Interplay of dFOXO and Two ETS-Family Transcription Factors Determines Lifespan in Drosophila melanogaster

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

Drosophila poly suggests a novel role for the Elongator complex in insulin receptor-target of rapamycin signalling

Multi-cellular organisms need to successfully link cell growth and metabolism to environmental cues during development. Insulin receptor–target of rapamycin (InR–TOR) signalling is a highly conserved pathway that mediates this link. Herein, we describe poly, an essential gene in Drosophila that mediates InR–TOR signalling. Loss of poly results in lethality at the third instar larval stage, but only after a stage of extreme larval longevity. Analysis in Drosophila demonstrates that Poly and InR interact and that poly mutants show an overall decrease in InR–TOR signalling, as evidenced by decreased phosphorylation of Akt, S6K and 4E-BP. Metabolism is altered in poly mutants, as revealed by microarray expression analysis and a decreased triglyceride : protein ratio in mutant animals. Intriguingly, the cellular distribution of Poly is dependent on insulin stimulation in both Drosophila and human cells, moving to the nucleus with insulin treatment, consistent with a role in InR–TOR signalling. Together, these data reveal that Poly is a novel, conserved (from flies to humans) mediator of InR signalling that promotes an increase in cell growth and metabolism. Furthermore, homology to small subunits of Elongator demonstrates a novel, unexpected role for this complex in insulin signalling

Cell type-specific modulation of healthspan by Forkhead family transcription factors in the nervous system

Reduced activity of insulin/insulin-like growth factor signaling (IIS) increases healthy lifespan among diverse animal species. Downstream of IIS, multiple evolutionarily conserved transcription factors (TFs) are required; however, distinct TFs are likely responsible for these effects in different tissues. Here we have asked which TFs can extend healthy lifespan within distinct cell types of the adult nervous system in Drosophila. Starting from published single-cell transcriptomic data, we report that forkhead (FKH) is endogenously expressed in neurons, whereas forkhead-box-O (FOXO) is expressed in glial cells. Accordingly, we find that neuronal FKH and glial FOXO exert independent prolongevity effects. We have further explored the role of neuronal FKH in a model of Alzheimer’s disease-associated neuronal dysfunction, where we find that increased neuronal FKH preserves behavioral function and reduces ubiquitinated protein aggregation. Finally, using transcriptomic profiling, we identify Atg17, a member of the Atg1 autophagy initiation family, as one FKH-dependent target whose neuronal overexpression is sufficient to extend healthy lifespan. Taken together, our results underscore the importance of cell type-specific mapping of TF activity to preserve healthy function with age

Characterization of Poly: a novel mediator of insulin receptor signalling in drosophila.

Poly is a novel, essential protein in Drosophila melanogaster, loss of function of which results in late larval lethality. Importantly, Poly is evolutionarily conserved with a human homologue. poly mutation was isolated in a P-element mutagenesis screen that aimed to generate a larger collection of single P-element induced mutants. Mutant poly larvae are characterized by extreme larval longevity without pupation, formation of melanotic masses, smaller imaginal discs and brains, and abnormal nuclear morphology in neuroblasts. During the course of my project, I attempted to identify cellular processes and pathways that Poly might be involved in. Interestingly, my data suggest that Poly is a novel interactor and regulator of Insulin receptor/target of rapamycin (InR/TOR) signalling in Drosophila. Linking environmental cues to cell growth and metabolism is an essential process that multicellular organisms need to accomplish successfully for normal development. InR/TOR signalling is a highly conserved pathway that mediates the link between the environment and cellular processes such as growth, metabolism and ageing. My analysis in Drosophila suggests that Poly interacts physically with the InR and mutation of Poly leads to an overall down-regulation of InR/TOR signalling in Drosophila as revealed by decreases in the phosphorylation levels of Akt, S6K and 4E-BP - all downstream effectors of this pathway. In addition, loss of poly results in constitutive activation of autophagy in Drosophila fat body and a decrease in stored triglyceride levels. Furthermore, I show that localisation and levels of Poly protein are dependent on insulin action in both Drosophila and human cells. Together, these data suggest that Poly is a novel mediator of InR signalling that promotes an increase in cell growth and metabolism. Taking into consideration the observed poly mutant phenotype, I also investigated the potential involvement of Poly during cell cycle progression and the Drosophila innate immune response. While my analysis suggests that poly loss of function does not have a direct effect on cell cycle progression, alteration of Poly has consequences on various aspects of the Drosophila innate immune response. Therefore, I conclude that the Drosophila innate immune response is a cellular process in which Poly plays a crucial role

Sex difference in pathology of the ageing gut mediates the greater response of female lifespan to dietary restriction

