p53 and PI3K/AKT signalings are up-regulated in flies with defects in the THO complex

The THO complex (THO) is an evolutionary conserved protein required for the formation of export-competent mRNP. The growing evidence indicates that the metazoan THO plays important roles in cell differentiation and cellular stress response. But the underlying mechanisms are poorly understood. Herein we examined the relevance of THO to cellular signaling pathways involved in cell differentiation and cellular stress response. When we examined the endogenous p53 level in the testis, it was sustained much longer during spermatogenesis in the THO mutant compared to that of wild-type. In flies with impaired THO, overexpression of p53 by eye-specific GAL4 not only enhanced p53-mediated retinal degeneration, but p53 level was also elevated compared to the control flies. Since the body size of the THO mutant flies was significantly larger than control flies, we also examined whether the PI3K/AKT signaling is enhanced in the mutant flies. The results showed that the endogenous level of phosphorylated AKT, which is the active form, was highly elevated in the THO mutants. Taken together our results suggested that both p53 and PI3K/AKT signalings are up-regulated in the flies with impaired THO.     

Role of the insulin/Tor signaling network in starvation-induced programmed cell death in Drosophila oogenesis

Amino-acid starvation leads to an inhibition of cellular proliferation and the induction of programmed cell death (PCD) in the Drosophila ovary. Disruption of insulin signaling has been shown to inhibit the progression of oogenesis, but it is unclear whether this phenotype mimics starvation. Here, we investigate whether the insulin-mediated phosphoinositide kinase-3 pathway regulates PCD in mid oogenesis. We reasoned that under well-fed conditions, disruption of positive components of the insulin signaling pathway within the germline would mimic starvation and produce degenerating egg chambers. Surprisingly, mutants did not mimic starvation but instead produced many abnormal egg chambers in which the somatic follicle cells disappeared and the germline persisted. These abnormal egg chambers did not show an induction of caspases and lysosomes like that observed in wild-type (WT) degenerating egg chambers. Egg chambers from insulin signaling mutants were resistant to starvation-induced PCD, indicating that a complete block in insulin-signaling prevents the proper response to starvation. However, target of rapamycin (Tor) mutants did show a phenotype that mimicked WT starvation-induced PCD, indicating an insulin independent regulation of PCD via Tor signaling. These results suggest that inhibition of the insulin signaling pathway is not sufficient to regulate starvation-induced PCD in mid oogenesis. Furthermore, starvation-induced PCD is regulated by Tor signaling converging with the canonical insulin signaling pathway.     

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO₂ output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.     

SIFamide and SIFamide Receptor Define a Novel Neuropeptide Signaling to Promote Sleep in Drosophila

SIFamide receptor (SIFR) is a Drosophila G protein-coupled receptor for the neuropeptide SIFamide (SIFa). Although the sequence and spatial expression of SIFa are evolutionarily conserved among insect species, the physiological function of SIFa/SIFR signaling remains elusive. Here, we provide genetic evidence that SIFa and SIFR promote sleep in Drosophila. Either genetic ablation of SIFa-expressing neurons in the pars intercerebralis (PI) or pan-neuronal depletion of SIFa expression shortened baseline sleep and reduced sleep-bout length, suggesting that it caused sleep fragmentation. Consistently, RNA interference-mediated knockdown of SIFR expression caused short sleep phenotypes as observed in SIFa-ablated or depleted flies. Using a panel of neuron-specific Gal4 drivers, we further mapped SIFR effects to subsets of PI neurons. Taken together, these results reveal a novel physiological role of the neuropeptide SIFa/SIFR pathway to regulate sleep through sleep-promoting neural circuits in the PI of adult fly brains.     

Expression of a constitutively active insulin receptor in Drosulfakinin (Dsk) neurons regulates metabolism and sleep in Drosophila

