InteractiveFly: GeneBrief

forkhead box, sub-group O: Biological Overview | Regulation | Developmental Biology | Effects of Mutation and Overexpression | Evolutionary Homologs | References


Gene name - forkhead box, sub-group O

Synonyms -

Cytological map position - 88A5--8

Function - transcription factor

Keywords - insulin receptor signaling pathway

Symbol - foxo

FlyBase ID: FBgn0038197

Genetic map position - 3R

Classification - Winged helix/forkhead sub-group 'O'

Cellular location - nuclear



NCBI links: HomoloGene | Entrez Gene

Recent literature
Spellberg, M. J. and Marr, M. T. (2015). FOXO regulates RNA interference in Drosophila and protects from RNA virus infection. Proc Natl Acad Sci U S A 112: 14587-14592. PubMed ID: 26553999
Summary:
Small RNA pathways are important players in posttranscriptional regulation of gene expression. These pathways play important roles in all aspects of cellular physiology from development to fertility to innate immunity. However, almost nothing is known about the regulation of the central genes in these pathways. The forkhead box O (FOXO) family of transcription factors is a conserved family of DNA-binding proteins that responds to a diverse set of cellular signals. FOXOs are crucial regulators of cellular homeostasis that have a conserved role in modulating organismal aging and fitness. This study shows that Drosophila FOXO (dFOXO) regulates the expression of core small RNA pathway genes. In addition, increased dFOXO activity results in an increase in RNA interference (RNAi) efficacy, establishing a direct link between cellular physiology and RNAi. Consistent with these findings, dFOXO activity is stimulated by viral infection and is required for effective innate immune response to RNA virus infection. This study reveals an unanticipated connection among dFOXO, stress responses, and the efficacy of small RNA-mediated gene silencing and suggests that organisms can tune their gene silencing in response to environmental and metabolic conditions.

Slade, J. D. and Staveley, B. E. (2016). Enhanced survival of Drosophila Akt1 hypomorphs during amino-acid starvation requires foxo. Genome 59(2):87-93. PubMed ID: 26783834
Summary:
Disordered eating includes any pattern of irregular eating that may lead to either extreme weight loss or obesity. The conserved insulin receptor signalling pathway acts to regulate energy balance and nutrient intake, and its central component Akt1 and endpoint effector foxo are pivotal for survival during nutritional stress. Recently generated Akt1 hypomorphic mutant lines exhibit a moderate decrease in lifespan when aged upon standard media, yet show a considerable increase in survival upon amino-acid starvation media. While the loss of foxo function significantly reduces the survival response to amino-acid starvation, a combination of these Akt1 hypomorphs and a null foxo mutation reveal a synergystic and severe reduction in lifespan upon standard media, and an epistatic relationship when undergoing amino-acid starvation. Evaluation of survivorship upon amino-acid starvation media of these double mutants indicate a phenotype similar to the original foxo mutant demonstrating the role of foxo in this Akt1 phenotype. These results indicate that the subtle manipulation of foxo through Akt1 can enhance survival during adverse nutrient conditions to model the ability of individuals to tolerate nutrient deprivation. Ultimately, a Drosophila model of disordered eating could generate new avenues to develop potential therapies for related human conditions.

Zhang, S., Guo, X., Chen, C., Chen, Y., Li, J., Sun, Y., Wu, C., Yang, Y., Jiang, C., Li, W. and Xue, L. (2016). dFoxO promotes Wingless signaling in Drosophila. Sci Rep 6: 22348. PubMed ID: 26936649
Summary:
The Wnt/β-catenin signaling is an evolutionarily conserved pathway that regulates a wide range of physiological functions, including embryogenesis, organ maintenance, cell proliferation and cell fate decision. Dysregulation of Wnt/β-catenin signaling has been implicated in various cancers, but its role in cell death has not yet been fully elucidated. This study shows that activation of Wg signaling induces cell death in Drosophila eyes and wings, which depends on dFoxO, a transcription factor known to be involved in cell death. In addition, dFoxO is required for ectopic and endogenous Wg signaling to regulate wing patterning. Moreover, dFoxO is necessary for activated Wg signaling-induced target genes expression. Furthermore, Arm is reciprocally required for dFoxO-induced cell death. Finally, dFoxO physically interacts with Arm both in vitro and in vivo. Thus, this study characterizes a previously unknown role of dFoxO in promoting Wg signaling, and shows that a dFoxO-Arm complex is likely involved in their mutual functions, e.g. cell death.

Webb, A. E., Kundaje, A. and Brunet, A. (2016). Characterization of the direct targets of FOXO transcription factors throughout evolution. Aging Cell [Epub ahead of print]. PubMed ID: 27061590
Summary:
FOXO transcription factors (FOXOs) are central regulators of lifespan across species, yet they also have cell-specific functions, including adult stem cell homeostasis and immune function. Whether FOXO targets are specific to cell types and species or conserved across cell types and throughout evolution remains uncharacterized. This study consisted of a analysis of direct FOXO targets across tissues and organisms, using data from mammals as well as C. elegans and Drosophila. FOXOs were shown to bind cell type-specific targets, which have functions related to that particular cell. Interestingly, FOXOs also share targets across different tissues in mammals, and the function and even the identity of these shared mammalian targets are conserved in invertebrates. Evolutionarily conserved targets show enrichment for growth factor signaling, metabolism, stress resistance, and proteostasis, suggesting an ancestral, conserved role in the regulation of these processes. Candidate cofactors at conserved FOXO targets were identified that change in expression with age, including CREB and ETS family factors. This analysis provides insight into the evolution of the FOXO network and highlights downstream genes and cofactors that may be important for FOXO's conserved function in adult homeostasis and longevity.
McLaughlin, C. N., Nechipurenko, I. V., Liu, N. and Broihier, H. T. (2016). A Toll receptor-FoxO pathway represses Pavarotti/MKLP1 to promote microtubule dynamics in motoneurons. J Cell Biol 214: 459-474. PubMed ID: 27502486
Summary:
FoxO proteins are evolutionarily conserved regulators of neuronal structure and function, yet the neuron-specific pathways within which they act are poorly understood. To elucidate neuronal FoxO function in Drosophila melanogaster, a screen was performed for FoxO's upstream regulators and downstream effectors. On the upstream side, genetic and molecular pathway analyses is presented indicating that the Toll-6 receptor, the Toll/interleukin-1 receptor domain adaptor dSARM, and FoxO function in a linear pathway. On the downstream side, it was found that Toll-6-FoxO signaling represses the mitotic kinesin Pavarotti/MKLP1 (Pav-KLP), which itself attenuates microtubule (MT) dynamics. In vivo functions were probed for this novel pathway, and it was found to be essential for axon transport and structural plasticity in motoneurons. Elevated expression of Pav-KLP underlies transport and plasticity phenotypes in pathway mutants, indicating that Toll-6-FoxO signaling promotes MT dynamics by limiting Pav-KLP expression. In addition to uncovering a novel molecular pathway, this work reveals an unexpected function for dynamic MTs in enabling rapid activity-dependent structural plasticity.
Kakanj, P., Moussian, B., Grönke, S., Bustos, V., Eming, S.A., Partridge, L. and Leptin, M. (2016). Insulin and TOR signal in parallel through FOXO and S6K to promote epithelial wound healing. Nat Commun 7: 12972. PubMed ID: 27713427
Summary:
The TOR and Insulin/IGF signalling (IIS) network controls growth, metabolism and ageing. Although reducing TOR or insulin signalling can be beneficial for ageing, it can be detrimental for wound healing, but the reasons for this difference are unknown. This study shows that IIS is activated in the cells surrounding an epidermal wound in Drosophila melanogaster larvae, resulting in PI3K activation and redistribution of the transcription factor FOXO. Insulin and TOR signalling are independently necessary for normal wound healing, with FOXO and S6K as their respective effectors. IIS is specifically required in cells surrounding the wound, and the effect is independent of glycogen metabolism. Insulin signalling is needed for the efficient assembly of an actomyosin cable around the wound, and constitutively active myosin II regulatory light chain suppresses the effects of reduced IIS. These findings may have implications for the role of insulin signalling and FOXO activation in diabetic wound healing.

Sears, J. C. and Broihier, H. T. (2016). FoxO regulates microtubule dynamics and polarity to promote dendrite branching in Drosophila sensory neurons. Dev Biol [Epub ahead of print]. PubMed ID: 27546375
Summary:
The size and shape of dendrite arbors are defining features of neurons and critical determinants of neuronal function. The molecular mechanisms establishing arborization patterns during development are not well understood, though properly regulated microtubule (MT) dynamics and polarity are essential. It has been found that FoxO regulates axonal MTs, raising the question of whether it also regulates dendritic MTs and morphology. This study demonstrates that FoxO promotes dendrite branching in all classes of Drosophila dendritic arborization (da) neurons. FoxO is required both for initiating growth of new branches and for maintaining existing branches. To elucidate FoxO function, MT organization was characterized in both foxO null and overexpressing neurons. FoxO was found to directs MT organization and dynamics in dendrites. Moreover, it is both necessary and sufficient for anterograde MT polymerization, which is known to promote dendrite branching. Lastly, FoxO promotes proper larval nociception, indicating a functional consequence of impaired da neuron morphology in foxO mutants. Together, these results indicate that FoxO regulates dendrite structure and function and suggest that FoxO-mediated pathways control MT dynamics and polarity.
Gruntenko, N. E., Adonyeva, N. V., Burdina, E. V., Karpova, E. K., Andreenkova, O. V., Gladkikh, D. V., Ilinsky, Y. Y. and Rauschenbach, I. Y. (2016). The impact of FOXO on dopamine and octopamine metabolism in Drosophila under normal and heat stress conditions. Biol Open [Epub ahead of print]. PubMed ID: 27754851
Summary:
The Forkhead BoxO transcription factor (FOXO) is a component of the insulin signalling pathway and plays a role in responding to adverse conditions, such as oxidative stress and starvation. In stressful conditions, FOXO moves from the cytosol to the nucleus where it activates gene expression programmes. This study shows that FOXO in Drosophila melanogaster responds to heat stress as it does to other stressors. The catecholamine signalling pathway is another component of the stress response. In Drosophila, dopamine and octopamine levels rise steeply under heat, nutrition and mechanical stresses, which is followed by a decrease in the activity of synthesis enzymes. This study demonstrates that the nearly twofold decline of FOXO expression in foxoBG01018 mutants results in dramatic changes in the metabolism of dopamine and octopamine and the overall response to stress. The absence of FOXO increases tyrosine decarboxylase activity, the first enzyme in octopamine synthesis, and decreases the enzymatic activity of enzymes in dopamine synthesis, alkaline phosphatase and tyrosine hydroxylase, in young Drosophila females. The juvenile hormone was identified as a mediator of FOXO regulation of catecholamine metabolism. These findings suggest that FOXO is a possible trigger for endocrinological stress reactions.
Gruntenko, N. E., et al. (2016). The impact of FOXO on dopamine and octopamine metabolism in Drosophila under normal and heat stress conditions. Biol Open. PubMed ID: 27754851
Summary:
FOXO is a component of the insulin signalling pathway and plays a role in responding to adverse conditions, such as oxidative stress and starvation. In stressful conditions, FOXO moves from the cytosol to the nucleus where it activates gene expression programmes. This study shows that FOXO in Drosophila melanogaster responds to heat stress as it does to other stressors. The catecholamine signalling pathway is another component of the stress response. In Drosophila, dopamine and octopamine levels rise steeply under heat, nutrition and mechanical stresses, which is followed by a decrease in the activity of synthesis enzymes. This study demonstrates that the nearly twofold decline of FOXO expression in foxoBG01018 mutants results in dramatic changes in the metabolism of dopamine and octopamine and the overall response to stress. The absence of FOXO increases tyrosine decarboxylase activity, the first enzyme in octopamine synthesis, and decreases the enzymatic activity of enzymes in dopamine synthesis, alkaline phosphatase and tyrosine hydroxylase, in young Drosophila females. This study identified the juvenile hormone as a mediator of FOXO regulation of catecholamine metabolism. These findings suggest that FOXO is a possible trigger for endocrinological stress reactions.
Blice-Baum, A.C., Zambon, A.C., Kaushik, G., Viswanathan, M.C., Engler, A.J., Bodmer, R. and Cammarato, A. (2017). Modest overexpression of FOXO maintains cardiac proteostasis and ameliorates age-associated functional decline. Aging Cell 16: 93-103. PubMed ID: 28090761
Summary:
Heart performance declines with age. Impaired protein quality control (PQC), due to reduced ubiquitin-proteasome system (UPS) activity, autophagic function, and/or chaperone-mediated protein refolding, contributes to cardiac deterioration. The transcription factor FOXO participates in regulating genes involved in PQC, senescence, and numerous other processes. In this study, a comprehensive approach, involving molecular genetics, novel assays to probe insect cardiac physiology, and bioinformatics, was utilized to investigate the influence of heart-restricted manipulation of dFOXO expression in the rapidly aging Drosophila melanogaster model. Modest dFOXO overexpression was cardioprotective, ameliorating nonpathological functional decline with age. This was accompanied by increased expression of genes associated predominantly with the UPS, relative to other PQC components, which was validated by a significant decrease in ubiquitinated proteins. RNAi knockdown of UPS candidates accordingly compromised myocardial physiology in young flies. Conversely, excessive dFOXO overexpression or suppression proved detrimental to heart function and/or organismal development. This study highlights D. melanogaster as a model of cardiac aging and FOXO as a tightly regulated mediator of proteostasis and heart performance over time.

Dobson, A.J., Ezcurra, M., Flanagan, C.E., Summerfield, A.C., Piper, M.D., Gems, D. and Alic, N. (2017). Nutritional programming of lifespan by FOXO inhibition on sugar-rich diets. Cell Rep 18: 299-306. PubMed ID: 28076775
Summary:
Consumption of unhealthy diets is exacerbating the burden of age-related ill health in aging populations. Such diets can program mammalian physiology to cause long-term, detrimental effects. This study shows that in Drosophila melanogaster, an unhealthy, high-sugar diet in early adulthood programs lifespan to curtail later-life survival despite subsequent dietary improvement. Excess dietary sugar promotes insulin-like signaling, inhibits dFOXO-the Drosophila homolog of forkhead box O (FOXO) transcription factors-and represses expression of dFOXO target genes encoding epigenetic regulators. Crucially, dfoxo is required both for transcriptional changes that mark the fly's dietary history and for nutritional programming of lifespan by excess dietary sugar, and this mechanism is conserved in Caenorhabditis elegans. The study implicates FOXO factors, the evolutionarily conserved determinants of animal longevity, in the mechanisms of nutritional programming of animal lifespan.

Polesello, C. and Le Bourg, E. (2017). A mild cold stress that increases resistance to heat lowers FOXO translocation in Drosophila melanogaster. Biogerontology [Epub ahead of print]. PubMed ID: 28677014
Summary:
Previous studies have shown that subjecting Drosophila melanogaster flies to a mild stress at young or middle age can increase lifespan and resistance to severe stresses throughout life and that the NF-kappaB-like transcription factor DIF, the 70 kDa heat-shock proteins, and the Drosophila Forkhead box class O (dFOXO) transcription factor could explain some of these effects. The present study showed that two dFOXO mutants do not survive longer heat if previously subjected to a mild cold stress, contrarily to wild-type flies. This cold pretreatment had nearly no effect on dFOXO nuclear translocation in wild-type males. Heat stress strongly increased dFOXO translocation, but this effect was lowered in cold-pretreated males. Because cold-pretreated wild-type males survived longer heat and had nevertheless a lower dFOXO translocation after this heat stress, one can conclude that dFOXO is required to resist heat but that the cold pretreatment makes that other mechanisms partly substitute to dFOXO translocation.
Borch Jensen, M., Qi, Y., Riley, R., Rabkina, L. and Jasper, H. (2017). PGAM5 promotes lasting FoxO activation after developmental mitochondrial stress and extends lifespan in Drosophila. Elife 6. PubMed ID: 28891792
Summary:
The mitochondrial unfolded protein response (UPRmt) has been associated with long lifespan across metazoans. In Caenorhabditis elegans, mild developmental mitochondrial stress activates UPRmt reporters and extends lifespan. This study shows that similar developmental stress is necessary and sufficient to extend Drosophila lifespan, and identify Phosphoglycerate Mutase 5 (PGAM5) as a mediator of this response. Developmental mitochondrial stress leads to activation of FoxO, via Apoptosis Signal-regulating Kinase 1 (ASK1) and Jun-N-terminal Kinase (JNK). This activation persists into adulthood and induces a select set of chaperones, many of which have been implicated in lifespan extension in flies. Persistent FoxO activation can be reversed by a high-protein diet in adulthood, through mTORC1 and GCN-2 activity. Accordingly, the observed lifespan extension is prevented on a high-protein diet and in FoxO-null flies. The diet-sensitivity of this pathway has important implications for interventions that seek to engage the UPRmt to improve metabolic health and longevity.
Erkosar, B., Kolly, S., van der Meer, J. R. and Kawecki, T. J. (2017). Adaptation to chronic nutritional stress leads to reduced dependence on microbiota in Drosophila melanogaster. MBio 8(5). PubMed ID: 29066546
Summary:
Numerous studies have shown that animal nutrition is tightly linked to gut microbiota, especially under nutritional stress. In Drosophila, microbiota are known to promote juvenile growth, development, and survival on poor diets, mainly through enhanced digestion leading to changes in hormonal signaling. This study shows that this reliance on microbiota is greatly reduced in replicated Drosophila populations that became genetically adapted to a poor larval diet in the course of over 170 generations of experimental evolution. Protein and polysaccharide digestion in these poor-diet-adapted populations became much less dependent on colonization with microbiota. This was accompanied by changes in expression levels of dFOXO transcription factor, a key regulator of cell growth and survival, and many of its targets. These evolutionary changes in the expression of dFOXO targets to a large degree mimic the response of the same genes to microbiota, suggesting that the evolutionary adaptation to poor diet acted on mechanisms that normally mediate the response to microbiota. This study suggests that some metazoans have retained the evolutionary potential to adapt their physiology such that association with microbiota may become optional rather than essential.
Artoni, F., Kreipke, R., Palmeira, O., Dixon, C., Goldberg, Z. and Ruohola-Baker, H. (2017). Loss of foxo rescues stem cell aging in Drosophila germ line. Elife 6. PubMed ID: 28925355
Summary:
Aging stem cells lose the capacity to properly respond to injury and regenerate their residing tissues. This study utilized the ability of Drosophila melanogaster germline stem cells (GSCs) to survive exposure to low doses of ionizing radiation (IR) as a model of adult stem cell injury and identified a regeneration defect in aging GSCs: while aging GSCs survive exposure to IR, they fail to reenter the cell cycle and regenerate the germline in a timely manner. Mechanistically, foxo and mTOR homologue Tor were identified as important regulators of GSC quiescence following exposure to ionizing radiation. foxo is required for entry in quiescence, while Tor is essential for cell cycle reentry. Importantly, it was further shown that the lack of regeneration in aging germ line stem cells after IR can be rescued by loss of foxo.
Kang, P., Chang, K., Liu, Y., Bouska, M., Birnbaum, A., Karashchuk, G., Thakore, R., Zheng, W., Post, S., Brent, C. S., Li, S., Tatar, M. and Bai, H. (2017). Drosophila Kruppel homolog 1 represses lipolysis through interaction with dFOXO. Sci Rep 7(1): 16369. PubMed ID: 29180716
Summary:
Transcriptional coordination is a vital process contributing to metabolic homeostasis. As one of the key nodes in the metabolic network, the forkhead transcription factor FOXO has been shown to interact with diverse transcription co-factors and integrate signals from multiple pathways to control metabolism, oxidative stress response, and cell cycle. Recently, insulin/FOXO signaling has been implicated in the regulation of insect development via the interaction with insect hormones, such as ecdysone and juvenile hormone. This study identified an interaction between Drosophila FOXO (dFOXO) and the zinc finger transcription factor Kruppel homolog 1 (Kr-h1), one of the key players in juvenile hormone signaling. Kr-h1 mutants show delayed larval development and altered lipid metabolism, in particular induced lipolysis upon starvation. Notably, Kr-h1 physically and genetically interacts with dFOXO in vitro and in vivo to regulate the transcriptional activation of insulin receptor (InR) and adipose lipase brummer (bmm). The transcriptional co-regulation by Kr-h1 and dFOXO may represent a broad mechanism by which Kruppel-like factors integrate with insulin signaling to maintain metabolic homeostasis and coordinate organism growth.
Tas, D., Stickley, L., Miozzo, F., Koch, R., Loncle, N., Sabado, V., Gnagi, B. and Nagoshi, E. (2018). Parallel roles of transcription factors dFOXO and FER2 in the development and maintenance of dopaminergic neurons. PLoS Genet 14(3): e1007271. PubMed ID: 29529025
Summary:
Forkhead box (FOXO) proteins are evolutionarily conserved, stress-responsive transcription factors (TFs) that can promote or counteract cell death. Mutations in FOXO genes are implicated in numerous pathologies, including age-dependent neurodegenerative disorders, such as Parkinson's disease (PD). However, the complex regulation and downstream mechanisms of FOXOs present a challenge in understanding their roles in the pathogenesis of PD. This study investigate the involvement of FOXO in the death of dopaminergic (DA) neurons, the key pathological feature of PD, in Drosophila. dFOXO null mutants exhibit a selective loss of DA neurons in the subgroup crucial for locomotion, the protocerebral anterior medial (PAM) cluster, during development as well as in adulthood. PAM neuron-targeted adult-restricted knockdown demonstrates that dFOXO in adult PAM neurons tissue-autonomously promotes neuronal survival during aging. dFOXO and the bHLH-TF 48-related-2 (FER2) act in parallel to protect PAM neurons from different forms of cellular stress. Remarkably, however, dFOXO and FER2 share common downstream processes leading to the regulation of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results indicate a role of dFOXO in neuroprotection and highlight the notion that multiple genetic and environmental factors interact to increase the risk of DA neuron degeneration and the development of PD.
Nowak, K., Gupta, A. and Stocker, H. (2018). FoxO restricts growth and differentiation of cells with elevated TORC1 activity under nutrient restriction. PLoS Genet 14(4): e1007347. PubMed ID: 29677182
Summary:
TORC1, a central regulator of cell survival, growth, and metabolism, is activated in a variety of cancers. Loss of the tumor suppressors PTEN and Tsc1/2 results in hyperactivation of TORC1. Tumors caused by the loss of PTEN, but not Tsc1/2, are often malignant and have been shown to be insensitive to nutrient restriction (NR). In Drosophila, loss of PTEN or Tsc1 results in hypertrophic overgrowth of epithelial tissues under normal nutritional conditions, and an enhanced TORC1-dependent hyperplastic overgrowth of PTEN mutant tissue under NR. This study demonstrates that epithelial cells lacking Tsc1 or Tsc2 also acquire a growth advantage under NR. The overgrowth correlates with high TORC1 activity, and activating TORC1 downstream of Tsc1 by overexpression of Rheb is sufficient to enhance tissue growth. In contrast to cells lacking PTEN, Tsc1 mutant cells show decreased PKB activity, and the extent of Tsc1 mutant overgrowth is dependent on the loss of PKB-mediated inhibition of the transcription factor FoxO. Removal of FoxO function from Tsc1 mutant tissue induces massive hyperplasia, precocious differentiation, and morphological defects specifically under NR, demonstrating that FoxO activation is responsible for restricting overgrowth of Tsc1 mutant tissue. The activation status of FoxO may thus explain why tumors caused by the loss of Tsc1-in contrast to PTEN-rarely become malignant.
BIOLOGICAL OVERVIEW

The Drosophila Insulin receptor (InR) regulates cell growth and proliferation through the PI3K/Akt pathway, which is conserved in metazoan organisms. The Drosophila forkhead-related transcription factor Foxo is a key component of the insulin signaling cascade. Foxo is phosphorylated by Akt upon insulin treatment, leading to cytoplasmic retention and inhibition of its transcriptional activity. Mutant Foxo lacking Akt phosphorylation sites no longer responds to insulin inhibition, remains in the nucleus, and is constitutively active. Foxo activation in S2 cells induces growth arrest and activates two key players of the InR/PI3K/Akt pathway: the translational regulator d4EBP/Thor (eukaryotic initiation factor 4E binding protein) and the InR itself. Induction of d4EBP likely leads to growth inhibition by Foxo, whereas activation of InR provides a novel transcriptionally induced feedback control mechanism. Targeted expression of Foxo in fly tissues regulates organ size by specifying cell number with no effect on cell size. These results establish Foxo as a key transcriptional regulator of the insulin pathway that modulates growth and proliferation (Puig, 2003).

During the development of multicellular organisms, growth is tightly regulated by controlling cell number and cell size so that each organ reaches its appropriate dimensions in relation to the size of the organism. Many studies indicate that growth and proliferation are coordinated but distinct processes and that cells progress through the cell cycle only when sufficient mass, size, and macromolecular biosynthesis have been reached. Organism growth is controlled by coordinating both cell cycle progression and survival, which is modulated by nutrient availability, growth factors, and temperature. Growth factors can stimulate cell division and survival by activating the insulin receptor, which in turn acts through two main signal transduction cascades: the Ras/MAP kinase and the PI3K/Akt kinase pathways. Insulin-mediated activation of PI3K increases production of 3'-phosphorylated phosphoinositide lipids (PIP3) that serve as second messengers to recruit Akt to the plasma membrane. Once properly localized in the membrane, Akt becomes activated by phosphorylation and in turn phosphorylates a number of downstream targets that ultimately regulate cell growth. For example, Akt stimulates protein synthesis through activation of the Target of rapamycin (TOR) kinase, which subsequently phosphorylates and inactivates the translational repressor eukaryotic initiation factor 4E-binding protein (4EBP) (Puig, 2003 and references therein).

Mammalian Akt, in addition to modulating translation, regulates transcription through the forkhead-related FOXO family of transcription factors FOXO1, FOXO3a, and FOXO4 (Burgering, 2002) by phosphorylating these proteins at three conserved serine/threonine residues. This leads to retention of FOXO transcription factors in the cytoplasm, thereby down-regulating RNA synthesis of specific target genes (Burgering, 2002) that affect cell cycle progression (Kops, 1999; Alvarez, 2001) and apoptosis (Brunet, 1999; Dijkers, 2000) and that modulate metabolic genes (Ayala, 1999; Durham, 1999; Guo, 1999; Hall, 2000; Nasrin, 2000; Schmoll, 2000; Nakae, 2001; Nadal, 2002). Thus, FOXO transcription factors play a critical role in regulating cell growth and survival (Puig, 2003).

Recent genetic studies in Drosophila and Caenorhabditis elegans show that the InR/PI3K/Akt signaling pathway is largely conserved in metazoans. In invertebrates, this pathway apparently plays an essential role in regulating life span as well as body, organ, and cell size. C. elegans can enter the dauer state when food is limited; when conditions improve, growth is stimulated by activating the InR/PI3K/Akt signaling pathway. Worms with mutations in this pathway are small, their organs have fewer cells, and they live longer. Interestingly, mutations in the transcription factor DAF-16/FOXO suppress this phenotype (Lin, 1997; Ogg, 1997), suggesting that this key transcription factor is negatively regulated by the InR/PI3K/Akt pathway (Burgering, 2002). In Drosophila, the InR/PI3K/Akt pathway is also thought to regulate body size and life span. Flies with heteroallelic combinations of InR mutations are reduced in size because of fewer and smaller cells. Mutations in other components of the dInR/dPI3K/dAkt pathway produce similar phenotypes. Although the identity and number of specific gene targets of the insulin signaling pathway in Drosophila remain unclear, one important downstream effector of insulin signaling appears to be the translational inhibitor d4EBP (Miron, 2001; Puig, 2003).

Despite the importance of the InR/PI3K/Akt pathway in regulating cell growth and proliferation in Drosophila, little is known about how signaling is controlled downstream of Akt. In C. elegans and mammals, a critical member of this pathway downstream of Akt is the transcription factor DAF-16/FOXO, which counteracts insulin signaling. However, the Drosophila equivalent of DAF-16/FOXO has thus far not been described. In addition, the mechanisms, if any, that are used to provide feedback regulation of the InR pathway are unknown. Drosophila Foxo proves to be a key transcriptional regulator that controls both downstream target genes responsible for growth as well as upstream feedback targets in the insulin signaling pathway (Puig, 2003).

Foxo functions in feedback regulation of the insulin signaling pathway. Surprisingly, Foxo transcriptionally activates downstream as well as upstream targets of the InR/PI3K/Akt signaling cascade, providing the first evidence for a transcriptional feedback mechanism in the InR pathway that regulates cell growth and proliferation. Furthermore, it was found that Foxo modulates the InR signaling pathway by transcriptionally activating two key elements of this signaling cascade: the downstream effector 4EBP and InR itself. Activation of InR provides an interesting way to modulate the InR/PI3K/Akt pathway via a feedback regulatory loop that may have important implications during development (Puig, 2003).

It has been shown that regulation of growth during development depends on the availability of nutrients and that food limitation decreases the Drosophila insulin-like peptide (DILP) levels. An activated InR pathway promotes growth, whereas mutations in this pathway impair normal development. For instance, flies mutant for chico, which encodes the Drosophila homolog of IRS1-4, are developmentally delayed, have severely reduced body size and increased fat accumulation. Likewise, mutations in several other components of the InR pathway produce related phenotypes. The finding that Foxo is involved with feedback activation of InR provides a novel mechanism for the cells to regulate growth by responding rapidly to changes in nutrient conditions. When nutrients are abundant, elevated levels of DILPs are secreted to activate the InR pathway, and the resulting downstream signaling promotes growth, in part by inhibiting Foxo. These favorable nutrient conditions allow growth and development. However, in a situation in which nutrients are limiting, DILPs are secreted at a reduced rate, the InR pathway is activated, and Foxo remains dephosphorylated, nuclear, and active. As a result, growth is inhibited, in part by Foxo activation of 4EBP. However, because Foxo is active when nutrients are limited, InR becomes up-regulated and primed to signal when triggered by changes in DILPs levels. In this way, when nutrient conditions change, the cells are highly sensitized and able to respond rapidly by turning on the mechanisms that stimulate growth, including shutting down Foxo via Akt phosphorylation. In addition, Foxo-mediated InR transcriptional activation presents a highly sensitive way to regulate the InR/PI3K/Akt signaling pathway in response to subtle developmental cues that modulate DILP levels. High levels of DILPs activate InR, which lead to the inhibition of its own transcription, turning off the pathway. Conversely, reduced levels of DILPs activate dInR transcription. This sensitizes the pathway and provides a mechanism to amplify growth factor signals by allowing detection of lower levels of DILPs. Once this pathway is activated, feedback regulation via Foxo dampens InR expression and signaling. Growth and development through the InR pathway is thus exquisitely balanced and regulated. Interestingly, overexpression of Foxo under the control of strong promoters (GMR, tubulin) in flies results in severe morphological defects. These results suggest that abnormally high levels of Foxo may produce growth arrest in developing organs. Surprisingly, flies with loss-of-function mutations in Foxo appear to develop normally, indicating that Foxo is not essential during fly development. These results suggest the existence of additional mechanisms that modulate insulin signaling and underscore the complexity of such developmental pathways (Puig, 2003).

In mammals, it has been reported that Akt promotes protein synthesis through TOR-mediated phosphorylation and subsequent inactivation of the translational inhibitor 4EBP. Hypophosphorylated 4EBP interacts strongly with eIF4E, providing a mechanism for Akt to regulate EBP via Tor. In flies, a similar mechanism has been reported. In contrast, the role of Foxo in transcriptionally modulating d4EBP has not been previously described. The finding that Foxo directly regulates 4EBP transcription provides an alternative and parallel mechanism for Akt to inhibit 4EBP function. Under conditions in which the insulin pathway is active, Akt sequesters Foxo in the cytoplasm, and 4EBP transcription is turned off. When the InR/PI3K/Akt pathway is inactive, Foxo is free to stimulate transcription of 4EBP and inhibit protein synthesis. Overexpression of 4EBP has been shown to slow growth in mammalian cells, and this study shows that Foxo overexpression leads to growth arrest in Drosophila S2 cells. It is therefore likely that up-regulation of 4EBP by Foxo contributes to the observed growth arrest. However, additional mechanisms (i.e., induction of apoptosis by Foxo), which could also contribute to the observed phenotype, cannot be ruled out (Puig, 2003).

It has been well documented that the InR/PI3K/Akt pathway regulates cell number and cell size in Drosophila. However, the precise mechanisms by which the insulin pathway controls these parameters remain unknown. Mutations in some members of this pathway affect cell size as well as cell number, whereas mutations in other members appear to affect only cell size. For example, mutations in both InR and PI3K produce smaller flies with reduced numbers of cells and smaller cell size. Mutations in the negative regulator PTEN produce bigger cells and increased proliferation. In contrast, mutations in Akt produce smaller organs without affecting cell number, only cell size. Overexpression of mutant 4EBP with increased binding affinity for eIF4E produces flies with smaller and fewer cells. Thus, until now, none of the components of the Drosophila InR pathway has been found to regulate only cell number without influencing cell size. It was therefore intriguing to find that overexpression of Foxo produces a reduction in cell number without any measurable effect on cell size. This is reminiscent of the transcription factor c-myc, which in mammals regulates cell number without altering cell size but in Drosophila affects both cell size and number. Taken together, the results of this study reveal the species specific complexity of the mechanisms that regulate cell growth and proliferation. Indeed, the results suggest that the InR/PI3K/Akt pathway is far from being a simple linear cascade. Instead, Akt appears to regulate numerous targets, each one with its own set of downstream effectors. In addition, it is conceivable that Foxo may be regulated by kinases other than Akt. In mammals, FOXO4 has been shown to be regulated by the Ras/MAP kinase pathway (Kops, 1999), and a similar mechanism may exist in flies. Interestingly, microarray experiments identified >200 genes in addition to 4EBP that are up-regulated by Foxo, which increases the complexity of transcriptional regulation affected by Foxo. Additional studies will be necessary to determine the multiple mechanisms by which the insulin signaling cascade dictates cell number and size during development of the metazoan body plan (Puig, 2003).

Genetic studies in C. elegans and Drosophila have led to two models regarding the output of the insulin pathway. (1) The complete epistasis of daf-16, coding for the Foxo homolog, over the insulin pathway mutants daf-2, age-1, akt-1 and akt-2 suggests that the primary function of PKB/AKT is to inactivate FOXO transcription factors (Paradis, 1998). (2) It has been proposed that the TSC tumor suppressor complex is the major target of PKB in the regulation of cell growth in Drosophila. Analysis of Drosophila Foxo indicates that it is indeed a critical PKB target, but that it mediates only one aspect of PKB function. Several lines of evidence support this model. (1) The effects of ectopic overexpression of Foxo and hFOXO3a in the developing Drosophila eye are altered by Dp110 and PKB signaling as well as by nutrient levels. Under conditions of lowered insulin signaling, the phenotypes resulting from expression of foxo and hFOXO3a are dramatically enhanced. This situation was mimicked by expressing a PKB-insensitive phosphorylation mutant, suggesting that endogenous PKB signaling is required to mitigate the effects of ectopically expressed Foxo and hFOXO3a. (2) The physiological relevance of Foxo in PKB signaling is most vividly demonstrated by the observation that the larval lethality associated with the complete loss of PKB is rescued by foxo mutations to the extent that some flies develop to pharate adults. The lethality associated with loss of PKB function is therefore to a large extent due to the hyperactivation of Foxo. (3) Loss of Foxo function suppresses the effects of insulin-signaling mutations only partially; Foxo mediates a reduction in cell number but not in cell size in response to reduced insulin signaling (Jünger, 2003).

Genetic analysis of the control of body size in Drosophila has revealed two classes of mutations. Flies carrying mutations in chico or viable allelic combinations of Inr, Dp110, and PKB are reduced in body size by up to 50% owing to a reduction in both cell size and cell number. Conversely, flies mutant for S6K exhibit a more moderate reduction in body size, caused almost exclusively by a reduction in cell size. This suggests that the pathways controlling cell number and cell size bifurcate at or below PKB. Although foxo single mutants have no obvious size phenotype, loss of foxo substantially suppresses the cell-number reduction observed in insulin-signaling mutants. It appears that Foxo mediates the repression of proliferation in flies mutant for Inr, chico, Dp110, and PKB without being required for the reduction in cell size. Chico-Foxo double mutant flies even have slightly smaller cells than chico mutants, suggesting that removal of Foxo permits cell-cycle acceleration under conditions of impaired insulin signaling. The pathway controlling body size in response to insulin therefore bifurcates at the level of PKB: PKB controls cell number by inhibiting Foxo function and PKB controls cell size, at least under some conditions, by regulating S6K activity by phosphorylation of TSC2 (Jünger, 2003).

The signaling systems controlling cell size and cell number are tightly interconnected. Genetic and biochemical analyses have revealed five different links between the TSC-TOR-S6K pathway and the Inr-PKB-Foxo pathway. (1) Under conditions of unnaturally high insulin-signaling activity (that is, following the oncogenic activation of PKB) PKB phosphorylates and inactivates TSC2, resulting in increased activation of S6K. Under normal culture conditions this regulation does not seem critical, however, loss of dPKB function does not lower dS6K activity in larval extracts. (2) Under physiological conditions, PDK1 regulates PKB as well as S6K. (3) S6K itself downregulates dPKB activity in a negative feedback loop. (4) Under severe starvation conditions, nuclear Foxo presumably activates target genes that reduce cell proliferation. One of these target genes is 4E-BP, which encodes an inhibitor of translation initiation. When conditions improve, the insulin and TOR signaling pathways can stimulate translation by disrupting the 4E-BP/eIF4E complex via phosphorylation of 4E-BP, and in parallel by repressing FOXO-dependent 4E-BP expression. (5) Under even more severe starvation or stress conditions, full activation of Foxo upregulates expression of the insulin receptor itself, thus rendering the cell hypersensitive to low insulin levels. These multiple positive and negative interactions ensure a continuous fine adjustment of the growth rate to changing environmental conditions (Jünger, 2003).

Genetic dissection of signaling by insulin and its target DAF-16 has been pioneered in C. elegans and has helped to unravel the role of this pathway in dauer formation and longevity. The same pathway with the homologous nuclear targets operates in flies in the control of cell growth and proliferation, processes that do not involve insulin signaling in worms. Dauer formation and possibly longevity affect the entire organism and do not depend on cell-autonomous functions of the insulin signaling pathway. The cell-growth phenotype in Drosophila, however, depends on the cell-autonomous functioning of the insulin-signaling cascade. Insects enter diapause in response to diverse environmental cues (nutrients, day length or temperature) and arrest development or the aging process in a manner similar to dauer formation in worms. Ageing, and possibly diapause, is also under the control of the insulin pathway in Drosophila. It has recently been shown that heterozygous IGF-1R mutant mice also exhibit a prolonged lifespan. It therefore appears that the function of the insulin pathway, its components, and possibly at least some of its targets, have been conserved throughout evolution (Jünger, 2003).

The longevity phenotype of IGF-1R-deficient mice is associated with enhanced resistance to oxidative stress. It is likely that this phenomenon is due to hyperactivation of FOXO proteins, since several studies have shown that FOXO transcription factors play a role in the oxidative-stress response in mammalian cells as well as in C. elegans. The observation that foxo mutant flies are hypersensitive to oxidative stress confirms that, in addition to their role in insulin signaling, the role of FOXO proteins in protecting against cellular stress is highly conserved. The mechanism by which Foxo confers oxidative-stress resistance is not yet known. In a microarray experiment, several genes encoding cytochrome P450 enzymes were identified as Foxo target gene candidates. Since it has been shown that cytochrome P450 enzymes reduce the toxic effects of paraquat in mice, they might partially mediate the protective effect of Foxo. Furthermore, it remains to be established whether the regulation of Foxo by insulin is required for Foxo's protective properties. It is tempting to speculate that distinct stress-induced signaling pathways activate Foxo under conditions of cellular stress, in addition to the negative input from the insulin cascade, since several stress-induced phosphorylation sites are conserved between hFOXO3a and Foxo. This view is supported by the observation that overexpression of a FOXO variant that cannot be inactivated by PKB elicits cell death, a phenotype not observed in larval tissues lacking insulin-signaling components. This result argues that Foxo induces cellular responses that are independent of insulin (Jünger, 2003).

The emerging model postulates that positive and negative inputs converge on FOXO proteins in response to different environmental conditions, making them central and important integrators controlling cellular (cell-cycle progression) and organismal adaptations (dauer formation, diapause and longevity. Elucidating the positive inputs that converge on FOXO, by mutating conserved phosphorylation sites in the single Drosophila homolog of this class, should help arrive at a better understand Foxo's integrator function (Jünger, 2003).

FoxO limits microtubule stability and is itself negatively regulated by microtubule disruption

Transcription factors are essential for regulating neuronal microtubules (MTs) during development and after axon damage. In this paper, a novel neuronal function for Drosophila melanogaster FoxO was identified in limiting MT stability at the neuromuscular junction (NMJ). foxO loss-of-function NMJs displayed augmented MT stability. In contrast, motor neuronal overexpression of wild-type FoxO moderately destabilized MTs, whereas overexpression of constitutively nuclear FoxO severely destabilized MTs. Thus, FoxO negatively regulates synaptic MT stability. FoxO family members are well-established components of stress-activated feedback loops. It is hypothesized that FoxO might also be regulated by cytoskeletal stress because it was well situated to shape neuronal MT organization after cytoskeletal damage. Indeed, levels of neuronal FoxO were strongly reduced after acute pharmacological MT disruption as well as sustained genetic disruption of the neuronal cytoskeleton. This decrease was independent of the dual leucine zipper kinase-Wallenda pathway and required function of Akt kinase. A model is presented wherein FoxO degradation is a component of a stabilizing, protective response to cytoskeletal insult (Nechipurenko, 2012).

Until recently, cell survival constituted the best-defined neuronal function for FoxOs. FoxO1, FoxO3, and FoxO6 are widely expressed in the developing and adult rodent brain. They have been implicated in establishment of polarity as RNAi-mediated knockdown of FoxO in central neurons promotes aberrant distribution of MAPs. Remarkably, all neuronal processes express both axonal and dendritic MAPs after FoxO knockdown. Furthermore, recent evidence demonstrates that the foxO homologue daf-16 regulates neuronal morphology in C. elegans. Daf-18/PTEN modulates the phosphoinositide 3-kinase signaling pathway to activate Daf-16/FoxO and promote developmental axon outgrowth in the AIY sensory interneuron. Together, these studies strongly suggest that FoxO family members are conserved regulators of neuronal morphology. (Nechipurenko, 2012).

What are the relevant FoxO transcriptional targets that mediate its effect on MT organization? Because Futsch distribution is sensitive to FoxO levels, and futsch mutations suppress foxO LOF phenotypes at the NMJ, FoxO could transcriptionally repress Futsch. However, this hypothesis is not favored, since total Futsch protein levels remain unchanged in foxO LOF and GOF animals. A reasonable model to explain the observed NMJ phenotypes is that FoxO up-regulates transcription of MT-destabilizing proteins or, alternatively, represses expression of MT-stabilizing molecules. It will be critical to identify the downstream effectors of foxO in this context. Bearing on this issue, mammalian FoxO1 has recently been reported to act in a complex with SnoN1 to repress expression of MAP Doublecortin in the brain (Nechipurenko, 2012).

Notably, a link between FoxO and MT stability has also been alluded to in the context of endothelial cell differentiation. FoxO1-deficient endothelial cells display thickening of MT bundles accompanied by expansion of the MT network into the cell periphery -- a set of phenotypes in agreement with those presented in this study (Nechipurenko, 2012).

In the current study, the prediction is that FoxO-regulated targets promote MT dynamics at the NMJ. Proper maintenance of dynamic MTs is crucial for axon outgrowth, guidance, and branching. Disruption of pre- or postsynaptic MT networks using genetic or pharmacological approaches also interferes with synaptic differentiation. The FoxO-dependent phenotypes described in this study underscore the significance of properly regulated MT behavior for synaptogenesis. However, the relationship between NMJ growth and MT stability is complex. Ample precedent exists for a positive correlation between MT stability and NMJ growth. Yet, there are also examples of increased MT stability associated with decreased NMJ growth. Supporting the importance of a dynamic MT population in neurite growth, the Knot transcription factor drives expansive dendritic elaboration in a class of sensory neurons by promoting expression of the MT-destabilizing protein Spastin. The differential effects of overexpression of wild-type and constitutively nuclear forms of FoxO on NMJ growth argue that although moderately destabilized MTs promote inappropriate growth, severely destabilized MTs compromise NMJ organization (Nechipurenko, 2012).

This study has demonstrated that FoxO is subject to regulation by MT destabilization. Because foxO NMJs display elevated MT stability, a reduction in neuronal FoxO in response to cytoskeletal stress is predicted to promote MT stabilization. Although FoxO is often tied to stress signaling, stress typically drives an increase, not a decrease, in nuclear FoxO levels. The observed down-regulation of FoxO is unexpected and supports an intimate and reciprocal relationship between FoxO and MTs. Notably, FoxO3a levels are reduced in rat dorsal root ganglia neurons after sciatic nerve crush. In addition, FoxO1, FoxO3, and FoxO4 emerged from a microarray analysis of genes regulated by retrograde signaling after sciatic nerve lesion. Consistent with these data, all three FoxO family members were found to be rapidly down-regulated after injury. These findings argue that FoxO family members represent conserved components of the neuronal injury response (Nechipurenko, 2012).

Subcellular localization of FoxO proteins is a primary mechanism for regulating their activity and is controlled via extensive posttranslational modifications. FoxOs are also known to be subject to ubiquitin-mediated degradation. The E3 ubiquitin ligases Skp2 and MDM2 are required for ubiquitination of mammalian FoxO1 and FoxO3, respectively. E3 ligase-dependent ubiquitination and degradation of FoxO proteins depend on FoxO phosphorylation by several kinases, including Akt in human primary tumors and cancer cell lines. This study demonstrates rapid attenuation of FoxO levels in response to cytoskeletal stress. These data strongly argue that FoxO is subject to active degradation in this context. To define the upstream regulatory events driving FoxO degradation, it will be essential to identify the relevant ubiquitin ligase (Nechipurenko, 2012).

This study has demonstrated that FoxO regulation after MT destabilization requires the Akt kinase. Furthermore, levels of the active phosphorylated form of Akt are elevated after acute MT disruption, raising the issue of whether Akt activity is regulated by diverse forms of cytoskeletal damage. In fact, Akt is activated in mammalian neurons after both physical damage and treatment with chemotherapeutic drugs. It will be crucial to identify signaling events upstream of Akt activation to define the signaling cascade controlling FoxO activity (Nechipurenko, 2012).

This work establishes that FoxO controls MT stability at the NMJ and is itself regulated by MT disruption. Neuronal MT organization is shaped by intra- and extracellular cues that modify both its structural and mechanical attributes. Several pathways modulate MT behavior by acting locally for example, through modification of MAPs or tubulin. This study presents in vivo evidence that synaptic MT dynamics are also controlled at the transcriptional level. Such regulatory mechanisms would allow for precise coordinated control of MT behavior in response to diverse cues. Proper MT regulation is essential for neuronal morphogenesis, synaptic maturation, and plasticity—and MT dysfunction is tied to motor neuron and neurodegenerative diseases. Given a single FoxO orthologue in the fly and extensive evolutionary conservation, Drosophila represents an ideal system for mapping FoxO-dependent regulatory circuits responsible for modulating MT stability in response to developmental and environmental stimuli (Nechipurenko, 2012).

Minibrain/Dyrk1a regulates food intake through the Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals

Feeding behavior, one of the most essential activities in animals, is tightly regulated by neuroendocrine factors. Drosophila short neuropeptide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY) regulate food intake. Understanding the molecular mechanism of sNPF and NPY signaling is critical to elucidate feeding regulation. This study found that minibrain (mnb) and the mammalian ortholog Dyrk1a, target genes of sNPF and NPY signaling, regulate food intake in Drosophila and mice. In Drosophila neuronal cells and mouse hypothalamic cells, sNPF and NPY modulated the mnb and Dyrk1a expression through the PKA-CREB pathway. Increased Dyrk1a activated Sirt1 to regulate the deacetylation of FOXO, which potentiated FOXO-induced sNPF/NPY expression and in turn promoted food intake. Conversely, AKT-mediated insulin signaling suppressed FOXO-mediated sNPF/NPY expression, which resulted in decreasing food intake. Furthermore, human Dyrk1a transgenic mice exhibited decreased FOXO acetylation and increased NPY expression in the hypothalamus, and increased food intake. These findings demonstrate that Mnb/Dyrk1a regulates food intake through the evolutionary conserved Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals (Hong, 2012).

The production of sNPF and NPY in sNPFnergic and hypothalamic neurons of flies and mammals respectively, is increased during fasting. These neuropeptides are secreted to produce paracrine and endocrine effects but also feedback upon their synthesizing neurons where they respectively induce mnb and Dyrk1a gene expression through the PKA-CREB pathway. This Mnb/Dyrk1a kinase phosphorylates and activates the Sir2/Sirt1 deacetylase, which in turn deacetylates and activates the FOXO transcription factor. Among its many potential targets, FOXO then increases sNPF/NPY mRNA expression. Negative controls modulate the positive feedback of sNPF/NPY. Feeding activates the insulin receptor-PI3K-AKT pathway. FOXO becomes phosphorylated and transcriptionally inactivated by translocation to the cytoplasm. In this state the induction of sNPF/NPY by FOXO is decreased. Because sNPF and NPY are orexogenic, their positive feedback during fasting should reinforce the propensity for food intake whereas the negative regulation of sNPF and NPY mRNA during feeding condition would then contribute to satiety (Hong, 2012).

FOXO family transcriptional factors are involved in metabolism, longevity, and cell proliferation. FOXO is in part regulated in these processes by post-transcriptional modifications including phosphorylation and acetylation. In many model systems, the ligand activated Insulin-PI3K-AKT pathway phosphorylates FOXO to inactivate this transcription factor by moving it to the cytoplasm. The cytoplasmic localization of FOXO is mediated by 14-3-3 chaperone proteins in Drosophila and mammals. FOXO may also be acetylated, as is FoxO1 of mice, by the CREB-binding protein (CBP)/p300 acetylase and this inhibits FOXO transcriptional function by suppressing its DNA-binding affinity. Such FoxO1 acetylation can be reversed by SirT1 to help activate the FoxO1 transcription factor. This study describes for Drosophila how dFOXO in sNPFR1 neurons regulates the expression of sNPF and food intake. This mechanism parallels how hypothalamic FoxO1 regulates food intake through its control of orexigenic NPY and Agrp in rodents. Post-transcriptional modification of FOXO is central to these controls in both animals. sNPF and NPY expression is increased when FOXO is deacetylated by Sir2/Sirt1, while sNPF and NPY are decreased when FOXO is phosphorylated via the Insulin-PI3K-AKT pathway. Post-transcriptional modifications of FOXO proteins play a critical role for controlling food intake through the sNPF and NPY expression in flies and rodents (Hong, 2012).

Mnb/Dyrk1a participate in olfactory learning, circadian rhythm, and the development of the nervous system and brain. Mnb and Dyrk1a proteins contain a nuclear targeting signal sequence, a protein kinase domain, a PEST domain, and a serine/threonine rich domain. The kinase domains are evolutionary well-conserved from flies to humans. In Down syndrome (DS), chromosome 21 trisomy gives patients three copies of a critical region that includes the Mnb/Dyrk1a; trisomy of this region is associated with anomalies of both the nervous and endocrine systems. DS patients often show high Body Mass Index due to the increased fat mass. Children with DS have elevated serum leptin coupled with leptin resistance, both of which contribute to the obesity risk common to DS patients. This study found a novel function of Mnb/Dyrk1a that may underlay this metabolic condition of DS patients. Mnb/Dyrk1a regulates food intake in flies and mice. This is controlled by sNPF/NPY-PKA-CREB upstream signaling and thus produces downstream affects upon Sir2/Sirt1-FOXO-sNPF/NPY. Fasting not only increases the expression of mnb, but also of sNPF, suggesting that Mnb kinase activates a positive feedback loop where Sir2-dFOXO induces sNPF gene expression. Notably, fasting increases Sirt1 deacetylase activity and localizes FoxO1 to the nucleus in the orexogenic AgRP neurons of the mouse hypothalamus. Increased dosage of Dyrk1a in DS patients may reinforce the positive feedback by NPY and disrupt the balance between hunger and satiety required to maintain a healthy body mass (Hong, 2012).

Insulin produced in the pancreas affects the hypothalamus to regulate feeding in mammals. Insulin injected into the intracerebroventrical of the hypothalamus reduces food intake while inhibiting insulin receptors of the hypothalamic ARC nucleus causes hyperphasia and obesity in rodent models. This study showed a similar pattern for Drosophila where overexpression of insulin-like peptide (Dilp2) at insulin producing neurons decreased food intake while food intake was increased by inhibiting the insulin receptor in sNPFR1 expressing neurons. Likewise, during fasting, serum insulin and leptin levels are decreased in mammals, as is mRNA for insulin-like peptides of Drosophila. Thus, the mechanism by which insulin and insulin receptor signaling suppresses food intake is conserved from fly to mammals in at least some important ways (Hong, 2012).

Previous work has shown how sNPF signaling regulates Dilp expression through ERK in IPCs and controls growth in Drosophila (Lee, 2008). This study shows that sNPF signaling regulates mnb expression through the PKA-CREB pathway in non-IPC neurons and controls food intake. Since sNPF works through the sNPFR1 receptor, sNPFR1 in IPCs and non-IPCs neurons might transduce different signals and thereby modulate different phenotypes. Four Dilps (Dilp1, 2, 3, and 5) are expressed in the IPCs of the brain. Interestingly, levels of Dilp1 and 2 mRNA are reduced in the sNPF mutant, which has small body size, but this study finds only Dilp3 and 5 mRNA levels are reduced upon 24 h fasting. Likewise, only Dilp5 is reduced when adult flies are maintained on yeast-limited diets. In addition, Dilp1 and 2 null mutants show slight reduced body weights but Dilp3 and Dilp5 null mutants do not. These results suggest that Dilp1 and 2 behave like a mammalian insulin growth factor for size regulation while Dilp3 and 5 act like a mammalian insulin for the regulation of metabolism. However, in the long term starvation, Dilp2 and Dilp5 mRNA levels are reduced and Dilp3 mRNA expression is increased (Hong, 2012).

During fasting, sNPF but not sNPFR1 mRNA expression was increased in samples prepared from fly heads increasing food intake. In contrast, in feeding, the high level of insulin signaling reduced sNPF but not sNPFR1 mRNA expression and suppresses food intake. Interestingly, in the antenna of starved flies, sNPFR1 but not sNPF mRNA expression is increased and induces presynaptic facilitation, which results in effective odor-driven food search. However, high insulin signaling suppresses sNPFR1 mRNA expression and prevents presynaptic facilitation in DM1 glomerulus. These results indicate that starvation-mediated or insulin signaling-mediated sNPF-sNPFR1 signaling plays a critical role in Drosophila feeding behavior including food intake and food search even though the fine tuning is different (Hong, 2012).

This study presents a molecular mechanism for how sNPF and NPY regulate food intake in Drosophila and mice. A system of positive feedback regulation for sNPF and NPY signaling is described that increases food intake and a mode of negative regulation for sNPF and NPY by the insulin signaling that suppresses food intake. Modifications of the FOXO protein play a critical role for regulating sNPF and NPY expression, resulting in the control of food intake (Hong, 2012).

Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in Drosophila, chromatin immunoprecipitation-sequencing analysis was performed with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a) within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling (Bai, 2013).

Insulin/IGF-1 signaling modulates longevity in many animals. Genetic analysis in C. elegans and Drosophila shows that insulin/IGF-1 signaling requires the DAF-16/FOXO transcription factor to extend lifespan, while in humans several polymorphisms of FoxO3A are associated with exceptional longevity. Although many downstream effectors of FOXO have been identified through genome-wide studies, the targets of FOXO responsible for longevity assurance upon reduced insulin signaling are largely unknown. This study found 273 genes targeted by Drosophila FOXO using ChIP-Seq with two long-lived insulin mutant genotypes. Focused was placed on daw, an Activin ligand, which is transcriptionally repressed by FOXO upon reduced insulin/IGF signaling. Inactivation of daw and of its downstream signaling partners babo and Smox extend lifespan. These results are reminiscent of observations from C. elegans where reduced TGF-β/dauer signaling extends longevity. Notably, the lifespan extension of TGF-β/dauer mutants (e.g. daf-7 (e1372) mutants) can be suppressed by daf-16 mutants, suggesting that TGF-β signaling intersects with the insulin/IGF-1 pathway for longevity in C. elegans. In phylogenetic analysis, DAF-7, Daw and mammalian Activin-like proteins share common ancestry. Activin signaling, in response to insulin/IGF-1, may thus represent a taxonomically conserved longevity assurance pathway (Bai, 2013).

Longevity benefits of reduced Activin (TGF-β/dauer) in C. elegans were resolved only when the matricide or 'bagging' (due to progeny hatching within the mother) was prevented by treating daf-7(e1372) mutants with 5-fluorodeoxyuridine (FUdR) to block progeny development. This approach made it possible to distinguish the role of Activin in somatic aging from the previously recognized influence of BMP (Sma/Mab signaling) upon reproductive aging in C. elegans. Activin, of course, is a somatically expressed regulatory hormone of mammalian menstrual cycles that induces follicle-stimulating hormone (FSH) in the pituitary gland. In young females, FSH is suppressed within a cycle when maturing follicles secrete the related TGF-β hormone Inhibin. In mammalian reproductive aging, the effect of Activin in the pituitary becomes unopposed as the stock of primary follicles declines, thus inducing elevated production of FSH. This study now found that reduced Activin but not BMP signaling favors somatic persistence in Drosophila. These parallels between reproductive and somatic aging among invertebrate models and humans suggest that unopposed Activin signaling is pro-aging while favoring reproduction (Bai, 2013).

Reduced insulin/IGF signaling extends lifespan through interacting autonomous and non-autonomous actions. Reducing IIS in some distal tissues has been shown to slow aging because this reduces insulin secretion from a few neurons: reducing IIS by increasing dFOXO in fat body or muscle extends Drosophila fly lifespan while decreasing IPC production of systemically secreted DILP2. This study has identified Activin as a direct, downstream target of insulin/dFOXO signaling within muscles that has the capacity to non-autonomously regulate lifespan. Knockdown of Activin in muscle but not in fat body is sufficient to prolong lifespan. RNAi for muscle Activin signaling led to decreased circulating DILP2 and increased peripheral insulin signaling. Muscle is thus proposed to produce a signaling factor, a myokine, which impacts organism-wide aging and metabolism (Bai, 2013).

Aging muscle may produce different myokine-like signals in response to their physiological state. Aged muscles degenerate in many ways including changes in composition, mitochondria, regenerative potential and within-cell protein homeostasis. Protein homeostasis is normally maintained, at least in part, by autophagy. Loss of macroautophagy and chaperone-mediated autophagy with age will accelerate the accumulation of damaged proteins. Expression of Atg8a in Drosophila CNS is reported to extend lifespan by 56% (Simonsen, 2008), while recent studies find elevated autophagy in long-lived mutants including those of the insulin/IGF-1 signaling pathway. The current results show that insulin/IGF signaling can regulate autophagy through its control of Activin via dFOXO. Poly-ubiquitinated proteins accumulate in aging Drosophila while lysosome activity and macroautophagy decline. Muscle performance with age (flight, climbing) was preserved by inactivating Activin within this tissue. This genetic treatment also reduced the accumulation of protein aggregates. These effects are mediated by blocking the transcription factor Smox, which otherwise represses Atg8a. Smox directly regulates Atg8a through its conserved Smad binding motif (AGAC AGAC). These results, however, contrast with an observation where TGF-β1 promotes autophagy in mouse mesangial cells (Bai, 2013).

Insulin/IGF-1 signaling is a widely conserved longevity assurance pathway. The data indicate that reduced insulin/IGF-1 retards aging at least in part through its FOXO-mediated control of Activin. Furthermore, affecting Activin only in muscle is sufficient to slow its functional decline as well as to extend lifespan. Autophagy within aging muscle controls these outcomes, and it is now found that Activin directly regulates autophagy through Smox-mediated repression of Atg8a. If extrapolated to mammals, pharmaceutical manipulations of Activin may reduce age-dependent muscle pathology associated with impaired autophagy, and potentially increase healthy and total lifespan through beneficial signaling derived from such preserved tissue (Bai, 2013).

Acute fasting regulates retrograde synaptic enhancement through a 4E-BP-dependent mechanism

While beneficial effects of fasting on organismal function and health are well appreciated, little is known about the molecular details of how fasting influences synaptic function and plasticity. Genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, these findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. These results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits (Kauwe, 2016).

Many forms of dietary restriction can reduce cellular stress, improve organismal health, and in many instances extend lifespan in a number of model organisms. A major cellular function that is highly sensitive to nutrient intake from yeast to mammals is cap-dependent translation under the regulation of the target of rapamycin (TOR). TOR promotes cap-dependent translation primarily through phosphorylation of 4E-BPs (eukaryotic initiation factor 4E binding proteins) and p70 S6Ks (S6 ribosomal protein kinases). Phosphorylation of 4E-BP suppresses its ability to bind and inhibit the interaction between eIF4E (eukaryotic initiation factor 4E) and the initiation factor eIF4G, a critical step for translation initiation. In addition to the regulation by TOR, 4E-BP undergoes upregulation in response to dietary restriction and starvation. Together these two responses result in a strong inhibition of protein synthesis and act as a metabolic brake. Multiple lines of evidence suggest that fasting-induced increase in ketone bodies influences neuronal excitability and aspects of neurotransmitter release; however, little is known about how different forms of dietary restriction, by influencing protein translation, can exert an effect on the regulation of synaptic function and plasticity (Kauwe, 2016).

At the Drosophila larval neuromuscular junction (NMJ), the genetic removal of GluRIIA, one of five glutamate receptor subunits, reduces the postsynaptic response to unitary release of neurotransmitter. As a result of this reduced response to neurotransmitter, a retrograde signal is triggered in the postsynaptic muscle that ultimately leads to a compensatory enhancement in presynaptic release from the motor neuron, a process that is conserved at the vertebrate NMJs. The maintenance of this homeostatic synaptic compensation or retrograde synaptic enhancement is highly sensitive to postsynaptic cap-dependent translation in Drosophila; mutations in either Target of Rapamycin (TOR) or eIF4E can dominantly suppress the synaptic compensation in GluRIIA mutants (Penney, 2012). Interestingly, postsynaptic overexpression of TOR or S6K, in an otherwise wild-type muscle, is also sufficient to trigger a retrograde enhancement in presynaptic neurotransmitter release, suggesting that normal synaptic strength may be affected by a postsynaptic signal from the muscle (Penney, 2012; Kauwe, 2016 and references therein).

Previous findings have demonstrated that postsynaptic translation plays a critical role in the regulation of retrograde synaptic enhancement at the NMJ. Therefore, in light of the effect of dietary restriction on TOR-dependent translation, this study set out to investigate the consequence of nutrient restriction on retrograde synaptic compensation in GluRIIA mutants. Electrophysiological analysis indicates that acute fasting, but not amino acid restriction, blocks this retrograde synaptic compensation. This block is not merely due to reduced TOR activity, but rather a result of transcriptional upregulation of postsynaptic 4E-BP under the control of the transcription factor Foxo. These results indicate that the retrograde regulation of synaptic strength at the NMJ depends on the balance between 4E-BP and TOR (Kauwe, 2016).

A few hours of fasting can have a strong impact on retrograde synaptic enhancement at the Drosophila larval NMJ. Removal of food source acutely activates 4E-BP transcription in postsynaptic muscles in a Foxo-dependent manner, thereby leading to the inhibition of retrograde synaptic enhancement at the NMJ. The results indicate that Foxo and 4E-BP act cell autonomously in postsynaptic muscles to exert a retrograde negative regulation on presynaptic neurotransmitter release. Future studies are needed to test whether fasting-induced alterations in insulin signaling underlie the transcriptional upregulation of 4E-BP via its effect on Foxo in postsynaptic muscles. While 4E-BP-mediated suppression of synaptic enhancement as a result of fasting could be considered undesirable during development, it can be beneficial under conditions of abnormally high synaptic activity. As such, 4E-BP-mediated inhibition of retrograde synaptic enhancement and the subsequent dampening of circuit activity might provide an explanation for the beneficial effects of fasting in reducing seizures in some cases. Similarly, in cases where dysregulation of TOR activity is thought to underlie abnormal circuit activity, such as in TSC models, intermittent fasting could potentially dampen the increase in synaptic release through a 4E-BP-dependent inhibition, thereby stabilizing neuronal circuits (Kauwe, 2016).

In addition to its role as a molecular responder to stress, 4E-BP exerts a constitutive negative regulation on presynaptic neurotransmitter release at the NMJ. Electrophysiological analysis of loss-of-function mutant larvae indicates that 4E-BP functions in postsynaptic muscles to constitutively provide a retrograde negative influence on synaptic strength. In light of these findings, a two-pronged scheme is proposed for the retrograde regulation of synaptic strength at the NMJ. On the one hand, a positive input from TOR is mediated through S6K/eIF4A and eIF4E to enhance postsynaptic translation. Synaptic compensation in GluRIIA mutant larva appears to rely mostly on this axis as evidenced by strong sensitivity to S6K heterozygosity and no change in the proportion of phosphorylated 4E-BP versus non-phosphorylated 4E-BP levels. Opposing this positive input, 4E-BP inhibits translation by sequestering eIF4E and adjusting the degree of retrograde compensation. Indeed, loss of 4E-BP leads to a strong increase in quantal content that is highly sensitive to eIF4E heterozygosity but not sensitive to S6K heterozygosity. The balance between these two forces reveals itself also when 4E-BP loss-of-function mutants are rescued by a non-phosphorylatable 4E-BP transgene. In this combination TOR can no longer inhibit 4E-BP, and this study finds that the presynaptic neurotransmitter release is lower than wild-type, similarly to what is observed in TOR hypomorphic mutants (Penney, 2012). A working model is proposed in which the negative force of 4E-BP is under constant check via phosphorylation by TOR, and the positive input from TOR/S6K is constitutively countered by 4E-BP’s ability to sequester eIF4E, a dynamic duel that ensures a tight regulation of synaptic strength (Kauwe, 2016).

Parallel roles of transcription factors dFOXO and FER2 in the development and maintenance of dopaminergic neurons

Forkhead box (FOXO) proteins are evolutionarily conserved, stress-responsive transcription factors (TFs) that can promote or counteract cell death. Mutations in FOXO genes are implicated in numerous pathologies, including age-dependent neurodegenerative disorders, such as Parkinson's disease (PD). However, the complex regulation and downstream mechanisms of FOXOs present a challenge in understanding their roles in the pathogenesis of PD. This study investigate the involvement of FOXO in the death of dopaminergic (DA) neurons, the key pathological feature of PD, in Drosophila. dFOXO null mutants exhibit a selective loss of DA neurons in the subgroup crucial for locomotion, the protocerebral anterior medial (PAM) cluster, during development as well as in adulthood. PAM neuron-targeted adult-restricted knockdown demonstrates that dFOXO in adult PAM neurons tissue-autonomously promotes neuronal survival during aging. dFOXO and the bHLH-TF 48-related-2 (FER2) act in parallel to protect PAM neurons from different forms of cellular stress. Remarkably, however, dFOXO and FER2 share common downstream processes leading to the regulation of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results indicate a role of dFOXO in neuroprotection and highlight the notion that multiple genetic and environmental factors interact to increase the risk of DA neuron degeneration and the development of PD (Tas, 2018).

This study demonstrates that dFOXO is tissue-autonomously required for the maintenance of DA neurons in the PAM cluster during aging. Evidence is presented that dFOXO and FER2 act in parallel pathways to protect PAM neurons from different forms of cellular stress. However, dFOXO and FER2 partly share downstream pathways leading to the control of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results highlight the notion that multiple genetic and environmental risk factors interact and affect DA neuron survival. Importantly, genome-wide association studies (GWAS) and functional studies in mammals implicated FOXO family TFs, including FOXO1, FOXO3, FOXA1 and FOXA2, in the maintenance of DA neurons and in PD. The current results are in accordance with these studies and further suggest that dfoxo loss of function offers a valuable tool to study the pathogenesis of sporadic PD (Tas, 2018).

In mammals, although constitutive activation of FOXO3 induces loss of DA neurons in the SN, the expression of a dominant negative FOXO3 causes oxidative damage that leads to DA neuron loss. Nevertheless, both the dominant-negative form and mild activation of FOXO3 are neuroprotective in mice overexpressing α-Synuclein. Thus, FOXO3 can be protective or detrimental to DA neurons in the substantia nigra (SN) depending on its activity levels and genetic background. Likewise, in Drosophila, previous studies have shown paradoxical roles for dFOXO in the survival of DA neurons in various PD models. dFOXO overexpression has been shown to ameliorates mitochondrial abnormality and protects DA neurons in Pink1 null mutants. Conversely, dFOXO mediates the death of DA neurons by inducing apoptosis in DJ-1β loss-of-function mutants and in flies overexpressing dLRRK (Tas, 2018).

The apparent paradox concerning the role of FOXOs suggests that the activity of FOXO factors should be tightly regulated in order to exert neuroprotective function, i.e., activity levels of FOXO factors that are too high or too low are both detrimental to DA neurons. Alternatively, the differences in the reagents and experimental conditions used to examine the role of FOXOs in prior studies may have contributed to the differences in the interpretation of the results. A number of previous experiments in Drosophila studies mentioned above used global overexpression of dFOXO and tissues other than DA neurons, such as eyes, muscles and wings, were mainly analyzed to evaluate its effect. Furthermore, for dfoxo loss of function experiments, these studies used dfoxo21 or dfoxo25, which contain nucleotide transversions resulting in premature stop codons but nevertheless are not null alleles (Tas, 2018).

The present study used a genuine null allele of dfoxo, dfoxoΔ94, to examine whether endogenous dFOXO is protective or detrimental to DA neurons. The results demonstrating that dFOXO is protective to DA neurons in the PAM cluster under basal conditions are in accordance with the report by Koh (2012). Curiously, however, that study observed the loss of DA neurons in the DL1 (dorso lateral 1) cluster, which corresponds to the PPL1 cluster in the nomenclature for adult DA neurons. Additionally, by PAM neuron-targeted constitutive and adult-restricted dfoxo RNAi, the current study shows that dFOXO expression within adult PAM neurons is required for the maintenance of PAM neurons in aged flies (Tas, 2018).

Overexpression of dfoxo in PAM neurons prevents the developmental impairment and age-dependent loss of PAM neurons in Fer22 mutants. Conversely, Fer2 overexpression ameliorates the effect of dfoxoΔ94 mutation on the development and maintenance of PAM neurons. Since Fer2 and dfoxo do not transcriptionally regulate each other, the reciprocal rescue suggests that their downstream mechanisms partly overlap. In line with this interpretation, this study showed that autophagy and mitochondrial morphology are commonly impaired in PAM neurons of dfoxoΔ94 and Fer22 mutants (Tas, 2018).

Mounting evidence indicates that FOXO factors regulate autophagy by controlling the expression of Atg genes in flies and mammals. FOXOs also regulate factors controlling mitophagy and mitochondrial remodeling in mammals. Therefore, dFOXO may regulate autophagy and mitophagy in PAM neurons, although dysregulation in autophagy and mitochondrial morphology in dfoxoΔ94 could be secondary effects of cellular damage. Uncovering genetic pathways downstream of dFOXO and FER2 and how they intersect will yield valuable information, especially because the current results suggest that targeted overexpression of dfoxo or Fer2 in DA neurons may confer protection against DA neuron demise in various genetic models of PD (Tas, 2018).

Consistent with the known role of PAM neurons in controlling locomotion, startle-induced climbing ability in dfoxoΔ94 and Fer22 mutants is significantly improved by the expression of dfoxo with R58E02-GAL4. However, R58E02>dfoxo does not rescue the shortened lifespan of dfoxoΔ94 and even further reduces the lifespan of Fer22. Therefore, neuroprotective role of dFOXO is independent of its role in longevity regulation. Many fly models of PD show lifespan shortening, which is likely caused by the systemic effect of mitochondrial impairment and/or elevated oxidative stress levels rather than DA neuron demise. Lifespan shortening of dfoxoΔ94 and Fer22 mutants may be similarly attributed to the impairment in mitochondrial biology or (in the case of Fer22) oxidative stress regulation in cells other than PAM neurons (Tas, 2018).

Given that mitochondrial dysfunction and oxidative stress are tightly linked and both implicated in neurodegeneration, it is surprising that no evidence was found that PAM degeneration in dfoxoΔ94 is associated with chronic or acute oxidative stress, unlike Fer22 mutants. The results also show no evidence that amino acid intake during adulthood is relevant for survival of PAM neurons. Then, how is dFOXO signaling activated during adulthood to promote PAM neuron survival in aged flies? Aging is associated with loss of proteostasis and FOXOs play a key role in cellular proteostasis. Consistent with the findings in other tissues, autophagy levels in PAM neurons decrease with age, and this is accelerated in dfoxoΔ94. Thus, age-dependent decrease in basal activity of autophagy might be an intracellular stress signal that leads to the activation of dFOXO in PAM neurons (Tas, 2018).

