Drosophila melanogaster

Methionine sulfoxide reductase (MSR) is an important antioxidant to help mitigate oxidative stress that contributes to age-associated neurodegenerative diseases, such as Alzheimer’s Disease and Parkinson’s Disease. In MSR deficient Drosophila melanogaster (fruit flies), larvae show a developmental delay like that seen when wild-type larvae are reared on nutrient deficit culture medium. These investigators further showed that serotonin levels were depressed in these nutrient deficient larvae. The overarching aim of this study was to better understand the role of serotonin in MSR regulated physiology.
Supplementing food with serotonin partially rescued the slower mouth hook movements (MHM) observed in the MSR-deficient flies. However, supplementation with serotonin altering drugs that cross the blood brain barrier (5-hydroxytryptophan, fluoxetine, or paravi chlorophenylalanine) did not rescue MHM and caused impairments to the growth of larvae during development. This study indicates that serotonin regulates feeding behavior partially through the regulation of MSR production but acts independently to regulate development.

Proper regulation of sleep and metabolism are critical to the survival of all organisms. In humans, dysregulation of sleep is linked to metabolic syndrome, including hypertension, hyperglycemia and hyperlipidemia. However, the mechanisms regulating interactions between sleep and metabolism are poorly understood. Although the fruit fly, Drosophila melanogaster, bears little anatomical resemblance to humans, it shares similar genetics essential in understanding normal development and disease in humans. From humans to flies, many disease-related genes and pathways are highly conserved, rendering the fruit fly ideal to understanding the interactions between sleep and metabolism. Therefore, using the fruit fly provides a framework for understanding how genes function between sleep and metabolism. During starvation, both humans and rats reduce their sleep. Similarly, previous studies have shown that fruit flies also suppress sleep to forage for food, further showing that sleep and metabolism are intricately tied to one another and that they are highly conserved across species. To further explore the interactions between sleep and metabolism, I have conducted multiple genetic screens to identify novel regulators of sleep-metabolism interactions. These experiments led to the identification of the mRNA binding protein translin (trsn) as being required for starvation-induced sleep suppression. A second screen that targeted metabolic genes from a genome-wide association study identified the ion channel accessory protein uncoordinated 79 (unc79) as a critical regulator of both sleep duration and starvation resistance. The genes function in different regions of the brain and suggest complex neural circuitry is likely to underlie regulation of sleep metabolism interactions. Taken together, a mechanistic understanding of how different genes function to regulate sleep in flies will further our understanding of how sleep and metabolism is regulated in humans.

Insect grooming has various functions, including defense against parasites and pathogens, cleaning of dust particles, and maintenance of sensory receptors. The hierarchy of grooming behavior suggests that cleaning one body part is more crucial than the other, the priority order more specifically being eyes, antennae, abdomen, then wings, followed by the thorax. Histamine is an extensively studied neurotransmitter found in the central nervous system of many animals. In Drosophila, histamine is found in both the peripheral and central nervous systems and is necessary for visual and mechanosensory behaviors. Histamine-gated chloride channel 1 (HisCl1) and Ora transientless (Ort) are two characterized histamine receptors, both of which are vital for visual signaling in the fly.

Diseases such as epilepsy, pain, and neurodegenerative disorders are associated with changes in neuronal dysfunction due to an imbalance of excitation and inhibition. This work details a novel electroconvulsive seizure assay for C. elegans using the well characterized cholinergic and GABAergic excitation and inhibition of the body wall muscles and the resulting locomotion patterns to better understand neuronal excitability. The time to recover normal locomotion from an electroconvulsive seizure could be modulated by increasing and decreasing inhibition. GABAergic deficits and a chemical proconvulsant resulted in an increased recovery time while anti-epileptic drugs decreased seizure duration. Successful modulation of excitation and inhibition in the new assay led to the investigation of a cGMP-dependent protein kinase (PKG) which modulates potassium (K+) channels, affecting neuronal excitability, and determined that increasing PKG activity decreases the time to recovery from an electroconvulsive seizure. The new assay was used as a forward genetic screening tool using C. elegans and several potential genes that affect seizure susceptibility were found to take longer to recover from a seizure. A naturally occurring polymorphism for PKG in D. melanogaster confirmed that both genetic and pharmacological manipulation of PKG influences seizure duration. PKG has been implicated in stress tolerance, which can be affected by changes in neuronal excitability associated with aging, so stress tolerance and locomotor behavior in senescent flies was investigated. For the first time, PKG has been implicated in aging phenotypes with high levels of PKG resulting in reduced locomotion and lifespan in senescent flies. The results suggest a potential new role for PKG in seizure susceptibility and aging.

