Adrian Rothenfluh

double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation

We have isolated three alleles of a novel Drosophila clock gene, double-time (dbt). Short- (dbtS) and long-period (dbtL) mutants alter both behavioral rhythmicity and molecular oscillations from previously identified clock genes, period and timeless. A third allele, dbtP, causes pupal lethality and eliminates circadian cycling of per and tim gene products in larvae. In dbtP mutants, PER proteins constitutively accumulate, remain hypophosphorylated, and no longer depend on TIM proteins for their accumulation. We propose that the normal function of DOUBLETIME protein is to reduce the stability and thus the level of accumulation of monomeric PER proteins. This would promote a delay between per/tim transcription and PER/TIM complex function, which is essential for molecular rhythmicity.

The Drosophila Clock Gene double-time Encodes a Protein Closely Related to Human Casein Kinase Iε

The cloning of double-time (dbt) is reported. DOUBLETIME protein (DBT) is most closely related to human casein kinase Iepsilon. dbtS and dbtL mutations, which alter period length of Drosophila circadian rhythms, produce single amino acid changes in conserved regions of the predicted kinase. dbtP mutants, which eliminate rhythms of per and tim expression and constitutively overproduce hypophosphorylated PER proteins, abolish most dbt expression. dbt mRNA appears to be expressed in the same cell types as are per and tim and shows no evident oscillation in wild-type heads. DBT is capable of binding to PER in vitro and in Drosophila cells, suggesting that a physical association of PER and DBT regulates PER phosphorylation and accumulation in vivo.

Guidelines on nicotine dose selection for in vivo research

RationaleThis review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure.ObjectivesThis review capitalizes on the authors’ collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models.ResultsSeven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses.ConclusionsThe selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose–response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.

Phosphorylation of period is influenced by cycling physical associations of double-time, period, and timeless in the Drosophila clock

The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the PERIOD protein. Whereas the period (per), timeless (tim), and dClock (dClk) genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and DBT protein are constitutively expressed. Robust circadian changes in DBT subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include PER, TIM, and DBT, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of PER is strongly enhanced when TIM is removed from PER/TIM/DBT complexes. The varying associations of PER, DBT and TIM appear to determine the onset and duration of nuclear PER function within the Drosophila clock.

A TIMELESS-independent function for PERIOD proteins in the Drosophila clock

The mutation timeless(UL) generates 33 hr rhythms, prolonged nuclear localization of PERIOD/TIMELESS(UL) protein complexes, and protracted derepression of period (per) and timeless (tim) transcription. Light-induced elimination of TIM(UL) from nuclear PER/TIM(UL) complexes gives strong downregulation of per and tim expression. Thus, in the absence of TIM, nuclear PER can function as a potent negative transcriptional regulator. Two additional studies support this role for PER: (1) Drosophila expressing PER that constitutively localizes to nuclei produce dominant behavioral arrhythmicity, and (2) constitutively nuclear PER represses dCLOCK/CYCLE-mediated transcription of per in cultured cells without TIM. Conversion of PER/TIM heterodimers to nuclear PER proteins appears to be required to complete transcriptional repression and terminate each circadian molecular cycle.

Distinct Behavioral Responses to Ethanol Are Regulated by Alternate RhoGAP18B Isoforms

In most organisms, low ethanol doses induce increased activity, while high doses are sedating. To investigate the underlying mechanisms, we isolated Drosophila mutants with altered ethanol responsiveness. Mutations in white rabbit (whir), disrupting RhoGAP18B, are strongly resistant to the sedating effects of ethanol. This resistance can be suppressed by reducing the levels of Rho1 or Rac, implicating these GTPases in the behavioral response to ethanol. Indeed, expression of constitutively active forms of Rho1 or Rac1 in adult flies results in ethanol resistance similar to that observed in whir mutants. The whir locus produces several transcripts, RA-RD, which are predicted to encode three distinct RhoGAPs that share only the GAP domain. The RC transcript mediates the sedating effects of ethanol, while the RA transcript regulates its stimulant effects. Thus, distinct RhoGAPs, encoded by the same gene, regulate different manifestations of acute ethanol intoxication.

Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila

BACKGROUND Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells, and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization, and function of Rab GTPases in an organism with a brain. RESULTS We report the surprising discovery that half of all Drosophila Rabs function specifically or predominantly in distinct subsets of neurons in the brain. Furthermore, functional profiling of the GTP/GDP-bound states reveals that these neuronal Rabs are almost exclusively active at synapses and the majority of these synaptic Rabs specifically mark synaptic recycling endosomal compartments. Our profiling strategy is based on Gal4 knockins in large genomic fragments that are additionally designed to generate mutants by ends-out homologous recombination. We generated 36 large genomic targeting vectors and transgenic rab-Gal4 fly strains for 25 rab genes. Proof-of-principle knockout of the synaptic rab27 reveals a sleep phenotype that matches its cell-specific expression. CONCLUSIONS Our findings suggest that up to half of all Drosophila Rabs exert specialized synaptic functions. The tools presented here allow systematic functional studies of these Rabs and provide a method that is applicable to any large gene family in Drosophila.

Drugs, flies, and videotape: the effects of ethanol and cocaine on Drosophila locomotion

Drosophila melanogaster has been introduced recently as a model organism in which to study the mechanisms by which drugs of abuse change behavior and by which the nervous system changes upon repeated drug exposure. Surprising similarities between flies and mammals have begun to emerge at the behavioral, neurochemical and molecular levels.

The Genetics of Behavioral Alcohol Responses in Drosophila

Drosophila melanogaster is commonly found near rotting or fermenting fruit, reflected in its name pomace, or vinegar fly. In such environments, flies often encounter significant levels of ethanol. Three observations have made Drosophila a very promising model organism to understand the genetic contributions to the behavioral responses to alcohol. First, similar to higher vertebrates, flies show hyperactivation upon exposure to a low to medium dose of alcohol, while high doses can lead to sedation. In addition, when given a choice, flies will actually prefer alcohol-containing food over regular food. Second, the genes and biochemical pathways implicated in controlling these behavioral responses in flies are also participating in determining alcohol responses, and drinking behavior in mammals. Third, the fact that flies have been studied genetically for over one hundred years means that an exceptional repertoire of genetic tools are at our disposal. Here, we will review some of these tools and experimental approaches, survey the methods for, and measures after Drosophila ethanol exposure, and discuss the different molecular components and functional pathways involved in these behavioral responses to alcohol.

Rsu1 regulates ethanol consumption in Drosophila and humans

Significance Genetic factors play a major role in the development of human addiction. Identifying these genes and understanding their molecular mechanisms are necessary first steps in the development of targeted therapeutic intervention. Here, we have isolated the gene encoding Ras suppressor 1 (Rsu1) in an unbiased genetic screen for altered ethanol responses in the vinegar fly, Drosophila melanogaster. Our behavioral, genetic, and biochemical experiments show that Rsu1 links signaling from the integrin cell adhesion molecule to the small GTPase Rac1 in adult neurons to regulate actin dynamics and alcohol consumption preference. We also show that variants in human RSU1 associate with altered drinking and brain activation during a reward prediction task, thereby validating the predictive power of our approach. Alcohol abuse is highly prevalent, but little is understood about the molecular causes. Here, we report that Ras suppressor 1 (Rsu1) affects ethanol consumption in flies and humans. Drosophila lacking Rsu1 show reduced sensitivity to ethanol-induced sedation. We show that Rsu1 is required in the adult nervous system for normal sensitivity and that it acts downstream of the integrin cell adhesion molecule and upstream of the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase to regulate the actin cytoskeleton. In an ethanol preference assay, global loss of Rsu1 causes high naïve preference. In contrast, flies lacking Rsu1 only in the mushroom bodies of the brain show normal naïve preference but then fail to acquire ethanol preference like normal flies. Rsu1 is, thus, required in distinct neurons to modulate naïve and acquired ethanol preference. In humans, we find that polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption, across phyla.