Laurent Seugnet

Novel Notch alleles reveal a Deltex-dependent pathway repressing neural fate

BACKGROUND The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. RESULTS Here, we describe new alleles of Notch that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. We show that the loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. CONCLUSIONS Our results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices.

D1 Receptor Activation in the Mushroom Bodies Rescues Sleep Loss Induced Learning Impairments in Drosophila

BACKGROUND Extended wakefulness disrupts acquisition of short-term memories in mammals. However, the underlying molecular mechanisms triggered by extended waking and restored by sleep are unknown. Moreover, the neuronal circuits that depend on sleep for optimal learning remain unidentified. RESULTS Learning was evaluated with aversive phototaxic suppression. In this task, flies learn to avoid light that is paired with an aversive stimulus (quinine-humidity). We demonstrate extensive homology in sleep-deprivation-induced learning impairment between flies and humans. Both 6 hr and 12 hr of sleep deprivation are sufficient to impair learning in Canton-S (Cs) flies. Moreover, learning is impaired at the end of the normal waking day in direct correlation with time spent awake. Mechanistic studies indicate that this task requires intact mushroom bodies (MBs) and requires the dopamine D1-like receptor (dDA1). Importantly, sleep-deprivation-induced learning impairments could be rescued by targeted gene expression of the dDA1 receptor to the MBs. CONCLUSIONS These data provide direct evidence that extended wakefulness disrupts learning in Drosophila. These results demonstrate that it is possible to prevent the effects of sleep deprivation by targeting a single neuronal structure and identify cellular and molecular targets adversely affected by extended waking in a genetically tractable model organism.

Identification of a biomarker for sleep drive in flies and humans

It is a common experience to sacrifice sleep to meet the demands of our 24-h society. Current estimates reveal that as a society, we sleep on average 2 h less than we did 40 years ago. This level of sleep restriction results in negative health outcomes and is sufficient to produce cognitive deficits and reduced attention and is associated with increased risk for traffic and occupational accidents. Unfortunately, there is no simple quantifiable marker that can detect an individual who is excessively sleepy before adverse outcomes become evident. To address this issue, we have developed a simple and effective strategy for identifying biomarkers of sleepiness by using genetic and pharmacological tools that dissociate sleep drive from wake time in the model organism Drosophila melanogaster. These studies have identified a biomarker, Amylase, that is highly correlated with sleep drive. More importantly, both salivary Amylase activity and mRNA levels are also responsive to extended waking in humans. These data indicate that the fly is relevant for human sleep research and represents a first step in developing an effective method for detecting sleepiness in vulnerable populations.

The perilipin homologue, lipid storage droplet 2, regulates sleep homeostasis and prevents learning impairments following sleep loss.

Starvation, which is common in the wild, appears to initiate a genetic program that allows fruitflies to remain awake without the sleepiness and cognitive impairments that typically follow sleep deprivation.

Notch Signaling Modulates Sleep Homeostasis and Learning after Sleep Deprivation in Drosophila

The role of the transmembrane receptor Notch in the adult brain is poorly understood. Here, we provide evidence that bunched, a negative regulator of Notch, is involved in sleep homeostasis. Genetic evidence indicates that interfering with bunched activity in the mushroom bodies (MBs) abolishes sleep homeostasis. Combining bunched and Delta loss-of-function mutations rescues normal homeostasis, suggesting that Notch signaling may be involved in regulating sensitivity to sleep loss. Preventing the downregulation of Delta by overexpressing a wild-type transgene in MBs reduces sleep homeostasis and, importantly, prevents learning impairments induced by sleep deprivation. Similar resistance to sleep loss is observed with Notch(spl-1) gain-of-function mutants. Immunohistochemistry reveals that the Notch receptor is expressed in glia, whereas Delta is localized in neurons. Importantly, the expression in glia of the intracellular domain of Notch, a dominant activated form of the receptor, is sufficient to prevent learning deficits after sleep deprivation. Together, these results identify a novel neuron-glia signaling pathway dependent on Notch and regulated by bunched. These data highlight the emerging role of neuron-glia interactions in regulating both sleep and learning impairments associated with sleep loss.

Aversive phototaxic suppression: evaluation of a short-term memory assay in Drosophila melanogaster.