Dysregulation of sleep and metabolism has enormous health consequences. Sleep
loss is linked to increased appetite and insulin insensitivity, and epidemiological studies
link chronic sleep deprivation to obesity-related disorders. Interactions between sleep and
metabolism involve the integration of signalling from brain regions regulating sleep,
feeding, and metabolism, as well as communication between the brain and peripheral
organs. In this series of studies, using the fruit fly as a model organism, we investigated
how feeding information is processed to regulate sleep, and how peripheral tissues
regulate sleep through the modulation of energy stores.
In order to address these questions, we performed a large RNAi screen to identify
novel genetic regulators of sleep and metabolism. We found that, the mRNA/DNA
binding protein, Translin (trsn), is necessary for the acute modulation of sleep in
accordance with feeding state. Flies mutant for trsn or selective knockdown of trsn in
Leucokinin (Lk) neurons abolishes starvation-induced sleep suppression. In addition, genetic silencing of Lk neurons or a mutation in the Lk locus also disrupts the integration
between sleep and metabolism, suggesting that Lk neurons are active during starvation.
We confirmed this hypothesis by measuring baseline activity during fed and starved
states. We found that LHLK neurons, which have axonal projections to sleep and
metabolic centers of the brain, are more active during starvation. These findings suggest
that LHLK neurons are modulated in accordance with feeding state to regulate sleep.
Finally, to address how peripheral tissues regulate sleep, we performed an RNAi
screen, selectively knocking down genes in the fat body. We found that knockdown of
Phosphoribosylformylglycinamidine synthase (Ade2), a highly conserved gene involved
the biosynthesis of purines, regulates sleep and energy stores. Flies heterozygous for two
Ade2 mutations are short sleepers and this effect is partially rescued by restoring Ade2 to
the fly fat body. These findings suggest Ade2 functions within the fat body to promote
both sleep and energy storage, providing a functional link between these processes.
Together, the experimental evidence presented here provides an initial model for how the
peripheral tissues communicate to the brain to modulate sleep in accordance with
metabolic state.

The oxidation of methionine (Met) into methionine sulfoxide (met-(o)) leads to deleterious modifications to a variety of cellular constituents. These deleterious alterations can be reversed by enzymes known as methionine sulfoxide reductases (Msr). The Msr (MsrA and MsrB) family of enzymes have been studied extensively for their biological roles in reducing oxidized Met residues back into functional Met. A wide range of studies have focused on Msr both in vivo and in vitro using a variety of model organisms. More specifically, studies have noted numerous processes affected by the overexpression, under expression, and silencing of MsrA and MsrB. Collectively, the results of these studies have shown that Msr is involved in lifespan and the management of oxidative stress. More recent evidence is emerging that supports existing biological functions of Msr and theorizes the involvement of Msr in numerous biological pathways.

The human brain functions within a narrow range of temperatures and
variations outside of this range incur cellular damage and death and, ultimately,
death of the organism. Other organisms, like the poikilotherm Drosophila
melanogaster, have adapted mechanisms to maintain brain function over wide
ranges in temperature and, if exposed to high temperatures where brain function
is no longer supported, these animals enter a protective coma to promote survival
of the organism once the acute temperature stress is alleviated.
This research characterized the role of different neuronal cell types,
including glia, in the protection of brain function during acute hyperthermia,
specifically looking at two protective pathways: the heat shock protein (HSP)
pathway and the cGMP-dependent protein kinase G (PKG) pathway. Whole
animal behavioral assays were used in combination with tissue-specific genetic
manipulation of protective pathways to determine the specific cell types sufficient to confer protection of neuronal function during acute hyperthermia. Using the
neuromuscular junction (NMJ) preparation, calcium imaging techniques were
combined with pharmacological and genetic manipulations to test the hypothesis
that alterations in ion channel conductance via endogenous mechanisms
regulating the cellular response to high temperature stress alter neuronal function.
Expression of foraging RNAi to inhibit PKG expression in neurons or glia
demonstrated protection of function during acute hyperthermia measured
behaviorally through the extension of locomotor function. This extension of
function with the tissue-specific inhibition of PKG was also confirmed at the cellular
level using the genetically encoded calcium indicator (GECI), GCaMP3, to image
calcium dynamics at the NMJ, where preparations expressing foraging RNAi could
continue to elicit changes in calcium dynamics in response to stimulation. Over
the course of this study, the mechanism underlying a novel glial calcium wave in
the peripheral nervous system was characterized in order to elucidate glia’s role in
the protection of neuronal function during acute hyperthermia.

