Search

Drosophila larvae need to adapt their metabolism to reach a critical body size to pupate. This process needs food resources and has to be tightly adjusted to control metamorphosis timing and adult size. Nutrients such as amino acids either directly present in the food or obtained via protein digestion play key regulatory roles in controlling metabolism and growth. Amino acids act especially on two organs, the fat body and the brain, to control larval growth, body size developmental timing and pupariation. The expression of specific amino acid transporters in fat body cells, and in the brain through specific neurons and glial cells is essential to activate downstream molecular signaling pathways in response to amino acid levels. In this review, we highlight some of these specific networks dependent on amino acid diet to control DILP levels, and by consequence larval metabolism and growth.

International audience; Oxysterols are molecules derived by the oxidation of cholesterol and can be formed either by auto-oxidation, enzymatically or by both processes. Among the oxysterols formed by auto-oxidation, 7-ketocholesterol and 7beta-hydroxycholesterol are the main forms generated. These oxysterols, formed endogenously and brought in large quantities by certain foods, have major cytotoxic properties. They are powerful inducers of oxidative stress, inducing dysfunction of organelles (mitochondria, lysosomes and peroxisomes) that can cause cell death. These molecules are often identified in increased amounts in common pathological states such as cardiovascular diseases, certain eye conditions, neurodegenerative disorders and inflammatory bowel diseases. To oppose the cytotoxic effects of these molecules, it is important to know their biological activities and the signaling pathways they affect. Numerous cell models of the vascular wall, eye, brain, and digestive tract have been used. Currently, to counter the cytotoxic effects of 7-ketocholesterol and 7beta-hydroxycholesterol, natural molecules and oils, often associated with the Mediterranean diet, as well as synthetic molecules, have proved effective in vitro. Bioremediation approaches and the use of functionalized nanoparticles are also promising. At the moment, invertebrate and vertebrate models are mainly used to evaluate the metabolism and the toxicity of 7-ketocholesterol and 7beta-hydroxycholesterol. The most frequently used models are mice, rats and rabbits. In order to cope with the difficulty of transferring the results obtained in animals to humans, the development of in vitro alternative methods such as organ / body-on-a-chip based on microfluidic technology are hopeful integrative approaches.

Indexation en cours. PMCID: PMC6819134; International audience; When facing microbes, animals engage in behaviors that lower the impact of the infection. We previously demonstrated that internal sensing of bacterial peptidoglycan reduces Drosophila female oviposition via NF-kB pathway activation in some neurons (Kurz et al., 2017). Although we showed that the neuromodulator octopamine is implicated, the identity of the involved neurons, as well as the physiological mechanism blocking egg-laying, remained unknown. In this study, we identified few ventral nerve cord and brain octopaminergic neurons expressing an NF-kB pathway component. We functionally demonstrated that NF-kB pathway activation in the brain, but not in the ventral nerve cord octopaminergic neurons, triggers an egg-laying drop in response to infection. Furthermore, we demonstrated via calcium imaging that the activity of these neurons can be directly modulated by peptidoglycan and that these cells do not control other octopamine-dependent behaviors such as female receptivity. This study shows that by sensing peptidoglycan and hence activating NF-kB cascade, a couple of brain neurons modulate a specific octopamine-dependent behavior to adapt female physiology status to their infectious state.

Summary Dietary leucine has been suspected to play an important role in insulin release, a hormone that controls satiety and metabolism. The mechanism by which insulin-producing cells (IPCs) sense leucine and regulate insulin secretion is still poorly understood. In Drosophila, insulin-like peptides (DILP2 and DILP5) are produced by brain IPCs and are released in the hemolymph after leucine ingestion. Using Ca2+-imaging and ex vivo cultured larval brains, we demonstrate that IPCs can directly sense extracellular leucine levels via minidiscs (MND), a leucine transporter. MND knockdown in IPCs abolished leucine-dependent changes, including loss of DILP2 and DILP5 in IPC bodies, consistent with the idea that MND is necessary for leucine-dependent DILP release. This, in turn, leads to a strong increase in hemolymph sugar levels and reduced growth. GDH knockdown in IPCs also reduced leucine-dependent DILP release, suggesting that nutrient sensing is coupled to the glutamate dehydrogenase pathway., Graphical Abstract, Highlights • IPCs directly sense extracellular leucine levels via minidiscs (MND) • MND knockdown in IPCs abolishes loss of DILP2 and DILP5 • This leads to a strong increase in hemolymph sugar levels and reduces growth • GDH knockdown in IPCs reduces leucine-dependent DILP release, Manière et al. find that leucine induces the disappearance of two DILPs in Drosophila IPCs. Minidiscs (MND) is the primary leucine sensor, and downregulation has consequences for glycemia and growth. The authors propose that the leucine/MND pathway represents a conserved mechanism for insulin release.