This study reveals an unexpected crosstalk between two pathways mediated by two TFs, dFOXO and FER2, in the development and maintenance of DA neurons in the PAM cluster. Importantly, both genes are also required for the proper development of PAM neurons. This is in line with the fact that several mammalian TFs required for DA neuron development play critical roles in the maintenance of adult midbrain DA neurons. dFOXO homologs FOXA1 and A2 fall within this category, suggesting that TFs having dual roles in the development and maintenance of DA neurons is an evolutionarily conserved mechanism of neuroprotection. Furthermore, the data suggest that loss of dfoxo expression before adulthood has lasting detrimental effect on the survival of PAM neurons in aging flies, which may be partly regulated non-cell-autonomously by dFOXO in the larval fat body or in other tissues. In conclusion, this study provides a starting point to investigate TF networks underlying the link between aberrant neural development and neurodegeneration, which will present new opportunities to better understand the etiology of sporadic PD (Tas, 2018).


REGULATION

Signaling upstream of Foxo

If the Foxo protein is a bona fide ortholog of mammalian FOXO, insulin should regulate its activity by phosphorylation via a cascade involving Akt (Brunet, 1999; Kops, 1999). Also, phosphorylation of the three specific serine/threonine residues should sequester this transcription factor in the cytoplasm. To test these properties, S2 cells were grown with or without insulin, and endogenous Foxo was detected by Western blot analysis. A single band migrating with molecular mass 113 kD was recognized by both N- and C-terminal Foxo antibodies in the untreated samples. In contrast, the insulin-treated sample revealed the presence of two bands, one migrating with the mobility of 113 kD and a second band with slower mobility. A similar insulin-induced shift was obtained with transfected Foxo-V5 expressed in S2 cells and detected with a V5 antibody. These results suggest that the slower-migrating band may correspond to a phosphorylated form of Foxo (Puig, 2003).

To establish that the slower-migrating form of Foxo induced by insulin treatment is indeed caused by Akt-catalyzed phosphorylation, a mutant form of Foxo was constructed in which all three putative dAkt phosphorylation sites (T44, S190, and S259) were mutated to alanine (FoxoA3). Both wild-type (Foxo-V5) and mutant (FoxoA3-V5) proteins were expressed in S2 cells. After transient expression, the cells were subjected to three different treatments in parallel: insulin; pretreatment with LY294002 (a specific inhibitor of PI3K that counteracts the effects of insulin) followed by insulin treatment, or no treatment control. Extracts derived from cells treated with insulin contained the slower-migrating form of wild-type Foxo when compared with control cells. Pretreatment with the PI3K inhibitor LY294002 reduced the amount of the slower-migrating form of Foxo. In contrast, no slower-migrating species was observed for the triple alanine mutant (FoxoA3) when comparing control, insulin-treated, and LY294002 + insulin-treated samples. To further confirm that the slower-migrating form of Foxo is caused by phosphorylation, cell extracts were incubated with calf intestinal phosphatase (CIP). Western blot analysis showed that the slower-migrating form of Foxo is quantitatively converted to the 113-kD form after CIP treatment. Together, these results indicate that Foxo is phosphorylated by insulin treatment and that this phosphorylation depends on the presence of the dAkt consensus residues T44, S190, and S259 (Puig, 2003).

To test how Foxo subcellular localization is affected by insulin-mediated phosphorylation, S2 cells expressing either wild-type Foxo or mutant FoxoA3 were incubated for 48 h in the absence of serum. Then insulin was added, and localization of transfected Foxo was determined by confocal microscopy after staining with the V5 antibody. When S2 cells are incubated in the absence of serum and insulin, both Drosophila Foxo and dFoxoA3 (constitutively active Drosophila Foxo in which all three putative Akt phosphorylation sites have been mutated to alanine) are found predominantly in the nucleus. After insulin treatment, Foxo is localized in the cytoplasm. In contrast, mutant FoxoA3 remains nuclear even after insulin treatment. This result is consistent with the idea that subcellular localization of Foxo is regulated by insulin (Puig, 2003).

Is Foxo phosphorylation regulated through the PI3K/Akt pathway? A constitutively active form of Drosophila Akt was used in which a myristoylation signal has been fused to the N terminus of Akt. Myr-Akt tagged with V5 epitope was cotransfected in S2 cells grown in the absence of serum and insulin with either Foxo or dFoxoA3, and the phosphorylation state of both proteins was analyzed by Western blot analysis. In the absence of Akt, both Foxo and dFoxoA3 remain unphosphorylated. When Myr-Akt is present in the cells, Foxo but not dFoxoA3 becomes phosphorylated even in the absence of insulin. This result indicates that Myr-dAkt can phosphorylate Foxo in S2 cells. To assess the effect of Foxo phosphorylation by Myr-Akt, use was made of a reporter construct containing four tandem FOXO4-binding sites upstream of the alcohol dehydrogenase distal core promoter driving the luciferase gene (pGL4xFRE). In the absence of Myr-Akt, cells cotransfected with wild-type or mutant Foxo constructs incubated without serum display comparable luciferase activity. In contrast, when Myr-Akt is present, cells cotransfected with wild-type Foxo display luciferase activity that is reduced by more than 65%, whereas activity of the mutant FoxoA3 remains essentially unchanged (Puig, 2003).

These results suggest that insulin induces Foxo phosphorylation through Akt, which leads to cytoplasmic localization and transcriptional inactivation of Foxo. To further confirm that insulin inhibits Foxo activity through Akt, RNAi experiments were performed. S2 cells transfected with either Foxo or FoxoA3 and cotransfected with the luciferase reporter pGL4xFRE were grown in the presence of insulin and treated with dsRNA directed against Drosophila Akt. As a control, dsRNA against lactose repressor (lacI) was used. As expected, Foxo activity is not inhibited by insulin when cells are depleted of Drosophila Akt by dsRNA treatment, but it is inhibited in the lacI control. These results confirm that Akt mediates insulin inhibition of Foxo (Puig, 2003).

PGRP-SC2 promotes gut immune homeostasis to limit commensal dysbiosis and extend lifespan

Interactions between commensals and the host impact the metabolic and immune status of metazoans. Their deregulation is associated with age-related pathologies like chronic inflammation and cancer, especially in barrier epithelia. Maintaining a healthy commensal population by preserving innate immune homeostasis in such epithelia thus promises to promote health and longevity. This study shows that, in the aging intestine of Drosophila, chronic activation of the transcription factor Foxo reduces expression of peptidoglycan recognition protein SC2 (PGRP-SC2), a negative regulator of IMD/Relish innate immune signaling, and homolog of the anti-inflammatory molecules PGLYRP1-4. This repression causes deregulation of Rel/NFκB activity, resulting in commensal dysbiosis, stem cell hyperproliferation, and epithelial dysplasia. Restoring PGRP-SC2 expression in enterocytes of the intestinal epithelium, in turn, prevents dysbiosis, promotes tissue homeostasis, and extends lifespan. These results highlight the importance of commensal control for lifespan of metazoans and identify SC-class PGRPs as longevity-promoting factors (Jasper, 2014).

Transcriptional feedback control of insulin receptor by dFOXO/FOXO1

The insulin signaling pathway evolved to allow a fast response to changes in nutrient availability while keeping glucose concentration constant in serum. This study shows that, both in Drosophila and mammals, insulin receptor (InR) represses its own synthesis by a feedback mechanism directed by the transcription factor dFOXO/FOXO1. In Drosophila, dFOXO is responsible for activating transcription of dInR, and nutritional conditions can modulate this effect. Starvation up-regulates mRNA of dInR in wild-type but not dFOXO-deficient flies. Importantly, FOXO1 acts in mammalian cells like its Drosophila counterpart, up-regulating the InR mRNA level upon fasting. Mammalian cells up-regulate the InR mRNA in the absence of serum, conditions that induce the dephosphorylation and activation of FOXO1. Interestingly, insulin is able to reverse this effect. Therefore, dFOXO/FOXO1 acts as an insulin sensor to activate insulin signaling, allowing a fast response to the hormone after each meal. These results reveal a key feedback control mechanism for dFOXO/FOXO1 in regulating metabolism and insulin signaling (Puig, 2005).

It is well known that the expression and activity of the InR can be regulated by a wide variety of factors and that changes in the numbers of receptor molecules plays a pivotal role in several physiologic and pathologic states. The lowered sensitivity of cells to insulin and the hyperinsulinemia observed in obesity and type II diabetes mellitus is often accompanied by a reduced number of insulin receptors. Insulin is thought to down-regulate its own receptor by a variety of mechanisms that can influence its synthesis as well as degradation. Interestingly, it has been shown that the number of InR molecules correlates with nutritional conditions both in tissue culture cells and in animals. Thus, levels of InR in growing HepG2 cells are relatively low, and they increase substantially if cells are starved. In addition, states of chronic hyperinsulinemia produce a reduction in the number of InR present in the plasma membrane. InR mRNA levels also change in animals depending on fasting-feeding conditions. For example, rats fed a high-fat diet display a decreased number of InR molecules in liver plasma membranes, and InR mRNA levels in rat skeletal muscle or liver increase after fasting, returning to normal levels after insulin treatment or refeeding. Interestingly, tissues other than muscle or liver might have similar regulation. For example, mRNA and protein levels of rat intestinal InR increase up to 230% in fasting conditions, and these effects are fully reversed by refeeding. Similar observations have been made in other organisms. These effects indicate a nutritional influence on the abundance of the InR. Importantly, insulin levels in serum change in parallel to nutrient availability, both in flies and mammals. Thus, when nutrients are high—that is, after a meal—insulin levels increase, while they decrease upon fasting. In Drosophila it has been shown that the InR/PI3K pathway coordinates cellular metabolism with nutritional conditions. Inhibiting this pathway phenocopies the cellular and organismal effects of starvation, while activating it bypasses the nutritional requirements for cell growth. The InR/PI3K pathway regulates the activity of FOXO1 in mammals, C. elegans, and Drosophila, so nutrient activation of the PI3K pathway results in inactivation of FOXO1 by phosphorylation. However, despite this accumulated base of information, the molecular mechanism linking FOXO1 and InR expression had not been revealed (Puig, 2005).

This study shows that mammalian FOXO1 and its Drosophila counterpart dFOXO directly regulate insulin-signaling response to nutritional conditions through a feedback mechanism that involves activation of transcription from the InR promoter. Incubating C2C12 cells with a balanced salt solution or with serum-free medium up-regulates insulin receptor mRNA. Under these conditions, FOXO1 becomes dephosphorylated and actively binds to the InR promoter. When insulin is added to the medium, InR mRNA is down-regulated, even in the absence of serum, vitamins, amino acids, and glucose. Concomitantly, phosphorylation of FOXO1 increases and binding to InR promoter decreases. These results indicate that FOXO1 regulates InR transcription through a direct feedback mechanism that senses insulin levels in serum, which is, in turn, a reflection of nutrient load. It is important to note that, at this point, it cannot be ruled out that the increased InR protein levels caused by FOXO1 could be due to other mechanisms in addition to increased transcription from the InR promoter (i.e., affecting mRNA stability, or protein translation) (Puig, 2005).

In Drosophila a similar mechanism occurs. Incubation of S2 cells with complete medium keeps dFOXO phosphorylated and inactive, while incubation in HBSS dephosphorylates dFOXO. dInR mRNA is up-regulated only when dFOXO is dephosphorylated and active. In addition, wild-type flies starved for 4 d up-regulate dInR, and this effect requires an intact dfoxo gene. These studies indicate that in Drosophila, the PI3K/Akt pathway also senses insulin levels and regulates binding of dFOXO to the dInR promoter accordingly. These results underscore the importance of the InR/PI3K/Akt pathway in sensing nutrients and insulin, a function that has been conserved during evolution. They also highlight the role of FOXO1 as a sensor for insulin levels, promoting accumulation of InR in the absence of insulin, thereby allowing a fast response to the hormone after each meal. Under conditions in which insulin levels are chronically elevated, for example, in obese animals or patients, down-regulation of InR transcription would occur and insulin sensitivity would be impaired. These results establish the FOXO1 transcription factor as a key player in a feedback control mechanism that regulates metabolism and insulin signaling (Puig, 2005).

The results show that in conditions in which insulin levels are low, mammalian FOXO1 activates InR. Interestingly, it was observed that FOXO1 also activates the insulin receptor substrate-2 (IRS-2) promoter under fasting conditions, and, since it occurs with InR, insulin is sufficient to reverse this effect. FOXO1 binds IRS-2 promoter in vitro and in vivo and activates IRS-2 transcription when muscle or liver cells are fasted. In addition, FOXO1 activates IRS-2 promoter in luciferase assays, and this activation depends on the presence of a consensus FRE present in the IRS-2 promoter, because mutating this FRE abolishes FOXO1-dependent activation. Thus, FOXO1 regulation of IRS-2 is parallel to InR regulation. It has also been reported that SREBPs compete with FOXO transcription factors for binding to the IRS-2 promoter in liver; while SREBPs inhibit IRS-2 production, FOXO1 was found to activate IRS-2 transcription. It was also found that fasting promotes binding of FOXO1 to the FRE of the IRS-2 promoter. Therefore, these findings strongly support the conclusions that FOXO1 regulates insulin signaling through a feedback mechanism that impinges on the insulin receptor and at least one of its substrates, IRS-2. After a meal, high levels of insulin peptide hormone activate its cognate receptor, which leads to repression of InR and IRS-2 transcription, resulting in subsequent dampening of the pathway by reducing the number of receptors on the cell surface and by limiting its ability to signal downstream through IRS-2. Conversely, fasting causes reduced levels of InR signaling, which in turn activates FOXO1, leading to increased transcription of InR and IRS-2. Once this transcription mechanism is activated, feedback regulation and phosphorylation of FOXO1 via the insulin signaling cascade automodulates InR expression. Insulin sensitivity could, therefore, be significantly affected by FOXO1 regulation. Regulation of insulin sensitivity by a feedback loop through FOXO1 would allow the cells to keep an exquisite metabolic balance between feeding and fasting states, permitting a faster response of the tissues to insulin changes. This feedback mechanism could well be disrupted in pathological states with abnormally increased insulin levels as is found in the case of insulin-resistant diabetes (Puig, 2005).

Activated FOXO-mediated insulin resistance is blocked by reduction of TOR activity

Reducing insulin/IGF signaling allows for organismal survival during periods of inhospitable conditions by regulating the diapause state, whereby the organism stockpiles lipids, reduces fertility, increases stress resistance, and has an increased lifespan. The Target of Rapamycin (TOR) responds to changes in growth factors, amino acids, oxygen tension, and energy status; however, it is unclear how TOR contributes to physiological homeostasis and disease conditions. This study shows that reducing the function of Drosophila TOR results in decreased lipid stores and glucose levels. Importantly, this reduction of TOR activity blocks the insulin resistance and metabolic syndrome phenotypes associated with increased activity of the insulin responsive transcription factor, FOXO. Reduction in TOR function also protects against age-dependent decline in heart function and increases longevity. Thus, the regulation of TOR activity may be an ancient 'systems biological' means of regulating metabolism and senescence, that has important evolutionary, physiological, and clinical implications (Luong, 2006).

The major cause of metabolic syndrome (defined as a cluster of metabolic abnormalities such as elevated glucose and lipid levels, related to a state of insulin resistance) and diabetes in humans is reduction of insulin signaling, but the underlying pathways and mechanisms are not completely understood. Likewise, caloric excess can lead to nutrient toxicity and desensitization of insulin signaling. Thus, dysregulation of energy homeostasis can lead to metabolic disturbances and predisposition to a variety of endocrine diseases including diabetes, cardiovascular disease, and cancer (Luong, 2006).

One major system that regulates energy homeostasis in higher metazoa is the insulin/IGF pathway. The functionally conserved components of the insulin/IGF pathway like insulin, the insulin receptor (InR), insulin receptor substrate (IRS), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB, a.k.a. Akt) and the forkhead transcription factor FOXO have been shown to be involved in glucose and lipid homeostasis. Loss of insulin signaling in the periphery and in pancreatic β cells can lead to hyperglycemia and diabetes. For example, disruption of the InsR gene in the pancreatic β cells reduces islet size and insulin secretion. IRS1 knockout mice are hyperglycemic, but their pancreatic β cells hypertrophy to compensate for increased peripheral insulin resistance. In contrast, IRS2 knockout mice are diabetic because their pancreatic β cells are absent due to increased cell death. Additionally, systemic loss of insulin signaling in metazoans leads to elevated lipids as seen in the Daf-2 mutant worms, Chico/IRS mutant flies, and IRS2 ablated mice (Luong, 2006 and references therein).

Many of these insulin/IGF-mediated metabolic effects depend on the winged helix transcription factor, FOXO. FOXO was first identified in the worm, C. elegans as Daf-16, a mutation that can suppress the increased lipid levels and longevity caused by loss of Daf-2, the worm InR ortholog. There is a single evolutionarily conserved Drosophila FOXO ortholog and three mammalian FOXO genes (FOXO1, FOXO3a, and FOXO4). FOXO1 controls glucose homeostasis in both peripheral tissues and pancreatic β cells. For example, expression of a constitutively activated FOXO1 (resistant to insulin/IGF-mediated inactivation) in liver and pancreatic β cells causes hepatic insulin resistance and loss of pancreatic β cells via increased apoptosis, whereas reduction of FOXO1 function can reverse the loss of pancreatic β cells and hyperglycemia seen in the IRS2 ablated mice. Thus, FOXO is a critical mediator of insulin signaling in both insulin sending and receiving tissues (Luong, 2006).

The Tuberous Sclerosis Complex (TSC1-2)/Target of Rapamycin (TOR) pathway responds to changes in insulin/IGF levels, amino acid levels, energy charge, lipid status, mitochondrial metabolites, and oxygen tension by adjusting cell growth. In addition to its well-defined role in controlling cell growth, the TSC1-2/TOR pathway may also potentially be a critical regulator of glucose and lipid homeostasis as TSC1-2/TOR signaling functionally interacts with the insulin/IGF pathway. A role for TOR signaling in glucose and lipid homeostasis in mammalian systems is demonstrated by the S6K1 knockout mice. These mice are hyperglycemic caused by diminished insulin secretion due to reduced pancreatic β cell mass. This result is in keeping with studies that show that rapamycin treatment leads to decreased levels of translation, growth, and survival of pancreatic. However, the mS6K1 mutant mice have low lipid levels because of adipocytes that have increased fatty acid β-oxidation. Additionally, the mS6K1 mutant mice show enhanced glucose uptake upon exogenous insulin addition due to insulin hypersensitivity in peripheral tissues via loss of a negative feedback loop on IRS. Thus, TOR signaling via S6K can modulate insulin sensitivity by altering Ser307 and Ser636/639 phosphorylation and IRS protein levels (Luong, 2006).

There are additional levels where TSC1-2/TOR signaling may positively and negatively regulate insulin signaling. There are data that suggest that the IRS Ser302 site is required for signaling to TOR and S6K. Thus, ser/thr phosphorylation of the IRS proteins may mediate both positive and negative signals for energy homeostasis. Furthermore, Akt/PKB activity may also be directly regulated by the nutrient-sensitive TOR pathway. Although the insulin/IGF pathway can signal to the TSC1-2/TOR pathway, recent evidence suggests that TOR may directly control Akt/PKB function because Akt/PKB activation depends on TORC2 complex-specific TOR Ser473 phosphorylation of Akt/PKB. Thus, these studies suggest that dysregulation of TSC1-2/TOR signaling may contribute to the pathological progression of metabolic syndrome and diabetes, yet the direct role and function of TOR is unclear in this context (Luong, 2006).

Another functionally conserved energy homeostatic pathway is the AMP-activated protein kinase (AMPK) pathway. This pathway responds to altered energy states caused by cellular stresses like mitochondrial dysfunction, anti-diabetic drugs, and exercise. Activation of the energy sensing AMPK pathway by activated AMPK as well as metformin or AICAR treatment results in decreased lipogenesis and gluconeogenesis via both central. Activated AMPK can phosphorylate TSC2, which inhibits TOR signaling, while loss of AMPK activity causes an increase in TOR signaling. However, the requirement of TOR function for the AMPK energy response is not known. These effects may also be mediated by targets including glycogen synthase, hormone-sensitive lipase, acetyl-CoA carboxylase-2, HMG-CoA reductase, p300, and p53; the different roles of these proteins in the AMPK-mediated low energy response are not well known. Furthermore, activation of AMPK leads to IRS Ser-789 phosphorylation and enhancement of insulin signaling, which suggests that the AMPK response can act separately from the TOR pathway to enhance insulin signaling. Clearly, there is a great need to understand the regulation of TSC1-2/TOR signaling as it relates to the maintenance of energy homeostasis because TOR function is implicated in both insulin/IGF and AMPK signaling (Luong, 2006).

Although TOR occupies a central node that governs catabolic or anabolic responses to different nutritional and energy states, the resultant metabolic effects of altering TOR function in a metazoan are incompletely and poorly understood. This study examines in detail the function of Drosophila TOR in terms of energy homeostasis and senescent responses. Reduction of TOR function is show to result in decreased glucose and lipid levels with concomitant increase of DILP2 from the insulin producing cells. A reduction of TOR function can block activated FOXO-mediated insulin resistance and metabolic syndrome phenotypes. Taken together, these data indicate that TOR function is required for the maintenance of energy homeostasis and organismal senescence. The additional ramifications of this study are that reduction of TOR function may have clinical utility for treating metabolic syndrome and insulin resistance (Luong, 2006).

In contrast to the elevated lipid levels caused by reduction of systemic insulin signaling, the dTOR7/P mutant (containing a P-element insert into TOR) does not show increased lipid levels. Instead, the dTOR7/P mutant shows decreased lipid levels of the fat body that depend on the function of a lipase involved in lipid metabolism. Elevated ketone bodies were observed in the hypoglycemic dTOR7/P mutant, which is indicative of the increased utilization of lipids. Studies in mammalian cardiac tissue have shown that ketone bodies provide their high energy electrons directly to complex I, the NADH dehydrogenase multienzyme complex, of the mitochondrial electron transport chain. Thus, the altered lipid levels show that TOR has a critical role in determining the fate of fats (Luong, 2006).

It has also been shown that 4EBP is involved in lipid metabolism because the increased lipid levels caused by rapamycin treatment are blocked by a 4EBP mutant. Furthermore, loss of the melted mutant has lower lipid levels, due to lowered triglyceride production. This effect is due to increased 4EBP protein levels via FOXO activation in the fatbody. However, there is no change in glucose levels. In this respect, the melted mutant resembles the FIRKO mouse because it shows decreased triglyceride levels without a change in glucose levels. The dTOR7/P mutant has a different lipid phenotype than the one caused by rapamycin treatment, which suggests that rapamycin alters TOR function in a different manner than the dTOR7/P mutant. Additionally, a novel hypomorphic dTOR FAT domain allele in combination with the dTORP allele also shows low glucose and lipid levels, which suggests that partial reduction of TOR activity represents a unique phenotypic class of TOR metabolic effects versus an allele specific phenotype. Although rapamycin affects TORC1 directly, TORC2 may be altered indirectly via TOR depletion and blocking of TORC2 assembly. Thus, it is not currently clear if the effects of rapamycin are due to inhibiting TORC1 and/or TORC2. It is known that rapamycin can impair pancreatic β cell function because it causes decreased growth and survival. Thus, rapamycin treatment can lead to elevated glucose and lipid levels, possibly as a result of systemic insulin loss (Luong, 2006).

Evidence shows that DILP2 levels are increased in the IPCs of the dTOR7/P mutant and the dTOR7/P mutant has lowered glucose levels. Thus, reduction of TOR function can lead to increased DILP2 levels and a reduction of glucose levels. Recent studies with the Drosophila miRNA-278 mutant also showed elevated DILP levels, yet displayed fatbody-mediated insulin resistance as shown by elevated d4EBP and glucose levels. It is believed that the dTOR7/P mutant represents an insulin-sensitized state because the dTOR7/P mutant shows decreased levels of the insulin resistance marker 4EBP, the dTOR7/P mutant shows decreased glucose levels, and, as discussed below, the dTOR7/P mutant blocks activated FOXO-mediated insulin resistance phenotypes. Thus, the dTOR mutant phenotype resembles a whole-animal 'insulin-sensitized' state that can function below the level of constitutive FOXO activity (Luong, 2006).

Overexpression of activated FOXO in peripheral and IPC tissues results in elevated glucose and lipid levels. Although TOR signaling can alter insulin signaling upstream of FOXO, reducing TOR function is able to reverse these effects. Thus, the results show that reduction of TOR activity can block the activated FOXO-mediated insulin resistance and metabolic syndrome phenotypes. These results suggest that strategies to dampen, reduce, or block TOR signaling may be able to overcome insulin resistance (i.e., hyperglycemia and hypertriglyceridemia) below the level of increased FOXO activity in mammalian systems (Luong, 2006).

Although FOXO has >100 potential targets that might contribute to the metabolic phenotype, this study identified Fatty acid synthase (FAS) and DILP2 as candidate mediators of the TOR effect on the FOXO metabolic phenotypes. The effect on FAS is interesting because it is upregulated by FOXO overexpression and in an IRS/chico mutant and may be an important determinant of the lipid levels. It has also been shown that activation of daf-16/FOXO can decrease the mRNA levels of a worm insulin gene, ins-7 . This result is consistent with results showing that DILP2 mRNA levels are decreased and reducing TOR activity can reverse this FOXO-mediated reduction of DILP2. These results might have parallels with a role for FOXO and TOR in the regulation of insulin levels in mammals (Luong, 2006).

A selective and unexpected regulation of TOR effectors is also seen: loss of 4EBP protein and a mild effect on S6K Ser389 phosphorylation. It has been recently shown that the 4EBP gene is a target of FOXO in Drosophila and thus may represent one of the TOR targets responsible for contributing to the FOXO-mediated metabolic phenotypes. It has also been shown that daf-15/Raptor is a target of daf-16/FOXO in C. elegans and may also contribute to the TOR metabolic and senescent phenotypes. Raptor may also account for the selective difference in the regulation of 4EBP and S6K function by TOR because Raptor binds to both S6K and 4EBP and loss of 4EBP may allow for more S6K binding to Raptor for TOR-mediated phosphorylation. Thus, these results suggest that reduction of TOR function may have selective effects on translation (Luong, 2006).

Reduction of TOR function does not provide resistance against acute stresses or cause sterility. This result is in contrast to the yeast TOR1 mutant, which shows elevated stress resistance, and the d4EBP mutant, which shows stress and starvation sensitivity. Nevertheless, the dTOR7/P mutant has an increased lifespan. This result is in keeping with the yeast, worm and fly studies that show that loss of TOR signaling can increase lifespan, as a major mediator of caloric restriction. Thus, alterations of TOR signaling contribute to the regulation of lifespan (Luong, 2006).

It is also seen that reduction of TOR activity prevents age-dependent functional decline of heart performance. It is not currently clear how TOR is regulating these organ and organismal responses, but the altered lipid metabolism may underlie these changes. For example, changes in lipid metabolism can both autonomously and non-autonomously affect heart function. Thus, reduction of TOR function may reallocate energy stores preferentially for the control of ‘long-term’ responses such as lifespan and organ maintenance. Importantly, there are many potential links between changes in energy homeostasis with alterations in aging and organ senescence. Channelling diverse stimuli like amino acids, growth factors, oxygen tension, and energy charge into the TOR pathway may be an economic method to mobilize fuel stores like lipids to counteract these fluctuations (Luong, 2006).

The conservation of basic mechanisms between Drosophila and mammals is well established. It has been shown that disruption of insulin signaling in non-mammalian systems like Drosophila results in altered glucose and lipid levels. Reducing TOR function can reverse activated FOXO-mediated insulin resistance phenotypes induced in both insulin producing and insulin receiving tissues, and thus this study provides the first direct evidence that reducing TOR function may have a clinical benefit to counter insulin resistance, metabolic syndrome, and/or diabetes. Furthermore, altering TOR signaling may underlie the benefits of various diet and nutritional regimens. These results demonstrate the utility of using the powerful genetics of this system to unravel the complex pathways involved in maintaining glucose and lipid homeostasis. In unraveling the complex genetic network of TOR and InR signaling, although far from completion, the Drosophila model has been indispensable in finding critical components and uncovering functionally important genetic interactions between these two pathways. Thus, the basic mechanisms controlling glucose and lipid homeostasis, including mechanisms by which the TSC1-2/TOR pathway influences insulin signaling as well as the influence of TSC1-2/TOR signaling on peripheral tissue and IPC physiology, are also functionally conserved (Luong, 2006).

This study has described a new use for reducing TOR activity to block insulin resistance, metabolic syndrome, and diabetic-like phenotypes downstream of activated FOXO, underlining the utility of the Drosophila model to identify and analyze components and compounds that block insulin resistance and metabolic syndrome phenotypes as well as pathological aspects of aging and organ senescence (Luong, 2006).

Phosphatidylinositol 3-kinase and Akt nonautonomously promote perineurial glial growth in Drosophila peripheral nerves

Drosophila peripheral nerves, structured similarly to their mammalian counterparts, comprise a layer of motor and sensory axons wrapped by an inner peripheral glia (analogous to the mammalian Schwann cell) and an outer perineurial glia (analogous to the mammalian perineurium). Growth and proliferation within mammalian peripheral nerves are increased by Ras pathway activation: loss-of-function mutations in Nf1, which encodes the Ras inhibitor neurofibromin, cause the human genetic disorder neurofibromatosis, which is characterized by formation of neurofibromas (tumors of peripheral nerves). However, the signaling pathways that control nerve growth downstream of Ras remain incompletely characterized. This study shows that expression specifically within the Drosophila peripheral glia of the constitutively active RasV12 increases perineurial glial thickness. Using chromosomal loss-of-function mutations and transgenes encoding dominant-negative and constitutively active proteins, it was shown that this nonautonomous effect of RasV12 is mediated by the Ras effector phosphatidylinositol 3-kinase (PI3K) and its downstream kinase Akt. The nonautonomous, growth-promoting effects of activated PI3K are suppressed by coexpression within the peripheral glia of FOXO, a transcription factor inhibited by Akt-dependent phosphorylation. It is suggested that Ras-PI3K-Akt activity in the peripheral glia promotes growth of the perineurial glia by inhibiting FOXO. In mammalian peripheral nerves, the Schwann cell releases several growth factors that affect the proliferative properties of neighbors. Some of these factors are oversecreted in Nf1 mutants. These results raise the possibility that neurofibroma formation in individuals with neurofibromatosis might result in part from a Ras-PI3K-Akt-dependent inhibition of FOXO within Schwann cells (Lavery, 2007).

Activating Ras specifically within the peripheral glia was sufficient to increase growth of the perineurial glia. In addition, activating the Ras effector PI3K, but not Raf or Ral, within the peripheral glia was sufficient to increase perineurial glial growth, and inhibiting PI3K activity in the peripheral glia, but not perineurial glia, suppressed the growth-promoting effects of activated Ras. It was also found that activity within the peripheral glia of the PI3K-activated kinase Akt was both necessary and sufficient to mediate the growth-promoting effects of PI3K. Finally, it was found that overexpression within the peripheral glia of FOXO, the forkhead-box transcription factor that is phosphorylated and inactivated by Akt-dependent phosphorylation, was sufficient to suppress the growth-promoting effects of PI3K. Together, these results suggest that Ras activity in the peripheral glia activates nonautonomous growth via the PI3K and Akt-dependent inhibition of FOXO. This observation is consistent with the previous observations that Nf1 mouse Schwann cells oversecrete growth factor(s) that cause increased recruitment of mast cells into the peripheral nerve and is consistent in part with the observation that the proliferation defects of Nf1 mutant mouse or human cells requires hyperactivation of Tor in a PI3K- and Akt-dependent manner (Lavery, 2007).

Perineurial glial growth in Drosophila peripheral nerves is regulated by several genes. These genes include Nf1, which is the Drosophila ortholog of human Nf1, push, which is thought to encode an E3 ubiquitin ligase and two genes implicated in neurotransmitter signaling: amnesiac, which is thought to encode a neuropeptide similar to vertebrate pituitary adenylate cyclase-activating polypeptide, and inebriated (ine), which encodes a member of the Na+/Cl-dependent neurotransmitter transporter family. Some of these genes might regulate perineurial glial growth via the activity of Ras or PI3K within peripheral glia. For example, mutations in push, but not ine, enhance the perineurial glial growth phenotype of RasV12 expressed in peripheral glia. These observations are consistent with the possibility that the activity of ine regulates Ras-GTP levels within peripheral glia. In contrast, push might regulate PI3K in a Ras-independent manner or act in the perineurial glia to regulate sensitivity to peripheral glial growth factors. Additional experiments will be required to distinguish between these possibilities (Lavery, 2007).

There are several lines of evidence from mice and humans suggesting that cell nonautonomous growth regulation, as a consequence of intercellular signaling, underlies neurofibroma formation. First, although neurofibromas arise in individuals heterozygous for Nf1 after loss of Nf1+ from cell(s) within peripheral nerves, neurofibromas are heterogeneous and contain cells that are not clonally related, such as Schwann cells, perineurial cells, and fibroblasts. These observations suggest that neurofibromas arise when a core of Nf1 cells cause overproliferation of their heterozygous neighbors via nonautonomous means. Second, neurofibroma formation in mice and humans requires a homozygous Nf1 mutant genotype in Schwann cells but not other cells within the tumor. Third, Ras-GTP levels in Schwann cells from the mouse Nf1 knock-out mutant are uniformly elevated. In contrast, only a subset of Schwann cells from human neurofibromas exhibit elevated Ras- GTP levels; the possibility has been raised that this subset, but not other Schwann cells from the tumor, is homozygous for Nf1. In this view, these Nf1 cells recruited neighboring Schwann cells that were heterozygous for Nf1 into the tumor by nonautonomous means, such as by the excessive release of one or more growth factors. Fourth, it has been demonstrated that Nf1 Schwann cells oversecrete the ligand for the c-Kit receptor. This oversecretion increased migration of mast cells into peripheral nerves and might be an essential step in neurofibroma formation. These Schwann cells also oversecrete additional factors whose physiological role remains unclear. The molecular mechanisms by which neurofibromin regulates the synthesis or release of these molecules remain incompletely understood. The current observations that Ras activity in the peripheral glia promotes growth nonautonomously via the PI3K- and Akt-dependent inhibition of FOXO might provide insights into the mechanisms by which peripheral nerve growth is regulated nonautonomously by the mammalian Schwann cell (Lavery, 2007).

By hyperactivating Ras, Nf1 mutations could in principle cause tumors via any of several Ras effector pathways. In addition, the diverse types of tumors observed in individuals with neurofibromatosis could result from hyperactivation of distinct Ras effector pathways. The Raf pathway has been viewed as a more relevant effector pathway than the PI3K pathway, mostly because the importance of Ras in the activation of PI3K under physiological conditions remains controversial. In particular, although it is clear that the oncogenic RasV12 mutant is sufficient to activate PI3K, it has sometimes been difficult to demonstrate that wild-type Ras is necessary for PI3K activation. Presumably, this difficulty reflects the fact that PI3K can be activated by Ras-independent as well as Ras-dependent mechanisms, such as direct activation by activated receptor tyrosine kinases or by PIKE-L (phosphatidylinositol kinase enhancer). However, more recently, it has been demonstrated that PI3K and Akt are hyperactivated in several Nf1 mutant cell types and that this hyperactivation is Ras dependent. Furthermore, PI3K activation plays an essential functional role in Nf1-mediated growth defects, as is demonstrated by the observation that PI3K- and Akt-dependent Tor activation is necessary for the proliferation defects of Nf1 mutants to occur: application of rapamycin, a Tor inhibitor, attenuates the ability of Nf1 mutant cells to proliferate. These observations demonstrate that PI3K and Akt play key roles in at least some aspects of Nf1-induced tumor growth (Lavery, 2007).

The results are consistent with these observations. By comparing the effects on perineurial glial growth of peripheral–glial expression of activated Raf, PI3K, or Ral, it was possible to demonstrate that activation of PI3K, not Raf or Ral, is sufficient to promote perineurial glial growth and that PI3K activity in the peripheral glia is necessary to observe the nonautonomous effect of activated Ras on perineurial glial growth. It was similarly shown that Akt activity os necessary and sufficient to mediate the growth-promoting effects of PI3K. However, previous studies have observed that Tor activation is critical for the PI3K- and Akt-dependent growth regulation of Nf1 mutant cells, this study observed a critical role for the PI3K- and Akt-dependent inhibition of the transcription factor FOXO. It is possible that the phenotype observed in previous studies reflects the well characterized ability of PI3K–Tor to activate growth cell autonomously, whereas the phenotype reported in this study reflects nonautonomous growth regulation. In this view, PI3K and Akt regulate autonomous and nonautonomous growth via the Tor and FOXO pathways, respectively (Lavery, 2007).

FOXO presumably inhibits the growth-promoting effects of PI3K and Akt by transcriptional regulation of target genes. Candidate FOXO target genes include those encoding the molecules oversecreted by Nf1 Schwann cells, whereas other targets might be represented in the distinct transcript profiles exhibited by Nf1 Schwann cells or malignant peripheral nerve sheath tumors compared with wild-type Schwann cells. For example, Schwann cells from neurofibromas, but not normal Schwann cells, express the epidermal growth factor (EGF) receptor. Other potential targets include genes encoding growth factors, although ectopic expression within the peripheral glia of two candidate genes, Hedgehog and the EGF ligands spitz and gurken, failed to induce perineurial glial growth. Additional experiments will be required to identify the FOXO targets that regulate nonautonomous growth in peripheral nerves (Lavery, 2007).

Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila

Animals use the insulin/TOR signaling pathway to mediate their response to fluctuations in nutrient availability. Energy and amino acids are monitored at the single-cell level via the TOR branch of the pathway and systemically via insulin signaling to regulate cellular growth and metabolism. Using a combination of genetics, expression profiling, and chromatin immunoprecipitation, this study examined nutritional control of gene expression and identified the transcription factor Myc as an important mediator of TOR-dependent regulation of ribosome biogenesis. myc was also identified as a direct target of FOXO, and genetic evidence is provided that Myc has a key role in mediating the effects of TOR and FOXO on growth and metabolism. FOXO and TOR also converge to regulate protein synthesis, acting via 4E-BP and Lk6, regulators of the translation factor eIF4E. This study uncovers a network of convergent regulation of protein biosynthesis by the FOXO and TOR branches of the nutrient-sensing pathway (Teleman, 2008).

The global transcriptional analysis reported in this study has revealed a surprising degree of interconnectedness between the two branches of the nutrient-sensing pathway. Insulin, acting through PI3K and Akt, feeds into the FOXO and TORC1 branches of the pathway, whereas energy levels (AMP/ATP) and amino acids act directly on the TORC1 branch. How are these inputs integrated to maintain energy balance? It was previously known that 4E-BP is transcriptionally regulated by FOXO and posttranslationally regulated by TOR. This study has identified the protein kinase Lk6 as a second direct FOXO target. Thus, there appear to be two parallel, independent mechanisms by which the TOR and FOXO branches of the insulin signaling pathway converge to regulate eIF4E activity and hence cellular protein translation. This 'belt and suspenders' approach to translational control might be important to make the system robust (Teleman, 2008).

A key finding of this study is the identification of Myc as a point of convergent regulation by the FOXO and TOR branches of the pathway. myc mRNA levels are controlled by FOXO in a tissue-specific manner. In addition, Myc protein levels are dependent on TORC1. Why use two independent means to control Myc levels? Transcription alone would limit the speed with which the system can respond to changing nutritional conditions. This might be detrimental, particularly as conditions worsen. Regulation of Myc activity by TORC1 permits a rapid response to changes in energy levels or amino acid availability and could serve to fine tune the nutritional response in the cell by controlling translational outputs. This parallels the situation with 4E-BP, albeit with a slightly different logic. Reduced insulin signaling allows FOXO to enter the nucleus and increase 4E-BP expression and at the same time alleviates TORC1-mediated inhibition of the existing pool of 4E-BP. A subsequent increase in energy or amino acid levels would permit rapid reinhibition of 4E-BP and thus allow a flexible response during the time needed for the pool of protein elevated in response to reduced insulin levels to decay (Teleman, 2008).

In yeast, TORC1 is known to regulate ribosome biogenesis through different nuclear RNA polymerases. It has been shown that yeast TORC1 can bind DNA directly at the 35S rDNA promoter and activate Pol I-mediated transcription in a rapamycin-sensitive manner. Moreover, yeast TORC1 is known regulate Pol II-dependent RP gene expression by controlling the nuclear localization of the transcription factor SFP1 and CRF1, a corepressor of the forkhead transcription factor FHL1. In Drosophila, TORC1 has recently been reported to regulate a set of protein-coding genes involved in ribosome assembly. This study has identified Myc as the missing link mediating TORC1-dependent regulation of this set of genes. Indeed, the fact that more than 90% of TORC1-activated genes contain E boxes suggests that Myc might be the main mediator of this transcriptional program. This connection suggests that expression of Myc targets as a whole should be responsive to nutrient conditions. Indeed, this study found that 33% of direct Myc targets -- defined as genes reported to be bound by Myc when assayed by DNA adenine methyltransferase ID (DamID) in Kc cells and to be regulated by myc overexpression in larvae -- are downregulated upon nutrient deprivation. This is a significant enrichment of 4-fold relative to all genes in the genome, despite the comparison being based on correlating data from different tissue types (Teleman, 2008).

It seems reasonable that cellular translation rates need to be dampened if the TOR branch of the pathway senses low amino acid levels. As ribosome biogenesis is energetically expensive, it may be advantageous to link ribosome biogenesis and translational control via TORC1. This dual regulation is well reflected in tissue growth, since this study observed that Myc, the regulator of ribosome biogenesis, is essential for tissue growth driven by the TOR pathway but not sufficient to drive growth in the absence of TOR activity. The FOXO branch of the pathway senses reduced insulin or mitogen levels. FOXO is also highly responsive to oxidative and other stresses and would integrate this information into the cellular control of translation. The data support the notion of a network in which TOR and FOXO regulate protein biosynthesis by converging on Myc to regulate ribosome biogenesis and on eIF4E activity via 4E-BP and Lk6 to regulate translation initiation (Teleman, 2008).

The work presented in this study complements a previous study in which larvae were either starved completely or starved for amino acids only, while having a supply of energy in the form of sugar. A significant and positive correlation (~0.4) indicates general agreement between the two data sets, but they differ in two ways. The current goal was to explore the regulatory network by which insulin controls cellular transcription. Individual tissues were isolated rather than assaying the whole animal. Genes found to be regulated in a previous but not in the current assays may be regulated in tissues other than muscle or adipose tissue. Conversely, genes identified only by the current study might be regulated oppositely in different tissues or might only be regulated in a subset of tissues and so be missed in a whole-animal analysis (Teleman, 2008).

Is Myc also involved in nutritional signaling networks in mammals? No similar rapid downregulation of c-myc was seen in response to rapamycin in human cell lines, suggesting that the mechanism by which TOR signaling controls gene expression may differ between phyla. This is further supported by the fact that the sets of genes reported to be rapamycin regulated also appear to be largely distinct in Drosophila and mammalian cells, with the caveat that different cell types were used in the two analyses. Although the mechanism does not appear to be identical in mammals, there are several suggestions in the literature of a connection between c-Myc and nutritional signaling. For example, dMyc and c-Myc share the ability to regulate ribosome biogenesis, although the specific target genes through which they do so are different. There is also evidence that mammalian c-myc expression in liver is regulated by nutrition and that transgenic expression of c-myc in liver affects metabolism, i.e., glucose uptake and gluconeogenesis. Furthermore, it has been reported that FOXO3 represses Myc activity in colon cancer cells by inducing members of the Mad/Mxi family, which are known to antagonize Myc. The current data suggest that Max and Mnt are not transcriptionally regulated by insulin or FOXO in Drosophila, whereas myc is. This is similar to what has been reported in murine lymphoid cells, in which c-myc expression is regulated by the FOXO homolog FKHRL1. These parallels between the fly and mammalian systems suggest a broader connection between insulin signaling and activity of the Myc/Mnt/Max network. Although some features may be different in the two systems, the similarities merit further investigation (Teleman, 2008).

Finally, this work has revealed a surprising amount of tissue specificity in the transcriptional response to insulin signaling. Roughly half of the genes regulated by insulin in adipose tissue or in muscle were not significantly regulated in the other tissue. Furthermore, 155 genes were differentially regulated in the two tissues (i.e., upregulated in one tissue and downregulated in the other). This likely reflects the roles of the different tissues in the organism's response to nutrient deprivation. Further work will elucidate the underlying molecular mechanisms (Teleman, 2008).

Sir2 mediates apoptosis through JNK-dependent pathways in Drosophila: Sir2 can activate a proapoptotic function of FOXO

Increased expression of the histone deacetylase sir2 has been reported to extend the life span of diverse organisms including yeast, Caenorhabditis elegans, and Drosophila melanogaster. A small molecule activator of Sir2, resveratrol, has also been suggested to extend the fitness and survival of these simple model organisms as well as mice fed high calorie diets. However, other studies in yeast have shown that Sir2 itself may prevent life extension, and high expression levels of Sir2 can be toxic to yeast and mouse cells. This conflicting evidence highlights the importance of understanding the mechanisms by which Sir2 expression or activation affects survival of organisms. To investigate the downstream signaling pathways affected by Sir2 in Drosophila, transgenic flies were generated expressing sir2. Overexpression of sir2 in Drosophila promotes caspase-dependent but p53-independent apoptosis that is mediated by the JNK and FOXO signaling pathways. Furthermore, a loss-of-function sir2 mutant partially prevents apoptosis induced by UV irradiation in the eye. Together, these results suggest that Sir2 normally participates in the regulation of cell survival and death in Drosophila (Griswold, 2008).

Drosophila has five sir2-like genes, with sir2 being most homologous to the yeast sir2, C. elegans sir2.1, and human sirt1. Ubiquitous overexpression of sir2 in Drosophila by using EP lines has been reported to extend life span. To understand the consequences of Drosophila sir2 overexpression and to identify its downstream signaling pathways, transgenic flies were generated that can express sir2 in the Drosophila eye, a well established system for characterizing signaling pathways. The use of the gmr-Gal4 driver line to overexpress this gene in developing eyes causes a phenotype, specifically a lack of pigmentation and a rough, bristled appearance. The severity of this phenotype correlates well with dosage of sir2. Consistent with the known pattern of gmr-Gal4 expression, Sir2 expression was seen in the developing eye imaginal disc in cells posterior to the morphogenic furrow. The endogenous Sir2 is also found at a low level in whole heads of Drosophila as well as in photoreceptor cells posterior to the morphogenic furrow and regions of the antennal disc. Ubiquitous sir2 overexpression using the actin-5C-Gal4 or the pan-neuronal driver elav-gal4 resulted in premature death during development, suggesting that Sir2 affects survival in other cell types as well. Additionally, it was found that the overexpressed Sir2 was enzymatically functional because it increased NAD+-dependent deacetylase activity in both larval eye imaginal discs and adult fly heads (Griswold, 2008).

To verify whether the observed phenotype is Sir2-specific, transgenic flies were generated to express a Sir2 paralog, CG5085, which shares 43% identity and 63% similarity in amino acid sequence in the deacetylase domain. CG5085 is indeed a functional Sir2 deacetylase family member because recombinant CG5085 demonstrates NAD+-dependent deacetylase activity. However, although structurally and enzymatically similar, when overexpressed by using the gmr-Gal4 driver, CG5085 did not alter the phenotype of the eye. Furthermore, overexpression of lacZ or the Drosophila G protein-coupled receptor methuselah had no effect on eye morphology. This indicates that the eye phenotype in the transgenic flies overexpressing sir2 is indeed Sir2-specific and not due to either general overexpression of proteins or the deacetylase activity itself (Griswold, 2008).

The finding that overexpression of sir2 results in a deleterious effect on various tissues of the fly contrasts with a previously reported effect of sir2 overexpression on longevity. To clarify this contradictory result, a sir2 overexpression line (EP2300) used in the previous report was crossed with the gmr-Gal4 driver but no defective eye phenotype was found, although it highly expressed sir2. It is plausible that the insertion of EP2300 could affect genes neighboring sir2, thus leading to modification of the eye phenotype. EP2300 is inserted in a 500-bp region upstream of a chaperone gene dnaJ-H, and an increase was observed in the transcription level of dnaJ-H in adult heads when EP2300 was crossed with the gmr-Gal4 driver. Because overexpression of dnaJ-H can suppress the effects of toxic proteins in the eye, transgenic flies were generated overexpressing UAS-sir2 and UAS-dnaJ-H together in the eye, and up-regulation of dnaJ-H was found to ameliorate the defective phenotype caused by sir2 overexpression. This result suggests an explanation for the lack of an eye phenotype in EP2300 line despite an increase in sir2 expression. Coexpression of sir2 and dnaJ-H by EP2300 raises the possibility that the reported effects of this line may give a misleading picture of the role of sir2 in Drosophila aging (Griswold, 2008).

Based on the defective eye phenotype, it was hypothesized that sir2 expression may cause cell death. Thus, the developing eye in third-instar larval imaginal discs was examined by using acridine orange staining, a vital dye that detects dying cells, and the TUNEL assay, which identifies cells undergoing programmed cell death. Staining with acridine orange showed an increase in dying cells in the posterior part of eye discs overexpressing sir2, suggesting that sir2 overexpression causes cell death. Numerous TUNEL-positive cells in the imaginal discs with sir2 overexpression were also found, whereas the control showed few positive cells, indicating that the phenotype is mediated by apoptotic cell death in the developing eye. In addition, in vitro caspase-3 activity in the eye imaginal disc overexpressing sir2 increased 1.5-fold when compared with the control. This increase in caspase-3 activity was also verified by immunostaining for active caspase-3 in the imaginal discs; overexpression of sir2 showed an increase in the number of more intensely stained cells (Griswold, 2008).

A previous report has characterized mammalian Sir2 as an apoptosis inhibitor through its deacetylation of p53. In Drosophila, the role of p53 is still under investigation, although it has been shown to regulate cell death in response to stress, similar to its mammalian homolog. However, the relationship of p53 and Sir2 in Drosophila has not been explored. To determine whether p53 and Sir2 are in the same genetic pathway to cause apoptosis in the eye, p53 and sir2 were coexpressed. The eyes of the flies overexpressing both p53 and sir2 were more severely affected than either p53 or sir2 overexpressed alone. However, overexpression of dominant negative p53 constructs did not rescue the sir2 phenotype. This suggests that the sir2 overexpression effect is p53-independent and that p53 and sir2 overexpression work in parallel to induce cell death in Drosophila (Griswold, 2008).

Sir2 can alter the activity of mammalian FOXO3a by deacetylation. Additionally, to increase life span in C. elegans, overexpression of sir2.1 requires DAF-16, the FOXO3a transcription factor homolog. No such direct link between Sir2 and FOXO has been established in Drosophila; however, overexpression of foxo in the Drosophila eye exhibits a defective eye phenotype. Because the results show that sir2 overexpression induces apoptotic cell death in the Drosophila eye, whether FOXO activity might be involved in the induction of apoptosis as a result of sir2 overexpression in Drosophila was investigated. sir2 was overexpressed in a foxo null mutant background, and a less severe eye phenotype was found, suggesting a genetic interaction between Sir2 and FOXO in cell death pathways in Drosophila (Griswold, 2008).

Recently, it was shown that the foxo-induced defective eye phenotype can be modulated through the JNK signaling pathway. Furthermore, increased activation of JNK is associated with an apoptotic eye phenotype in Drosophila. Because JNK signaling interacts with FOXO and influences apoptotic pathways, whether the JNK pathway is also involved in the Sir2-induced eye phenotype was examined. The transcription level of the JNK phosphatase puc, a downstream target of the JNK signaling pathway, is increased in the heads of flies overexpressing sir2, suggesting an increase in JNK-dependent transcription. Inhibition of this signaling pathway by overexpression of bskDN, a dominant negative form of Drosophila JNK, resulted in a major improvement of the eye phenotype caused by sir2 overexpression. Additionally, inhibition of JNK signaling by coexpression of puc with sir2 demonstrated a significant rescue in the eye, consistent with a report that constitutive overexpression of puc can rescue the eye phenotype caused by increased JNK activity. These rescue flies do not reduce the level of Sir2 below that expressed by coexpression of lacZ. Together, these results suggest that sir2 overexpression requires JNK signaling to induce cell death in the eye (Griswold, 2008).

In summary, the evidence that endogenous Sir2 in the Drosophila eye plays a role in apoptosis is consistent with the finding that sir2 overexpression induces apoptotic cell death in the eye imaginal discs. An important issue is thus the identification of signaling pathways that mediate these effects. JNK signaling is implicated by phenotypic alleviation via the expression of a dominant negative JNK or an inhibitor JNK signaling. Also, loss of function of foxo can ameliorate the eye phenotype induced by sir2 overexpression, suggesting that sir2 can activate a proapoptotic function of FOXO. These pathways may intersect at JNK to induce the proapoptotic function of FOXO. The result of these pathways is the observed increase in proapoptotic gene expression of reaper, grim, and hid. This in turn leads to increased caspase activity and ultimately cell death (Griswold, 2008).

The results show that sir2 overexpression in Drosophila does not necessarily promote longevity, and endogenous Sir2 plays a critical role in regulating cell survival and death in the animal. Hence, it will be of future interest to study the signaling pathways induced by sir2 expression that lead to JNK activation as well as the relationship between Sir2 and FOXO in modulating apoptotic and survival pathways in Drosophila. Together, these will determine pathways affected by sir2 expression and give insights as to how it can mediate both cell survival and cell death (Griswold, 2008).

Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling

Insulin and insulin growth factor have central roles in growth, metabolism and ageing of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain. This study shows that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. These effects are mediated through activation of extracellular signal-related kinase (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan (Lee, 2008).

Neuropeptides regulate a wide range of animal behaviours related to nutrition. In particular, mammalian NPY produced in the hypothalamus of the brain controls food consumption. NPY injection in the hypothalamus of rats produces hyperphagia and obese phenotypes. The Drosophila orthologue of NPY is sNPF. This peptide is expressed in the nervous system and it regulates food intake and body size; overexpression of sNPF produces bigger and heavier flies. Likewise, the G-protein-coupled receptor of sNPF (sNPFR1) is expressed in neurons and shows significant similarity with vertebrate NPY receptors. In mammals, however, little is known about how NPY and sNPF systemically modulate growth, metabolism and lifespan. This study shows that these neuropeptides control expression of insulin-like peptides and subsequently affect insulin signalling in target tissues (Lee, 2008).

Initially the effects of sNPF and sNPFR1 on body size were characterized by measuring the length of flies from head to abdomen. The body size of sNPF hypomorphic Drosophila mutants (sNPFc00448) was 23% of that of the wild-type, whereas overexpressing two copies of the sNPF in the sensory neurons and sensory structures of the nervous systems by MJ94-Gal4 (MJ94>2XsNPF) increased body size by 24%. Similar changes were seen in the overall size of adult wings, which resulted from changes in both cell size and number. Effects on body size were associated with sNPF expression levels: relative to wild type, sNPF levels were 3.5-fold higher in MJ94>2XsNPF and less than half of the wild type in sNPFc00448. In contrast to the effect of sNPF on body size, there was little effect on size from repression or overexpression of the sNPF receptor in MJ94-expressing cells (Lee, 2008).

Drosophila insulin-like peptides (Dilps) modulate growth and adult size; therefore, whether sNPF has a role in insulin-producing neurons was tested. For positive controls, Dilp2 was overexpressed in insulin-producing cells (IPCs) through Dilp2-Gal4, which increased body size, and the IPCs were ablated by expression of Dilp2>reaper to decrease body size. To investigate sNPF signalling, sNPFR1 was overexpressed in the IPCs and a 10% increase in body size was observed. Conversely, expression of the sNPFR1 dominant-negative mutant (Dilp2>sNPFR1-DN) reduced body size by 14%. Manipulation of the sNPF ligand with IPCs expressing Dilp2-Gal4, however, did not affect body size: flies overexpressing sNPF (Dilp2>2XsNPF) or in which sNPF was silenced by RNAi (Dilp2>sNPF-Ri) were of similar size to the wild type. Taken together, these results suggest that sNPF peptide may be secreted from MJ94-expressing sensory neurons and activate sNPFR1 of Dilp2-expressing IPCs (Lee, 2008).

To assess this model, the sNPF ligand and sNPFR1 receptor were visualized in the larval brain. Seven IPCs were detected in each brain hemisphere using the marker Dilp2-Gal4>nGFP. Neurons containing sNPF peptide in the axon terminal and cell body (sNPFnergic neurons) were stained adjacent to these IPCs. As expected, sNPFR1 receptors were localized in the plasma membrane of IPCs marked with Dilp2>DsRed. sNPFR1 was also localized in the neurons of the larval brain hemispheres, sub-oesophagus ganglion, ventral abdominal neurons and descending axons in the ventral ganglion (Lee, 2008).

To study genetic interactions between sNPFR1 and Dilps in IPCs, Dilp1 and Dilp2 interference mutants were generated in the sNPFR1 overexpression background. In contrast to the 10% body size increase by sNPFR1 overexpression in IPCs (Dilp2>sNPFR1), inhibition of Dilp1 and Dilp2 in IPCs (Dilp2>Dilp1-Ri and Dilp2>Dilp2-Ri) generated reduced body size by 10% and 15%, respectively. Inhibition of Dilp1 and Dilp2 with sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+Dilp1-Ri and Dilp2> sNPFR1+Dilp2-Ri) also generated a reduction in body size of 8% and 13%, indicating that Dilp1 and Dilp2 are downstream genes of sNPFR1 in IPCs for regulating body size (Lee, 2008).

To test whether sNPF regulates Dilp expression in larval IPCs, expression of Dilp1, 2, 3 and 5 were assessed in sNPF mutants. Neuronal overexpression of sNPF (MJ94>2XsNPF) markedly increased expression of Dilp2 in IPCs; it also produced novel Dilp2 expression outside of these cells. As expected, reduction of sNPF by MJ94>sNPF-Ri inhibited expression of Dilp2. In common with Dilp2, the expression of Dilp1 was positively regulated by sNPF overexpression and reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPFc00448). Consistent with the model, expression of Dilp1 and Dilp2 was increased more than fourfold with overexpression of the receptor in IPCs (Dilp2>sNPFR1) and decreased by half with inhibition of the receptor gene in IPCs (Dilp2>sNPFR1-DN). Larval IPCs also express Dilp3 and Dilp5. Expression of Dilp3 was reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPFc00448) but expression of Dilp5 was not regulated by any sNPF mutants. There are few functions known to distinguish these various insulin-like peptides. Nutrition-dependent growth regulation is associated with expression of Dilp3 and Dilp5, but not with that of Dilp2. Recent reports show that Dilp2 is reduced in long-lived flies expressing dFOXO or Jun-N-terminal kinase (JNK), whereas Dilp5 is uniquely upregulated upon dietary restriction that increases lifespan (Lee, 2008).

To investigate how Drosophila sNPF regulates Dilp expression, the activation of Drosophila MAP kinase signalling, which includes the action of ERK (encoded by Rolled) and JNK, was measured. sNPF overexpression with MJ94-Gal4 increased phospho-activated pERK relative to basal ERK1/2. Expression of the receptor protein sNPFR1 in IPCs also increased pERK. There were no detectable changes in phospho-activated pJNK in these sNPF and sNPFR1 mutants. Next, whether ERK activation in IPCs was sufficient to induce Dilp expression was tested. Expression of a constitutively active ERK in IPCs (Dilp2>rolledSEM) increased expression of Dilp1 and Dilp2 more than threefold, and both transcripts were repressed less than half by the expression of an ERK inhibitory phosphatase DMKP-3 in IPCs (Dilp2>DMKP-3). In addition, the inhibition of ERK with the sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+DMKP-3) also repressed expression of Dilp1 and Dilp2 compared with that of sNPFR1 overexpression in IPCs (Dilp2>sNPFR1). These results indicate that sNPF and sNPFR1 signalling regulate ERK activation in IPCs, which in turn modulates expression of Dilp1 and Dilp2 (Lee, 2008).

To further examine the effect of sNPF on Dilp, Drosophila CNS-derived neural BG2-c6 cells, which endogenously express sNPFR1 were treated with a synthetic sNPF peptide. Dilp1 and Dilp2 were induced within 15 min, and the elevated transcript persisted for 1 h. Concomitant with this gene expression, sNPF-treated cells activated ERK. Importantly, sNPF did not induce Dilp expression significantly when cells were treated with ERK-specific kinase MEK inhibitor PD98059. To compare the functional conservation of sNPF and NPY in the regulation of insulin expression, similar tests were conduced with rat insulinoma INS-1 cells, which express NPY receptors NPYR1 and NPRY2. When treated with the human NPY peptide, expression of insulin1 and insulin2 and ERK was activated within 15 min. Furthermore, treatment with the MEK inhibitor PD98059 and NPY abolished the induction of insulin1 and insulin2. Together, these findings suggest that the regulation of insulin expression by sNPF or NPY through ERK is evolutionarily conserved in Drosophila and mammals (Lee, 2008).

To verify that sNPF induction of Dilp expression has a physiological consequence, insulin signals at a target tissue, the Drosophila fat body were examined. Fat body cells in flies with neuronal overexpression of sNPF (MJ94>2XsNP) were 42% larger than in the control, whereas inhibition of sNPF by MJ94>sNPF-Ri and sNPFc00448 reduced cell size by 38% and 51% respectively. These differences in size correspond to changes in insulin signal transduction within the cells. Overexpression of sNPF (MJ94>sNPF and MJ94>2XsNPF) leads to phosphorylation and activation of Akt in the fat body, whereas the opposite effect was seen with neuronal inhibition of sNPF (MJ94>sNPF-Ri and sNPFc00448). Activated Akt represses the transcription factor dFOXO by phosphorylation and subsequent cytoplasmic localization. In wild-type flies, dFOXO localized equally in the cytoplasm and nucleus. As predicted, neuronal induction of sNFP (MJ94>2XsNPF) increased the cytoplasmic localization of dFOXO, whereas inhibition of sNPF (MJ94>sNPF-Ri and sNPFc00448) yielded fat body cells with dFOXO predominantly localized in the nucleus. Finally, dFOXO induces expression of the translational inhibitor d4E-BP, and, consistent with the current observations, expression of d4E-BP was elevated in animals where sNPF was inhibited (MJ94>sNPF-Ri and sNPFc00448) and reduced in animals where sNPF was overexpressed (MJ94>2XsNPF) (Lee, 2008).

Besides cell growth, Drosophila insulin-like peptides modulate aspects of metabolism and ageing. For instance, ablation of the IPCs reduces animal size, elevates the level of haemolymph carbohydrates.Therefore trehalose and glucose were assessed in sNPF mutant flies. As predicted, both carbohydrates were reduced upon sNPF overexpression, and both were elevated in sNPF hypomorphs. Also the lifespan of sNPF mutants was measured. As expected, inhibition of sNPF by MJ94>sNPF-Ri increased median lifespan by 16-21%, whereas sNPF overexpression (MJ94>2XsNPF) did not affect lifespan in flies (Lee, 2008).

Overall, the effects on Dilp1 and Dilp2 expression in IPCs regulated by sNPF are associated with cellular, carbohydrate and lifespan responses that are predicted to be caused by changes in the actual level of available insulin peptides. It is concluded that sNPF ultimately regulates insulin secretion from the IPC to affect target tissue insulin/dFOXO signalling and thus modulate growth, metabolism and lifespan (Lee, 2008).

Regulation of food consumption by neuropeptides is a critical step for interventions for managing obesity and metabolic syndromes. Mammalian NPY is known to positively regulate appetite and has thus been thought to promote weight gain primarily by affecting food intake. Thus study revealed a novel physiological role for NPY that is conserved by sNPF of Drosophila. These neuropeptides can affect growth, metabolism and lifespan by modulating ERK-regulated transcription of insulin-like peptides. In Drosophila, sNPFnergic and IPC neurons are adjacent in the brain. This study found, however, that pancreatic β-cells are also responsive to NPY, which is of hypothalamic origin. Although the hypothalamic neurosecretory cells and responding pancreatic endocrine cells are spatially distinct in mammals, recent developmental analysis suggests a parallel developmental pathway for hypothalamic neurosecretory cells and the IPCs of Drosophila, raising the possibility of a common molecular mechanism for β-cell formation. This would suggest that β-cells are not only evolutionarily tied to the hypothalamic neurosecretory cells but also that they retain their functional relationship to their hypothalamic origin by regulating insulin in response to the neuropeptide NPY (Lee, 2008).

RNAi screening for kinases and phosphatases identifies FoxO regulators

FoxO transcription factors are key regulators of growth, metabolism, life span, and stress resistance. FoxOs integrate signals from different pathways and guide the cellular response to varying energy and stress conditions. FoxOs are modulated by several signaling pathways, e.g., the insulin-TOR signaling pathway and the stress induced JNK signaling pathway. This study reports a genome wide RNAi screen of kinases and phosphatases aiming to find regulators of dFoxO activity in Drosophila S2 cells. By using a combination of transcriptional activity and localization assays several enzymes were identified that modulate dFoxO transcriptional activity, intracellular localization and/or protein stability. Importantly, several currently known dFoxO regulators were found in the screening, confirming the validity of the approach. In addition, several interesting new regulators were identified, including protein kinase C and glycogen synthase kinase 3beta, two proteins with important roles in insulin signaling. Furthermore, several mammalian orthologs of the proteins identified in Drosophila also regulate FOXO activity in mammalian cells. These results contribute to a comprehensive understanding of FoxO regulatory processes (Mattila, 2008).

By using a combination of transcriptional reporter and localization assays, twenty one dFoxO regulators were discovered. Some positive hits from the screen had an effect in dFoxO activity, localization, and protein stability, whereas other hits affected only transcriptional activity, suggesting that more mechanisms beyond subcellular localization and degradation are used by cells to regulate dFoxO activity. In addition to the 18 proteins that affected dFoxO transcriptional activity, the screening produced three more hits. Two of them seem to affect only dFoxO localization (dgkd and ptp69d), and one, neurospecific receptor kinase (nrk), affected exclusively dFoxO protein stability. It is possible that these proteins regulate dFoxO transcription on specific promoters in conjunction with other activators and that such factors are missing in Drosophila S2 cells. This would explain their lack of effect on the dInR promoter. Alternatively, they could affect dFoxO stability resulting in a net effect of dFoxO protein accumulation in the nucleus (Mattila, 2008).

Initially, the screening strategy was designed to identify both positive and negative regulators of dFoxO activity; however, no dFoxO repressors were found. Putative dFoxO repressors were present in the primary hit list of 31 targets, but those were later excluded in the secondary screen. This surprising observation suggests that the screen may be biased against dFoxO repressors. dFoxO is a well known inhibitor of protein biosynthesis in vivo, so under conditions of increased dFoxO activity, a reduction of general translation is expected that could affect GFP and luciferase translation too. Therefore, it is hypothesized that in the case of enhanced dFoxO activity it is possible that the concomitant inhibition of protein biosynthesis overruled a slight increase in reporter accumulation. This would explain the lack of dFoxO repressors among the targets of the screen. Moreover, the design of the screening based on S2 cells excludes the identification of regulatory mechanisms specific for other cell types, and instances where dFoxO is acting as a cofactor thereby regulating transcription indirectly (Mattila, 2008).

The results demonstrate that Drosophila PKC53E isoform is a dFoxO activator. Similar results were obtained in mammalian cells pointing out that the interaction is conserved. PKC isoforms have very important roles in insulin signaling, and each of the isoforms has been shown to be activated by insulin stimulation or conditions important for effective insulin stimulation. Importantly, PKC isoforms can both activate or inhibit insulin signaling: Atypical PKC isoforms are required for insulin-stimulated glucose transport in muscle and adipocytes. In contrast, certain conventional and novel PKC isoforms are known to antagonize insulin signaling in vertebrates. This interaction is implicated in the pathogenesis of free fatty acid mediated insulin resistance. Drosophila possesses six PKC isoforms whose role in this context has not yet been addressed. PKC53E homolog is closest to human conventional PKCα. Interestingly, it has been shown that PKCα inhibits insulin signaling through binding and phosphorylation of IRS1. Thus, PKCα would serve as a constitutively active inhibitory regulator of the insulin cascade through its association with IRS1. On stimulation with insulin, PKCα would dissociate from IRS1, thus releasing this protein from its down-regulated state. This would open the 'gate' for transmission of the insulin signal. It has been found that dFoxO/FOXO1 increases insulin sensitivity by up-regulating insulin receptor transcription. The observation that Drosophila PKCα activates dFoxO adds an additional twist in the complex regulatory network that dFoxO has on insulin signaling. Interestingly, in the experimental system used in this study AKT dependent dFoxO bandshift and AKT Ser-505 phosphorylation was not affected by PKC53E, indicating that PKC53E regulation of dFoxO is independent of AKT signaling (Mattila, 2008).

Another well known enzyme implicated in the control of metabolism identified as a regulator of dFoxO transcriptional activity is the Drosophila ortholog of Glycogen synthase kinase 3β (GSK-3β, Shaggy). GSK-3β is a regulator of glucose metabolism through the phosphorylation and inhibition of glycogen synthase, the rate limiting enzyme of glycogen deposition. GSK-3β is inhibited by AKT, so it was not surprising to see that GSK-3β activates dFoxO. GSK-3β protein level and activity is elevated in type II diabetic skeletal muscle cells reflecting the impairment of whole body glucose uptake characteristic to this disease. In addition, selective inhibition of GSK-3β by lithium chloride represses the expression of g6pase and pepck in rat hepatoma cells, both known targets of FoxO. Taken together, these observations suggest that some of the metabolic effects of GSK-3β are achieved by directly modulating dFoxO activity (Mattila, 2008).

An interesting dFoxO regulator is Polo-like kinase. Polo-like kinases (Plks) are known regulators of cell cycle progression. In addition, Plks have a role in the protection against cellular stress through the transcription factor HSF1. Recently it was proposed that an intricate tradeoff between lifespan and cancer results from opposing effects of enzymes regulating FoxO and p53 activity. Plks are known to inhibit p53 transcriptional activity, so the results raise the possibility that Plks mediate the common but opposing regulators of p53 and FoxO. Interestingly, FoxOs are necessary in the completion of the cell cycle, which is partly mediated by cell cycle dependent activation of Plk expression by FOXO3a. The results show that Drosophila dFoxO is regulated by Polo, suggesting the existence of a positive feedback mechanism that has a role in achieving periodic M-phase gene expression and proper cell cycle exit (Mattila, 2008).

dFoxO localization was affected by eight modulators; however, band shifts demonstrated that none of these proteins phosphorylated dFoxO in the three conserved Ser/Thr amino acids known to regulate nuclear/cytoplasmic status through AKT. This observation raises the possibility that some of the newly identified dFoxO regulators could affect dFoxO nuclear/cytoplasmic localization by phosphorylating dFoxO in additional residues that do not alter its electrophoretic mobility, or that dFoxO regulation by these proteins is indirect. Further studies will be needed to clarify this point (Mattila, 2008).

In summary, this study has identified 21 dFoxO modulators. The results underscore the complexity underlying dFoxO regulation and establish dFoxO as a transcription factor controlled exquisitely by an intricate network of kinases and phosphatases achieving a perfect balance of activity. This balance ensures the correct execution of key cellular processes in metabolism, response to stress, and life span (Mattila, 2008).

Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies

Sestrins are conserved proteins that accumulate in cells exposed to stress, potentiate adenosine monophosphate-activated protein kinase (AMPK), and inhibit activation of Target of rapamycin (TOR). The abundance of Drosophila sestrin (dSesn) is increased upon chronic TOR activation through accumulation of reactive oxygen species that cause activation of c-Jun amino-terminal kinase and transcription factor Forkhead box O (FoxO). Loss of dSesn resulted in age-associated pathologies including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration, and cardiac malfunction, which were prevented by pharmacological activation of AMPK (see AMP-activated protein kinase) or inhibition of TOR. Hence, dSesn appears to be a negative feedback regulator of TOR that integrates metabolic and stress inputs and prevents pathologies caused by chronic TOR activation that may result from diminished autophagic clearance of damaged mitochondria, protein aggregates, or lipids (Lee, 2010).

Target of rapamycin (TOR) is a key protein kinase that regulates cell growth and metabolism to maintain cellular and organismal homeostasis. Insulin and insulin-like growth factors are major TOR activators that operate through phosphoinositide 3-kinase (PI3K) and the protein kinase AKT. Conversely, adenosine monophosphate-activated protein kinase (AMPK), which is activated upon energy depletion, caloric restriction (CR), or genotoxic damage, is a stress-responsive inhibitor of TOR activation. TOR stimulates cell growth and anabolism by increasing protein and lipid synthesis through p70 S6 kinase (S6K), eukaryotic translation initiation factor 4E-binding protein (4E-BP), and sterol response element binding protein (SREBP) and by decreasing autophagic catabolism through phosphorylation-mediated inhibition of ATG1 protein kinase. Persistent TOR activation is associated with diverse pathologies such as cancer, diminished cardiac performance, and obesity-associated metabolic diseases. Conversely, inhibition of TOR prolongs life span and increases quality of life by reducing the incidence of age-related pathologies. The antiaging effects of CR could be due to inhibition of TOR (Lee, 2010 and references therein).

Sestrins (Sesns) are highly conserved proteins that accumulate in cells exposed to stress, lack obvious domain signatures, and have poorly defined physiological functions. Mammals express three Sesns, whereas Drosophila melanogaster and Caenorhabditis elegans have single orthologs. In vitro, Sesns exhibit oxidoreductase activity and may function as antioxidants. Independently of their redox activity, Sesns lead to AMPK-dependent inhibition of TOR signaling and link genotoxic stress to TOR regulation (Badanov, 2008). However, Sesns are also widely expressed in the absence of exogenous stress, and in Drosophila, expression of Drosophila sestrin (dSesn) is increased upon maturation and aging. Given the redundancy between mammalian Sesns, the importance of Sesns as regulators of TOR function was tested in Drosophila. Both gain- and loss-of-function dSesn mutants were created. Analysis of these mutants revealed that dSesn is an important negative feedback regulator of TOR whose loss results in various TOR-dependent, age-related pathologies (Lee, 2010).