Seizures are a symptom of epilepsy, characterized by spontaneous firing due to an imbalance of excitatory and inhibitory features. While mammalian seizure models
receive the most attention, the simplicity and tractability of invertebrate model systems, specifically C. elegans and D. melanogaster, have many advantages in understanding
the molecular and cellular mechanisms of seizure behavior. This research explores C. elegans and D. melanogaster as electroconvulsive seizure models to investigate
methods to both modulate and better understand seizure susceptibility. A common underlying feature of seizures in mammals, worms, and flies involves regulating
excitation and inhibition. The C. elegans locomotor circuit is regulated via well characterized GABAergic and cholingeric motoneurons that innervate two rows of
dorsal and ventral body wall muscles. In this research, we developed an electroconvulsive seizure assay which utilizes the locomotor circuit as a behavioral read out of neuronal function. When inhibition is decreased in the circuit, for example by decreasing GABAergic input, we find a general increase in the time to recovery from a seizure. After establishing the contribution of excitation and inhibition to seizure recovery, we explored a ubiquitin ligase, associated with comorbidity of an X-linked Intellectual Disorder and epilepsy in humans, and established that the worm homolog, eel-1, contributes to seizure susceptibility similarly to the human gene. Next, we investigated a cGMP-dependent protein kinase (PKG) that functions in the nervous system of both worms and flies and determined that increasing PKG activity, decreases the time to recovery from an electroconvulsive seizure. These experiments suggest a potential novel role for a major protein, PKG, in seizure susceptibility and that the C. elegans and D. melanogaster electroconvulsive seizure assays can be used to investigate possible genes involved in seizure susceptibility and future therapeutic to treat epilepsy.

Drosophila melanogaster tolerates several hours of anoxia (the absence of
oxygen) by entering a protective coma. A burst of reactive oxygen species (ROS) is
produced when oxygen is reintroduced to the cells. ROS causes oxidative damage to
critical cellular molecules, which contribute to aging and development of certain agerelated
conditions. The amino acid, methionine, is susceptible to oxidation, although this
damage can be reversed by methionine sulfoxide reductases (Msr). This project
investigates the effect of Msr-deficiency on anoxia tolerance in Drosophila throughout
the lifespan of the animal. The data show that the time for recovery from the
protective comma as well as the survival of the animals lacking any Msr activity
depends on how quickly the coma is induced by the anoxic conditions. Insight into
the roles(s) of Msr genes under anoxic stress can lead us to a path of designing
therapeutic drugs around these genes in relation to stroke.

The venom of cone snails is a potent cocktail of peptides, proteins, and other small molecules. Several of the peptides (conopeptides and conotoxins) target ion channels and receptors and have proven useful as biochemical probes or pharmaceutical leads. In this study, the venom of a fish-hunting cone snail, Conus purpurascens was analyzed for intraspecific variability; α-conotoxins from the venom were isolated by high performance liquid chromatography, identified by mass spectrometry and nuclear magnetic resonance, and tested in a electrophysiological assay in Drosophila melanogaster; the effects of diet change on venom composition was investigated. It has been determined that each specimen of C. purpurascens expresses a distinct venom, resulting in the expression of more than 5,000 unique conopeptides across the species. α- conotoxin PIA was shown to inhibit the Dα7 nicotinic acetylcholine receptor.