Abstract Women live on average longer than men but have greater levels of late-life morbidity. We have uncovered a substantial sex difference in the pathology of the aging gut in Drosophila. The intestinal epithelium of the aging female undergoes major deterioration, driven by intestinal stem cell (ISC) division, while lower ISC activity in males associates with delay or absence of pathology, and better barrier function, even at old ages. Males succumb to intestinal challenges to which females are resistant, associated with fewer proliferating ISCs, suggesting a trade-off between highly active repair mechanisms and late-life pathology in females. Dietary restriction reduces gut pathology in aging females, and extends female lifespan more than male. By genetic sex reversal of a specific gut region, we induced female-like aging pathologies in males, associated with decreased lifespan, but also with a greater increase in longevity in response to dietary restriction

Interplay of dFOXO and Two ETS-Family Transcription Factors Determines Lifespan in <i>Drosophila melanogaster</i>

<div><p>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 <i>in vivo</i> is unclear. Here, we identify an E-twenty six (ETS)-family transcriptional repressor, <i>Anterior open</i> (<i>Aop</i>), as regulated by the single <i>Drosophila melanogaster</i> 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, <i>Pointed</i> (<i>Pnt</i>), 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 <i>dfoxo</i>, such as the secreted, non-sensory, odorant binding protein (<i>Obp99b</i>), 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.</p></div

<i>Aop</i> prevents the detrimental effects of <i>dfoxo</i> and <i>Pnt</i> co-activation.

<p><b>A</b> Survival of female flies expressing <i>dfoxo</i>, <i>Aop^RNAi</i> or both under the control of <i>S₁106</i> in the presence or absence or RU486. Log-rank test revealed significant effect of RU486 for <i>S₁106>dfoxo</i> (p<10⁻⁴; total dead/censored: − RU486 145/0, + RU486 139/6; median/maximum lifespan: − RU486: 77/87, + RU486 80/90) and <i>S₁106>dfoxo Aop^RNAi</i> (p<10⁻⁴; total dead/censored: − RU486 145/2, + RU486 155/2; median/maximum lifespan: − RU486: 74/90, + RU486 65/79) but not <i>S₁106>Aop^RNAi</i> (p = 0.5; total dead/censored: − RU486 142/0, + RU486 147/4; median/maximum lifespan: − RU486: 72/86, + RU486 74/87). CHP analysis showed that the response to RU486 in <i>S₁106>dfoxo Aop^RNAi</i> was significantly different from the response in <i>S₁106>dfoxo</i> (p<5×10⁻¹⁵) and <i>S₁106>Aop^RNAi</i> (p<10⁻⁷) females. <b>B</b> Survival of female flies expressing <i>dfoxo</i>, <i>PntP1 and Aop^ACT</i> under the control of <i>S₁106</i> in the presence or absence or RU486. Log-rank test revealed significant effect of RU486 for <i>S₁106>Pnt^P1 dfoxo</i> (p<10⁻⁴; total dead/censored: − RU486 138/6, + RU486 145/2; median/maximum lifespan: − RU486: 77/90, + RU486 63/83) but not <i>S₁106>Pnt^P1</i> (p = 0.06; total dead/censored: − RU486 147/0, + RU486 143/2; median/maximum lifespan: − RU486: 84/92, + RU486 79/90) or <i>S₁106>Pnt^P1 dfoxo Aop^ACT</i> (p = 0.1; total dead/censored: − RU486 147/3, + RU486 142/3; median/maximum lifespan: − RU486: 77/90, + RU486 77/83). CHP analysis showed that the response to RU486 in <i>S₁106>Pnt^P1</i> was significantly different from the response in <i>S₁106>Pnt^P1 dfoxo</i> (p<10⁻¹⁵) but not in <i>S₁106>Pnt^P1 dfoxo Aop^ACT</i> (p = 0.5) females. <b>C</b> Survival of <i>TIGS>dfoxo</i> female flies in the presence or absence of RU486. Log-rank test detected no significant differences (p = 0.3; total dead/censored: − RU486 152/1, + RU486 149/2; median/maximum lifespan: − RU486: 77/87, + RU486 77/86). <b>D</b> Survival of female flies expressing <i>dfoxo</i>, <i>Pnt^P1 and Aop^ACT</i> under the control of <i>TIGS</i> in the presence or absence or RU486. Log-rank test revealed significant effect of RU486 for <i>TIGS>Pnt^P1</i> (p<10⁻⁴; total dead/censored: − RU486 140/2, + RU486 136/7; median/maximum lifespan: − RU486: 74/86, + RU486 35/50), <i>TIGS>Pnt^P1 dfoxo</i> (p<10⁻⁴; total dead/censored: − RU486 146/3, + RU486 148/1; median/maximum lifespan: − RU486: 74/85, + RU486 32/45) and <i>TIGS>Pnt^P1 dfoxo Aop^ACT</i> (p = 0.1; total dead/censored: − RU486 143/1, + RU486 155/1; median/maximum lifespan: − RU486: 72/85, + RU486 35/55). CHP analysis showed that the response to RU486 in <i>TIGS>Pnt^P1</i> was significantly different from the response in <i>TIGS>Pnt^P1 dfoxo</i> (p = 2×10⁻⁴) but not in <i>TIGS>Pnt^P1 dfoxo Aop^ACT</i> (p = 0.8) females.</p

<i>dfoxo</i> and <i>Pnt</i> act synergistically on lipid metabolism.