Drosophila melanogaster is increasingly being used to model human conditions that are associated with cognitive deficits including fragile‐X syndrome, Alzheimer’s disease, Parkinson’s disease, sleep loss, etc. With few exceptions, cognitive abilities that are known to be modified in these conditions in humans have not been evaluated in fly models. One reason is the absence of a simple, inexpensive and reliable behavioral assay that can be used by laboratories that are not expert in learning and memory. Aversive phototaxic suppression (APS) is a simple assay in which flies learn to avoid light that is paired with an aversive stimulus (quinine/humidity). However, questions remain about whether the change in the fly’s behavior reflects learning an association between light and quinine/humidity or whether the change in behavior is because of nonassociative effects of habituation and/or sensitization. We evaluated potential effects of sensitization and habituation on behavior in the T‐maze and conducted a series of yoked control experiments to further exclude nonassociative effects and determine whether this task evaluates operant learning. Together these experiments indicate that a fly must associate the light with quinine/humidity to successfully complete the task. Next, we show that five classic memory mutants are deficient in this assay. Finally, we evaluate performance in a fly model of neurodegenerative disorders associated with the accumulation of Tau. These data indicate that APS is a simple and effective assay that can be used to evaluate fly models of human conditions associated with cognitive deficits.

Identification of Genes Associated with Resilience/Vulnerability to Sleep Deprivation and Starvation in Drosophila

BACKGROUND AND STUDY OBJECTIVES Flies mutant for the canonical clock protein cycle (cyc(01)) exhibit a sleep rebound that is ∼10 times larger than wild-type flies and die after only 10 h of sleep deprivation. Surprisingly, when starved, cyc(01) mutants can remain awake for 28 h without demonstrating negative outcomes. Thus, we hypothesized that identifying transcripts that are differentially regulated between waking induced by sleep deprivation and waking induced by starvation would identify genes that underlie the deleterious effects of sleep deprivation and/or protect flies from the negative consequences of waking. DESIGN We used partial complementary DNA microarrays to identify transcripts that are differentially expressed between cyc(01) mutants that had been sleep deprived or starved for 7 h. We then used genetics to determine whether disrupting genes involved in lipid metabolism would exhibit alterations in their response to sleep deprivation. SETTING Laboratory. PATIENTS OR PARTICIPANTS Drosophila melanogaster. INTERVENTIONS Sleep deprivation and starvation. MEASUREMENTS AND RESULTS We identified 84 genes with transcript levels that were differentially modulated by 7 h of sleep deprivation and starvation in cyc(01) mutants and were confirmed in independent samples using quantitative polymerase chain reaction. Several of these genes were predicted to be lipid metabolism genes, including bubblegum, cueball, and CG4500, which based on our data we have renamed heimdall (hll). Using lipidomics we confirmed that knockdown of hll using RNA interference significantly decreased lipid stores. Importantly, genetically modifying bubblegum, cueball, or hll resulted in sleep rebound alterations following sleep deprivation compared to genetic background controls. CONCLUSIONS We have identified a set of genes that may confer resilience/vulnerability to sleep deprivation and demonstrate that genes involved in lipid metabolism modulate sleep homeostasis.

Differential activation of immune factors in neurons and glia contribute to individual differences in resilience/vulnerability to sleep disruption

Individuals frequently find themselves confronted with a variety of challenges that threaten their wellbeing. While some individuals face these challenges efficiently and thrive (resilient) others are unable to cope and may suffer persistent consequences (vulnerable). Resilience/vulnerability to sleep disruption may contribute to the vulnerability of individuals exposed to challenging conditions. With that in mind we exploited individual differences in a flys ability to form short-term memory (STM) following 3 different types of sleep disruption to identify the underlying genes. Our analysis showed that in each category of flies examined, there are individuals that form STM in the face of sleep loss (resilient) while other individuals show dramatic declines in cognitive behavior (vulnerable). Molecular genetic studies revealed that Antimicrobial Peptides, factors important for innate immunity, were candidates for conferring resilience/vulnerability to sleep deprivation. Specifically, Metchnikowin (Mtk), drosocin (dro) and Attacin (Att) transcript levels seemed to be differentially increased by sleep deprivation in glia (Mtk), neurons (dro) or primarily in the head fat body (Att). Follow-up genetic studies confirmed that expressing Mtk in glia but not neurons, and expressing dro in neurons but not glia, disrupted memory while modulating sleep in opposite directions. These data indicate that various factors within glia or neurons can contribute to individual differences in resilience/vulnerability to sleep deprivation.