Persistent TOR activation in wing discs by a constitutively active form of the insulin receptor (InRCA) resulted in prominent dSesn protein accumulation, which is not seen in a dSesn-null larvae. InRCA also induced accumulation of dSesn RNA, indicating that dSesn accumulation is due to increased transcription or mRNA stabilization. Since dSesn accumulation was restricted to cells in which TOR was activated, the response is likely to be cell autonomous. dSesn was also induced when TOR was chronically activated by overexpression of the small guanine triphosphatase Rheb, clonal loss of phosphatase and tensin homolog (PTEN), or tuberous sclerosis complex 1 (TSC1). Dominant-negative PI3K (PI3KDN) or TOR (TORDN) inhibited dSesn accumulation caused by overexpression of InRCA, but inactive ribosomal S6 protein kinase (S6K, S6KDN) and hyperactive 4E-BP (4E-BPCA), two downstream TOR effectors, did not. Furthermore, dorsal-specific expression of activated S6KCA or loss of 4E-BP activity failed to induce dSesn expression, indicating that TOR regulates expression of dSesn through different effector(s) (Lee, 2010).

In mammals, transcription of Sesn genes is increased in cells exposed to oxidative stress, and reactive oxygen species (ROS) accumulation, detected by oxidation of dihydroethidium (DHE), was observed in the same region of the imaginal discs in which InRCA or Rheb were expressed. InRCA-induced accumulation of ROS was blocked by coexpression of either PI3KDN or TORDN, but not S6KDN or 4E-BPCA, revealing TOR’s role in ROS accumulation. Wing-disc clones in which TOR was activated by loss of TSC1 also exhibited ROS accumulation, confirming that TOR-dependent ROS accumulation is cell-autonomous. Expression of the ROS scavengers catalase or peroxiredoxin inhibited InRCA-induced accumulation of dSesn. Feeding animals with vitamin E, an antioxidant, also prevented dSesn induction caused by TSC1 loss (Lee, 2010).

Forkhead box O (FoxO) and p53 are ROS-activated transcription factors that control mammalian Sesn genes. The dSesn locus contains eight perfect FoxO-response elements, a frequency 25 times higher than that expected on the basis of random distribution. Overexpressed FoxO or p53 could both increase expression of the dSesn gene. However, InRCA caused accumulation of dSesn in a p53-null background, but not in a FoxO-null background, indicating that TOR-activated FoxO is likely to be the regulator of dSesn gene transcription. Accumulation of dSesn in response to Rheb overexpression was also FoxO-dependent (Lee, 2010).

In dorsal wing disc cells, where ROS accumulated in response to InRCA, c-Jun N-terminal kinase (JNK), a protein kinase that phosphorylates FoxO, was also activated. JNK activation was diminished in cells overexpressing catalase, suggesting that it depends on TOR-induced accumulation of ROS. Mitogen-activated protein kinase kinase 7-mediated activation of JNK also resulted in accumulation of dSesn, as did overexpression of mammalian STE20-like kinase 1 (MST1), another protein kinase that phosphorylates FoxO. However, only JNKDN (but not Mst1DN) inhibited InRCA-mediated accumulation of dSesn. Collectively, these data suggest that dSesn transcription is increased upon chronic TOR activation through ROS-dependent activation of JNK and FoxO (Lee, 2010).

To determine effects of dSesn on cell growth, a major function of TOR, dSesn was overexpressed in dorsal wings. This resulted in a dose-dependent phenotype in which the wing bends upward, indicating suppressed dorsal tissue growth. A dSesnC86S variant, in which the cysteine required for oxidoreductase activity was mutated (C86S, Cys86->Ser86), still conferred this phenotype when expressed in amounts similar to those of wild-type dSesn (dSesnWT). Cell number and size were measured in a dorsal wing region defined by the L3, L4, C1, and C2 veins. Although the size of this area was significantly reduced by dSesn expression, the cell number remained unchanged, showing that decreased cell size can account for dSesn suppression of tissue growth. Overexpression of dSesn also reduced cell size in larval wing discs and adult eyes. Thus, dSesn inhibits cell growth without affecting cell proliferation and does so independently of its redox activity (Lee, 2010).

When dSesn was expressed with InRCA or Rheb, it suppressed the hyperplastic phenotypes caused by these TOR activators. Both eye and individual ommatidia sizes were significantly reduced. dSesn also inhibited InRCA- or Rheb-induced phosphorylation of TOR targets S6K and 4E-BP. In mammalian cells, dSesn enhanced AMPK-induced phosphorylation of TSC2 and inhibited S6K activity through TSC2, just as mSesn2 does (Budanov, 2008). In Drosophila wings, dSesn-induced growth suppression was attenuated by reduced gene dosage of TSC1, TSC2, or AMPK, although reduced dosage of these genes alone did not affect normal growth. Expression of mSesn1/2 in flies also reduced normal and InRCA-induced hyperplastic growth (Lee, 2010).

Expression of InR, constitutively active PI3K (PI3KCA), AKT, or S6KCA in dorsal cells of the wing caused an overgrowth phenotype in which the wing bends downward. dSesn expression reversed this effect of overexpressed InR, PI3KCA, and AKT, but not that of S6KCA, suggesting that dSesn inhibits TOR downstream of AKT. Conversely, dorsal wing-specific expression of PTEN and InRDN, PI3KDN, or S6KDN caused wings to bend upward, and this effect was enhanced by dSesn (Lee, 2010).

Although dSesn-null flies did not exhibit developmental abnormalities, the growth-promoting effect of overexpressed InR or AKT was enhanced in dSesn-null background, suggesting that endogenous dSesn restricts TOR activation and its growth-promoting effect. Loss of dSesn, however, did not enhance S6K-stimulated cell growth or decrease growth suppression by overexpressed InRDN or S6KDN. These findings indicate that Sesn is an evolutionarily conserved inhibitor of TOR signaling that acts via the AMPK-TSC2 axis (Lee, 2010).

Fat bodies from dSesn-null third-instar larvae contained more lipids than did those of WT animals. dSesn-null adults also contained more triglycerides, which were decreased after ectopic expression of dSesnWT or dSesnCS. Thus, the TOR-inhibitory function of dSesn, rather than its antioxidant activity, appears to affect metabolic control. Congruently, dSesn-null fat bodies showed decreased AMPK and increased TOR activities. Pharmacological manipulation strengthened this conclusion; feeding dSesn-null mutants with AMPK-activators such as 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) or metformin, or the TOR-inhibitor rapamycin reduced triglyceride accumulation (Lee, 2010).

Expression of the gene-encoding transcription factor dSREBP and its targets, which encode fatty acyl coenzyme A (CoA) synthetase, fatty acid synthase, acetyl CoA carboxylase, and acetyl CoA synthetase, was significantly increased (20 to 70%) in dSesn-null mutants. However, the peroxisome proliferator-activated receptor γ coactivator 1 (dPGC-1) gene and some lipolytic genes showed decreased expression. This is consistent with reports that dSREBP and dPGC-1 (spargel; CG9809)are inversely regulated by TOR and AMPK to properly control lipid metabolism (Lee, 2010).

Age-related decline in heart performance is another phenotype associated with TOR hyperactivity in insects and mammals. In WT flies, the heart beats in a highly regular manner, but in dSesn-null mutants, heart function was compromised, as manifested by arrhythmia and decreased heart rate. Slowing of heart rate reflected expansion of the diastolic period, as observed in aged or TOR-activated flies. These defects were largely prevented by feeding flies AICAR or rapamycin, indicating that they are caused by low activity of AMPK or high TOR activity. Vitamin E feeding or catalase expression suppressed the arrhythmia caused by loss of dSesn, but not the decrease in heart rate, suggesting that TOR-induced oxidative stress contributes to the arrhythmic phenotype. Analysis of F-actin revealed structural disorganization of myofibrils in dSesn-null hearts, suggesting that cardiac muscle degeneration may cause some of the functional defects in dSesn-null hearts. Reflecting this structural abnormality, dSesn-null hearts were dilated during both the diastolic and systolic phases, and this was prevented by AICAR or rapamycin (Lee, 2010).

Heart-specific depletion of dSesn caused cardiac malfunction similar to that seen in dSesn-null mutants. Heart-specific depletion of AMPK also caused cardiac malfunction, but this was not alleviated by AICAR administration, supporting the notion that dSesn maintains normal heart physiology through AMPK activation (Lee, 2010).

dSesn mRNA and protein are abundant in the adult thorax, which is mostly composed of Mesoderm. mSesn1 is also highly expressed in skeletal muscle (Velasco-Miguel, 1999). Therefore, whether dSesn has a role in maintaining muscle homeostasis was tested. 20-day-old dSesn-null flies showed degeneration of thoracic muscles with loss of sarcomeric structure, including discontinued Z discs, disappearance of M bands, scrambled actomyosin arrays, and diffused sarcomere boundaries. Such defects are only partially observed in very old WT flies (~90 days) and were not found in young (5-day-old) dSesn-null muscles. Thus, the dSesn-null skeletal muscle appears to undergo accelerated age-related degeneration (Lee, 2010).

Despite its normal appearance, muscle from 5-day-old dSesn-null flies exhibited mitochondrial abnormalities, including a rounded shape, occasional enlargement, and disorganization of cristae, which were also observed in 20-day-old mutants. Mitochondrial dysfunction can result in excessive generation of ROS leading to other abnormalities. dSesn-null muscles exhibited increased accumulation of ROS, revealed by more intense DHE fluorescence and reduced cis-aconitase activity, which was associated with muscle cell death. Furthermore, the muscle defects were prevented by vitamin E feeding, underscoring the role of ROS in muscle degeneration (Lee, 2010).

Expression of exogenous dSesnCS, devoid of redox activity, prevented muscle degeneration, suggesting again that regulation of AMPK-TOR by dSesn, rather than intrinsic redox activity, is of importance. Feeding animals with AMPK activators prevented muscle degeneration in dSesn-null mutants, and depletion of AMPK in skeletal muscles caused severe degeneration of mitochondrial and sarcomeric structures. Treatment of animals with rapamycin also prevented muscle degeneration in dSesn-null flies. Thus, dSesn-dependent control of AMPK-TOR signaling is essential for prevention of mitochondrial dysfunction and maintenance of muscle homeostasis during aging (Lee, 2010).

It was noticed that dSesn-null muscles accumulated polyubiquitin aggregates, which are hallmarks of defective autophagy. To test whether decreased autophagy brought about by excessive and prolonged TOR activity might cause muscle degeneration, expression was silenced of ATG1, an essential component of the autophagic machinery, which is inhibited by TOR. This caused a decline in cardiac performance, as well as degeneration and mitochondrial abnormalities in skeletal muscle. These results suggest that TOR up-regulation caused by dSesn loss inhibits autophagy needed to eliminate ROS-producing dysfunctional mitochondria, which may contribute to muscle degeneration. Consistent with this view, ATG1 silencing resulted in ROS accumulation in wing discs (Lee, 2010).

These results identify Sesn as a negative feedback regulator of TOR function. In mammalian cells, increased expression of mSesns in response to genotoxic stress leads to inhibition of TOR activity through activation of AMPK (Budanov, 2008). This study now shows that transcription of the dSesn gene is increased upon chronic TOR activation through JNK and FoxO in a manner dependent on ROS accumulation. Although transient InR activation inhibits FoxO through its phosphorylation by AKT, this study finds that chronic TOR activation overcomes this inhibition and results in nuclear translocation of FoxO, which increases dSesn transcription. In turn, dSesn suppresses metabolic dysfunction and age-related tissue degeneration brought about by hyperactivated TOR. Although dSesn can inhibit TOR-stimulated cell growth, this analysis points to its most important function being the maintenance of metabolic homeostasis and prevention of TOR-induced tissue degeneration. The three major functions of dSesn revealed by this study -- suppression of lipid accumulation, prevention of cardiac malfunction, and protection of muscle from age-related degeneration -- are adversely affected by obesity, lack of exercise, and aging, which make a disproportional contribution to health problems in developed and rapidly developing societies (Lee, 2010).

Whereas TOR controls cell growth mostly through inhibition of 4E-BP and activation of S6K kinase, its ability to induce dSesn expression depends on ROS accumulation, which the results suggest is a pathophysiological aberration caused by TOR hyperactivation that is normally antagonized by dSesn. However, the previously described redox function of Sesn is not required for its protective role. TOR-induced accumulation of ROS has been observed in yeast and hematopoietic cells, but the molecular mechanism underlying this phenomenon and its physiological and pathophysiological importance were unknown. The current results suggest that TOR-stimulated production of ROS, which is needed for accumulation of dSesn, is independent of two of the major TOR targets (4E-BP and S6K) and instead may result from TOR-mediated inhibition of physiological autophagy, a process that eliminates ROS-producing dysfunctional mitochondria. Nonetheless, inhibition of 4E-BP also contributes to the pro-aging effects of TOR by suppressing translation of several mitochondrial proteins and by accelerating age-related cardiac malfunction at young ages, which is reminiscent of the observed cardiac defects seen in dSesn-null flies. Although TOR activates SREBP, which may contribute to lipid accumulation in dSesn-null flies, autophagy promotes lipid elimination. Thus, decreased autophagy may also contribute to triglyceride accumulation. Hence, the different degenerative phenotypes exhibited by dSesn-null flies are due to the cumulative effects of several biochemical and cell biological defects caused by hyperactive TOR, including reduced autophagy and reduced function of 4E-BP. Both basal physiologic autophagy and 4E-BP function are enhanced by calorie restriction, which prevents aging-related pathologies. In the future, it will be of interest to determine the contribution of Sesn to these antiaging effects (Lee, 2010).

Liver Med23 ablation improves glucose and lipid metabolism through modulating FOXO1 activity

Mediator complex is a molecular hub integrating signaling, transcription factors, and RNA polymerase II (RNAPII) machinery. Mediator MED23 is involved in adipogenesis and smooth muscle cell differentiation, suggesting its role in energy homeostasis. Through the generation and analysis of a liver-specific Med23-knockout mouse, this study found that liver Med23 deletion improved glucose and lipid metabolism, as well as insulin responsiveness, and prevented diet-induced obesity. Remarkably, acute hepatic Med23 knockdown in db/db mice significantly improved the lipid profile and glucose tolerance. Mechanistically, MED23 participates in gluconeogenesis and cholesterol synthesis through modulating the transcriptional activity of FOXO1, a key metabolic transcription factor. Indeed, hepatic Med23 deletion impaired the Mediator and RNAPII recruitment and attenuated the expression of FOXO1 target genes. Moreover, this functional interaction between FOXO1 and MED23 is evolutionarily conserved, as the in vivo activities of dFOXO in larval fat body and in adult wing can be partially blocked by Med23 knockdown in Drosophila. Collectively, these data revealed Mediator MED23 as a novel regulator for energy homeostasis, suggesting potential therapeutic strategies against metabolic diseases (Chu, 2014).

Pelle modulates dFoxO-mediated cell death in Drosophila

Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole Drosophila IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. This study reports a novel function of pll in modulating apoptotic cell death, which is independent of the Toll pathway. It was found that loss of pll results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of pll up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-pll induced cell death. Furthermore, loss of pll activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene Thor/4E-BP. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of pll in cell death, but also sheds light on the mechanism of IRAKs in cell survival/death during tumorigenesis (Wu, 2015).

Drosophila melanogaster has emerged as an excellent model organism to study apoptotic cell death and has made significant contribution to understand cell death regulation and its role in development. While mammalian IRAKs function as the mediator of IL-1Rs/TLRs signal transduction in the immune and inflammatory responses, Pll, the sole Drosophila orthologue of IRAKs, has been implicated as a central regulator of Toll pathway involved in embryonic dorsal/ventral patterning, innate immune response, muscle development and axon guidance. This work identified a Toll pathway independent function of Pll in modulating caspase-mediated cell death in animal development (Wu, 2015).

Previous studies have suggested that Drosophila wing vein formation is a result of cell fate specification regulated by multiple signaling pathways including Notch, Hedgehog, EGF (epidermal growth factor) and BMP (bone morphogenetic proteins) pathways. The present study found that knock-down pll along the A/P compartment boundary of the developing wing (ptc>pll-IR) resulted in extensive cell death in the wing disc and a loss-of-ACV phenotype in the adult wing, implying a potential role of cell death in vein patterning. Consistent with this notion, the loss-of-ACV phenotype is rescued by blocking apoptotic cell death, suggesting cell death is responsible for the loss of ACV. To investigate whether cell death is able to impede vein patterning, apoptosis was initiated by expressing the pro-apoptotic protein Grim under the control of ptc-Gal4. ptc>Grim caused extensive cell death in tissue ablation between L3 and L4 in the adult wing. In most cases, L3 and L4 were fused in the proximal area where ACV is located. To adjust Grim expression and cell death, Tub-Gal80ts that represses Gal4 activity was added in a temperature sensitive manner. At 25°C, Tub-Gal80ts partially blocks ptc-Gal4 activity and allows limited Grim expression and therefore, cell death, between L3 and L4. Intriguingly, under this condition, the loss-of-ACV phenotype was observed to be accompanied by a slight reduction of area between L3 and L4, suggesting that both reduced area and loss-of-ACV phenotypes are consequences of cell death. The loss-of-ACV phenotype is more sensitive to cell death, since weak cell death is sufficient to generate the phenotype, whereas stronger cell death is required to delete tissue between L3 and L4. Consistent with this notion, while ptc>pll-IR flies reared at 25°C only displayed the loss-of-ACV phenotype , those raised at 29°C also showed reduced area between L3 and L4, which was caused by a reduction in cell number, but not cell size (Wu, 2015).

Pll regulates caspase activation and cell death through dFoxO. Mechanistically, loss of pll promotes the nuclear translocation of dFoxO, which otherwise is retained in the cytoplasm by phosphorylation. A number of kinases, including AKT, IκK and JNK, have been reported to phosphorylate FoxO and regulate its nuclear-cytoplasmic trafficking. This study provides evidence that Pll is another dFoxO kinase that phosphorylates dFoxO and inhibits its nuclear localization. Thus, it would be very interesting to check whether a similar interaction is conserved between IRAKs and FoxOs in mammal (Wu, 2015).

The FoxO family proteins have been implicated in multiple important biological processes, including cell death and tumor suppression. It has been reported that conditional deletion of FoxO1, FoxO3 and FoxO4 simultaneously results in the development of hemangiomas and thymic lymphomas, and IκB kinase represses FoxO3a activity to promote human breast tumorigenesis and acute myeloid leukemia (AML). IRAKs also show altered expression level in tumors and surrounding stroma, and participate in tumor initiation and progression, yet the underlying mechanisms remain poorly understood. Thus, the inhibitory effect of Pll on dFoxO activity in Drosophila provides a beneficial framework for a better understanding of mammalian IRAKs’ crucial roles in tumor development (Wu, 2015).

As the Toll/NF-κB pathway is not implicated in loss-of-pll triggered dFoxO-dependent cell death, it was of interest to learn about what are the pathways or factors act upstream of Pll to regulate its role in cell death. Since dFoxO has been reported as a downstream transcription factor in the JNK and Insulin pathways in Drosophila, it was asked whether Pll is also involved in these pathways. Activation of JNK signaling between L3 and L4 by expressing Egr (Drosophila TNF) or Hep (Drosophila JNK Kinase), or depleting puc (encoding a JNK inhibitor), produced similar loss-of-ACV and reduced area phenotypes as that of pll depletion, yet the phenotypes were not affected by gain or loss of pll. Inactivation of the Insulin pathway by expressing a dominant negative form of PI3K, or knocking-down PI3K or Akt, resulted in diminished area between L3 and L4, which remained unaffected by gain or loss of pll. The Hippo pathway, known to play a crucial role in regulating cell death and organ size, was also examined. Up-regulation of Hippo pathway in distinct wing areas by different Gal4 drivers led to various small wing or wing tissue ablation phenotypes, which were not altered by changing Pll level. Finally, dMyc, the fly homolog of c-Myc that regulates cell growth and cell death in Drosophila, and the cell polarity gene scribble (scrib), whose depletion promotes cell death were also examined. Depletion of dMyc triggered wing phenotype and loss of scrib induced cell death are both independent of Pll. Thus, while Pll directly regulates dFoxO-mediated caspase-dependent cell death in development, the upstream factors modulating Pll activity remain unknown, which deserve further investigation (Wu, 2015)

Targets of Activity

Foxo regulates cell cycle arrest possibly by transcriptionally activating genes implicated in cell division or in cell growth. As an initial attempt to identify target genes of Foxo, DNA microarrays were used to assess gene expression profiles in S2 cells stably transfected with mutant Foxo and grown in the presence of insulin. Cells expressing wild-type Foxo or untransfected S2 cells subjected to the same treatment were assayed as controls (Puig, 2003).

Two-hundered and seventy-seven genes were found to be up-regulated in Foxoa3-expressing cells when compared with Foxo-expressing cells or untransfected S2 cells. Interestingly, two genes that were consistently and specifically up-regulated in these conditions were the Drosophlia InR gene (13.5-fold) and the Drosophila 4EBP/Thor gene (25-fold). Both genes have been implicated in the regulation of cell growth by insulin. To confirm that InR and 4EBP are bona fide transcriptional targets of Foxo, the same experiment described above was performed but in the presence of cycloheximide to inhibit translation. As expected, both InR and 4EBP continue to be transcriptionally activated (2.5- and 3.1-fold, respectively) by FOXOA3 but not Foxo in the insulin-repressed state. This result suggests that Foxo, when released from control by the insulin/dAkt cascade, is involved in transcription from the InR and 4EBP promoters (Puig, 2003).

To confirm these microarray results and to independently quantitate the increase in mRNA transcription, RNase protection assays were performed with mRNAs extracted from cells stably transfected with either Foxo or FoxoA3. Indeed, FoxoA3 stimulates transcription of Drosophila 4EBP and InR by 16.3- and 11-fold, respectively. A time-course experiment confirmed that Drosophila InR mRNA increases rapidly upon FoxoA3 expression: 3 h after CuSO4 addition, there is already an 8-fold increase, reaching 20-fold after 9 h of CuSO4 induction. Similar results were obtained for Drosophila 4EBP. These experiments suggest that Foxo expression specifically activates both Drosophila InR and 4EBP transcription, thus unmasking an important feedback control mechanism in this pathway involving Foxo and InR (Puig, 2003).

Having obtained evidence that exogenously transfected Foxo responds to insulin and regulates both the downstream target gene 4EBP and the feedback control target InR, it was of interest to know if endogenous Foxo would also activate transcription of these genes. The PI3K inhibitor LY294002 was used to activate endogenous Foxo or insulin to deactivate it. S2 cells grown in the absence of serum for 48 h were treated either with LY294002 or insulin. Total RNA was extracted and RNase protection was performed to detect Drosophila InR and 4EBP mRNAs. Both mRNA levels are significantly increased after LY294002 treatment (5.3-fold for dInR and 4-fold for d4EBP) when compared with insulin treatment. This result provides further evidence indicating that the PI3K–Akt pathway regulates InR and 4EBP transcription via Foxo (Puig, 2003).

It was of interest to determine whether Foxo directly binds to the promoters of Drosophila 4EBP and InR. To identify the DNA region recognized by Foxo in these two promoters, a 1708-bp fragment of the 4EBP promoter and a 1562-bp fragment of the InR promoter were inserted into a luciferase reporter vector. When transfected into S2 cells, these fragments responded to Foxo activation (3-fold for 4EBP, >200-fold for InR. A series of deletions lacking upstream sequences still responded to Foxo activation, albeit more weakly, suggesting that Foxo can bind the DNA in a region close to the start of transcription (485 bp for the d4EBP promoter and 194 bp for the dInR promoter). In contrast, Foxo completely fails to activate a reporter construct in which upstream activating sequences (UAS) for the transcription factor GAL4 are fused to the luciferase gene, confirming that transcription activation is specific for both 4EBP and InR promoters (Puig, 2003).

Interestingly, 125 bp upstream of the transcription start site of the d4EBP promoter there are three tandem copies of a putative FOXO4 recognition element (FRE). These elements are reminiscent of the ones present in the human glucose-6-phosphatase promoter, previously shown to bind FOXO4 (Yang, 2002). This was reassuring because Foxo and FOXO4 share 85% identity in the core of the forkhead DNA-binding domain. Similarly, several putative FRE sequences appear in the InR promoter in the region comprising nucleotides -1434 to -70 (Puig, 2003).

To determine whether Foxo binds these putative FREs, band shift experiments were performed with a 113-bp DNA probe encompassing the 4EBP FRE motifs and with 12 separate DNA probes (ranging from 100 to 150 bp) spanning a region of 1.4 kb from the InR promoter. Purified recombinant Foxo expressed in Escherichia coli efficiently binds the 113-bp FRE-containing fragment from the 4EBP promoter compared with control DNA fragments. Furthermore, Foxo binding to the 4EBP promoter fragment can be efficiently competed with an unlabeled 113-bp 4EBP promoter fragment but not with nonspecific DNA. Similarly, purified recombinant Foxo binds efficiently to 5 out of 12 of the DNA fragments located within the InR promoter. As expected, each of the five DNA fragments bound by Foxo contains putative FREs. Thus, Foxo can specifically bind to both promoters in vitro. To determine whether Foxo also binds these same DNA regions in vivo, chromatin immunoprecipitation (ChIP) experiments were performed with S2 cells expressing either Foxo or dFoxoA3. Cells were incubated with serum, and Foxo expression was induced with the addition of CuSO4. After 6 h, cells were cross-linked with formaldehyde, and extracts were prepared and immunoprecipitated. After reversal of cross-links, DNA was recovered, and PCR was performed with primers encompassing regions containing putative FREs in both promoters. The results indicate that Foxo can directly bind to both the 4EBP and InR promoters in vivo. These results establish that Foxo can specifically bind the 4EBP and InR promoters both in vitro and in vivo (Puig, 2003).

To demonstrate that Foxo can directly activate transcription of these promoters in vitro, the constructs were used that contain 485 bp of the 4EBP promoter region and 514 bp of the InR promoter region, respectively. Addition of purified recombinant Foxo to in vitro reactions activates transcription of these promoters by at least 3-fold (4EBP) and 5.5-fold (InR), which is comparable to the activation observed in vivo. Under in vitro transcription conditions, activation of the 4EBP promoter by Foxo becomes rapidly saturated with increasing amounts of Foxo. As expected, Foxo also activates (up to sixfold) a synthetic promoter bearing four FOXO4-binding sites placed upstream of the alcohol dehydrogenase distal promoter. Together these results show that transcription of 4EBP and InR can be directly activated by Foxo in vitro (Puig, 2003).

Drosophila embryonic Kc167 cells respond to insulin stimulation with upregulated activities of PKB and S6K. mRNA profiling experiments were performed using the Affymetrix GeneChip system to measure on a genome-wide scale the transcriptional changes induced by insulin in these cells. On the basis of the currently held model that FOXO transcription factors are transcriptional activators that are negatively regulated by insulin, potential Foxo target genes were expected to be repressed in Kc167 cells upon insulin stimulation. Foxo target gene candidates were selected that are transcriptionally downregulated by a factor of two or more upon insulin stimulation and whose promoter regions contain one or more conserved forkhead-response elements (FHREs) with the consensus sequence (G/A)TAAACAA. Three of these candidate gene products are each involved in one of two biological processes known to be negatively regulated by insulin, namely gluconeogenesis (PEPCK) and lipid catabolism (CPTI and long-chain-fatty-acid-CoA-ligase). The remaining candidates are involved in stress responses (cytochrome P450 enzymes), DNA repair (DNA polymerase iota), transcription and translation control (4E-BP and CDK8), and cell-cycle control (centaurin gamma and CG3799). Several of the insulin-repressed genes have been reported to be transcriptionally induced in Drosophila larvae under conditions of complete starvation (4E-BP and PEPCK) or sugar-only diet (CPTI and long-chain-fatty-acid-CoA-ligase) (Jünger, 2003).

4E-BP was chosen for further investigation, because it has previously been reported to be insulin-regulated at the level of protein phosphorylation, but not at the level of gene expression. The 4E-BP gene encodes a translational repressor and was initially identified as the immune-compromised Thor mutant in a genetic screen for genes involved in the innate immune response to bacterial infection. There are several FHREs in the genomic region around the 4E-BP locus. The 4E-BP protein is negatively regulated by insulin through LY294002- and rapamycin-sensitive phosphorylation, suggesting involvement of the Dp110 and TOR signaling pathways. Phosphorylation of 4E-BP leads to the dissociation of 4E-BP from its binding partner, the translation initiation factor eIF4E, which then participates in the formation of a functional initiation complex. Positive transcriptional regulation of 4E-BP by Foxo, which corresponds to negative transcriptional regulation by insulin, would be a complementary mechanism of regulation (Jünger, 2003).

Whether overexpression of endogenous foxo can induce transcriptional upregulation of the 4E-BP gene was investigated. On the basis of overexpression results, the Dp110DN-Foxo coexpression was used to efficiently activate Foxo. Eye imaginal discs from Dp110DN-expressing third instar larvae display a low level of basal 4E-BP transcription throughout the disc, which is not induced by the driver construct alone. Coexpression of foxo elicits a dramatic upregulation of 4E-BP transcription posterior to the morphogenetic furrow. Consistent with this observation, it was possible to induce expression of the 4E-BP enhancer trap line Thor1 with human FOXO3a-TM . It remains unclear, however, whether regulation of d4E-BP expression by Foxo is of physiological relevance (Jünger, 2003).

Overexpression of 4E-BP partially suppresses the PKB overexpression phenotype, but since ectopic expression experiments have to be interpreted with some caution, whether loss of 4E-BP function suppresses the cell-number reduction in insulin-signaling mutants as does loss of Foxo function was investigated. Double-mutant flies were generated for PKB and 4E-BP and it was observed that the Thor1 mutation slightly but significantly suppressed the reduced cell-number phenotype in a dose-dependent manner. The Thor1 mutation itself had no effect on ommatidial number compared to wild-type flies, so additive effects of d4E-BP and dPKB can be ruled out. These observations strongly argue that under conditions of reduced insulin-signaling activity, the Foxo-dependent reduction in cell number is in part mediated by the transcriptional upregulation of its target 4E-BP. Microarray studies in both mammalian and Drosophila cells imply that FOXO transcription factors exert their physiological functions by modulating expression of large sets of target genes (Jünger, 2003).

The messenger RNA 5' cap-binding protein eIF4E is regulated by its binding protein (4E-BP), a downstream target of phosphatidylinositol-3-OH kinase [PI(3)K] signaling. Drosophila 4E-BP (d4E-BP) activity becomes critical for survival under dietary restriction and oxidative stress, and is linked to life span. The Drosophila forkhead transcription factor (dFOXO) activates d4E-BP transcription. Ectopic expression of d4E-BP in dFOXO-null flies restores oxidative stress resistance to control levels. Thus, d4E-BP is an important downstream effector of a dFOXO phenotype, and regulation of translation by eIF4E is vital during environmental stress (Tettweiler, 2005).

A rapid response is a crucial early line of defense in preventing cellular death in situations of stress. Translational regulation allows an organism to generate quick responses to environmental cues by controlling the expression of protein from existing cellular mRNAs. Translation initiation of most eukaryotic mRNAs requires binding of eIF4F, a protein complex made up of eIF4A, eIF4G, and eIF4E, to the 5' cap structure. eIF4E activity is highly regulated both by Mnk1/Mnk2-dependent phosphorylation and by repressor proteins termed eIF4E-binding proteins (4E-BPs), which compete with eIF4G for the same binding site on eIF4E. 4E-BPs themselves are negatively regulated by phosphorylation, and are downstream effectors of the PI3K/TOR pathway. Under nutritionally favorable conditions, the evolutionarily conserved TOR pathway is active and results in 4E-BP phosphorylation. This prevents 4E-BP binding to eIF4E, thus upregulating translation. Conversely, poor nutrition causes inhibition of the TOR pathway, such that unphosphorylated 4E-BP represses translation through eIF4E binding (Tettweiler, 2005 and references therein).

Whether d4E-BP is essential under starvation and oxidative stress conditions was investigated, because dFOXO activates the transcription of d4E-BP and d4E-BP mRNA levels increase upon starvation. Evidence is provided that d4E-BP activity is linked to life span, since overexpression of dFOXO is linked to increased longevity. This work establishes that d4E-BP is the critical effector of the dFOXO-induced stress-sensitive phenotype (Tettweiler, 2005).

FoxO transcription factors and TORC1 are conserved downstream effectors of Akt. This study unraveled regulatory circuits underlying the interplay between Akt, FoxO, and mTOR. Activated FoxO1 inhibits mTORC1 by TSC2-dependent and TSC2-independent mechanisms. First, FoxO1 induces Sestrin3 (Sesn3) gene expression. Sesn3, in turn, inhibits mTORC1 activity in Tsc2-proficient cells. Second, FoxO1 elevates the expression of Rictor, leading to increased mTORC2 activity that consequently activates Akt. In Tsc2-deficient cells, the elevation of Rictor by FoxO increases mTORC2 assembly and activity at the expense of mTORC1, thereby activating Akt while inhibiting mTORC1. FoxO may act as a rheostat that maintains homeostatic balance between Akt and mTOR complexes' activities. In response to physiological stresses, FoxO maintains high Akt activity and low mTORC1 activity. Thus, under stress conditions, FoxO inhibits the anabolic activity of mTORC1, a major consumer of cellular energy, while activating Akt, which increases cellular energy metabolism, thereby maintaining cellular energy homeostasis (Chen, 2010; see graphical abstract).

High-resolution dynamics of the transcriptional response to nutrition in Drosophila: a key role for dFOXO

A high-resolution time series of transcript abundance was generated to describe global expression dynamics in response to nutrition in Drosophila. Nonparametric change-point statistics revealed that within 7 h of feeding upon yeast, transcript levels changed significantly for approximately 3,500 genes or 20% of the Drosophila genome. Differences as small as 15% were highly significant, and 80% of the changes were <1.5-fold. Notably, transcript changes reflected rapid downregulation of the nutrient-sensing insulin and target of rapamycin pathways, shifting of fuel metabolism from lipid to glucose oxidation, and increased purine synthesis, TCA-biosynthetic functions and mitochondria biogenesis. To investigate how nutrition coordinates these transcriptional changes, feeding-induced expression changes were compared with those induced by the insulin-regulated transcription factor dFOXO in Drosophila S2 cells. Remarkably, 28% (995) of the nutrient-responsive genes were regulated by activated dFOXO, including genes of mitochondrial biogenesis and a novel homolog of mammalian peroxisome proliferator-gamma coactivator-1 (PGC-1), a transcriptional coactivator implicated in controlling mitochondrial gene expression in mammals. These data implicate dFOXO as a major coordinator of the transcriptional response to nutrients downstream of insulin and suggest that mitochondria biogenesis is linked to insulin signaling via dFOXO-mediated repression of a PGC-1 homolog (Gershman, 2007).

Given the change in mRNA encoding proteins involved in intermediary metabolism upon refeeding, transcriptional changes were expected in genes underlying mitochondrial function. Indeed, many genes whose products directly participate in mitochondrial substrate transport and ATP synthesis were coordinately upregulated. These included at least 13 genes encoding proteins with functions in electron transport and thirteen substrate carriers. Indeed, of 232 genes assigned Gene Ontology (GO) functions associated with mitochondria, 54% changed with refeeding, and 91% of these were increased (Gershman, 2007).

Most strikingly, there was a marked and rapid increase in transcripts encoding genes with roles in mitochondrial biogenesis, such as membrane preprotein translocases, chaperones, mitochondrial DNA binding proteins, and nuclear encoded mitochondrial ribosome proteins. Of 60 nuclear encoded mitochondrial ribosomal genes on the array, expression of 54 was increased an average of 44% at 5-7 h after refeeding. The induction of mitochondrial ribosomal proteins contrasts with the relative stasis of transcripts encoding proteins of the cytoplasmic ribosome. Of the 39 cytoplasmic ribosome genes represented on the array, mRNA for seven were moderately increased and two were reduced. An expansion of mitochondria within cells may reflect de novo adipogenesis in adult fat body when females first feed on yeast, similar to what is observed in mammalian 3T3-L1 cells that increase mitochondrial density as they differentiate into adipocytes. The observed increase in mitochondrial gene expression may also reflect activity in skeletal muscle cells, given that nutrients have been shown to regulate expression of mitochondrial oxidative phosphorylation genes in mammalian muscle (Gershman, 2007).

How nutrients regulate mitochondrial capacity is poorly understood. Many nutrient-induced transcripts encoding proteins of mitochondria biogenesis were broadly repressed in cells expressing dFOXO-A3. Of the 54 mRNAs for mitochondrial ribosome proteins that increased upon refeeding, 42 were downregulated by dFOXO-A3. Likewise, of the 25 upregulated genes with functions in mitochondrial biogenesis, 14 were repressed in dFOXO-A3 cells. In contrast, only six of the 53 nutrient-regulated, nuclear-encoded genes for mitochondrial function were inversely regulated by dFOXO-A3. Therefore, insulin, presumably via dFOXO, has a strong and selective impact on genes associated with mitochondrial biogenesis (Gershman, 2007).

One candidate to mediate mitochondrial biogenesis is PGC-1. PGC-1α of mammals stimulates mitochondrial biogenesis in muscle, brown adipose tissue, and adipogenic 3T3-L1 cells. Interestingly, a mRNA of the single ortholog of a PGC-like peptide in D. melanogaster (CG9809, Spargel) increases upon refeeding, and declines in dFOXO-A3 cells. CG9809 encodes a predicted protein of 1,088 amino acids that exhibits 68 or 52% homology with mammalian PGC-1α or PGC-1ß, respectively, in their COOH-terminal RNA-binding motif. Other domains in common with its mammalian homologs include an arginine-serine-rich domain located NH2-terminal to the RNA-binding motif, an acidic NH2-terminal domain and leucine-rich motifs, although the canonical LXXLL nuclear receptor binding motif is not present. However, the LXXLL motif is not absolutely required for coactivator binding, and CG9809 does contain a variant of this motif near its COOH terminus, FXXLL, which has been reported to function in nuclear receptor binding. Notably, this sequence is conserved in all mammalian PGC-1 family members. Beside a parallel based on sequence, orthologous targets of PGC-1 were elevated in refed flies and were reduced by dFOXO-A3 in S2 cells, including mitochondrial transcription factor A. Consistent with these data, insulin has been reported to upregulate PGC-1α and PGC-1ß mRNA levels in human muscle, although the mechanism was unclear. These data suggest that insulin signaling may be a conserved regulator of normal mitochondrial biogenesis via FOXO-mediated control of PGC-1-like cofactors. Importantly, this suggests a mechanistic basis for the prevalent mitochondrial dysfunction in humans with insulin resistance and diabetes (Gershman, 2007).

dFOXO regulates transcription of a Drosophila acid lipase

Insulin resistance is a major feature of pathological states such as obesity and diabetes. A consequence of insulin resistance is enhanced lipolysis, which causes excessive release of free fatty acids and deregulates fatty acid homeostasis. The transcription factor FOXO1 has a central role in the regulation of glucose levels by insulin: reduced insulin signaling causes FOXO1 activation, which increases hepatic glucose production by activating transcription of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. The results of this study suggest an additional role for FOXO transcription factors: the regulation of lipid homeostasis by insulin. In flies, dFOXO regulates lipase 4 (dLip4), a Drosophila homologue of human acid lipases. dFOXO binds and activates the dLip4 promoter, in vitro and in vivo, and regulates dLip4 expression. In addition, dLip4 mRNA expression in flies is dependent on dFOXO. The results support a model where dFOXO acts as a key modulator of lipid metabolism by insulin signaling and integrates insulin responses to glucose and lipid homeostasis (Vihervaara, 2008).

Regulation of insulin sensitivity occurs by a feedback loop through dFOXO/FOXO1 allowing cells to keep an exquisite metabolic balance between feeding and fasting states, permitting a faster response of the tissues to insulin changes. In conditions of limited nutrients, the amount of insulin (which mimics nutrient state) in serum is low, insulin signaling is reduced, and dFOXO/FOXO1 is activated. In these conditions, the body needs to produce energy to maintain basal metabolism and vital functions. By activating phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and dLip4, dFOXO coordinates production of glucose by gluconeogenesis and release of FFA by increasing lipolysis of stored lipids. In the absence of nutrients, when insulin levels drop, dFOXO increases lipolysis by activating dLip4, which would release FFA as a source of energy. In contrast, when nutrients are abundant, insulin is secreted, dFOXO is repressed, and glucose and FFA are deposited as energy stores (converted into glycogen and triglycerides, respectively. Thus, dFOXO would have a central role in coordinating the degradation and synthesis of energy stores in the form of lipid-FFA and glycogen-glucose. In this way, gluconeogenesis and lipolysis, key metabolic processes involved in energy homeostasis, are coordinated with nutritional status to produce a balanced outcome. This regulatory mechanism could well be disrupted in pathological states with abnormally increased insulin levels as is found in the case of insulin-resistant diabetes (Vihervaara, 2008).

Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies

Sestrins are conserved proteins that accumulate in cells exposed to stress, potentiate adenosine monophosphate-activated protein kinase (AMPK), and inhibit activation of Target of rapamycin (TOR). The abundance of Drosophila sestrin (dSesn) is increased upon chronic TOR activation through accumulation of reactive oxygen species that cause activation of c-Jun amino-terminal kinase and transcription factor Forkhead box O (FoxO). Loss of dSesn resulted in age-associated pathologies including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration, and cardiac malfunction, which were prevented by pharmacological activation of AMPK (see AMP-activated protein kinase) or inhibition of TOR. Hence, dSesn appears to be a negative feedback regulator of TOR that integrates metabolic and stress inputs and prevents pathologies caused by chronic TOR activation that may result from diminished autophagic clearance of damaged mitochondria, protein aggregates, or lipids (Lee, 2010).

Target of rapamycin (TOR) is a key protein kinase that regulates cell growth and metabolism to maintain cellular and organismal homeostasis. Insulin and insulin-like growth factors are major TOR activators that operate through phosphoinositide 3-kinase (PI3K) and the protein kinase AKT. Conversely, adenosine monophosphate-activated protein kinase (AMPK), which is activated upon energy depletion, caloric restriction (CR), or genotoxic damage, is a stress-responsive inhibitor of TOR activation. TOR stimulates cell growth and anabolism by increasing protein and lipid synthesis through p70 S6 kinase (S6K), eukaryotic translation initiation factor 4E-binding protein (4E-BP), and sterol response element binding protein (SREBP) and by decreasing autophagic catabolism through phosphorylation-mediated inhibition of ATG1 protein kinase. Persistent TOR activation is associated with diverse pathologies such as cancer, diminished cardiac performance, and obesity-associated metabolic diseases. Conversely, inhibition of TOR prolongs life span and increases quality of life by reducing the incidence of age-related pathologies. The antiaging effects of CR could be due to inhibition of TOR (Lee, 2010 and references therein).

Sestrins (Sesns) are highly conserved proteins that accumulate in cells exposed to stress, lack obvious domain signatures, and have poorly defined physiological functions. Mammals express three Sesns, whereas Drosophila melanogaster and Caenorhabditis elegans have single orthologs. In vitro, Sesns exhibit oxidoreductase activity and may function as antioxidants. Independently of their redox activity, Sesns lead to AMPK-dependent inhibition of TOR signaling and link genotoxic stress to TOR regulation (Badanov, 2008). However, Sesns are also widely expressed in the absence of exogenous stress, and in Drosophila, expression of Drosophila sestrin (dSesn) is increased upon maturation and aging. Given the redundancy between mammalian Sesns, the importance of Sesns as regulators of TOR function was tested in Drosophila. Both gain- and loss-of-function dSesn mutants were created. Analysis of these mutants revealed that dSesn is an important negative feedback regulator of TOR whose loss results in various TOR-dependent, age-related pathologies (Lee, 2010).

Persistent TOR activation in wing discs by a constitutively active form of the insulin receptor (InRCA) resulted in prominent dSesn protein accumulation, which is not seen in a dSesn-null larvae. InRCA also induced accumulation of dSesn RNA, indicating that dSesn accumulation is due to increased transcription or mRNA stabilization. Since dSesn accumulation was restricted to cells in which TOR was activated, the response is likely to be cell autonomous. dSesn was also induced when TOR was chronically activated by overexpression of the small guanine triphosphatase Rheb, clonal loss of phosphatase and tensin homolog (PTEN), or tuberous sclerosis complex 1 (TSC1). Dominant-negative PI3K (PI3KDN) or TOR (TORDN) inhibited dSesn accumulation caused by overexpression of InRCA, but inactive ribosomal S6 protein kinase (S6K, S6KDN) and hyperactive 4E-BP (4E-BPCA), two downstream TOR effectors, did not. Furthermore, dorsal-specific expression of activated S6KCA or loss of 4E-BP activity failed to induce dSesn expression, indicating that TOR regulates expression of dSesn through different effector(s) (Lee, 2010).

In mammals, transcription of Sesn genes is increased in cells exposed to oxidative stress, and reactive oxygen species (ROS) accumulation, detected by oxidation of dihydroethidium (DHE), was observed in the same region of the imaginal discs in which InRCA or Rheb were expressed. InRCA-induced accumulation of ROS was blocked by coexpression of either PI3KDN or TORDN, but not S6KDN or 4E-BPCA, revealing TOR’s role in ROS accumulation. Wing-disc clones in which TOR was activated by loss of TSC1 also exhibited ROS accumulation, confirming that TOR-dependent ROS accumulation is cell-autonomous. Expression of the ROS scavengers catalase or peroxiredoxin inhibited InRCA-induced accumulation of dSesn. Feeding animals with vitamin E, an antioxidant, also prevented dSesn induction caused by TSC1 loss (Lee, 2010).

Forkhead box O (FoxO) and p53 are ROS-activated transcription factors that control mammalian Sesn genes. The dSesn locus contains eight perfect FoxO-response elements, a frequency 25 times higher than that expected on the basis of random distribution. Overexpressed FoxO or p53 could both increase expression of the dSesn gene. However, InRCA caused accumulation of dSesn in a p53-null background, but not in a FoxO-null background, indicating that TOR-activated FoxO is likely to be the regulator of dSesn gene transcription. Accumulation of dSesn in response to Rheb overexpression was also FoxO-dependent (Lee, 2010).

In dorsal wing disc cells, where ROS accumulated in response to InRCA, c-Jun N-terminal kinase (JNK), a protein kinase that phosphorylates FoxO, was also activated. JNK activation was diminished in cells overexpressing catalase, suggesting that it depends on TOR-induced accumulation of ROS. Mitogen-activated protein kinase kinase 7-mediated activation of JNK also resulted in accumulation of dSesn, as did overexpression of mammalian STE20-like kinase 1 (MST1), another protein kinase that phosphorylates FoxO. However, only JNKDN (but not Mst1DN) inhibited InRCA-mediated accumulation of dSesn. Collectively, these data suggest that dSesn transcription is increased upon chronic TOR activation through ROS-dependent activation of JNK and FoxO (Lee, 2010).

To determine effects of dSesn on cell growth, a major function of TOR, dSesn was overexpressed in dorsal wings. This resulted in a dose-dependent phenotype in which the wing bends upward, indicating suppressed dorsal tissue growth. A dSesnC86S variant, in which the cysteine required for oxidoreductase activity was mutated (C86S, Cys86->Ser86), still conferred this phenotype when expressed in amounts similar to those of wild-type dSesn (dSesnWT). Cell number and size were measured in a dorsal wing region defined by the L3, L4, C1, and C2 veins. Although the size of this area was significantly reduced by dSesn expression, the cell number remained unchanged, showing that decreased cell size can account for dSesn suppression of tissue growth. Overexpression of dSesn also reduced cell size in larval wing discs and adult eyes. Thus, dSesn inhibits cell growth without affecting cell proliferation and does so independently of its redox activity (Lee, 2010).

When dSesn was expressed with InRCA or Rheb, it suppressed the hyperplastic phenotypes caused by these TOR activators. Both eye and individual ommatidia sizes were significantly reduced. dSesn also inhibited InRCA- or Rheb-induced phosphorylation of TOR targets S6K and 4E-BP. In mammalian cells, dSesn enhanced AMPK-induced phosphorylation of TSC2 and inhibited S6K activity through TSC2, just as mSesn2 does (Budanov, 2008). In Drosophila wings, dSesn-induced growth suppression was attenuated by reduced gene dosage of TSC1, TSC2, or AMPK, although reduced dosage of these genes alone did not affect normal growth. Expression of mSesn1/2 in flies also reduced normal and InRCA-induced hyperplastic growth (Lee, 2010).

Expression of InR, constitutively active PI3K (PI3KCA), AKT, or S6KCA in dorsal cells of the wing caused an overgrowth phenotype in which the wing bends downward. dSesn expression reversed this effect of overexpressed InR, PI3KCA, and AKT, but not that of S6KCA, suggesting that dSesn inhibits TOR downstream of AKT. Conversely, dorsal wing-specific expression of PTEN and InRDN, PI3KDN, or S6KDN caused wings to bend upward, and this effect was enhanced by dSesn (Lee, 2010).

Although dSesn-null flies did not exhibit developmental abnormalities, the growth-promoting effect of overexpressed InR or AKT was enhanced in dSesn-null background, suggesting that endogenous dSesn restricts TOR activation and its growth-promoting effect. Loss of dSesn, however, did not enhance S6K-stimulated cell growth or decrease growth suppression by overexpressed InRDN or S6KDN. These findings indicate that Sesn is an evolutionarily conserved inhibitor of TOR signaling that acts via the AMPK-TSC2 axis (Lee, 2010).

Fat bodies from dSesn-null third-instar larvae contained more lipids than did those of WT animals. dSesn-null adults also contained more triglycerides, which were decreased after ectopic expression of dSesnWT or dSesnCS. Thus, the TOR-inhibitory function of dSesn, rather than its antioxidant activity, appears to affect metabolic control. Congruently, dSesn-null fat bodies showed decreased AMPK and increased TOR activities. Pharmacological manipulation strengthened this conclusion; feeding dSesn-null mutants with AMPK-activators such as 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) or metformin, or the TOR-inhibitor rapamycin reduced triglyceride accumulation (Lee, 2010).

Expression of the gene-encoding transcription factor dSREBP and its targets, which encode fatty acyl coenzyme A (CoA) synthetase, fatty acid synthase, acetyl CoA carboxylase, and acetyl CoA synthetase, was significantly increased (20 to 70%) in dSesn-null mutants. However, the peroxisome proliferator-activated receptor γ coactivator 1 (dPGC-1) gene and some lipolytic genes showed decreased expression. This is consistent with reports that dSREBP and dPGC-1 (spargel; CG9809)are inversely regulated by TOR and AMPK to properly control lipid metabolism (Lee, 2010).

Age-related decline in heart performance is another phenotype associated with TOR hyperactivity in insects and mammals. In WT flies, the heart beats in a highly regular manner, but in dSesn-null mutants, heart function was compromised, as manifested by arrhythmia and decreased heart rate. Slowing of heart rate reflected expansion of the diastolic period, as observed in aged or TOR-activated flies. These defects were largely prevented by feeding flies AICAR or rapamycin, indicating that they are caused by low activity of AMPK or high TOR activity. Vitamin E feeding or catalase expression suppressed the arrhythmia caused by loss of dSesn, but not the decrease in heart rate, suggesting that TOR-induced oxidative stress contributes to the arrhythmic phenotype. Analysis of F-actin revealed structural disorganization of myofibrils in dSesn-null hearts, suggesting that cardiac muscle degeneration may cause some of the functional defects in dSesn-null hearts. Reflecting this structural abnormality, dSesn-null hearts were dilated during both the diastolic and systolic phases, and this was prevented by AICAR or rapamycin (Lee, 2010).

Heart-specific depletion of dSesn caused cardiac malfunction similar to that seen in dSesn-null mutants. Heart-specific depletion of AMPK also caused cardiac malfunction, but this was not alleviated by AICAR administration, supporting the notion that dSesn maintains normal heart physiology through AMPK activation (Lee, 2010).

dSesn mRNA and protein are abundant in the adult thorax, which is mostly composed of Mesoderm. mSesn1 is also highly expressed in skeletal muscle (Velasco-Miguel, 1999). Therefore, whether dSesn has a role in maintaining muscle homeostasis was tested. 20-day-old dSesn-null flies showed degeneration of thoracic muscles with loss of sarcomeric structure, including discontinued Z discs, disappearance of M bands, scrambled actomyosin arrays, and diffused sarcomere boundaries. Such defects are only partially observed in very old WT flies (~90 days) and were not found in young (5-day-old) dSesn-null muscles. Thus, the dSesn-null skeletal muscle appears to undergo accelerated age-related degeneration (Lee, 2010).

Despite its normal appearance, muscle from 5-day-old dSesn-null flies exhibited mitochondrial abnormalities, including a rounded shape, occasional enlargement, and disorganization of cristae, which were also observed in 20-day-old mutants. Mitochondrial dysfunction can result in excessive generation of ROS leading to other abnormalities. dSesn-null muscles exhibited increased accumulation of ROS, revealed by more intense DHE fluorescence and reduced cis-aconitase activity, which was associated with muscle cell death. Furthermore, the muscle defects were prevented by vitamin E feeding, underscoring the role of ROS in muscle degeneration (Lee, 2010).

Expression of exogenous dSesnCS, devoid of redox activity, prevented muscle degeneration, suggesting again that regulation of AMPK-TOR by dSesn, rather than intrinsic redox activity, is of importance. Feeding animals with AMPK activators prevented muscle degeneration in dSesn-null mutants, and depletion of AMPK in skeletal muscles caused severe degeneration of mitochondrial and sarcomeric structures. Treatment of animals with rapamycin also prevented muscle degeneration in dSesn-null flies. Thus, dSesn-dependent control of AMPK-TOR signaling is essential for prevention of mitochondrial dysfunction and maintenance of muscle homeostasis during aging (Lee, 2010).

It was noticed that dSesn-null muscles accumulated polyubiquitin aggregates, which are hallmarks of defective autophagy. To test whether decreased autophagy brought about by excessive and prolonged TOR activity might cause muscle degeneration, expression was silenced of ATG1, an essential component of the autophagic machinery, which is inhibited by TOR. This caused a decline in cardiac performance, as well as degeneration and mitochondrial abnormalities in skeletal muscle. These results suggest that TOR up-regulation caused by dSesn loss inhibits autophagy needed to eliminate ROS-producing dysfunctional mitochondria, which may contribute to muscle degeneration. Consistent with this view, ATG1 silencing resulted in ROS accumulation in wing discs (Lee, 2010).

These results identify Sesn as a negative feedback regulator of TOR function. In mammalian cells, increased expression of mSesns in response to genotoxic stress leads to inhibition of TOR activity through activation of AMPK (Budanov, 2008). This study now shows that transcription of the dSesn gene is increased upon chronic TOR activation through JNK and FoxO in a manner dependent on ROS accumulation. Although transient InR activation inhibits FoxO through its phosphorylation by AKT, this study finds that chronic TOR activation overcomes this inhibition and results in nuclear translocation of FoxO, which increases dSesn transcription. In turn, dSesn suppresses metabolic dysfunction and age-related tissue degeneration brought about by hyperactivated TOR. Although dSesn can inhibit TOR-stimulated cell growth, this analysis points to its most important function being the maintenance of metabolic homeostasis and prevention of TOR-induced tissue degeneration. The three major functions of dSesn revealed by this study -- suppression of lipid accumulation, prevention of cardiac malfunction, and protection of muscle from age-related degeneration -- are adversely affected by obesity, lack of exercise, and aging, which make a disproportional contribution to health problems in developed and rapidly developing societies (Lee, 2010).

Whereas TOR controls cell growth mostly through inhibition of 4E-BP and activation of S6K kinase, its ability to induce dSesn expression depends on ROS accumulation, which the results suggest is a pathophysiological aberration caused by TOR hyperactivation that is normally antagonized by dSesn. However, the previously described redox function of Sesn is not required for its protective role. TOR-induced accumulation of ROS has been observed in yeast and hematopoietic cells, but the molecular mechanism underlying this phenomenon and its physiological and pathophysiological importance were unknown. The current results suggest that TOR-stimulated production of ROS, which is needed for accumulation of dSesn, is independent of two of the major TOR targets (4E-BP and S6K) and instead may result from TOR-mediated inhibition of physiological autophagy, a process that eliminates ROS-producing dysfunctional mitochondria. Nonetheless, inhibition of 4E-BP also contributes to the pro-aging effects of TOR by suppressing translation of several mitochondrial proteins and by accelerating age-related cardiac malfunction at young ages, which is reminiscent of the observed cardiac defects seen in dSesn-null flies. Although TOR activates SREBP, which may contribute to lipid accumulation in dSesn-null flies, autophagy promotes lipid elimination. Thus, decreased autophagy may also contribute to triglyceride accumulation. Hence, the different degenerative phenotypes exhibited by dSesn-null flies are due to the cumulative effects of several biochemical and cell biological defects caused by hyperactive TOR, including reduced autophagy and reduced function of 4E-BP. Both basal physiologic autophagy and 4E-BP function are enhanced by calorie restriction, which prevents aging-related pathologies. In the future, it will be of interest to determine the contribution of Sesn to these antiaging effects (Lee, 2010).

A Drosophila insulin-like peptide promotes growth during nonfeeding states

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. This study shows 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 (Slaidina, 2009).

Growth relies on the ability of cells and organisms to access nutrients. Nutrients can be obtained from diverse sources, such as from the environment through feeding, or from internal stores as with early embryos that develop from large eggs. Accordingly, because alternate sources of nutrients are used during specific periods of development, organisms must be able to adapt their metabolic and growth programs to changes in the developmental or environmental energy context (Slaidina, 2009).

In complex animal species, growth is controlled by intermingled paracrine and endocrine regulatory processes, with organ and tissue growth governed by specific genetic programs that determine the target size and relative proportions of the species. The output of these genetic programs is further modified by environmental cues, including nutrition. Variations in nutritional input can influence growth and metabolism via insulin/IGF signaling (IIS). In particular, when nutrients are abundant, IIS is maximally active and growth is limited solely by the organ-intrinsic program; upon nutrient shortage, in contrast, IIS becomes limiting and restricts the growth and metabolic parameters accordingly (Slaidina, 2009).

In mammals, the IIS system is split into two complementary and interacting subsystems that govern growth, metabolism, reproduction, and longevity. The first of these corresponds to circulating insulin levels, which control carbohydrate and fat metabolism, and the second is the GH/IGF-I axis, which regulates cell and tissue growth. Starvation lowers circulating IGF-I, in part through decreased transcription of the IGF-I gene in the liver; this suggests that one major way in which starvation can affect growth is by reducing levels of circulating growth factors (Slaidina, 2009).

The function of IIS in growth control is remarkably conserved in insects, and in particular in Drosophila, where seven Drosophila insulin-like peptides (DILPs) have been identified. The various DILP genes are expressed in different larval and adult tissues, suggesting that they carry nonredundant functions. In particular, DILP1, -2, -3, and -5 are expressed in specialized neurosecretory cells located in each brain hemisphere, called the insulin-producing cells (IPCs). Genetic ablation of these cells leads to severe larval growth deficits, hypertrehalosemia, and increased lifespan (Slaidina, 2009).

One major role for IIS in insects is to couple growth with the animal's energy status. Indeed, total nutrient deprivation downregulates DILP3 and DILP5 transcription in the IPCs, although DILP2 expression remains unchanged. Recent results indicate that variations in nutritional information are relayed by a nutrient sensor operating in the fat body, a larval organ that shares metabolic functions with the vertebrate white fat and liver. In particular, it has been shown that amino acid restriction triggers fat body-specific inhibition of the TOR complex1 (TORC1), a major cell-based nutrient-sensing pathway. Inhibition of TORC1 in the fat body systemically reduces larval growth in part by blocking Dilp secretion from the brain IPCs. Therefore, in line with the decreased levels of circulating IGF-I in vertebrates, starvation affects Drosophila growth by severely reducing brain-specific DILP function (Slaidina, 2009).

Interestingly, previous work has shown that protein starvation causes the growth arrest of endoreplicative larval tissues (ERTs), while only slowing the growth and proliferation of cells in the larval brain and in imaginal discs. Similarly, generally reduced TOR signaling in the larva, which in many respects mimics the starvation state, strongly inhibits ERT growth, while generally sparing the imaginal tissues (ITs) that form the adult structures. This suggests a protection mechanism whereby, under adverse nutrition conditions, the fat body allows larval resources to be reallocated to high-priority tissues like the imaginal discs. Significantly, such a mechanism would require that some ILPs are produced during starvation and activate IIS in the tissues that continue to grow (Slaidina, 2009).

Feeding arrest is also a programmed event during development. At the end of the larval period, animals undergo a stereotyped behavior called the wandering stage, when they migrate away from the food and prepare for pupariation. This developmentally induced starvation precedes the long pupal feeding arrest. During pupal development, larval tissues undergo intense remodeling. This process involves a major reallocation of resources, as future adult tissues form from ITs in a process that uses either nutrient stores that had accumulated in fat cells during larval life, or energy obtained from the degradation of obsolete larval tissues. Since organisms do not feed during this stage, no global growth or weight gain is observed; nevertheless, because tissue remodeling involves cell growth and proliferation, growth-promoting pathways presumably come into play. The paradox of pursuing a growth program in a nonfeeding organism that is subjected to catabolic regulation could be circumvented by the induction of growth-promoting hormones upon feeding arrest (Slaidina, 2009).

This study presents the characterization of a particular DILP, DILP6, which promotes growth during nonfeeding stages. The DILP6 gene is expressed in fat body cells and is strongly induced during the wandering larval and pupal periods, as well as upon starvation. Reduced DILP6 function results in a growth deficit during pupal development and an increased sensitivity to starvation in young adults. The sudden increase of DILP6 expression at the onset of pupal development requires an endocrine signal that is provided by the steroid hormone ecdysone. In parallel, starvation increases DILP6 expression through dFoxO-mediated feedback regulation of IIS. Therefore, DILP6 constitutes an IGF-like peptide with a specialized role in promoting growth during developmentally or environmentally induced nonfeeding states (Slaidina, 2009).

During the successive stages of development, organisms use alternate sources of nutrients to support tissue growth and morphogenesis. In Drosophila, embryonic tissues develop using maternal stores accumulated in the egg in the form of yolk. Larval development follows, with a major growth program relying on the animals' capacity to obtain nutrients from the environment. Finally, during the pupal stage, animals do not feed, and a large quantity of nutrients stored in fat cells allows pupae to prolong growth and finalize the development of adult structures. On top of these basic developmental strategies, feeding larvae have evolved additional buffering mechanisms to protect growing tissues from sudden variations in environmental energy supplies. Notably, brain ILPs promote larval growth and allow the coupling of growth to nutritional input. Their expression and secretion from brain IPCs decrease upon starvation, and several brain DILPs show only residual expression in the pupa. Therefore, there must be a distinct set of growth inducers that take the lead to activate growth in the pupa and upon nutritional stress. In both of these contexts, a physiological switch takes place that triggers the activation of DILP6, a member of a distinct class of ILPs devoted to growth during nonfeeding periods (Slaidina, 2009).

The DILP1 and DILP3 genes are also expressed during pupal development, suggesting that they may act in concert with DILP6. Individual knockout of either of these two DILP genes produces only marginal growth defects, suggesting that there is a high level of redundancy between them or with DILP6. The observation that DILP1 expression increases two-fold in DILP6 mutant larvae suggests a possible compensatory mechanism that could partially suppress the growth impairment observed in DILP6 mutants. The functional class of ILPs represented by DILP6 may be conserved in other insect species, as an ecdysone-induced, fat-body-specific ILP has recently been described in Bombyx mori (Slaidina, 2009).

The developmental and environmental induction of DILP6 involves overlapping mechanisms. First, in response to nutrient deprivation, the IIS component, dFoxO, provokes a burst of DILP6 transcription, thereby linking DILP6 expression with the nutritional status of the animal. This represents a feedback regulation on IIS, as dFoxO, an inhibitor in the IIS pathway, induces the expression of DILP6, an activator of IIS. Interestingly, expression of DILP3 in the adult was also recently shown to depend on dFoxO function, suggesting that other DILP genes in this subclass are subjected to similar controls (Slaidina, 2009).

DILP6 does not appear to be effective as a paracrine/autocrine factor for fat cells. Indeed, fat cells of starved larvae, which express high levels of DILP6, undergo extensive autophagic transformation, even though autophagy has been shown to be blocked in these cells by IIS activation. In addition, overexpression of DILP6 in the fat body of starved larvae does not prevent autophagy (Slaidina, 2009).

More generally, ERTs present stronger growth inhibition in response to starvation than do ITs. The role of a starvation-specific ILP that is induced upon nutritional stress could be to reroute energy stores toward high-priority organs and tissues, such as those responsible for the formation of the future adult. The specific action of DILP6 on imaginal cells could contribute to this diversified behavior, although this would require that ITs are more receptive to the DILP6 signal than are ERTs, at least upon starvation. Such differences in the response of ERTs and ITs to the DILP signal, combined with the production of specific DILPs upon starvation, could constitute a bona fide mechanism for the specific allocation of spare resources to ITs under nutritional stress. However, the mechanisms for such a biased response need to be elucidated (Slaidina, 2009).

At the end of larval development, animals stop feeding and prepare for pupal development. This study shows that tissue remodeling in the pupa involves IIS-dependent growth, and that DILP6 is specifically expressed and required for growth during this period. The transition from larval to pupal development is controlled by the steroid hormone ecdysone (20E), and it was also shown that 20E is required for proper DILP6 induction at the larval/pupal transition. In view of the absence of obvious EcR/Usp binding sites in the 5' region of the DILP6 gene, as well as a previous demonstration that EcR signaling controls dFoxO nuclear localization, it is hypothesized that dFoxO could mediate the ecdysone-dependent expression of DILP6. However, both genetics and ex vivo experiments on dissected fat bodies indicate that, although dFoxO appears to contribute to the developmental induction of DILP6 at the larval/pupal transition, it is not required for the 20E-induced expression of DILP6. In an accompanying manuscript, Okamoto (2009) reports that 20E-induced expression of DILP6 is not affected by cycloheximide, suggesting that the transcriptional induction of DILP6 by EcR/Usp is direct (Slaidina, 2009).

It has been previously shown that ecdysone has a growth-inhibitory function during larval development. Indeed, increased basal levels of circulating ecdysone in larvae can reduce the growth rate and, conversely, decreased basal ecdysone levels can increase the growth rate. Although the mechanisms underlying this relationship are not yet fully understood, this study has established that the levels of ecdysone produced experimentally in these experiments remain close to basal levels, and are insufficient to modify DILP6 expression. Therefore, while basal levels of ecdysone can inhibit systemic growth through an unknown mechanism, high ecdysone levels at the larval/pupal transition can induce DILP6, and thus systemically activate IIS (Slaidina, 2009).

One puzzling observation reported in this study is that the modification of DILP6 expression in pupae can alter the adult mass as well as the resistance of animals to starvation at eclosion. How can DILP6 overexpression in pupae increase adult mass if the mass of the pupa is fixed at the end of larval life? One possible explanation is that DILP6 participates in a tradeoff between the construction of adult tissues and the maintenance of energy stores in the pupa. Indeed, the levels of both TAG and glycogen stores in the young adult are affected by DILP6 levels in the pupa. In this line, recent reports indicate that, under optimal conditions, not all nutrients are used by the pupa, and part of the energy is conserved to provide sustenance during the early period of adult life that precedes feeding. Some larval fat body cells are still present in early adults, and provide energy until feeding begins. Suppressing the death of these cells increases the energy stores and enhances the resistance of young adults to starvation (Aguila, 2007). DILP6 knockdown in pupae has a similar effect: less energy is used by the pupa to build tissues, meaning that the adult ecloses with a smaller body, but with greater energy stores to help overcome early nutritional stress. DILP6 overexpression has the opposite effect. The current results therefore indicate that DILP6 sets the energy balance in pupae by promoting tissue growth, while sparing an energy pool that can be used by the young adult (Slaidina, 2009).

DILP6 shares some specific features with vertebrate IGF-I that distinguish both of them from insulin. DILP6 peptide sequence does not present obvious cleavage sites for an internal C peptide (Brogiolo, 2001). It is produced in the fat body, a tissue sharing common functions with the vertebrate liver, where IGF-I is mainly produced. DILP6 mutant animals present growth defects without obvious metabolic changes, suggesting that DILP6 might have an exclusive growth function. Finally, the induction of growth factor production under conditions of energy stress is also relevant to cancer biology. Indeed, IGF-I and IGF-II are frequently expressed within neoplastic tissue. It is suspected that they act as autocrine and paracrine growth factors within tumors, allowing tumor cells to evade nutritional shortage and acquire survival properties (Pollak, 2008). The induction of DILP6 under starvation and its preferential targeting to ITs instead of ERTs could represent an interesting parallel to the induction of IGFs in tumor cells, where the selective action of growth factors can promote growth and survival of specific tissues in a nonfavorable environment (Slaidina, 2009).

dDOR is an EcR coactivator that forms a feed-forward loop connecting insulin and ecdysone signaling

Mammalian DOR (for 'Diabetes- and Obesity Regulated') was discovered as a gene whose expression is misregulated in muscle of Zucker diabetic rats. Because no DOR loss-of-function mammalian models are available, this study analyzed the in vivo function of DOR by studying flies mutant for Drosophila DOR (dDOR). dDOR is a novel coactivator of ecdysone receptor (EcR) that is needed during metamorphosis. dDOR binds EcR and is required for maximal EcR transcriptional activity. In the absence of dDOR, flies display a number of ecdysone loss-of-function phenotypes such as impaired spiracle eversion, impaired salivary gland degradation, and pupal lethality. Furthermore, dDOR knockout flies are lean. dDOR expression is inhibited by insulin signaling via FOXO. This work uncovers dDOR as a novel EcR coactivator. It also establishes a mutual antagonistic relationship between ecdysone and insulin signaling in the fly fat body. Furthermore, because ecdysone signaling inhibits insulin signaling in the fat body, this also uncovers a feed-forward mechanism whereby ecdysone potentiates its own signaling via dDOR (Francis, 2010).

Thyroid hormone receptor (TR) is an important regulator of development and metabolism in animals. TR is a type II nuclear hormone receptor (NR). It resides in the nucleus and binds DNA regardless of ligand binding, and it heterodimerizes with retinoid X receptor (RXR). In the absence of ligand, TR is complexed with corepressors to inhibit transcription, whereas in the presence of ligand, it binds coactivators and activates transcription. One recently discovered TR coactivator is DOR. DOR was first identified as a gene that is downregulated in muscle of diabetic rats. DOR was then shown to have two functions. It acts as a coactivator of thyroid hormone receptor TRα1, binding TRα1 and impacting its transcriptional activity. Furthermore, DOR has a second life outside the nucleus, as a regulator of autophagy. Together, these data implicate DOR as a regulator of NR function and of metabolism. However, no DOR mutant animals have yet been reported, and the in vivo function of DOR remains to be studied (Francis, 2010).

Drosophila has 18 nuclear receptors, including ecdysone receptor (EcR). EcR shares many commonalities with type II NRs, in that it heterodimerizes with the fly RXR homolog USP, binds DNA constitutively, complexes with either coactivators or corepressors depending on its state of ligand binding, and can form a functional complex with mammalian RXR. The EcR/USP complex senses and responds to the hormone 20-hydroxyecdysone (20E) to regulate developmental timing and metabolism. 20E triggers all developmental transitions, such as the molts from one larval stage to the next, and many events occurring during metamorphosis. These include termination of larval feeding, apoptosis, and elimination of larval salivary glands and larval fat body, as well as many morphological changes in tissues that will give rise to the adult fly. Several EcR corepressors and coactivators have been identified and characterized, including Alien, SMRTER, bonus, Trithorax-related gene (TRR), Taiman, and rigor mortis. However the coactivator(s) of EcR required for proper pupal development and metamorphosis remain to be described (Francis, 2010).

Interestingly, crosstalk has recently come to light between ecdysone signaling and insulin signaling, which regulates the growth and metabolism of animals. Ecdysone regulates insulin signaling and vice versa. In particular, in the fat body of the fly, ecdysone signaling inhibits PI3K activity and thereby insulin signaling, suggesting an antagonistic relationship between these two hormonal signaling pathways. The molecular mechanisms underlying these regulatory events, however, are not fully understood (Francis, 2010).

In order to study the function of DOR in an in vivo animal model, the Drosophila genome was searched for homologs of human DOR (hDOR). A BLAST search through all predicted Drosophila proteins with the sequence of hDOR yielded CG11347 as the top hit, which was rename Drosophila DOR (dDOR) (Francis, 2010).

The dDOR locus is predicted to encode six different transcripts, giving rise to three different polypeptides. The -RA, -RB, -RD, and -RE isoforms encode a 387 amino acid protein hereafter referred to as DORlong, whereas the -RC isoform encodes a shorter protein, of 273 amino acids, referred to as DORshort. The -RF isoform encodes an even shorter protein similar to DORshort but lacking 44 amino acids at the N terminus. While performing RT-PCR with oligonucleotides specific for the long isoform, the presence of two differently sized PCR products was detected. Sequencing revealed that one of the products corresponded to the predicted 'long' isoform. The second product corresponded to an unannotated isoform consisting of the 'long' isoform plus a 90 bp extension of the third exon, resulting from use of an alternate splice donor. As a result, 30 amino acids are inserted in the middle of the dDORlong protein. Contained in these 30 amino acids is the sequence FENLL, which is similar to the LXXLL nuclear-receptor-interacting motif found in nuclear receptor coactivators. This FENLL sequence aligns to the transactivation domain motif of human DOR (LEDLL) when the two proteins are aligned to each other. This isoform is referred to as dDORFENLL. The domain of dDORFENLL surrounding the FENLL sequence has 75% identity and 85% homology to human DOR. Three isoforms of dDOR were studied in this work (Francis, 2010).