International audience; Amino-acid transporters are involved in functions reportedly linked to the sleep/wake cycle: neurotransmitter synthesis and recycling, the regulation of synaptic strength, protein synthesis and energy metabolism. In addition, the existence of bidirectional relationships between extracellular content, transport systems and sleep/wake states is receiving emerging support. Nevertheless, the connection between amino-acid transport and sleep/wake regulation remains elusive. To address this question, we used Drosophilamelanogaster and investigated the role of LAT1 (Large neutral Amino-acid Transporter 1) transporters. We show that the two Drosophila LAT1-like transporters: JhI-21 and minidiscs (Mnd) are required in dopaminergic neurons for sleep/wake regulation. Down-regulating either gene in dopaminergic neurons resulted in higher daily sleep and longer sleep bout duration during the night, suggesting a defect in dopaminergic transmission. Since LAT1 transporters can mediate in mammals the uptake of L-DOPA, a precursor of dopamine, we assessed amino-acid transport efficiency by L-DOPA feeding. We find that downregulation of JhI-21, but not Mnd, reduced the sensitivity to L-DOPA as measured by sleep loss. JhI-21 downregulation also attenuated the sleep loss induced by continuous activation of dopaminergic neurons. Since LAT1 transporters are known to regulate TOR (Target Of Rapamycin) signaling, we investigated the role of this amino-acid sensing pathway in dopaminergic neurons. Consistently, we report that TOR activity in dopaminergic neurons modulates sleep/wake states. Altogether, this study provides evidence that LAT1 mediated amino-acid transport in dopaminergic neurons, is playing a significant role in sleep/wake regulation, and is providing several entry points to elucidate the role of nutrients such as amino-acids in sleep/wake regulation.

Summary In Drosophila, ecdysone hormone levels determine the timing of larval development. Its production is regulated by the stereotypical rise in prothoracicotropic hormone (PTTH) levels. Additionally, ecdysone levels can also be modulated by nutrition (specifically by amino acids) through their action on Drosophila insulin-like peptides (Dilps). Moreover, in glia, amino-acid-sensitive production of Dilps regulates brain development. In this work, we describe the function of an SLC7 amino acid transporter, Sobremesa (Sbm). Larvae with reduced Sbm levels in glia remain in third instar for an additional 24 hr. These larvae show reduced brain growth with increased body size but do not show reduction in insulin signaling or production. Interestingly, Sbm downregulation in glia leads to reduced Ecdysone production and a surprising delay in the rise of PTTH levels. Our work highlights Sbm as a modulator of both brain development and the timing of larval development via an amino-acid-sensitive and Dilp-independent function of glia., Graphical Abstract, Highlights • Glia express the SLC7 amino acid transporter Sobremesa, which controls development • Sobremesa downregulation in glia leads to contrasting effects: small brain and big body size • Sobremesa downregulation results in reduced ecdysone production • Sobremesa downregulation causes a delayed rise in PTTH, Galagovsky et al. reveal a molecular and cellular pathway linking metabolism to developmental growth control. This pathway governs body and nervous system growth timing and involves an amino acid transporter expressed in glia.

International audience; Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinsons disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein-protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain.

Changes in synaptic physiology underlie neuronal network plasticity and behavioral phenomena, which are adjusted during development. The Drosophila larval glutamatergic neuromuscular junction (NMJ) represents a powerful synaptic model to investigate factors impacting these processes. Amino acids such as glutamate have been shown to regulate Drosophila NMJ physiology by modulating the clustering of postsynaptic glutamate receptors and thereby regulating the strength of signal transmission from the motor neuron to the muscle cell. To identify amino acid transporters impacting glutmatergic signal transmission, we used Evolutionary Rate Covariation (ERC), a recently developed bioinformatic tool. Our screen identified ten proteins co-evolving with NMJ glutamate receptors. We selected one candidate transporter, the SLC7 (Solute Carrier) transporter family member JhI-21 (Juvenile hormone Inducible-21), which is expressed in Drosophila larval motor neurons. We show that JhI-21 suppresses postsynaptic muscle glutamate receptor abundance and that JhI-21 expression in motor neurons regulates larval crawling behavior in a developmental stage-specific manner.

