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SummaryReduced insulin/insulin-like growth factor (IGF) signaling may be a natural way for the reduction of dietary nutrients to extend lifespan. While evidence challenging this hypothesis is accumulating with Caenorhabditis elegans , for Drosophila melanogaster it is still thought that insulin/IGF and the mechanisms of dietary restriction (DR) might as yet function through overlapping mechanisms. Here, we aim to understand this potential overlap. We found that over-expression of dFOXO in head fat body extends lifespan and reduces steady-state mRNA abundance of insulin-like peptide-2 under conditions of high dietary yeast, but not when yeast is limiting. In contrast, conditions of DR that increase lifespan change only insulin-like peptide-5 ( ilp5 ) mRNA abundance. Thus, reduction of ilp5 mRNA is associated with longevity extension by DR, while reduction of insulin-like peptide-2 is associated with the diet-dependent effects of FOXO over-expression upon lifespan. To assess whether reduction of ilp5 is required for DR to extend lifespan, we blocked its diet-dependent change with RNAi. Loss of the ilp5 dietary response did not diminish the capacity of DR to extend lifespan. Finally, we assessed the capacity of DR to extend lifespan in the absence of dFOXO, the insulin/IGF-responsive transcription factor. As with the knockdown of ilp5 diet responsiveness, DR was equally effective among genotypes with and without dFOXO. It is clear from many Drosophila studies that insulin/IGF mediates growth and metabolic responses to nutrition, but we now find no evidence that this endocrine system mediates the interaction between dietary yeast and longevity extension.

BackgroundJuvenile hormone (JH) has been demonstrated to control adult lifespan in a number of non-model insects where surgical removal of the corpora allata eliminates the hormone’s source. In contrast, little is known about how juvenile hormone affects adult Drosophila melanogaster. Previous work suggests that insulin signaling may modulate Drosophila aging in part through its impact on juvenile hormone titer, but no data yet address whether reduction of juvenile hormone is sufficient to control Drosophila life span. Here we adapt a genetic approach to knock out the corpora allata in adult Drosophila melanogaster and characterize adult life history phenotypes produced by reduction of juvenile hormone. With this system we test potential explanations for how juvenile hormone modulates aging.ResultsA tissue specific driver inducing an inhibitor of a protein phosphatase was used to ablate the corpora allata while permitting normal development of adult flies. Corpora allata knockout adults had greatly reduced fecundity, inhibited oogenesis, impaired adult fat body development and extended lifespan. Treating these adults with the juvenile hormone analog methoprene restored all traits toward wildtype. Knockout females remained relatively long-lived even when crossed into a genotype that blocked all egg production. Dietary restriction further extended the lifespan of knockout females. In an analysis of expression profiles of knockout females in fertile and sterile backgrounds, about 100 genes changed in response to loss of juvenile hormone independent of reproductive state.ConclusionsReduced juvenile hormone alone is sufficient to extend the lifespan of Drosophila melanogaster. Reduced juvenile hormone limits reproduction by inhibiting the production of yolked eggs, and this may arise because juvenile hormone is required for the post-eclosion development of the vitellogenin-producing adult fat body. Our data do not support a mechanism for juvenile hormone control of longevity simply based on reducing the physiological costs of egg production. Nor does the longevity benefit appear to function through mechanisms by which dietary restriction extends longevity. We identify transcripts that change in response to juvenile hormone independent of reproductive state and suggest these represent somatically expressed genes that could modulate how juvenile hormone controls persistence and longevity.

Insulin/IGF signaling (IIS) regulates essential processes including development, metabolism, and aging. The Drosophila genome encodes eight insulin/IGF‐like peptide (dilp) paralogs, including tandem‐encoded dilp1 and dilp2. Many reports show that longevity is increased by manipulations that decrease DILP2 levels. It has been shown that dilp1 is expressed primarily in pupal stages, but also during adult reproductive diapause. Here, we find that dilp1 is also highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Here, we study dilp1 and dilp2 single and double mutants to describe epistatic and synergistic interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1‐dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 − dilp2 double mutants. On the other hand, dilp1 and dilp2 redundantly or synergistically interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. These interactions do not correlate with patterns for how dilp1 and dilp2 affect longevity and AKH. Thus, repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro‐longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. Our data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro‐longevity insulin‐like peptide.

We have cloned the Drosophila gene discs degenerate-4 (dd4) and find that it encodes a component of the ␥-tubulin ring complex (␥TuRC) homologous to Spc98 of budding yeast. This provides the first opportunity to study decreased function of a member of the ␥-tubulin ring complex, other than ␥-tubulin itself, in a metazoan cell. ␥-tubulin is no longer at the centrosomes but is dispersed throughout dd4 cells and yet bipolar metaphase spindles do form, although these have a dramatically decreased density of microtubules. Centrosomin (CNN) remains in broad discrete bodies but only at the focused poles of such spindles, whereas Asp (abnormal spindle protein) is always present at the presumptive minus ends of microtubules, whether or not they are focused. This is consistent with the proposed role of Asp in coordinating the nucleation of mitotic microtubule organizing centers. The centrosome associated protein CP190 is partially lost from the spindle poles in dd4 cells supporting a weak interaction with ␥-tubulin, and the displaced protein accumulates in the vicinity of chromosomes. Electron microscopy indicates not only that the poles of dd4 cells have irregular amounts of pericentriolar material, but also that they can have abnormal centrioles. In six dd4 cells subjected to serial sectioning centrioles were missing from one of the two poles. This suggests that in addition to its role in nucleating cytoplasmic and spindle microtubules, the ␥TuRC is also essential to the structure of centrioles and the separation of centrosomes.