<p><b>A</b> Top three most significantly enriched GO categories within genes bound by FLAG-AOP^ACT alone or FLAG-AOP^ACT and dFOXO, as determined by EASE analysis. The intensity of red indicates log-10 derived p value associated with the enrichment. The complete GO analysis is given in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004619#pgen.1004619.s010" target="_blank">Dataset S1</a></b>. <b>B</b><i>CG6296</i> mRNA was quantified relative to <i>Act</i> by qPCR in the females of the indicated genotypes, induced or not with RU486. Boxplots show log-10 derived relative expression with − RU486 values set to zero. Data were analysed with a linear model and the effects of RU486, genotype and their interaction were significant (n = 4, p<10⁻⁴), with <i>S₁106>dfoxo Pnt^P1</i> + RU486 condition being different to all others (t-test , p<10⁻⁴). <b>C</b> The levels of TAG were quantified under the same conditions as in B. Data were analysed with a linear model and the effects of RU486 (p = 2×10⁻⁴), genotype (p<10⁻⁴) and their interaction (p = 0.01) were significant (n = 8, where 10 measurements were made and the lowest and highest measurement removed from each group). The <i>S₁106>dfoxo Pnt^P1</i> + RU486 condition was different from all others (t-test , p<0.05). The genotypes are denoted with the same colours in B and C, with the legend given in B.</p

<i>Aop</i> extends lifespan.

<p><b>A</b> Survival of <i>S₁106>Aop^ACT</i> female flies in the presence or absence or RU486. Log-rank test detected significant differences (p = 2×10⁻¹⁰; total dead/censored: − RU486 145/1, + RU486 129/3; median/maximum lifespan: − RU486: 74/87, + RU486 84/97). <b>B</b> Median lifespan extension achieved by RU486 feeding in <i>S₁106>Aop^ACT</i> (6 experiments) or <i>TIGS>Aop^ACT</i> (3 experiments) females. Log-rank test detected significant extension (p<0.05) of lifespan in 6 out of 6 <i>S₁106>Aop^ACT</i> and in 1 out of 3 <i>TIGS>Aop^ACT</i> trials. In one <i>TIGS>Aop^ACT</i> trial lifespan was shortened. <b>C</b> Haemolymph glucose and trehalose in <i>S₁106>Aop^ACT</i> females fed or not RU486, where RU486 had a significant effect in each case (t-test, p = 0.01 and 0.02 respectively; n = 8 where 10 measurements were made and the highest and lowest measurement removed from each group). <b>D</b> Survival of <i>S₁106>Aop^ACT</i> or dfoxoΔ/Δ <i>S₁106>Aop^ACT</i> female flies in the presence or absence or RU486. Log-rank test detected significant differences for both: <i>S₁106>Aop^ACT</i> (p<10⁻⁴; total dead/censored: − RU486 139/3, + RU486 136/7; median/maximum lifespan: − RU486: 77/87, + RU486 86/94) and dfoxoΔ/Δ <i>S₁106>Aop^ACT</i> (p<10⁻⁴; total dead/censored: − RU486 138/0, + RU486 136/4; median/maximum lifespan: − RU486: 56/81, + RU486 72/90). CHP analysis revealed significant effects of RU486 (p<10⁻¹⁵) and <i>dfoxo</i> (p<10⁻¹⁵) but no significant difference in the response to RU486 between the two lines (p = 0.2).</p

dFOXO targets in the adult gut and fat body.

<p><b>A</b> Proportional Venn diagram showing the sets of genes that were differentially regulated by <i>dfoxo</i> induction in the gut or the fat body. The number of genes that were bound by GFP-dFOXO within each differentially expressed-gene set are given in black. p values for significant set overlaps are indicated. <b>B</b> Biological process GO categories differentially regulated (p<10⁻¹⁰) in the fat body or gut upon induction of <i>dfoxo</i> as determined by Catmap analysis. Any redundant categories (overlap by more than 75%) were removed, retaining the most specific category. The full list is given in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004619#pgen.1004619.s010" target="_blank">Dataset S1</a></b>. The intensity of red shows the log₁₀-transformed p-value associated with differential regulation for each category.</p