The ability of organisms to sense their nutritional environment and adjust their behavior accordingly is critical for survival. Insulin-like peptides (ilps) play major roles in controlling behavior and metabolism; however, the tissues and cells that insulin acts on to regulate these processes are not fully understood. In the fruit fly, Drosophila melanogaster, insulin signaling has been shown to function in the fat body to regulate lipid storage, but whether ilps act on the fly brain to regulate nutrient storage is not known. In this study, we manipulate insulin signaling in defined populations of neurons in Drosophila and measure glycogen and triglyceride storage. Expressing a constitutively active form of the insulin receptor (dInR) in the insulin-producing cells had no effect on glycogen or triglyceride levels. However, activating insulin signaling in the Drosulfakinin (Dsk)-producing neurons led to triglyceride accumulation and increased food consumption. The expression of ilp2, ilp3 and ilp5 was increased in flies with activated insulin signaling in the Dsk neurons, which along with the feeding phenotype, may cause the triglyceride storage phenotypes observed in these flies. In addition, expressing a constitutively active dInR in Dsk neurons resulted in decreased sleep in the fed state and less starvation-induced sleep suppression suggesting a role for insulin signaling in regulating nutrient-responsive behaviors. Together, these data support a role for insulin signaling in the Dsk-producing neurons for regulating behavior and maintaining metabolic homeostasis.     

SLOB, a SLOWPOKE channel binding protein, regulates insulin pathway signaling and metabolism in Drosophila

There is ample evidence that ion channel modulation by accessory proteins within a macromolecular complex can regulate channel activity and thereby impact neuronal excitability. However, the downstream consequences of ion channel modulation remain largely undetermined. The Drosophila melanogaster large conductance calcium-activated potassium channel SLOWPOKE (SLO) undergoes modulation via its binding partner SLO-binding protein (SLOB). Regulation of SLO by SLOB influences the voltage dependence of SLO activation and modulates synaptic transmission. SLO and SLOB are expressed especially prominently in median neurosecretory cells (mNSCs) in the pars intercerebralis (PI) region of the brain; these cells also express and secrete Drosophila insulin like peptides (dILPs). Previously, we found that flies lacking SLOB exhibit increased resistance to starvation, and we reasoned that SLOB may regulate aspects of insulin signaling and metabolism. Here we investigate the role of SLOB in metabolism and find that slob null flies exhibit changes in energy storage and insulin pathway signaling. In addition, slob null flies have decreased levels of dilp3 and increased levels of takeout, a gene known to be involved in feeding and metabolism. Targeted expression of SLOB to mNSCs rescues these alterations in gene expression, as well as the metabolic phenotypes. Analysis of fly lines mutant for both slob and slo indicate that the effect of SLOB on metabolism and gene expression is via SLO. We propose that modulation of SLO by SLOB regulates neurotransmission in mNSCs, influencing downstream insulin pathway signaling and metabolism.     

Insulin stimulates glucose metabolism via the pentose phosphate pathway in Drosophila Kc cells

Drosophila melanogaster has become a prominent and convenient model for analysis of insulin action. However, to date very little is known regarding the effect of insulin on glucose uptake and metabolism in Drosophila. Here we show that, in contrast to effects seen in mammals, insulin did not alter [(3)H]2-deoxyglucose uptake and in fact decreased glycogen synthesis ( approximately 30%) in embryonic Drosophila Kc cells. Insulin significantly increased ( approximately 1.5-fold) the production of (14)CO(2) from D-[1-(14)C]glucose while the production of (14)CO(2) from D-[6-(14)C]glucose was not altered. Thus, insulin-stimulated glucose oxidation did not occur via increasing Krebs cycle activity but rather by stimulating the pentose phosphate pathway. Indeed, inhibition of the oxidative pentose phosphate pathway by 6-aminonicotinamide abolished the effect of insulin on (14)CO(2) from D-[U-(14)C]glucose. A corresponding increase in lactate production but no change in incorporation of D-[U-(14)C]glucose into total lipids was observed in response to insulin. Glucose metabolism via the pentose phosphate pathway may provide an important source of 5'-phosphate for DNA synthesis and cell replication. This novel observation correlates well with the fact that control of growth and development is the major role of insulin-like peptides in Drosophila. Thus, although intracellular signaling is well conserved, the metabolic effects of insulin are dramatically different between Drosophila and mammals.     

Energy-Dependent Modulation of Glucagon-Like Signaling in Drosophila via the AMP-Activated Protein Kinase

Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks.     