In order to measure the relative abundance of the three isoforms in vivo, quantitative RT-PCR was performed with isoform-specific primers on RNA extracted from animals of various stages of development. The most abundant isoform is the long one, followed by the FENLL isoform (roughly half the level of the long isoform), whereas the short isoform is expressed at comparatively low levels. This relative expression of the three isoforms is also observed in fat body of wandering third-instar larvae whereas in fat body of early pupae the FENLL isoform strongly predominates. Indeed, the FENLL isoform is highly enriched in fat body of early pupae when compared to the rest of the body (Francis, 2010).

Because expression of human DOR is misregulated in rats, via an unknown mechanism, upon development of diabetes, it was asked whether expression of Drosophila DOR is also regulated by nutritional conditions. Given that the FENLL isoform of dDOR is responsible for the metabolic defects of dDOR mutants, attention was focused on the FENLL isoform. Third-instar larvae were either fasted or fed for 18 hr and then assayed dDORFENLL mRNA levels in fat body by quantitative RT-PCR. When control larvae were fasted, dDORFENLL expression in fat body increased > 2-fold. One important signaling pathway that is inhibited upon fasting is insulin. It was therefore asked whether dDORFENLL expression is inhibited by insulin, because this would explain its upregulation upon fasting. Explanted fat bodies were tested in the presence or absence of 5 μg/ml insulin and dDORFENLL expression levels were assayed by quantitative RT-PCR. In the presence of insulin, dDORFENLL expression decreased by 73%. dDORFENLL expression levels also decreased by 59% in S2 cells treated with 1 μM insulin for 2 hr (Francis, 2010).

One transcription factor mediating much of the transcriptional output of the insulin pathway is FOXO. FOXO activity is suppressed by insulin signaling. Whether regulation of dDORFENLL expression is mediated by FOXO was tested by studying animals containing the FOXO21/25 null allele combination. FOXO21/25 mutants were starved and it was found that the fasting-induced upregulation of dDORFENLL expression in fat body was strongly impaired, indicating that this transcriptional regulation is FOXO dependent. The transcriptional regulation of dDORFENLL is analogous to that of a canonical FOXO target gene, 4E-BP. 4E-BP expression is suppressed by insulin in vivo in fat bodies and increases in vivo in fat body upon fasting of wild-type animals but does not increase upon fasting of FOXO mutant animals. It was therefore asked whether dDOR is also a direct transcriptional target of FOXO. In Drosophila, FOXO targets sites are preferentially located within 1 kb of the target promoter. The dDOR promoter region was screened and a perfect consensus FOXO binding site (GTAAACAA) was found 230 nt upstream of the transcription start site of the –RA and –RB transcripts. To test whether FOXO binds this site in vivo, chromatin immunoprecipitation (ChIP) of endogenous FOXO from third-instar larvae was performed. Two negative controls were performed: a mock ChIP using preimmune serum from wild-type animals, and a ChIP using anti-FOXO antibody from FOXO21/25 null mutant animals. Quantitative PCR (qPCR) on the immunoprecipitated material revealed that the promoter region of 4E-BP, an established direct target of FOXO, was strongly enriched in the FOXO ChIP from wild-type animals, but not in the negative controls. Strikingly, the promoter region of dDOR-RA/B was also strongly enriched in the FOXO ChIP but not in the negative controls, indicating that FOXO binds the dDOR promoter in vivo. As a negative control, the genomic region of mir-278 was not enriched in the FOXO ChIP. Together, these data indicate that expression of dDORFENLL is inhibited by insulin signaling as a direct target of FOXO, and identify a molecular mechanism by which insulin signaling inhibits ecdysone signaling in the fat body. Because dDOR is involved in linking nutrient signaling to EcR signaling, whether dDOR mutants have impaired fitness upon nutrient deprivation was tested. Upon removal of food (but not water), dDOR knockout animals died more rapidly than controls.

Thus dDOR functions as a novel coactivator of the ecdysone receptor that plays an important role during metamorphosis. Clearly not all EcR functions are impaired in DOR mutants. For instance, very little lethality is seen during larval stages of development, indicating that larval molts are occurring properly. It is possible that different EcR coactivators are important for different aspects of EcR signaling, for instance with rigor mortis plays an important role in the regulation of larval molts. Alternatively, because induction of EcR target genes is reduced but not completely eliminated in dDOR knockout animals, this could reflect the differential sensitivity of various biological processes to the degree of EcR activation. Future work may shed more light on this issue. Interesting in this context is that it was possible to rescue the lethality of DOR knockouts by feeding 20E. This suggests that either DOR knockouts also have low ecdysone titers due to impaired expression of E75A, which is involved in an ecdysone feed-forward production pathway, or because the elevated ecdysone titers achieved by supplying exogenous 20E allow other coactivators to compensate for DOR loss of function (Francis, 2010).

This work identifies a new link between ecdysone signaling and insulin signaling. It was previously known that ecdysone signaling inhibits insulin signaling in the fat body. This study shows, conversely, that insulin signaling also inhibits ecdysone signaling. When insulin signaling is high, FOXO activation is low and dDOR expression is low. Conversely, when insulin signaling drops, this allows FOXO to become active, resulting in elevated levels of dDOR expression and maximal activation of EcR target genes. In sum, this study found that there is a mutual antagonistic relationship between insulin signaling and ecdysone signaling in the fat body, possibly creating a system with two equilibrium states -- high ecdysone/low insulin and low ecdysone/high insulin. This makes biological sense because insulin plays an anabolic role in the fat body, whereas ecdysone plays a catabolic role, encouraging lipid mobilization and autophagy. By identifying dDOR as a direct FOXO target, this study has shed light on the molecular mechanism by which part of this antagonistic relationship is achieved (Francis, 2010).

A second consequence of the regulation of dDOR by FOXO is the creation of a feed-forward regulatory mechanism. When ecdysone signaling is activated, it inhibits insulin signaling and activates FOXO, causing increased expression of dDOR. This results in potentiation of the ecdysone signal. This type of mechanism may be important for the dramatic activation of the ecdysone pathway at the end of larval development. Indeed, ecdysone signaling has several autoregulatory positive feedback loops, including EcR-dependent transcription of the EcR gene and downregulation of a microRNA, miR-14, which inhibits EcR expression (Francis, 2010).

DOR was first identified as a gene whose expression is aberrant in Zucker diabetic rats. Until DOR knockout mice are analyzed, it is possible that this aberrant regulation is either a cause or a consequence of the diabetes. Because dDOR knockout flies have reduced triglyceride and elevated glycogen stores, it is tempting to speculate that aberrant DOR expression in mammals might actually cause metabolic defects and not simply be a consequence of them. Although DOR expression was downregulated in muscle of diabetic rats, this study found a 2-fold increase in hDOR expression in adipose tissue of type 2 diabetic patients. This indicates that regulation of DOR expression -- and hence the effect on metabolism -- in conditions of metabolic disease in mammals is likely to be tissue specific and complex. The reduction in triglycerides in dDOR knockout flies is also interesting in light of the antagonistic relationship between ecdysone signaling and insulin signaling in the fly. Previous work has shown that flies with systemically reduced insulin signaling have elevated triglyceride levels. Therefore, the leanness of dDOR knockouts would be consistent with increased systemic insulin signaling in dDOR knockout animals (Francis, 2010).

Intriguingly, dDOR shares a number of features with its mammalian homolog. Like hDOR, dDOR functions as a nuclear hormone coactivator. Whereas hDOR binds TRα1, dDOR binds EcR. TRα1 and EcR are similar in that they both form heterodimeric complexes with RXR/USP. In fact, EcR can form a functional complex with the human USP homolog RXR in mammalian cells. Furthermore, EcR and TRα1 both play catabolic roles in some contexts. For instance, ecdysone signaling induces autophagy and lipid mobilization in the fat body and programmed cell death in salivary glands during metamorphosis. Likewise, thyroid hormones increase basal metabolic rates, induce fat mobilization, and enhance fatty acid oxidation. A second similarity between dDOR and hDOR is that both are transcriptionally regulated by nutritional inputs. DOR expression is misregulated in diabetic rats, whereas dDOR expression changes depending on whether the animals are feeding or fasting. Because this study found that regulation of dDOR expression is insulin and FOXO dependent, this raises the possibility that the transcriptional effect on DOR in diabetic rats may also be insulin dependent. A third similarity is that both hDOR and dDOR have two separable functions -- as a nuclear hormone receptor coactivator, and as a regulator of autophagy. This makes particular biological sense within the context of the fat body, where ecdysone signaling induces autophagy during metamorphosis. Therefore, the dual functions of dDOR work in parallel, both by potentiating ecdysone signaling and by interacting with the autophagy proteins Atg8a/b (Francis, 2010).

In sum, this work discovers dDOR as a novel EcR coactivator required during fly metamorphosis. Furthermore, it identifies dDOR as a novel component of a gene regulatory network integrating ecdysone and insulin signaling to regulate fly development and metabolism (Francis, 2010).

Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signalling

FoxO transcription factors, inhibited by insulin/insulin-like growth factor signalling (IIS), are crucial players in numerous organismal processes including lifespan. Using genomic tools, this study uncovered 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. The part of the IIS transcriptional response directly controlled by dFOXO and the indirect effects were identified in this study, and it was 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. Genome-wide evolutionary conservation of dFOXO targets was identified between the fly and the worm Caenorhabditis elegans, enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor (Alic, 2011).

Using ChIP-chip this study has defined >1400 genomic locations occupied by dFOXO in the adult fly. Interestingly, these locations are distinct from those observed by others in larvae. It is possible that the differences between the adult data and the published larval data stem from differences in protocols (e.g. the antibody used) or even experimental design (e.g. sex of the flies used). Importantly, however, this study showed that the observed differences between S2 cells and adults, in the case of the promoter (P1) and the coding region of the Drosophila InR, represent true biological differences. It is not surprising that dFOXO would occupy different locations during development and in the adult fly. A similar observation has been made for a number of transcriptional events, and even the dInR gene alone is transcribed from three promoters under tight spatio-temporal control. Furthermore, some differences will stem from cell- and tissue-specificity of dFOXO action. Indeed, FoxO factors are known to elicit tissue-specific transcriptional changes in the mouse, and the same tissue-restricted action by dFOXO on the transcription of the myc gene has been observed in Drosophila larvae. By binding to different locations in a spatially and temporally determined manner, dFOXO would be able to orchestrate different responses to suit its function in different life stages and tissues. Interestingly, a substantial portion of dFOXO bound in transcribed regions. In yeast, forkhead factors regulate Pol II elongation, and dFOXO may perform a similar function (Alic, 2011).

dFOXO was observed to be bound to a number of genes encoding IIS signalling components. Furthermore, dfoxo may also exert feedback onto other pathways that regulate it: dFOXO was bound near the genes encoding PP2A-B′, 14-3-3ε and JNKKKs (slpr and TAK1), among others. PP2A, 14-3-3ε and JNK have all been shown to regulate FoxO activity. A number of these dFOXO-activated genes is also activated on over-expression of superoxide dismutase, suggesting that dFOXO, like its mammalian counterparts, may be redox regulated. Interestingly, binding was detected to only the intracellular components of IIS such as chico, Lnk and Akt, while the genes with altered expression level in dfoxo-/- include extracellular cell-to-cell signalling molecules, such as those encoded by dilp3, dilp6 and Imp-L2. The latter genes have a more localised expression pattern, for example dilp3 is expressed in only ~14 cells in the whole adult fly. It is possible that genes such as dilp3 are also bound and directly regulated by dFOXO but that this was not observed in the whole fly ChIP-chip due to a very small number of cells in which this binding occurs (Alic, 2011).

4E-BP (a.k.a. Thor) has been shown to be bound by dFOXO in larvae, and its regulation has been reported as consistent with dFOXO acting as a direct activator of its expression. On the other hand, dFOXO binding was not observed in the vicinity of this gene in adults, and the 4E-BP transcript is actually elevated in a dfoxo null. It is possible that dFOXO is required for direct activation of this gene in only a limited number of cells/tissues in the adult, thus escaping detection by ChIP-chip on whole animals. Furthermore, the role of dFOXO in 4E-BP regulation may be sexually dimorphic. Alternatively, 4E-BP might be a target of a different forkhead factor in the adult female fly. Indeed, Forkhead (Fkh, the fly FoxA orthologue) is able to activate transcription of 4E-BP in larvae. Since dfoxo nulls have reduced levels of TOR, and TOR is an inhibitor of Fkh activity, it is likely that Fkh is activated in dfoxo nulls leading to increased levels of the 4E-BP transcript. It remains to be established whether Fkh might indeed be directly binding to the 4E-BP locus in adult flies (Alic, 2011).

From the 1400 dFOXO-bound locations, using transcriptional profiling of dfoxo null flies under normal conditions or with reduced IIS, >700 direct transcriptional targets of dFOXO were identified in the adult. Several functions associated with these genes have been linked with FoxO biology previously, such as cell cycle, cytoskeleton organisation, negative regulation of gene expression such as translation and regulation of protein catabolism. dFOXO is known to be involved in the repression of protein synthetic machinery via myc in larvae but this study also revealed a significant regulation of ribosome biogenesis genes effected directly by dFOXO in the adult female. Other, previously unknown functions were identified, such as control of negative regulators of transcription and chromatin modifiers, hinting at the importance of dFOXO in establishment and maintenance of repressive chromatin states. Yet other functions were completely unexpected. For example, dFOXO appears as a positive regulator of sexual reproduction, including oogenesis, in an IIS mutant. This surprising finding is backed up by phenotypic epistasis analysis that shows removal of dfoxo to exacerbate the fecundity defect of several IIS mutants. Hence, dFOXO actually positively regulates some aspects of IIS. Indeed, one of the most surprising findings of this study is that dFOXO is directly required for expression of several components of IIS and interacting pathways, including TOR and Sos, in the wild-type fly, with consequences for the downstream signalling events. Importantly, this is not just simple feedback in response to alteration in the levels of insulin/IGF-like signal, but rather dFOXO is active in the normal adult and its activity promotes signalling through the IIS pathway. This observation can also explain why dfoxo deletion is lethal in combination with certain IIS mutants, since the combined reduction in IIS will be too great for the flies to survive. This potentiation of IIS by FoxOs could also explain why mice with reduced IIS through mutation of IRS1 have mild insulin resistance but preserved old-age glucose homoeostasis. In this case, the mild insulin resistance would be the primary effect of the mutation of IRS1, while the resulting activation of FoxOs would be responsible for sustained IIS in old age and thus for the observed preservation of glucose homoeostasis (Alic, 2011).

dFOXO directly regulates an extensive second tier of regulators; throughout this study different transcriptional and post-transcriptional regulators were repeatedly encountered as predominant dFOXO targets. This aspect of dFOXO biology is also conserved in the worm. Indeed, some of the potential secondary effectors are directly conserved between the worm and the fly, such as the nuclear hormone receptor dHR96/daf-12, highlighting their importance. This study also illustrates the role this second tier of regulators may play. dFOXO is directly required for the maintenance of GATAd mRNA levels in both the wild-type and IIS-compromised flies, and this in effect may constitute an IIS feed-forward loop, since GATAd in turn may be an important transcriptional repressor in response to reduced IIS. Hopefully, subsequent studies will demonstrate the existence of such a feed-forward loop (Alic, 2011).

Since daf-16 is strictly required for all phenotypic outputs of reduced IIS in the worm, and also appears strictly required for the transcriptional response to reduced IIS, it was very surprising to find that dFOXO was only required for part of the transcriptional response to reduced IIS in the fly. On the other hand, this is in accordance with phenotypic epistasis experiments in the fly where lifespan extension and xenobiotic resistance are dependent on dfoxo, while lowered fecundity and body size, delayed development and resistance to paraquat are not. This implies that phenotypes such as fecundity are negatively regulated via other factors in the fly. This study indicates that GATA factors are the most likely candidates for mediating transcriptional repression in response to reduced IIS. Studies in the worm have also revealed the presence of a GATA-recognition sequence in the promoters of IIS-regulated genes. Furthermore, at least one of the 14 worm GATA TF (elt-3) is regulated by IIS, and reduced function in any of the three GATA TFs (elt-3, egr-1, egl-27) blocks the lifespan extension by a daf-2 mutant. The role of GATA factors in lifespan in other organisms awaits examination. At the same time, this study reveals the potential involvement of other forkhead factors, besides dFOXO, in the transcriptional activation response to IIS reduction. Fkh is the prime suspect, since it is regulated by TOR signalling in the fly, and Foxa2 is involved in the IIS response in mammals. Indeed, Foxa2 is directly inactivated by Akt via phosphorylation of a single site that is conserved in the fly Fkh. While this study provides hints, further work will be needed to determine the identity of other TFs involved in the fly IIS response (Alic, 2011).

This study reveals that the transcriptional response to IIS in the fly is clearly more complex than that in the worm. The parallel genetic study performed by (Slack (2011) shows that the genes directly regulated by dFOXO must still effect the lifespan extension by reduction in IIS. Importantly, this study has now identified these genes. Their characterisation is the next step towards understanding the physiological and molecular changes that can extend animal lifespan, keeping in mind that it is now crucial to determine the architecture of the mammalian response to reduced IIS (Alic, 2011).

Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster

Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. This study shows that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis; the mechanism for this protection was dissected. Upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, PDGF- and VEGF-related factor 1 (Pvf1), a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer (Xing, 2015).

A form of programmed cell death, apoptosis, is characterized as controlled, caspase-induced degradation of cellular compartments to terminate the activity of the cell. Apoptosis plays a vital role in various processes including normal cell turnover, proper development and function of the immune system and embryonic development. Apoptosis is also induced by upstream signals, such as DNA double-strand breaks (DSB), to destruct severely damaged cells. DSB activate ATM checkpoint kinase and Chk2 kinase-dependent p53 phosphorylation and induction of repair genes. However, if DSB are irreparable, p53 activation will result in pro-apoptotic gene expression and cell death. However, aggressive cancers contain cells that show inability to undergo apoptosis in response to stimuli that trigger apoptosis in sensitive cells. This feature is responsible for the resistance to anticancer therapies, as well as the relapse of tumours after treatment, yet the molecular mechanism of this resistance is poorly understood (Xing, 2015).

As the cell type that constantly regenerates and gives rise to differentiated cell types in a tissue, stem cells share high similarities with cancer stem cells, including unlimited regenerative capacity and resistance to genotoxic agents. Adult stem cells in model organisms such as Drosophila melanogaster, have been utilized to study stem cell biology and for conducting drug screens, thanks to their intrinsic niche, which provides authentic in vivo microenvironment. This study shows that Drosophila adult stem cells are resistant to radiation/chemical-induced apoptosis, and the mechanism for this protection was dissected. A previously reported cell survival gene with a human homologue, pineapple eye (pie) , acts in both stem cells and in differentiating cells to repress the transcription factor FOXO. Elevated FOXO levels in pie mutants lead to apoptosis in differentiating cells, but not in stem cells, indicating the presence of an additional anti-apoptotic mechanism(s) in the latter. We show that this mechanism requires Tie, encoding a homologue of human receptor tyrosine kinase Tie-2, and its target, bantam, encoding a microRNA. The downstream effector of FOXO, Tie and ban, is show to be Hid, encoding a Smac/DIABLO orthologue. Knocking down the ligand Pvf1/PDGF/VEGF/Ang in differentiating daughter cells made stem cells more sensitive to radiation-induced apoptosis, suggesting that Pvf1 from the apoptotic differentiating daughter cells protects stem cells (Xing, 2015).

This study shows that an anti-apoptotic gene, pie, is required for stem cell self-renewal but not for resistance to apoptosis, indicating a compensatory anti-apoptotic mechanism in stem cells. The cell cycle marker profile of pie GSCs resembles that of InR deficient GSCs, leading to the finding that pie controls GSC, as well as ISC self-renewal/division through FOXO protein levels. Surprisingly, pie targets FOXO as well in differentiating cells, failing to explain why the loss of pie does not induce apoptosis in stem cells. However, while the upregulation of FOXO leads to the upregulation of its apoptotic target Hid in differentiating cells, in adult stem cells Hid is not upregulated. Hence additional regulatory pathway is in place to repress Hid and thereby apoptosis in stem cells. This study identified Tie-receptor as the key gatekeeper for the process in the GSCs. The signal (Pvf1) from the dying daughter cells activates Tie in GSCs to upregulate bantam microRNA that represses Hid, thereby protecting the stem cells. Bantam is known to repress apoptosis and activate the cell cycle. However, while protected from apoptosis in this manner, the stem cells do not activate the cell cycle but rather stay in protective quiescence through FOXO activity. When the challenge is passed, stem cells repopulate the tissue (Xing, 2015).

The mammalian pie homologue, G2E3 was reported to be an ubiquitin ligase with amino terminal catalytic PHD/RING domains. G2E3 is essential for early embryonic development (Brooks, 2008). Importantly, microarray data show significant enrichment of G2E3 expression levels in human embryonic stem (ES) cell lines. These observations suggest a critical role of G2E3 in embryonic development, potentially in maintaining the pluripotent capacity. Since FOXO is shown to be an important ESC regulator, it will be interesting to test whether defects in G2E3 result in changes in FOXO levels. Furthermore, future studies are required to test whether human ES cells also are protected from apoptosis due to external signals from dying neighbouring cells (Xing, 2015).

The cell cycle defects of pie mutant stem cells, such as abnormal cell cycle marker profile, can be a consequence of elevated FOXO levels, since FOXO is a transcription factor with wide array of target genes, many of which are involved with cell cycle progress, such as the cyclin-dependent kinase inhibitor p21/p27 (Dacapo in Drosophila). This may be critical when bantam function is considered in the stem cells. Bantam is known to function as anti-apoptotic and cell cycle inducing microRNA. While in GSC bantam is critical through its anti-apoptotic function as a Hid repressor, it has no capacity to induce GSC cell cycle after irradiation. In a challenging situation, such as irradiation, an additional protection mechanism for the tissue is to keep the stem cell in a quiescent state during challenge. bantam's pro-cell division activity may be dampened by FOXO's capacity to upregulate p21/Dacapo (Xing, 2015).

The FOXO family is involved in diverse cellular processes such as tumor suppression, stress response and metabolism. The FOXO group of human Forkhead proteins contains four members: FOXO1, FOXO3a, FOXO4, and FOXO6. Studies to elucidate their function in various stem cell types in vivo using knockout mice have shown some potential redundancy of FOXO proteins. Recent publications have demonstrated a requirement for some of the FOXO family members in mouse hematopoietic stem cell proliferation, mouse neural stem cells, leukaemia stem cells and human and mouse ES cells in vitro. However, FOXO is shown to be dispensable in the early embryonic development in mouse. Drosophila genome has only one FOXO, allowing a definitive study of FOXO's function in stem cells. This study now demonstrates that tight regulation of FOXO protein levels is essential for in vivo GSC and ISC self-renewal in Drosophila. While the loss of FOXO function generates supernumerary stem cells, inappropriately high level of FOXO results in stem cell loss. Under challenge, such as exposure to irradiation, stem cells depleted of FOXO fail to stay quiescent and become more sensitive to the damage, leading to the loss of GSC population. These data demonstrate the importance of the balanced FOXO expression level for stem cell fate (Xing, 2015).

Previous studies have shown that multiple adult stem cell types manage to avoid cell death in response to severe DNA damage. This work has studied the mechanisms that stem cells utilize to avoid apoptosis in absence of pie and revealed that apoptosis is protected through a receptor, Tie and its target miRNA bantam that can repress the pro-apoptotic gene Hid. The ligand for Tie is likely secreted from the dying neighbours since Tie is essential in GSC only after irradiation challenge, IR induces Tie's potential ligand Pvf1 expression in cystoblasts and knockdown of Pvf1 in cystoblasts eliminates stem cells' protection against apoptosis. Further studies will reveal whether the same protective pathway is utilized in other stem cells. Community phenomenon have been described previously around dying cells: compensatory proliferation, Phoenix rising, bystander effect and Mahakali. While Bystander effect describes dying cells inducing death in the neighbours, compensatory proliferation, Phoenix rising and Mahakali describe positive effects in cells neighbouring the dying cells. The present work shows that adult stem cell can survive but show no immediate induction of proliferation when neighboured by dying cells. However, since adult stem cells can repopulate the tissue when death signals have passed, it is proposed that in adult stem cells these phenomenon merge. First, the GSCs survive by bantam repressing the apoptotic inducer, Hid, and later repopulate the tissue by activating cell cycle. Recent findings have suggested that p53 might play an important role in re-entry to cell cycle in stem cells51. The results from the current studies shed light on the general understanding of stem cell behaviour in response to surrounding tissue to ensure the normal tissue homeostasis. It is also plausible that cancer stem cells hijack these normal capacities of stem cells (Xing, 2015).

APLP1 promotes dFoxO-dependent cell death in Drosophila.

The amyloid precursor like protein-1 (APLP1), an engineered human gene introduced into the Drosophila genome, belongs to the amyloid precursor protein family that also includes the Drosophila amyloid precursor protein (APP) and the amyloid precursor like protein-2 (APLP2). Though the three proteins share similar structures and undergo the same cleavage processing by α-, β- and γ-secretases, APLP1 shows divergent subcellular localization from that of APP and APLP2, and thus, may perform distinct roles in vivo. While extensive studies have been focused on APP, which is implicated in the pathogenesis of Alzheimer’s disease (AD), the functions of APLP1 remain largely elusive. This study reports that the expression of APLP1 in Drosophila induces cell death and produces developmental defects in wing and thorax. This function of APLP1 depends on the transcription factor dFoxO, as the depletion of dFoxO abrogates APLP1-induced cell death and adult defects. Consistently, APLP1 up-regulates the transcription of dFoxO target hid and reaper-two well known pro-apoptotic genes. Thus, the present study provides the first in vivo evidence that APLP1 is able to induce cell death, and that FoxO is a crucial downstream mediator of APLP1’s activity (Wang, 2015).

Amyloid pecursor like protein-1 (APLP1) is a mammalian paralog of amyloid precursor protein (APP). While APP has been extensively studied for its involvement in the Alzheimer’s disease, few studies have been directed to APLP1 and its in vivo functions remain largely unknown. This study investigated the in vivo functions of APLP1 using Drosophila as a model organism. It was found that ectopic expression of APLP1 induces cell death and developmental defects in the nervous and non-nervous system. Genetic studies characterize the transcription factor dFoxO as a critical downstream factor that mediates APLP1’s activity, for the depletion of dFoxO significantly suppresses APLP1-induced cell death in larval discs and associated phenotypes in adults. Further, it was shown that APLP1 is able to up-regulate the transcription of dFoxO target genes hid and reaper (Wang, 2015).

APLP1 has been reported to function mainly in the nervous system, as high expression level of APLP1 is detected in the developing central and peripheral nervous systems, yet a weak expression signal of APLP1 is also observed in organs like heart, lung, liver and kidney in mouse embryos, implying a role of APLP1 in the development of non-neuronal tissues. Consistent with this explanation, RNAi mediated knockdown of APLP1 in WI-38 and MCF7 cells dramatically reduced the proliferation of these cells. This study showed that expression of APLP1 could induce cell death and developmental defects in both neuronal and non-neuronal systems in Drosophila, and thus, provides further evidence for the function of APLP1 in non-neuronal cells (Wang, 2015).

Earlier studies have also shown that loss of APLP1 diminishes stress induced apoptosis in neuroblastoma cells, whereas ectopic expression of APLP1 moderately enhances cell death upon stress stimulation. However, expression of APLP1 alone is not sufficient to induce neuroblastoma cell death, suggesting APLP1 induces cell death in a context dependent manner. Data from this study demonstrates that APLP1 by itself is sufficient to induce cell death. APLP1 has been reported to be a direct transcriptional target of the p53 tumor suppressor, which suggests a possible involvement of APLP1 in p53-induced cell death. p53 is known to interact with the transcriptional factor FoxO, and MDM2 is known to act downstream of p53 to promote FoxO ubiquitination and degradation. In the present study, it was shown that FoxO mediates APLP1-induced cell death. The exact relationship between APLP1, FoxO and p53 in cell death will require further investigation. Overall, this study highlights a novel function of APLP1 in promoting FoxO-mediated cell death in vivo, which will shed light on the role of APLP1 in mammalian cells (Wang, 2015).

Protein Interactions

Melted modulates Foxo and Tor activity

The insulin/PI3K signaling pathway controls both tissue growth and metabolism. Melted has been identified as a new modulator of this pathway in Drosophila. Melted interacts with both Tsc1 and Foxo and can recruit these proteins to the cell membrane. Evidence is provided that in the melted mutant, Tor activity is reduced and Foxo is activated. The melted mutant condition mimics the effects of nutrient deprivation in a normal animal, producing an animal with 40% less fat than normal (Teleman, 2005).

As a means to identify possible functions of Melted, the Eukaryotic Linear Motif server) was used to look for functional motifs conserved between fly and human Melted. The only conserved motifs found in the N-terminal region of these proteins were two Forkhead-associated domain ligand domains (LIG_FHA_1). Forkhead transcription factors FoxA2, FoxA3, FoxC2, and FoxO1 are involved in glucose and fat metabolism. Insulin signaling activates Akt, which phosphorylates Foxo and leads to its retention in the cytoplasm. It was therefore asked if Melted affects the subcellular localization of a Foxo-GFP fusion protein. Foxo-GFP is predominantly nuclear in the absence of insulin stimulation in serum-starved S2 cells and increases in the cytoplasm after insulin stimulation. In serum-starved cells cotransfected to express Melted, Foxo-GFP is still primarily nuclear, but much of the nonnuclear protein appears at the membrane colocalized with Melted. Upon insulin stimulation, a robust increase in the level of Foxo-GFP was observed at the cell membrane. The interaction was confirmed by coimmunoprecipitation of Melted with Foxo in insulin-stimulated S2 cells (Teleman, 2005).

The observation that insulin stimulation induces a shift toward membrane localization of Foxo in the presence of Melted in S2 cells raised the possibility that melted regulates Foxo activity in vivo. To address this, expression of the Foxo target 4E-BP was examined in wild-type and melted mutant animals. Under fed conditions, insulin signaling is active and 4E-BP transcript levels are relatively low. In wild-type flies that were starved for 24 hr to reduce insulin levels and thereby activate Foxo, 4E-BP transcript increased ~4-fold. In starved flies lacking Melted, 4E-BP transcript increased over 25-fold. This increase in 4EBP transcription was absent in the starved melted/Foxo double mutant, confirming that it is Foxo dependent. Thus, in the absence of Melted, Foxo activity is higher than normal, suggesting that Melted limits Foxo activity in vivo (Teleman, 2005).

To determine whether the elevated Foxo activity observed in melted mutants contributes to the lean phenotype of these animals, the normalized triglyceride levels of melted mutant and melted foxo double-mutant flies were compared. Reducing Foxo activity suppresses the leanness of the melted mutant to a considerable degree, reaching near normal fat levels. The rescue was highly statistically significant. foxo mutants did not show higher-than-normal fat levels compared to wild-type. These observations suggest that Melted acts by regulating Foxo activity to control expression of genes important in fat metabolism (Teleman, 2005).

The Tor pathway integrates information on cellular nutritional status and stress from the heterodimeric Tsc1/2 complex. melted mutants exhibit reduced Tor activity. By recruiting Foxo to the membrane in an insulin-regulated manner Melted influences expression of Foxo targets. By reducing Tor activity and at the same time increasing Foxo activity, the melted mutant mimics the effects of nutrient deprivation in a normal animal, producing a lean phenotype (Teleman, 2005).

To determine whether Tor activity affects fat accumulation, the effects were tested of increasing Tor activity in wild-type and melted mutant adipose tissue. Use was made of a UAS-Tor transgene that can provide Tor activity in vivo when expressed at appropriate levels. It was confirmed that expression of UAS-Tor under ppl-Gal4 control in adipose tissue leads to increased total body fat, as does increasing PI3K activity. In contrast, a comparable elevation of Tor expression in melted mutant flies has no effect on fat levels. Both this result and the significant rescue caused by removal of Foxo indicate that in the melted mutant, the Foxo branch of the pathway becomes limiting for fat accumulation. In view of this finding, it was next asked whether elevated Tor pathway activity could increase fat levels in the melted mutant if Foxo activity was simultaneously reduced. To do so, use was made of the catalytic subunit of PI3K (Dp110) to inactivate Foxo and simultaneously activate Tor. The fat body driver lsp2-Gal4 or the UAS-Dp110 transgenes have little effect on their own in the melted mutant background, but when combined, the elevated PI3K activity in the fat body increases fat levels of the melted mutant. The effect is stronger than that of removing Foxo only, increasing fat levels to above normal. Taken together, these observations suggest that the Tor branch of the pathway contributes to the control of fat levels under conditions in which Foxo activity levels are low. This is normally the case in feeding animals in which insulin levels are relatively high (Foxo activity is elevated under starvation conditions: as seen by comparing 4E-BP levels in fed versus starved wild-type and foxo mutant flies). Under conditions in which insulin levels are low or in the melted mutant, in which Foxo activity is elevated, the effects of Foxo appear to dominate (Teleman, 2005).

The nitric oxide-cyclic GMP pathway regulates FoxO and alters dopaminergic neuron survival in Drosophila

Activation of the forkhead box transcription factor FoxO is suggested to be involved in dopaminergic (DA) neurodegeneration in a Drosophila model of Parkinson's disease (PD), in which a PD gene product LRRK2 activates FoxO through phosphorylation. In the current study that combines Drosophila genetics and biochemical analysis, it was shown that cyclic guanosine monophosphate (cGMP)-dependent kinase II (cGKII) also phosphorylates FoxO at the same residue as LRRK2, and Drosophila orthologues of cGKII and LRRK2, DG2/For and dLRRK, respectively, enhance the neurotoxic activity of FoxO in an additive manner. Biochemical assays using mammalian cGKII and FoxO1 reveal that cGKII enhances the transcriptional activity of FoxO1 through phosphorylation of the FoxO1 S319 site in the same manner as LRRK2. A Drosophila FoxO mutant resistant to phosphorylation by DG2 and dLRRK (dFoxO S259A corresponding to human FoxO1 S319A) suppressed the neurotoxicity and improved motor dysfunction caused by co-expression of FoxO and DG2. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) also increased FoxO's activity, whereas the administration of a NOS inhibitor L-NAME suppressed the loss of DA neurons in aged flies co-expressing FoxO and DG2. These results strongly suggest that the NO-FoxO axis contributes to DA neurodegeneration in LRRK2-linked PD (Kanao, 2012).

Genetic dissection reveals that Akt is the critical kinase downstream of LRRK2 to phosphorylate and inhibit FOXO1, and promotes neuron survival

Leucine-rich repeat kinase 2 (LRRK2) is a complex kinase and mutations in LRRK2 are perhaps the most common genetic cause of Parkinson's disease (PD). However, the identification of the normal physiological function of LRRK2 remains elusive. This study shows that LRRK2 protects neurons against apoptosis induced by the Drosophila genes grim, hid and reaper. Genetic dissection reveals that Akt is the critical downstream kinase of LRRK2 that phosphorylates and inhibits FOXO1, and thereby promotes survival. Like human LRRK2, Drosophila lrrk also promotes neuron survival; lrrk loss-of-function mutant displays reduced cell numbers, which can be rescued by LRRK2 expression. Importantly, LRRK2 G2019S and LRRK2 R1441C mutants impair the ability of LRRK2 to activate Akt, and result in a failure of preventing apoptotic death. Ectopic expression of a constitutive active form of Akt hence is sufficient to rescue this functional deficit. These data establish that LRRK2 can protect neurons from apoptotic insult through a survival pathway in which LRRK2 signals to activate Akt, and then inhibits FOXO1. These results might indicate that a therapeutic pathway to promote neuron survival and to prevent neurodegeneration in Parkinson's disease (Chuang, 2014).

Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis

Despite their fundamental importance for body size regulation, the mechanisms that stop growth are poorly understood. In Drosophila melanogaster, growth ceases in response to a peak of the molting hormone ecdysone that coincides with a nutrition-dependent checkpoint, critical weight. Previous studies indicate that insulin/insulin-like growth factor signaling (IIS)/Target of Rapamycin (TOR) signaling in the prothoracic glands (PGs) regulates ecdysone biosynthesis and critical weight. This study elucidates a mechanism through which this occurs. This study shows that Forkhead Box class O (FoxO), a negative regulator of IIS/TOR, directly interacts with Ultraspiracle (Usp), part of the ecdysone receptor. While overexpressing FoxO in the PGs delays ecdysone biosynthesis and critical weight, disrupting FoxO-Usp binding reduces these delays. Further, feeding ecdysone to larvae eliminates the effects of critical weight. Thus, nutrition controls ecdysone biosynthesis partially via FoxO-Usp prior to critical weight, ensuring that growth only stops once larvae have achieved a target nutritional status (Koyama, 2014).


DEVELOPMENTAL BIOLOGY

Antagonistic actions of ecdysone and of insulins, acting through Foxo and 4E-BP, determine final size in Drosophila

All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. The larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size (Colombani, 2005).

In metazoans, the insulin/IGF signaling pathway (IIS) plays a key role in regulating growth and metabolism. In Drosophila, a family of insulin-like molecules called Dilps activates a unique insulin receptor (InR) and a conserved downstream kinase cascade that includes PI3-kinase (PI3K) and Akt/PKB. Recent genetic experiments have established that this pathway integrates extrinsic signals such as nutrition with the control of tissue growth during larval stages. The larval period is critical for the control of animal growth, since it establishes the size at which maturation occurs and, consequently, the final adult size. Maturation is itself a complex process that is controlled by the steroid 20-hydroxyecdysone (20E). Peaks of 20E determine the timing of all developmental transitions, from embryo to larva, larva to pupa, and pupa to adult. Ecdysteroids are mainly produced by the prothoracic gland (PG), a part of a composite endocrine tissue called the ring gland. Final adult size thus mainly depends on two parameters: the speed of growth (or growth rate), which is primarily controlled by IIS, and the overall duration of the growth period, which is limited by the onset of the larval-pupal transition and timed by peaks of ecdysone secretion. Very little is known concerning the mechanisms that coordinate these two parameters during larval development (Colombani, 2005).

To investigate the function of ecdysone in controlling organismal growth, a genetic approach was developed that allowed modulation basal levels of ecdysteroids in Drosophila. The initial rationale was to modify the mass of the ring gland in order to change the level of ecdysteroid production. For this goal, the levels of PI3- kinase activity were manipulated in the PG by crossing P0206-Gal4 (P0206>), a line with specific Gal4 expression in the PG and corpora allata (CA), with flies carrying UAS constructs allowing expression of either wild-type (PI3K) or dominant-negative (PI3KDN) PI3-kinase. As expected, these crosses produced dramatic autonomous growth effects in the ring gland, and particularly in the PG: tissue size was increased upon PI3K activation and decreased upon inhibition. Surprisingly, the changes in ring gland growth were accompanied by opposite effects at the organismal level. P0206>PI3K animals (with large ring glands) showed reduced growth at all stages of development and produced emerging adults with reduced size and body weight (78% of wt). Conversely, reducing PI3K activity in the ring gland of P0206>PI3KDN animals led to increased growth and produced adults with 17% greater weight on average. Adult size increase was attributable to an increase in cell number in the wing and the eye. Adult size reduction was accompanied by a decrease in cell number in the wing and in cell size in the eye (Colombani, 2005).

Importantly, the timing of embryonic and larval development of these animals was comparable to control. Both the L2 to L3 transition as well as the cessation of feeding (wandering) occurred at identical times. Further, animals entered pupal development at the same time, except for P0206>PI3KDN animals, which showed a 1-2 hrs delay intrinsic to the UAS-PI3KDN line itself. The duration of pupal development was slightly modified, however, as adult emergence was delayed in P0206>PI3K animals and advanced in P0206>PI3KDN animals, albeit by less than 4 hours following 10 days of development. In contrast, the speed of larval growth was found to be increased in P0206>PI3KDN animals and decreased in the P0206>PI3K animals background at the earliest stage that could be measured (early L2 instar). Because none of these effects were observed when PI3K levels were modified specifically in the CA using the Aug21- Gal4 driver, it was concluded that the observed phenotypes are solely due to PI3K modulation in the PG. Together, these results demonstrate that manipulating PI3K levels in the PG induces non-autonomous changes in the speed of larval growth (growth rate effects), without changing the timing of larval development (Colombani, 2005).

To investigate whether these effects could be attributed to changes in 20E levels, ecdysteroid titers were measured in third instar larvae of the different genotypes. Early after ecdysis into third instar (74hrs AED) ecdysteroids are present at basal level. They accumulate to an intermediate plateau around 90hrs AED and reach peak levels before pupariation (120hrs AED). Because early L3 levels are below the detection limit of the EIA assay, ecdysteroid titers were measured at the intermediate plateau (90hrs AED). In these conditions, a very modest increase of ecdysteroids was observed in P0206>PI3K animals larvae and a small but significant decrease in P0206>PI3KDN animals animals. This was further confirmed by measuring the transcript levels of a direct target of 20E, E74B, which responds to low/moderate levels of 20E. However, in early L3 larvae with basal ecdysteroid levels (74hrs AED), differences in E74B transcripts were clearly visible, with a 1.9-fold increase seen for P0206>PI3K animals and a 1.7-fold decrease for P0206>PI3KDN animals. This establishes that basal circulating levels of 20E are modified in response to manipulation of PI3K levels in the PG. It also suggests that the differences observed on basal 20E level off with the strong global increase of ecdysteroids in mid/late L3 (Colombani, 2005).

Several related lines of evidence strengthen these results: (1) the increase in growth rate and size observed in P0206>PI3KDN animals can be efficiently reverted by adding 20E to their food; (2) feeding wild-type larvae 20E recapitulates the effects observed in P0206>PI3K animals animals; (3) ubiquitous silencing of EcR using an inducible EcR RNAi construct results in a growth increase similar to that observed in P0206>PI3KDN larvae. Finally, the phantom (phm) and disembodied (dib) genes, which are specifically expressed in the PG and encode hydroxylases required for ecdysteroid biosynthesis, showed 1.65- and 2.2- fold increased expression, respectively, upon PI3K activation in the ring gland. This supports the notion that 20E biosynthesis is mildly induced in these experimental conditions. In line with previous results, neither 20E treatment nor EcR silencing has any effect on developmental timing. Overall, the results indicate that manipulating basal levels of 20E signaling in various ways modifies the larval growth rate without affecting the timing of the larval transitions (Colombani, 2005).

Variations in ecdysone levels in animals with different sized ring glands could be due to changes in the PG tissue mass or, alternatively, to a specific effect of PI3K signaling in the secreting tissue. To distinguish between these two possibilities, extra growth was induced in the PG using either dMyc or CyclinD/Cdk4, two potent growth inducers in Drosophila. Although the size of the larval ring gland was markedly increased under these conditions, no effect on pupal or adult size was observed, implying that the size of the ring gland is not the critical factor in the control of body size. Instead, it is likely that the InR/PI3K signaling pathway can specifically activate ecdysone production from the PG (Colombani, 2005).

The possibility was tested that ecdysone signaling opposes the growth-promoting effects of IIS in the larva. To test this, larvae were fed 20E and xPI3K activity was assessed in vivo using a GFP-PH fusion (tGPH) as a marker. Membrane tGPH localization shows a marked decrease in the fat body of 20E-fed animals, and this effect can be reverted by specifically silencing EcR in the fat body. This indicates that ecdysone-induced growth inhibition correlates with decreased IIS, and is mediated through the nuclear receptor EcR. Conversely, larvae with PI3KDN expression in the PG show a 4.2-fold increase in the global levels of dPKB/Akt activity, as measured by the phosphorylation levels of serine 505. In Drosophila cells (as in other metazoan cells) high levels of PI3K/AKT activity cause the transcription factor dFOXO to be retained in the cytoplasm, while low PI3K/AKT activity allows dFOXO to enter the nucleus where it promotes 4E-BP transcription. In larvae with ectopic PI3K expression in the PG, a strong increase is observed in nuclear dFOXO in fat body cells. Similar results were obtained by feeding larvae with 20E. Conversely, inactivation of EcR signaling in fat body cells was carried out using the clonal over-expression of a dominant-negative form of EcR (EcRF645A). In these conditions, a reduction was observed in the accumulation of dFOXO in the nuclei of EcRF645A-expressing cells. As an expected consequence of the increased nuclear dFOXO, global accumulation of 4E-BP transcripts was consistently higher in P0206>PI3K animals as well as in 20E-fed early L3 larvae as compared to control animals, and reduced in arm>EcR-RNAi animals. Together, these results indicate that ecdysone-dependent inhibition of larval growth correlates with a general alteration of insulin/IGF signaling, and a relocalization of dFOXO into the cell nuclei. To more directly test the role of dFOXO in the growth-inhibitory function of ecdysone signaling, the effects of increasing ecdysone levels were examined in a dFOXOmutant genetic background. Although homozygous dFOXO21 animals do not display a detectable growth phenotype, introducing the dFOXO21 mutation was sufficient to totally revert the growth defects of P0206>PI3K animals animals, either when homozygous or heterozygous. This data establishes that dFOXO is required for 20E-mediated growth inhibition (Colombani, 2005).

The endocrine activities of the brain and the fat body have previously been implicated in the humoral control of larval growth. In order to test for possible roles of these two organs in mediating the systemic growth effects of ecdysone, EcR expression was silenced specifically in the brain cells that produce insulins (IPCs) or in the fat body. While specific expression of EcR RNAi in the IPCs fails to reproduce the overgrowth observed in armGal4>EcR-RNAi animals, EcR silencing in the fat body elicits an acceleration of larval growth and a remarkable increase in pupal size. Moreover, no detectable delay in the larval timing was observed in pplGal4>EcR-RNAi animals. Thus, specifically reducing 20E signaling in the fat body is sufficient to recapitulate the systemic effects of global EcR silencing. This demonstrates that the fat body is a major target for ecdysone, and that this tissue can act to relay the 20E growth-inhibitory signal to all larval tissues (Colombani, 2005).

In summary, these results establish an additional role for 20E in modulating animal growth rates. This function is mediated by an antagonistic interaction with IIS that ultimately targets dFOXO function. A similar antagonistic interaction between 20E and insulin signaling controls developmentally-regulated autophagy in Drosophila larva (Colombani, 2005).

Although a direct effect of ecdysone on the cellular growth rate of all larval tissues cannot be ruled out, the experiments reveal a key role for the fat body in relaying ecdysone-dependent growth control signals. Together with previous work, these data suggest that various inputs such as nutrition and ecdysone converge on this important regulatory organ, which then controls the general IIS to modulate organismal growth (Colombani, 2005).

How then is growth connected to developmental timing? The finding that 20E can modulate growth rates in addition to developmental transitions places this hormone in a central position for coordinating these two key processes and controlling organismal size (Colombani, 2005).

JNK, targeting Foxo, extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling

Aging of a eukaryotic organism is affected by its nutrition state and by its ability to prevent or repair oxidative damage. Consequently, signal transduction systems that control metabolism and oxidative stress responses influence life span. When nutrients are abundant, the insulin/IGF signaling pathway promotes growth and energy storage but shortens life span. The transcription factor Foxo, which is inhibited by insulin/IGF signaling, extends life span in conditions of low insulin/IGF signaling activity. Life span can also be increased by activating the stress-responsive Jun-N-terminal kinase (JNK) pathway. JNK requires Foxo to extend life span in Drosophila. JNK antagonizes insulin/IGF signaling, causing nuclear localization of Foxo and inducing its targets, including growth control and stress defense genes. JNK and Foxo also restrict insulin/IGF signaling activity systemically by repressing insulin/IGF ligand expression in neuroendocrine cells. The convergence of JNK signaling and IIS on Foxo provides a model to explain the effects of stress and nutrition on longevity (Wang, 2005).

These data suggest Foxo is a convergence point for insulin/IGF signaling (IIS) and JNK signaling. Through its responsiveness to these two pathways, Foxo is well positioned to integrate information about environmental stress and nutrient availability and to elicit appropriate biological responses. Such a system would ensure that growth could proceed in an unrestrained manner when energy resources are available and the cell is not exposed to external insults (IIS is active, JNK is off, and Foxo is repressed). However, in situations of low food availability or an adverse environment, IIS would cease to signal, or JNK would be activated, resulting in translocation of Foxo to the nucleus. The ensuing Foxo-induced gene expression has several effects at the cell as well as the organism level and is likely to counteract premature senescence. The induction of genes such as thor can reduce cell growth, presumably to limit the cell’s anabolic expenses in adverse situations. Other target genes, such as the small heat shock protein l(2)efl, are expected to have a direct role in allaying damage inflicted by environmental insults and may prevent the accumulation of toxic protein aggregates. The suppression of dilp2 expression by JNK and Foxo in insulin-producing cells, in contrast, is likely to control growth, metabolism, and stress responses systemically by downregulating IIS in all responsive tissues in a coordinated fashion (Wang, 2005).

The interaction between JNK and Foxo is thus expected to influence stress tolerance and life span at two levels. In peripheral tissues, JNK activates Foxo and prevents senescence cell-autonomously. Such a mechanism is exemplified by the recent finding that Foxo overexpression prevents age-dependent decline of cardiac performance. Systemic control of IIS by JNK-mediated activation of Foxo in IPCs, in contrast, would serve to coordinate cellular responses to changes in the environment throughout the organism. These data indicate that this latter mechanism plays a significant role in the regulation of life span by JNK and Foxo. The identification of this endocrine function of JNK/Foxo signaling supports and extends the proposed role of JNK signaling on longevity and demonstrates a role for IPCs in life span regulation. In addition to controlling growth and metabolism, IPCs may thus act as a coordination point for the organism’s stress response by downregulating Dilp production in response to oxidative stress and JNK activation. In target tissues, such a mechanism would induce protective gene expression by the second, cell-autonomous tier of Foxo signaling. Interestingly, the effects of IPC-specific JNK activation on longevity and growth are separable. Life span can be extended by moderately increased JNK activity in IPCs when growth effects are yet not evident. This finding is consistent with observations by others who showed that the extension of life span in IIS loss-of-function situations is not a mere consequence of small body size (Wang, 2005).

How did such a multilayered regulation of IIS activity by JNK evolve? It is tempting to speculate that localized activation of Foxo is required to prevent cellular damage and ultimately senescence in conditions in which stressful insults are confined to specific tissues. Such localized insults could, for example, be inflicted by reactive oxygen species that are produced in the environment of amyloid deposits in Alzheimer’s disease as well as by mechanical and oxidative stress experienced by particularly active tissues such as the heart. Systemic regulation of Foxo activity, in contrast, is expected to be an important response mechanism to coordinate metabolism and stress defenses throughout the organism upon changes in the environment. A good example for such a mechanism is the induction of diapause in invertebrates in response to environmental stress or food deprivation. Accordingly, sensory neurons expressing the insulin-like peptide DAF-28 are required for the induction of the dauer larval stage in response to environmental cues in C. elegans (Wang, 2005).

Systemic and tissue-autonomous effects of JNK/Foxo signaling may be connected in multiple ways. The data indicate that JNK and Foxo interact in IPCs to repress dilp2 expression, ultimately activating Foxo in Dilp2 target tissues in a coordinated fashion. Since JNK is be activated in IPCs even under normal culture conditions, it is likely that this systemic control of IIS activity by JNK and Foxo plays a critical role in life span regulation. It is, however, also possible that the cell-autonomous protective function of JNK/Foxo signaling is most critical for the survival of specific tissues as the organism ages, thus extending life of the organism by preventing the loss of indispensable cells or tissues. In addition, stress and the JNK-mediated activation of Foxo in peripheral tissues may signal back to IPCs to initiate a systemic response. In Drosophila, such a mechanism has been documented in the case of the fatbody. Activation of Foxo in this tissue relays a signal to the IPCs, causing them to curb Dilp2 production, a process that has been proposed to require Foxo activity. The exact nature of this feedback signaling mechanism in flies is unclear, but it is reminiscent of the complex signaling interactions between β cells and insulin target tissues in mammals. Further studies are required to shed light on the relative contributions of JNK/Foxo signaling in IPCs or Dilp target tissues to life span regulation (Wang, 2005).

JNK-mediated modulation of IIS activity is likely to be evolutionarily conserved. Inhibitory crosstalk from JNK to IIS in mammalian cells has been found to occur by JNK-mediated phosphorylation and inhibition of IRS-1. This interaction is responsible for obesity-induced insulin resistance in mice. Whether mammalian homologs of Foxo take part in this pathology remains to be determined. A second possible mechanism for JNK/IIS pathway interaction is the direct phosphorylation and activation of Foxo by JNK. Such a mechanism is supported by the observation that in mouse cells JNK can phosphorylate the DFoxo homolog Foxo4 in response to oxidative stress. The physiological relevance of this phosphorylation event has not yet been addressed. The JNK target residues on IRS-1 and Foxo4 are not conserved in the Drosophila homologs Chico and DFoxo, and further studies are thus required to determine whether JNK-Foxo crosstalk in Drosophila is mediated via homologous mechanisms (Wang, 2005).

The systemic regulation of IIS activity by JNK and Foxo appears to be conserved as well. It has been suggested that C. elegans Daf16/Foxo regulates life span (at least in part) by reducing the expression of insulin-like peptides. In mammals, pancreatic β cells (the counterparts of IPCs) reduce their production of insulin in response to oxidative stress-mediated JNK activation. Conversely, dephosphorylation of JNK by MAPK phosphatase 1 can induce insulin expression in these cells. Reducing circulating insulin levels by JNK-mediated Foxo activation may thus be a general mechanism that balances growth and metabolism with stress defense and damage repair (Wang, 2005).

Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila

Drosophila larval skeletal muscles are single, multinucleated cells of different sizes that undergo tremendous growth within a few days. The mechanisms underlying this growth in concert with overall body growth are unknown. The size of individual muscles correlates with the number of nuclei per muscle cell and with increasing nuclear ploidy during development. Inhibition of Insulin receptor (InR; Insulin-like receptor) signaling in muscles autonomously reduces muscle size and systemically affects the size of other tissues, organs and indeed the entire body, most likely by regulating feeding behavior. In muscles, InR/Tor signaling, Foxo and dMyc (Diminutive) are key regulators of endoreplication, which is necessary but not sufficient to induce growth. Mechanistically, InR/Foxo signaling controls cell cycle progression by modulating dmyc expression and dMyc transcriptional activity. Thus, maximal dMyc transcriptional activity depends on InR to control muscle mass, which in turn induces a systemic behavioral response to allocate body size and proportions (Demontis, 2009).

Therefore, interplay between InR/Tor signaling, Foxo and dMyc activity regulates muscle growth that occurs during Drosophila larval development, in part via the induction of endoreplication. Interestingly, the extent of muscle growth is sensed systemically, regulates feeding behavior and, in turn, influences the size of other tissues and indeed the whole body. Thus, the growth of a single tissue is sensed systemically via modulating a whole-organism behavior (Demontis, 2009).

dMyc, as well as activation of InR signaling, can promote endoreplication in muscles, whereas Foxo and inhibitors of dMyc and of InR/Tor have the opposite effect. dMyc is likely to regulate the expression of genes required for multiple G-S and S-G transitions during endoreplication, similar to vertebrate Myc, which regulates key cell-cycle regulators including cyclin D2, cyclin E, and the cyclin kinase inhibitors p21 and p27 (Cdkn1a and Cdkn1b, respectively). Indeed, aberrant levels of Cyclin E block muscle growth, indicating that proper muscle growth requires tight control of the expression and activity of endoreplication genes. Further, endoreplication is also modulated by Foxo, which is activated in conditions of nutrient starvation, impaired InR/Tor signaling and by other cell stressors. Foxo presumably regulates cell cycle progression at least in part by modulating the expression of evolutionarily conserved Foxo/Myc-target genes, such as dacapo (the Drosophila p21/p27 homolog) and Cyclin E, that regulate the G1-S transition. Interestingly, Foxo and Myc might control different steps in the activation of common target genes (Demontis, 2009).

In addition, it was found that active Foxo can also inhibit dMyc protein activity and regulates dmyc gene expression. Mechanistically, Foxo could influence dMyc activity in several ways. First, it might physically interact with dMyc, although no evidence was found to support this notion. Second, Foxo could regulate the expression of genes that target dMyc for proteasomal degradation, including several ubiquitin E3 ligases that are induced by Foxo during muscle atrophy in mice and humans. However, by analyzing dMyc protein levels by western blot, no significant dMyc protein instability was found upon Foxo overexpression. Third, Foxo might promote the expression of transcriptional regulators that oppose dMyc function, including Mad/Mnt, although no substantial increase in dmnt mRNA levels was detected upon Foxo activation in muscles. Possibly, the expression of other dMyc regulators might be affected by Foxo. Future experiments will be needed to dissect the Foxo-dMyc interaction (Demontis, 2009).

Finally, by manipulating muscle growth and/or endoreplication, it was found that in muscles the ratio of cell size to nuclear size is not constant, and increased nuclear size and DNA content, indicative of ploidy, is necessary but not sufficient to drive growth. Usually, an increase in cell size is matched by an increase in nuclear size, which commonly parallels increases in nuclear ploidy. However, the current findings indicate that in muscles, dMyc-driven variation in nuclear size and ploidy is permissive but not sufficient for substantial growth, even in the presence of increased biogenesis of nucleoli and expression of genes involved in protein translation. This is different from fat body cells, in which dmyc overexpression induces endoreplication and proportional cell growth. Thus, additional instructive signals, possibly modulating protein synthesis, mitochondriogenesis, ribosome biogenesis, sarcomere assembly, and other anabolic responses must be concomitantly received to promote maximal muscle growth. Therefore, increases in cell size and nuclear ploidy are surprisingly uncoupled during muscle growth (Demontis, 2009).

Little is known about the mechanisms that control and coordinate cell, organ and body size, and in particular how muscle growth is matched with the growth of other tissues and of the entire organism. Inhibition of InR/Tor signaling and dMyc activity in muscles impairs, in addition to muscle mass, the size of the entire body and of other internal organs. Similarly, overexpression of Cyclin E in muscles also results in autonomous and systemic growth defects, indicating that, at least in some cases, modulation of muscle growth by means independent from InR signaling can be sensed systemically. In the larva, endoreplicating tissues and organs (gut, salivary glands, epidermis, fat body) are severely affected, whereas non-endoreplicating tissues (brain and imaginal discs) are less affected, indicating distinct tissue responsiveness to this regulation. Similarly, inhibition of Tor signaling in the fat body also primarily affects the size of endoreplicating tissues (Demontis, 2009).

Non-autonomous regulation of tissue size may rely on humoral factors (e.g. hormone-binding proteins, hormones, metabolites) produced by muscles in response to achieving a certain mass. However, alternative models are possible. In particular, decreased and increased larval feeding, respectively, were observed upon inhibition and activation of InR signaling in muscles. This whole-organism behavioral adaptation is possibly due to decreased and increased efficiency of smaller and bigger muscles, respectively, and to regulated expression of neuropeptides that hormonally control feeding behavior. As a consequence of the regulation of feeding behavior, nutrient uptake is decreased and larval growth is blocked in the cells of endoreplicating tissues, which are extremely sensitive to poor nutritional conditions, and to a lesser extent in non-endoreplicating tissues, which are more resistant to limited nutritional supply. In turn, increased or decreased size of non-muscle tissues arise as a consequence of abnormal feeding. Thus, muscle size coordinates with the size of other organs and of the entire body, at least in part via a systemic, behavioral response. Distinct tissues are differently sensitive to this regulation, resulting in altered body proportions (Demontis, 2009).

Understanding the mechanisms regulating muscle mass is of special interest because they underline the etiology of several human diseases. Directly relevant to these studies, both MYC and InR (INSR) signaling have been found to regulate muscle growth and maintenance in humans. Further, muscle atrophy is triggered by FOXO activation in several pathological conditions. In addition, MYC function has been implicated in heart hypertrophy, a process that is conversely regulated by FOXO (Demontis, 2009).

The findings that Foxo functionally antagonizes dMyc during the growth of Drosophila muscles suggest that these factors might also interact similarly in humans. Consistent with this hypothesis, FOXO and MYC regulate, in opposite fashions, the atrophic and hypertrophic programs in human skeletal muscles and cardiomyocytes, and display complementary gene expression and activity in these contexts (Demontis, 2009).

Finally, the finding that during larval development, inhibition of InR signaling in muscles has profound systemic effects might also reflect physiological conditions found in humans. Indeed, defective responsiveness of muscles to Insulin during type II diabetes has autonomous effects on muscle maintenance that are associated with systemic effects on the metabolism of the entire organism, contributing to the improper control of glycemia and the development of metabolic syndrome. This study has identified feeding behavior as part of the systemic response that in Drosophila senses perturbations in muscle mass. These findings might help further elucidate the signals involved in metabolic and growth homeostasis, which may be conserved across evolution (Demontis, 2009).

Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation

Reactive oxygen species (ROS), produced during various electron transfer reactions in vivo, are generally considered to be deleterious to cells. In the mammalian haematopoietic system, haematopoietic stem cells contain low levels of ROS. However, unexpectedly, the common myeloid progenitors (CMPs) produce significantly increased levels of ROS. The functional significance of this difference in ROS level in the two progenitor types remains unresolved. This study shows that Drosophila multipotent haematopoietic progenitors, which are largely akin to the mammalian myeloid progenitors, display increased levels of ROS under in vivo physiological conditions, which are downregulated on differentiation. Scavenging the ROS from these haematopoietic progenitors by using in vivo genetic tools retards their differentiation into mature blood cells. Conversely, increasing the haematopoietic progenitor ROS beyond their basal level triggers precocious differentiation into all three mature blood cell types found in Drosophila, through a signalling pathway that involves JNK and FoxO activation as well as Polycomb downregulation. It is concluded that the developmentally regulated, moderately high ROS level in the progenitor population sensitizes them to differentiation, and establishes a signalling role for ROS in the regulation of haematopoietic cell fate. These results lead to a model that could be extended to reveal a probable signalling role for ROS in the differentiation of CMPs in mammalian haematopoietic development and oxidative stress response (Owusu-Ansah, 2009).

The Drosophila lymph gland is a specialized haematopoietic organ which produces three blood cell types -- plasmatocytes, crystal cells and lamellocytes -- with functions reminiscent of the vertebrate myeloid lineage. During the first and early second larval instars, the lymph gland comprises only the progenitor population. However, by late third instar, multipotent stem-like progenitor cells become restricted to the medial region of the primary lymph gland lobe, in an area referred to as the medullary zone; whereas a peripheral zone, referred to as the cortical zone, contains differentiated blood cells. By late third instar, the progenitors within the medullary zone are essentially quiescent, whereas the mature, differentiated population in the cortical zone proliferates extensively. The posterior signalling centre is a group of about 30 cells that secretes several signalling molecules and serves as a stem-cell niche regulating the balance between cells that maintain 'stemness' and those that differentiate (Owusu-Ansah, 2009).

Although several studies have identified factors that regulate the differentiation and maintenance of Drosophila blood cells and the stem-like progenitor population that generates them, intrinsic factors within the stem-like progenitors are less explored. Interrogation of these intrinsic factors is the central theme of this investigation. It was observed that by the third instar, the progenitor population in the normal wild-type lymph gland medullary zone contains significantly increased ROS levels compared with their neighbouring differentiated progeny that express mature blood cell markers in the cortical zone. ROS are not increased during the earlier larval instars but increase as the progenitor cells become quiescent and subside as they differentiate. This first suggested that the rise in ROS primes the relatively quiescent stem-like progenitor cells for differentiation. ROS was reduced by expressing antioxidant scavenger proteins GTPx-1 or catalase, specifically in the progenitor cell compartment using the GAL4/UAS system, and it was found that suppressing increased ROS levels in haematopoietic progenitors significantly retards their differentiation into plasmatocytes. As a corollary, mutating the gene encoding the antioxidant scavenger protein superoxide dismutase (Sod2) led to a significant increase in differentiated cells and decrease in progenitors (Owusu-Ansah, 2009).

ROS levels in cells can be increased by the genetic disruption of complex I proteins of the mitochondrial electron transport chain, such as ND75 and ND42. Unlike in wild type, where early second-instar lymph glands exclusively comprise undifferentiated cells, mitochondrial complex I depletion triggers premature differentiation of the progenitor population. This defect is even more evident in the third instar, where a complete depletion of the progenitors is seen as primary lobes are populated with differentiated plasmatocytes and crystal cells. The third differentiated cell type, the lamellocyte, defined by the expression of the antigen L1, is rarely observed in the wild-type lymph gland but is abundantly seen in the mutant. Finally, the secondary and tertiary lobes, largely undifferentiated in wild type, also embark on a robust program of differentiation upon complex I depletion. Importantly, the phenotype resulting from ND75 disruption can be suppressed by the co-expression of the ROS scavenger protein GTPx-1, which provides a causal link between increased ROS and the premature differentiation phenotype. It is concluded that the normally increased ROS levels in the stem-like progenitors serve as an intrinsic factor that sensitizes the progenitors to differentiation into all three mature cell types. Any further increase or decrease in the level of ROS away from the wild-type level enhances or suppresses differentiation respectively (Owusu-Ansah, 2009).

In unrelated systems, increased ROS levels have been demonstrated to activate the JNK signal transduction pathway. Consequently, it was tested whether the mechanism by which the progenitors in the medullary zone differentiate when ROS levels increase could involve this pathway. The gene puckered (puc) is a downstream target of JNK signalling and its expression has been used extensively to monitor JNK activity. Although puc transcripts are detectable by reverse transcriptase PCR (RT- PCR), the puc-lacZ reporter is very weakly expressed in wild type. After disruption of ND75, however, a robust transcriptional upregulation of puc-lacZ expression can be seen, indicating that JNK signalling is induced in these cells in response to high ROS levels. The precocious progenitor cell differentiation caused by mitochondrial disruption is suppressed upon expressing a dominant negative version of basket (bsk), the sole Drosophila homologue of JNK. This suppression is associated with a decrease in the level of expression of the stress response gene encoding phosphoenol pyruvate carboxykinase; quantitatively a 68% suppression of the ND75 crystal cell phenotype was observed when JNK function was removed as well. Although disrupting JNK signalling suppressed differentiation, ROS levels remain increased in the mutant cells, as would be expected from JNK functioning downstream of ROS (Owusu-Ansah, 2009).

In several systems and organisms, JNK function can be mediated by activation of FoxO as well as through repression of Polycomb activity. FoxO activation can be monitored by the expression of its downstream target Thor, using Thor-lacZ as a transcriptional read-out. This reporter is undetectable in wild-type lymph glands although Thor transcripts are detectable by RT-PCR; however, the reporter is robustly induced when complex I is disrupted, suggesting that the increase in ROS that is mediated by loss of complex I activates FoxO. To monitor Polycomb de-repression, a Polycomb reporter was used that expresses lacZ when Polycomb proteins are downregulated. Although undetectable in wild-type lymph glands, disrupting ND75 leads to lacZ expression suggesting that Polycomb activity is downregulated by the altered ROS and resulting JNK activation. Direct FoxO overexpression causes a remarkable advancement in differentiation to a time as early as the second instar, never seen in wild type. By early third instar, the entire primary and secondary lobes stained for plasmatocyte and crystal cell markers when FoxO is expressed in the progenitor population. Unlike with ROS increase, no a significant increase in lamellocytes was found upon FoxO overexpression. However, downregulating the expression of two polycomb proteins, Polyhomeotic Proximal (Php-x) and Enhancer of Polycomb [E(Pc)], that function downstream of JNK, markedly increased lamellocyte number without affecting plasmatocytes and crystal cells. When FoxO and a transgenic RNA interference (RNAi) construct against E(Pc) are expressed together in the progenitor cell population, differentiation to all three cell types is evident. It is concluded that FoxO activation and Polycomb downregulation act combinatorially downstream of JNK to trigger the full differentiation phenotype: an increase in plasmatocytes and crystal cells due to FoxO activation, and an increase in lamellocytes primarily due to Polycomb downregulation (Owusu-Ansah, 2009).

This analysis of ROS in the wild-type lymph gland highlights a previously unappreciated role for ROS as an intrinsic factor that regulates the differentiation of multipotent haematopoietic progenitors in Drosophila. Any further increase in ROS beyond the developmentally regulated levels, owing to oxidative stress, will cause the progenitors to differentiate into one of three myeloid cell types. It has been reported that the ROS levels in mammalian haematopoietic stem cells is low but that in the CMPs is relatively high. The Drosophila haematopoietic progenitors give rise entirely to a myeloid lineage and therefore are functionally more similar to CMPs than they are to haematopoietic stem cells. It is therefore a remarkable example of conservation to find that they too have high ROS levels. The genetic analysis makes it clear that the high ROS in Drosophila haematopoietic progenitors primes them towards differentiation. It will be interesting to determine whether such a mechanism operates in mammalian CMPs. In mice, as in flies, a function of FoxO is to activate antioxidant scavenger proteins. Consequently, deletion of FoxO increases ROS levels in the mouse haematopoietic stem cell and drives myeloid differentiation. However, even in the mouse haematopoietic system, FoxO function is dose and context dependent, as ROS levels in CMPs are independent of FoxO. Thus, although the basic logic of increased ROS in myeloid progenitors is conserved between flies and mice, the exact function of FoxO in this context may have diverged (Owusu-Ansah, 2009).

Past work has hinted that ROS can function as signalling molecules at physiologically moderate levels. This work supports and further extends this notion. Although excessive ROS is damaging to cells, developmentally regulated ROS production can be beneficial. The finding that ROS levels are moderately high in normal Drosophila haematopoietic progenitors and mammalian CMPs raises the possibility that wanton overdose of antioxidant products may in fact inhibit the formation of cells participating in the innate immune response (Owusu-Ansah, 2009).

Insulin signals control the competence of the Drosophila female germline stem cell niche to respond to Notch ligands

Adult stem cells reside in specialized microenvironments, or niches, that are essential for their function in vivo. Stem cells are physically attached to the niche, which provides secreted factors that promote their self-renewal and proliferation. Despite intense research on the role of the niche in regulating stem cell function, much less is known about how the niche itself is controlled. Previous work has shown that insulin signals directly stimulate germline stem cell (GSC) division and indirectly promote GSC maintenance via the niche in Drosophila. Insulin-like peptides are required for maintenance of cap cells (a major component of the niche that are directly attached to GSCs through E-cadherin) via modulation of Notch signaling, and they also control attachment of GSCs to cap cells and E-cadherin levels at the cap cell-GSC junction. This study has further dissected the molecular and cellular mechanisms underlying these processes. Insulin and Notch ligands were shown to directly stimulate cap cells to maintain their numbers and indirectly promote GSC maintenance. It is also reported that insulin signaling, via phosphoinositide 3-kinase and FOXO, intrinsically controls the competence of cap cells to respond to Notch ligands and thereby be maintained. Contrary to a previous report, it was also found that Notch ligands originated in GSCs are not required either for Notch activation in the GSC niche, or for cap cell or GSC maintenance. Instead, the niche itself produces ligands that activate Notch signaling within cap cells, promoting stability of the GSC niche. Finally, insulin signals control cap cell-GSC attachment independently of their role in Notch signaling. These results are potentially relevant to many systems in which Notch signaling modulates stem cells and demonstrate that complex interactions between local and systemic signals are required for proper stem cell niche function (Hsu, 2011).

The Notch pathway plays a central role in many stem cell systems, and how systemic signals impact Notch signaling in stem cell niches is a question of wide relevance to stem cell biology. Notch controls cap cell number in the Drosophila female GSC niche, and recent studies showed that insulin-like peptides control Notch signaling in the niche (Hsu, 2009), although the underlying cellular mechanisms remained unclear. This study dissected the specific cellular requirements for Notch pathway components and the insulin receptor and reveals that insulin signaling controls cell–cell communication via Notch signaling within the niche (Hsu, 2011).

To summarize, from this study in combination with previous work, a fairly complex model emerges of how insulin-like peptides -- systemic signals influenced by diet -- impact the function of GSCs and their niche through multiple mechanisms. In adult females under favorable nutritional conditions, insulin-like peptides signal directly to GSCs via PI3K to inhibit FOXO and thereby increase their division rates by promoting progression through G2. In parallel to this direct effect on GSC proliferation, insulin-like peptides also act directly on cap cells (a major cellular component of the GSC niche) to control two separate processes. Stimulation of the insulin pathway, also via PI3K inhibition of FOXO, within cap cells intrinsically increase their responsiveness to the Notch ligand Delta (likely at a step upstream of nuclear translocation of the intracellular domain of Notch), which is likely produced by neighboring cap cells. (A similar process likely occurs during niche formation in larval/pupal stages, although in this case, Delta produced in basal terminal filament cells clearly contributes to the specification of cap cells.) Notch signaling within cap cells leads to their maintenance and, indirectly, to GSC maintenance. Independently of its effect on Notch signaling, insulin/PI3K/FOXO pathway activation in cap cells intrinsically promotes stronger cap cell-GSC adhesion (presumably via E-cadherin; Hsu, 2009), which also promotes GSC maintenance. Further, aging also appears to influence insulin signaling levels in Drosophila females (Hsu, 2009), suggesting that physiological changes caused by diverse factors can impinge on this GSC regulatory network. Together, these studies underscore the importance of investigating how whole organismal physiology impacts stem cell function via effects on stem cells and on their niche, potentially via changes in local signaling (Hsu, 2011).

