A multicomponent screen for feeding behaviour and nutritional status in Drosophila to interrogate mammalian appetite-related genes.

OBJECTIVE: More than 300 genetic variants have been robustly associated with measures of human adiposity. Highly penetrant mutations causing human obesity do so largely by disrupting satiety pathways in the brain and increasing food intake. Most of the common obesity-predisposing variants are in, or near, genes expressed highly in the brain, but little is known of their function. Exploring the biology of these genes at scale in mammalian systems is challenging. We sought to establish and validate the use of a multicomponent screen for feeding behaviour phenotypes, taking advantage of the tractable model organism Drosophila melanogaster. METHODS: We validated a screen for feeding behaviour in Drosophila by comparing results after disrupting the expression of centrally expressed genes that influence energy balance in flies to those of 10 control genes. We then used this screen to explore the effects of disrupted expression of genes either a) implicated in energy homeostasis through human genome-wide association studies (GWAS) or b) expressed and nutritionally responsive in specific populations of hypothalamic neurons with a known role in feeding/fasting. RESULTS: Using data from the validation study to classify responses, we studied 53 Drosophila orthologues of genes implicated by human GWAS in body mass index and found that 15 significantly influenced feeding behaviour or energy homeostasis in the Drosophila screen. We then studied 50 Drosophila homologues of 47 murine genes reciprocally nutritionally regulated in POMC and agouti-related peptide neurons. Seven of these 50 genes were found by our screen to influence feeding behaviour in flies. CONCLUSION: We demonstrated the utility of Drosophila as a tractable model organism in a high-throughput genetic screen for food intake phenotypes. This simple, cost-efficient strategy is ideal for high-throughput interrogation of genes implicated in feeding behaviour and obesity in mammals and will facilitate the process of reaching a functional understanding of obesity pathogenesis.

Calpain inhibition mediates autophagy-dependent protection against polyglutamine toxicity.

Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington's disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington's disease.

Comparative efficacy of a proprietary topical ibuprofen gel and oral ibuprofen in acute soft tissue injuries: a randomized, double-blind study.

The efficacy of a novel, proprietary topical formulation of ibuprofen 5% gel (Ibugel) and ibuprofen 400 mg tablets (1200 mg daily) was compared in a double-blind, double-dummy, parallel group study in patients with acute soft tissue injuries. Patients received either active gel plus placebo tablets (n = 50) or active tablets plus placebo gel (n = 50) for at least 7 days. The gel was applied and one tablet was taken three times daily. The two treatments showed similar efficacy. There were no significant differences between the groups for either the primary efficacy endpoint, the median time for the injury to be rated as 'completely better' by the patients (>14 days active gel, 13.5 days active tablets; P = 0.59), or for other efficacy measures including the times to clinically significant relief from pain at rest or on movement and swelling. In summary, ibuprofen gel shows similar efficacy to oral ibuprofen 400 mg and may offer improved tolerability.

Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila.

Amphiphysins 1 and 2 are enriched in the mammalian brain and are proposed to recruit dynamin to sites of endocytosis. Shorter amphiphysin 2 splice variants are also found ubiquitously, with an enrichment in skeletal muscle. At the Drosophila larval neuromuscular junction, amphiphysin is localized postsynaptically and amphiphysin mutants have no major defects in neurotransmission; they are also viable, but flightless. Like mammalian amphiphysin 2 in muscles, Drosophila amphiphysin does not bind clathrin, but can tubulate lipids and is localized on T-tubules. Amphiphysin mutants have a novel phenotype, a severely disorganized T-tubule/sarcoplasmic reticulum system. We therefore propose that muscle amphiphysin is not involved in clathrin-mediated endocytosis, but in the structural organization of the membrane-bound compartments of the excitation-contraction coupling machinery of muscles.

The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila.

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.

Dribble, the Drosophila KRR1p homologue, is involved in rRNA processing.

The Drosophila dribble (dbe) gene encodes a KH domain protein, homologous to yeast KRR1p. Expression of dbe transcripts is ubiquitous during embryogenesis. Overexpressed Dribble protein is localized in the nucleus and in some cell types in a subregion of the nucleolus. Homozygous dbe mutants die at first instar larval stage. Clonal analyses suggest that dbe(+) is required for survival of dividing cells. In dbe mutants, a novel rRNA-processing defect is found and accumulation of an abnormal rRNA precursor is detected.

Rapsynoid/partner of inscuteable controls asymmetric division of larval neuroblasts in Drosophila.

Asymmetric cell division generates daughter cells with different developmental fates. In Drosophila neuroblasts, asymmetric divisions are characterized by (1) a difference in size between the two daughter cells and (2) an asymmetric distribution of cell fate determinants, including Prospero and Numb, between the two daughter cells. In embryonic neuroblasts, the asymmetric localization of cell fate determinants is under the control of the protein Inscuteable (Insc), which is itself localized asymmetrically as an apical crescent. Here, we describe a new Drosophila protein, Rapsynoid (Raps), which interacts in a two-hybrid assay with the signal transduction protein Galpha(i). We show that Raps is localized asymmetrically in dividing larval neuroblasts and colocalizes with Insc. Moreover, in raps mutants, the asymmetric divisions of neuroblasts are altered: (1) Insc is no longer asymmetrically localized in the dividing neuroblast; and (2) the neuroblast division produces two daughter cells of similar sizes. However, the morphologically symmetrical divisions of raps neuroblasts still lead to daughter cells with different fates, as shown by differences in gene expression. Our data show that Raps is a novel protein involved in the control of asymmetric divisions of neuroblasts.