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

PMCID: PMC3617446 ; http://www.frontiersin.org/; International audience; Odors are key sensory signals for social communication and food search in animals including insects. Drosophila melanogaster, is a powerful neurogenetic model commonly used to reveal molecular and cellular mechanisms involved in odorant detection. Males use olfaction together with other sensory modalities to find their mates. Here, we review known olfactory signals, their related olfactory receptors, and the corresponding neuronal architecture impacting courtship. OR67d receptor detects 11-cis-Vaccenyl Acetate (cVA), a male specific pheromone transferred to the female during copulation. Transferred cVA is able to reduce female attractiveness for other males after mating, and is also suspected to decrease male-male courtship. cVA can also serve as an aggregation signal, maybe through another OR. OR47b was shown to be activated by fly odors, and to enhance courtship depending on taste pheromones. IR84a detects phenylacetic acid (PAA) and phenylacetaldehyde (PA). These two odors are not pheromones produced by flies, but are present in various fly food sources. PAA enhances male courtship, acting as a food aphrodisiac. Drosophila males have thus developed complementary olfactory strategies to help them to select their mates.

International audience; Many animals attract mating partners through the release of volatile sex pheromones, which can convey information on the species, gender and receptivity of the sender to induce innate courtship and mating behaviours by the receiver. Male Drosophila melanogaster fruitflies display stereotyped reproductive behaviours towards females, and these behaviours are controlled by the neural circuitry expressing male-specific isoforms of the transcription factor Fruitless (FRU(M)). However, the volatile pheromone ligands, receptors and olfactory sensory neurons (OSNs) that promote male courtship have not been identified in this important model organism. Here we describe a novel courtship function of Ionotropic receptor 84a (IR84a), which is a member of the chemosensory ionotropic glutamate receptor family, in a previously uncharacterized population of FRU(M)-positive OSNs. IR84a-expressing neurons are activated not by fly-derived chemicals but by the aromatic odours phenylacetic acid and phenylacetaldehyde, which are widely found in fruit and other plant tissues that serve as food sources and oviposition sites for drosophilid flies. Mutation of Ir84a abolishes both odour-evoked and spontaneous electrophysiological activity in these neurons and markedly reduces male courtship behaviour. Conversely, male courtship is increased--in an IR84a-dependent manner--in the presence of phenylacetic acid but not in the presence of another fruit odour that does not activate IR84a. Interneurons downstream of IR84a-expressing OSNs innervate a pheromone-processing centre in the brain. Whereas IR84a orthologues and phenylacetic-acid-responsive neurons are present in diverse drosophilid species, IR84a is absent from insects that rely on long-range sex pheromones. Our results suggest a model in which IR84a couples food presence to the activation of the fru(M) courtship circuitry in fruitflies. These findings reveal an unusual but effective evolutionary solution to coordinate feeding and oviposition site selection with reproductive behaviours through a specific sensory pathway.

author can archive publisher's version/PDF (embargo 6 months) ; 1529-2401 (Electronic ISSN); International audience; To sense myriad environmental odors, animals have evolved multiple, large families of divergent olfactory receptors. How and why distinct receptor repertoires and their associated circuits are functionally and anatomically integrated is essentially unknown. We have addressed these questions through comprehensive comparative analysis of the Drosophila olfactory subsystems that express the ionotropic receptors (IRs) and odorant receptors (ORs). We identify ligands for most IR neuron classes, revealing their specificity for select amines and acids, which complements the broader tuning of ORs for esters and alcohols. IR and OR sensory neurons exhibit glomerular convergence in segregated, although interconnected, zones of the primary olfactory center, but these circuits are extensively interdigitated in higher brain regions. Consistently, behavioral responses to odors arise from an interplay between IR- and OR-dependent pathways. We integrate knowledge on the different phylogenetic and developmental properties of these receptors and circuits to propose models for the functional contributions and evolution of these distinct olfactory subsystems.