The endosymbiont Wolbachia increases insulin/IGF-like signalling in Drosophila

Insulin/IGF-like signalling (IIS) is an evolutionarily conserved pathway that has diverse functions in multi-cellular organisms. Mutations that reduce IIS can have pleiotropic effects on growth, development, metabolic homeostasis, fecundity, stress resistance and lifespan. IIS is also modified by extrinsic factors. For instance, in the fruitfly Drosophila melanogaster, both nutrition and stress can alter the activity of the pathway. Here, we test experimentally the hypothesis that a widespread endosymbiont of arthropods, Wolbachia pipientis, can alter the degree to which mutations in genes encoding IIS components affect IIS and its resultant phenotypes. Wolbachia infection, which is widespread in D. melanogaster in nature and has been estimated to infect 30 per cent of strains in the Bloomington stock centre, can affect broad aspects of insect physiology, particularly traits associated with reproduction. We measured a range of IIS-related phenotypes in flies ubiquitously mutant for IIS in the presence and absence of Wolbachia. We show that removal of Wolbachia further reduces IIS and hence enhances the mutant phenotypes, suggesting that Wolbachia normally acts to increase insulin signalling. This effect of Wolbachia infection on IIS could have an evolutionary explanation, and has some implications for studies of IIS in Drosophila and other organisms that harbour endosymbionts.     

Effects of starvation and desiccation on energy metabolism in desert and mesic Drosophila

Energy availability can limit the ability of organisms to survive under stressful conditions. In Drosophila, laboratory experiments have revealed that energy storage patterns differ between populations selected for desiccation and starvation. This suggests that flies may use different sources of energy when exposed to these stresses, but the actual substrates used have not been examined. We measured lipid, carbohydrate, and protein content in 16 Drosophila species from arid and mesic habitats. In five species, we measured the rate at which each substrate was metabolized under starvation or desiccation stress. Rates of lipid and protein metabolism were similar during starvation and desiccation, but carbohydrate metabolism was several-fold higher during desiccation. Thus, total energy consumption was lower in starved flies than desiccated ones. Cactophilic Drosophila did not have greater initial amounts of reserves than mesic species, but may have lower metabolic rates that contribute to stress resistance.     

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.     

Selective modulation of task performance by octopamine in honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) division of labour

Octopamine treatment has previously been shown to increase honey bee foraging behaviour. We determined the effects of octopamine on other tasks to learn how octopamine affects division of labour in honey bee colonies. Octopamine treatment did not increase the rate of corpse removal from the hive, suggesting that elevated brain levels of octopamine do not act to increase the performance of all flight-related tasks. Octopamine treatment also did not increase attendance in the queen's retinue, suggesting that elevated brain levels of octopamine do not act to increase responsiveness to all olfactory stimuli. Consistent with these findings, octopamine treatment enhanced the foraging response to brood pheromone but not the cell capping response, a component of brood care. These results demonstrate a relatively specific form of neuromodulation by octopamine in the regulation of division of labour in honey bee colonies.     

The Perilipin Homologue,Lipid Storage Droplet 2, Regulates Sleep Homeostasis and Prevents Learning Impairments Following Sleep Loss

Extended periods of waking result in physiological impairments in humans, rats, and flies. Sleep homeostasis, the increase in sleep observed following sleep loss, is believed to counter the negative effects of prolonged waking by restoring vital biological processes that are degraded during sleep deprivation. Sleep homeostasis, as with other behaviors, is influenced by both genes and environment. We report here that during periods of starvation, flies remain spontaneously awake but, in contrast to sleep deprivation, do not accrue any of the negative consequences of prolonged waking. Specifically, the homeostatic response and learning impairments that are a characteristic of sleep loss are not observed following prolonged waking induced by starvation. Recently, two genes, brummer (bmm) and Lipid storage droplet 2 (Lsd2), have been shown to modulate the response to starvation. bmm mutants have excess fat and are resistant to starvation, whereas Lsd2 mutants are lean and sensitive to starvation. Thus, we hypothesized that bmm and Lsd2 may play a role in sleep regulation. Indeed, bmm mutant flies display a large homeostatic response following sleep deprivation. In contrast, Lsd2 mutant flies, which phenocopy aspects of starvation as measured by low triglyceride stores, do not exhibit a homeostatic response following sleep loss. Importantly, Lsd2 mutant flies are not learning impaired after sleep deprivation. These results provide the first genetic evidence, to our knowledge, that lipid metabolism plays an important role in regulating the homeostatic response and can protect against neuronal impairments induced by prolonged waking.     

Genetic analysis of mitochondrial protein misfolding in Drosophila melanogaster

Protein misfolding has a key role in several neurological disorders including Parkinson's disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control.     