Notch signaling requires direct cell-cell contact because Notch ligands are membrane-bound proteins that induce Notch activation in neighboring cells. In addition to transactivating Notch in adjacent cells, the Notch ligand Delta also inhibits Notch in cis, thus creating a potent switch between high Delta expression/low Notch activity and high Notch activity/low Delta expression (Sprinzak, 2010). Differential Notch activation often underlies binary cell fate decisions. For example, during Drosophila sensory organ development, cells with high levels of Delta and low Notch activity become neurons, while those with elevated Notch activity and low Delta become epidermal cells (Hsu, 2011).

In the Drosophila GSC niche, Notch activity is detected in all cap cells, and Dl-lacZ is expressed in all terminal filament cells. A subset of cap cells also expresses Dl-lacZ, suggesting that some cap cells may express Delta and have high Notch activity simultaneously. The basal terminal filament cell, in which Dl is required for cap cell formation, does not contact all cap cells directly, and it was also found that Dl and Ser are not required within GSCs for cap cell formation or maintenance. It is therefore proposed that cap cells may signal to each other via Delta to activate Notch signaling, and that, in cap cells, Delta might not consistently act in cis to inhibit Notch activation (Hsu, 2011).

The observation that a subset of cap cells can express Dl-lacZ and Notch activity simultaneously is consistent with recent findings. Human eosinophils express both Notch and its ligands, and autocrine Notch signaling controls their migration and survival (Radke, 2009). Similarly, Notch is co-expressed with its ligands in rat hepatocytes following partial hepatectomy and also in normal human breast cells, although it is unclear if autocrine signaling occurs. It is therefore conceivable that Delta expressed in cap cells may stimulate Notch signaling via both paracrine and autocrine manners (Hsu, 2011).

Alternatively, Notch ligands might be secreted from terminal filament cells to stimulate Notch signaling in all cap cells and thereby promote their maintenance. In fact, a soluble form of Delta capable of stimulating Notch has been identified in Drosophila S2 cell cultures, and the ADAM disintegrin metalloprotease Kusbanian is required for the production of soluble Delta in culture. Further, Dl and kuzbanian genetically interact, raising the possibility that soluble forms of ligands might modulate Notch signaling in vivo (Hsu, 2011).

neur encodes an E3 ubiquitin ligase that mediates the endocytosis of Notch ligands in signal-sending cells, thereby enhancing their signaling strength. Contrary to a previous report, this study found no evidence that Notch ligands produced from GSCs are required for self-renewal. In contrast, neur is intrinsically required for GSC maintenance. Similarly, in the Drosophila testis, neur, but not Dl and Ser, is required for GSC maintenance, further indicating that Neuralized maintains GSCs via a Notch-independent pathway (Hsu, 2011).

neur mutant cysts exhibit large and highly branched fusomes, another Notch-independent phenotype. In principle, this aberrant fusome morphology might result from a defect in fusome growth and/or partitioning, or be secondary to an excessive number of cyst division rounds. Nevertheless, the close association of some of these abnormal fusomes with the cap cell interface suggests that fusome defects might lead to GSC loss. Ubiquitination regulates many processes, including protein degradation and vesicular trafficking. It is therefore possible that Neuralized ubiquitinates specific substrates that regulate fusome-related vesicular trafficking during cyst division. Future studies should test whether E3 ligase activity is indeed required for the role of neur in early germline cysts, identify key ubiquitination targets, and elucidate the molecular mechanisms they regulate (Hsu, 2011).

Under low insulin signaling, the FOXO transcriptional factor is required for extended longevity, reduced rates of proliferation, and stress resistance, among other processes. FOXOs are conserved from yeast to humans, and they control many target genes, different subsets of which modulate distinct processes. Drosophila FOXO negatively controls GSC division when insulin signaling is low (Hsu, 2008). It was also shown that insulin signaling modulates niche-stem cell interactions and Notch signaling in the niche (to control cap cell number), and that insulin signaling declines as females become older, leading to stem cell loss (Hsu, 2009). This study has shown that FOXO is required to negatively regulate Notch signaling within cap cells under low insulin activity and that FOXO also modulates the physical interaction between cap cells and GSCs. The multiplicity of FOXO roles in stem cell regulation is further underscored by studies in other stem cell systems. For example, FOXOs regulate several processes, including cell cycle progression, oxidative stress, and apoptosis, in the hematopoietic stem cell compartment, thereby influencing stem cell number and activity. It will be important to investigate how the specificity of FOXO is controlled and also whether or not FOXO regulates other stem cell niches, perhaps acting as a mediator of changes in niche size and/or activity during aging or cancer development (Hsu, 2011).

This study suggests a potentially novel mechanism by which the Notch and insulin pathways interact. In the Drosophila female GSC niche, insulin signaling does not control ligand transcription, and it is not required for ligand function (i.e., Dl is required in basal terminal filament cells during cap cell formation, but InR is not). Instead, both InR and N are cell autonomously required for cap cell maintenance, and insulin receptor function (via repression of FOXO) is required for proper Notch signaling. Expression of the intracellular domain of Notch rescues the low cap cell and GSC numbers of InR mutants (Hsu, 2009), and ovarian Notch expression does not appear altered in InR mutants. Therefore, it is speculated that FOXO inhibits the ability of cap cells to respond to Notch ligands by regulating a target that negatively regulates the series of proteolytic events responsible for the release of the intracellular domain of Notch. It cannot, however, be rulef out that Notch and FOXO normally interact at the level of target gene regulation but that overexpression of the intracellular domain of Notch overrides the normal inhibition by FOXO (Hsu, 2011).

These findings contrast with other types of interactions between FOXO and Notch that have been reported. During muscle differentiation in myoblast cultures, FOXO promotes (instead of antagonizing) Notch activity via a physical interaction that leads to activation of Notch target genes. Positive interactions between Notch and PI3K signaling have also been reported. Specifically, activation of the PI3K pathway potentiates Notch-dependent responses in CHO cells, T-cells, and hippocampal neurons. The suggested mechanism, however, involves the inactivation of GSK3 by Akt phosphorylation upstream of FOXO, which is distinct from the involvement of FOXO in the insulin-Notch signaling interaction within the GSC niche. These examples illustrate the diversity of modes of interaction between Notch and insulin signaling. It is conceivable that the positive interaction that is describe between insulin and Notch signaling pathways in the GSC niche may occur in other stem cell niches (Hsu, 2011).

Deregulated Notch signaling is associated with many types of cancers and, in some cases, it is thought that altered Notch signaling promotes cancer development by overstimulating the self-renewal of normal stem cells (Wang, 2009). Hyperactivation of insulin/IGF pathway is also linked to increased cancer risk and poor cancer prognosis. The Notch and insulin/IGF pathways have been reported to interact in cancerous cells via yet another mechanism. Specifically, upregulation of the Notch ligand Jagged 1 leads to PI3K activation in human papillomavirus-induced cancer lines. It is speculated that additional types of interactions between Notch and insulin/IGF signaling, such as the positive regulation of Notch activity by the insulin/PI3K/FOXO pathway that occurs in the Drosophila GSC niche, may also contribute to cancer progression (Hsu, 2011).

The insulin receptor is required for the development of the Drosophila peripheral nervous system

The Insulin Receptor (InR) in Drosophila presents features conserved in its mammalian counterparts. InR is required for growth; it is expressed in the central and embryonic nervous system and modulates the time of differentiation of the eye photoreceptor without altering cell fate. This study shows that the InR is required for the formation of the peripheral nervous system during larval development and more particularly for the formation of sensory organ precursors (SOPs) on the fly notum and scutellum. SOPs arise in the proneural cluster that expresses high levels of the proneural proteins Achaete (Ac) and Scute (Sc). The other cells will become epidermis due to lateral inhibition induced by the Notch (N) receptor signal that prevents its neighbors from adopting a neural fate. In addition, misexpression of the InR or of other components of the pathway (PTEN, Akt, FOXO) induces the development of an abnormal number of macrochaetes, which are Drosophila mechanoreceptors. These data suggest that InR regulates the neural genes ac, sc and sens. The FOXO transcription factor, which becomes localized in the cytoplasm upon insulin uptake, displays strong genetic interaction with the InR and is involved in Ac regulation. The genetic interactions between the epidermal growth factor receptor (EGFR), Ras and InR/FOXO suggest that these proteins cooperate to induce neural gene expression. Moreover, InR/FOXO is probably involved in the lateral inhibition process, since genetic interactions with N are highly significant. These results show that the InR can alter cell fate, independently of its function in cell growth and proliferation (Dutrieux, 2013).

A model is proposed in which the InR receptor plays a role in the development of the peripheral nervous system mainly through FOXO cell localization independently of its role in proliferation and apoptosis. The role of the InR/FOXO pathway appears early in PNS development before SOP formation. The use of different mutants involved in growth indicates that the TOR pathway does not play a major role in the phenotypes observed. The results using genetic and molecular methods strongly suggest that InR/FOXO controls the level of proneuronal genes such as ac, sc and Sens early in PNS development. This explains the interaction observed with N55e11 (Dutrieux, 2013).

Several arguments indicate that the phenotypes observed when InR is overexpressed are not due, at least for the most part, to proliferation, growth or lack of apoptosis. First using anti-PH3 staining that allows to visualize mitotic cells, no extra mitoses are observed in the clusterOverexpression of genes such as dE2F1, or dacapo did not lead to a significant increase or decrease in the number of macrochaetes. In addition co-expression of these genes with InR indicates no interaction. Moreover, the effects of InR and FOXO when overexpressed on respectively the increase and the decrease in cell number, could be estimated by the number of Ac-positive cells in the DC and SC clusters. No significant differences were observed between the control and the overexpressed strain (either InR or FOXO) in the number of cells positive for Ac. If the possibility that proliferation is somehow involved in cluster size cannot be discarded, it does not account for the effects observed since the ratio of Sens-positive cells when InR is overexpressed over the control strain is much higher than the ratio of Ac-positive cells. A similar role for FOXO in apoptosis could also be discarded on the same basis. No clear interactions were observed between FOXO and genes involved in inhibition of apoptosis like diap1 (Dutrieux, 2013).

Along the same line it has been shown that the InR/TOR pathway plays a role in controlling the time of neural differentiation. This has been observed in photoreceptor formation but also in the chordotonal organs of the leg that develop on the same basis as thoracic bristles. The dynamic formation of the SOPs, particularly after a block of InR signaling was undertaken. No differences were observed before the end third larval instar in the test and in the overexpressed strain. Only an increase in the number of positive Sens stained cells are observed in the sca>InR strain (Dutrieux, 2013).

Using Pros staining that marks pIIb cells, this study shows that staining appears in the late third instar larvae at the level of DC SOPs in sca>InR; this is not observed in the control strain. In addition in sca>FOXO RNAi wing discs it also leads to Pros staining. This indicates that the time of differentiation is advanced in the InR strain through the absence of nuclear FOXO. However it was verified that in very early third instar larvae the first scutellar SOP appears at the same time in the control and in the overexpressed strains and that no differences were observed in mid third instar (Dutrieux, 2013).

In addition the observations show that the increase in the number of macrochaetes in sca>InR is independent of the TOR pathway since none of the members induces a similar phenotype as does InR or interacts either with InR or FOXO in this process. However, some interactions were observed with raptor and Rheb that could be the consequence for the latter of its role in PIIa and PIIb formation regulating N (Dutrieux, 2013).

Are InR and FOXO acting on the same target in SOP formation? Several arguments are in favor of this possibility. First underexpression experiments (InR clones, InR RNAi or FOXO RNAi overexpression and FOXO homozygotes and even heterozygotes,) induce exactly opposite phenotypes. This is also true for overexpression experiments with InR and hFOXO3a-TM. Moreover overexpression of both transgenes leads to an intermediate phenotype, very different from the control phenotype. Finally, overexpression of InR in a heterozygote FOXO mutant background leads to an increase in the number of macrochaetes compared to InR alone. FOXO null flies are fully viable and do not usually display any phenotype. However an increase in the number of pDC and aSC macrochaetes is observed in some FOXO homozygotes and even heterozygotes that are nor observed in the control strain. This could indicate that FOXO function is in part dispensable. Even if the InR/FOXO double heterozygote is completely normal, the double null mutant InR/FOXO shows either an excess or a lack of macrochaetes, that is in favor of the hypothesis that InR acts through FOXO. FOXO null clones do not display any phenotype comparable to FOXO RNAi overexpression. However overexpression of InR in a FOXO null clone leads to stronger phenotypes than overexpression of InR alone in a clone. Yet, it cannot be excluded that part of the InR overexpression phenotype is not due to the absence of FOXO or its cytoplasmic retention (Dutrieux, 2013).

The absence of FOXO, using FOXO RNAi, or its retention in the cytoplasm by InR or Akt overexpression produces the same neurogenic phenotypes that are exactly the opposite when nuclear hFOXO3a-TM is overexpressed. In addition overexpression of both hFOXO3a-TM and InR leads to a decrease in the number of highly positive Ac and Sens expressing cells compared to overexpression of InR alone. Finally, overexpression of FOXO RNAi in dpp regulatory sequences, induces Ac expression. All these results should be explained by the same molecular process. One possibility would be that InR/FOXO regulates one or several neural genes involved in cluster formation and maintenance. The results are in favor of the hypothesis that genes of the Ac/Sc complex could be regulated by InR. Either InR via nuclear FOXO represses the Ac/Sc pathway, or FOXO activates a repressor of the pathway (Dutrieux, 2013).

Since it has been well established that InR induces cell proliferation, it remains possible that these functions could affect the size of the proneural clusters when the genes are overexpressed. However, when the number of the Ac-positive cells in the DC and SC clusters in the different genotypes was estimated, it was not significantly different (Dutrieux, 2013).

Several relevant arguments exist suggesting that InR is necessary for SOP formation and regulation of neural gene expression. (1) The phenotype of the overexpression experiments either with InR or with InR RNAi suggests that InR perturbs the normal pattern of singling out a cell in the proneural cluster that will become an SOP. The fact that the sensitive period occurs in the late second/beginning third instar is in accordance with this hypothesis. The phenotype of the InR null clones comfort this hypothesis. (2) When InR is overexpressed the level of Ac is significantly higher. This is confirmed by the IMARIS technique that estimated that in this genotype, the number of cells with the highest scores (106 and 107 units) is larger than in the control strain. These 'highly Ac-positive cells' seem to also be Sens positive cells indicating a correlation between the two events. (3) In sca>InR the level of Sens, measured by the IMARIS technique is higher than in the test raising the possibility that InR regulates several neural genes independently. However another possibility would be that this high Sens expression level would be indirectly due to the induction by InR of a Sens-positive regulator such as sc. (4) Several sc enhancers are regulated by InR, the sc promoter, and the SRV and DC enhancers. As sc is auto-regulated through its different enhancers, it is difficult to evaluate if a specific enhancer is involved although the effect on the 3.8 kb sc promoter is the most striking. For FOXO the absence of FOXO using the FOXO RNAi strain shows that Ac is induced. The double expression of InR and hFOXO3a-TM produces an intermediate phenotype and decreases the effects of InR, on Ac and Sens expression. The results using the sc enhancers when hFOXO3a-TM is overexpressed showed that only a decrease in the expression of the SRV enhancer is observed. However, the phenotypes observed in sca>hFOXO3a-TM agree with the hypothesis of repression of ac and sc by hFOXO3a-TM. As expected, overexpression of FOXO RNAi induces sc-lacZ enhancer. (5) Overexpression of both InR and sc leads to a significant increase in the effect of a single transgene. This indicates that both transgenes have a common target; one of them could be sc itself. An opposite effect is observed with constitutively active hFOXO3a-TM. This favors the model whereby InR and FOXO act in opposite ways on the sc target in SOP formation. (6) Highly significant genetic interactions are observed between sc and InR, and sc and FOXO. (7) Another gene charlatan (chn) which is both upstream and downstream of sc, strongly interacts genetically with InR (Dutrieux, 2013).

Lateral inhibition is determined by the activity of the N receptor. When N is mutated, cell fate changes and extra macrochaete singling appear. Using the N deletion (N55e11) to test possible genetic interaction with InR and with FOXO in heterozygote females, interaction was observed with the InR RNAi strain. Moreover strong interaction is observed with InR overexpression. This indicates that InR impairs lateral inhibition and cooperates with N in this process. In parallel, as for Inr overexpression, the absence of nuclear FOXO either using FOXO25 homozygotes (or even heterozygotes) or FOXO RNAi overexpression induces an increase in the neurogenic phenotype. With this latter strain, tufted microchaetes were observed, indicating that FOXO could also act later in development. Overexpression of hFOXO3a-TM displays highly significant interaction with N55e11 as the neurogenic phenotype is increased compared to overexpression in a wild type background. However, overexpression of InR RNAi in a N55e11 heterozygote background leads to a significant increase but only at the level of aSC, raising the possibility of a local interaction or appearing at a specific time for the different clusters (Dutrieux, 2013).

Moreover the fact that there is no differences when Suppressor of Hairless (Su(H)) which transduces the N pathway, is expressed with or without the InR, indicates that lateral inhibition is not affected. In addition in the InR strain, Sens stained cells were clearly individualized and separated from one another. These results clearly indicate that InR and FOXO act with N on the choice of the cell that will become an SOP (Dutrieux, 2013).

EGFR has also been implicated in macrochaete development. Indeed EGFR mutants and EGFR null clones display macrochaete phenotypes. This could be explained since in EGFR hypomorphic mutants the level of Sc is reduced in some clusters and increased in others suggesting a different requirement of EGFR for the different SOPs. If RasV12 was overexpressed with an ubiquitous driver, sc was ectopically expressed. Thus, Ac/Sc induction by Ras overrules lateral inhibition due to N. Moreover N downregulation enhances EGFR signaling. A model has been established of antagonist interaction between EGFR and N in which Ac/Sc activates both pathways that in turn act on the same SOP specific enhancers (Dutrieux, 2013).

Moreover, the InR/TOR pathway regulates the expression of some of the components of the EGFR signaling pathway such as argos, rhomboid and pointed. The results suggest that both the InR and the EGFR/Ras pathways induce sc in a synergic manner and this further overrules the lateral inhibition mechanism induced by N. The fact that overexpression of RasV12 in an InR null heterozygote background significantly lowers the phenotype observed with RasV12 only, is in agreement with this hypothesis. The interactions observed with the EGFR RNAi strain seem to be FOXO independent (Dutrieux, 2013).

Taken together these results show that InR and several components of the pathway such as PTEN, Akt and FOXO are involved in PNS development independently of their role in growth, proliferation and delay in the time of neural differentiation. The function of InR in PNS development seems to be independent of TOR/4E-BP. FOXO cytoplasmic retention either by InR activation or by the use of FOXO RNAi produces opposite phenotypes suggesting that nuclear FOXO could be a repressor of PNS development. These results using antibody staining and reporters of sc enhancers indicate that InR targets are the neural genes ac, sc and sens. However, as most of these neural genes display a complex co-regulation, it is difficult to demonstrate whether or not sc is the primary target of the pathway. A strong interaction is observed between the EGFR/Ras pathways and InR suggesting that both could act together to induce neural gene expression and this would explain the strong interaction observed between InR/FOXO and N (Dutrieux, 2013).

Misregulation of an adaptive metabolic response contributes to the age-related disruption of lipid homeostasis in Drosophila

Loss of metabolic homeostasis is a hallmark of aging and is commonly characterized by the deregulation of adaptive signaling interactions that coordinate energy metabolism with dietary changes. The mechanisms driving age-related changes in these adaptive responses remain unclear. This study characterized the deregulation of an adaptive metabolic response and the development of metabolic dysfunction in the aging intestine of Drosophila. Activation of the insulin-responsive transcription factor Foxo in intestinal enterocytes was found to be required to inhibit the expression of evolutionarily conserved lipases as part of a metabolic response to dietary changes. This adaptive mechanism becomes chronically activated in the aging intestine, mediated by changes in Jun-N-terminal kinase (JNK) signaling. Age-related chronic JNK/Foxo activation in enterocytes is deleterious, leading to sustained repression of intestinal lipase expression and the disruption of lipid homeostasis. Changes in the regulation of Foxo-mediated adaptive responses thus contribute to the age-associated breakdown of metabolic homeostasis (Karpac, 2013).

This work identifies Foxo-mediated repression of intestinal lipases as a critical component of an adaptive response to dietary changes in Drosophila. Interestingly, misregulation of this metabolic response also contributes to the age-associated breakdown of lipid homeostasis, as elevated JNK signaling leads to chronic Foxo activation and subsequent disruption of lipid metabolism due to chronic repression of lipases. This age-related deregulation of an adaptive metabolic response is reminiscent of insulin resistance-like phenotypes in vertebrates, which can also be triggered by chronic activation of JNK, and thus highlights the antagonistic pleiotropy inherent in metabolic regulation. The adaptive nature of signaling interactions that drive pathology (such as JNK-mediated insulin resistance) has remained elusive in many instances, and this work provides a model for age-related changes in an adaptive regulatory process that ultimately lead to a pathological outcome. It is believed that this system can serve as a productive model to address a number of interesting questions with relevance to the loss of metabolic homeostasis in aging organisms (Karpac, 2013).

In mammals, JNK has been shown to promote insulin resistance both cell-autonomously and systemically (through inflammation), subsequently affecting lipid homeostasis in various tissues. The current results further introduce a mechanism by which JNK can alter cellular and systemic lipid metabolism through the regulation of lipases, independent of changes in IIS. Thus, JNK-mediated Foxo activation in select tissues may be able to alter intracellular lipid metabolism, changing metabolic fuel substrates and disrupting metabolic homeostasis in other tissues without altering insulin responsiveness (Karpac, 2013).

Whereas the current data show that Foxo activation leads to the transcriptional repression of intestinal lipases, especially LipA/Magro, it remains unclear if this control is direct or indirect. Foxo is classically described as an activator of transcription, but recent reports have shown that Foxo can transcriptionally repress genes through direct association with promoters. The promoter regions of LipA/Magro and CG6295 do not contain conserved Foxo transcription factor binding sites, suggesting that the regulation of these genes may be indirect, potentially through Foxo-regulated expression of secondary effectors. Thus, tissue-specific control of lipid homeostasis by IIS/Foxo might be achieved through the regulation of lipogenic or lipolytic transcription factors that can elicit global and direct changes in cellular lipid metabolism. Previous reports have shown that the nuclear receptor dHR96, a critical regulator of lipid and cholesterol homeostasis, promotes lipA/magro expression. However, dhr96 expression is upregulated in aging intestines, suggesting that the age-related repression of intestinal lipases is not merely due to decreases in dHR96 levels. dhr96 transcript levels are strongly induced in genetic conditions where Foxo is activated and intestinal lipases are repressed, again suggesting that Foxo does not mediate its effects on lipase transcription by antagonizing dhr96 expression. Furthermore, age-related changes that are independent of JNK/Foxo activation may also contribute to the repression of intestinal lipase expression and disruption of lipid metabolism, such as an age-associated decline in feeding/food intake (Karpac, 2013).

The reasons for the increase in JNK and Foxo activity in aging enterocytes remain to be explored. Buchon (2009) has also shown that age-related activation of JNK in the intestinal epithelium is dependent on the presence of commensal bacteria, as maintaining animals axenically reduces activation of JNK in the first 30 days of life. Thus, bacteria-induced inflammation and subsequent JNK activation appears to be a likely cause, in part, for age-related increases in Foxo activity. In a separate study, however, this laboratory found that Foxo activation still occurs in intestines of old (40-day-old), axenically reared flies, suggesting that age-related activation of Foxo may also occur through JNK-independent processes. Supporting this idea, the results show that inhibiting JNK function in enterocytes can attenuate, although not completely inhibit, this Foxo activation. Additional factors, such as sirtuins or histone deacetylases, recently shown to deacetylate and activate Foxo in response to endocrine signals, may also lead to age-related increases in intestinal Foxo activity (Karpac, 2013).

Interactions between JNK and IIS/Foxo are mediated by various mechanisms. In mammals, JNK phosphorylates the insulin receptor substrate (IRS), inhibiting insulin signaling transduction. Whereas JNK has clearly been shown to antagonize IIS (activate Foxo) in C. elegans and Drosophila, that exact mechanism by which Foxo activation is achieved may be divergent in mammals. For example, no IRS homolog has been identified in worms, and the JNK phosphorylation site in mammalian IRS is not conserved in flies. The current data show that JNK-mediated Foxo activation in the aging fly intestine is not achieved through IIS antagonism upstream of Akt, suggesting either a direct interaction between Foxo and JNK or changes in other regulators of Foxo. Recent studies have shown that JNK-mediated phosphorylation of 14-3-3 proteins results in the release of their binding partners, including Foxo. The conservation of 14-3-3 proteins between vertebrates and invertebrates makes 14-3-3 an interesting candidate in promoting Foxo function via JNK in the aging fly intestine. This chronic intestinal Foxo activation and subsequent metabolic changes, provide a physiological system in Drosophila to genetically dissect the crosstalk between IIS/Foxo and JNK signaling. Detailed analysis of these signaling interactions promises to provide important insight into the pleiotropic effects of IIS/Foxo function and the pathogenesis of age-related metabolic diseases (Karpac, 2013).

The data further reveal the pleiotropic consequences of Foxo activation in regard to healthspan and longevity in Drosophila. Overexpressing Foxo in the fat body or muscle of flies leads to lifespan extension. The data presented here show that chronic Foxo activation in intestinal enterocytes disrupts lipid metabolism by deregulating intestinal lipases and thus highlight how cell- and tissue-specific consequences of Foxo function play an important role in determining either the beneficial (i.e., lifespan extension) or pathological (i.e., disruption of lipid metabolism) outcome of Foxo activation (Karpac, 2013).

Recent work in C. elegans has begun to explore the relationship between lipid metabolism and longevity, revealing that increases in intestinal lipase expression can extend lifespan. The beneficial effects of elevated lipase expression appear to be mediated by increases in specific types of fatty acids, which can activate autophagy and lead to lifespan extension. The current study identifies Foxo-mediated repression of intestinal lipases as a critical component of an adaptive response to dietary changes in Drosophila. Interestingly, misregulation of this metabolic response also contributes to the age-associated breakdown of lipid homeostasis, as elevated JNK signaling leads to chronic Foxo activation and subsequent disruption of lipid metabolism due to chronic repression of lipases. This age-related deregulation of an adaptive metabolic response is reminiscent of insulin resistance-like phenotypes in vertebrates, which can also be triggered by chronic activation of JNK, and thus highlights the antagonistic pleiotropy inherent in metabolic regulation. The adaptive nature of signaling interactions that drive pathology (such as JNK-mediated insulin resistance) has remained elusive in many instances, and the current work provides a model for age-related changes in an adaptive regulatory process that ultimately lead to a pathological outcome. This system can serve as a productive model to address a number of interesting questions with relevance to the loss of metabolic homeostasis in aging organisms (Karpac, 2013).

The results further introduce a mechanism by which JNK can alter cellular and systemic lipid metabolism through the regulation of lipases, independent of changes in IIS. Thus, JNK-mediated Foxo activation in select tissues may be able to alter intracellular lipid metabolism, changing metabolic fuel substrates and disrupting metabolic homeostasis in other tissues without altering insulin responsiveness (Karpac, 2013).

Whereas the data show that Foxo activation leads to the transcriptional repression of intestinal lipases, especially LipA/Magro, it remains unclear if this control is direct or indirect. Foxo is classically described as an activator of transcription, but recent reports have shown that Foxo can transcriptionally repress genes through direct association with promoters. The promoter regions of LipA/Magro and CG6295 do not contain conserved Foxo transcription factor binding sites, suggesting that the regulation of these genes may be indirect, potentially through Foxo-regulated expression of secondary effectors. Thus, tissue-specific control of lipid homeostasis by IIS/Foxo might be achieved through the regulation of lipogenic or lipolytic transcription factors that can elicit global and direct changes in cellular lipid metabolism. Previous reports have shown that the nuclear receptor dHR96, a critical regulator of lipid and cholesterol homeostasis, promotes lipA/magro expression. However, dhr96 expression is upregulated in aging intestines (data not shown), suggesting that the age-related repression of intestinal lipases is not merely due to decreases in dHR96 levels. dhr96 transcript levels are strongly induced in genetic conditions where Foxo is activated and intestinal lipases are repressed, again suggesting that Foxo does not mediate its effects on lipase transcription by antagonizing dhr96 expression. Furthermore, age-related changes that are independent of JNK/Foxo activation may also contribute to the repression of intestinal lipase expression and disruption of lipid metabolism, such as an age-associated decline in feeding/food intake (Karpac, 2013).

The reasons for the increase in JNK and Foxo activity in aging enterocytes remain to be explored. Age-related activation of JNK in the intestinal epithelium is dependent on the presence of commensal bacteria, as maintaining animals axenically reduces activation of JNK in the first 30 days of life. Thus, bacteria-induced inflammation and subsequent JNK activation appears to be a likely cause, in part, for age-related increases in Foxo activity. In a separate study, however, it was found that Foxo activation still occurs in intestines of old (40-day-old), axenically reared flies, suggesting that age-related activation of Foxo may also occur through JNK-independent processes. Supporting this idea, the current results show that inhibiting JNK function in enterocytes can attenuate, although not completely inhibit, this Foxo activation. Additional factors, such as sirtuins or histone deacetylases, recently shown to deacetylate and activate Foxo in response to endocrine signals, may also lead to age-related increases in intestinal Foxo activity (Karpac, 2013).

Interactions between JNK and IIS/Foxo are mediated by various mechanisms. In mammals, JNK phosphorylates the insulin receptor substrate (IRS), inhibiting insulin signaling transduction. JNK has also been shown to directly phosphorylate and activate Foxo in mammalian cell culture, that exact mechanism by which Foxo activation is achieved may be divergent in mammals. For example, no IRS homolog has been identified in worms, and the JNK phosphorylation site in mammalian IRS is not conserved in flies. The data show that JNK-mediated Foxo activation in the aging fly intestine is not achieved through IIS antagonism upstream of Akt, suggesting either a direct interaction between Foxo and JNK or changes in other regulators of Foxo. Recent studies have shown that JNK-mediated phosphorylation of 14-3-3 proteins results in the release of their binding partners, including Foxo. This chronic intestinal Foxo activation and subsequent metabolic changes, provide a physiological system in Drosophila to genetically dissect the crosstalk between IIS/Foxo and JNK signaling. Detailed analysis of these signaling interactions promises to provide important insight into the pleiotropic effects of IIS/Foxo function and the pathogenesis of age-related metabolic diseases (Karpac, 2013).

The data further reveal the pleiotropic consequences of Foxo activation in regard to healthspan and longevity in Drosophila. Overexpressing Foxo in the fat body or muscle of flies leads to lifespan extension. Overexpression of selected cytoprotective Foxo target genes in stem cells, on the other hand, is sufficient to prevent age-associated dysplasia and extend lifespan. The data presented here show that chronic Foxo activation in intestinal enterocytes disrupts lipid metabolism by deregulating intestinal lipases and thus highlight how cell- and tissue-specific consequences of Foxo function play an important role in determining either the beneficial (i.e., lifespan extension) or pathological (i.e., disruption of lipid metabolism) outcome of Foxo activation (Karpac, 2013).

Recent work in C. elegans has begun to explore the relationship between lipid metabolism and longevity, revealing that increases in intestinal lipase expression can extend lifespan. The beneficial effects of elevated lipase expression appear to be mediated by increases in specific types of fatty acids, which can activate autophagy and lead to lifespan extension. Interventions that promote lipid homeostasis with age, such as JNK/Foxo inhibition in intestinal enterocytes, might thus affect healthspan and/or longevity through means other than primarily maintaining energy homeostasis (Karpac, 2013).

Cell-nonautonomous effects of dFOXO/DAF-16 in aging

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. This study shows 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, the current results pinpoint FoxO-to-other signaling as an important mechanism through which localized FoxO activity ameliorates aging (Alic, 2014).

Insulin signaling regulates neurite growth during metamorphic neuronal remodeling

Although the growth capacity of mature neurons is often limited, some neurons can shift through largely unknown mechanisms from stable maintenance growth to dynamic, organizational growth (e.g. to repair injury, or during development transitions). During insect metamorphosis, many terminally differentiated larval neurons undergo extensive remodeling, involving elimination of larval neurites and outgrowth and elaboration of adult-specific projections. This study shows in the fruit fly that a metamorphosis-specific increase in insulin signaling promotes neuronal growth and axon branching after prolonged stability during the larval stages. FOXO, a negative effector in the insulin signaling pathway, blocks metamorphic growth of peptidergic neurons that secrete the neuropeptides CCAP and bursicon. RNA interference and CCAP/bursicon cell-targeted expression of dominant-negative constructs for other components of the insulin signaling pathway (InR, Pi3K92E, Akt1, S6K) also partially suppresses the growth of the CCAP/bursicon neuron somata and neurite arbor. In contrast, expression of wild-type or constitutively active forms of InR, Pi3K92E, Akt1, Rheb, and TOR, as well as RNA interference for negative regulators of insulin signaling (PTEN, FOXO), stimulate overgrowth. Interestingly, InR displays little effect on larval CCAP/bursicon neuron growth, in contrast to its strong effects during metamorphosis. Manipulations of insulin signaling in many other peptidergic neurons revealed generalized growth stimulation during metamorphosis, but not during larval development. These findings reveal a fundamental shift in growth control mechanisms when mature, differentiated neurons enter a new phase of organizational growth. Moreover, they highlight strong evolutionarily conservation of insulin signaling in neuronal growth regulation (Gu, 2014).

It is well established that insulin/insulin-like signaling (IIS) is crucial for regulating cell growth and division in response to nutritional conditions in Drosophila. However, most studies have focused on growth of the body or individual organs, and comparatively little is known about the roles of IIS during neuronal development, particularly in later developmental stages. Drosophila InR transcripts are ubiquitously expressed throughout embryogenesis, but are concentrated in the nervous system after mid-embryogenesis and remain at high levels there through the adult stage. This suggests that IIS plays important roles in the post-embryonic nervous system. Recently, analysis of Drosophila motorneurons, mushroom body neurons, and IPCs has revealed important roles of PI3K and Rheb in synapse growth or axon branching. These studies revealed some growth regulatory functions of IIS in the CNS, but they have not explored whether the control of neuronal growth by IIS is temporally regulated (Gu, 2014).

This study has shown that IIS strongly stimulates organizational growth of neurons during metamorphosis, whereas the effects of IIS on larval neurons are comparatively modest. Recently, similar results have been reported in mushroom body neurons, in which the TOR pathway strongly promoted axon outgrowth of γ-neurons after metamorphic pruning. Expression of FOXO or reduction of InR function had no significant effect on larval growth of the CCAP/bursicon neurons, or on the soma size of many other larval neurons. Thus, while IIS has been shown to regulate motorneuron synapse expansion in larvae, the current findings indicate that IIS may not play a major role in regulating structural growth in many larval neurons. This is consistent with a recent report that concluded that the Drosophila larval CNS is insensitive to changes in IIS (Gu, 2014).

When InRact was used to activate IIS without ligand, a modest but significant increase was seen in the soma size of the more anterior CCAP/bursicon neurons during larval development. This result indicates that the IIS pathway is present and functional in these larval neurons, but the ligand for InR is either absent or inactive. During metamorphosis, unlike in larvae, downregulation of IIS by altering the level of InR or downstream components of the pathway significantly reduced CCAP/bursicon neuron growth. Thus, the results suggest that IIS is strongly upregulated during metamorphosis to support post-embryonic, organizational growth of diverse peptidergic neurons, and this activation may at least in part be due to the presence of as yet unidentified InR ligands during metamorphosis (Gu, 2014).

Attempts were to identify this proposed InR ligand source by eliminating, in turn, most of the known sources of systemic DILPs. None of these manipulations had any effect on metamorphic growth of the CCAP/bursicon neurons. These results are consistent with three possible mechanisms. First, there may be a compensatory IIS response to loss of some dilp genes. For example, a compensatory increase in fat body DILP expression has been observed in response to ablation of brain dilp genes. Second, the growth may be regulated by another systemic hormone (e.g. DILP8) that was not tested, or by residual DILP peptides in the RNAi knockdown animals. Third, a local insulin source may be responsible for stimulating metamorphic outgrowth of the CCAP/bursicon neurons. Consistent with this view, a recent report showed that DILPs secreted from glial cells were sufficient to reactivate neuroblasts during nutrient restriction without affecting body growth, while overexpression of seven dilp genes (dilp1-7) in the IPCs had no effect on neuroblast reactivation under the same conditions. It seems likely that glia or other local DILP sources play an important role in regulating metamorphic neuron growth, but further experiments will be needed to test this model (Gu, 2014).

When IIS was manipulated in the CCAP/bursicon neurons, changes were observed in cell body size (and sometimes shape) and in the extent of branching in the peripheral axon arbor. Although this study focused analysis of neurite growth on the peripheral axons, which are easily resolved in fillet preparations, corresponding changes were also observed in the size and complexity of the central CCAP/bursicon neuron arbor. These IIS manipulations (both upregulation or downregulation) resulted in the above structural changes as well as wing expansion defects, suggesting that the normal connectivity of the CCAP/bursicon neurons was required for proper functioning of this cellular network. This model is consistent with the observation of two subsets of morphologically distinct bursicon-expressing neurons (the BSEG and BAG neurons), which are activated sequentially to control central and peripheral aspects of wing expansion. The BSEG neurons project widely within the CNS to trigger wing expansion behavior as well as secretion of bursicon by the BAG neurons. In turn, the BAG neurons send axons into the periphery to secrete bursicon into the hemolymph to control the process of tanning in the external cuticle. Therefore, manipulation of IIS within these neurons, and the changes in morphology that result, may disrupt the wiring and function of this network. However, because the possibility cannot be ruled out that these IIS manipulations also altered neuronal excitability, synaptic transmission, or neuropeptide secretion, this study relied on measurements of cellular properties (and not wing expansion rates) when assessing the relative effects of different IIS manipulations on cell growth (Gu, 2014).

The results indicate that IIS is critical for organizational growth, which occurs during insect metamorphosis but is also seen during neuronal regeneration in other systems. However, the regenerative ability of many neurons is age-dependent and context-dependent; immature neurons possess a more robust regenerative capacity, while the regenerative potential of many mature neurons is largely reduced. In particular, the adult vertebrate CNS displays very limited regeneration, in marked contrast to the regeneration abilities displayed by the peripheral nervous system. Recent studies in mice suggest that age-dependent inactivation of mTOR contributes to the reduced regenerative capacity of adult corticospinal neurons, and activation of mTOR activity through PTEN deletion promoted robust growth of corticospinal tract axons in injured adult mice. The current genetic experiments demonstrate a requirement for activity of TOR, as well as several other IIS pathway components both upstream and downstream of TOR, in controlling organizational growth of many peptidergic neurons. This suggests that under certain conditions, the activation of IIS may be a crucial component of the conversion of mature neurons to a more embryonic-like state, in which reorganizational growth either after injury or as a function of developmental stage is possible. Given the strong evolutionary conservation of these systems and the powerful genetic tools available to identify novel regulatory interactions in Drosophila, studies on the control of organizational growth in this species hold great promise for revealing factors that are crucial for CNS regeneration (Gu, 2014). >

FOXO regulates RNA interference in Drosophila and protects from RNA virus infection

Small RNA pathways are important players in posttranscriptional regulation of gene expression. These pathways play important roles in all aspects of cellular physiology from development to fertility to innate immunity. However, almost nothing is known about the regulation of the central genes in these pathways. The forkhead box O (FOXO) family of transcription factors is a conserved family of DNA-binding proteins that responds to a diverse set of cellular signals. FOXOs are crucial regulators of cellular homeostasis that have a conserved role in modulating organismal aging and fitness. This study shows that Drosophila FOXO (dFOXO) regulates the expression of core small RNA pathway genes. In addition, increased dFOXO activity results in an increase in RNA interference (RNAi) efficacy, establishing a direct link between cellular physiology and RNAi. Consistent with these findings, dFOXO activity is stimulated by viral infection and is required for effective innate immune response to RNA virus infection. This study reveals an unanticipated connection among dFOXO, stress responses, and the efficacy of small RNA-mediated gene silencing and suggests that organisms can tune their gene silencing in response to environmental and metabolic conditions (Spellberg, 2015).

Despite its importance, almost nothing is known about how the protein components of the small RNA pathways are transcriptionally regulated in the cell. Currently only studies addressing the transcriptional regulation of germ-line small RNA pathways (piRNA) have been reported. Nothing is reported on the transcriptional regulation of the protein components of the dominant somatic cell small RNA pathways, the miRNA and siRNA pathways. This study found dFOXO at the promoters of many core small RNA pathway genes. Components of the miRNA, siRNA, and piRNA pathways are all bound by dFOXO, suggesting an integrated control of the small RNA pathways (Spellberg, 2015).

The current work focused on the core small RNA pathway genes dominant in somatic cells. The transcription of the Ago2, Ago1, and Dcr2 genes were found to be increased during dFOXO activation. The effect of this dFOXO activation is augmented RNAi efficacy even with an unchanged limiting level of the dsRNA trigger. This result suggests the RNA-mediated gene silencing response is not constant but it is tunable to cellular physiology. This notion is consistent with previous work showing enhanced RNAi-based phenotypes in a daf-2/INR mutant of C. elegans and greater knockdown of target genes with dsRNA in Drosophila S2 cells after serum starvation. Both of these conditions increase FOXO activity (Spellberg, 2015).

It is interesting to note that Dcr1, the core miRNA dicer, does not seem to be a dFOXO target. This finding is despite the fact that the core miRNA argonaute, Ago1, is a dFOXO target. There is limited evidence for Ago1 involvement in inhibiting viral replication. However, there is evidence showing changes in the miRNA RISC and enhanced silencing by miRNAs under serum-starved conditions. This effect is achieved through the increased recruitment of GW182 (Gawky) to the miRNA RISC. Based on dFOXO ChIP data, GW182 is also a dFOXO target. Rather than dealing directly with a viral infection, dFOXO's up-regulation of these miRNA factors may be a stress responsive mechanism to repress translation initiation, a previously described role for dFOXO during stress (Spellberg, 2015).

dFOXO was found to be activated by viral infection to a comparable level as another well-defined physiological signal, serum starvation. Activated dFOXO can decrease viral load in cell culture and is required for effective resistance to RNA virus infection. The FOXO family of transcription factors responds to a multitude of cellular and extracellular signals. The current study shows dFOXO provides a link among cellular physiology, the RNAi pathway, and innate immunity enhancing the effectiveness of silencing and allowing the RNAi pathway to respond dynamically to changes in cellular homeostasis (Spellberg, 2015).

The importance of the RNAi pathway for viral immunity in invertebrates is well defined. However, the role of RNAi in viral immunity for mammals is still an open question. The mammalian cellular innate immune system differs from lower organisms, relying strongly on the IFN response during a viral infection. However, in cell types that lack a fully developed IFN response, RNAi plays an important role in viral defense. Additionally, several viruses that infect mammalian cells contain genes that suppress the RNAi response. This result suggests an ongoing battle between RNAi-based innate immunity and viruses. There is a growing appreciation for the role of FOXOs in mammalian immune regulation. If conservation of the function of FOXO-small RNA regulation exists in mammals, there are potential therapeutic benefits (Spellberg, 2015).


EFFECTS OF MUTATION

Foxo is a key regulator of the insulin signaling pathway. As expected, Foxo activity is inhibited via the InR/PI3K/Akt pathway. It was also found that Foxo activates transcription of a major downstream target (4EBP) of this pathway. In Drosophila, this pathway has been linked to cell size and cell number regulation. It was of interest to discover whether Foxo expression in vivo would affect these same parameters. To address this point, transgenic flies that overexpressed Foxo were generated by using the UAS/GAL4 system. Foxo expression was directed to the eye by using eyeless-GAL4 earller than wild type. Interestingly, the reduction in eye size (35%) was caused by a reduction in cell number (673 ± 24 ommatidia in control eyes vs. 438 ± 50 ommatidia in ey-GAL4/UAS-Foxo), but no significant change in cell size was observed (85.8 ± 5.9 area units/ommatidia in control eyes vs. 91.2 ± 4.8 area units/ommatidia in ey-GAL4/UAS-Foxo). When GMR-GAL4, which directs expression in cells posterior to the morphogenetic furrow was used to drive Foxo expression, a more severe phenotype was observed. Many ommatidia were lost, and the remaining ommatidia lacked bristles and appeared disorganized, altering the general structure of the eye. These results suggest that Foxo overexpression can severely affect normal development of the eye (Puig, 2003).

The effect of Foxo overexpression was tested in an organ other than the eye. dpp-GAL4 was used to direct expression of Foxo in the wing region encompassed by the third and fourth longitudinal veins. Foxo overexpression results in a significant reduction of compartment size. This reduction in size is caused by a reduction in cell number but not in cell size (71 ± 7.5 cells/area unit in the control vs. 71 ± 5.5 cells/area unit in dpp-GAL4/UAS-Foxo). Ectopic expression of Foxo in the wing using MS1096-GAL4 produces a more striking reduction in wing size, again because of loss of cell number with no significant variation in cell size (70.6 ± 6.5 cells/area unit in the control vs. 74.8 ± 11.8 cells/area unit in MS1096-GAL4/UAS-Foxo) (Puig, 2003).

Akt can phosphorylate and inactivate Foxo in S2 cells. It was therefore important to know whether Akt also inhibits the phenotypic effects of Foxo in flies expressing Foxo, Akt, or both. Indeed, Akt expression partially rescues the eye phenotype observed with Foxo, showing that both these proteins interact genetically. These results provide further evidence of both proteins acting in the same pathway (Puig, 2003).

Forkhead transcription factors belonging to the FOXO subfamily are negatively regulated by protein kinase B (PKB) in response to signaling by insulin and insulin-like growth factor in Caenorhabditis elegans and mammals. In Drosophila, the insulin-signaling pathway regulates the size of cells, organs, and the entire body in response to nutrient availability, by controlling both cell size and cell number. A genetic characterization has been reported for foxo, the only Drosophila FOXO ortholog. Ectopic expression of foxo and human FOXO3a induces organ-size reduction and cell death in a manner dependent on phosphoinositide (PI) 3-kinase and nutrient levels. Surprisingly, flies homozygous for foxo null alleles are viable and of normal size. They are, however, more sensitive to oxidative stress. Furthermore, Foxo function is required for growth inhibition associated with reduced insulin signaling. Loss of Foxo suppresses the reduction in cell number but not the cell-size reduction elicited by mutations in the insulin-signaling pathway. By microarray analysis and subsequent genetic validation, 4EBP, which encodes a translation inhibitor, has been identified as a relevant Foxo target gene. These results show that Foxo is a crucial mediator of insulin signaling in Drosophila, mediating the reduction in cell number in insulin-signaling mutants. It is proposed that in response to cellular stresses, such as nutrient deprivation or increased levels of reactive oxygen species, Foxo is activated and inhibits growth through the action of target genes such as 4EBP (Jünger, 2003).

To assess whether Foxo has a key function in insulin signaling like that of DAF-16 in C. elegans, tests were performed to see whether overexpression of wild-type or mutant forms of hFOXO3a and foxo could antagonize insulin signaling. Elimination of the three PKB consensus phosphorylation sites in mammalian FOXO3a prevents its phosphorylation, subsequent binding to 14-3-3 proteins, and sequestration in the cytoplasm. This leads to constitutive nuclear localization of the mutant FOXO3a and transcriptional activation of its target genes. Assuming that blocking the PKB signal would have the same activating effect on Drosophila Foxo, wild-type and triple PKB-phosphorylation-mutant (TM) variants of both foxo and human FOXO3a were expressed. Furthermore, an EP transposable element insertion was identified in the second foxo intron, which permits the GAL4-induced overexpression of endogenous foxo. The GMR-Gal4 construct was used to drive UAS-dependent expression in postmitotic cells in the eye imaginal disc. While expression of wild-type hFOXO3a in the developing eye did not result in a visible phenotype, triple mutant constitutively active hFOXO3a-TM expression causes pupal lethality. Few escaper flies eclosed and displayed a strong necrotic eye phenotype. A block of cell differentiation and necrosis was also observed when hFOXO3a-TM was expressed in cell clones in the developing eye (Jünger, 2003).

Assuming that the lack of a phenotype observed upon UAS-hFOXO3a expression is due to hFOXO3a inactivation by endogenous Drosophila Inr signaling in the eye disc, the same experiment was performed in a background of reduced insulin signaling. Indeed, in the presence of a dominant-negative (DN) form of Dp110 (encoding the PI 3-kinase catalytic subunit), hFOXO3a expression induces a necrotic phenotype similar to the one observed with the hyperactive phosphorylation mutant. To confirm that hFOXO3a is responsive to Drosophila insulin signaling and rule out artificial coexpression effects, hFOXO3a was expressed in flies mutant for either dPKB or Dp110, and similar phenotypes were observed as those seen upon coexpression of Dp110DN. Drosophila FOXO has qualitatively similar, but stronger effects. Expressing the wild-type form of foxo causes a weak eye-size reduction and disruption of the ommatidial pattern even in a wild-type background, and the phenotype is strongly affected by Dp110DN as well. The UAS-Foxo-TM transgene appears to cause lethality even in the absence of a Gal4 driver, as no viable transgenic lines were obtained with this construct. Furthermore, the effects of nutrient deprivation on FOXO-expressing tissues was examined. If nutrient availability is limited, FOXO should be more active in response to lowered insulin signaling. Indeed, the overexpression phenotypes of both hFOXO3a and foxo are enhanced under conditions of starvation. Drosophila larvae that are starved until 70 h after egg laying (AEL) die within a few days. But if the onset of nutrient deprivation occurs after they have surpassed the metabolic '70 h change', they survive and develop into small adult flies. Therefore larvae expressing hFOXO3a or foxo (under GMR control) were subjected to either protein starvation (sugar as the only energy source) or complete starvation, starting 80-90 h AEL, and the effect on the adult's eyes was analyzed. Both phenotypes are progressively exacerbated by protein starvation and complete starvation, the latter condition being accompanied by early adult or larval lethality, in the case of hFOXO3a or foxo, respectively. The resulting phenotypes are due to the foxo transgenes, because wild-type control flies that have been starved during development display only a body-size reduction while maintaining normal proportions and normal eye structure (Jünger, 2003).

The foxo overexpression phenotype does not appear to be caused by the activation of any of the known cell-death pathways. Expression of the caspase inhibitors p35 or DIAP1, or of p21, an inhibitor of p53-induced apoptosis, and loss of eiger, which encodes the Drosophila homolog of tumor necrosis factor (TNF), did not suppress the eye phenotype. In agreement with these results, it was observed in a parallel study that the GMR-Foxo overexpression phenotype is insensitive to caspase inhibitors, and is not accompanied by increased acridine-orange-detectable apoptosis in the imaginal disc (Kramer, 2003). It therefore remains unclear whether high levels of nuclear Foxo induce a specific caspase-independent cell-death program or whether nuclear accumulation of overexpressed Foxo leads to secondary necrosis in a rather nonspecific fashion. Furthermore, the necrotic eye phenotype does not reflect the phenotype observed following a complete block in insulin signaling. Loss-of-function mutations in insulin-signaling components reduce cell size and cell number but do not increase cell death in larval tissues. In summary, these overexpression experiments are consistent with a model in which, under normal conditions, excess Foxo transcription factor is phosphorylated by PKB and kept inactive in the cytoplasm. Under conditions of reduced insulin-signaling activity or nutrient deprivation, Foxo or hFOXO3a protein translocates to the nucleus and induces growth arrest and necrosis (Jünger, 2003).

foxo loss-of-function mutants are viable, have no overgrowth phenotype and are hypersensitive to oxidative stress. Although these overexpression experiments did not reveal the physiological function of Foxo, they provided an entry point for isolation of loss-of-function mutations. Use of the EP35-147 element permits the generation of the necrotic eye phenotype by driving expression of endogenous foxo in the presence of Dp110DN. Homozygous EP males were mutagenized, mated to homozygous GMR-Gal4 UAS-Dp110DN females and then the F1 generation was screened for reversion of the strong gain-of-function phenotype and its associated semilethality. Several loss-of-function alleles of foxo were isolated and molecularly characterized. Two such revertants were characterized. In Foxo21 and Foxo25, the codons for W95 and W124 within the forkhead domain are mutated to stop codons, respectively, so they are assumed to be null alleles of Foxo (Jünger, 2003).

Because FOXO transcription factors have been proposed to be the primary effectors of insulin signaling, on the basis of epistasis of daf-16 over daf-2 in C. elegans, it seemed reasonable to expect an overgrowth phenotype in Foxo-/- flies as is observed in dPTEN loss-of-function mutants. Surprisingly, foxo loss-of-function mutants are homozygous-viable and display no obvious phenotype under normal culturing conditions. Thus, foxo is not essential for development. Only close inspection of the foxo mutants reveals that their wing size is significantly reduced. But cellular and organismal growth are unaffected by foxo mutations. To assess whether Foxo-mutant tissue grows to a different size than wild-type tissue, the Foxo21 and Foxo25 alleles were recombined onto the FRT82 chromosome and genetic mosaic flies were induced with the ey-Flp/FRT system. When the eye and head capsule were composed almost exclusively of Foxo-/- tissue, no head-size difference was observed compared to the control fly with a head homozygous for the FRT82 chromosome without the foxo mutation. This is consistent with experience from extensive genetic screens carried out for recessive growth mutations. An ey-Flp-screen on the right arm of chromosome 3 did not reveal any mutations in foxo based on an altered head-size phenotype (Jünger, 2003).

It was next asked whether cell size, like organ size, is not affected by the loss of Foxo. For this purpose, a heat shock-inducible Flp construct was used to generate clones of homozygous Foxo-/-photoreceptor cells and wild-type cells within one adult eye. The cells lacking Foxo are marked by the absence of pigment granules. Consistent with the absence of a 'bighead' phenotype, Foxo-/- cells and wild-type cells have the same size. Similarly, no significant difference in the body weight of mutant and control flies was observed. In contrast, flies with a viable heteroallelic combination of dPTEN loss-of-function alleles are significantly bigger than wild-type flies. Taken together, these results argue that with the exception of the slight wing-size reduction, Foxo is not required to control cellular, tissue, or organismal growth in a wild-type background (Jünger, 2003).

A critical role has been reported for mammalian and C. elegans FOXO proteins in resistance against various cellular stresses, in particular oxidative stress, DNA damage and cytokine withdrawal. The stress resistance of adult foxo mutant flies was tested by measuring survival time following different challenges. Among starvation on water, oxidative-stress challenge, bacterial infection, heat shock, and heavy-metal stress, the only condition for which hypersensitivity was observed is oxidative stress. When placed on hydrogen-peroxide-containing food, foxo mutant flies display a significantly reduced survival time compared to control flies. A very similar effect is elicited by paraquat feeding. These observations are consistent with the paraquat hypersensitivity of daf-16 mutants in C. elegans, suggesting that a role for FOXO proteins in protecting against oxidative stress is conserved across species (Jünger, 2003).

The growth-deficient phenotypes of Inr, chico, Dp110 and PKB mutants are significantly suppressed by loss of Foxo. Genetic epistasis experiments were performed to examine whether the growth phenotypes of Inr-signaling mutants are dependent on Foxo function. For this purpose, either double-mutant flies were generated or the double-mutant effect only in the head was investigated using the ey-Flp/FRT system. In contrast to the absence of a growth phenotype in single foxo mutant flies, lack of Foxo significantly suppresses the growth-deficient phenotype observed in flies mutant for the insulin receptor substrate (IRS) homolog chico. Flies mutant for chico are smaller because they have fewer and smaller cells. Loss of one foxo copy dominantly suppresses the cell-number reduction in chico mutant flies without affecting cell size. The suppression is more pronounced when both copies of foxo are removed in a chico mutant background. In this situation, the chico small body-size phenotype is partially suppressed. Homozygous chico-Foxo double-mutant flies have more, and even slightly smaller, cells than homozygous chico single mutants. It seems that removal of foxo accelerates the cell cycle at the expense of cell size in a chico background (Jünger, 2003).

It was next asked whether foxo interacts with other components of the Drosophila insulin-signaling pathway. The ey-Flp/FRT system was used to generate heterozygous insulin-signaling mutant flies with heads homozygous for each mutation. Removal of Inr, Dp110 or PKB leads to a characteristic 'pinhead' phenotype, which is substantially suppressed by the presence of a foxo loss-of-function allele on the same FRT chromosome as the insulin-signaling mutation. In all three cases, a partial rather than a complete rescue of the tissue growth repression was observed, consistent with the finding that foxo mutations affect only the cell-number aspect of the chico phenotype. Surprisingly, loss of Foxo dramatically delays lethality in PKB mutants. Complete loss of PKB leads to larval lethality in the early third instar, but homozygous PKB-Foxo double mutants are able to develop into pharate adults of reduced size, most of which fail to eclose. The lethality associated with the complete loss of PKB is therefore largely due to hyperactivation of Foxo (Jünger, 2003).

It was also observed that foxo interacts with the tumor suppressors TSC1 and PTEN. Tissue-specific removal of either gene from the head leads to a bighead phenotype. The TSC1-/- bighead phenotype is enhanced by loss of Foxo. This observation is consistent with the recently reported negative feedback loop between Drosophila S6K and PKB. Mutant TSC1 larvae have elevated levels of S6K activity, which in turn downregulates PKB activity. This reduction in PKB activity probably leads to enhanced activation of Foxo, which in turn partially mitigates the overgrowth phenotype by slowing down proliferation. The TSC1 phenotype can therefore be enhanced by loss of the inhibitory function of Foxo. Unexpectedly, the PTEN-/- bighead phenotype is slightly suppressed by foxo mutations. From the current model, it would be expected that in a PTEN mutant PKB activity is high and Foxo is to a large extent inactive in the cytoplasm. Thus, removal of Foxo function should have no effect on the PTEN phenotype. At present, it is possible only to speculate about possible explanations for this observation. In a parallel study, it has been shown that Foxo can induce transcription of DInr (Puig, 2003). It may be that in a dPTEN-mutant background Foxo activates Inr expression in a negative-feedback loop. In this model, concomitant loss of Foxo would alleviate the PTEN overgrowth phenotype by lowering Inr levels. Another possible explanation is that Foxo has additional functions when localized to the cytoplasm or during its nuclear export, such as interacting with other proteins. Loss of Foxo might affect the function of interaction partners that have a role in PTEN signaling (Jünger, 2003).

In summary, this epistasis analysis provides strong genetic evidence that Foxo is required to mediate the organismal growth arrest that is elicited in insulin-signaling mutants (Jünger, 2003).

Studies with several components of the Drosophila InR/PI3K/Akt pathway have shown that insulin promotes growth and proliferation by activating PI3K and Akt. Foxo activity is inhibited by Akt upon insulin treatment. It was therefore interesting to ask whether induction of Foxo would inhibit growth in S2 cells. To do so, S2 cells stably transfected with either Foxo or mutant dFoxoA3 (Constitutively active Drosophila Foxo in which all three putative Akt phosphorylation sites were mutated to alanine) were cultured in the presence of serum and insulin for 48 h. Then exogenous Foxo and dFoxoA3 expression was induced with CuSO4 for a period of 8 h. Subsequently, Cu2+ was removed, and cells were then allowed to divide for 1 wk with samples taken every 12 h to measure cell numbers. Cells stably transfected with the wild-type protein proliferate at the same rate irrespective of Foxo induction. In contrast, cells stably transfected with the mutant protein, dFoxoA3, display a significantly slower growth rate when compared with the same cells grown without dFoxoA3 induction. Because insulin was present throughout these experiments (inactivating Foxo but not dFoxoA3), these findings suggest that the constitutively active dFoxoA3 can induce cell arrest. Importantly, cells expressing dFoxoA3 arrest growth during the first 44 h after induction when dFoxoA3 is present. After 44 h, when dFoxoA3 has apparently been turned over, cells recover and start dividing normally again. FACS analysis of samples taken during the different time points indicates that S2 cells arrest their growth at G2/M. These results indicate that activation of Foxo can induce cell cycle arrest and this effect is mitigated by insulin (Puig, 2003).

Expression of Drosophila FOXO regulates growth and can phenocopy starvation

Components of the insulin signaling pathway are important regulators of growth. The FOXO transcription factors regulate cellular processes under conditions of low levels of insulin signaling. Expression of Drosophila Foxo during early larval development causes inhibition of larval growth and alterations in feeding behavior. Inhibition of larval growth is reversible upon discontinuation of Foxo expression. Expression of Foxo during the third larval instar or at low levels during development leads to the generation of adults that are reduced in size. Analysis of the wings and eyes of these small flies indicates that the reduction in size is due to decreases in cell size and cell number. Overexpression of Foxo in the developing eye leads to a characteristic phenotype with reductions in cell size and cell number. This phenotype can be rescued by co-expression of upstream insulin signaling components, PI3K and Akt, however, this rescue is not seen when Foxo is mutated to a constitutively active form. It is concluded that Drosophila Foxo is conserved in both sequence and regulatory mechanisms when compared with other FOXO homologs. The establishment of Drosophila as a model for the study of FOXO transcription factors should prove beneficial in determining the biological role of these signaling molecules. The alterations in larval development seen upon overexpression of Foxo closely mimic the phenotypic effects of starvation, suggesting a role for Foxo in the response to nutritional adversity. This work has implications in the understanding of cancer and insulin related disorders, such as diabetes and obesity (Kramer, 2003).

Ectopic expression of Foxo during development phenocopies starvation and alters feeding behavior. Drosophila larvae feed continuously for about 5 days after egg laying (AEL). During this time the appetite and growth rate of the larvae is enormous. If young larvae are deprived of food, they do not grow and tend to disperse randomly. When the food supply is replenished, the larvae immediately move towards it and continue eating until they are close to pupation. If the food supply is depleted, the larvae will disperse again. The UAS/Gal4 ectopic expression system was used to overexpress Foxo in the developing larvae under the control of the ActGal4 driver. This results in complete developmental arrest of the larvae, which remain as first instar for up to 7 days, similar to the life expectancy of starved larvae. This trend was also seen using a constitutively active version of murine Foxo1 (mFoxo1) containing an alanine substitution at the T1 (T24A), and S1 (S253A) Akt phosphorylation sites (mFoxo1-AA). In addition, larvae expressing Foxo and mFoxo1-AA are often found to be wandering far from their food supply. Feeding behavior was monitored by assessing the number of larvae away from their food at 48 and 72 hours after egg laying (AEL). Larvae expressing Foxo and mFoxo1-AA show a 3-4 fold increase in wandering over larvae expressing Gal4 alone. Thus, Foxo expression drastically alters feeding behavior and is able to induce a starvation type response in larvae that have an adequate food supply (Kramer, 2003).

In Drosophila, PI3K consists of an adaptor subunit, dp60, and a catalytic subunit, dp110. Unexpectedly, expression of an inhibitory or 'dominant negative' version of dp110 (UAS-dPI3K-DN) under the control of the ActGal4 does not lead to increased larval wandering. Expression of this construct also does not appear to inhibit larval growth, whereas other negative regulators of insulin signaling do. It is possible that the level of expression of this construct is not high enough under the control of the ActGal4 driver to have a complete dominant negative effect (Kramer, 2003).

Starved larvae that are developmentally arrested are able to resume growth upon acquisition of food. Whether larvae that are expressing Foxo could resume growth upon termination of Foxo expression was analyzed. To do this the hsGal4 driver was used. Foxo was expressed in the larvae by heat shock treatment (HST) for 10 minutes every 24 hours. This treatment is sufficient to inhibit growth while allowing controls to survive to adulthood with a 48 hour delay in the time to pupation. When Foxo expression was discontinued after 2, 4, and 6 days of HST, developmentally arrested larvae were able to recover with decreased levels of survival as time progressed. Significant lethality was observed in controls as well suggesting that low survival rate is partially due to the expression of Gal4, which can induce apoptosis, or the HST itself. Nevertheless, developmental arrest caused by Foxo is clearly reversible since these individuals could be returned to their normal path of development (Kramer, 2003).

The formation of dauer larvae in C. elegans is a developmental response to nutrient limitation. The dauer larvae provides a temporary defense mechanism allowing the nematode to persevere until nutrients are available, at which point development can continue. Interestingly, constitutive activation of Daf-16 by mutation of its Akt phosphorylation sites to alanine residues causes obligatory dauer larvae formation. A similar result was found in the Drosophila larvae using the constitutively active mFoxo1-AA. This construct has an effect similar to that of Foxo when expressed under the control of ActGal4, and hsGal4. Upon removal from HST, larvae expressing mFoxo1-AA do not resume growth but remain in a state of developmental arrest until death. Although a few larvae did survive to adulthood after 2 days of HST, none of the larvae were able to continue development after 4, or 6 days of HST. Out of 450 larvae examined at all time points, only 10 expressing mFoxo1-AA survived, when compared to 110 and 180 for larvae expressing Foxo, and Gal4 alone, respectively. Presumably this occurs because Akt is unable to deactivate mFoxo1-AA, allowing it to continue functioning long after expression is induced. Taken together, this data suggests that Foxo is evolutionarily conserved in function, possibly playing a role in the response to nutritional adversity, as seen in the formation of dauer larvae in C. elegans (Kramer, 2003).

Expression of Foxo in the third instar larvae causes significant lethality, however, rare flies that do survive are much smaller than control flies, showing a phenotype similar to that caused by mutations in chico, Akt and Inr. Expression of Foxo under the control of the ubiquitous low level Gal4 drivers, armadillo-Gal4, and hsGal4 (raised at 25°C with no heat shock) had very little effect on growth. In contrast, increasing expression of Foxo using the hsGal4 driver in flies raised at 29°C leads to the development of small adults, which are approximately half the weight of control flies. Analysis of the wings of these flies shows that the wing area was reduced by nearly one third and that this reduction was due to a decrease in both cell size and cell number. SEM analysis of the eyes reveals reductions in both ommatidia number and ommatidia area, which reflect cell number and cell size, respectively. These results implicate Foxo in the control of body size through alterations in cell size and cell number (Kramer, 2003).

When Foxo is expressed in the developing eye under the control of the GMR-Gal4 driver, the eye is smaller, lacking many ommatidia and nearly all of the mechanosensory bristles. The remaining ommatidia are arranged in the typical hexahedral array and cross sectional analysis reveals that all of the normal photoreceptor cells are present. Thus, it appears that Foxo expression causes a reduction in the number of cells but does not interfere with cellular differentiation and the organization of the ommatidia themselves. This eye phenotype was used to test for interactions between Foxo and other components of the insulin signaling pathway (Kramer, 2003).

Expression of PI3K-DN under the control of GMR-Gal4 leads to the formation of relatively normal eyes with fewer and smaller cells. When Foxo is co-expressed in the developing eye with PI3K-DN, the eye is nearly obliterated. In contrast, co-expression of Akt, and wild type PI3K with Foxo causes nearly complete rescue of the phenotype, restoring the ommatidia and nearly all of the mechanosensory bristles. Thus, diminishing insulin signaling (through overexpression of PI3K-DN) allows for greater activity of Foxo, and enhancing insulin signaling (through overexpression of Akt or PI3K) leads to inhibition of Foxo activity. Similar results were obtained using a Murine Foxo1 (mFoxo1) construct, indicating that the regulatory mechanisms between these two proteins is conserved and that they are functionally interchangeable (Kramer, 2003).

The constitutively active mFoxo1-AA construct was also expressed in the developing eye. Expression of this construct causes a phenotype similar to that of Foxo and mFoxo1, with characteristic lack of ommatidia and mechanosensory bristles. When mFoxo1-AA is co-expressed with Drosophila PI3K-DN, the eye is nearly obliterated, as seen with Foxo and mFoxo1. Co-expression of mFoxo1-AA with PI3K leads to a partial rescue of the phenotype, with still an obvious lack of ommatidia and mechanosensory bristles. In contrast, co-expression of mFoxo1-AA with Drosophila Akt does not cause rescue of the ommatidia or mechanosensory bristles, indicating that this construct is not responsive to Akt signaling. The partial rescue of the Foxo phenotype by PI3K appears to be mediated through alterations in cell size rather than cell number, since there is still an obvious lack of ommatidia and mechanosensory bristles. This data indicates that inactivation of Foxo is required for the full effects of growth mediated by PI3K and Akt (Kramer, 2003).

To examine the effect of Foxo overexpression on cell size, the area of the ommatidia was measured. Expression of Foxo, mFoxo1, and mFoxo1-AA causea a significant reduction in the area of the ommatidia. Expression of PI3K causes a significant increase in ommatidia size over wild type. This result is consistent with previous studies showing that PI3K affects cell size in a cell autonomous manner. Co-expression of Foxo, mFoxo1, and mFoxo1-AA with dPI3K has no significant effect on the enlarged ommatidia. Thus, it appears that FOXO proteins have a very minimal effect on cell size in the presence of high levels of PI3K. Surprisingly, this is the case even with the mFoxo1-AA construct, which is only partially responsive to PI3K signaling. This indicates that the Drosophila PI3K mediated increase in cell size can occur through Akt independent mechanisms (Kramer, 2003).

Expression of Akt in the developing eye causes a significant increase in ommatidia size, similar to that seen with PI3K. Co-expression of Drosophila Akt with either Foxo or mFoxo1, cause a slight, but insignificant decrease in the size of the enlarged ommatidia. However, co-expression of Drosophila Akt with mFoxo1-AA results in ommatidia that are approximately the same size as the ommatidia in eyes expressing Gal4 alone, and significantly smaller than the ommatidia in eyes expressing Akt alone. This indicates that the deactivation of FOXO by Akt is essential for Akt to induce an increase in cell size (Kramer, 2003).

The lack of ommatidia and mechanosensory bristles caused by Foxo expression suggests a reduction in cell number during eye development. Reduction of cell number can occur through either increased cell death, or decreased of cell proliferation. The Drosophila inhibitors of apoptosis, Diap1 and Diap2, and the baculovirus inhibitor of apoptosis, p35, were unable to rescue the phenotype caused by Foxo expression. In addition, acridine orange staining of eye imaginal discs expressing Foxo showed no increase in apoptosis when compared to controls. Drosophila Epidermal Growth Factor Receptor (Egfr) signaling acts to protect differentiated cells from death during eye development. It was thought that the pro-survival effects of Egfr may be sufficient to suppress the phenotype caused by Foxo overexpression. Co-expression of Egfr with Foxo, however, does not rescue the Foxo phenotype since ommatidia and bristles are clearly still missing. Conversely, Foxo does not appear to affect the phenotype of Egfr overexpression since the general disorganization of the ommatidia appears to be the same. Thus, it appears that these two mechanisms are acting independently. Taken together, these results suggest that Foxo overexpression does not cause cell death during eye development because direct inhibitors of the apoptotic machinery (p35 and Diap1/2) and a known cell survival factor (Egfr) are unable to rescue the Foxo phenotype (Kramer, 2003).

Since inhibition of apoptosis could not rescue the phenotype caused by Foxo overexpression in the eye, an examination was performed to see if activating the cell cycle could inhibit the phenotype. Expression of the E2F and Dp transcription factors has been shown to promote cell proliferation in the wing imaginal disc. Co-expression of E2F and Dp with Foxo is not sufficient to rescue the Foxo phenotype. Overexpression of constitutively active Drosophila Ras1 (dRas1V12) has been shown to induce ectopic cell proliferation and G1/S progression in the Drosophila wing disc. Co-expression of dRas1V12 with Foxo is lethal, a constitutively active version of Drosophila Ras2 (dRas2V14) was used. Although Ras2 has not been characterized for its role in cell cycle control, it is possible that it has a similar function to Ras1. Expression of UAS-dRas2V14 under the control of GMR-Gal4 led to extreme overgrowth of the eye, lack of ommatidial organization, and the formation of huge ommatidia. Co-expression of dRas2V14 with Foxo is sufficient to restore many of the ommatidia and mechanosensory bristles lost through overexpression of Foxo alone. A similar effect was observed upon co-expression of dRas2V14 with mFoxo1. In contrast, the loss of ommatidia and bristles seen upon over expression of mFoxo1-AA is not rescued by dRas2V14 . This suggests that dRas2V14 inhibits Foxo via a Akt phosphorylation dependent mechanism (Kramer, 2003).

Thus, Foxo as a negative controller of growth and organism size, which is regulated by components of the Drosophila insulin signaling pathway, PI3K and Akt. Through overexpression studies in the developing eye, it has been shown that Foxo is regulated by PI3K and Akt in a manner that is consistent with the regulatory mechanisms deduced through studies in C. elegans and mammalian cell culture. In addition, overexpression of Foxo in the larvae reduces larval growth, phenocopies the effects of nutritional stress, and causes alterations in feeding behavior. With this in mind, it is proposed that Foxo is involved in the response of Drosophila larvae to nutritional stress (Kramer, 2003).

The FOXO homologs appear to play an evolutionarily conserved role in the control of cellular processes under conditions of low levels of insulin signaling. These experiments have provided three lines of evidence supporting the conservation of this mechanism in Drosophila. (1) Foxo shows strong sequence homology to Daf-16 and the human FOXO homologs. One significant characteristic is the high conservation of the three consensus Akt phosphorylation sites, suggesting that Drosophila Akt is most likely able to phosphorylate Foxo in vivo, as shown biochemically with the mammalian FOXO homologs. (2) These experiments show that Foxo and mFoxo1 cause nearly identical phenotypic responses when overexpressed in the developing Drosophila eye. This suggests that the activity of these proteins is highly conserved as is observed when the C. elegans FOXO homolog, Daf-16, is expressed in mammalian cell culture. (3) The phenotypic effects of FOXO overexpression can be modulated by alterations in the insulin signaling pathway. Reduced insulin signaling leads to a drastic enhancement of the phenotype that results from expression of FOXO factors. In contrast, increased insulin signaling tends to mask these phenotypes, in a manner that is dependent on the integrity of the Akt phosphorylation sites. As a result, it is believed that regulation of FOXO is conserved in Drosophila, and that this will be a very useful system in elucidating the function of FOXO transcription factors in a model organism (Kramer, 2003).

