decrease in lifespan.
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Chemical changes in aging Drosophila melanogaster.
Age (Dordr)
PUBLISHED: 04-14-2009
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The “Green Theory” of aging proposes that organismal lifespan is limited by the failure to repair molecular damage generated by a broad range of metabolic processes. Two specific predictions arise from this: (1) that these processes will produce a wide variety of stable but dysfunctional compounds that increase in concentration with age, and (2) that organisms maintained under conditions that extend lifespan will display a reduced rate of accumulation of such “molecular rubbish”. To test these predictions, novel analytical techniques were developed to investigate the accumulation of damaged compounds in Drosophila melanogaster. Simple preparative techniques were developed to produce digests of whole D. melanogaster for use in three-dimensional (3D) fluorimetry and 1H NMR spectrometry. Cohorts of Drosophila maintained under normal conditions showed an age-related increase in signals consistent with damage whereas those maintained under conditions of low temperature and dietary restriction did not. 1H NMR revealed distinct age-associated spectral changes that will facilitate the identification of novel compounds that both increase and decrease during aging in this species. These findings are consistent with the predictions of the “Green Theory”.
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Endocrine regulation of aging and reproduction in Drosophila.
Mol. Cell. Endocrinol.
PUBLISHED: 04-03-2009
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Hormonal signals can modulate lifespan and reproductive capacity across the animal kingdom. The use of model organisms such as worms, flies and mice has been fundamentally important for aging research in the discovery of genetic alterations that can extend healthy lifespan. The effects of mutations in the insulin and insulin-like growth factor-like signaling (IIS) pathways are evolutionarily conserved in that they can increase lifespan in all three animal models. Additionally, steroids and other lipophilic signaling molecules modulate lifespan in diverse organisms. Here we shall review how major hormonal pathways in the fruit fly Drosophila melanogaster interact to influence reproductive capacity and aging.
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Insulin/IGF-like signalling, the central nervous system and aging.
Biochem. J.
PUBLISHED: 01-23-2009
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Enormous strides in understanding aging have come from the discovery that mutations in single genes can extend healthy life-span in laboratory model organisms such as the yeast Saccharomyces, the fruit fly Drosophila melanogaster, the nematode worm Caenorhabditis elegans and the mouse. IIS [insulin/IGF (insulin-like growth factor)-like signalling] stands out as an important, evolutionarily conserved pathway involved in the determination of lifespan. The pathway has diverse functions in multicellular organisms, and mutations in IIS can affect growth, development, metabolic homoeostasis, fecundity and stress resistance, as well as lifespan. The pleiotropic nature of the pathway and the often negative effects of its disruption mean that the extent, tissue and timing of IIS manipulations are determinants of a positive effect on lifespan. One tissue of particular importance for lifespan extension in diverse organisms is the CNS (central nervous system). Although lowered IIS in the CNS can extend lifespan, IIS is also widely recognized as being neuroprotective and important for growth and survival of neurons. In the present review, we discuss our current understanding of the role of the nervous system in extension of lifespan by altered IIS, and the role of IIS in determination of neuronal function during aging. The nervous system can play both endocrine and cell-autonomous roles in extension of lifespan by IIS, and the effects of IIS on lifespan and neuronal function can be uncoupled to some extent. Tissue-specific manipulation of IIS and the cellular defence mechanisms that it regulates will better define the ways in which IIS affects neuronal and whole-organism function during aging.
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Invertebrate models of age-related muscle degeneration.
Biochim. Biophys. Acta
PUBLISHED: 01-16-2009
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Functional and structural deterioration of muscles is an inevitable consequence of ageing in a wide variety of animal species. What underlies these changes is a complex network of interactions between the muscle-intrinsic and muscle-extrinsic factors, making it very difficult to distinguish between the cause and the consequence. Many of the genes, structures, and processes implicated in mammalian skeletal muscle ageing are preserved in invertebrate species Drosophila melanogaster and Caenorhabditis elegans. The absence in these organisms of mechanisms that promote muscle regeneration, and substantially different hormonal environment, warrant caution when extrapolating experimental data from studies conducted in invertebrates to mammalian species. The simplicity and accessibility of these models, however, offer ample opportunities for studying age-related myopathologies as well as investigating drugs and therapies to alleviate them.