Cystine-glutamate transporters import cystine into cells for glutathione synthesis and protection from oxidative stress, but also export significant amounts of glutamate. Increasing evidence suggests that 'ambient extracellular glutamate' secreted by cystine-glutamate transporters in the nervous system modulates glutamatergic synapse strength and behavior. To date, the only cystine-glutamate transporter mutants examined behaviorally are Drosophila genderblind mutants. These animals contain loss-of-function mutations in the 'genderblind' gene, which encodes an xCT subunit essential for cystine-glutamate transporter function. Genderblind was named based on a mutant courtship phenotype: male genderblind mutants are attracted to normally aversive male pheromones and thus court and attempt to copulate with both male and female partners equally. However, genderblind protein is expressed in many parts of the fly brain and thus might be expected to also regulate other behaviors, including behaviors not related to male courtship or chemosensation. Here, we show that genderblind mutants display faster recovery and increased negative geotaxis after strong mechanical stimuli (e.g., they climb faster and farther after vial banging). This phenotype is displayed by both males and females, consistent with strong genderblind expression in both sexes.

International audience; One of the most widely used transgenic animal models in biology is Drosophila melanogaster, the fruit fly. Chemical information from this exceedingly small organism is usually accomplished by studying populations to attain sample volumes suitable for standard analysis methods. This paper describes a direct sampling technique capable of obtaining 50-300 nL of hemolymph from individual Drosophila larvae. Hemolymph sampling performed under mineral oil and in air at 30 s intervals up to 120 s after piercing larvae revealed that the effect of evaporation on amino acid concentrations is insignificant when the sample was collected within 60 s. Qualitative and quantitative amino acid analyses of obtained hemolymph were carried out in two optimized buffer conditions by capillary electrophoresis with laser-induced fluorescence detection after derivatizing with fluorescamine. Thirteen amino acids were identified from individual hemolymph samples of both wild-type (WT) control and the genderblind (gb) mutant larvae. The levels of glutamine, glutamate, and taurine in the gb hemolymph were significantly lower at 35%, 38%, and 57% of WT levels, respectively. The developed technique that samples only the hemolymph fluid is efficient and enables accurate organism-level chemical information while minimizing errors associated with possible sample contaminations, estimations, and effects of evaporation compared to the traditional hemolymph-sampling techniques.

1097-6256 (Print) Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't; Mate choice is an evolutionarily critical decision that requires the detection of multiple sex-specific signals followed by central integration of these signals to direct appropriate behavior. The mechanisms controlling mate choice remain poorly understood. Here, we show that the glial amino-acid transporter genderblind controls whether Drosophila melanogaster males will attempt to mate with other males. Genderblind (gb) mutant males showed no alteration in heterosexual courtship or copulation, but were attracted to normally unappealing male species-specific chemosensory cues. As a result, genderblind mutant males courted and attempted to copulate with other Drosophila males. This homosexual behavior could be induced within hours using inducible RNAi, suggesting that genderblind controls nervous system function rather than its development. Consistent with this, and indicating that glial genderblind regulates ambient extracellular glutamate to suppress glutamatergic synapse strength in vivo, homosexual behavior could be turned on and off by altering glutamatergic transmission pharmacologically and/or genetically.

We hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, we identified and functionally characterized a novelDrosophilaxCT gene, which we subsequently named “genderblind” (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination ofgbcauses a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering,gbmutants show a large (200–300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additionalin situpharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, our results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamatein vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitizedin vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses.

0001-8244 (Print) Journal Article Research Support, Non-U.S. Gov't; Brain maturation, a developmental process influenced by both endogenous and environmental factors, can affect sexual behavior. In vertebrates and invertebrates, sexual maturation is under the influence of hormones and neuromodulators, but the role of developmental genes in this process is still poorly understood. We report that prospero (pros), a gene crucial for nervous system development, can change the age of onset of sexual behavior in Drosophila melanogaster males: adult males carrying a single copy of several pros mutations court females and mate at a younger age than control males. However, these pros mutations had no effect on female sexual receptivity and did not alter other male phenotypes related to mating behavior. The Pros protein was detected in several brain and sensory structures of immature adult males, some of which are normally involved in the regulation of male specific behaviors. Our data suggest that the altered pros expression affects the age of onset of male mating behavior.