Results show that ectopic Foxo expression can mediate reduction in cell size and cell number. However, the mechanisms by which these reductions occur are still unclear. Net reduction in cell number may occur through decreased cell proliferation or increased apoptosis. Insulin and other growth factors that activate PI3K and Akt have been implicated as potent survival factors in mammalian cell culture. They prevent cell death, in part, by inhibition of FOXO factors and it has been shown that FOXO3a can upregulate expression of the pro-apoptotic protein Bim. In Drosophila, reduction of insulin signaling can lead to apoptosis in the developing embryo. It is possible that this increase in apoptosis is a result of Foxo activation, however, when Foxo is expressed in the developing eye there is no apparent increase in apoptosis, nor is the phenotype suppressed by inhibition of caspases, or by co-expression of a known cell survival factor, Egfr. These apparent discrepancies may be the result of tissue specific differences. In mammalian cell culture, induction of cell death by FOXO factors seems to be limited to non-transformed hematopoietic cell lineages. In Drosophila, loss of Akt function, inhibition of PI3K, or overexpression of PTEN, all induce cell death in the embryo. However, in imaginal disc cells lacking PI3K function, there is no increase in apoptosis. Thus, the cells in the embryo and imaginal discs may react differently to reduced levels of insulin signaling. Although no induction of apoptosis upon Foxo expression is observed, it is possible that increased levels of Foxo activity (e.g., through dominant negative inhibition of PI3K) do cause apoptosis (Kramer, 2003).

Studies in mammalian cell culture have implicated FOXO factors in control of the cell cycle through increased expression of the cyclin dependent kinase inhibitor p27Kip1. It is possible that the reduction of cell number seen upon Foxo expression is a result of cell cycle inhibition. Co-expression of an activated version of Drosophila Ras2 (dRas2V14) is sufficient to increase cell number in the presence of Foxo. Ras1 has been shown to induce growth in Drosophila imaginal discs through activation of PI3K and the transcription factor Myc. Although there is very little information available about Ras2, it is possible that the function of Ras2 overlaps with that of Ras1. Expression of dRas2V14 in the developing eye does cause a phenotype that suggests overgrowth of cells, and the dRas2V14 interaction with Foxo appears to be dependent on Akt signaling. This is not surprising considering that Ras1 and mammalian Ras have been shown to activate PI3K signaling. Interestingly, increasing the cell cycle through overexpression of the transcription factors E2F and Dp does not rescue the cell number deficit seen upon overexpression of Foxo. This suggests the possibility that activation of Foxo may override the function of other growth promoting factors, such as dMyc, which mediates Ras1 induced G1/S progression. Supporting this, it has been observed that increased growth as mediated by Akt is entirely dependent on its ability to inactivate Foxo. Furthermore, increased growth mediated by PI3K appears to be dependent on Foxo inactivation with respect to increased cell number, but not cell size. In humans, inactivation of FOXO factors may play an important role in tumor suppression by down regulating expression of D-type cyclins, thus inhibiting cell cycle progression and transformation. It will be interesting to test the interactions between Foxo and other cell cycle promoters to determine the extent of Foxo dominance over cell proliferation (Kramer, 2003).

In addition to its effect on cell number, Foxo is able to control cell size. The ability of Akt to increase cell size is dependent on Foxo inactivation, however, Drosophila PI3K does not need to inactivate Foxo to increase cell size. The difference between dPI3K and Akt might be attributed to greater activity of the UAS-dPI3K transgene. However, expression of these constructs individually yields very similar results indicating that this is probably not the case. This suggests that PI3K may control size through Akt-independent mechanisms. One possibility is through the positive growth regulator, S6k. Akt appears to increase growth through inhibition of a TSC1/TSC2 (tuberous sclerosis) complex. This complex acts through inhibition of TOR (target of rampamycin), which promotes growth through activation of S6K. Although it appears that Akt can upregulate growth through S6K, S6K activity is not reduced in larvae lacking Akt or PI3K. These results do not necessarily suggest that PI3K and Akt can not activate S6K, since S6K levels may be maintained through amino acid signals. S6K activity is dependent on phosphoinositide dependent kinase (PDK1), which interacts genetically with Akt, PI3K, PTEN, and Inr. Thus, it is possible that PI3K can modulate S6K activity through PDK1, independently of Akt (Kramer, 2003).

Studies in C. elegans indicate that insulin signaling is a critical mediator of longevity and stress resistance. One of the most well-studied stress responses is the Daf-16 mediated formation of the dauer larvae under conditions of starvation and/or crowding. Several lines of evidence indicate that Foxo may play a similar role in Drosophila larvae. (1) Drosophila larvae are deprived of food prior to 70 hours AEL, they live in a state of developmental arrest for several days before death. However, when starved after 70 hours AEL, the larvae are able to develop into adults that are reduced in size. This alteration in developmental response has been termed the '70 hour change'. and is likely determined by the minimum size required for a Drosophila larvae to enter pupation. The '70 hour change' can be mimicked through overexpression of Foxo at different stages of larval development, in the presence of ample food. For example, ubiquitous high level expression of Foxo in the early larvae (i.e., before 70 hours AEL) leads to developmental arrest, whereas heat shock induced expression of Foxo during the third instar (i.e. ,after 70 hours AEL) leads to the development of small adults. (2) The normal development of starved larvae can resume upon the acquisition of food. Similarly, developmental arrest caused by expression of Foxo prior to the '70 hour change' can be reversed if Foxo expression is discontinued. Developmental arrest caused by expression of mFoxo1-AA before the '70 hour change' is not reversible, suggesting a constitutive starvation type response as seen in C. elegans when Daf-16 phosphorylation sites are mutated. Interestingly, the reversibility of FOXO induced arrest has also been observed in mammalian cell culture. (3) Under conditions of poor nutrition or crowding larval development does not cease, but the larval period is extended and small adults are produced. This effect has been replicated through low level expression of Foxo during the course of development. (4) Feeding behavior is drastically altered in larvae expressing Foxo, causing them to wander away from their food. These larvae are often found crawling on the sides and lids of Petri dishes. This response may provide a selective advantage in the search for food as seen in C. elegans dauer larvae, which often crawl up to the highest point possible in hopes of attaching to passing organisms that could move the larvae to new locations with better food supply. Taken together, these results suggest that Foxo activity may act to promote survival during times of nutritional stress in a manner that recapitulates the formation of dauer larvae in C. elegans. It is tempting to speculate that Foxo plays a role in response to other forms of stress, as observed with Daf-16. Mammalian FOXO factors have been implicated in the protective response to oxidative stress and FOXO factors are upregulated in response to caloric restriction in rat skeletal muscle. Thus, it is possible that FOXO factors provide an evolutionarily conserved switch, by which an organism can alter its developmental program in order to promote survival under harsh conditions (Kramer, 2003).

Activation of insulin signaling causes larvae to wander away from their food. A similar effect was observed through overexpression of Foxo, which acts in opposition to insulin signaling. It is possible that hyperactivation of insulin signaling may lead to depletion of the hemolymph by increasing the cellular uptake of nutrients. This would lead to increased hunger and cause the larvae to wander in search of food. Since PI3K activity is lost under conditions of starvation, it stands to reason that Foxo would be active under these conditions. Being a transcription factor, endogenous Foxo could activate a host of genes under conditions of starvation leading to a 'genetic starvation profile'. Indeed gene expression is drastically altered upon starvation. Thus, Foxo may induce larval wandering through expression of a sub-set of genes which are normally active during starvation, whereas activation of insulin signaling may induce larval wandering by causing physiological changes that lead to a false sense of starvation (Kramer, 2003).

Foxo and aging

Insulin-IGF receptor (InR) signaling has a conserved role in regulating lifespan, but little is known about the genetic control of declining organ function. This study describes progressive changes of heart function in aging fruit flies: from one to seven weeks of a fly's age, the resting heart rate decreases and the rate of stress-induced heart failure increases. These age-related changes are minimized or absent in long-lived flies when systemic levels of insulin-like peptides are reduced and by mutations of the only receptor, InR, or its substrate, Chico. Moreover, interfering with InR signaling exclusively in the heart, by overexpressing the phosphatase PTEN or the forkhead transcription factor FOXO, prevents the decline in cardiac performance with age. Thus, insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its systemic effect on lifespan. The aging fly heart is a model for studying the genetics of age-sensitive organ-specific pathology (Wessells, 2004).

In Drosophila ageing is slowed when insulin-like signalling is reduced: life expectancy is extended by more than 50% when the insulin-like receptor (InR) or its receptor substrate (Chico) are mutated, or when insulin-producing cells are ablated. But it has yet to be resolved whether insulin affects ageing, or whether insulin signals regulate ageing directly or indirectly through secondary hormones. C. elegans lifespan is also extended when insulin signalling is inhibited in certain tissues, or when repressed in adult worms, and this requires the forkhead transcription factor (FOXO) encoded by daf-16. The Drosophila insulin-like receptor mediates phosphorylation of Foxo, the equivalent of nematode Daf-16 and mammalian FOXO3a. Drosophila Foxo regulates ageing when activated in the adult pericerebral fat body. It is further shown that this limited activation of Foxo reduces expression of the Drosophila insulin-like peptide dilp-2 synthesized in neurons, and represses endogenous insulin-dependent signalling in peripheral fat body. These findings suggest that autonomous and non-autonomous roles of insulin signalling combine to control ageing (Hwangbo, 2004).

To investigate whether activated Foxo affects ageing in Drosophila, foxo was conditionally expressed in specific adult tissues. Without ligand binding at the insulin-like receptor, Foxo remains unphosphorylated and is transported to the nucleus where it promotes factors that retard cell growth and proliferation. Drosophila was transformed with UAS-constructs, containing either a wild-type full-length complementary DNA of foxo (UAS-foxo) or Foxo with the three protein kinase B (PKB) phosphorylation sites mutated to permit insulin-insensitive nuclear transport (UAS-foxo-TM). Expression of these constructs in the eye disc reduced growth, as has previously been reported for independent transformants of UAS-foxo and for a phosphorylation-site mutant of human FOXO3a. The constitutive expression of UAS-foxo or UAS-foxo-TM killed larvae when promoted from actin-GAL4, or when expressed from fat body (adh-GAL4) or neurons (ELAV-GAL4). Therefore, conditional expression of foxo is required to bypass developmental lethality as well as to study its impact on ageing exclusively in the adult stage (Hwangbo, 2004).

The mifepristone inducible-GAL4 system (annotated P{Switch} and GeneSwitch) was used to drive the expression of UAS constructs in defined adult tissues. Ingested mifepristone strongly induces reporter expression at all ages, and the compound alone has no effect on adult survival. Adult survival is not improved when UAS-foxoTM is induced by a pan-neuronal driver (ELAV-GeneSwitch), or in glial cells (P{Switch} MB221) or neurolemma (P{Switch} S113). Thus, broadly activated dFOXO in neuron-associated cells is not sufficient to slow ageing; however, it may do so if expressed in subsets of cells within these tissues. Similarly, expression of UAS-foxo-TM or UAS-foxo did not affect survival when induced with the P{Switch} strain S1106, an efficient promoter in the fat body. In contrast, survival is significantly increased in both sexes when foxo is induced with the P{Switch} strain S132, which is also expressed in fat body. Multiple independent inserts of UAS-foxo-TM and of wild-type UAS-foxo increase median lifespan by as much as 35% when induced with 25microg/ml mifepristone and 56% when induced by 50microg/ml mifepristone; averaged across trials, lifespan was increased by 15.5% in males and 19.4% in females. Because the phosphatase and tensin homologue protein (PTEN) antagonizes phosphatidylinositol-3-OH kinase activity, which promotes nuclear localization of endogenous Foxo and inhibits Tor function, UAS-Pten was induced with S1106 and S132. Survival was unaffected when Pten was expressed from S1106 but increased by about 20% when expressed from SS13232. Together, these data demonstrate that Foxo activated in a specific tissue can regulate lifespan in adult flies (Hwangbo, 2004).

To understand how S132 but not S1106 can improve lifespan, their patterns of expression were compared in adult tissue. S1106 is expressed in fat body of the thorax, abdomen and occasionally in the cavity surrounding the mouthparts. In contrast, S132 is expressed in fat body of the head and not in the abdomen or thorax; S32 uniquely appears in the pericerebral fat body located above the brain. The insertion site of S132 maps to the first intron of bunched. Although bunched was identified in egg follicles, S132 does not express in these cells, perhaps because it is inserted near two intronic open reading frames. It is concluded that specifically activated foxo in the adult head fat body is sufficient to slow ageing. Surprisingly, although UAS-foxo-TM (expressing activated Foxo) induced by S132 increases longevity it does not affect fecundity. In contrast, when adults are challenged with an acute oxidative stress agent (paraquat), survival is improved when UAS-foxo-TM is induced by S132 (in the head fat body) but not by S1106, in agreement with long-lived insulin signalling mutants of C. elegans and Drosophila that are often stress resistant. Similarly, lipids are frequently elevated in C. elegans and Drosophila insulin-signalling mutants and as anticipated, when foxo-TM is expressed in the head fat body, lipid aggregates appear in this tissue. Remarkably, in the same animals, lipids also accumulated in the peripheral fat tissue even though this construct is not expressed outside the head (Hwangbo, 2004).

To understand how Foxo-TM that is expressed exclusively in the head fat body can regulate integrated physiological traits such as ageing, stress resistance and lipid metabolism, the cellular location of the Foxo protein was followed in the head and peripheral fat body. Without transgene induction, endogenous Foxo is distributed throughout the cytoplasm in all cells. On expression of UAS-foxo-TM, antibody-labelled Foxo was increased in both the cytoplasm and nuclei of the targeted tissue. Notably, activated Foxo induced by S132 in head fat body also increases endogenous Foxo nuclear localization of peripheral fat body, in agreement with the pattern of lipid accumulation. In contrast, Foxo-TM expressed in peripheral fat body does not affect endogenous Foxo in the head. Since endogenous Foxo in peripheral tissue can become localized in the nucleus in response to decreased insulin signalling, these results suggest that Foxo that is activated in the head fat body retards systemic levels of the insulin ligand (Hwangbo, 2004).

Cells in the pars intercebralis of the adult brain synthesize insulin peptides. To test whether activated Foxo in the head fat body influences insulin production messenger RNA levels of the seven Drosophila insulin-like peptides (dilp) was measured. Complementary DNA was prepared from the heads of S132 /UAS-foxo-TM adults fed mifepristone or treated as controls. In a preliminary screen, multiple independent samples were specifically analysed for dilp message abundance using microarrays: dilp-2 alone is reduced in response to activated Foxo in the head fat body. These samples were used to perform quantitative polymerase chain reaction with reverse transcription (RT-PCR) to determine robustly the relative abundance of dilp message originating in the adult brain: the dilp-2 message decreased nearly threefold whereas dilp-3 and dilp-5 were unchanged. Therefore, insulin signalling within the head fat body influences transcription of one specific dilp of the neuronal insulin-producing cells (Hwangbo, 2004).

Studies across model systems have established that insulin-like signalling can control lifespan non-autonomously from a limited set of cells or a specific tissue. In C. elegans, these cells may occur primarily in the intestine and secondarily in neurons. In mice, a disrupted insulin receptor in the adipose tissue across all life stages alters adult adipose morphology, decreases fasted insulin levels and modestly increases adult survival. This study shows with the fly that activated Foxo in the head fat body is sufficient to increase both male and female lifespan, to increase resistance to oxidative challenge and to alter whole-animal lipid metabolism. Therefore, in Drosophila systemic secondary signals must function downstream of Foxo, activated in the head fat body. Both juvenile hormone and 20-hydroxyecdysone are reduced in Drosophila mutants of InR, and both these hormones have the potential to regulate lifespan. Candidates for secondary hormonal signals have yet to be identified in C. elegans but these may involve sterols because Daf-9, a cytochrome P450 related to mammalian steroidogenic hydroxylases, functions downstream of Daf-2 but upstream of Daf-12, which encodes a putative nuclear hormone receptor (Hwangbo, 2004).

The data suggest that an insulin peptide itself may function as one secondary messenger of insulin-regulated ageing in D. melanogaster. A similar model is emerging for C. elegans: the peptide encoded by ins-7 accelerates ageing and is a systemic agonist of the daf-2 encoded receptor, whereas functional DAF-16 in the intestine non-autonomously activates DAF-16 in distant tissues and is sufficient to increase lifespan. This study finds that Dilp-2 is uniquely reduced when Foxo is activated in the head fat body of Drosophila; this transcriptional change will decrease the amount of circulating insulin peptide released from insulin-producing neurons. Notably, ablation of neuronal cells expressing Dilp-2 is sufficient to retard demographic ageing and to retard functional decline of the adult heart. Ageing of the heart, however, can also be delayed when insulin signalling is inhibited exclusively within the cardiac tissue itself, including heart-specific expression of activated Foxo. If senescence of tissue and systems throughout the adult is regulated autonomously by insulin, decreased circulation of this peptide downstream of regulatory insulin action within the head fat body could extend lifespan by reducing the mortality risk associated with degeneration of the soma as a whole (Hwangbo, 2004).

JNK extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling

Aging of a eukaryotic organism is affected by its nutrition state and by its ability to prevent or repair oxidative damage. Consequently, signal transduction systems that control metabolism and oxidative stress responses influence life span. When nutrients are abundant, the insulin/IGF signaling (IIS) pathway promotes growth and energy storage but shortens life span. The transcription factor Foxo, which is inhibited by IIS, extends life span in conditions of low IIS activity. Life span can also be increased by activating the stress-responsive Jun-N-terminal kinase (JNK) pathway. This study shows that JNK requires Foxo to extend life span in Drosophila. JNK antagonizes IIS, causing nuclear localization of Foxo and inducing its targets, including growth control and stress defense genes. JNK and Foxo also restrict IIS activity systemically by repressing IIS ligand expression in neuroendocrine cells. The convergence of JNK signaling and IIS on Foxo provides a model to explain the effects of stress and nutrition on longevity (Wang, 2005).

The data suggest Foxo as a convergence point for IIS and JNK signaling. Through its responsiveness to these two pathways, Foxo is well positioned to integrate information about environmental stress and nutrient availability and to elicit appropriate biological responses. Such a system would ensure that growth could proceed in an unrestrained manner when energy resources are available and the cell is not exposed to external insults (IIS is active, JNK is off, and Foxo is repressed). However, in situations of low food availability or an adverse environment, IIS would cease to signal, or JNK would be activated, resulting in translocation of Foxo to the nucleus. The ensuing Foxo-induced gene expression has several effects at the cell as well as the organism level and is likely to counteract premature senescence. The induction of genes such as thor can reduce cell growth, presumably to limit the cell's anabolic expenses in adverse situations. Other target genes, such as the small heat shock protein l(2)efl, are expected to have a direct role in allaying damage inflicted by environmental insults and may prevent the accumulation of toxic protein aggregates. The suppression of dilp2 expression by JNK and Foxo in insulin-producing cells (IPCs), in contrast, is likely to control growth, metabolism, and stress responses systemically by downregulating IIS in all responsive tissues in a coordinated fashion (Wang, 2005).

The interaction between JNK and Foxo is thus expected to influence stress tolerance and life span at two levels. In peripheral tissues, JNK activates Foxo and prevents senescence cell-autonomously. Such a mechanism is exemplified by the recent finding that Foxo overexpression prevents age-dependent decline of cardiac performance (Wessells, 2004). Systemic control of IIS by JNK-mediated activation of Foxo in IPCs, in contrast, would serve to coordinate cellular responses to changes in the environment throughout the organism. The data indicate that this latter mechanism plays a significant role in the regulation of life span by JNK and Foxo. The identification of this endocrine function of JNK/Foxo signaling supports and extends the proposed role of JNK signaling on longevity and demonstrates a role for IPCs in life span regulation. In addition to controlling growth and metabolism, IPCs may thus act as a coordination point for the organism's stress response by downregulating Dilp production in response to oxidative stress and JNK activation. In target tissues, such a mechanism would induce protective gene expression by the second, cell-autonomous tier of Foxo signaling. Interestingly, the effects of IPC-specific JNK activation on longevity and growth are separable. Life span can be extended by moderately increased JNK activity in IPCs when growth effects are yet not evident. This finding is consistent with observations that the extension of life span in IIS loss-of-function situations is not a mere consequence of small body size (Wang, 2005).

How did such a multilayered regulation of IIS activity by JNK evolve? It is tempting to speculate that localized activation of Foxo is required to prevent cellular damage and ultimately senescence in conditions in which stressful insults are confined to specific tissues. Such localized insults could, for example, be inflicted by reactive oxygen species that are produced in the environment of amyloid deposits in Alzheimer's disease as well as by mechanical and oxidative stress experienced by particularly active tissues such as the heart. Systemic regulation of Foxo activity, in contrast, is expected to be an important response mechanism to coordinate metabolism and stress defenses throughout the organism upon changes in the environment. A good example for such a mechanism is the induction of diapause in invertebrates in response to environmental stress or food deprivation. Accordingly, sensory neurons expressing the insulin-like peptide DAF-28 are required for the induction of the dauer larval stage in response to environmental cues in C. elegans (Wang, 2005).

Systemic and tissue-autonomous effects of JNK/Foxo signaling may be connected in multiple ways. The data indicate that JNK and Foxo interact in IPCs to repress dilp2 expression, ultimately activating Foxo in Dilp2 target tissues in a coordinated fashion. Since JNK was found to be activated in IPCs even under normal culture conditions, it is likely that this systemic control of IIS activity by JNK and Foxo plays a critical role in life span regulation. It is, however, also possible that the cell-autonomous protective function of JNK/Foxo signaling is most critical for the survival of specific tissues as the organism ages, thus extending life of the organism by preventing the loss of indispensable cells or tissues. In addition, stress and the JNK-mediated activation of Foxo in peripheral tissues may signal back to IPCs to initiate a systemic response. In Drosophila, such a mechanism has been documented in the case of the fatbody. Activation of Foxo in this tissue relays a signal to the IPCs, causing them to curb Dilp2 production, a process that has been proposed to require Foxo activity (Hwangbo, 2004). The exact nature of this feedback signaling mechanism in flies is unclear, but it is reminiscent of the complex signaling interactions between β cells and insulin target tissues in mammals. Further studies are required to shed light on the relative contributions of JNK/Foxo signaling in IPCs or Dilp target tissues to life span regulation (Wang, 2005).

JNK-mediated modulation of IIS activity is likely to be evolutionarily conserved. Inhibitory crosstalk from JNK to IIS in mammalian cells has been found to occur by JNK-mediated phosphorylation and inhibition of IRS-1. This interaction is responsible for obesity-induced insulin resistance in mice. Whether mammalian homologs of Foxo take part in this pathology remains to be determined. A second possible mechanism for JNK/IIS pathway interaction is the direct phosphorylation and activation of Foxo by JNK. A recent study supports such a mechanism, showing that in mouse cells JNK can phosphorylate the DFoxo homolog Foxo4 in response to oxidative stress. The physiological relevance of this phosphorylation event has not yet been addressed. The JNK target residues on IRS-1 and Foxo4 are not conserved in the Drosophila homologs Chico and DFoxo, and further studies are thus required to determine whether JNK-Foxo crosstalk in Drosophila is mediated via homologous mechanisms (Wang, 2005).

The systemic regulation of IIS activity by JNK and Foxo appears to be conserved as well. It has been suggested that C. elegans Daf16/Foxo regulates life span (at least in part) by reducing the expression of insulin-like peptides. In mammals, pancreatic β cells (the counterparts of IPCs) reduce their production of insulin in response to oxidative stress-mediated JNK activation. Conversely, dephosphorylation of JNK by MAPK phosphatase 1 can induce insulin expression in these cells. Reducing circulating insulin levels by JNK-mediated Foxo activation may thus be a general mechanism that balances growth and metabolism with stress defense and damage repair (Wang, 2005).

Akt and FOXO dysregulation contribute to infection-induced wasting in Drosophila
Studies in Drosophila have taught us a great deal about how animals regulate the immediate innate immune response, but we still know little about how infections cause pathology. This study examines the pathogenesis associated with Mycobacterium marinum infection in the fly. M. marinum is closely related to M. tuberculosis, which causes tuberculosis in people. A microarray analysis shows that metabolism is profoundly affected in M. marinum-infected flies. A genetic screen identifies foxo mutants as slower-dying after infection than wild-type flies. FOXO activity is inhibited by the insulin effector kinase Akt; it was shown that Akt activation is systemically reduced as a result of M. marinum infection. Additionally, flies infected with Mycobacterium marinum undergo a process like wasting: they progressively lose metabolic stores, in the form of fat and glycogen. They also become hyperglycemic. In contrast, foxo mutants exhibit less wasting. In people, many infections—including tuberculosis—can cause wasting, much as is seen in Drosophila. This study is the first examination of the metabolic consequences of infection in a genetically tractable invertebrate and gives insight into the metabolic consequences of mycobacterial infection, implicating impaired insulin signaling as a key mediator of these events. These results suggest that the fly can be used to study more than the immediate innate immune response to infection; it can also be used to understand the physiological consequences of infection and the immune response (Dionne, 2006).

This study shows that flies infected with M. marinum undergo a process like wasting. This wasting response is in part the consequence of systemic failure of Akt activation and consequent activation of the transcription factor FOXO, resulting in a diabetes-like state. The observed failure of Akt activation is not caused by traditional insulin resistance mechanisms. Data suggest that mycobacterial infection causes a systemic reduction in Akt activation, either by reducing the level of circulating insulin or by increasing the turnover of activated Akt, or both. This results in excessive Gsk-3 and FOXO activity, which causes the progressive loss of energy stores. The study argues that the impaired insulin signaling seen in M. marinum-infected Drosophila is likely to represent a common response to many infections in many hosts, including humans, and moreover is likely a significant cause of disease morbidity (Dionne, 2006).

It was shown that foxo mutants are longer-lived when infected with M. marinum. This makes an intriguing contrast with earlier observations that flies overexpressing foxo are longer-lived than wild-type flies when uninfected. Moreover, foxo mutant flies die more rapidly than wild-type flies under conditions of oxidative stress. These observations suggest that death from old age or oxidative stress is mechanistically different from death from M. marinum infection (Dionne, 2006). 

In humans, insulin is among the most important anabolic signals, and it is also a satiety signal: type-1 diabetics (who progressively lose the insulin-producing cells of the pancreas) exhibit increases in appetite and consequently in food intake, even as they are progressively losing body mass. In fly larvae, insulin-like peptides (ILPs) appear to play a role roughly analogous to the metabolic role seen in humans: Larvae in which the insulin receptor (InR) or PI3 kinase (PI3K) are overexpressed accumulate excessive levels of fat and show reduced feeding. Conversely, larvae that lack ILP-producing cells (IPCs) in the brain become hyperglycemic (Dionne, 2006). 

In adult flies, the metabolic role of ILPs appears to be more complex. The loss of ILP signaling via IPC ablation or loss-of-function mutations in chico (the fly homolog of mammalian IRS proteins) or InR results in increased triglyceride and glycogen storage—although, in the case of IPC ablation, this increase in energy stores is still accompanied by hyperglycemia. Studies using a temperature-sensitive InR allele show that the critical period for the increase in metabolic storage is during pupariation, indicating that the storage effect should be regarded as a developmental defect rather than a physiological one. This study is the first examination of the results of insulin inhibition in wild-type adult flies without developmental perturbation. It suggests that the insulin signaling pathway acts in adult flies to drive glucose uptake and energy storage in a manner analogous to its action in mammals and larval Drosophila (Dionne, 2006). 

Immune responses pose significant costs for the host. One hypothesis suggests that the primary cost of immune responses is energetic: that metabolic energy used by the immune system is being taken from other important systems. This has been easiest to observe in cases where animals are placed under energy constraints and then forced to raise an immune response: In these situations, the induced immune responses have easily observable deleterious effects on other physiological processes. Conversely, this cost is also visible as immunosuppression in animals that are carrying out other energy-intensive activities (Dionne, 2006). 

These observations suggest that there should be mechanisms for direct control of energy allocation to the immune response. Data from this study indicate that Akt and Foxo form an important component in this regulation. The study speculates that the systemic disruption of insulin signaling may be a mechanism by which insects reduce energy allocation to nonimmune tissues; moreover, a similar mechanism might operate in mammals. The clinical literature is rich with examples of metabolic changes resulting from a variety of infections. Tuberculosis and other chronic infections can be associated with slow wasting of fatty and lean tissues and glucose intoleranc; acute bacteremia tends to be associated with rapid wasting and full-scale hyperglycemia. Resting insulin levels are an excellent predictor of survival in septic patients, and aggressive treatment of septic hyperglycemia with exogenous insulin dramatically increases survival. Wasting alone could be accounted for simply by the energetic cost of the immune response; however, infections often cause hyperglycemia as well, suggesting that systemic changes in metabolic regulation may be the underlying cause of infection-induced wasting. That is, wasting may be a pathological consequence of regulated energy reallocation (Dionne, 2006).

Other possibilities should not be overlooked: in particular, the possibility that the metabolic changes observed in the fly might be part of a pathogenic strategy on the part of the bacterium. Organisms living in the circulation can easily double their local glucose concentration simply by degrading circulating insulin. In this reading, the fact that a wide variety of infections cause hyperglycemia in mammals would be a result of the fact that this strategy is such an attractive one for a pathogen that it has been selected many times independently. However, the apparent connection between increased levels of proinflammatory cytokines and cachexia in mammals leads to the connection that infection-induced wasting is primarily a consequence of the host response—though one that is ripe for exploitation by some classes of pathogen (Dionne, 2006).

A Drosophila model for age-associated changes in sleep:wake cycles

One of the most consistent behavioral changes that occurs with age in humans is the loss of sleep consolidation. This can be quite disruptive and yet little is known about its underlying basis. To better understand the effects of aging on sleep:wake cycles, this problem was studied in Drosophila. By assaying flies of different ages as well as monitoring individual flies constantly over the course of their lifetime, it was found that the strength of sleep:wake cycles decreased and that sleep became more fragmented with age in Drosophila. These changes in sleep:wake cycles became faster or slower with manipulations of ambient temperature that decreased or increased lifespan, respectively, demonstrating that they are a function of physiological rather than chronological age. The effect of temperature on lifespan was not mediated by changes in overall activity level or sleep amount. Flies treated with the oxidative stress-producing reagent paraquat showed a breakdown of sleep:wak cycles similar to that seen with aging, leading to a proposal that the accumulation of oxidative damage with age contributes to the changes in rhythm and sleep. Together, these findings establish Drosophila as a valuable model for studying age-associated sleep fragmentation and breakdown of rhythm strength, and indicate that these changes in sleep:wake cycles are an integral part of the physiological aging process (Koh, 2006).

Aging is associated with an accumulation of oxidative damage. Young flies lacking the FOXO protein, which protects cells from oxidative stress in mammalian tissue culture, show rhythm phenotypes. In particular, foxo mutant flies are unable to sustain sleep:wake rhythms in the presence of the oxidative stress-producing agent paraquat. These data suggested that an increase in oxidative damage can cause deterioration of sleep:wake cycles. To determine whether paraquat can produce similar effects in wild-type flies, flies were maintained on food containing 1 mM paraquat throughout life. At this low dosage, wild-type flies could live on average for ~1 month, which is approximately half of their normal lifespan. For the first 3 weeks of treatment, paraquat had little effect on sleep:wake cycles. At ~3 weeks of age, however, compared with control flies, flies treated with paraquat showed a faster rate of decrease in rhythm strength and average duration of sleep bouts and a faster rate of increase in sleep bout numbers. Paraquat treatment also promoted faster changes in overall activity and total sleep amount in females. These measures showed little change in males after ~3 weeks and were relatively unaffected by paraquat treatment. These results resemble those found in flies whose aging process was accelerated with high ambient temperature and suggest that the effects of increased oxidative stress on sleep:wake cycles are similar to those that occur with aging (Koh, 2006).

FOXO-regulated transcription restricts overgrowth of Tsc mutant organs

FOXO is thought to function as a repressor of growth that is, in turn, inhibited by insulin signaling. However, inactivating mutations in Drosophila melanogaster FOXO result in viable flies of normal size, which raises a question over the involvement of FOXO in growth regulation. Previously, a growth-suppressive role for FOXO under conditions of increased target of rapamycin (TOR) pathway activity was described. This study further characterizes this phenomenon. Tuberous sclerosis complex 1 mutations cause increased FOXO levels, resulting in elevated expression of FOXO-regulated genes, some of which are known to antagonize growth-promoting pathways. Analogous transcriptional changes are observed in mammalian cells, which implies that FOXO attenuates TOR-driven growth in diverse species (Harvey, 2008).

To investigate mechanisms by which the TOR pathway controls tissue growth, transcriptional profiles were analyzed of tissue lacking Tsc1, which leads to hyperactivation of the TOR pathway and excessive growth. Eye-antennal imaginal discs from third instar Drosophila larvae were generated that were composed almost entirely of tissue derived from one of two different genotypes: Tsc1 or wild-type isogenic control. Three biologically independent first strand cDNA samples from each genotype were hybridized to microarray chips. Expression levels of 157 genes were elevated 1.5-fold or more, whereas 211 genes were repressed 1.5-fold or more when compared with control tissue. These genes have been implicated in diverse cellular functions including metabolism, membrane transport, stress response, cell growth, and cell structure (Harvey, 2008).

Observed transcriptional changes were validated for several genes using Drosophila gene-enhancer trap lines. The UAS-Gal4 system was used to activate the TOR pathway in a specific tissue domain by driving expression of Rheb under the control of the glass multiple reporter (GMR) promoter. Induction of astray (aay) and 4E-BP (both of which were found to be elevated in Tsc1 tissue by microarray analysis) were observed in the GMR expression domain (posterior to the morphogenetic furrow) when Rheb was misexpressed but were not induced when the negative control Gal4 gene was misexpressed. QPCR was also used to confirm expression changes observed in Tsc1 tissue for charybdis (chrb), scylla (scy), phosphoenolpyruvate carboxy kinase, 4E-BP, and aay (Harvey, 2008).

Intriguingly, several gene products whose expression was elevated in Tsc1 tissue have been implicated in tissue growth controlled by the insulin and TOR pathways, including 4E-BP, Chrb, and Scy. 4E-BP is a repressor of cap-dependent translation. Upon phosphorylation by TOR, 4E-BP dissociates from eIF4E, allowing assembly of the initiation complex at the mRNA cap structure, ribosome recruitment, and subsequent translation. Scy and Chrb, and their mammalian orthologues REDD1 and REDD2, inhibit insulin and TOR signaling in response to hypoxia and energy stress and restrict growth during Drosophila development. The finding that inhibitors of growth are highly expressed in Tsc1 tissue led to the hypothesis that such genes are transcriptionally induced as part of a feedback loop that restricts tissue growth under conditions of excessive TOR activity. Feedback loops are an important activity-modulating feature of many signaling pathways, including the TOR and insulin pathways (Harvey, 2008).

To examine the mechanism whereby transcription of growth inhibitors is induced in response to TOR hyperactivation, attempts were made to determine which transcription factors were responsible for their expression. One obvious candidate was FOXO, a member of the forkhead transcription factor family, which has a well-established role as an effector of insulin signaling. If FOXO has a role in inducing expression of negative regulators of growth in Tsc tissue, then expression of some of those genes should be elevated under conditions of increased FOXO activity. To investigate this hypothesis, the expression profiles were examined of Tsc1 LOF tissue and Drosophila S2 cells expressing FOXOA3, a mutant version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt. This analysis revealed that 25 genes were up-regulated 1.5-fold or greater in both Tsc1 LOF and FOXO GOF expression profiles, which represents a highly significant degree of overlap as determined by calculation of the hypergeometric distribution. A highly statistically significant P value strongly suggests that there is a functional overlap between these two datasets that cannot be explained by random variation (Harvey, 2008).

Interestingly, two genes previously implicated in tissue growth regulated by the insulin and TOR pathways 4E-BP and scy were elevated in both microarray experiments, whereas the chrb growth-inhibiting gene was not. Thus, a subset of genes elevated in Tsc1 tissue appears to respond to FOXO activity and was investigated further (Harvey, 2008).

4E-BP is a well-characterized FOXO target gene. To determine whether FOXO could directly activate transcription of genes that were elevated in Tsc1 tissue other than 4E-BP, focus was placed on scy and the phosphoserine phosphatase aay (one of the most highly elevated transcripts in each microarray experiment). scy and aay both possess consensus FOXO recognition elements (FREs) in their promoters comparable to those found in dInR and 4E-BP promoters. Therefore, whether these genes are bona fide FOXO targets was examined by measuring their expression in Drosophila S2 cells misexpressing FOXOA3 in the presence of insulin. aay and scy mRNAs were up-regulated 19.4- and 4.3-fold, respectively, relative to a control gene, actin, as determined by QPCR (Harvey, 2008).

Luciferase reporter assays in S2 cells were used to determine whether the aay promoter region containing putative FREs was sensitive to FOXO activity. Luciferase activity dependent on the aay promoter was strongly induced by FOXOA3. In addition, using in vitro band shift assays, it was demonstrated that FOXO directly binds to the aay promoter, indicating that FOXO likely activates expression of aay by directly binding to the FRE. Surprisingly, in parallel luciferase reporter assays, activation of the scy promoter by FOXO could not be demonstrated, despite the fact that strong binding of FOXO to the putative scy FRE was observed using in vitro band-shift assays. A possible explanation is that the scy-promoter construct lacked the minimal promoter elements required for transcription of luciferase (Harvey, 2008).

TOR pathway hyperactivation caused by Tsc deficiency has been shown to strongly repress activity of Akt. FOXO is normally inactivated by Akt-dependent phosphorylation, which restricts nuclear entry of FOXO and leads to its ubiquitin-dependent destruction. Therefore, in response to TOR pathway hyperactivation, it was predicted that reduced Akt activity would cause FOXO protein to accumulate. To examine this hypothesis, expression of FOXO protein was analyzed in mosaic Tsc1 imaginal discs. It was found that FOXO protein was markedly increased in Tsc1 clones when compared with neighboring wild-type tissue (Harvey, 2008).

In addition, FOXO protein appeared to be mostly nuclear in Tsc1 tissue and cytoplasmic in wild-type tissue. Consistent with this observation, nuclear localization of the mouse FOXO orthologue FOXO1 is observed in endothelial cells of Tsc2 mutant hemangiomas, whereas FOXO1 is mostly cytoplasmic in normal cells. FOXO mRNA levels are unchanged in Tsc1 tissue as determined by microarray analysis, which suggests that changes in translation or stability of FOXO protein account for its accumulation in Tsc1 tissue. The presence of increased FOXO protein in the nuclei of Tsc1 cells is consistent with the hypothesis that FOXO is responsible for increased expression of some of the growth inhibitors that are up-regulated in Tsc1 cells (Harvey, 2008).

To determine whether FOXO was necessary for transcriptional induction of genes that were elevated in Tsc1 tissue, QPCR analysis was used to measure 4E-BP, aay, and scy expression in Tsc1 and Tsc1-FOXO double mutant eye-antennal imaginal discs. Consistent with microarray analysis, increased expression of 4E-BP, aay, and scy was observed in Tsc1 tissue. In Tsc1-FOXO tissue, however, 4E-BP was expressed at approximately equivalent amounts as in wild-type tissue, whereas aay and scy expression was only partially reduced. This demonstrates that elevated expression of 4E-BP in Tsc1 tissue is dependent on the FOXO transcription factor and provides evidence that FOXO activity increases when the TOR pathway is hyperactivated. Expression of aay and scy appear to be partially dependent on FOXO but are likely stimulated by additional transcription factors in Tsc1 tissue (Harvey, 2008).

Next, attempts were made to determine whether FOXO is required to limit growth of tissues with increased TOR pathway activity. In addition, a potential role was examined for another transcription factor, HIF-1, for retardation of TOR-driven growth. HIF-1 is a dual-subunit transcription factor consisting of α and β subunits that functions in response to insulin/TOR signaling and drives transcription of the growth-inhibiting genes scy and chrb, both of which are elevated in Tsc1 tissue (Harvey, 2008).

Drosophila possesses several HIF-1α subunits and a sole HIF-1β subunit, tango (tgo), which partners with each HIF-1α subunit. If FOXO and/or HIF-1 are required to induce expression of genes that limit tissue growth when the TOR pathway is hyperactivated, one might predict that Tsc1-FOXO and/or Tsc1-tgo double mutant tissue would possess a greater capacity to grow than Tsc1 tissue alone. To test this hypothesis, the size was examined of Drosophila eyes comprised almost entirely of the following genotypes: control, tgo, FOXO, Tsc1, Tsc1-tgo, and Tsc1-FOXO. Mutant eyes were created by driving mitotic recombination of chromosomes bearing flipase recognition target (FRT) sites and the appropriate gene mutations, specifically in developing Drosophila eye-antennal imaginal discs. Eyes lacking either tgo or FOXO were approximately the same size as control eyes, whereas Tsc1 eyes were considerably larger. Tsc1-tgo double mutant eyes did not exhibit a further increase in size, which suggests that HIF-1 is not required to inhibit tissue growth in response to Tsc1 loss. In contrast, Tsc1-FOXO double mutant eyes were substantially larger than Tsc1 eyes. This finding is particularly significant in light of the finding that eyes lacking FOXO were indistinguishable in size from wild-type eyes. Thus, it appears that FOXO is normally dispensable for control of eye size, but when growth control is altered by virtue of increased TOR activity, FOXO partially offsets the increased tissue growth. These findings are consistent with observations that FOXO protein accumulates in Tsc1 tissue and that transcriptional profiles of FOXO GOF and Tsc1 LOF cells overlap significantly (Harvey, 2008).

Because individual components of the insulin and TOR pathways are highly conserved among eukaryotes, important regulatory mechanisms that control tissue growth via these pathways are also likely to be conserved. To investigate this idea, transcriptional control was analyzed of mouse orthologues of genes that were elevated in D. melanogaster Tsc1 tissue. Initially, Northern blotting analysis was performed on Tsc2 primary mouse embryonic fibroblasts (MEFs; derived on a p53 background to overcome premature senescence induced by Tsc2 loss). It is reasonable to predict that transcriptional changes that occur because of loss of either Tsc1 or Tsc2 should be very similar because TSC1 and TSC2 function together in an obligate fashion, and mutation of either gene leads to almost indistinguishable phenotypes. It was found that several gene expression changes observed in Drosophila Tsc1 tissue are conserved in Tsc2 MEFs (Harvey, 2008).

The homologues of aay, heat shock protein (hsp) 23, scy, and chrb (PSPH, hsp 27, REDD1, and REDD2, respectively) were all significantly up-regulated in Tsc2 MEFs when compared with control MEFs and expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. To demonstrate that these expression changes were a specific consequence of Tsc2 loss, Tsc2 expression was reconstituted in Tsc2 null cells, which substantially suppressed mammalian TOR activity and expression of these genes. Interestingly, expression of phosphoenolpyruvate carboxy kinase and 4E-BP1/2 was not altered between wild-type and Tsc2 cells, which might reflect tissue- or species-specific differences in the transcriptome of Drosophila epithelial cells and MEFs (Harvey, 2008).

To determine whether the mode of transcription of these genes was also conserved in mammals, expression was analyzed of the scy homologue REDD1. Like scy, mammalian REDD1 orthologues possess a putative consensus FRE within their proximal promoters. Cotransfection of a version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt (TM-FKHRL-1) induced robust activation of a mouse REDD1 reporter construct in primary MEFs. To determine whether induction was mediated through the identified FRE, a mutant reporter was created lacking this sequence. Deletion of the REDD1 FRE consistently reduced FOXO-mediated induction of the REDD1 promoter. Finally, to directly assess whether FOXO-dependent transcription was activated in mammalian cells lacking Tsc2, activity of the REDD1 promoter reporter or the corresponding mutant FRE reporter was examined in wild-type and Tsc2 MEFs. As predicted, the wild-type REDD1 promoter exhibited robust activation in Tsc2 cells compared with wild-type cells, and this activation was substantially reduced by deletion of the FRE. Together, these findings provide evidence that transcriptional changes resulting from Tsc1/Tsc2 deficiency are conserved in diverse species (Harvey, 2008).

This study has identified of an evolutionary conserved transcriptional program important for restricting tissue overgrowth driven by excessive activation of the TOR pathway. The FOXO transcription factor plays a key role in this transcriptional response, likely by stimulating expression of several growth inhibitory genes. Thus, although the requirement for FOXO in restricting growth under normal development conditions appears dispensable, this is no longer the case under conditions of excessive TOR activation. These findings have important implications for cancer syndromes that arise because of inappropriate TOR pathway activation, such as the human hamartomatous syndrome, tuberous sclerosis. TOR-dependent feedback inhibition is thought to contribute to the benign nature of Tsc1 and Tsc2 tumors (Ma, 2005; Manning, 2005). Conceivably, inactivating mutations in FOXO family transcription factors and/or FOXO target genes that possess growth-inhibiting properties could promote further growth in normally benign Tsc1 and Tsc2 tumors (Harvey, 2008).

FOXO-dependent regulation of innate immune homeostasis

The innate immune system represents an ancient host defence mechanism that protects against invading microorganisms. An important class of immune effector molecules to fight pathogen infections are antimicrobial peptides (AMPs) that are produced in plants and animals. In Drosophila, the induction of AMPs in response to infection is regulated through the activation of the evolutionarily conserved Toll and immune deficiency (IMD) pathways. This study shows that AMP activation can be achieved independently of these immunoregulatory pathways by the transcription factor FOXO, a key regulator of stress resistance, metabolism and ageing. In non-infected animals, AMP genes are activated in response to nuclear FOXO activity when induced by starvation, using insulin signalling mutants, or by applying small molecule inhibitors. AMP induction is lost in foxo null mutants but enhanced when FOXO is overexpressed. Expression of AMP genes in response to FOXO activity can also be triggered in animals unable to respond to immune challenges due to defects in both the Toll and IMD pathways. Molecular experiments at the Drosomycin promoter indicate that FOXO directly binds to its regulatory region, thereby inducing its transcription. In vivo studies in Drosophila, but also studies in human lung, gut, kidney and skin cells indicate that a FOXO-dependent regulation of AMPs is evolutionarily conserved. These results indicate a new mechanism of cross-regulation of metabolism and innate immunity by which AMP genes can be activated under normal physiological conditions in response to the oscillating energy status of cells and tissues. This regulation seems to be independent of the pathogen-responsive innate immunity pathways whose activation is often associated with tissue damage and repair. The sparse production of AMPs in epithelial tissues in response to FOXO may help modulating the defence reaction without harming the host tissues, in particular when animals are suffering from energy shortage or stress (Becker, 2009).

In Drosophila, eight families of AMPs have been described. During infection, their induction depends on members of the nuclear factor-κB (NF-κB) family of inducible transactivators. They are regulated by two distinct signalling cascades, the Toll and IMD pathways, which are highly conserved in evolution and show strong parallels to the mammalian Toll-like receptors (TLR) and tumour necrosis factor receptor pathways. Under non-infected conditions, AMPs are synthesized constitutively in specific tissues, in particular in barrier epithelia in both insects and mammals where they probably confer a first line of defence against opportunistic and pathogenic bacteria. The regulatory mechanisms promoting constitutive and inducible AMP expression in barrier epithelia in combination with systemically induced gene expression are not well understood (Becker, 2009).

The cytohesin Steppke was recently identified as an essential component of insulin/insulin-like growth factor signalling (ILS) in both Drosophila (Fuss, 2006) and mice (Hafner, 2006). While studying the phenotype of steppke (step) mutant larvae, a significant upregulation of different AMP genes was observed in non-infected animals. Other mutants of the ILS cascade, including the Drosophila insulin receptor substrate homologue chico, likewise showed an upregulation of AMP genes in non-infected conditions. Because it is known that AMP transcription is highly sensitive to variations in the developmental stage, allelic combinations of step and chico mutants were carefully compared with wild-type controls at different stages and it was determined that the effects were not due to developmental delay of the ILS mutants. To exclude developmental effects further, AMP regulation in the adult stage was examined. Feeding adult wild-type flies for 8-10 days with SecinH3, which was previously shown to result in the downregulation of ILS, resulted in an induction of AMP levels (Becker, 2009).

Starvation is a stress situation known to phenocopy the growth defects of genetic ILS mutants and reflects the physiological condition of an animal searching for food. In a series of nutrient deprivation experiments, it was found that AMP expression was strongly induced in wild-type larvae after starvation. These data were further corroborated by recent gene expression studies showing that AMP genes are induced upon starvation in adult flies (Bauer, 2006). AMP gene expression could also be induced in Drosophila S2 cells after starvation in PBS or by growth factor removal. Together, these results indicate that reduction of ILS by pathway-specific mutation, feeding chemical inhibitors or by starvation results in AMP activation in tissue culture cells, larvae and adult flies (Becker, 2009).

The signal transducer of the ILS cascade, the forkhead transcription factor FOXO, has a pivotal role in adapting metabolism to nutrient conditions. When energy levels are low and ILS is reduced, FOXO enters the nucleus, resulting in enhanced target gene expression. To test whether the observed activation of AMP expression was dependent on FOXO activity, foxo21/W24 null mutants were analysed. It was found that AMP upregulation was lost in foxo mutant larvae after starvation and in adults after SecinH3 feeding. In control experiments, lipase 3 was shown to be properly regulated in starving foxo mutants, demonstrating that a FOXO-independent response to nutrient deprivation is still functional. In contrast, overexpression of FOXOTM, a dominant active form of the protein expressed in the nucleus, resulted in a strong induction of AMP expression in both normal fed larvae and adults. Together, the loss- and gain-of-function data demonstrate a prominent role of nuclear FOXO in the activation of AMP genes (Becker, 2009).

Sequence analysis of the regulatory regions identified highly conserved FOXO/forkhead consensus binding sites in most of the AMP genes, especially in the Drosomycin (Drs) promoter. Therefore, focus was placed on Drs. In situ hybridization showed that Drs is strongly upregulated in wild-type larvae after starvation, but not in foxo mutants, indicating that regulation of Drs is FOXO-specific. A fragment of the Drs promoter, which reproduces the endogenous Drs expression in vivo, contains a cluster of five putative FOXO binding sites and in addition a NF-κB site. This regulatory region was used to drive luciferase expression, and an upregulation of reporter gene activity and endogenous Drs expression was found when FOXO-GFP was overexpressed in S2 cells. In contrast, luciferase expression was lost in this assay when the cluster of FOXO binding sites, but not the NF-κB binding site, was deleted. The same response of luciferase expression was found in transgenic larvae after starvation as well as FOXOTM overexpression, further corroborating the functionality of these FOXO binding sites in vivo. Moreover, electrophoretic mobility band shift and supershift assays (EMSA) showed that FOXO binds directly to the FOXO sites in the Drs promoter. To test the in vivo significance of these FOXO sites further, each binding site or combinations of them was mutated. It was found that mutating the site at position -990 already strongly reduced FOXO-GFP-dependent luciferase expression, whereas it was entirely lost when four of five sites were mutated. Similar results were obtained by using just the isolated cluster of putative FOXO binding sites to drive luciferase expression, either containing wild-type or mutated sites. Together, these data provide strong evidence for an essential role of FOXO for Drs regulation (Becker, 2009).

These results show a direct regulation of AMP genes by FOXO that is dependent on the energy status of the cells. To exclude that this effect depends on a cross-regulation triggering the immunity pathways, double mutants for Relish (Rel) and spätzle (spz) were used in which both the IMD and Toll pathways are defective. It has been shown that NF-κB responses after bacterial challenge fail in these mutants. When nuclear FOXO activity was induced in Rel,spz mutant larvae by starvation, an upregulation of Drs expression was detectable. Similarly, feeding SecinH3 to adult Rel,spz double mutants resulted in an upregulation of different AMPs (<2">Fig. 2k). Together, these data show that FOXO can activate AMP expression independently of the NF-κB-derived innate immune pathways. To test whether the IMD and Toll pathways require FOXO to operate, adult foxo mutants were infected and it was found that NF-κB-derived AMP expression was strongly induced, enabling survival of the animals. This further indicates that the NF-κB pathways are operating independently of FOXO in activating AMP expression and that NF-κB signalling and not FOXO seems to be essential after an acute infection (Becker, 2009).

To obtain further insight into where the FOXO-dependent mechanism of AMP induction is operating, tissues of wild-type larvae were isolated that were either yeast fed or starved, and the expression of AMPs was analyzed. Focus was placed on barrier tissues, in which AMPs are known to be expressed in both insects and mammals, and on the fat body, which is a major site for the regulation of both energy homeostasis and systemic innate immunity in insects. All tissues showed a starvation-dependent upregulation of multiple AMPs. In agreement with this, enhanced Drs promoter activity was found in several tissues, most prominently in trachea and fat body after starvation of flies carrying a Drs-GFP reporter transgene. When FOXO-GFP was overexpressed in a mosaic pattern in the fat body using the FLP/GAL4 technique, it was found by using in situ hybridization that the FOXO-GFP-expressing cells showed a strong upregulation of Drs expression and a reduced cell size as compared to neighbouring cells not expressing FOXO-GFP. These data demonstrate that the FOXO-dependent mechanism of AMP gene regulation functions cell-autonomously (Becker, 2009).

Because FOXO is conserved from worms to humans, tests were performed to see whether a FOXO-dependent regulation of AMPs may also occur in mammalian cells. The regulation of defensin β-1 (also known as DEFB1), defensin β-3 (DEFB103A), defensin α-1 (DEFA1) and drosomycin-like defensin was examined in several human cell lines. In all these cell types, expression of AMPs was ILS-dependent, as already observed in Drosophila. This indicates that the FOXO-dependent regulation of AMP genes is conserved (Becker, 2009).

These results demonstrate a new mechanism of cross-regulation between metabolism and innate immunity, in which FOXO directly regulates AMP genes in non-infected animals in response to the oscillating energy and stress status of cells. This regulation is independent of the pathogen-responsive innate immunity pathways and seems to be evolutionarily conserved in mammals. In the course of an infection, high levels of AMPs are induced by the NF-κB-dependent immunity pathways to ensure bacterial clearance. However, AMP induction is normally downregulated within several hours, as continuous upregulation is detrimental to the host tissues. In the Drosophila gut epithelium, the induction of NF-κB-dependent AMP expression is repressed by the intestinal homeobox gene caudal, thereby regulating symbiotic interactions of commensal bacteria with the intestinal epithelium. Similarly, TLR signalling is downregulated in the mammalian intestine to avoid chronic inflammation in response to constant exposure of the host cells to microorganisms. The FOXO-dependent mechanism ensures the sparse production of AMPs, which may help maintaining and strengthening the defence barrier, in particular when animals are suffering from energy shortage or stress. This would be consistent with previous studies reporting that genetic mutants of the ILS pathway, which have an extended life span, such as chico mutants in Drosophila or daf-2, age-1 and akt-1 mutants in Caenorhabditis elegans, show enhanced pathogen resistance. It is also conceivable that interactions of the host with severe pathogens can be independent of FOXO-dependent immune and barrier functions. Along these lines it is therefore not surprising that foxo mutants can be more resistant to some infections, as exemplified by Mycobacterium marinum, a model organism for tuberculosis disease. Taken together, these data reveal a new evolutionarily conserved mechanism of cross-regulation of metabolism and innate immunity, which allows the adaptation of organismal defence to environmental conditions (Becker, 2009).

Inactivation of both Foxo and reaper promotes long-term adult neurogenesis in Drosophila

Adult neurogenesis occurs in specific locations in the brains of many animals, including some insects, and relies on mitotic neural stem cells. In mammals, the regenerative capacity of most of the adult nervous system is extremely limited, possibly because of the absence of neural stem cells. This study shows that the absence of adult neurogenesis in Drosophila results from the elimination of neural stem cells (neuroblasts) during development. Prior to their elimination, their growth and proliferation slows because of decreased insulin/PI3 kinase signaling, resulting in nuclear localization of Foxo. These small neuroblasts are typically eliminated by caspase-dependent cell death, and not exclusively by terminal differentiation as has been proposed. Eliminating Foxo, together with inhibition of reaper family proapoptotic genes, promotes long-term survival of neuroblasts and sustains neurogenesis in the adult mushroom body (mb), the center for learning and memory in Drosophila. Foxo likely activates autophagic cell death, because simultaneous inhibition of ATG1 (autophagy-specific gene 1) and apoptosis also promotes long-term mb neuroblast survival. mb neurons generated in adults incorporate into the existing mb neuropil, suggesting that their identity and neuronal pathfinding cues are both intact. Thus, inhibition of the pathways that normally function to eliminate neural stem cells during development enables adult neurogenesis (Siegrist, 2010).

These findings demonstrate that two pathways act in concert to eliminate mb neuroblasts and terminate neurogenesis. Downregulation of insulin/PI3 kinase signaling occurs first and may activate both autophagy and a program of caspase-dependent cell death. In the absence of one of these cell death pathways, mb neuroblasts persist, but only transiently. Thus a fail-safe mechanism likely exits to ensure mb neuroblast elimination, similar to salivary gland cells (Siegrist, 2010).

The reduction in growth that precedes neuroblast apoptosis appears to be developmentally regulated since it occurs at an earlier time in central brain neuroblasts than in mushroom body neuroblasts. This may be due to either local differences in microenvironments or differences in the ability of neuroblasts to respond to circulating insulin-like peptides. Moreover, the extended survival of mb neuroblasts under these conditions, but not other central brain neuroblasts, suggests that additional mechanisms such as terminal differentiation still function to ensure elimination of most neuroblasts. Indeed, during mammalian development, neural progenitors are eliminated via cell death and by terminal differentiation. The relative importance of death and differentiation for neuroblast elimination may be lineage dependent. Finally because cricket adult mb neuroblasts proliferate in response to insulin in explant cultures, a common mechanism may regulate adult neurogenesis among insects and possibly in more distantly related metazoans. These findings may represent an important first step towards devising ways to manipulate the regenerative capacity of adult brains in diverse species and provide insight into how aberrantly persisting neural stem cells behave in vivo (Siegrist, 2010).

FOXO/4E-BP signaling in Drosophila muscles regulates organism-wide proteostasis during aging

The progressive loss of muscle strength during aging is a common degenerative event of unclear pathogenesis. Although muscle functional decline precedes age-related changes in other tissues, its contribution to systemic aging is unknown. This study shows that muscle aging is characterized in Drosophila by the progressive accumulation of protein aggregates that associate with impaired muscle function. The transcription factor FOXO and its target 4E-BP remove damaged proteins at least in part via the autophagy/lysosome system, whereas foxo mutants have dysfunctional proteostasis. Both FOXO and 4E-BP delay muscle functional decay and extend life span. Moreover, FOXO/4E-BP signaling in muscles decreases feeding behavior and the release of insulin from producing cells, which in turn delays the age-related accumulation of protein aggregates in other tissues. These findings reveal an organism-wide regulation of proteostasis in response to muscle aging and a key role of FOXO/4E-BP signaling in the coordination of organismal and tissue aging (Demontis, 2010).

By using a number of behavioral, genetic and molecular assays, this study has described a mechanism in the pathogenesis of muscle aging that is based on the loss of protein homeostasis (proteostasis) and the resulting decrease in muscle strength (see FOXO/4E-BP Signaling in Muscles Controls Proteostasis and Systemic Aging). Increased activity of Pten and the transcription factor FOXO is sufficient to delay this process, while foxo null animals experience accelerated loss of proteostasis during muscle aging. Pten and FOXO induce multiple protective responses, including the expression of folding chaperones, and the regulator of protein translation 4E-BP that has a pivotal role in preserving proteostasis. FOXO and 4E-BP preserve muscle function at least in part by sustaining the basal activity of the autophagy/lysosome system, which removes aggregates of damaged proteins. However, additional mechanisms may be involved. For example, the proteasome system may degrade damaged proteins and thus avoid their accumulation in aggregates. Thus, perturbation in proteasome assembly and subunit composition may contribute to muscle aging in response to FOXO activity. In addition, while overexpression of a single chaperone had limited effects, interventions to effectively limit the extent of protein damage are likely to delay the decay in proteostasis by decreasing the workload for the proteasome and autophagy systems (Demontis, 2010).

By comparing the accumulation of poly-Ubiquitinated proteins in aggregates of aging muscles, retinas, brains, and adipose tissue, this study has found that reduced protein homeostasis is a general feature of tissue aging that is particularly prominent in muscles. The observation that muscle aging is characterized by loss of proteostasis further suggests some similarity between muscle aging and neurodegenerative diseases, many of which are characterized by the accumulation of protein aggregates (Demontis, 2010).

Mechanical, thermal, and oxidative stressors occur during muscle contraction and therefore muscle proteins may be particularly susceptible to damage in comparison with other tissues. While the current findings refer to the loss of proteostasis in the context of normal aging, it is likely that a better understanding of this process will likely help cure muscle pathologies associated with aging, as some of the underlying mechanisms of etiology may be shared. For example, most cases of inclusion body myositis (IBM) arise over the age of 50 years, defining aging as a major risk factor for the pathogenesis of this disease. Interestingly, muscle weakness in patients with IBM is characterized by the accumulation of protein aggregates, which we have now described to occur in the context of regular muscle aging in Drosophila. Thus, FOXO may interfere with the pathogenesis of muscle degenerative diseases in addition to muscle aging. Studies in animal disease models of IBM will be needed to test this hypothesis (Demontis, 2010).

There is an apparent contradiction between the current findings and data describing the FOXO-dependent induction of muscle atrophy in mice, a serious form of age-related muscle degeneration that results in decreased muscle strength. The observation that different degrees of FOXO activation can promote stress resistance or rather cell death could explain why FOXO activity can be protective or rather detrimental during muscle aging. In particular, while physiologic FOXO activation can preserve protein homeostasis and muscle function, its excessive activation may lead to decreased muscle function due to hyper-activation of protein turnover pathways. Consistent with this view, the macroautophagy pathway has also been involved in both muscle atrophy as well as in the preservation of muscle sarcomere organization, highlighting the importance of fine tuning the degree of activation of stress resistance pathways to maintain muscle homeostasis. In addition, the output of FOXO activity may radically differ in growing versus pre-existing myofibers. In particular, the current study indicates that FOXO protects pre-existing myofibers against age-dependent changes in proteostasis, while it also blunts developmental muscle growth in flies (Demontis, 2009), as observed in mammals. Thus, deleterious effects of FOXO activation as observed in mammalian muscles may result from the inhibition of growth of novel myofibers in post-natal development and adulthood, a process which is thought to be limited to development in Drosophila (Demontis, 2010).

An interesting observation of this study is that interventions that decrease muscle aging also extend the lifespan of the organism. In particular, this work raises the prospect that the extent of muscle aging may be a key determinant of systemic aging. Reduced muscle proteostasis may be detrimental per se for life expectancy, presumably due to the involvement of muscles in a number of key physiological functions. Consistent with this view, overexpression in muscles of aggregation-prone human Huntington’s disease proteins is sufficient to decrease lifespan. Moreover, FOXO signaling in muscles regulates proteostasis in other tissues, via inhibition of feeding behavior and decreased release of Insulin from producing cells, that in turn promote 4E-BP activity systemically. Thus, it is proposed that FOXO/4E-BP signaling in muscles regulates lifespan and remotely controls aging events in other tissues by bringing about some of the protection associated with decreased food intake (Demontis, 2010).

In mammals, muscles produce a number of cytokines involved in the control of systemic metabolism. For example, Interleukin-6 (IL-6) is produced by muscles and has been proposed to control glucose homeostasis and feeding behavior through peripheral and brain mechanisms. Thus, a muscle-based network of systemic aging as observed in flies may occur in humans (Demontis, 2010).

This study supports the common belief that preserving muscle function is beneficial for overall aging and the notion that muscles are central tissues to coordinate organism-wide processes, including aging and metabolic homeostasis. Moreover, the observation that FOXO signaling in muscles influences aging events in other tissues suggests that the systemic regulation of aging relies on tissue-to-tissue communication, which may provide the basis for interventions to extend healthy lifespan (Demontis, 2010).

dFOXO-independent effects of reduced insulin-like signaling in Drosophila

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. This study 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. 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. These 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 (Slack, 2011).

Silent information regulator 2 (Sir2) and Forkhead box O (FOXO) complement mitochondrial dysfunction and dopaminergic neuron loss in Drosophila PTEN-induced kinase 1 (PINK1) null mutant

PTEN-induced kinase 1 (PINK1), which is associated with early onset Parkinson disease, encodes a serine-threonine kinase that is critical for maintaining mitochondrial function. Moreover, another Parkinson disease-linked gene, parkin, functions downstream of PINK1 in protecting mitochondria and dopaminergic (DA) neuron. In a fly genetic screening, knockdown of Sir2 blocked PINK1 overexpression-induced phenotypes. Consistently, ectopic expression of Sir2 successfully rescued mitochondrial defects in PINK1 null mutants, but unexpectedly, failed in parkin mutants. In further genetic analyses, deletion of FOXO nullified the Sir2-induced mitochondrial restoration in PINK1 null mutants. Moreover, overexpression of FOXO or its downstream target gene such as SOD2 or Thor markedly ameliorated PINK1 loss-of-function defects, suggesting that FOXO mediates the mitochondrial protecting signal induced by Sir2. Consistent with its mitochondria-protecting role, Sir2 expression prevented the DA neuron loss of PINK1 null mutants in a FOXO-dependent manner. Loss of Sir2 or FOXO induced DA neuron degeneration, which is very similar to that of PINK1 null mutants. Furthermore, PINK1 deletion had no deleterious effect on the DA neuron loss in Sir2 or FOXO mutants, supporting the idea that Sir2, FOXO, and PINK1 protect DA neuron in a common pathway. Overall, these results strongly support the role of Sir2 and FOXO in preventing mitochondrial dysfunction and DA neuron loss, further suggesting that Sir2 and FOXO function downstream of PINK1 and independently of Parkin (Koh, 2012).

From these findings, the following model is proposed for PINK1-mediated mitochondrial protection. To protect mitochondria, PINK1 translocates Parkin to mitochondria and activates its E3 ubiquitin ligase activity. In mitochondria, Parkin ubiquitinates mitochondrial proteins such as voltage-dependent anion channel 1 (VDAC1) and mitofusin (Mfn) to regulate mitochondrial remodeling process. In addition to the direct action in mitochondria, PINK1 transduces signals to the cytosol and activates Sir2. Sir2 deacetylates FOXO and induces the FOXO-dependent transcription of mitochondrial protective genes including SOD2 and Thor in the nucleus. The expressed proteins locate to the cytosol or mitochondria and play their roles such as scavenging harmful reactive oxygen species (ROS) and enhancing production of mitochondrial proteins. Through the direct regulation of mitochondrial protein turnover and the induction of mitochondrial protective gene expression, PINK1 can efficiently protect cells from mitochondrial damages (Koh, 2012).

Fragile X Protein is required for inhibition of insulin signaling and regulates glial-dependent neuroblast reactivation in the developing brain

Fragile X syndrome (FXS) (FXS) is the most common form of inherited mental disability and known cause of autism. It is caused by loss of function for the RNA binding protein FMRP, which has been demonstrated to regulate several aspects of RNA metabolism including transport, stability and translation at synapses. Recently, FMRP has been implicated in neural stem cell proliferation and differentiation both in cultured neurospheres as well as in vivo mouse and fly models of FXS. Previous studies have shown that FMRP deficient Drosophila neuroblasts upregulate Cyclin E, prematurely exit quiescence, and overproliferate to generate on average 16% more neurons. This study further investigated FMRP's role during early development using the Drosophila larval brain as a model. Using tissue specific RNAi it was found that FMRP is required sequentially, first in neuroblasts and then in glia, to regulate exit from quiescence as measured by Cyclin E expression in the brain. Furthermore, the hypothesis was tested that FMRP controls brain development by regulating the insulin signaling pathway, which has been recently shown to regulate neuroblast exit from quiescence. The data indicate that phosphoAkt, a readout of insulin signaling, is upregulated in dFmr1 brains at the time when FMRP is required in glia for neuroblast reactivation. In addition, dFmr1 interacts genetically with dFoxO, a transcriptional regulator of insulin signaling. These results provide the first evidence that FMRP is required in vivo, in glia for neuroblast reactivation and suggest that it may do so by regulating the output of the insulin signaling pathway (Callan, 2012).

Although there is a clear role for FMRP in the glia, its precise function in these cells remains poorly understood. It was previously shown that FMRP is expressed in glia during embryonic development but appears downregulated postnatally in the mouse brain. Co-culture of glia and hippocampal neurons demonstrated that glial cells contribute to the neuroanatomical defects found in the Fragile X brain, albeit the factors responsible are yet to be identified. This work provides the first in vivo evidence for FMRP's requirement in glial cells, and future work will focus on using more restricted glial Gal4 drivers to dissect the contribution of different glial types to regulating neuroblast reactivation in the dFmr1 mutant brains (Callan, 2012).

Several mRNA targets have been predicted or confirmed for FMRP. Given FMRP's complex tissue specific and temporal requirements during development it is likely that more remain to be identified and confirmed in vivo. Some clues as to the possible pathways and targets regulated by FMRP in the developing brain come from recent reports that glia can provide cues (in the form of secreted dILPs) to neighboring neuroblasts awaiting a reactivation signal. These findings together with the current data showing that FMRP is required sequentially in neuroblasts, then in glia, for proper neuroblast reactivation suggest a model whereby FMRP may control the timing and/or levels of insulin signaling in the brain by acting in different tissues at different times during development. While more work is needed to fully validate this model and to identify the direct mRNA targets of FMRP during brain development, initial tests of the model were carried out by evaluating the levels of pAkt, a readout of insulin signaling in the brain. This work shows that indeed, at the time when FMRP is required in glia (12-18 h ALH), more cells belonging to neuroblast lineages express pAkt. Coupled with the genetic interaction discovered between dFmr1 and dFoxO, a downstream effector as well as inhibitor of insulin signaling, it is suggested that FMRP controls neural stem cell behavior by directly regulating components of insulin signaling. Notably, PI3K, an upstream activator of Akt has been previously shown to be a target of FMRP in the context of mGluR signaling at synapses. Thus for its autonomous function in neuroblasts, FMRP could regulate PI3K directly, while later, in glia, for its nonautonomous function, FMRP could control (directly or indirectly) the expression of the dILPs secreted by glial cells. Notably, in vertebrates, Insulin Growth Factor-1 (IGF-1) and PI3K/Akt can also promote cell-cycle progression in neural stem cells, thus raising the possibility that the current findings in the fly model may be highly relevant to the molecular mechanisms underlying FXS. While more work is needed to elucidate FMRP's role in the communication between the glial niche and neuroblasts, it is tempting to speculate that FMRP may regulate similar signaling cassettes and molecules (i.e., PI3K) in different developmental contexts. The Drosophila model offers unique opportunities to dissect tissue specific regulation such as glial versus neuroblast specific mRNA targets in future experiments (Callan, 2012).

Lowered insulin signalling ameliorates age-related sleep fragmentation in Drosophila

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. This study 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. These findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans (Metaxakis, 2014).

Sleep syndromes are highly prevalent in elderly humans and, with a continuing increase in life expectancy and a greater proportion of elderly people worldwide, effective treatments with fewer side effects are becoming increasingly needed. Sleep in flies shares striking similarities with sleep in humans, including an age-related reduction in sleep quality. This study used Drosophila to examine age-related sleep pathologies and to suppress these pathologies through genetic and pharmacological perturbation of insulin/IGF and TOR signaling (Metaxakis, 2014).

This study has shown that the highly conserved IIS pathway, with roles in growth and development, metabolism, fecundity, stress resistance, and lifespan, also affects sleep patterns in Drosophila. Reduced IIS increases and consolidates night sleep, while decreasing day sleep and inducing day activity. Interestingly, dilp2-3 double mutant flies as well as flies with neuron or fat-body-specific down-regulation of IIS showed no obvious or only mild sleep phenotypes in a previous study, suggesting that a strong and/or systemic reduction in IIS activity may be necessary to induce the activity and sleep phenotypes. Consistently, dilp2-3 double mutant flies have very mild growth, lifespan, and metabolic phenotypes compared to the dilp2-3,5 triple mutant flies used in this study. Reduced IIS activity resulted in increased sleep consolidation in young flies. Importantly, reduced IIS ameliorated the age-related decline in sleep consolidation seen in wild-type flies, thus showing that it is malleable. Contrary to the increased sleep consolidation with reduced IIS, high calorie diets have been reported to accelerate sleep fragmentation. Furthermore, dietary sugar affects sleep pattern in flies. Taken together, these findings reveal a role of nutrition and metabolism in sleep regulation and age-related sleep decline in flies (Metaxakis, 2014).

In humans, several studies suggest a link between nutrition and sleep. The amino acid tryptophan can promote sleep, possibly by affecting synthesis of the sleep regulators serotonin and melatonin. Also, the carbohydrate/fat content of the diet seemingly affects sleep parameters. However, most of these studies are based on correlational methods and small sample size, and it is not yet clear how diet affects sleep. Interestingly, sleep duration can affect metabolism, risk for obesity and diabetes, and even food preference. These findings associate sleep and metabolism; thus, manipulation of nutrient-sensing pathways, such as IIS and TOR signalling, may affect activity and sleep in humans (Metaxakis, 2014).

The transcription factor FoxO is an important downstream mediator of IIS. In C. elegans all aspects of IIS are dependent on daf-16, the worm ortholog of foxO. In contrast, in Drosophila IIS-mediated lifespan extension is dependent on dfoxo, whereas several phenotypes of reduced IIS are dfoxo-independent. Activity and sleep were unaffected by the loss of dfoxo in wild-type flies. In contrast, under low IIS conditions loss of dfoxo specifically affected daytime behaviour, with night time behaviours unaffected. Reduced IIS therefore affects day and night sleep and activity through distinct mechanisms. It also uncouples the effects of IIS on lifespan and on night sleep consolidation, since dfoxo is essential for extended longevity of flies with reduced IIS. dFOXO has been previously shown to increase neuronal excitability, possibly via transcription of ion channel subunits or other (Metaxakis, 2014).