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Effect of a standardised dietary restriction protocol on multiple laboratory strains of Drosophila melanogaster.
PLoS ONE
PUBLISHED: 01-01-2009
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Outcomes of lifespan studies in model organisms are particularly susceptible to variations in technical procedures. This is especially true of dietary restriction, which is implemented in many different ways among laboratories.
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Using answer set programming to integrate RNA expression with signalling pathway information to infer how mutations affect ageing.
PLoS ONE
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A challenge of systems biology is to integrate incomplete knowledge on pathways with existing experimental data sets and relate these to measured phenotypes. Research on ageing often generates such incomplete data, creating difficulties in integrating RNA expression with information about biological processes and the phenotypes of ageing, including longevity. Here, we develop a logic-based method that employs Answer Set Programming, and use it to infer signalling effects of genetic perturbations, based on a model of the insulin signalling pathway. We apply our method to RNA expression data from Drosophila mutants in the insulin pathway that alter lifespan, in a foxo dependent fashion. We use this information to deduce how the pathway influences lifespan in the mutant animals. We also develop a method for inferring the largest common sub-paths within each of our signalling predictions. Our comparisons reveal consistent homeostatic mechanisms across both long- and short-lived mutants. The transcriptional changes observed in each mutation usually provide negative feedback to signalling predicted for that mutation. We also identify an S6K-mediated feedback in two long-lived mutants that suggests a crosstalk between these pathways in mutants of the insulin pathway, in vivo. By formulating the problem as a logic-based theory in a qualitative fashion, we are able to use the efficient search facilities of Answer Set Programming, allowing us to explore larger pathways, combine molecular changes with pathways and phenotype and infer effects on signalling in in vivo, whole-organism, mutants, where direct signalling stimulation assays are difficult to perform. Our methods are available in the web-service NetEffects: http://www.ebi.ac.uk/thornton-srv/software/NetEffects.
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Genetics of longevity in model organisms: debates and paradigm shifts.
Annu. Rev. Physiol.
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Discovering the biological basis of aging is one of the greatest remaining challenges for science. Work on the biology of aging has discovered a range of interventions and pathways that control aging rate. A picture is emerging of a signaling network that is sensitive to nutritional status and that controls growth, stress resistance, and aging. This network includes the insulin/IGF-1 and target of rapamycin (TOR) pathways and likely mediates the effects of dietary restriction on aging. Yet the biological processes upon which these pathways act to control life span remain unclear. A long-standing guiding assumption about aging is that it is caused by wear and tear, particularly damage at the molecular level. One view is that reactive oxygen species (ROS), including free radicals, generated as by-products of cellular metabolism, are a major contributor to this damage. Yet many recent tests of the oxidative damage theory have come up negative. Such tests have opened an exciting new phase in biogerontology in which fundamental assumptions about aging are being reexamined and revolutionary concepts are emerging. Among these concepts is the hyperfunction theory, which postulates that processes contributing to growth and reproduction run on in later life, leading to hypertrophic and hyperplastic pathologies. Here we reexamine central concepts about the nature of aging.
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Activation of AMPK by the putative dietary restriction mimetic metformin is insufficient to extend lifespan in Drosophila.
PLoS ONE
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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.
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Detrimental effects of RNAi: a cautionary note on its use in Drosophila ageing studies.
PLoS ONE
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RNA interference (RNAi) provides an important tool for gene function discovery. It has been widely exploited in Caenorhabditis elegans ageing research because it does not appear to have any non-specific effects on ageing-related traits in that model organism. We show here that ubiquitous, adult-onset activation of the RNAi machinery, achieved by expressing a double stranded RNA targeting GFP or lacZ for degradation, or by increasing expression of Dicer substantially reduces lifespan in Drosophila melanogaster. Induction of GFPRNAi construct also alters the response of lifespan to nutrition, exacerbating the lifespan-shortening effects of food containing a high quantity of yeast. Our study indicates that activation of the RNAi machinery may have sequence-independent side-effects on lifespan, and that caution needs to be exercised when employing ubiquitous RNAi in Drosophila ageing studies. However, we also show that RNAi restricted to certain tissues may not be detrimental to lifespan.