Summary Although extensively studied in C. elegans, no work has yet demonstrated for Drosophila melanogaster whether reduced insulin/IGF signaling (IIS) requires the FOXO transcription factor (foxo) to extend lifespan. Here we conduct genetic epistasis analysis to determine if foxo is required for chico mutants (insulin receptor substrate) to reduce age-specific mortality and thus extend lifespan. The mutant chico1 allele strongly extends lifespan relative to wildtype sibs. A mutant of foxo eliminates most of this chico survival benefit. In addition, we used a factorial proportional hazard analysis to formally study the main effects of chico and of foxo, and to determine how these genes interact to influence mortality. We document that foxo indeed contributes to how chico increases lifespan, but part of the convergence in survival between chico genotypes in the foxo mutant background may occur because chico mutation exacerbates the negative effects of foxo mutation.

Mutations of the Drosophila melanogaster insulin/IGF signaling system slow aging, while also affecting growth and reproduction. To understand this pleiotropy, we produced an allelic series of single codon substitutions in the Drosophila insulin receptor, InR. We generated InR substitutions using homologous recombination and related each to emerging models of receptor tyrosine kinase structure and function. Three mutations when combined as trans-heterozygotes extended lifespan while retarding growth and fecundity. These genotypes reduced insulin-stimulated Akt phosphorylation, suggesting they impede kinase catalytic domain function. Among these genotypes, longevity was negatively correlated with egg production, consistent with life history trade-off theory. In contrast, one mutation (InR  353) was located in the kinase insert domain, a poorly characterized element found in all receptor tyrosine kinases. Remarkably, wildtype heterozygotes with InR  353 robustly extended lifespan without affecting growth or reproduction and retained capacity to fully phosphorylate Akt. The Drosophila insulin receptor kinase insert domain contains a previously unrecognized SH2 binding motif. We propose the kinase insert domain interacts with SH2-associated adapter proteins to affect aging through mechanisms that retain insulin sensitivity and are independent of reproduction.

Protein-only (prion) epigenetic elements confer unique phenotypes by adopting alternate conformations that specify new traits. Given the conformational flexibility of prion proteins, protein-only inheritance requires efficient self-replication of the underlying conformation. To explore the cellular regulation of conformational self-replication and its phenotypic effects, we analyzed genetic interactions between [PSI ؉ ], a prion form of the S. cerevisiae Sup35 protein (Sup35 [PSI ؉ ] ), and the three N ␣ -acetyltransferases, NatA, NatB, and NatC, which collectively modify ϳ50% of yeast proteins. Although prion propagation proceeds normally in the absence of NatB or NatC, the [PSI ؉ ] phenotype is reversed in strains lacking NatA. Despite this change in phenotype, [PSI ؉ ] NatA mutants continue to propagate heritable Sup35 [PSI ؉ ] . This uncoupling of protein state and phenotype does not arise through a decrease in the number or activity of prion templates (propagons) or through an increase in soluble Sup35. Rather, NatA null strains are specifically impaired in establishing the translation termination defect that normally accompanies Sup35 incorporation into prion complexes. The NatA effect cannot be explained by the modification of known components of the [PSI ؉ ] prion cycle including Sup35; thus, novel acetylated cellular factors must act to establish and maintain the tight link between Sup35 [PSI ؉ ] complexes and their phenotypic effects. INTRODUCTIONThe transmission of phenotypes from one individual to another is a fundamental process in biology. Much of our understanding of these events arises from decades of study on nucleic acid metabolism, but new traits may also be passed between individuals without changes in nucleic acid content through a number of epigenetic mechanisms. One particularly intriguing example of such a process is the prion phenomenon, in which the activity of a protein is altered in a heritable way to transmit a new phenotype. How is such a feat accomplished? In 1967, Griffith proposed that some proteins, now known as prions (Prusiner, 1982), can adopt more than one stable form in vivo (Griffith, 1967). Since a protein's structure determines its function, two cells containing the same protein but in diffrent physical states will have distinct phenotypes. This protein-based process has been linked to a number of previously inexplicable events, including the development and spread of the transmissible spongiform encephalopathies in mammals (Prusiner, 1982) and the non-Mendelian inheritance of some traits in fungi (Wickner, 1994).Protein-based traits can only become transmissible, however, if the inherent structural flexibility of prion proteins can be constrained by regulatory mechanisms to create an epigenetic element. For example, if each newly synthesized prion polypeptide chain independently folded to a unique form, all cells would display the same phenotype, which would reflect the average of the accessible states. The appearance of distinct protein-based phenotypes suggests that ...