Characterisation of the gene for Drosophila amphiphysin.

A sequence similarity search of the Drosophila nucleotide database using vertebrate amphiphysin as a query identified a cDNA that encodes a Drosophila amphiphysin. The predicted protein has conserved sequence domains that should enable it to dimerise and bind to dynamin. Structural modelling suggests that the Src-homology-3 (SH3) domains of vertebrate and Drosophila amphiphysins are highly similar, supporting the putative ability of the latter to bind dynamin. However, the fly amphiphysin shows less conservation to sequences in the vertebrate amphiphysins that bind other endocytic components such as clathrin, AP-2 and endophilin. Amphiphysin is a single-copy gene that maps to position 49B on polytene chromosomes. Messenger RNA of this amphiphysin is expressed widely during embryogenesis and has elevated expression in a number of sites including the foregut, hindgut and epidermis, but not in the central nervous system. Taken together, these data are consistent with a role for Drosophila amphiphysin in endocytosis, but the details of this role may differ from that of vertebrate amphiphysins.

Transgenic inhibitors identify two roles for protein kinase A in Drosophila development.

We have initiated an analysis of protein kinase A (PKA) in Drosophila using transgenic techniques to modulate PKA activity in specific tissues during development. We have constructed GAL4/UAS-regulated transgenes in active and mutant forms that encode PKAc, the catalytic subunit of PKA, and PKI(1-31), a competitive inhibitor of PKAc. We present evidence that the wild-type transgenes are active and summarize the phenotypes produced by a number of GAL4 enhancer-detector strains. We compare the effects of transgenes encoding PKI(1-31) with those encoding PKAr*, a mutant regulatory subunit that constitutively inhibits PKAc because of its inability to bind cyclic AMP. Both inhibitors block larval growth, but only PKAr* alters pattern formation by activating the Hedgehog signaling pathway. Therefore, transgenic PKI(1-31) should provide a tool to investigate the role of PKAc in larval growth regulation without concomitant changes in pattern formation. The different effects of PKI(1-31) and PKAr* suggest two distinct roles, cytoplasmic and nuclear, for PKAc in Hedgehog signal transduction. Alternatively, PKAr* may target proteins other than PKAc, suggesting a role for free PKAr in signal transduction, a role inhibited by PKAc in reversal of the classical relationship of these subunits.

Sexual behaviour: Courting dissatisfaction.

A nuclear receptor, the product of the dissatisfaction gene, has been found to regulate Drosophila sexual behaviour, probably via its action in a small subset of neurons. The results shed new light on the genetic determination of sexual behaviour.

loco encodes an RGS protein required for Drosophila glial differentiation.

In Drosophila, glial cell development depends on the gene glial cells missing (gcm). gcm activates the expression of other transcription factors such as pointed and repo, which control subsequent glial differentiation. In order to better understand glial cell differentiation, we have screened for genes whose expression in glial cells depends on the activity of pointed. Using an enhancer trap approach, we have identified loco as such a gene. loco is expressed in most lateral CNS glial cells throughout development. Embryos lacking loco function have an normal overall morphology, but fail to hatch. Ultrastructural analysis of homozygous mutant loco embryos reveals a severe glial cell differentiation defect. Mutant glial cells fail to properly ensheath longitudinal axon tracts and do not form the normal glial-glial cell contacts, resulting in a disruption of the blood-brain barrier. Hypomorphic loco alleles were isolated following an EMS mutagenesis. Rare escapers eclose which show impaired locomotor capabilities. loco encodes the first two known Drosophila members of the family of Regulators of G-protein signalling (RGS) proteins, known to interact with the alpha subunits of G-proteins. loco specifically interacts with the Drosophila alphai-subunit. Strikingly, the interaction is not confined to the RGS domain. This interaction and the coexpression of LOCO and Galphai suggests a function of G-protein signalling for glial cell development.

Identification and characterization of kraken, a gene encoding a putative hydrolytic enzyme in Drosophila melanogaster.

Kraken, a novel Drosophila gene isolated from a 4-8-h-old Drosophila embryo cDNA library, shows homology to a family of serine hydrolases whose common feature is that they all catalyse breakage of substrates with a carbonyl-containing group. It is a single-copy gene with at least two introns and maps to position 21D on polytene chromosomes. kraken is a member of a conserved gene family. Messenger RNA of kraken is expressed ubiquitously in early embryogenesis. Later, it is concentrated in the foregut and the posterior midgut primordium. Towards the end of embryogenesis, expression of kraken is confined to the gastric caeca. During the third-instar larval stage, kraken is expressed at low levels in the gastric caeca and parts of the gut, and at higher levels in the fat body. We suggest a role for Kraken in detoxification and digestion during embryogenesis and larval development.