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Ageing as a risk factor for disease.
Curr. Biol.
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Age is the main risk factor for the prevalent diseases of developed countries: cancer, cardiovascular disease and neurodegeneration. The ageing process is deleterious for fitness, but can nonetheless evolve as a consequence of the declining force of natural selection at later ages, attributable to extrinsic hazards to survival: ageing can then occur as a side-effect of accumulation of mutations that lower fitness at later ages, or of natural selection in favour of mutations that increase fitness of the young but at the cost of a higher subsequent rate of ageing. Once thought of as an inexorable, complex and lineage-specific process of accumulation of damage, ageing has turned out to be influenced by mechanisms that show strong evolutionary conservation. Lowered activity of the nutrient-sensing insulin/insulin-like growth factor/Target of Rapamycin signalling network can extend healthy lifespan in yeast, multicellular invertebrates, mice and, possibly, humans. Mitochondrial activity can also promote ageing, while genome maintenance and autophagy can protect against it. We discuss the relationship between evolutionarily conserved mechanisms of ageing and disease, and the associated scientific challenges and opportunities.
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Experimental analysis of risk factors for ulcerative dermatitis in mice.
Exp. Dermatol.
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Ulcerative dermatitis (UD) is a severe inflammatory skin disorder with an unknown aetiology. Recently, insulin receptor substrate 1 KO mice were shown to be fully resistant to UD. In this study, we showed that high-fat diet (HFD) feeding significantly increased incidence of UD in wild type (WT) C57BL/6 mice, as did lithium-mediated inhibition of GSK3-?, which is a key negative regulator of IRS1. In contrast to WT mice, resistance to UD was fully preserved in HFD-fed Irs1-KO mice. Our results identify IRS1 as a key determinant of UD pathogenesis and establish a direct link between diet composition, obesity-induced inflammation and chronic ulceration.
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Ageing increases vulnerability to a?42 toxicity in Drosophila.
PLoS ONE
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Age is the major risk factor for many neurodegenerative diseases, including Alzheimers Disease (AD), for reasons that are not clear. The association could indicate that the duration or degree of exposure to toxic proteins is important for pathology, or that age itself increases susceptibility to protein toxicity. Using an inducible Drosophila model of AD, we investigated these possibilities by varying the expression of an A?42 transgene in neurons at different adult ages and measuring the effects on A?42 levels and associated pathological phenotypes. Acute induction of Arctic A?42 in young adult flies resulted in rapid expression and clearance of mRNA and soluble Arctic A?42 protein, but in irreversible expression of insoluble Arctic A?42 peptide. Arctic A?42 peptide levels accumulated with longer durations of induction, and this led to a dose-dependent reduction in negative geotaxis and lifespan. For a standardised level of mRNA expression, older flies had higher levels of Arctic A?42 peptide and associated toxicity, and this correlated with an age-dependent reduction in proteasome activity. Equalising A?42 protein at different ages shortened lifespan in correlation with the duration of exposure to the peptide, suggesting that A?42 expression accumulates damage over time. However, the relative reduction in lifespan compared to controls was greater in flies first exposed to the peptide at older ages, suggesting that ageing itself also increases susceptibility to A?42 toxicity. Indeed older flies were more vulnerable to chronic A?42 toxicity even with a much lower lifetime exposure to the peptide. Finally, the persistence of insoluble A?42 in both young and old induced flies suggests that aggregated forms of the peptide cause toxicity in later life. Our results suggest that reduced protein turnover, increased duration of exposure and increased vulnerability to protein toxicity at later ages in combination could explain the late age-of-onset of neurodegenerative phenotypes.
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The place of genetics in ageing research.