It is suggested that a possible such regulator could be octopaminergic signalling, known to promote arousal in Drosophila. Octopamine, the arthropod equivalent of noradrenaline, regulates several behavioural/physiological processes, including glycogenolysis and fat metabolism, as well as synaptic and behavioural plasticity. Moreover, octopamine can affect sleep by acting on insulin-producing cells in the fly brain, thus linking IIS and sleep/activity. Indeed, this study found that IIS mutants have increased octopamine levels and, importantly, pharmacological inhibition of octopaminergic signalling reverted the increased day activity of IIS mutants. Noteworthy, mRNA expression of octopamine biosynthetic enzymes was not changed, but tyramine levels were significantly reduced, suggesting that increased translation, reduced degradation, or increased activity of the tyramine-β hydroxylase regulates octopamine levels in IIS mutants. In contrast to day activity, increased lifespan of IIS mutants was not affected by pharmacological inhibition of octopaminergic signalling, thus separating longevity from the day activity phenotype (Metaxakis, 2014).

The effect of reduced IIS on day sleep/activity was mediated through AKH, the equivalent of human glucagon, an antagonist of. In flies, AKH coordinates the response to hunger through mobilizing energy stores and increasing food intake, as well as inducing a starvation-like hyperactivity. Loss of AKH receptor (AkhR) abrogated the increased activity of IIS mutants without affecting night sleep. These results demonstrate that day and night phenotypes of IIS mutants can be uncoupled, suggesting that the increased night sleep of IIS mutants is not just a compensatory consequence of increased day activity (Metaxakis, 2014).

dilp2-3,5 mutants have increased octopamine levels, and loss of AkhR in the dilp2-3,5 mutant background reduced their octopamine level back to wild-type levels, suggesting that AkhR-mediated regulation of octopamine controls day hyperactivity in IIS mutants. In support of these findings, octopaminergic cells mediate the increased activity effect of AKH in other insects. Flies lacking dFOXO did not respond to chemically induced AKH release, suggesting that AKH affects activity through dFOXO. Therefore, it is suggested that dFOXO and AkhR act through overlapping mechanisms to enhance octopaminergic signalling and induce activity (Metaxakis, 2014).

In flies, AkhR is highly expressed in fat body and its loss alters lipid and carbohydrate store levels. Therefore, AkhR might indirectly enhance octopaminergic signalling through alterations in lipid and carbohydrate metabolism. In support of this idea, lipid metabolism affects sleep homeostasis in flies. Additionally, AkhR expression in octopaminergic cells could regulate octopamine synthesis and release in flies. Interestingly, expression of AkhR is altered in dfoxo mutants, thus implicating dFOXO in AkhR regulation. Both are highly expressed in fat body, an important organ for metabolism in flies, and fat-body-specific insulin receptor may regulate AkhR function through dFOXO activation (Metaxakis, 2014).

In larval motor neurons, dFOXO increases neuronal excitability and octopamine increases glutamate release, suggesting there is at least a spatial functional link between the two. Thus, together with a possible role in AkhR synthesis, dFOXO could act downstream of octopamine to increase activity (Metaxakis, 2014).

To determine the mechanism underlying the IIS-dependent amelioration of age-related sleep decline, downstream components and genetic interactors of IIS were investigated. One such interactor that affects health and ageing is TORC1. TORC1 is a major regulator of translation, through S6K, 4E-BP, and of autophagy, through ATG1. Inhibiting TOR signalling, and thus translation, by rapamycin treatment in wild-type flies recapitulated the sleep features of IIS mutants, even in old flies. This rescue of sleep quality was blocked by ubiquitous expression of activated S6K, suggesting that reduced S6K activity is required for the rescue. These findings, together with previous results showing S6K to regulate hunger-driven behaviours, highlight the importance of S6K as a regulator of behaviour in flies. Thus, manipulating TOR signalling can improve sleep quality through S6K (Metaxakis, 2014).

In mammals, rapamycin treatment has beneficial effects on behaviour throughout lifespan. Although complete block of TOR activity is detrimental for long-term memory, a moderate decrease through rapamycin treatment can improve cognitive function, abrogate age-related cognitive deterioration, and reduce anxiety and depression. Moreover, increased TOR activity throughout development is detrimental for neuronal plasticity and memory. In flies, rapamycin prevents dopaminergic neuron loss in mutants with parkinsonism. Although the role of TOR in brain function has not been well studied in flies, the advantageous effect of rapamycin in both mammalian brain function and sleep in flies may be mediated through common neurophysiological mechanisms (Metaxakis, 2014).

Gene expression studies have suggested that protein synthesis is up-regulated during sleep, which may be an essential stage in macromolecular biosynthesis. Consistent with this, inhibiting protein synthesis in specific brain domains prolongs sleep duration in mammals, suggesting that sleep is maintained until specific levels of biosynthesis occur and aids in explaining the ubiquitously conserved need for sleep. Brief cycloheximide treatment has been shown to prolong night sleep and increased consolidation in flies, indicating an evolutionarily conserved role for protein synthesis inhibition on sleep regulation. Contrary to reduced IIS, cycloheximide reduced day activity, possibly due to the global effect of cycloheximide on protein synthesis or due to toxic defects in flies' physiology. Decreased protein synthesis rates may enhance the necessity for increased sleep duration, to allow sufficient synthesis of proteins and other macromolecules during sleep, allowing organisms to be healthy and functional during the day (Metaxakis, 2014).

Alternatively, the effect of protein synthesis inhibition on night sleep could be the result of reduced expression of specific sleep regulators. This study found that DopR1 and dilp2-3,5 mutants share night phenotypes and that rapamycin did not affect sleep of DopR1 mutants, suggesting that TOR acts on dopaminergic signalling to affect night sleep. Reduced IIS elevated expression of DopR1, independently of dFOXO, in accordance with data from mammals. This effect may be feedback caused by down-regulation of dopaminergic signalling in IIS mutants, although not through direct regulation of DopR. Under normal physiological conditions, dopamine signalling is determined by the level of extracellular dopamine and the rate of DAT-mediated dopamine clearance from the synaptic cleft. The rate of dopamine clearance is dependent on the turnover rate of DAT and the number of functional transporters at the plasma membrane. This study found that reduced IIS and rapamycin treatment induced increased expression of DAT, suggesting an increased rate of dopamine clearance from the synaptic cleft, and thus a reduction in the amplitude of dopamine signalling, without changes in total dopamine levels. DAT function and IIS have recently been linked in mammals. DAT function increases upon insulin stimulation and is diminished on insulin depletion, through alterations in DAT membrane localization. However, IIS-dependent regulation of DAT subcellular localization in Drosophila has not yet been demonstrated. The current data suggest down-regulating dopaminergic signalling, either by loss of DopR1 or increasing DAT levels, is beneficial for sleep quality. In agreement with this it was shown that artificially increasing dopaminergic signalling, through short-term methamphetamine treatment, increases both day and night activity and reduces night sleep, and reverts the beneficial effect of reduced IIS on night behaviours. In mammals, cocaine administration, which enhances dopaminergic signalling, increases TOR activity. Also, rapamycin blocks cocaine-induced locomotor sensitization. Interestingly, cocaine stimulates S6K phosphorylation in rat brains, and this effect is blocked by rapamycin. Taken together, these results show that in flies and mammals dopaminergic and IIS/TOR signalling may interact in similar ways (Metaxakis, 2014).

In conclusion, reduced IIS extends lifespan in diverse organisms. This study has have shown that it can also ameliorate age-related sleep fragmentation, but that the mechanisms by which it does so are distinct from those by which it extends lifespan. Reduced IIS affected day activity and sleep phenotypes through increased octopaminergic signalling, but enhanced octopaminergic signalling did not increase lifespan. Similarly, in Drosophila increased lifespan from reduced IIS requires dfoxo, but the night sleep phenotypes of IIS mutants were independent of this transcription factor. Reduced IIS thus acts through multiple pathways to ameliorate different aspects of loss of function during ageing. IIS links metabolism and behaviour through its components, such as S6K and dFOXO, which act through different neuronal circuits and neurons to affect sleep. The strong evolutionary conservation of these circuits and their functions suggests that pharmacological manipulation of IIS effectors could be beneficial in treatments of sleep syndromes in humans (Metaxakis, 2014).

A rapid one-generation genetic screen in a Drosophila model to capture rhabdomyosarcoma effectors and therapeutic targets

Rhabdomyosarcoma (RMS) is an aggressive childhood malignancy of neoplastic muscle-lineage precursors that fail to terminally differentiate into syncytial muscle. The most aggressive form of RMS, Alveolar-RMS (A-RMS), is driven by misexpression of the PAX-FOXO1 oncoprotein, which is generated by recurrent chromosomal translocations that fuse either the PAX3 or PAX7 gene (homologs of Drosophila Paired) to FOXO1. The molecular underpinnings of PAX-FOXO1-mediated RMS pathogenesis remain unclear, however, and clinical outcomes poor. This study reports a new approach to dissect RMS, exploiting a highly efficient Drosophila PAX7-FOXO1 model uniquely configured to uncover PAX-FOXO1 RMS genetic effectors in only one generation. With this system, a comprehensive deletion screen was performed against the Drosophila autosomes, and mutation of Mef2, a myogenesis lynchpin in both flies and mammals, was demonstrated to dominantly suppresses PAX7-FOXO1 pathogenicity and act as a PAX7-FOXO1 gene target. Additionally, mutation of mastermind, a gene encoding a MEF2 transcriptional co-activator, was shown to similarly suppress PAX7-FOXO1, further pointing towards MEF2 transcriptional activity as a PAX-FOXO1 underpinning. These studies show the utility of the PAX-FOXO1 Drosophila system as a robust one-generation (F1) RMS gene discovery platform and demonstrate how Drosophila transgenic conditional expression models can be configured for the rapid dissection of human disease (Galindo, 2014: PubMed).

Given the critical role that the PAX-FOXO1 fusion oncoprotein plays in RMS, this study focuses on PAX-FOXO1 as an entry-point for designing a transgenic Drosophila RMS-related model that would be amenable to forward genetic screening and RMS gene discovery. To bypass the issue of cumbersome multigenerational screening schemes that would normally be required, a Gal80 X-linked chromosomal transgene was incorporated to generate a viable screening Gal4/UAS-PAX-FOXO1 master stock that allows for the rapid identification of PAX-FOXO1 genetic modifiers in a single genetic cross (Galindo, 2014).

With this platform, new PAX-FOXO1 pathogenesis underpinnings were probed. Though very similar in molecular structure, PAX3-FOXO1− and PAX7-FOXO1−positive RMS demonstrate differing clinical behaviors, as PAX3-FOXO1 tumors are more common and notoriously aggressive. Consequently, PAX3-FOXO1 is the PAX-FOXO1 fusion most commonly investigated in vertebrate models. This study focuses on PAX7-FOXO1 in the Drosophila system, which demonstrates phenotypes that are better penetrant and experimentally tractable due to the fact that human PAX7 demonstrates slightly greater sequence identity to fly PAX3/7 than does human PAX3. Additionally, as no other animal models of PAX7-FOXO1 presently exist, the fly PAX7-FOXO1 model also conveniently serves as a complement to vertebrate PAX3-FOXO1 models (Galindo, 2014).

The extent to which observations from the PAX7-FOXO1 fly model would impact the clinically more aggressive PAX3-FOXO1 RMS subtype, as well as PAX-FOXO1-negative (embryonal) RMS, is unknown. Notably, previous studies show that genetic modifiers identified from the Drosophila system impact PAX3-FOXO1 RMS oncogenesis and tumorigenesis. Furthermore, unpublished studies suggest that fly PAX7-FOXO1 genetic modifiers are similarly involved in Embryonal RMS. These findings provide marked validation for the applicability and value of this genetic fly system to human RMS (Galindo, 2014).

Interestingly, though PAX7-FOXO1 induces expression of the late myogenic differentiation marker MHC, PAX-FOXO1 RMS myoblasts in culture and in vivo demonstrate only partial differentiation with little-to-no MHC expression. In considering this discrepancy, it should be first noted that PAX-FOXO1 is a relatively weak driver of RMS in culture and in vivo and requires additional/sequential genetic aberrations to induce oncogenic transformation. Thus, secondary mutations might be necessary to force the strength of RMS myoblast differentiation-arrest seen in human RMS tumors; by contrast, the PAX7-FOXO1 model of this study differs in that the system is free of any additional background mutations. Second, earlier studies show that expression of PAX3-FOXO1 in mouse embryonic cultured cells induces the formation of MHC-positive myocytes and myotube, similar to the Drosophila system in this study as the da-Gal4/UAS-PAX7-FOXO1 expression system targets undifferentiated embryonic primordia. Uncovering of the genetic/molecular sequence of RMS pathogenesis and the cell(s) origin will shed further insight into the underlying mechanisms that account for the myoblast differentiation arrest phenotypes seen in RMS in vivo (Galindo, 2014).

The differentiation and fusion of myoblasts into postmitotic, syncytial muscle requires that the bHLH myogenic regulatory factors (MRFs: Myf5, Mrf4, MyoD, and Myogenin) interact with E-proteins, which drive and regulate critical aspects of myogenic fate determination. The MRFs subsequently interact with the MEF2 transcription factors that, although lacking intrinsic myogenic activity, cooperate with the MRFs to synergistically activate muscle-specific genes and the downstream myogenic terminal differentiation program. Vertebrates possess four MEF2 family member genes (-A, -B, -C, -D), which demonstrate complex overlapping spatial and temporal expression patterns in embryonic and adult tissues, with greatest expression levels seen in striated muscle and brain. Because of genetic redundancy and overlapping expression patterns of the MEF2 genes, interrogating individual MEF2 gene activity in mammals is experimentally challenging, with loss-of-function mutation studies revealing only limited insights into MEF2 gene function in tissues in which the MEF2 genes do not overlap/compensate. Conveniently, flies possess only one Mef2 gene (D-Mef2) and serve as an excellent model system to delineate MEF2’s critical role in myogenesis. The study speculates that the lack of Mef2 redundancy in flies provides a marked experimental advantage in isolating D-Mef2 as a PAX7-FOXO1 effector. Similarly, the identification of mam was also likely facilitated by the fact that flies possess one mam gene, whereas mammals contain three mam orthologs. Thus, the study proposes that the comparative lack of genetic compensation/redundancy is an attractive advantage to Drosophila as a disease model system (Galindo, 2014).

The study suggests that further interrogation of MEF2 in RMS will open new avenues for RMS chemotherapy, which for high-risk disease has not improved for decades. For example, since MEF2 activity is tightly governed by class IIa histone deacetylases, histone deacetylase inhibitors are now ripe for preclinical testing as new anti-RMS agents. Additionally, it was found that the MEF2 cofactor Mastermind, which interacts with MEF2C and mediates crosstalk between Notch signals during myogenic differentiation, similarly influences PAX-FOXO1 pathogenicity in flies. Interestingly, Mastermind-specific, cell-permeable peptide inhibitors have been shown to block the progression of T-cell acute lymphoblastic leukemia in mice in vivo and thus are also new agents available for RMS preclinical testing. Further characterization of MEF2 in RMS cell and mouse models will continue to refine both our understanding and the potential targeting of MEF2 activity in RMS (Galindo, 2014).

In conclusion, the study postulates that: 1) The Drosophila PAX7-FOXO1 model is uniquely configured for the quick uncovering of new RMS genetic effectors with one simple genetic screening cross; 2) a putative PAX-FOXO1-to-MEF2/MASTERMIND axis underlies A-RMS; and 3) Drosophila conditional expression models are an efficient and powerful gene discovery platform for the rapid dissection of human disease (Galindo, 2014).

Gr33a modulates Drosophila male courtship preference

In any gamogenetic species, attraction between individuals of the opposite sex promotes reproductive success that guarantees their thriving. Consequently, mate determination between two sexes is effortless for an animal. However, choosing a spouse from numerous attractive partners of the opposite sex needs deliberation. In Drosophila melanogaster, both younger virgin females and older ones are equally liked options to males; nevertheless, when given options, males prefer younger females to older ones. Non-volatile cuticular hydrocarbons, considered as major pheromones in Drosophila, constitute females' sexual attraction that act through males' gustatory receptors (Grs) to elicit male courtship. To date, only a few putative Grs are known to play roles in male courtship. This study reports that loss of Gr33a function or abrogating the activity of Gr33a neurons does not disrupt male-female courtship, but eliminates males' preference for younger mates. Furthermore, ectopic expression of human amyloid precursor protein (APP; see Drosophila Appl) in Gr33a neurons abolishes males' preference behavior. Such function of APP is mediated by the transcription factor Forkhead box O (dFoxO). These results not only provide mechanistic insights into Drosophila male courtship preference, but also establish a novel Drosophila model for Alzheimer's disease (AD) (Xue, 2015).

To avoid futile reproductive efforts, an animal must distinguish conspecifics from other species and differentiate the sex of conspecific partners. It must also determine the most suitable mates from large amounts of available partners in order to maximize reproductive efficiency. Evolution endows Drosophila melanogaster males with the instinct to discriminate conspecifics from other Drosophila species1 and to discern females from males. It also bestows on them the ability to select the most favorable mates among masses of desirable virgin females (Xue, 2015).

In Drosophila, non-volatile cuticular hydrocarbons (CHCs) have been recognized as a type of major sex pheromone, which convey information of an individual such as species and sex. Female-specific CHCs are detected by male gustatory receptors (Grs), a chemosensory receptor family mainly responsible for detecting non-volatile chemicals, during tapping and licking steps in stereotypical male courtship behavior. So far, only a few Grs, including Gr32a, Gr33a and Gr39a are reported to be engaged in Drosophila male courtship behavior. While Gr32a acts to assist males to discriminate conspecifics from other species, Gr39a is required for males to distinguish females from males. On the other hand, Gr33a functions to inhibit male-male courtship. Despite these findings, roles of the Grs in males' choices for the most favorable mates have remained largely unknown (Xue, 2015).

A previous study (Hu, 2014) set a paradigm of choice model in which both options (younger virgin females and older ones) are proved to be attractive to Drosophila males, but males still intensely prefer younger mates to older ones. Using this model, this study explored the mechanisms by which males bias their potential mates. Gene loss-of-function, gain-of-function, and cell-inactivation experiments demonstrated that Gr33a and Gr33a neurons are essential for males' preference for younger mates. Since the previous data indicated that pan-neuronal expression of human amyloid precursor protein (APP) ablates males' preference for younger mates, this study sought to investigate whether APP expression in Gr33a neurons would affect this behavior. Indeed, it was found that Gr33a neurons-specific expression of APP abolished males' preference for younger mates, and this function of APP is mediated by the transcription factor forkhead box O (dFoxO) (Xue, 2015).

Drosophila male courtship choice has been frequently applied for studying decision making in animals, yet most of the past studies have focused on male courtship choices between likes and dislikes, such as court towards females vs. males, or virgin vs. non-virgin females. Previous study has characterized a choice behavior between two equally-liked options: mature virgin females, whether younger or older, were similarly attractive to naive males; nevertheless, when given the option, males turn out to be picky and prefer younger virgin females to older ones. This study found that a gustatory receptor, Gr33a, is necessary for males' preference for younger mates. Gr33a is thought to be necessary to inhibit homosexual behavior; its role in heterosexual behavior, however, is rarely pondered. This study has revealed the critical role of Gr33a in males' preference for younger mates. Furthermore, ectopic expression of APP in Gr33a neurons eliminates males' preference behavior, and such function is mediated by dFoxO, a recently reported downstream factor of APP27. Therefore, this work demonstrates the genetic interaction of APP and dFoxO in Gr33a neurons, which modulates males' preference for younger mates. APP is identified as a potential causative protein of AD, a common progressive neurodegenerative disorder, in which cognitive decline is the prime symptom. Although Drosophila has long been utilized for building AD models to investigate the pathogenesis and possible cure for AD, accepted Drosophila AD models are limited to locomotion model and life span model, which have little correlation with cognitive ability. The current findings, however, have offered the possibility for establishing a novel Drosophila AD model that is related to cognitive ability (Xue, 2015).

In all CHCs produced by files, 7, 11-HD and 7, 11-ND have been identified as female specific aphrodisiac pheromones to Drosophila melanogaster males. GC and MS studies suggest that both 7, 11-HD and 7, 11-ND are expressed at lower concentration in younger virgin females than the older ones. Hence, it appears unlikely that 7, 11-HD or 7, 11-ND is the cause that leads males to court younger virgin females more vigorously than the older ones. On the contrary, since Gr33a has been reported as a receptor of aversive odors, it is more likely that older females produce certain aversive odors that can be recognized by males and repel them. Consistent with this explanation, this study found that the concentrations of most detected CHCs are significantly higher on the older virgin females than the younger ones. Nevertheless, at this stage it was not possible to identify the CHC(s) that serves as the aversive pheromone to males. Besides, it cannot be excluded that younger virgin females produce unknown attractive pheromones other than 7, 11-HD or 7, 11-ND. However, all CHCs detected on younger virgin females also present on older ones at a similar or higher lever. Thus, the tentative conclusion is drawn that younger virgin females do not produce more attractive pheromones than the older ones. The results, taken together, unravel the role of bitter sensory Gr33a neurons in males' preference for younger mates and infer that older females might produce certain aversive odors that cause males to turn to younger mates (Xue, 2015).


EVOLUTIONARY HOMOLOGS

C. elegans Daf-16, the insulin pathway, response to stress and life span

In mammals, insulin signalling regulates glucose transport together with the expression and activity of various metabolic enzymes. In the nematode Caenorhabditis elegans, a related pathway regulates metabolism, development and longevity. Wild-type animals enter the developmentally arrested dauer stage in response to high levels of a secreted pheromone, accumulating large amounts of fat in their intestines and hypodermis. Mutants in DAF-2 (a homolog of the mammalian insulin receptor) and AGE-1 (a homolog of the catalytic subunit of mammalian phosphatidylinositol 3-OH kinase) arrest development at the dauer stage. Moreover, animals bearing weak or temperature-sensitive mutations in daf-2 and age-1 can develop reproductively, but nevertheless show increased energy storage and longevity. Null mutations in daf-16 suppress the effects of mutations in daf-2 or age-1; lack of daf-16 bypasses the need for this insulin receptor-like signalling pathway. The principal role of DAF-2/AGE-1 signalling is thus to antagonize DAF-16. daf-16 is widely expressed and encodes three members of the Fork head family of transcription factors. The DAF-2 pathway acts synergistically with the pathway activated by a nematode TGF-beta-type signal, DAF-7, suggesting that DAF-16 cooperates with nematode SMAD proteins in regulating the transcription of key metabolic and developmental control genes. The probable human orthologs of DAF-16, FKHR and AFX, may also act downstream of insulin signalling and cooperate with TGF-beta effectors in mediating metabolic regulation. These genes may be dysregulated in diabetes (Ogg, 1997).

The wild-type Caenorhabditis elegans nematode ages rapidly, undergoing development, senescence, and death in less than 3 weeks. In contrast, mutants with reduced activity of the gene daf-2, a homolog of the insulin and insulin-like growth factor receptors, age more slowly than normal and live more than twice as long. These mutants are active and fully fertile and have normal metabolic rates. The life-span extension caused by daf-2 mutations requires the activity of the gene daf-16. daf-16 appears to play a unique role in life-span regulation and encodes a member of the hepatocyte nuclear factor 3 (HNF-3)/forkhead family of transcriptional regulators. In humans, insulin down-regulates the expression of certain genes by antagonizing the activity of HNF-3, raising the possibility that aspects of this regulatory system have been conserved (Lin, 1997).

A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. Two C. elegans Akt/PKB homologs, akt-1 and akt-2, transduce insulin receptor-like signals that inhibit dauer arrest and AKT-1 and AKT-2 signaling are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1, revealed by a genetic screen, as well as increased dosage of wild-type akt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 and akt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation (Paradis, 1998).

C. elegans insulin-like signaling regulates metabolism, development, and life span. This signaling pathway negatively regulates the activity of the forkhead transcription factor DAF-16. daf-16 encodes multiple isoforms that are expressed in distinct tissue types and are probable orthologs of human FKHRL1, FKHR, and AFX. Human FKHRL1 can partially replace DAF-16, proving the orthology. In mammalian cells, insulin and insulin-like growth factor signaling activate AKT/PKB kinase to negatively regulate the nuclear localization of DAF-16 homologs. The absence of AKT consensus sites on DAF-16 is sufficient to cause dauer arrest in daf-2 plus animals, proving that daf-16 is the major output of insulin signaling in C. elegans. FKHR, FKRHL1, and AFX may similarly be the major outputs of mammalian insulin signaling. daf-2 insulin signaling, via AKT kinases, negatively regulates DAF-16 by controlling its nuclear localization. Surprisingly, daf-7 TGF-beta signaling also regulates DAF-16 nuclear localization specifically at the time when the animal makes the commitment between diapause and reproductive development. daf-16 function is supported by the combined action of two distinct promoter/enhancer elements, whereas the coding sequences of two major DAF-16 isoforms are interchangeable. Together, these observations suggest that the combined effects of transcriptional and posttranslational regulation of daf-16 transduce insulin-like signals in C. elegans and perhaps more generally (Lee, 2001).

Evolutionary models of aging propose that a trade-off exists between the resources an organism devotes to reproduction and growth and those devoted to cellular maintenance and repair, such that an optimal life history always entails an imperfect ability to resist stress. Yet, since environmental stressors, such as caloric restriction or exposure to mild stress, can increase stress resistance and life span, it is possible that a common genetic mechanism could regulate the allocation of resources in response to a changing environment. Consistent with predictions of evolutionary trade-off models, nematodes carrying an integrated DAF-16::GFP transgene grow and reproduce more slowly yet are more stress resistant and longer lived than controls carrying the integration marker alone. The nuclear localization of the DAF-16::GFP fusion protein responds to environmental inputs as well as genetic. Environmental stresses, such as starvation, heat, and oxidative stress, cause rapid nuclear localization of DAF-16. In conditions rich in food, DAF-16::GFP is inhibited from entry into the nucleus by daf-2 and akt-1/akt-2, both components of insulin-like signaling in nematodes. It is suggested that changes in the subcellular localization of DAF-16 by environmental cues allows for rapid reallocation of resources in response to a changing environment at all stages of life (Henderson, 2001).

The lifespan of Caenorhabditis elegans is regulated by the insulin/insulin-like growth factor (IGF)-1 receptor homolog DAF-2, which signals through a conserved phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Mutants in this pathway remain youthful and active much longer than normal animals and can live more than twice as long. This lifespan extension requires DAF-16, a forkhead/winged-helix transcription factor. DAF-16 is thought to be the main target of the DAF-2 pathway. Insulin/IGF-1 signaling is thought to lead to phosphorylation of DAF-16 by AKT activity, which in turn shortens lifespan. The DAF-2 pathway prevents DAF-16 accumulation in nuclei. Disrupting Akt-consensus phosphorylation sites in DAF-16 causes nuclear accumulation in wild-type animals, but, surprisingly, has little effect on lifespan. Thus the DAF-2 pathway must have additional outputs. Lifespan in C. elegans can be extended by perturbing sensory neurons or germ cells. In both cases, lifespan extension requires DAF-16. Both sensory neurons and germline activity regulate DAF-16 accumulation in nuclei, but the nuclear localization patterns are different. Together these findings reveal unexpected complexity in the DAF-16-dependent pathways that regulate aging (Lin, 2001).

Aging and limited life span are fundamental biological phenomena observed in a variety of species. Approximately 55 genes have been identified that can extend longevity when altered in Caenorhabditis elegans. These genes include an insulin-like receptor (daf-2) and a phosphatidylinositol 3-OH kinase (age-1) regulating a forkhead transcription factor (daf-16), as well as genes mediating metabolic throughput, sensory perception, and reproduction. Moreover, these mutant alleles both extend life span and increase resistance to ultraviolet (UV) radiation, heat, and oxidative stress, though the stress resistance of clk-1 is controversial. With the exception of old-1 and perhaps some other genes, all of the life-extension alleles are hypomorphic or nullomorphic. The OLD-1 transmembrane tyrosine kinase (formerly TKR-1) is expressed in a variety of tissues, is stress inducible, and is a positive regulator of longevity and stress resistance. The transcription of old-1 is upregulated in long-lived age-1 and daf-2 mutants and is upregulated in response to heat, UV light, and starvation. Both RT-PCR and analysis of an OLD-1::GFP tag suggest that old-1 expression is dependent on daf-16. Importantly, old-1 is required for the life extension of age-1 and daf-2 mutants. This study reveals a new system for specifying longevity and stress resistance and suggests possible mechanisms for mediating life extension by dietary restriction and hormesis (Murakami, 2001).

The daf-2 insulin-like receptor pathway regulates development and life-span in Caenorhabditis elegans. Reduced DAF-2 signaling leads to changes in downstream targets via the daf-16 gene, a fork-head transcription factor which is regulated by DAF-2, and results in extended life-span. This paper describes the identification of genes whose expression is controlled by the DAF-2 signaling cascade. dao-1, dao-2, dao-3, dao-4, dao-8 and dao-9 are down-regulated in daf-2 mutant adults compared to wild-type adults, whereas dao-5, dao-6 and dao-7 are up-regulated. The latter genes are negatively regulated by DAF-2 signaling and positively regulated by DAF-16. Positive regulation by DAF-2 on dao-1, dao-4 and dao-8 is mediated by DAF-16, whereas daf-16 mediates only part of DAF-2 signaling for dao-2 and dao-9. Regulation by DAF-2 is most likely DAF-16 independent for dao-3 and hsp-90. RNA levels of dao-5 and dao-6 show elevated expression in daf-2 adults, as well as being strongly expressed in dauer larvae. In contrast, hsp-90 transcript levels are low in daf-2 mutant adults though they are enriched in dauer larvae, indicating overlapping but not identical mechanisms of efficient life maintenance in stress-resistant dauer larvae and long-lived daf-2 mutant adults. dao-1, dao-8 and dao-9 are homologs of the FK506 binding proteins that interact with the mammalian insulin pathway. dao-3 encodes a putative methylenetetrahydrofolate dehydrogenase. DAO-5 shows 33 % identity with human nucleolar phosphoprotein P130. dao-7 is similar to the mammalian ZFP36 protein. Distinct regulatory patterns of dao genes implicate their diverse positions within the signaling network of DAF-2 pathway, and suggest they have unique contributions to development, metabolism and longevity (Yu, 2001).

Oxidative damage shortens the life span of the nematode Caenorhabditis elegans, even in an age-1 mutant that is characterized by a long life and oxygen resistance. Daily short-term exposure (3 h) to hyperoxia further extends the life span of age-1, a phenomenon known as an adaptive response. age-1 also shows resistance to paraquat and heat. Acute hyperoxic treatment does not extend the life spans of wild type, daf-16 or mev-1. daf-16 mutants have a slightly shorter life span compared to wild type and are sensitive to heat and paraquat. The daf-16 phenotype resembles that of mev-1 showing a short life and oxygen sensitivity. mRNA levels were measured of superoxide dismutase genes (sod-1 through 4), catalase genes (clt-1 and ctl-2), known to encode anti-oxidant enzymes, and found that these levels were elevated in age-1 young adults. However, in daf-16 and mev-1, the expression of sod-1, sod-2 and sod-3 genes was lower rather than in wild type. Conversely, ctl-1 and ctl-2 genes expression was significantly elevated in daf-16 and mev-1. This suggests that DAF-16, a forkhead/winged-helix transcription factor, whose expression is suppressed by AGE-1, phosphoinositide 3-kinase (PI3-kinase), regulates anti-oxidant genes as well as energy metabolism under atmospheric conditions. However, the level of gene expression of SOD and catalase was not elevated by short-term exposure to 90% oxygen in wild type, mev-1, daf-16 and even age-1. This suggests that SOD and catalase do not play a role in the adaptive response against oxidative stress under hyperoxia, at least under these experimental conditions (Yanase, 2002).

Signaling from the DAF-2/insulin receptor to the DAF-16/FOXO transcription factor controls longevity, metabolism, and development in disparate phyla. To identify genes that mediate the conserved biological outputs of daf-2/insulin-like signaling, comparative genomics were used to identify 17 orthologous genes from Caenorhabditis and Drosophila, each of which bears a DAF-16 binding site in the promoter region. One-third of these DAF-16 downstream candidate genes are regulated by daf-2/insulin-like signaling in C. elegans, and RNA interference inactivation of the candidates show that many of these genes mediate distinct aspects of daf-16 function, including longevity, metabolism, and development (Lee, 2003).

Ageing is a fundamental, unsolved mystery in biology. DAF-16, a FOXO-family transcription factor, influences the rate of ageing of Caenorhabditis elegans in response to insulin/insulin-like growth factor 1 (IGF-I) signalling. Using DNA microarray analysis, DAF-16 has been found to affect expression of a set of genes during early adulthood, the time at which this pathway is known to control ageing. Many of these genes influence the ageing process. The insulin/IGF-I pathway functions cell non-autonomously to regulate lifespan, and these findings suggest that this pathway signals other cells, at least in part, by feedback regulation of an insulin/IGF-I homolog. Furthermore, these findings suggest that the insulin/IGF-I pathway ultimately exerts its effect on lifespan by upregulating a wide variety of genes, including cellular stress-response, antimicrobial and metabolic genes, and by downregulating specific life-shortening genes (Murphy, 2003).

The Caenorhabditis elegans transcripion factor HSF-1, which regulates the heat-shock response, also influences aging. Reducing hsf-1 activity accelerates tissue aging and shortens life-span, and hsf-1 overexpression extends lifespan. HSF-1, like the transcription factor DAF-16, is required for daf-2-insulin/IGF-1 receptor mutations to extend life-span. This is because HSF-1 and DAF-16 together activate expression of specific genes, including genes encoding small heat-shock proteins, which in turn promote longevity. The small heat-shock proteins also delay the onset of polyglutamine-expansion protein aggregation, suggesting that these proteins couple the normal aging process to this type of age-related disease (Hsu, 2003).

In Caenorhabditis elegans, an insulin-like signaling pathway, which includes the daf-2 and age-1 genes, controls longevity and stress resistance. Downregulation of this pathway activates the forkhead transcription factor DAF-16, whose transcriptional targets are suggested to play an essential role in controlling the phenotypes governed by this pathway. The genes that have the DAF-16 consensus binding element (DBE) within putative regulatory regions have been surveyed. One such gene, termed scl-1, is a positive regulator of longevity and stress resistance. Expression of scl-1 is upregulated in long-lived daf-2 and age-1 mutants and is undetectable in a short-lived daf-16 mutant. SCL-1 is a putative secretory protein with an SCP domain and is homologous to the mammalian cysteine-rich secretory protein (CRISP) family. scl-1 is required for the extension of the life span of daf-2 and age-1 mutants, and downregulation of scl-1 reduces both life span and stress resistance of this animal. SCL-1, whose expression is dependent on DAF-16, is the first example of a putative secretory protein that positively regulates longevity and stress resistance (Ookuma, 2003).

The highly conserved target-of-rapamycin (TOR) protein kinases control cell growth in response to nutrients and growth factors. In mammals, TOR has been shown to interact with raptor (regulatory associated protein of mTOR: potential Drosophila homolog CG4320) to relay nutrient signals to downstream translation machinery. Raptor associates in a near stoichiometric ratio with mTOR to form a complex that functions as the nutrient sensor. It was proposed that raptor acts as a scaffold to bridge TOR with its putative phosphorylation targets. In C. elegans, mutations in the genes encoding CeTOR and raptor result in dauer-like larval arrest, implying that CeTOR regulates dauer diapause. The daf-15 (raptor) and let-363 (CeTOR) mutants shift metabolism to accumulate fat, and raptor mutations extend adult life span. daf-15 transcription is regulated by DAF-16, a FOXO transcription factor that is in turn regulated by daf-2 insulin/IGF signaling. This is a new mechanism that regulates the TOR pathway. Thus, DAF-2 insulin/IGF signaling and nutrient signaling converge on DAF-15 (raptor) to regulate C. elegans larval development, metabolism and life span (Jia, 2004).

Insulin/IGF-1 signaling (IIS) regulates aging in worms, flies, and mice through a well-characterized, highly conserved core set of components. IIS also regulates early developmental decisions, the reproductive status of the animal, innate immunity, and stress-resistance functions. In C. elegans, the sole insulin/IGF-1 receptor, DAF-2, negatively regulates the FOXO transcription factor, DAF-16. A new component of the IIS longevity pathway, SMK-1, specifically influences DAF-16-dependent regulation of the aging process in C. elegans by regulating the transcriptional specificity of DAF-16 activity. Localization analysis of DAF-16 places SMK-1 downstream of DAF-16's phosphorylation-dependent relocation to the nucleus. Physiological and transcription analyses indicate that smk-1 is required for the innate immune, UV, and oxidative stress but not the thermal stress functions of DAF-16. SMK-1 therefore plays a role in longevity by modulating DAF-16 transcriptional specificity without affecting other processes regulated by IIS (Wolff, 2006).

In C. elegans, removing the germ cells extends life span by triggering the nuclear localization and activation of the DAF-16/FOXO transcription factor in the intestine. This study identifies and analyzes genes required to extend life span as a consequence of germline ablation. The reproductive system communicates with the intestine through lipophilic-hormone signaling and a gene called kri-1 is likely to act in the intestine to promote DAF-16 nuclear localization in response to this signal. This lipophilic-signaling pathway and kri-1 are not required for DAF-16's nuclear localization and life-span extension in animals with decreased insulin/IGF-1 signaling. Thus, this pathway specifically enables the integration of cues from the reproductive system with central DAF-16-activation pathways to influence the aging of the animal (Berman, 2006).

The kri-1 gene encodes a conserved protein with ankyrin repeats that is constitutively expressed in the pharynx and intestine throughout postembryonic stages. Like daf-12, an orphan nuclear receptor that regulates dauer diapause, reproductive development, fat metabolism, and life span, kri-1 mutations suppress the increase in life span associated with germline loss but do not affect the life span of wild-type animals. In addition, kri-1 mutants have no significant effect on the life span of germline-deprived daf-2 mutants (daf-2 encodes an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans) or on their Daf-c (dauer constitutive) phenotype, indicating that kri-1 acts independently of DAF-2 in the reproductive-signaling pathway. Upon germline ablation, DAF-16 in the intestine translocates from the cytoplasm to the nucleus, where its activity accounts for the entire increase in life span. By using a DAF-16::GFP fusion protein, kri-1 is shown to be required for DAF-16 nuclear localization in the intestinal cells of germline-deficient animals. A less dramatic but significant reduction in nuclear DAF-16::GFP was also seen in daf-9 or daf-12 mutants. The nuclear localization of DAF-16 seen in daf-2 mutants, however, is not affected by kri-1, daf-9 (encoding a CYP2 cytochrome P450 enzyme involved in metabolizing steroid hormones), or daf-12 mutations. This indicates that the roles of kri-1 and lipophilic hormone production on DAF-16 nuclear localization are specific to the reproductive pathway and act largely independently of insulin signaling (Berman, 2006; Beckstead, 2006).

A constitutively nuclear active DAF-16 protein was used to perform epistasis tests with kri-1, daf-9, and daf-12 mutations to address the long-standing question of how DAF-9 and DAF-12 contribute to the longevity of germline-deficient animals. As expected, nuclear DAF-16 extends the life span of daf-16 mutants that lack a germline. Similarly, nuclear DAF-16 rescues the increase in longevity seen upon germline ablation of kri-1 mutants, demonstrating that a key function for KRI-1 is to facilitate this localization of DAF-16 in the intestine. Interestingly, a daf-12 null mutation strongly blocked the longevity of germline-deficient animals that express nuclear DAF-16, indicating that DAF-12 can control longevity independently of DAF-16. Even more remarkable, a strong daf-9 allele has no effect on the longevity of germline-deficient animals that express nuclear DAF-16, indicating that daf-9 acts upstream from DAF-16 and that DAF-12 has functions in the germline-longevity pathway that are independent of lipophilic hormone signaling. The identification of dafachronic-acid ligands for DAF-12 provides new directions for these studies. It will be interesting to determine how the hormone affects DAF-16 nuclear localization and adult life span in both wild-type and mutant worms (Berman, 2006; Beckstead, 2006).

Development is typically studied as a continuous process under laboratory conditions, but wild animals often develop in variable and stressful environments. C. elegans larvae hatch in a developmentally arrested state (L1 arrest) and initiate post-embryonic development only in the presence of food (E. coli in lab). In contrast to the well-studied dauer arrest, L1 arrest occurs without morphological modification, although larvae in L1 arrest are more resistant to environmental stress than developing larvae. Consistent with its role in dauer formation and aging, insulin/insulin-like growth factor (IGF) signaling is shown to regulate L1 arrest. daf-2 insulin/IGF receptor mutants have a constitutive-L1-arrest phenotype when fed and extended survival of L1 arrest when starved. Conversely, daf-16/FOXO mutants have a defective-arrest phenotype, failing to arrest development and dying rapidly when starved. DAF-16 is required for transcription of the cyclin-dependent kinase inhibitor cki-1 in stem cells in response to starvation, accounting for the failure of daf-16/FOXO mutants to arrest cell division during L1 arrest. Other developmental events such as cell migration, cell fusion, and expression of the microRNA lin-4, a temporal regulator of post-embryonic development, are also observed in starved daf-16/FOXO mutants. These results suggest that DAF-16/FOXO promotes developmental arrest via transcriptional regulation of numerous target genes that control various aspects of development (Baugh, 2006).

Dietary restriction (DR) is the most effective environmental intervention to extend lifespan in a wide range of species. However, the molecular mechanisms underlying the benefits of DR on longevity are still poorly characterized. AMP-activated protein kinase (AMPK; see Drosophila SNF1A/AMP-activated protein kinase) is activated by a decrease in energy levels, raising the possibility that AMPK might mediate lifespan extension by DR. By using a novel DR assay that was developed and validated in C. elegans, it was found that AMPK is required for this DR method to extend lifespan and delay age-dependent decline. It was found that AMPK exerts its effects in part via the FOXO transcription factor DAF-16. FOXO/DAF-16 is necessary for the beneficial effects of this DR method on lifespan. Expression of an active version of AMPK in worms increases stress resistance and extends longevity in a FOXO/DAF-16-dependent manner. Lastly, it was found that AMPK activates FOXO/DAF-16-dependent transcription and phosphorylates FOXO/DAF-16 at previously unidentified sites, suggesting a possible direct mechanism of regulation of FOXO/DAF-16 by AMPK. This study shows that an energy-sensing AMPK-FOXO pathway mediates the lifespan extension induced by a novel method of dietary restriction in C. elegans (Greer, 2007).

Previous genetic evidence suggested that the C. elegans TGF-β Dauer pathway is responsible solely for the regulation of dauer formation, with no role in longevity regulation, whereas the insulin/IGF-1 signaling (IIS) pathway regulates both dauer formation and longevity. A significant longevity-regulating activity by the TGF-β Dauer pathway has been discovered that is masked by an egg-laying (Egl) phenotype; mutants in the pathway display up to 2-fold increases in life span. The expression profiles of adult TGF-β mutants overlap significantly with IIS pathway profiles: Adult TGF-β mutants regulate the transcription of many DAF-16-regulated genes, including genes that regulate life span, the two pathways share enriched Gene Ontology categories, and a motif previously associated with DAF-16-regulated transcription (the DAE, or DAF-16-associated element) is overrepresented in the promoters of TGF-β regulated genes. The TGF-β Dauer pathway's regulation of longevity appears to be mediated at least in part through insulin interactions with the IIS pathway and the regulation of DAF-16 localization. Together, these results suggest there are TGF-β-specific downstream targets and functions, but that the TGF-β and IIS pathways might be more tightly linked in the regulation of longevity than has been previously appreciated (Shaw, 2007).

Genetic and RNA interference (RNAi) screens for life span regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in Caenorhabditis elegans longevity. This pathway converges on the DAF-16 transcription factor and may regulate life span by controlling the expression of a large number of genes, including free-radical detoxifying genes, stress resistance genes, and pathogen resistance genes. A genome-wide RNAi screen was conducted to identify genes necessary for the extended life span of daf-2 mutants and ~200 gene inactivations were identified that shorten daf-2 life span. Some of these gene inactivations dramatically shorten daf-2 mutant life span but less dramatically shorten daf-2; daf-16 mutant or wild-type life span. Molecular and behavioral markers for normal aging and for extended life span in low insulin/IGF1 (insulin-like growth factor 1) signaling were assayed to distinguish accelerated aging from general sickness and to examine age-related phenotypes. Detailed demographic analysis, molecular markers of aging, and insulin signaling mutant test strains were used to filter progeric gene inactivations for specific acceleration of aging. Highly represented in the genes that mediate life span extension in the daf-2 mutant are components of endocytotic trafficking of membrane proteins to lysosomes. These gene inactivations disrupt the increased expression of the DAF-16 downstream gene superoxide dismutase sod-3 in a daf-2 mutant, suggesting trafficking between the insulin-like receptor and DAF-16. The activities of these genes may normally decline during aging (Samuelson, 2007).

A conserved PTEN/FOXO pathway regulates neuronal morphology during C. elegans development

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is a conserved signal transduction cascade that is fundamental for the correct development of the nervous system. The major negative regulator of PI3K signaling is the lipid phosphatase DAF-18/PTEN, which can modulate PI3K pathway activity during neurodevelopment. This study identified a novel role for DAF-18 in promoting neurite outgrowth during development in C. elegans. DAF-18 modulates the PI3K signaling pathway to activate DAF-16/FOXO and promote developmental neurite outgrowth. This activity of DAF-16 in promoting outgrowth is isoform-specific, being effected by the daf-16b isoform but not the daf-16a or daf-16d/f isoform. It was also demonstrated that the capacity of DAF-16/FOXO in regulating neuron morphology is conserved in mammalian neurons. These data provide a novel mechanism by which the conserved PI3K signaling pathway regulates neuronal cell morphology during development through FOXO (Christensen, 2011).

Insulin/IGF1 sgnaling inhibits age-dependent axon regeneration

The ability of injured axons to regenerate declines with age, yet the mechanisms that regulate axon regeneration in response to age are not known. This study shows that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2. DAF-2's function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO. DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron-specific, and genetically regulated process. In addition, DAF-18/PTEN was found to inhibit regeneration independently of age and FOXO signaling via the TOR pathway. Finally, DLK-1, a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and required for both DAF-16- and DAF-18-mediated regeneration. Together, these data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR (Byrne, 2004).

Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans

Inhibition of DAF-2 (insulin-like growth factor 1 [IGF-1] receptor) or RSKS-1 (S6K), key molecules in the insulin/IGF-1 signaling (IIS) and target of rapamycin (TOR) pathways, respectively, extend lifespan in Caenorhabditis elegans. However, it has not been clear how and in which tissues they interact with each other to modulate longevity. This study demonstrates that a combination of mutations in daf-2 and rsks-1 produces a nearly 5-fold increase in longevity that is much greater than the sum of single mutations. This synergistic lifespan extension requires positive feedback regulation of DAF-16 (FOXO) via the AMP-activated protein kinase (AMPK) complex. Furthermore, germline was identified as the key tissue for this synergistic longevity. Moreover, germline-specific inhibition of rsks-1 activates DAF-16 in the intestine. Together, these findings highlight the importance of the germline in the significantly increased longevity produced by daf-2 rsks-1, which has important implications for interactions between the two major conserved longevity pathways in more complex organisms (Chen, 2013).

IIS and TOR pathways play conserved roles in modulating lifespan in multiple species. However, it is unclear how they might interactively modulate aging. This study set out to address this question by constructing a daf-2 rsks-1 double mutant, which has reduced function of IIS and an important branch of the TOR pathway. Surprisingly, the daf-2 rsks-1 double mutant showed a nearly 5-fold lifespan extension. This phenotype is defined as a synergistic lifespan extension, based on the observation that the longevity of the daf-2 rsks-1 double mutant is beyond the combined effects of rsks-1 and daf-2 single mutants. This synergistic longevity phenotype cannot be explained by the hypothesis that daf-2 and rsks-1 function in parallel to modulate lifespan independently, since an additive effect would be expected under such an assumption (Chen, 2013).

The synergistic longevity phenotype is different from what we previously reported; i.e., that rsks-1 RNAi further extended the daf-2 lifespan by 24%. One major difference in the experimental procedures used is that in the previous study, daf-2 animals were treated with rsks-1 RNAi only during adulthood, whereas in the current work, a double mutant was made that carries the putative null allele of rsks-1 throughout life (Chen, 2013).

When daf-2 animals were treated with rsks-1 RNAi for two generations, resulting in a more complete reduction in rsks-1 mRNA levels, a 54% further lifespan extension was observed. These results suggest that inhibition of rsks-1 during development is critical for the synergistic longevity phenotype. Consistently, inhibition of the RSKS-1 upstream activator LET-363/CeTOR in daf-2 during adulthood led to a 17% additive lifespan extension. Since let-363 is an essential gene, inhibition of which during development leads to larval arrest, a pharmaceutical approach was used to inhibit let-363 by treating animals with rapamycin. Rapamycin treatment throughout life extended the lifespan of N2 and daf-2 animals by 26% and 45%, respectively. There are multiple possible reasons why rapamycin treatment could not extend the lifespan of daf-2 animals as much as the rsks-1 deletion mutant did. One possibility is that rapamycin treatment did not fully block RSKS-1, which is required for the synergistic longevity. Another possibility is that since rapamycin treatment at this dosage has been shown to inhibit both TOR complex 1 and complex 2 activities (Robida-Stubbs et al., 2012), the drug might also affect other lifespan-determinant genes. Nevertheless, these results are consistent with the idea that inhibiting rsks-1 in daf-2 during development leads to a synergistic lifespan extension (Chen, 2013).

Previous studies showed that null mutants of age-1, which encodes a catalytic subunit of the phosphatidylinositol-3-kinase (PI3K) in the IIS pathway, exhibit an exceptional lifespan extension in a DAF-16-dependent manner (Chen, 2013).

Since the daf-2 mutations that were used in this study are not null alleles, one possible explanation for the synergistic longevity produced by daf-2 rsks-1 is that the rsks-1 deletion makes daf-2 mutant phenotypes more severe. This is unlikely to be true, because many aging-related phenotypes of daf-2 are not enhanced by the rsks-1 deletion.rsks-1 does not affect daf-2-mediated dauer arrest, and rsks-1 has a minor or even opposite effect on most stress resistance. Understanding why these phenotypes are uncoupled from the synergistically prolonged longevity produced by daf-2 rsks-1 will help to elucidate the basic mechanisms of aging (Chen, 2013).

TOR plays a conserved role in dietary restriction (DR)-mediated lifespan extension. The effect of nutrients on the synergistic longevity was tested using the DR-FD regimen (FD stands for food deprivation). The rsks-1 single mutant did not show a lifespan extension under DR, which is consistent with the idea that DR and reduced TOR signaling function through overlapping mechanisms to extend lifespan. Interestingly, the synergistic longevity produced by daf-2 rsks-1 is nutrient independent, suggesting that rsks-1 functions through unidentified mechanisms to further extend the lifespan of daf-2 animals (Chen, 2013).

To better understand the molecular mechanisms of the synergistic longevity produced by daf-2 rsks-1, this study set out to identify critical mediators by testing known regulators of IIS or rsks-1. Heat-shock factor 1 (HSF-1) is critical for daf-2-mediated lifespan extension. Inhibition of hsf-1 almost completely abolished the lifespan extension produced by daf-2 rsks-1. Lifespan extension via genetic or pharmaceutical inhibition of TOR requires the IIS downstream transcription factor SKN-1. Surprisingly, inhibition of skn-1 by RNAi had little effect on the synergistic longevity produced by daf-2 rsks-1. Similarly, inhibition of PHA-4, a FOXA transcription factor that is required for the rsks-1 single mutant-mediated lifespan extension, did not affect the lifespan of daf-2 rsks-1. This is further evidence that the mechanism of the synergistic longevity in the daf-2 rsks-1 double mutant is distinct from the lifespan extension caused by the single mutants (Chen, 2013).

Microarray studies were performed and genes were identified that are differentially expressed in daf-2 rsks-1. A genetic screen using RNAi helped identify the AMPK complex (see Drosophila AMP-activated protein kinase alpha subunit) as the key mediator of the synergistic longevity produced by daf-2 rsks-1. Quantitative analysis of the lifespan data indicated that suppression of the daf-2 rsks-1 lifespan by inhibition of AMPK was not due to general sickness. Instead, inhibition of AMPK suppressed the synergy part of the lifespan extension. Further analysis identified a positive feedback regulation of DAF-16 via AMPK in the daf-2 rsks-1 mutant. AMPK plays important roles in various cellular functions. Under energy-starved conditions, AMPK is activated to promote catabolism and thus ATP production. Further characterization of the role of AMPK in metabolism will enhance understanding of the synergistic longevity produced by daf-2 rsks-1 (Chen, 2013).

Both IIS and signals from the reproductive system have endocrine functions. Modulation of these pathways in one tissue leads to nonautonomous activation of DAF-16 in the intestine. To better understand how aging is coordinately modulated across multiple tissues, the involvement of key regulators of the daf-2 rsks-1-mediated synergistic longevity were tested by tissue-specific RNAi. It was found that rsks-1, daf-16, and aak-2 function in the germline to regulate the synergistic lifespan extension, which can also be suppressed by a genetic mutation that causes germline overproliferation or by inhibition of key mediators of germline signaling. In addition, inhibiting rsks-1 in the germline leads to nonautonomous activation of DAF-16 in the intestine. Previous studies on the tissue-specific requirements of key longevity determinants, including DAF-16, mainly employed transgenic rescue approaches. However, the traditional microinjection method creates transgenic lines with a high copy number of transgenes, which will be silenced in the germline. The results indicate that the germline is an important tissue for integrating signals from the IIS pathway and S6K for lifespan determination (Chen, 2013).

Similar to the rsks-1 single mutant, daf-2 rsks-1 animals showed significantly delayed, prolonged, and overall reduced reproduction. This is consistent with a recent study showing that RSKS-1 acts in parallel with the IIS pathway to play an essential role in establishing the germline stem cell/progenitor pool. Interestingly, RSKS-1 functions cell autonomously to regulate establishment of the germline progenitor. This effect is independent of its known suppressors in the regulation of lifespan. These findings suggest that the synergistic longevity of daf-2 rsks-1 cannot simply be linked with its functions in germline development and reproduction (Chen, 2013).

In C. elegans, the intestine carries out multiple nutrient-related functions and is the site for food digestion and absorption, fat storage, and immune response. DAF-16 is one of the essential transcription factors that function in the intestine to modulate lifespan. It was found that intestinal-specific inhibition of daf-16, aak-2, or hsf-1 largely abolishes the synergistic lifespan extension of daf-2 rsks-1. However, knockdown of rsks-1 in the intestine only has an additive effect on daf-2 lifespan, suggesting that rsks-1 may function through nonautonomous mechanisms to activate DAF-16 (Chen, 2013).

The hypodermis is considered as part of the epithelial system in C. elegans. It is involved in basic body plan establishment, cell fate specification, axon migration, apoptotic cells removal, and fat storage. Hypodermis-specific knockdown of rsks-1 in daf-2 also leads to synergistic lifespan extension, and that hypodermis-specific knockdown of daf-16 significantly reduces the synergistic lifespan extension. These results provide evidence for the important role of the hypodermis in lifespan determination. In future studies, it will be interesting to examine which biological functions of the hypodermis are involved in regulating the synergistic longevity by daf-2 rsks-1 (Chen, 2013).

Previous studies showed that muscle decline is one of the major physiological causes of aging in C. elegans. Neither rsks-1 nor the downstream regulators daf-16, hsf-1, and aak-2 seem to function in the muscle to modulate the synergistic lifespan extension. However, the possibility that these regulators may function in other tissues to nonautonomously regulate muscle functions in daf-2 rsks-1 cannot be ruled out. Characterization of age-dependent muscle decline in daf-2 rsks-1 will help to elucidate whether muscle functions are important for the synergistic lifespan extension (Chen, 2013).

There are limitations to assessing tissue-specific involvement of key regulators in lifespan determination by RNAi, such as uncertainty of knockdown efficiency and potential leakiness. It has been reported that in rrf-1 mutants, RNAi can be processed in certain somatic tissues, including the intestine, at least for the genes tested. However, the critical function of rsks-1 in the germline is unlikely to be an artifact, as rsks-1 knockdown in the intestine of daf-2 animals did not lead to synergistic lifespan extension. Moreover, inhibition of certain strong suppressors of daf-2 rsks-1 (e.g., hsf-1) in the intestine, but not in the germline, significantly decreased the synergistic lifespan extension produced by daf-2 rsks-1. Further analyses with single-copied, isoform-specific transgenic rescue will help to quantitatively determine the tissue-specific involvement of key regulators in the synergistic lifespan extension produced by daf-2 rsks-1 (Chen, 2013).

It has not been clear whether DAF-16 is quantitatively more active or is uniquely activated in certain tissues, such as the germline of daf-2 rsks-1. Although the AMPK-mediated positive feedback regulation of DAF-16 was identified based on genes that are expressed to a greater extent in daf-2 rsks-1 animals, it is speculated that the double mutant has some unique properties, as shown in dauer formation and various stress-tolerance assays. The data from the phenotypic analysis of the double mutant and epistasis analysis of tissue requirement of DAF-16 suggest that with the rsks-1 deletion, DAF-16 plays a more important role in certain tissues, such as the germline, to further extend the lifespan of daf-2. Characterization of the genes that are uniquely upregulated in daf-2 rsks-1 or those that are regulated independently of DAF-16 will help distinguish these models (Chen, 2013).

In conclusion, this study found that the daf-2 rsks-1 double mutant shows a synergistic lifespan extension, which is achieved through positive feedback regulation of DAF-16 by AMPK. Tissue- specific epistasis analysis suggests that this enhanced activation of DAF-16 is initiated by signals from the germline, and that the germline tissue may play a key role in integrating the interactions between daf-2 and rsks-1 to cause a synergistic lifespan extension. Since DAF-2, RSKS-1, AMPK, and DAF-16 are highly conserved molecules, similar regulation may also exist in mammals. Further characterization of the daf-2 rsks-1-mediated synergistic longevity will contribute to a better understanding of the molecular mechanisms of aging and age-related diseases (Chen, 2013).

A Caenorhabditis elegans developmental decision requires insulin signaling-mediated neuron-intestine communication

Adverse environmental conditions trigger C. elegans larvae to activate an alternative developmental program, termed dauer diapause, which renders them stress resistant. High-level insulin signaling prevents constitutive dauer formation. However, it is not fully understood how animals assess conditions to choose the optimal developmental program. This study shows that insulin-like peptide (ILP)-mediated neuron-intestine communication plays a role in this developmental decision. Consistent with, and extending, previous findings, it was shown that the simultaneous removal of INS-4, INS-6 and DAF-28 leads to fully penetrant constitutive dauer formation, whereas the removal of INS-1 and INS-18 significantly inhibits constitutive dauer formation. These ligands are processed by the proprotein convertases PC1/KPC-1 and/or PC2/EGL-3. The agonistic and antagonistic ligands are expressed by, and function in, neurons to prevent or promote dauer formation. By contrast, the insulin receptor DAF-2 and its effector, the FOXO transcription factor DAF-16, function solely in the intestine to regulate the decision to enter diapause. These results suggest that the nervous system normally establishes an agonistic ILP-dominant paradigm to inhibit intestinal DAF-16 activation and allow reproductive development. Under adverse conditions, a switch in the agonistic-antagonistic ILP balance activates intestinal DAF-16, which commits animals to diapause (Hung, 2014).

FOXO/DAF-16 activation slows down turnover of the majority of proteins in C. elegans

Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. However, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. This study examined turnover of individual proteins were studied in the long-lived insulin receptor/daf-2 mutant. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. This slowdown was most prominent for translation-related and mitochondrial proteins. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory (Dhondt, 2016).

Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens

The short day lengths of late summer program the mosquito Culex pipiens to enter a reproductive diapause characterized by an arrest in ovarian development and the sequestration of huge fat reserves. It is suggested that insulin signaling and FOXO (forkhead transcription factor), a downstream molecule in the insulin signaling pathway, mediate the diapause response. When RNAi was used to knock down expression of the insulin receptor in nondiapausing mosquitoes (those reared under long day lengths) the primary follicles were arrested in a stage comparable to diapause. The mosquitoes could be rescued from this developmental arrest with an application of juvenile hormone, an endocrine trigger known to terminate diapause in this species. When dsRNA directed against FOXO was injected into mosquitoes programmed for diapause (reared under short day lengths) fat storage was dramatically reduced and the mosquito's lifespan was shortened, results suggesting that a shutdown of insulin signaling prompts activation of the downstream gene FOXO, leading to the diapause phenotype. Thus, the results are consistent with a role for insulin signaling in the short-day response that ultimately leads to a cessation of juvenile hormone production. The similarity of this response to that observed in the diapause of Drosophila melanogaster (Tu, 2005; Williams, 2006) and in dauer formation of Caenorhabditis elegans suggests a conserved mechanism regulating dormancy in insects and nematodes (Sim, 2008).

Insulin signaling is essential for normal growth in insects, and arguably it is the most important regulator of insect growth and size. This pathway has been implicated in diverse roles including the immune response, apoptosis, longevity, and energy metabolism. In addition, suppression of insulin signaling has been implicated in the induction of adult diapause in Drosophila and in dauer formation of the nematode C. elegans. The results of this study suggest that insulin signaling is integral to diapause in the mosquito C. pipiens as well. This common theme across taxa thus suggests a conserved role for the insulin signaling pathway for developmental and reproductive arrests among insects and other invertebrates (Sim, 2008).

The fact that methoprene, a JH analog, can counter the ovarian arrest caused by the down-regulation of Culex InR indicates that insulin signaling has a significant role mediating JH synthesis in C. pipiens. Several lines of evidence indicate that JH synthesis is shut down during diapause in C. pipiens, and experiments rescuing the double-stranded RNAi InR shutdown of development with the JH analog methoprene support a causative link between insulin signaling and JH production. The responsiveness of InR mutants in Drosophila to JH also supports such a connection. In nondiapausing mosquitoes, the corpora allata synthesize JH immediately after adult eclosion, and JH titers reach peak activity during that first week. Knocking down the InR has likely blocked JH production in these long-day females, thus generating the diapause phenotype (Sim, 2008).

In C. elegans and Drosophila, insulin signals through a conserved PI3-kinase/Akt pathway to ultimately phosphorylate the FOXO protein and block the translocation of this protein into the nucleus. Thus, suppression of the insulin signal likely causes the FOXO protein to be translocated into the nucleus to initiate transcription of its downstream genes, some of which are known to be involved in key diapause characters such as the metabolic switch toward lipid storage and protection from reactive oxygen species. The results of this study suggest that these functional roles for FOXO are evident in diapausing C. pipiens as well. Suppression of FOXO by RNAi in diapausing mosquitoes resulted in loss of two key characters essential for successful overwintering: fat hypertrophy and extended lifespan. An antioxidant role is also suggested by the results elicited by a coinjection of dsFOXO and Mn(III)TBAP, an exogenous substitute for oxidoreductase: coinjection increased the lifespan and countered the mortality observed by an injection of dsFOXO alone. This result suggests that adding the oxidoreductase function enables the mosquito to cope with the stressful conditions of food shortage and environmental stress evoked by suppression of FOXO. Down-regulating the FOXO gene possibly impairs expression of oxidoreductases or small heat-shock proteins that enhance survival during diapause. The introduction of exogenous Mn(III)TBAP may, at least partially, compensate for the function of stress-responsive proteins that may be missing in FOXO RNAi mosquitoes (Sim, 2008).

In summary, these data from C. pipiens support the hypothesis that the insulin signaling pathway and forkhead transcription factor control key characters of diapause, including the metabolic switch to lipid storage, the halt in ovarian development, and enhanced overwintering survival. It is proposed that, under long day lengths, insulin signaling leads to the production of JH needed to prompt ovarian development, and, concurrently, FOXO is suppressed, thus preventing accumulation of fat stores. By contrast, in response to short day lengths, the insulin signaling pathway is shut down, which in turn halts synthesis of the JH needed for ovarian development and releases the suppression of FOXO, leading to accumulation of lipid and the stress tolerance characteristic of diapause. The concurrence of these observations with the proposed involvement of the insulin signaling pathway in other forms of dormancy suggests a mechanism common to diverse forms of developmental arrest (Sim, 2008).

Phosphorylation of FoxO family members, regulation of nuclear localization and degradation: FoxO homologs are direct targets of PKB/Akt

Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene (Brunet, 1999).

Although genetic analysis has demonstrated that members of the winged helix, or forkhead, family of transcription factors play pivotal roles in the regulation of cellular differentiation and proliferation, both during development and in the adult, little is known of the mechanisms underlying their regulation. The activation of phosphatidylinositol 3 (PI3) kinase by extracellular growth factors induces phosphorylation, nuclear export, and transcriptional inactivation of FKHR1, a member of the FKHR subclass of the forkhead family of transcription factors. Protein kinase B (PKB)/Akt, a key mediator of PI3 kinase signal transduction, phosphorylates recombinant FKHR1 in vitro at threonine-24 and serine-253. Mutants FKHR1(T24A), FKHR1(S253A), and FKHR1(T24A/S253A) are resistant to both PKB/Akt-mediated phosphorylation and PI3 kinase-stimulated nuclear export. These results indicate that phosphorylation by PKB/Akt negatively regulates FKHR1 by promoting export from the nucleus (Biggs, 1999).

The phosphatidylinositol-3-OH-kinase (PI(3)K) effector protein kinase B regulates certain insulin-responsive genes, but the transcription factors regulated by protein kinase B have yet to be identified. Genetic analysis in Caenorhabditis elegans has shown that the Forkhead transcription factor daf-16 is regulated by a pathway consisting of insulin-receptor-like daf-2 and PI(3)K-like age-1. Protein kinase B phosphorylates AFX, a human ortholog of daf-16, both in vitro and in vivo. Inhibition of endogenous PI(3)K and protein kinase B activity prevents protein kinase B-dependent phosphorylation of AFX and reveals residual protein kinase B-independent phosphorylation that requires Ras signalling towards the Ral GTPase. In addition, phosphorylation of AFX by protein kinase B inhibits its transcriptional activity. Together, these results delineate a pathway for PI(3)K-dependent signalling to the nucleus (Kops, 1999).

The regulation of intracellular localization of AFX, a human Forkhead transcription factor, was studied. AFX was recovered as a phosphoprotein from transfected COS-7 cells growing in the presence of FBS, and the phosphorylation was eliminated by wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase. AFX is phosphorylated in vitro by protein kinase B (PKB), a downstream target of PI 3-kinase, but a mutant protein in which three putative phosphorylation sites of PKB have been replaced by Ala is not recognized by PKB. In Chinese hamster ovary cells (CHO-K1) cultured with serum, the AFX protein fused with green fluorescence protein (AFX-GFP) is localized mainly in the cytoplasm, and wortmannin induces transient nuclear translocation of the fusion protein. The AFX-GFP mutant in which all three phosphorylation sites have been replaced by Ala is detected exclusively in the cell nucleus. AFX-GFP is in the nucleus when the cells are infected with an adenovirus vector encoding a dominant-negative form of either PI 3-kinase or PKB, whereas the fusion protein stays in the cytoplasm when the cells express constitutively active PKB. In CHO-K1 cells expressing AFX-GFP, DNA fragmentation is induced by the stable PI 3-kinase inhibitor LY294002, and the expression of the active form of PKB suppresses this DNA fragmentation. The phosphorylation site mutant of AFX-GFP enhances DNA fragmentation irrespective of the presence and absence of PI 3-kinase inhibitor. These results indicate that the nuclear translocation of AFX is negatively regulated through its phosphorylation by PKB (Takaishi, 1999).

AFX belongs to a subfamily of Forkhead transcription factors that are phosphorylated by protein kinase B (PKB), also known as Akt. Phosphorylation inhibits the transcriptional activity of AFX and changes the steady-state localization of the protein from the nucleus to the cytoplasm. The goal of this study was threefold: (1) to identify the cellular compartment in which PKB phosphorylates AFX, (2) to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and (3) to elucidate the mechanism by which phosphorylation alters the localization of AFX. Phosphorylation of AFX by PKB is shown to occur in the nucleus. In addition, nuclear export mediated by the export receptor, Crm1, is required for the inhibition of AFX transcriptional activity. Both phosphorylated and unphosphorylated AFX, however, bind Crm1 and can be exported from the nucleus. These results suggest that export is unregulated and that phosphorylation by PKB is not required for the nuclear export of AFX. AFX enters the nucleus by an active, Ran-dependent mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical nuclear localization signal (NLS). S193, contained within this atypical NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a negative charge at S193 by mutating the residue to glutamate reduces nuclear accumulation. PKB-mediated phosphorylation of AFX, therefore, attenuates the import of the transcription factor, which shifts the localization of the protein from the nucleus to the cytoplasm and results in the inhibition of AFX transcriptional activity (Brownawell, 2001).

In Caenorhabditis elegans, an insulin-like signaling pathway to phosphatidylinositol 3-kinase (PI 3-kinase) and AKT negatively regulates the activity of DAF-16, a Forkhead transcription factor. In mammalian cells, C. elegans DAF-16 is a direct target of AKT and AKT phosphorylation generates 14-3-3 binding sites and regulates the nuclear/cytoplasmic distribution of DAF-16 as previously shown for its mammalian homologs FKHR and FKHRL1. In vitro, interaction of AKT- phosphorylated DAF-16 with 14-3-3 prevents DAF-16 binding to its target site in the insulin-like growth factor binding protein-1 gene, the insulin response element. In HepG2 cells, insulin signaling to PI 3-kinase/AKT inhibits the ability of a GAL4 DNA binding domain/DAF-16 fusion protein to activate transcription via the insulin-like growth factor binding protein-1-insulin response element, but not the GAL4 DNA binding site, which suggests that insulin inhibits the interaction of DAF-16 with its cognate DNA site. Elimination of the DAF-16/14-3-3 association by mutation of the AKT/14-3-3 sites in DAF-16, prevents 14-3-3 inhibition of DAF-16 DNA binding and insulin inhibition of DAF-16 function. Similarly, inhibition of the DAF-16/14-3-3 association by exposure of cells to the PI 3-kinase inhibitor LY294002, enhances DAF-16 DNA binding and transcription activity. Surprisingly constitutively nuclear DAF-16 mutants that lack AKT/14-3-3 binding sites also show enhanced DNA binding and transcription activity in response to LY294002, pointing to a 14-3-3-independent mode of regulation. Thus, these results demonstrate at least two mechanisms, one 14-3-3-dependent and the other 14-3-3-independent, whereby PI 3-kinase signaling regulates DAF-16 DNA binding and transcription function (Cahill, 2001).

FKHR is a member of the FOXO subfamily of Forkhead transcription factors, which are important targets for insulin and growth factor signaling. FKHR contains three predicted protein kinase B phosphorylation sites (Thr-24, Ser-256, and Ser-319) that are conserved in other FOXO proteins. Phosphorylation of Ser-256 is critical for the ability of insulin and insulin-like growth factors to suppress transactivation by FKHR and for its exclusion from the nucleus. Ser-256 is located in a basic region of the FKHR DNA binding domain where phosphorylation may have direct effects on DNA binding and/or nuclear targeting. Phosphorylation of Ser-256 may also be required for the phosphorylation of Thr-24 and Ser-319. Evidence is provided that basic residues in the FKHR DNA binding domain are critical for DNA binding and that Ser-256 phosphorylation alters binding activity. Ser-256 phosphorylation also is critical for regulating nuclear/cytoplasmic trafficking; however, this effect requires Thr-24/Ser-319 phosphorylation. Transient transfection studies with reporter gene constructs in 293 cells reveal that the phosphorylation of Ser-256 can inhibit the function of FKHR independent of Thr-24/Ser-319 phosphorylation. Studies with GFP(1) fusion proteins indicate that Ser-256 phosphorylation is critical for nuclear exclusion of FKHR. However, this effect is disrupted when Thr-24 and Ser-319 are replaced by alanine, indicating that nuclear exclusion of FKHR also requires Thr-24/Ser-319 phosphorylation. Gel shift and fluorescence anisotropy studies reveal that basic residues at the C-terminal end of the FKHR DBD are important for DNA binding, and the introduction of a negative charge at the site of Ser-256 limits binding activity. Binding is rapid and reversible, providing an opportunity for the phosphorylation of Ser-256 and subsequent phosphorylation of Thr-24 and Ser-319 and nuclear exclusion of FKHR (Zhang, 2002).

Forkhead transcription factors of the FoxO-group are associated with cellular processes like cell cycle progression and DNA-repair. FoxO function is regulated by protein kinase B (PKB) via the phosphatidylinositol 3-kinase/PKB survival pathway. Phosphorylation of serine and threonine residues in specific PKB phosphorylation motifs leads to exclusion of FoxO-proteins from the nucleus, which excludes them from exerting transactivating activity. Members of the FoxO-group have three highly conserved regions containing a PKB phosphorylation motif. This study describes the cloning and characterization of a novel forkhead domain gene from mouse that appeared to be highly related to the FoxO group of transcription factors and was therefore designated FoxO6. The FoxO6 gene was mapped in region D1 on mouse chromosome 4. In humans, FOXO6 is located on chromosomal region 1p34.1. Embryonic expression of FoxO6 is most apparent in the developing brain, and FoxO6 is expressed in a specific temporal and spatial pattern. Therefore it is probably involved in regulation of specific cellular differentiation. In the adult animal FoxO6 expression is maintained in areas of the nucleus accumbens, cingulate cortex, parts of the amygdala, and in the hippocampus. Structure function analysis of FoxO6 compared with its group members shows that the overall homology is high, but surprisingly a highly conserved region containing multiple phosphorylation sites is lacking. In transfection studies, FoxO6 coupled to GFP showed an unexpected high nuclear localization after stimulation with growth factors, in contrast to the predominant cytosolic localization of FoxO1 and FoxO3. Nuclear export of FoxO6 is mediated through the phosphatidylinositol 3-kinase/PKB pathway. Using a chimeric approach it has been shown that the ability of FoxO6 to shuttle between nucleus and cytosol can be fully restored. In conclusion, the data presented here gives a new view on regulation of FoxO-function through multiple phosphorylation events and other mechanisms involved in the nuclear exclusion of FoxO-proteins (Jacobs, 2003).