Identification and characterization of the gene for Drosophila L3 ribosomal protein.

A cDNA clone that encodes a Drosophila homologue of ribosomal protein L3 was isolated from a Drosophila ovary gridded cDNA library. The Drosophila ribosomal protein L3 gene (RpL3) is highly conserved with ribosomal protein L3 genes in other organisms. It is a single copy gene and maps to position 86D5-10 on polytene chromosomes. A Minute gene in this region, M(3) 86D, is a possible candidate to encode RPL3. RPL3 message is expressed ubiquitously. A partial RPL8 cDNA clone was also isolated and mapped to 62F.

Identification and characterization of the gene for Drosophila S20 ribosomal protein.

A cDNA clone that encodes a Drosophila homologue of ribosomal protein S20 was isolated from a Drosophila ovary cDNA library. The Drosophila S20 gene (RpS20) is highly conserved with S20 genes in other organisms. It is a single copy gene and maps to position 92F-93A on polytene chromosomes. No Minute mutation in this location has been reported; at least five essential genes are possible candidates to encode RpS20. RpS20 message is expressed ubiquitously in embryos, but is expressed at high levels in the midgut.

Genetic feminization of pheromones and its behavioral consequences in Drosophila males.

Pheromones are intraspecific chemical signals important for mate attraction and discrimination. In the fruit fly Drosophila melanogaster, hydrocarbons on the cuticular surface of the animal are sexually dimorphic in both their occurrence and their effects: Female-specific molecules stimulate male sexual excitation, whereas the predominant male-specific molecule tends to inhibit male excitation. Complete feminization of the pheromone mixture produced by males was induced by targeted expression of the transformer gene in adult oenocytes (subcuticular abdominal cells) or by ubiquitous expression during early imaginal life. The resulting flies generally exhibited male heterosexual orientation but elicited homosexual courtship from other males.

Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies.

Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3',5' monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein alpha subunit (Galphas*) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when Galphas* was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of Galphas* did not affect gross MB morphology, and wild-type Galphas expression did not affect learning. Thus, olfactory learning depends on regulated Gs signaling in Drosophila MBs.

Mutations in shaking-B prevent electrical synapse formation in the Drosophila giant fiber system.

The giant fiber system (GFS) is a simple network of neurons that mediates visually elicited escape behavior in Drosophila. The giant fiber (GF), the major component of the system, is a large, descending interneuron that relays visual stimuli to the motoneurons that innervate the tergotrochanteral jump muscle (TTM) and dorsal longitudinal flight muscles (DLMs). Mutations in the neural transcript from the shaking-B locus abolish the behavioral response by disrupting transmission at some electrical synapses in the GFS. This study focuses on the role of the gene in the development of the synaptic connections. Using an enhancer-trap line that expresses lacZ in the GFs, we show that the neurons develop during the first 30 hr of metamorphosis. Within the next 15 hr, they begin to form electrical synapses, as indicated by the transfer of intracellularly injected Lucifer yellow. The GFs dye-couple to the TTM motoneuron between 30 and 45 hr of metamorphosis, to the peripherally synapsing interneuron that drives the DLM motoneurons at approximately 48 hr, and to giant commissural interneurons in the brain at approximately 55 hr. Immunocytochemistry with shaking-B peptide antisera demonstrates that the expression of shaking-B protein in the region of GFS synapses coincides temporally with the onset of synaptogenesis; expression persists thereafter. The mutation shak-B2, which eliminates protein expression, prevents the establishment of dye coupling shaking-B, therefore, is essential for the assembly and/or maintenance of functional gap junctions at electrical synapses in the GFS.

Inducible ternary control of transgene expression and cell ablation in Drosophila.

In Drosophila, P-GAL4 enhancer trap lines can target expression of a cloned gene, under control of a UASGAL element, to any cells of interest. However, additional expression of GAL4 in other cells can produce unwanted lethality or side-effects, particularly when it drives expression of a toxic gene product. To target the toxic gene product ricin A chain specifically to adult neurons, we have superimposed a second layer of regulation on the GAL4 control. We have constructed flies in which an effector gene is separated from UASGAL by a polyadenylation site flanked by two FRT sites in the same orientation. A recombination event between the two FRT sites, catalysed by yeast FLP recombinase, brings the effector gene under control of UASGAL. Consequently, expression of the effector gene is turned on in that cell and its descendants, if they also express GAL4. Recombinase is supplied by heat shock induction of a FLP transgene, allowing both timing and frequency of recombination events to be regulated. Using a lacZ effector (reporter) to test the system, we have generated labelled clones in the embryonic mesoderm and shown that most recombination events occur soon after FLP recombinase is supplied. By substituting the ricin A chain gene for lacZ, we have performed mosaic cell ablations in one GAL4 line that marks the adult giant descending neurons, and in a second which marks mushroom body neurons. In a number of cases we observed loss of one or both the adult giant descending neurons, or of subsets of mushroom body neurons. In association with the mushroom body ablations, we also observed misrouting of surviving axons.

Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila.

In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.