Nat. Rev. Genet.
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Rapidly increasing numbers of older people present many countries with growing social and economic challenges. Yet despite the far-reaching implications of ageing, its biological basis remains a topic of much debate. Recent advances in genomics have spurred research on ageing and lifespan in human populations, adding to extensive genetic studies being carried out in model organisms. But how far is ageing controlled by our genes? In this Viewpoint, six experts present their opinions and comment on future directions in ageing research.
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Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H2O2 in living Drosophila.
Nat Protoc
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The role of hydrogen peroxide (H(2)O(2)) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H(2)O(2) in vivo. Here we describe a method for assessing changes in H(2)O(2) within the mitochondrial matrix of living Drosophila. We use a ratiometric mass spectrometry probe, MitoB ((3-hydroxybenzyl)triphenylphosphonium bromide), which contains a triphenylphosphonium cation component that drives its accumulation within mitochondria. The arylboronic moiety of MitoB reacts with H(2)O(2) to form a phenol product, MitoP. On injection into the fly, MitoB is rapidly taken up by mitochondria and the extent of its conversion to MitoP enables the quantification of H(2)O(2). To assess MitoB conversion to MitoP, the compounds are extracted and the MitoP/MitoB ratio is quantified by liquid chromatography-tandem mass spectrometry relative to deuterated internal standards. This method facilitates the investigation of mitochondrial H(2)O(2) in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H(2)O(2) production in mouse and cell culture studies.
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Genome-wide transcription analysis of clinal genetic variation in Drosophila.
PLoS ONE
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Clinal variation in quantitative traits is widespread, but its genetic basis awaits identification. Drosophila melanogaster shows adaptive, clinal variation in traits such as body size along latitudinal gradients on multiple continents. To investigate genome wide transcription differentiation between North and South that might contribute to the clinal phenotypic variation, we compared RNA expression patterns during development of D. melanogaster from tropical northern and temperate southern populations using whole genome tiling arrays. We found that genes that were differentially expressed between the cline ends were generally associated with metabolism and growth, and experimental alteration of expression of a sample of them generally resulted in altered body size in the predicted direction, sometimes significantly so. We further identified the serpent (srp) transcription factor binding sites to be enriched near genes up-regulated in expression in the south. Analysis of clinal populations revealed a significant cline in the expression level of srp. Experimental over-expression of srp increased body size, as predicted from its clinal expression pattern, suggesting that it may be involved in regulating adaptive clinal variation in Drosophila. This study identified a handful of genes that contributed to clinal phenotypic variation through altered gene expression level, yet misexpression of individual gene led to modest body size change.
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Vascular endothelial insulin/IGF-1 signaling controls skin wound vascularization.
Biochem. Biophys. Res. Commun.
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Type 2 diabetes mellitus affects 6% of western populations and represents a major risk factor for the development of skin complications, of which impaired wound healing, manifested in e.g. "diabetic foot ulcer", is most prominent. Impaired angiogenesis is considered a major contributing factor to these non-healing wounds. At present it is still unclear whether diabetes-associated wound healing and skin vascular dysfunction are direct consequences of impaired insulin/IGF-1 signaling, or secondary due to e.g. hyperglycemia. To directly test the role of vascular endothelial insulin signaling in the development of diabetes-associated skin complications and vascular function, we inactivated the insulin receptor and its highly related receptor, the IGF-1 receptor, specifically in the endothelial compartment of postnatal mice, using the inducible Tie-2CreERT (DKO(IVE)) deleter. Impaired endothelial insulin/IGF-1 signaling did not have a significant impact on endothelial homeostasis in the skin, as judged by number of vessels, vessel basement membrane staining intensity and barrier function. In contrast, challenging the skin through wounding strongly reduced neo-angiogenesis in DKO(IVE) mice, accompanied by reduced granulation tissue formation reduced. These results show that endothelial insulin/IGF signaling is essential for neo-angiogenesis upon wounding, and imply that reduced endothelial insulin/IGF signaling directly contributes to diabetes-associated impaired healing.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.