Autophagy in Drosophila ovaries is induced by starvation and is required for oogenesis

Autophagy, an evolutionarily conserved lysosome-mediated degradation, promotes cell survival under starvation and is controlled by insulin/target of rapamycin (TOR) signaling. In Drosophila, nutrient depletion induces autophagy in the fat body. Interestingly, nutrient availability and insulin/TOR signaling also influence the size and structure of Drosophila ovaries, however, the role of nutrient signaling and autophagy during this process remains to be elucidated. Here, we show that starvation induces autophagy in germline cells (GCs) and in follicle cells (FCs) in Drosophila ovaries. This process is mediated by the ATG machinery and involves the upregulation of Atg genes. We further demonstrate that insulin/TOR signaling controls autophagy in FCs and GCs. The analysis of chimeric females reveals that autophagy in FCs, but not in GCs, is required for egg development. Strikingly, when animals lack Atg gene function in both cell types, ovaries develop normally, suggesting that the incompatibility between autophagy-competent GCs and autophagy-deficient FCs leads to defective egg development. As egg morphogenesis depends on a tightly linked signaling between FCs and GCs, we propose a model in which autophagy is required for the communication between these two cell types. Our data establish an important function for autophagy during oogenesis and contributes to the understanding of the role of autophagy in animal development.     

Unique and Shared Functions of Nuclear Lamina LEM Domain Proteins in Drosophila

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins.     

amnesiac regulates sleep onset and maintenance in Drosophila melanogaster

The adenylate cyclase/cAMP signaling pathway and adult mushroom bodies (MBs) have been shown to play an important role in sleep regulation in Drosophila. The amnesiac (amn) gene, encodes a neuropeptide that is homologous with vertebrate pituitary adenylate cyclase-activating peptide (PACAP), is expressed in dorsal paired medial (DPM) neurons and is required for the middle-term memory (MTM) in flies. However, the role of amn on regulation of sleep is as yet unknown. Here we provide evidence that amn plays a major role on sleep maintenance and onset in Drosophila. Flies with the amnesiac allele, loss-of-function amn^X8 mutation, showed a fragmented sleep pattern and short sleep latency. Moreover, homeostatic regulation was disrupted in amn^X8 mutants after sleep deprivation. Sleep maintenance was also influenced by disruption of neurotransmission in DPM neurons with increased sleep bout number and decreased sleep bout length. Furthermore, age-related sleep fragmentation and initiation were inhibited in amn^X8 mutant flies. These data suggest that amn is required in initiation and maintenance of sleep.     

Variation in sleep and metabolic function is associated with latitude and average temperature in Drosophila melanogaster

Regulation of sleep and metabolic homeostasis is critical to an animal's survival and under stringent evolutionary pressure. Animals display remarkable diversity in sleep and metabolic phenotypes; however, an understanding of the ecological forces that select for, and maintain, these phenotypic differences remains poorly understood. The fruit fly, Drosophila melanogaster, is a powerful model for investigating the genetic regulation of sleep and metabolic function, and screening in inbred fly lines has led to the identification of novel genetic regulators of sleep. Nevertheless, little is known about the contributions of naturally occurring genetic differences to sleep, metabolic phenotypes, and their relationship with geographic or environmental gradients. Here, we quantified sleep and metabolic phenotypes in 24 D. melanogaster populations collected from diverse geographic localities. These studies reveal remarkable variation in sleep, starvation resistance, and energy stores. We found that increased sleep duration is associated with proximity to the equator and elevated average annual temperature, suggesting that environmental gradients strongly influence natural variation in sleep. Further, we found variation in metabolic regulation of sleep to be associated with free glucose levels, while starvation resistance associates with glycogen and triglyceride stores. Taken together, these findings reveal robust naturally occurring variation in sleep and metabolic traits in D. melanogaster, providing a model to investigate how evolutionary and ecological history modulate these complex traits.     

Dopamine Dynamics and Signaling in Drosophila: An Overview of Genes,Drugs and Behavioral Paradigms

Changes in dopamine (DA) signaling have been implicated in a number of human neurologic and psychiatric disorders. Similarly, defects in DA signaling in the fruit fly, Drosophila melanogaster, have also been associated with several behavioral defects. As most genes involved in DA synthesis, transport, secretion, and signaling are conserved between species, Drosophila is a powerful genetic model organism to study the regulation of DA signaling in vivo. In this review, we will provide an overview of the genes and drugs that regulate DA biology in Drosophila. Furthermore, we will discuss the behavioral paradigms that are regulated by DA signaling in flies. By analyzing the genes and neuronal circuits that govern such behaviors using sophisticated genetic, pharmacologic, electrophysiologic, and imaging approaches in Drosophila, we will likely gain a better understanding about how this neuromodulator regulates motor tasks and cognition in humans.