0012-1606 (Print) Comparative Study Journal Article Research Support, Non-U.S. Gov't; Adaptive animal behaviors depend upon the precise development of the nervous system that underlies them. In Drosophila melanogaster, the pan-neural prospero gene (pros), is involved in various aspects of neurogenesis including cell cycle control, axonal outgrowth, neuronal and glial cell differentiation. As these results have been generally obtained with null pros mutants inducing embryonic lethality, the role of pros during later development remains poorly known. Using several pros-Voila (prosV) alleles, that induce multiple developmental and behavioral anomalies in the larva and in adult, we explored the relationship between these phenotypes and the variation of pros expression in 5 different neural regions during pre-imaginal development. We found that the quantity of pros mRNA spliced variants and of Pros protein varied between these alleles in a tissue-specific and developmental way. Moreover, in prosV1 and prosV13 alleles, the respective decrease or increase of pros expression, affected (i) neuronal and glial cell composition, (ii) cell proliferation and death and (iii) axonal-dendritic outgrowth in a stage and cellular context dependant way. The various phenotypic consequences induced during development, related to more or less subtle differences in gene expression, indicate that Pros level needs a precise and specific adjustment in each neural organ to allow its proper function.

0376-6357 (Print) Journal Article Research Support, Non-U.S. Gov't; The fruitfly Drosophila melanogaster is one of the most appropriate model organisms to study the genetics of behaviour. Here, we focus on prospero (pros), a key gene for the development of the nervous system which specifies multiple aspects from the early formation of the embryonic central nervous system to the formation of larval and adult sensory organs. We studied the effects on locomotion, courtship and mating behaviour of three mild pros mutations. These newly isolated pros mutations were induced after the incomplete excision of a transposable genomic element that, before excision, caused a lethal phenotype during larval development. Strikingly, these mutant strains, but not the strains with a clean excision, produced a high frequency of heterozygous flies, after more than 50 generations in the lab. We investigated the factors that could decrease the fitness of homozygotes relatively to heterozygous pros mutant flies. Flies of both genotypes had slightly different levels of fertility. More strikingly, homozygous mutant males had a lower sexual activity than heterozygous males and failed to mate in a competitive situation. No similar effect was detected in mutant females. These findings suggest that mild mutations in pros did not alter vital functions during development but drastically changed adult male behaviour and reproductive fitness.

The PGal4 transposon inserted upstream of the pan-neural gene prospero (pros) causes several neural and behavioral defects in the Voila(1) strain. The precise excision of the transposon simultaneously rescued all these defects whereas its unprecise excision created new pros(V) alleles, including the null allele pros(V17). Here, we describe the relationship between the genetic structure of pros locus, larval locomotion, and larval gustatory response. These two behaviors showed varying degrees of variation depending upon the pros allele. We also found a good relation between behavioral alteration, the level of Pros protein in the embryo, and the degree of disorganization in the larval neuromuscular junction. These data suggest that the complete development of the nervous system requires a full complement of Pros, and that a gradual decrease in the levels of this protein can proportionally alter the development and the function of the nervous system.

0016-6723 (Print) Journal Article Research Support, Non-U.S. Gov't; In Drosophila melanogaster, the PGal4 transposon inserted at the chromosomal site 86E1-2 is associated with the Voila1 allele that causes multiple phenotypes. Homozygous Voila1/1 flies rarely reach adulthood and heterozygous Voila1/+ adult males display strong homosexual courtship behaviour. Both normal behavioural and developmental phenotypes were rescued by remobilizing the PGal4 element. Yet, the rescue of heterosexual courtship and of adult viability did not occur in the same strains, indicating that these defects have different genetic origins. Furthermore, many strains showed a partial rescue of both characters. Molecular analysis revealed that the PGal4 transposon is inserted upstream of the 5'UTR of the prospero gene. The excision strains with no detectable fragment of the PGal4 transposon remaining showed a rescued viability for homozygote adults. Moreover, the developmental period with the highest homozygote lethality was correlated with the size of PGal4 element that remained inserted at the Voila locus. This suggests a relationship between developmental viability and the amount of DNA inserted within the promoter of prospero.