Forkhead transcription factor FKHR (Foxo1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. The fate has been investigated of FKHR after cells are stimulated by insulin. Insulin treatment is shown to decrease endogenous FKHR proteins in HepG2 cells; this decrease is inhibited by proteasome inhibitors. FKHR is ubiquitinated in vivo and in vitro, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination in vivo and in vitro. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation (Matsuzaki, 2003).

Growth factor receptors promote cell growth and survival by stimulating the activities of phosphatidylinositol 3-kinase and Akt/PKB. Akt activation causes proteasomal degradation of substrates that control cell growth and survival. Expression of activated Akt triggers proteasome-dependent declines in the protein levels of the Akt substrates tuberin, FOXO1, and FOXO3a. The addition of proteasome inhibitors stabilizes the phosphorylated forms of multiple Akt substrates, including tuberin and FOXO proteins. Activation of Akt triggers the ubiquitination of several proteins containing phosphorylated Akt substrate motifs. Together the data indicate that activated Akt stimulates proteasomal degradation of its substrates and suggest that Akt-dependent cell growth and survival are induced through the degradation of negative regulators of these processes (Plas, 2003).

The life span of C. elegans is extended by mutations that inhibit the function of sensory neurons. In this study, specific subsets of sensory neurons are shown to influence longevity. Certain gustatory neurons inhibit longevity, whereas others promote longevity, most likely by influencing insulin/IGF-1 signaling. Olfactory neurons also influence life span, and they act in a distinct pathway that involves the reproductive system. In addition, a putative chemosensory G protein-coupled receptor expressed in some of these sensory neurons inhibits longevity. Together these findings imply that the life span of C. elegans is regulated by environmental cues and that these cues are perceived and integrated in a complex and sophisticated fashion by specific chemosensory neurons (Alcedo, 2004).

These findings suggest that gustatory neurons are likely to influence life span by perturbing the insulin/IGF-1 pathway. One possibility is that these neurons sense cues that regulate the release of insulin/IGF-1-like hormones that influence the insulin/IGF-1 receptor DAF-2 activity. The C. elegans genome contains more than 30 insulin/IGF-1 homologs, and several of these are expressed in gustatory neurons. The model is favored that longevity-inhibiting ASI and ASG neurons exert their effects on life span by inhibiting the activities of the ASJ and ASK neurons. Thus, one possibility is that the ASI and ASG neurons prevent the longevity-promoting ASJ and ASK neurons from producing a DAF-2 antagonist. It is intriguing that double mutants that have defects in proteins thought to be required for neuronal insulin secretion and in daf-2 activity have an intermediate life span between those of the corresponding neurosecretory single mutants and daf-2 hypomorphic single mutants (Ailion et al., 1999). This finding is consistent with the idea that some sensory neurons might secrete DAF-2 antagonists (Alcedo, 2004).

A number of insulin-like peptides have now been implicated in the regulation of aging. One such candidate DAF-2 antagonist is ins-1, but this gene is expressed not only in longevity-promoting neurons but also in longevity-inhibiting neurons. At this point, the information available about specific insulin-like peptides does not suggest simple models that explain the data. However, this may change as more is learned about the functions of these proteins. For example, it is possible that other insulin/IGF-1-like peptides function as antagonists in the ASJ and ASK neurons but not in the ASI and ASG neurons, since other insulin/IGF-1-like peptides are expressed in ASJ (Alcedo, 2004).

These observations suggest that olfactory neurons act in a regulatory pathway distinct from gustatory neurons to affect life span. (1) The combined ablation of the gustatory ASI and olfactory AWA and AWC neurons increases life span more than does ablation of either ASI or of AWA and AWC neurons alone. (2) Killing ASJ and ASK suppresses the longevity of ASI-ablated animals but not that of olfactory neuron-ablated animals. (3) The life span extension produced by killing gustatory neurons is completely daf-16 dependent, whereas the life span extension produced by killing olfactory neurons is only partially daf-16 dependent (Alcedo, 2004).

Olfactory neurons may influence life span by perturbing an endocrine signaling pathway that involves the reproductive system. Previous findings have suggested that the germline of C. elegans generates a signal that inhibits longevity and is counterbalanced by a signal from the somatic gonad that promotes longevity. Like the olfactory neurons characterized, the somatic gonad of C. elegans affects life span, at least in part, in a daf-16-independent fashion. In addition, olfactory neurons are required for the somatic gonad to influence life span. In wild-type animals, killing the somatic gonad precursors completely prevents germline ablation from extending life span, but in animals lacking olfactory neurons, it does not. One possibility is that olfactory neurons regulate the release of a hormone that allows the somatic gonad to influence longevity. If this model is correct, then it implies that, under some environmental conditions, the somatic gonad signal is silenced and may no longer be able to counterbalance the signals from the animal's germline. Alternatively, these olfactory neurons could produce a longevity signal in response to a different signal from the somatic gonad. The somatic gonad appears to regulate a pathway that involves DAF-2. Thus, as with the gustatory neurons, it is possible that the olfactory neurons influence longevity by regulating the release of insulin-like peptides (Alcedo, 2004).

Why might sensory neurons influence longevity? One environmental condition, food limitation, is known to have a dramatic effect on life span in many organisms. Caloric restriction extends life span and also delays reproduction. When ample food is restored to calorically-restricted rats, they can reproduce, even at a time when the age-matched controls are post-reproductive or dead. Thus, this response to caloric restriction has obvious survival value, since it allows animals to postpone reproduction until conditions improve. Dauer formation, which is regulated, at least in part, by sensory cues, serves the same function in C. elegans -- it allows animals to postpone reproduction under harsh environmental conditions. No obvious changes were observed in the timing of reproduction in the neuron-ablated animals; however, it is possible that the environmental cues that influence the activities of these neurons in nature also influence other neurons that control reproduction. In this way, sensory cues could affect life span and reproduction coordinately. Alternatively, certain environmental conditions could favor a shorter post-reproductive life span to prevent the aging animals from competing for resources with their progeny. A population of worms that lacks parental competition for resources should, over time, develop a significant advantage relative to populations in which such competition takes place (Alcedo, 2004).

The odr-10 gene encodes an olfactory G protein-coupled receptor that senses diacetyl, an odorant sensed by AWA neurons. odr-10 null mutants are not long-lived, implying that neither diacetyl nor its receptor regulates life span. In contrast, decreasing the mRNA levels of the putative chemosensory G protein-coupled receptor str-2, through RNA-mediated interference, extends life span. This suggests that C. elegans' life span is influenced by its perception of an environmental cue -- as yet unidentified -- that is sensed by STR-2. The identification of sensory cues that influence life span, such as those sensed by STR-2, should make it possible to address this interesting question experimentally (Alcedo, 2004).

Despite genetic evidence establishing angiopoietin-1 (Ang-1) as an essential regulator of vascular development, the molecular mechanisms underlying Ang-1 function are almost completely uncharacterized. This study demonstrates that Ang-1, via Akt activation, is a potent inhibitor of the forkhead transcription factor FKHR (FOXO1), identifying a nuclear signaling pathway through which Ang-1 modulates gene expression. Microarray analysis was used to show that FKHR, whose function in endothelial cells has not previously been elucidated, regulates many genes associated with vascular destabilization and remodeling (including angiopoietin-2, an Ang-1 antagonist) and endothelial cell apoptosis (e.g., survivin, TRAIL). Ang-1 inhibits FKHR-mediated changes in gene expression and FKHR-induced apoptosis. Analysis of gene expression changes induced by an activated version of Akt confirms that FKHR is a major target through which Akt regulates transcription in endothelial cells. RNA interference was used to demonstrate that FKHR is required for the expression of genes (including Ang-2) that have important vascular functions. These data suggest a novel, tissue-specific role for the Akt/FKHR pathway in the vasculature and suggest a mechanistic basis for the previously described actions of Ang-1 as a regulator of endothelial cell survival and blood vessel stability (Daly, 2004).

Other signaling upstream of FoxO family members

AFX is a Forkhead transcription factor that induces a G(1) cell cycle arrest via upregulation of the cell cycle inhibitor p27(Kip1). Protein kinase B (PKB) phosphorylates AFX causing inhibition of AFX by nuclear exclusion. In addition, Ras, through the activation of the RalGEF-Ral pathway, induces phosphorylation of AFX. The Ras-Ral pathway provokes phosphorylation of threonines 447 and 451 in the C terminus of AFX. A mutant protein in which both threonines are substituted for alanines (T447A/T451A) still responds to PKB-regulated nuclear-cytoplasmic shuttling, but transcriptional activity and consequent G(1) cell cycle arrest are greatly impaired. Furthermore, inhibition of the Ral signaling pathway abolishes both AFX-mediated transcription and regulation of p27(Kip1), while activation of Ral augments AFX activity. From these results it is concluded that Ral-mediated phosphorylation of threonines 447 and 451 is required for proper activity of AFX-WT. Interestingly, the T447A/T451A mutation did not affect the induction of transcription and G(1) cell cycle arrest by the PKB-insensitive AFX-A3 mutant, suggesting that Ral-mediated phosphorylation plays a role in the regulation of AFX by PKB (De Ruiter, 2001).

The cytokine IL-2 plays a very important role in the proliferation and survival of activated T cells. These effects of IL-2 are dependent on signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. PI3K, through activation of protein kinase B/Akt, inhibits transcriptional activation by a number of forkhead transcription factors (FoxO1, FoxO3, and FoxO4). The role of these forkhead transcription factors in the IL-2-induced T cell proliferation and survival has been investigated. IL-2 regulates phosphorylation of FoxO3 in a PI3K-dependent fashion. Phosphorylation and inactivation of FoxO3 appears to play an important role in IL-2-mediated T cell survival, because mere activation of FoxO3 is sufficient to trigger apoptosis in T cells. Indeed, active FoxO3 can induce expression of IL-2-regulated genes, such as the cdk inhibitor p27(Kip1) and the proapoptotic Bcl-2 family member Bim. Furthermore, IL-2 triggers a rapid, PI3K-dependent, phosphorylation of FoxO1a in primary T cells. Thus, it is proposed that inactivation of FoxO transcription factors by IL-2 plays a critical role in T cell proliferation and survival (Stahl, 2002).

Myc synergizes with Ras and PI3-kinase in cell transformation, yet the molecular basis for this behavior is poorly understood. Myc is shown to recruit TFIIH, P-TEFb and Mediator to the cyclin D2 and other target promoters, while the PI3-kinase pathway controls formation of the preinitiation complex and loading of RNA polymerase II. The PI3-kinase pathway involves Akt-mediated phosphorylation of FoxO transcription factors. In a nonphosphorylated state, FoxO factors inhibit induction of multiple Myc target genes, Myc-induced cell proliferation and transformation by Myc and Ras. Abrogation of FoxO function enables Myc to activate target genes in the absence of PI3-kinase activity and to induce foci formation in primary cells in the absence of oncogenic Ras. It is suggested that the cooperativity between Myc and Ras is at least in part due to the fact that Myc and FoxO proteins control distinct steps in the activation of an overlapping set of critical target genes (Bouchard, 2004).

Forkhead transcription factors of the FOXO class are negatively regulated by PKB/c-Akt in response to insulin/IGF signalling, and are involved in regulating cell cycle progression and cell death. In contrast to insulin signalling, low levels of oxidative stress generated by treatment with H2O2 induce the activation of FOXO4. Upon treatment of cells with H2O2, the small GTPase Ral is activated and this results in a JNK-dependent phosphorylation of FOXO4 on threonine 447 and threonine 451. This Ral-mediated, JNK-dependent phosphorylation is involved in the nuclear translocation and transcriptional activation of FOXO4 after H2O2 treatment. In addition, it is shown that this signalling pathway is also employed by tumor necrosis factor alpha to activate FOXO4 transcriptional activity. FOXO members have been implicated in cellular protection against oxidative stress via the transcriptional regulation of manganese superoxide dismutase and catalase gene expression. The results reported here, therefore, outline a homeostasis mechanism for sustaining cellular reactive oxygen species that is controlled by signalling pathways that can convey both negative (PI-3K/PKB) and positive (Ras/Ral) inputs (Essers, 2004).

Forkhead box class O (FOXO) proteins are transcription factors that function downstream of the PTEN tumor suppressor and directly control the expression of genes involved in apoptosis, cell cycle progression, and stress responses. In the present study, FOXO1 is shown to interact with four and a half LIM 2 (FHL2) in prostate cancer cells. This interaction occurs in the nucleus and is enhanced by lysophosphatic acid. FHL2 decreases the transcriptional activity of FOXO1 and the expression of known FOXO target genes and inhibits FOXO1-induced apoptosis. Interestingly, SIRT1, a mammalian homolog of yeast Sir2, binds to and deacetylates FOXO1 and inhibits its transcriptional activity. FHL2 enhances the interaction of FOXO1 and SIRT1 and the deacetylation of FOXO1 by Sirtuin-1 (SIRT1). Overall, these data show that FHL2 inhibits FOXO1 activity in prostate cancer cells by promoting the deacetylation of FOXO1 by SIRT1 (Yang, 2005 ).

Splice variants of FoxO family members

Several studies indicate that FKHR and AFX, mammalian homologs of the Caenorhabditis elegans forkhead transcription factor DAF-16, function in the insulin signaling pathway. A novel AFX isoform, designated AFX zeta, has been discoved in which the first 16 amino acids of the forkhead domain are not present. PCR analysis shows that this isoform is most abundant in the liver, kidney, and pancreas. In HepG2 cells, overexpressed AFX zeta induces reporter gene activity through the insulin-responsive sequences of the phosphoenolpyruvate carboxykinase (PEPCK), IGFBP-1, and G6Pase promoters. AFX zeta-mediated stimulation was repressed by insulin treatment, by bisperoxovanadate treatment, and by overexpression of constitutively active protein kinase B (PKB). Insulin treatment and PKB overexpression result in phosphorylation of AFX zeta. Furthermore, an AMP-activated protein kinase activator, represses AFX zeta-dependent reporter activation. Taken together, these findings suggest that AFX zeta is a downstream target of both the phosphatidylinositol 3-kinase/PKB insulin signaling pathway and an AMP-activated protein kinase-dependent pathway (Yang, 2002).

Transcriptional targets of FoxO family members

Because overexpression of the glucose-6-phosphatase catalytic subunit (G-6-Pase) in both type 1 and type 2 diabetes may contribute to the characteristic increased rate of hepatic glucose production, whether the insulin response unit (IRU) identified in the mouse G-6-Pase promoter is conserved in the human promoter was investigated. A series of human G-6-Pase-chloramphenicol acetyltransferase (CAT) fusion genes was transiently transfected into human HepG2 hepatoma cells, and the effect of insulin on basal CAT expression was analyzed. The results suggest that the IRU identified in the mouse promoter is conserved in the human promoter, but that an upstream multimerized insulin response sequence (IRS) motif that is only found in the human promoter appears to be functionally inactive. The G-6-Pase IRU comprises two distinct promoter regions, designated A and B. Region B contains an IRS, whereas region A acts as an accessory element to enhance the effect of insulin, mediated through region B, on basal G-6-Pase gene transcription. The accessory factor binding region A is hepatocyte nuclear factor-1, and the forkhead protein FKHR is a candidate for the insulin-responsive transcription factor binding region B (Ayala, 1999).

Cell death is regulated mainly through apoptosis. Deregulation of apoptosis has been associated with cancer, autoimmune diseases and degenerative disorders. Many cells, particularly those of the hematopoietic system, have a default program of cell death and survival that is dependent on the constant supply of survival signals. The Bcl-2 family, which has both pro- and anti-apoptotic members, plays a critical role in regulating cell survival. One family member, the Bcl-2 interacting mediator of cell death (Bim), contains only a protein-interaction motif known as the BH3 domain, allowing it to bind pro-survival Bcl-2 molecules, neutralizing their function. Disruption of the bim gene results in resistance to apoptosis following cytokine withdrawal in leukocytes, indicating that regulation of the pro-apoptotic activity of Bim is critical for maintenance of the default apoptotic program. Withdrawal of cytokine results in upregulation of Bim expression concomitant with induction of the apoptotic program in lymphocytes. Activation of the forkhead transcription factor FKHR-L1, implicated in regulation of apoptosis in T lymphocytes, is sufficient to induce Bim expression. A mechanism is proposed by which cytokines promote lymphocyte survival by inhibition of FKHR-L1, preventing Bim expression (Dijkers, 2000).

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulin-responsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. The Caenorhabditis elegans protein DAF-16 binds the IGFBP-1-IRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX, activate transcription through the IGFBP-1.IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300/CREB-binding protein (CBP). DAF-16 and FKHR can interact with both the KIX and E1A/SRC interaction domains of p300/CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1.IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, it is concluded that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300/CBP/SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site (Nasrin, 2000).

Paclitaxel is used to treat breast cancers, but the mechanisms by which it induces apoptosis are poorly understood. Consequently, the role of the FoxO transcription factors in determining cellular response to paclitaxel has been examined. Western blotting has revealed that in a panel of 9 breast cancer cell lines expression of FoxO1a and FoxO3a correlates with the expression of the pro-apoptotic FoxO target Bim, which is associated with paclitaxel-induced apoptosis. In paclitaxel sensitive MCF-7 cells, the already high basal levels of FoxO3a and Bim protein increases dramatically after drug treatment, as does Bim mRNA; this increase correlates with apoptosis induction. This was not observed in MDA-231 cells which expressed low levels of FoxOs and Bim. In MCF-7 cells, maximal induction of Bim promoter is dependent on a FoxO binding site, suggesting that FoxO3a is responsible for the transcriptional upregulation of Bim. Gene silencing experiments show that siRNA specific for FoxO3a reduces the levels of FoxO3a and Bim protein as well as inhibits apoptosis in paclitaxel-treated MCF-7 cells. Furthermore, siRNA specific for Bim reduces the levels of Bim protein and inhibits apoptosis in paclitaxel-treated MCF-7 cells. This is the first demonstration that upregulation of FoxO3a by paclitaxel can result in increased levels of Bim mRNA and protein which can be a direct cause of apoptosis in breast cancer cells (Sunters, 2003).

FoxO family members and insulin signaling pathways

Insulin inhibits the expression of multiple genes in the liver containing an insulin response sequence (IRS) (CAAAA(C/T)AA), and protein kinase B (PKB) mediates this effect of insulin. Genetic studies in Caenorhabditis elegans indicate that daf-16, a forkhead/winged-helix transcription factor, is a major target of the insulin receptor-PKB signaling pathway. FKHR, a human homolog of daf-16, contains three PKB sites and is expressed in the liver. Reporter gene studies in HepG2 hepatoma cells show that FKHR stimulates insulin-like growth factor-binding protein-1 promoter activity through an IRS, and introduction of IRSs confers this effect on a heterologous promoter. Insulin disrupts IRS-dependent transactivation by FKHR, and phosphorylation of Ser-256 by PKB is necessary and sufficient to mediate this effect. Antisense studies indicate that FKHR contributes to basal promoter function and is required to mediate effects of insulin and PKB on promoter activity via an IRS. These results provide the first report that FKHR stimulates promoter activity through an IRS and that phosphorylation of FKHR by PKB mediates effects of insulin on gene expression. Signaling to FKHR-related forkhead proteins via PKB may provide an evolutionarily conserved mechanism by which insulin and related factors regulate gene expression (Guo, 1999).

The insulin response element (IRE) in the IGFBP-1 promoter, and in other gene promoters, contains a T(A/G)TTT motif essential for insulin inhibition of transcription. Studies presented here test whether FKHR may be the transcription factor that confers insulin inhibition through this IRE motif. Immunoblots using antiserum to the synthetic peptide FKHR413-430, RNase protection, and Northerns blots show that FKHR is expressed in HEP G2 human hepatoma cells. Southwestern blots, electromobility shift assays, and DNase I protection assays show that Escherichia coli-expressed GST-FKHR binds specifically to IREs from the IGFBP-1, PEPCK and TAT genes; however, unlike HNF3beta, another protein proposed to be the insulin regulated factor, GST-FKHR does not bind the insulin unresponsive G/C-A/C mutation of the IGFBP-1 IRE. When HEP G2 cells were cotransfected with FKHR expression vectors and with IGFBP-1 promoter plasmids containing either native or mutant IREs, FKHR expression induced a 5-fold increase in activity of the native IGFBP-1 promoter but no increase in activity of promoter constructs containing insulin unresponsive IRE mutants. These data suggest that FKHR, and/or a related family member, is the important T(G/A)TTT binding protein that confers the inhibitory effect of insulin on gene transcription (Durham, 1999).

Winged helix/forkhead (Fox) transcription factors have been implicated in the regulation of a number of insulin-responsive genes. The insulin response elements (IREs) of the phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes bind members of the FKHR and HNF3 subclasses of Fox proteins. Mutational analyses of the PEPCK and IGFBP-1 IREs reveal mutations that do not affect the binding of HNF3 proteins to these elements but do eliminate the ability of the IREs to mediate an insulin response. This dissociation of binding and function provides compelling evidence that HNF3 proteins, per se, are not insulin response proteins. The same approach was used here to determine if FKHRL1, a member of the FKHR subclass of Fox proteins, binds to the PEPCK and IGFBP-1 IREs in a manner that correlates with the ability of these elements to mediate an insulin response. Overexpression of FKHRL1 stimulates transcription from transfected reporter constructs that contain a multimerized PEPCK IRE or an IGFBP-1 IRE and this stimulation is repressed by insulin. There is a direct correlation between the ability of mutant versions of the PEPCK and IGFBP-1 IREs to bind FKHRL1 and their ability to mediate FKHRL1-induced transcription when FKHRL1 is overexpressed. However, under conditions where FKHRL1 is not overexpressed, there is a lack of correlation between FKHRL1 binding to mutant versions of the PEPCK and IGFBP-1 IREs and the ability of these elements to mediate an insulin response. Therefore, the PEPCK and IGFBP-1 IREs mediate FKHRL1-induced transcription and its inhibition by insulin when this protein is overexpressed, but at the normal cellular concentration of FKHRL1 the insulin response mediated by these elements must involve another protein (Hall, 2000).

The forkhead rhabdomyosarcoma transcription factor (FKHR) is a promising candidate to be the transcription factor that binds to the insulin response element of the insulin-like growth factor-binding protein-1 (IGFBP-1) promoter and mediates insulin inhibition of IGFBP-1 promoter activity. Cotransfection of mouse FKHR increases IGFBP-1 promoter activity 2-3-fold in H4IIE rat hepatoma cells; insulin inhibits FKHR-stimulated promoter activity approximately 70%. A C-terminal fragment of mouse FKHR (residues 208-652) that contains the transcription activation domain fused to a Gal4 DNA binding domain potently stimulated Gal4 promoter activity. Insulin inhibits FKHR fragment-stimulated promoter activity by approximately 70%. Inhibition is abolished by coincubation with the phosphatidylinositol-3 kinase inhibitor, LY294002. The FKHR 208-652 fragment contains two consensus sites for phosphorylation by protein kinase B (PKB)/Akt, Ser-253 and Ser-316. Neither site is required for insulin inhibition of promoter activity stimulated by the FKHR fragment, and overexpression of Akt does not inhibit FKHR fragment-stimulated Gal4 promoter activity. These results suggest that insulin- and phosphatidylinositol-3 kinase-dependent phosphorylation of another site in the fragment by a kinase different from PKB/Akt inhibits transcription activation by the fragment. Phosphorylation of this site also may be involved in insulin inhibition of transcription activation by full-length FKHR, but only after phosphorylation of Ser-253 by PKB/Akt (Tomizawa, 2000).

Glucose-6-phosphatase plays an important role in the regulation of hepatic glucose production, and insulin suppresses glucose-6-phosphatase gene expression. Recent studies indicate that protein kinase B and Forkhead proteins contribute to insulin-regulated gene expression in the liver. The role has been examined of protein kinase B and Forkhead proteins in mediating effects of insulin on glucose-6-phosphatase promoter activity. Transient transfection studies with reporter gene constructs demonstrate that insulin suppresses both basal and dexamethasone/cAMP-induced activity of the glucose-6-phosphatase promoter in H4IIE hepatoma cells. Both effects are partially mimicked by coexpression of protein kinase Balpha. Coexpression of the Forkhead transcription factor FKHR stimulates the glucose-6-phosphatase promoter activity via interaction with an insulin response unit (IRU), and this activation is suppressed by protein kinase B. Coexpression of a mutated form of FKHR that cannot be phosphorylated by protein kinase B abolishes the regulation of the glucose-6-phosphatase promoter by protein kinase B and disrupts the ability of insulin to regulate the glucose-6-phosphatase promoter via the IRU. Mutation of the insulin response unit of the glucose-6-phosphatase promoter also prevents the regulation of promoter activity by FKHR and protein kinase B but only partially impairs the ability of insulin to suppress both basal and dexamethasone/cAMP-stimulated promoter function. Taken together, these results indicate that signaling by protein kinase B to Forkhead proteins can account for the ability of insulin to regulate glucose-6-phosphatase promoter activity via the IRU and that other mechanisms that are independent of the IRU, protein kinase B, and Forkhead proteins also are important in mediating effects of insulin on glucose-6-phosphatase gene expression (Schmoll, 2000).

The transcription factor Foxo1 controls the expression of genes involved in fundamental cellular processes. In keeping with its important physiological roles, Foxo1 activity is negatively regulated in response to growth factors and cytokines that activate a phosphatidylinositol 3-kinase (PI 3-kinase) protein kinase B (PKB)/Akt pathway. PKB/Akt-mediated phosphorylation of Foxo1 has been shown to result in the inhibition of target gene transcription and to trigger the export of Foxo1 from the nucleus, which is generally believed to explain the subsequent decrease of transcription. In the present study, using a chimeric protein in which a C-terminal fragment of Foxo1 (amino acids 208-652) containing the transactivation domain is fused to the yeast Gal4 DNA binding domain, evidence is presented showing that insulin can directly regulate transactivation by Foxo1 in H4IIE rat hepatoma cells. Insulin inhibition of Foxo1-(208-652)-stimulated transactivation is mediated by PI 3-kinase but in contrast to full-length Foxo1, does not require either of the two PKB/Akt phosphorylation sites (Ser253 and Ser316) present in the protein fragment. Using mutational and deletion studies, two potential phosphorylation sites, Ser319 and Ser499, as well as a 15-amino acid region located between residues 350 and 364, are identified that are critical for insulin inhibition of transactivation by Foxo1-(208-652). It is concluded that the transcriptional activity of Foxo1 is regulated at different levels by insulin: transactivation, as well as DNA binding and nuclear exclusion. These different regulatory mechanisms allow the precise control of transcription of Foxo1 target genes by insulin (Perrot, 2003).

Peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) plays a major role in mediating hepatic gluconeogenesis in response to starvation, during which PGC-1 is induced by the cyclic AMP response element binding protein. Although it is observed that insulin counteracts PGC-1 transcription, the mechanism by which insulin suppresses the transcription of PGC-1 is still unclear. Forkhead transcription factor FKHR is shown to contribute to mediating the effects of insulin on PGC-1 promoter activity. Reporter assays demonstrate that insulin suppresses the basal PGC-1 promoter activity and coexpression of PKB mimics the effect of insulin in HepG2 cells. Insulin response sequences (IRSs) are addressed in the PGC-1 promoter as the direct target for FKHR in vivo. Coexpression of FKHR stimulates the PGC-1 promoter activity via interaction with the IRSs, while coexpression of FKHR (3A), in which the three putative PKB sites in FKHR are mutated, mainly abolishes the suppressive effect of PKB. Whereas deletion of the IRSs prevents the promoter stimulation by FKHR, that activity is still partially inhibited by insulin. These results indicate that signaling via PKB to FKHR can partly account for the effect of insulin to regulate the PGC-1 promoter activity via the IRSs (Daitoku, 2003).

The FOXO family of forkhead transcription factors stimulates the transcription of target genes involved in many fundamental cell processes, including cell survival, cell cycle progression, DNA repair, and insulin sensitivity. The activity of FOXO proteins is principally regulated by activation of protein kinase B (PKB)/Akt by insulin and other cytokines. PKB/Akt phosphorylates three consensus sites in FOXO proteins, leading to their export from the nucleus and the inhibition of FOXO-stimulated transcription. It has been widely accepted that the decreased transcription results from reduced abundance of FOXO proteins in the nucleus. In the present study Leu375 was mutated to alanine in the nuclear export signal of Foxo1 (mouse FOXO1), so that Foxo1 would remain in the nucleus of H4IIE rat hepatoma cells after insulin treatment. Would insulin still inhibit transcription stimulated by the Foxo1 mutant? Despite the retention of the Foxo1 mutant in the nucleus, insulin inhibits L375A-Foxo1-stimulated transcription to the same extent as transcription stimulated by wild-type Foxo1. Similar results were obtained using reporter plasmids containing the rat IGF-binding protein-1 promoter or a minimal promoter with three copies of the insulin response element to which FOXO proteins bind. It was concluded that insulin can inhibit Foxo1-stimulated transcription even when nuclear export of Foxo1 is prevented, indicating that insulin inhibition can occur by direct mechanisms that do not depend on altering the subcellular distribution of the transcription factor (Tsai, 2003).

Insulin-FOXO3 signaling modulates circadian rhythms via regulation of clock transcription

Circadian rhythms are responsive to external and internal cues, light and metabolism being among the most important. In mammals, the light signal is sensed by the retina and transmitted to the suprachiasmatic nucleus (SCN) master clock, where it is integrated into the molecular oscillator via regulation of clock gene transcription. The SCN synchronizes peripheral oscillators, an effect that can be overruled by incoming metabolic signals. As a consequence, peripheral oscillators can be uncoupled from the master clock when light and metabolic signals are not in phase. The signaling pathways responsible for coupling metabolic cues to the molecular clock are being rapidly uncovered. This study shows that insulin-phosphatidylinositol 3-kinase (PI3K)-Forkhead box class O3 (FOXO3) signaling is required for circadian rhythmicity in the liver via regulation of Clock. Knockdown of FoxO3 dampens circadian amplitude, an effect that is rescued by overexpression of Clock. Subsequently, binding of FOXO3 to two Daf-binding elements (DBEs) located in the Clock promoter area was demonstrated, implicating Clock as a transcriptional target of FOXO3. Transcriptional oscillation of both core clock and output genes in the liver of FOXO3-deficient mice is affected, indicating a disrupted hepatic circadian rhythmicity. Finally, it was shown that insulin, a major regulator of FOXO activity, regulates Clock levels in a PI3K- and FOXO3-dependent manner. These data point to a key role of the insulin-FOXO3-Clock signaling pathway in the modulation of circadian rhythms (Chaves, 2014)

FoxO family members interact with other transcription factors

The forkhead factor Foxo1 (or FKHR) was identified in a yeast two-hybrid screen as a peroxisome proliferator-activated receptor (PPAR) gamma-interacting protein. Foxo1 antagonized PPARgamma activity and vice versa indicating that these transcription factors functionally interact in a reciprocal antagonistic manner. One mechanism by which Foxo1 antagonizes PPARgamma activity is through disruption of DNA binding; Foxo1 inhibits the DNA binding activity of a PPARgamma/retinoid X receptor alpha heterodimeric complex. The Caenorhabditis elegans nuclear hormone receptor, DAF-12, interacts with the C. elegans forkhead factor, DAF-16, paralleling the interaction between PPARgamma and Foxo1. daf-12 and daf-16 have been implicated in C. elegans insulin-like signaling pathways, and PPARgamma and Foxo1 likewise have been linked to mammalian insulin signaling pathways. These results suggest a convergence of PPARgamma and Foxo1 signaling that may play a role in insulin action and the insulinomimetic properties of PPARgamma ligands. A more general convergence of nuclear hormone receptor and forkhead factor pathways may be important for multiple biological processes and this convergence may be evolutionarily conserved (Dowell, 2003).

Recent studies have suggested that the protection of cell apoptosis by AKT involves phosphorylation and inhibition of FKHR and related FOXO forkhead transcription factors and that androgens provide an AKT-independent cell survival signal in prostate cancer cells. This study reports receptor-dependent repression of FKHR function by androgens in prostate cancer cells. Transcriptional analysis demonstrates that activation of the androgen receptor caused an inhibition of both wild-type FKHR and a mutant in which all three known AKT sites were mutated to alanines, showing that the repression is AKT independent. In vivo and in vitro coprecipitation studies demonstrate that the repression is mediated through protein-protein interaction between FKHR and the androgen receptor. Mapping analysis localized the interacting domains to the carboxyl terminus between amino acids 350 and 655 of FKHR and to the amino-terminal A/B region and the ligand binding domain of the receptor. Further analysis demonstrates that the activated androgen receptor blocks FKHR's DNA binding activity and impairs its ability to induce Fas ligand expression and prostate cancer cell apoptosis and cell cycle arrest. These studies identify a new mechanism for androgen-mediated prostate cancer cell survival that appears to be independent of the activity of the receptor on androgen response element-mediated transcription and establish FKHR and related FOXO forkhead proteins as important nuclear targets for both AKT-dependent and -independent survival signals in prostate cancer cells (Li, 2003).

Smooth muscle cells (SMCs) modulate their phenotype between proliferative and differentiated states in response to physiological and pathological cues. Insulin-like growth factor-I stimulates differentiation of SMCs by activating phosphoinositide-3-kinase (PI3K)-Akt signaling. Foxo forkhead transcription factors act as downstream targets of Akt and are inactivated through phosphorylation by Akt. Foxo4 represses SMC differentiation by interacting with and inhibiting the activity of myocardin, a transcriptional coactivator of smooth muscle genes. PI3K/Akt signaling promotes SMC differentiation, at least in part, by stimulating nuclear export of Foxo4, thereby releasing myocardin from its inhibitory influence. Accordingly, reduction of Foxo4 expression in SMCs by siRNA enhances myocardin activity and SMC differentiation. It is concluded that signal-dependent interaction of Foxo4 with myocardin couples extracellular signals with the transcriptional program for SMC differentiation (Liu, 2005).

Cell cycle regulation by FoxO family members

The Forkhead transcription factors AFX, FKHR and FKHR-L1 are orthologs of DAF-16, a Forkhead factor that regulates longevity in Caenorhabditis elegans. Overexpression of these Forkhead transcription factors causes growth suppression in a variety of cell lines, including a Ras-transformed cell line and a cell line lacking the tumor suppressor PTEN. Expression of AFX blocks cell-cycle progression at phase G1, independent of functional retinoblastoma protein (pRb) but dependent on the cell-cycle inhibitor p27kip1. Indeed, AFX transcriptionally activates p27kip1, resulting in increased protein levels. It is concluded that AFX-like proteins are involved in cell-cycle regulation and that inactivation of these proteins is an important step in oncogenic transformation (Medema, 2000).

Cell cycle progression is a process that is tightly controlled by internal and external signals. Environmental cues, such as those provided by growth factors, activate early signals that promote cell cycle entry. Cells that have progressed past the restriction point become independent of growth factors, and cell cycle progression is then controlled endogenously. The phosphatidylinositol 3OH kinase (PI(3)K)/protein kinase B (PKB) pathway must be activated in G1 to inactivate forkhead transcription factors (FKH-TFs) and allow cell cycle entry. Subsequent attenuation of the PI(3)K/PKB pathway is required to allow transcriptional activation of FKH-TF in G2. FKH-TF activity in G2 controls mammalian cell cycle termination, since interference with FKH transcriptional activation by disrupting PI(3)K/PKB downregulation, or by expressing a transcriptionally inactive FKH mutant, induces cell accumulation in G2/M, defective cytokinesis, and delayed transition from M to G1 of the cell cycle. FKH-TFs regulate expression of mitotic genes such as cyclin B and polo-like kinase (Plk). These results support the important role of forkhead in the control of mammalian cell cycle completion, and suggest that efficient execution of the mitotic program depends on downregulation of PI(3)K/PKB and consequent induction of FKH transcriptional activity (Alvarez, 2001).

The Forkhead factors AFX (FOXO4) and FKHR-L1 (FOXO3a) directly control transcription of the retinoblastoma-like p130 protein and cause upregulation of p130 protein expression. Detailed analysis of p130 regulation demonstrates that following Forkhead-induced cell cycle arrest, cells enter G0 and become quiescent. This is shown by a change in phosphorylation of p130 to G0-specific forms and increased p130/E2F-4 complex formation. Most importantly, long-term Forkhead activation causes a sustained but reversible inhibition of proliferation without a marked increase in apoptosis. As for the activity of the Forkheads, protein levels of p130 are controlled by endogenous PI3K/PKB signaling upon cell cycle reentry. Surprisingly, not only nontransformed cells, but also cancer cells such as human colon carcinoma cells, are forced into quiescence by Forkhead activation. It is therefore proposed that Forkhead inactivation by PKB signaling in quiescent cells is a crucial step in cell cycle reentry and contributes to the processes of transformation and regeneration (Kops, 2002a).

The FoxO forkhead transcription factors FoxO4 (AFX), FoxO3a (FKHR.L1), and FoxO1a (FKHR) represent important physiological targets of phosphatidylinositol-3 kinase (PI3K)/protein kinase B (PKB) signaling. Overexpression or conditional activation of FoxO factors is able to antagonize many responses to constitutive PI3K/PKB activation including its effect on cellular proliferation. The FoxO-induced cell cycle arrest is partially mediated by enhanced transcription and protein expression of the cyclin-dependent kinase inhibitor p27(kip1). A p27(kip1)-independent mechanism has been identified that plays an important role in the antiproliferative effect of FoxO factors. Forced expression or conditional activation of FoxO factors leads to reduced protein expression of the D-type cyclins D1 and D2 and is associated with an impaired capacity of CDK4 to phosphorylate and inactivate the S-phase repressor pRb. Downregulation of D-type cyclins involves a transcriptional repression mechanism and does not require p27(kip1) function. Ectopic expression of cyclin D1 can partially overcome FoxO factor-induced cell cycle arrest, demonstrating that downregulation of D-type cyclins represents a physiologically relevant mechanism of FoxO-induced cell cycle inhibition (Schmidt, 2002).

The insulin-like growth factor I (IGF-I) stimulates muscle satellite cell proliferation. IGF-I-stimulated proliferation of primary satellite cells is associated with the activation of phosphatidylinositol 3'-kinase (PI3K)/Akt and the downregulation of a cell-cycle inhibitor p27Kip1. To understand mechanisms by which IGF-I signals the downregulation of p27Kip1 in rat skeletal satellite cells, the role of Forkhead transcription factor FoxO1 in transcriptional activity of p27Kip1 was examined. When primary rat satellite cells are transfected with a p27Kip1 promoter-reporter gene construct, IGF-I inhibits specific p27Kip1 promoter activity. Addition of LY294002, an inhibitor of PI3K, reverses the IGF-I-mediated downregulation of p27Kip1 promoter activity. Co-transfection of wild type (WT) FoxO1 into satellite cells increases p27Kip1 promoter activity in the absence of IGF-I supplementation. Addition of IGF-I reverses the induction of p27Kip1 promoter activity by WT FoxO1. When a mutated FoxO1 (without Thr24, Ser256, and Ser316 Akt phosphorylation sites) is used, IGF-I is no longer able to reverse the FoxO1 induced stimulation of p27Kip1 promoter activity that is seen when WT FoxO1 is present. When the satellite cells are treated with IGF-I, phosphorylation of Akt-Ser473 and FoxO1-Ser256 is increased. In addition, when the cells are pre-incubated with LY294002 before IGF-I stimulation, the phosphorylation of Akt-Ser473 and FoxO1-Ser256 is inhibited, implying that phosphorylation of Akt and FoxO1 is downstream of IGF-I-induced PI3K signaling. However, IGF-I does not induce phosphorylation of FoxO1 on residues Thr24 and Ser316. These results suggest that IGF-I induces the phosphorylation of Ser256 and inactivates FoxO1, thereby downregulating the activation of the p27Kip1 promoter. Thus, inactivation of FoxO1 by IGF-I plays a critical role in rat skeletal satellite cell proliferation through regulation of p27Kip1 expression (Machida, 2003).

Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. The involvement of the FOXO family of transcription factors and their downstream target p27Kip1 has been investigated in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12-EET induces a time- and dose-dependent decrease in p27Kip1 protein expression, whereas p21Cip1 is not significantly affected. This effect on p27Kip1 protein is associated with decreased mRNA levels as well as p27Kip1 promoter activity. 11,12-EET also stimulates the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominant-negative or an 'Akt-resistant'/constitutively active FOXO3a mutant reverses the 11,12-EET-induced down-regulation of p27Kip1, whereas transfection of a constitutive active Akt decreases p27Kip1 expression independent of the presence or absence of 11,12-EET. To determine whether these effects are involved in EET-induced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicits endothelial cell proliferation and this effect is inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. However, reducing FOXO expression using RNA interference attenuates p27Kip1 expression and stimulates endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt-dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27Kip1 (Potente, 2003).

FoxO Forkhead transcription factors have been shown to act as signal transducers at the confluence of Smad, PI3K, and FoxG1 pathways. Smad proteins activated by TGF-ß form a complex with FoxO proteins to turn on the growth inhibitory gene p21Cip1. This process is negatively controlled by the PI3K pathway, a known inhibitor of FoxO localization in the nucleus, and by the telencephalic development factor FoxG1, which binds to FoxO-Smad complexes and blocks p21Cip1 expression. It is suggested that the activity of this network confers resistance to TGF-ß-mediated cytostasis during the development of the telencephalic neuroepithelium and in glioblastoma brain tumor cells (Seoane, 2004).

FoxO family members and development

Activation of the transcription factor FKHR in various established cell lines induces cell cycle arrest followed by apoptosis. These effects are inhibited through activation of the phosphatidylinositol 3-kinase/Akt pathway, resulting in FKHR phosphorylation and its export from the nucleus, thus blocking its pro-apoptotic activity. FKHR regulates fusion of differentiating primary myoblasts. FKHR is localized in the cytoplasm of proliferating myoblasts, yet translocates to the nucleus by a phosphorylation-independent pathway following serum starvation, a condition that induces myoblast differentiation. FKHR phosphorylation during terminal differentiation appears to downregulate its fusion activity; a dominant-active non-phosphorylatable FKHR mutant dramatically augments the rate and extent of myotube fusion. However, this FKHR mutant exerts its effects only after other events initiated the differentiation process. Conversely, enforced expression of a dominant-negative FKHR mutant blocks myotube formation whereas wild-type FKHR has no effect. It is concluded that in addition to the role of FoxO proteins in regulating cell cycle progress and apoptosis, FKHR controls the rate of myotube fusion during myogenic differentiation (Bois, 2003).

Insulin-like growth factors promote myoblast differentiation through phosphoinositol 3-kinase and Akt signaling. Akt substrates required for myogenic differentiation are unknown. Forkhead transcription factors of the forkhead box gene, group O (Foxo) subfamily are phosphorylated in an insulin-responsive manner by phosphatidylinositol 3-kinase-dependent kinases. Phosphorylation leads to nuclear exclusion and inactivation. A constitutively active Foxo1 mutant inhibits differentiation of C2C12 cells and prevents myotube differentiation induced by constitutively active Akt. In contrast, a transcriptionally inactive mutant Foxo1 partially rescues inhibition of C2C12 differentiation mediated by wortmannin, but not by rapamycin, and is able to induce aggregation-independent myogenic conversion of teratocarcinoma cells. Inhibition of Foxo expression by siRNA results in more efficient differentiation, associated with increased myosin expression. These observations indicate that Foxo proteins are key effectors of Akt-dependent myogenesis (Hribal, 2003).

Foxo transcription factors have been implicated in diverse biological processes, including metabolism, cellular stress responses, and aging. Foxo3a minus female mice exhibit a distinctive ovarian phenotype of global follicular activation leading to oocyte death, early depletion of functional ovarian follicles, and secondary infertility. Foxo3a thus functions at the earliest stages of follicular growth as a suppressor of follicular activation. In addition to providing a molecular entry point for studying the regulation of follicular growth, these results raise the possibility that accelerated follicular initiation plays a role in premature ovarian failure, a common cause of infertility and premature aging in women (Castrillon, 2003).

Neuronal polarity is essential for normal brain development and function. However, cell-intrinsic mechanisms that govern the establishment of neuronal polarity remain to be identified. This study reports that knockdown of endogenous FOXO proteins in hippocampal and cerebellar granule neurons, including in the rat cerebellar cortex in vivo, reveals a requirement for the FOXO transcription factors in the establishment of neuronal polarity. The FOXO transcription factors, including the brain-enriched protein FOXO6, play a critical role in axo-dendritic polarization of undifferentiated neurites, and hence in a switch from unpolarized to polarized neuronal morphology. The gene encoding the protein kinase Pak1, which acts locally in neuronal processes to induce polarity, was identified as a critical direct target gene of the FOXO transcription factors. Knockdown of endogenous Pak1 phenocopies the effect of FOXO knockdown on neuronal polarity. Importantly, exogenous expression of Pak1 in the background of FOXO knockdown in both primary neurons and postnatal rat pups in vivo restores the polarized morphology of neurons. These findings define the FOXO proteins and Pak1 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal polarity, thus identifying a novel function for the FOXO transcription factors in a unique aspect of neural development (de la Torre-Ubieta, 2010).

FoxOs Are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis

Activated phosphoinositide 3-kinase (PI3K)-AKT signaling appears to be an obligate event in the development of cancer. The highly related members of the mammalian FoxO transcription factor family, FoxO1, FoxO3, and FoxO4, represent one of several effector arms of PI3K-AKT signaling, prompting genetic analysis of the role of FoxOs in the neoplastic phenotypes linked to PI3K-AKT activation. While germline or somatic deletion of up to five FoxO alleles produced remarkably modest neoplastic phenotypes, broad somatic deletion of all FoxOs engendered a progressive cancer-prone condition characterized by thymic lymphomas and hemangiomas, demonstrating that the mammalian FoxOs are indeed bona fide tumor suppressors. Transcriptome and promoter analyses of differentially affected endothelium identified direct FoxO targets and revealed that FoxO regulation of these targets in vivo is highly context-specific, even in the same cell type. Functional studies validated Sprouty2 and PBX1, among others, as FoxO-regulated mediators of endothelial cell morphogenesis and vascular homeostasis (Paik, 2007).

Phenotypic characterization shows that FoxO family functions in normal tissue homeostasis and cancer suppression are not only lineage-restricted, but also organ-specific. To gain additional insights into the mechanistic basis for such specificity, comparative transcriptome analyses was conducted of purified lung and liver ECs following pI-pC treatment of age-matched Mx-Cre+ and Mx-Cre mice (The Mx-Cre transgene achieves widespread somatic FoxO deletion in adult tissues). It was hypothesized that normalization against phenotypically unaffected lung ECs would provide an effective biological filter for identifying physiologically relevant FoxO targets versus secondary/bystander transcriptional events—the latter would include genes whose expression responds to FoxO regulation but who do not play a rate-limiting role in the observed phenotypes, as well as those whose expression might be altered by activation of Cre expression during FoxO deletion. Transcriptome profiles of liver ECs with and without functional FoxOs were compared with those of lung ECs to generate a list of 138 significantly differentially expressed genes—89 of which were upregulated and 49 downregulated in liver, but not in lung, ECs upon documented FoxO . Consistent with the observed in vivo phenotypes, several differentially expressed genes have validated roles in EC biology, angiogenesis, and tissue morphogenesis, such as XLKD1 (LYVE-1), VCAM1, angiopoietin-like 4 (ANGPTL4), adrenomedullin (ADM), thrombospondin1 (THBS1), and ID1, and extracellular matrix proteins such as fibrillin (FBN1) (Paik, 2007).

Next, it was reasoned that the identification of FoxO binding elements (BEs) in differentially expressed genes would provide more direct insights into FoxO's actions in the observed phenotypes. To that end, a systematic in silico analysis was conducted of the regulatory regions of these 138 genes to ascertain the presence of evolutionarily conserved FoxO consensus BEs. For each gene, the −8 kb to +2 kb region surrounding the transcription start site and the 0 to +5 kb region downstream of the transcription end site in the mouse genome was surveyed. A position-specific weight matrix (PWM) based on evolutionary conservation of canonical insulin-regulated FoxO targets (IGFBP1, G6PD, PEPCK) was constucted to characterize the FoxO binding motif (consensus = BBTRTTTTD). Potential FoxO BEs were filtered further by cross-species conservation with two independent methodologies. BEs that could be identified in mouse, human, and at least one other species were designated as a “3-species conserved” BE. By requiring at least one evolutionary conserved FoxO BE predicted by both methods, 21 putative direct targets of FoxO family were identified in liver ECs, 9 of which were downmodulated and 12 upmodulated upon FoxO deletion. Interestingly, several of these genes are highly relevant to cancer (e.g., TCF4), vascular biology (e.g., CTGF), or both, such as ID1 and ADM, two factors known to play critical roles in EC survival and to promote angiogenesis during development and tumorigenesis (Paik, 2007).

To validate the computational approach, attempts were made to document direct binding of FoxOs on predicted BEs by chromatin immunoprecipitation (ChIP) and to confirm expression modulation by quantitative RT-PCR and RISH in vitro and in vivo, respectively. Sprouty2 was verified as a direct FoxO target. First, using a mixture of anti-FoxO1/3/4 antibodies, DNA fragments spanning FoxO BEs from both the proximal and distal regions of Sprouty2 gene were more efficiently coimmunoprecipitated in the FoxO-expressing liver ECs versus those deficient for the FoxOs. Next, it was documented that Sprouty2 was significantly and reproducibly downregulated in independently derived Mx-Cre+ liver ECs, but not in Mx-Cre+ lung ECs. Mindful of the potential artifact introduced by culturing EC in vitro, RISH was performed in tissue sections to confirm that Sprouty2 mRNA levels were comparable in Mx-Cre and Mx-Cre+ lung EC but were significantly downmodulated in vascular beds of affected tissues, including liver, skeletal muscle, and uterus, in Mx-Cre+ mice 3 weeks after treatment with pI-pC (Paik, 2007).

For the other targets, similar validation studies were constructed. By RISH, 12 of 14 randomly selected putative targets exhibited transcriptional regulation by FoxO in vivo. Of note, Meis1 and Klf6, which did not confirm by RISH, were shown to be regulated by quantitative RT-PCR, pointing to the detection limits of RISH. Moreover, all 17 randomly selected putative targets (8 downmodulated and 9 upmodulated) showed expression modulation by quantitative RT-PCR in response to FoxO deletion in culture. Finally, all eight randomly selected putative targets (six downmodulated and two upmodulated) were validated to be true direct targets of FoxO by ChIP. Thus, eight of eight putative targets satisfied both ChIP and expression validation, indicating the robustness of the integrated computational-biological approach in the identification of direct targets of FoxOs in ECs in vivo (Paik, 2007).

TGF-beta-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia

Chronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO), supporting the proliferation or inhibiting the apoptosis of CML cells. Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy, imatinib does not deplete the leukaemia-initiating cells (LICs) that drive the recurrence of CML. Using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model, this study shows that Foxo3a has an essential role in the maintenance of CML LICs. Cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a+/+ and Foxo3a-/- mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore, TGF-beta is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-beta inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-beta inhibitor impaired their colony-forming ability in vitro. These results demonstrate a critical role for the TGF-beta-FOXO pathway in the maintenance of LICs, and strengthen understanding of the mechanisms that specifically maintain CML LICs in vivo (Naka, 2010).

FoxO family members, cell survival, stress response and longevity

The signaling pathway from phosphoinositide 3-kinase to the protein kinase Akt controls organismal life-span in invertebrates and cell survival and proliferation in mammals by inhibiting the activity of members of the FOXO family of transcription factors. Mammalian FOXO3a also functions at the G2 to M checkpoint in the cell cycle and triggers the repair of damaged DNA. By gene array analysis, FOXO3a was found to modulate the expression of several genes that regulate the cellular response to stress at the G2-M checkpoint. The growth arrest and DNA damage response gene Gadd45a appears to be a direct target of FOXO3a; Gadd45a mediates part of FOXO3a's effects on DNA repair. These findings indicate that in mammals FOXO3a regulates the resistance of cells to stress by inducing DNA repair and thereby may also affect organismal life-span (Tran, 2002).

Reactive oxygen species are required for cell proliferation but can also induce apoptosis. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. The PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans, and it is concluded that this model also extends to mammalian systems (Kops, 2002b).

Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, a cell line was used in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induce apoptosis, which is preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlates with the disruption of mitochondrial membrane integrity and cytochrome c release. This is preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduces the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone is sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim-/- mice exhibit reduced levels of apoptosis upon inhibition of PI3K/PKB signaling. These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB--mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death (Dijkers, 2002).

Members of the FOXO family of mammalian forkhead transcription factors, including AFX, FKHRL1, and FKHR, are homologs of DAF-16, which regulates genes that contribute both to longevity and to resistance to various stresses (including oxidative stress) in Caenorhabditis elegans. Mouse myoblastic C2C12 cell lines have been generated in which expression of a constitutively active form of AFX (AFX-TM) is inducible by Cre-mediated recombination at loxP sites. Forced expression of AFX-TM is shown to block cell cycle progression at the G(1) and G(2) phases, and FOXO family members regulate the expression of stress-inducible genes such as GADD45. AFX and FKHRL1 each directly activate the GADD45 promoter through interaction with FOXO binding motifs. Oxidative stress activates the GADD45 promoter in a FOXO-dependent manner, resulting in an increased abundance of GADD45 mRNA and protein as well as G(2) arrest. These responses were evident in cells in which the tumor suppressor protein p53 is inactivated. These results suggest that the FOXO family of transcription factors plays an important role in the regulation of GADD45 in response to oxidative stress and thereby contributes to G(2)-M checkpoint (Furukawa-Hibi, 2002).

Developing sympathetic neurons die by apoptosis when deprived of NGF. BIM, a BH3-only member of the BCL-2 family, is induced after NGF withdrawal in these cells and contributes to NGF withdrawal-induced death. The involvement of the Forkhead box, class O (FOXO) subfamily of Forkhead transcription factors was examined in the regulation of BIM expression by NGF. Overexpression of FOXO transcription factors induces BIM expression and promotes death of sympathetic neurons in a BIM-dependent manner. In addition, FKHRL1 (FOXO3a) directly activates the bim promoter via two conserved FOXO binding sites and that mutation of these sites abolishes bim promoter activation after NGF withdrawal. Finally, it has been shown that FOXO activity contributes to the NGF deprivation-induced death of sympathetic neurons (Gilley, 2003).

A forkhead-type transcription factor, DAF-16, is located in the most downstream part of the insulin signalling pathway via PI3K (phosphoinositide 3-kinase). It is essential for the extension of life-span and is also involved in dauer formation induced by food deprivation in Caenorhabditis elegans. The present study addresses whether or not FOXO members AFX, FKHR (forkhead homolog in rhabdomyosarcoma) and FKHRL1 (FKHR-like protein 1), mammalian counterparts of DAF-16, are involved in starvation stress. A remarkable selective induction of FKHR and FKHRL1 transcripts was found in skeletal muscle of mice during starvation. The induction of FKHR gene expression was observed at 6 h after food deprivation, peaked at 12 h, and returned to the basal level by 24 h of refeeding. The induction was also found in skeletal muscle of mice with glucocorticoid treatment. Moreover, the levels of PDK4 (pyruvate dehydrogenase kinase 4) gene expression were up-regulated through the direct binding of FKHR to the promoter region of the gene in C2C12 cells. These results suggest that FKHR has an important role in the regulation of energy metabolism, at least in part, through the up-regulation of PDK4 gene expression in skeletal muscle during starvation (Furuyama, 2003).

In C. elegans, the transcription factor DAF-16 promotes longevity in response to reduced insulin/IGF-1 signaling or germline ablation. This study asks how different tissues interact to specify the lifespan of the animal. Several tissues act as signaling centers. In particular, DAF-16 activity in the intestine, which also comprises the animal's adipose tissue, completely restores the longevity of daf-16 minus germline-deficient animals, and increases the lifespans of daf-16 minus insulin/IGF-1-pathway mutants substantially. These findings indicate that DAF-16 may control two types of downstream signals: DAF-16 activity in signaling cells upregulates DAF-16 in specific responding tissues, possibly via regulation of insulin-like peptides, and also evokes DAF-16-independent responses. It is suggested that this network of tissue interactions and feedback regulation allows the tissues to equilibrate and fine-tune their expression of downstream genes, which, in turn, coordinates the rate of tissue aging within the animal (Libina, 2003).

The signaling pathway of insulin/insulin-like growth factor-1/phosphatidylinositol-3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans. This regulatory process involves the activity of DAF-16, a forkhead transcription factor. Although reduction-of-function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells. Akt activity has been shown to increase along with cellular senescence; inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur. FOXO3a influences p53 activity by regulating the level of reactive oxygen species. These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt-induced senescence may be involved in vascular pathophysiology (Miyauchi, 2004).

To understand the role of FoxO family members in hematopoiesis, FoxO1, FoxO3, and FoxO4 were conditionally deleted in the adult hematopoietic system. FoxO-deficient mice exhibited myeloid lineage expansion, lymphoid developmental abnormalities, and a marked decrease of the lineage-negative Sca-1+, c-Kit+ (LSK) compartment that contains the short- and long-term hematopoietic stem cell (HSC) populations. FoxO-deficient bone marrow had defective long-term repopulating activity that correlated with increased cell cycling and apoptosis of HSC. Notably, there was a marked context-dependent increase in reactive oxygen species (ROS) in FoxO-deficient HSC compared with wild-type HSC that correlated with changes in expression of genes that regulate ROS. Furthermore, in vivo treatment with the antioxidative agent N-acetyl-L-cysteine resulted in reversion of the FoxO-deficient HSC phenotype. Thus, FoxO proteins play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment, a function that is required for its long-term regenerative potential (Tothova, 2007).

JNK regulates FoxO-dependent autophagy in neurons

The cJun N-terminal kinase (JNK) signal transduction pathway is implicated in the regulation of neuronal function. JNK is encoded by three genes that play partially redundant roles. This study reports the creation of mice with targeted ablation of all three Jnk genes in neurons. Compound JNK-deficient neurons are dependent on autophagy for survival. This autophagic response is caused by FoxO-induced expression of Bnip3 that displaces the autophagic effector Beclin-1 from inactive Bcl-XL complexes. These data identify JNK as a potent negative regulator of FoxO-dependent autophagy in neurons (Xu, 2011).

Studies of nonneuronal cells have implicated JNK in the induction of autophagy. Indeed, this study confirmed the conclusion that JNK can contribute to increased autophagy by examining primary mouse embryonic fibroblasts (MEFs) with compound JNK deficiency. The mechanism of JNK-induced autophagy may be mediated by phosphorylation of Bcl2 by JNK and the subsequent release of the autophagic effector Beclin-1. The sites of JNK phosphorylation on Bcl2 are conserved in the related protein Bcl-XL. This conservation suggests that phosphorylation of Bcl2 and Bcl-XL is functionally important. Phosphorylation of Bcl2 and Bcl-XL by JNK and other protein kinases may represent an important mechanism of autophagy regulation. Indeed, the properties of JNK as a stress-responsive kinase provide an elegant mechanism for coupling stress exposure to the induction of autophagy (Xu, 2011).

Studies of nonneuronal cells demonstrate that JNK is markedly activated from a low basal state when cells are exposed to stress. However, JNK is regulated very differently in neurons. JNK1 remains constitutively activated under basal conditions, while JNK2 and JNK3 exhibit low basal activity and are stress-responsive. The proautophagy role of JNK in nonneuronal cells has been reported to be mediated by JNK1. It is therefore intriguing that JNK1 is constitutively activated in neurons. Based on studies of nonneuronal cells, the constitutive activation of JNK1 in neurons should cause autophagy. A mechanism must therefore exist to prevent autophagy activation by constitutively activated JNK1 in neurons. Although the mechanism is unclear, these considerations indicate that neurons are refractory to the proautophagy JNK1 signaling pathway that has been identified in nonneuronal cells (Xu, 2011).

This analysis of compound JNK-deficient neurons demonstrates that JNK regulates neuronal autophagy. In contrast to the proautophagy role of JNK nonneuronal cells, neuronal JNK acts to suppress autophagy. Loss of neuronal JNK function causes engagement of a transcriptional program that leads to increased expression of autophagy-related genes and the induction of an autophagic response. One consequence of autophagy induction caused by JNK deficiency is improved neuronal survival (Xu, 2011).

FoxO transcription factors are implicated in the induction of both cell death (apoptosis) and cell survival (autophagy) responses. The results of this study identify JNK as a signaling molecule that may contribute to the coordination of these divergent responses to FoxO transcription factor activation (Xu, 2011).

FoxO activation in neurons leads to the expression of the target gene Bim, a proapoptotic BH3-only protein, and causes cell death. JNK activation in neurons promotes expression of Bim, most likely because JNK-dependent AP-1 activity is required for Bim expression. Moreover, JNK phosphorylates Bim on an activating site, and also causes the release of Bim from complexes with the anti-apoptotic Bcl2 family protein Mcl-1. Together, these processes initiate JNK-dependent apoptosis. JNK inhibition can therefore prevent neuronal cell death. Indeed, small molecule inhibitors of JNK cause neuroprotection in models of neurodegenerative disease (Xu, 2011).

Activation of FoxO transcription factors can also cause increased expression of autophagy-related genes, including Atg8/Lc3b, Atg12, and Bnip3. While JNK cooperates with FoxO to increase proapoptotic Bim expression, JNK deficiency prevents induction of Bim expression and promotes a survival response that is mediated by increased FoxO-dependent expression of the autophagy-related target genes Atg8/Lc3b, Atg12, and Bnip3. Indeed, inhibition of autophagy in JNK-deficient neurons causes rapid death. This neuronal survival response is relevant to stroke models in which neuronal death is mediated by a JNK-dependent mechanism (Xu, 2011).

Together, these data demonstrate that cross-talk between the FoxO and JNK signaling pathways leads to neuronal death. In contrast, loss of JNK promotes FoxO-induced survival mediated by increased autophagy. JNK therefore acts as a molecular switch that defines the physiological consequence of FoxO activation in neurons (Xu, 2011).

Thus, JNK is implicated in the induction of autophagy in nonneuronal cells. However, JNK1 is constitutively activated in neurons, and these cells are refractory to JNK-induced autophagy. Instead, JNK acts to suppress autophagy in neurons by inhibiting FoxO-induced expression of autophagy-related genes (e.g., Atg8/Lc3b, Atg12, and Bnip3) and increasing the expression of proapoptotic genes (e.g., Bim). JNK inhibition causes neuroprotection that is mediated by loss of proapoptotic gene expression and increased autophagy (Xu, 2011).

FoxO transcription factors suppress Myc-driven lymphomagenesis via direct activation of Arf

FoxO transcription factors play critical roles in cell cycle control and cellular stress responses, and abrogation of FoxO function promotes focus formation by Myc in vitro. Stable introduction of a dominant-negative FoxO moiety (dnFoxO) into Eµ-myc transgenic hematopoietic stem cells accelerates lymphoma development in recipient mice by attenuating Myc-induced apoptosis. When expressed in Eµ-myc; p53+/- progenitor cells, dnFoxO alleviates the pressure to inactivate the remaining p53 allele in upcoming lymphomas. Expression of the p53 upstream regulator p19Arf (alternative reading frame of p16INK, also called p14arf in humans and p19arf in mice) is virtually undetectable in most dnFoxO-positive Myc-driven lymphomas. It was found that FoxO proteins bind to a distinct site within the Ink4a/Arf locus and activate Arf expression. Moreover, constitutive Myc signaling induces a marked increase in nuclear FoxO levels and stimulates binding of FoxO proteins to the Arf locus. These data demonstrate that FoxO factors mediate Myc-induced Arf expression and provide direct genetic evidence for their tumor-suppressive capacity (Bouchard, 2007).

The FoxO subclass of forkhead-box transcription factors (consisting of FoxO1 (FKHR), FoxO3a (FKHRL1), FoxO4 (AFX), and FoxO6) regulates numerous cellular functions including proliferation, stress sensitivity, and survival; it has also been implicated in the regulation of organism life span. The members of this family activate gene expression via interaction with a specific DNA sequence, and known targets include the cell cycle regulating Kip1, the proapoptotic Bim, the DNA damage-responsive Gadd45a, and the oxidative stress-protective manganese superoxide dismutase genes. In addition, FoxO proteins can repress several cell cycle promoting genes (e.g., cyclin D1 and cyclin D2) in a manner that might be independent of direct DNA binding (Bouchard, 2007 and references therein).

In response to growth factor signaling and to oxidative stress, FoxO proteins are post-translationally modified by phosphorylation, acetylation, and ubiquitination; collectively, these modifications regulate FoxOs’ subcellular localization, transcriptional activity, and stability. Notably, all FoxO proteins are inhibited by protein kinase B/Akt-mediated phosphorylation that promotes their nuclear export and subsequent proteolytic degradation via ubiquitination by the SCFSkp2 complex. As a consequence, FoxO proteins mediate the induction of p27Kip1 and Bim expression in response to inhibition of the phosphatidylinositol-3-OH (PI3)-kinase/Akt pathway (Bouchard, 2007 and references therein).

Conditional codeletion of the FoxO1, FoxO3, and FoxO4 alleles uncovers a context-dependent cancer-prone phenotype characterized by thymic lymphomas forming in some and hemangiomas developing in most animals after a long latency, suggesting that FoxO proteins exert their tumor-suppressive capability in the presence of additional oncogenic mutations. In support of this view, Akt-mediated phosphorylation of FoxO proteins has been identified as the critical PI3-kinase signaling component that substitutes for oncogenic Ras in Myc-induced proliferation and focus formation in vitro. Furthermore, constitutive Akt signaling cooperates with Myc to accelerate B-cell lymphomagenesis; however, it remains unclear whether Akt-mediated phosphorylation of FoxO proteins contributes to Eµ-myc transgenic lymphoma formation in this setting (Bouchard, 2007).

Proapoptotic Arf/p53 signaling is known as the pivotal Myc-induced tumor-suppressive barrier. Eµ-myc transgenic mice lacking one p53 allele develop lymphomas that inactivate the remaining wild-type allele. Likewise, Eµ-myc; Arf+/- or Eµ-myc; Ink4a/Arf+/- mice produce tumors that lack expression of p19Arf. Primary Arf deletions protect cells from acquiring p53 mutations during lymphoma development. Similarly, introduction of strictly anti-apoptotic genes such as bcl2 or a dominant-negative form of caspase 9 into Eµ-myc; p53+/- hematopoietic stem cells alleviates the pressure to inactivate p53, thereby underscoring apoptosis as the critical p53-governed tumor suppressor function in Myc-driven lymphomagenesis (Bouchard, 2007).

Previous work has shown that p53 and FoxO3a share target genes and that FoxO3a can activate transcription via p53 sites, suggesting a potential collaboration of FoxO3a and p53 in tumor suppression. Although a direct interaction between FoxO3a and p53 proteins has been demonstrated under conditions of overexpression, the observed collaboration would be consistent with an as-yet-unidentified FoxO target acting upstream of p53. This study reports that FoxO factors elicit their tumor-suppressive potential as critical inducers of Arf during Myc-driven lymphomagenesis, providing further evidence for a close link between the FoxO and p53 tumor suppressor pathways (Bouchard, 2007).

Codependent activators direct myoblast-specific MyoD transcription

Although FoxO and Pax proteins represent two important families of transcription factors in determining cell fate, they had not been functionally or physically linked together in mediating regulation of a common target gene during normal cellular transcription programs. This study identified MyoD, a key regulator of myogenesis, as a direct target of FoxO3 and Pax3/7 in myoblasts. Cell-based assays and in vitro studies reveal a tight codependent partnership between FoxO3 and Pax3/7 to coordinately recruit RNA polymerase II and form a preinitiation complex (PIC) to activate MyoD transcription in myoblasts. The role of FoxO3 in regulating muscle differentiation is confirmed in vivo by observed defects in muscle regeneration caused by MyoD downregulation in FoxO3 null mice. These data establish a mutual interdependence and functional link between two families of transcription activators serving as potential signaling sensors and regulators of cell fate commitment in directing tissue specific MyoD transcription (Hu, 2008).

Sequence analysis revealed four potential FRE sites in a 6 kb region upstream of the myod transcription initiation site, which are between the DRR and PRR elements important for MyoD regulation during embryonic muscle development. ChIP experiments were performed to determine promoter occupancy of FoxOs at the myod gene using antibodies specifically directed against each of the three FoxO proteins. FoxO3 was efficiently detected at the myod promoter regions overlapping putative FRE -940 and -1598, but not at -1928 and -2351. In marked contrast to FoxO3, neither FoxO1 nor FoxO4 was detected significantly above background at the four potential FREs in C2C12 cells. As a control, the same FoxO1 and FoxO4 antibodies were used to successfully detect their occupancy at the p21 promoter, which is known to be regulated by these FoxOs. Indeed, it appears that FoxO3 selectively binds two of the putative FREs at the myod promoter in myoblasts, suggesting that FoxO3 binding may correspond to one of the important steps regulating MyoD expression. RNA polymerase II was also found at the myod promoter by ChIP, further confirming the correlation between FoxO3 promoter occupancy and transcription activation. Although FoxO4 appeared to have low transcriptional activity, little, if any, FoxO4 was detected at the myod promoter by ChIP in myoblasts. Taken together, these results suggest that at least FoxO3 is likely part of the active transcription machinery directly recruited to the myod promoter in myoblasts (Hu, 2008).

Since FoxO3 binds to putative FRE -940 and FRE -1598 of the myod promoter in C2C12 cells, whether FoxO3 is also able to recognize and bind these putative FREs in vitro was tested by electrophoretic mobility shift assays (EMSA). Purified recombinant FoxO3 efficiently bound to both the FRE -940 and FRE -1598 probes, but not to mutant probes, confirming that FoxO3 can bind to both FRE sequences specifically in vitro. Consistent with ChIP results, in luciferase reporter assays, mutations in the FREs at -940 or -1598 significantly debilitated myod transcription, while mutations in FRE -1928 and FRE -2351 had little or no effect on transcription activity. These results suggest that FRE -940 and -1598 bound by FoxO3 are critical for myod transcription activation. Taken together, these assays indicate that FoxO3 can specifically bind two FREs in the myod promoter and activate transcription (Hu, 2008).

Although FoxOs have previously been implicated functioning in muscle differentiation, skeletal muscle-specific genes directly targeted by FoxOs had not been identified. The current findings indicate that FoxO3 (but not FoxO1 or FoxO4) binds a subset of FREs in the myod promoter to work in concert with Pax3/7 in regulating cell type-specific transcription activation in myoblasts. The contribution of FoxO3 in directing myod transcription activation in vivo was further confirmed by the observed muscle regeneration defects in FoxO3 null mice (Hu, 2008).

Identification of FoxO3 as an important myod transcription activator may provide a handle to explore the potential signaling pathways governing muscle regeneration. Interestingly, FoxO1 was found to negatively regulate MyoD expression indirectly through the Delta-Notch pathway. The potential repression of MyoD by FoxO1 together with the results of direct activation of MyoD by FoxO3 suggests an intriguing mechanism to fine tune MyoD expression. Muscle differentiation may therefore utilize selected FoxOs in partnership with Pax3/7 to integrate inputs from multiple signaling pathways (Hu, 2008).

The apparent Kd of FoxO3 and Pax3/7 binding individually to the DNA elements in the myod promoter is on the order of 10−7M in vitro, which is rather modest compared to a typical DNA binding protein, such as GAL4 (apparent Kd, ~10−11M). This is consistent with the finding that neither FoxO3 nor Pax3/7 alone binds promoter DNA efficiently to form a stable DNA-activator complex capable of recruiting a functional PIC. It appears that to efficiently assemble an active PIC via FoxO3 and Pax3/7 at the myod promoter both protein-DNA and protein-protein interactions mediated by this hitherto unknown activator partnership must take place to trigger transcription activation synergistically. This may therefore represent a useful and efficient combinatorial mechanism to direct cell type-specific transcription while utilizing two activators shared by many cell types. This codependence is reminiscent of the mechanism utilized by Sox2 and Pax6 to drive lens-specific transcription of δ-crystallin gene (Hu, 2008).

Although under normal conditions FoxO3 is predominantly detected occupying the FRE at -1598 of the myod promoter, curiously, it was found that some FoxO4 can be detected at this site in myoblasts when Pax3/7 is depleted. It is known that Pax3/7 is not expressed in myotubes, but MyoD expression persists in myotubes. It will be interesting to survey the identity of FoxOs binding to the myod promoter in myotubes versus myoblasts and explore additional mechanisms involved in cell type-specific transcription activation during later stages of myogenesis. Intriguingly, there is a switching of the core transcription machinery from the canonical holo-TFIID to a TRF3/TAF3 complex during myoblasts differentiation to myotubes (Deato, 2007). While FoxO3 and Pax3/7 appear to work in concert with TFIID at the MyoD promoter in myoblasts, it will be important to identify the key activator(s) that function together with the TRF3/TAF3 complex in myotubes (Hu, 2008).


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Search PubMed for articles about Drosophila forkhead box, sub-group O

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