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Although sleep appears to be broadly conserved in animals, the physiological functions of sleep remain unclear. In this study, we sought to identify a physiological defect common to a diverse group of short-sleeping Drosophila mutants, which might provide insight into the function and regulation of sleep. We found that these short-sleeping mutants share a common phenotype of sensitivity to acute oxidative stress, exhibiting shorter survival times than controls. We further showed that increasing sleep in wild-type flies using genetic or pharmacological approaches increases survival after oxidative challenge. Moreover, reducing oxidative stress in the neurons of wild-type flies by overexpression of antioxidant genes reduces the amount of sleep. Together, these results support the hypothesis that a key function of sleep is to defend against oxidative stress and also point to a reciprocal role for reactive oxygen species (ROS) in neurons in the regulation of sleep.

At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. Here, we design a forward genetic screen to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac ( inc ), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, we find that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in mono-ubiquitination. Finally, we show that Peflin, a Ca 2+ -regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca 2+ signaling and control of Cul3/Inc activity in the postsynaptic compartment. Our study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses. The authors use a forward genetic screen to discover postsynaptic factors required for homeostatic synaptic plasticity at the Drosophila neuromuscular junction. They identify insomniac and the ubiquitin ligase Cul3 , genes involved in sleep regulation, to be necessary for retrograde homeostatic signalling at this synapse.

Mutations of the Cullin-3 (Cul3) E3 ubiquitin ligase are associated with autism and schizophrenia, neurological disorders characterized by sleep disturbances and altered synaptic function. Cul3 engages dozens of adaptor proteins to recruit hundreds of substrates for ubiquitination, but the adaptors that impact sleep and synapses remain ill-defined. Here we implicate Insomniac (Inc), a conserved protein required for normal sleep and synaptic homeostasis in Drosophila , as a Cul3 adaptor. Inc binds Cul3 in vivo, and mutations within the N-terminal BTB domain of Inc that weaken Inc-Cul3 associations impair Inc activity, suggesting that Inc function requires binding to the Cul3 complex. Deletion of the conserved C-terminus of Inc does not alter Cul3 binding but abolishes Inc activity in the context of sleep and synaptic homeostasis, indicating that the Inc C-terminus has the properties of a substrate recruitment domain. Mutation of a conserved, disease-associated arginine in the Inc C-terminus also abolishes Inc function, suggesting that this residue is vital for recruiting Inc targets. Inc levels are negatively regulated by Cul3 in neurons, consistent with Inc degradation by autocatalytic ubiquitination, a hallmark of Cullin adaptors. These findings link Inc and Cul3 in vivo and support the notion that Inc-Cul3 complexes are essential for normal sleep and synaptic function. Furthermore, these results indicate that dysregulation of conserved substrates of Inc-Cul3 complexes may contribute to altered sleep and synaptic function in autism and schizophrenia associated with Cul3 mutations.

Odor attraction in walking Drosophila melanogaster is commonly used to relate neural function to behavior, but the algorithms underlying attraction are unclear. Here, we develop a high-throughput assay to measure olfactory behavior in response to well-controlled sensory stimuli. We show that odor evokes two behaviors: an upwind run during odor (ON response), and a local search at odor offset (OFF response). Wind orientation requires antennal mechanoreceptors, but search is driven solely by odor. Using dynamic odor stimuli, we measure the dependence of these two behaviors on odor intensity and history. Based on these data, we develop a navigation model that recapitulates the behavior of flies in our apparatus, and generates realistic trajectories when run in a turbulent boundary layer plume. The ability to parse olfactory navigation into quantifiable elementary sensori-motor transformations provides a foundation for dissecting neural circuits that govern olfactory behavior. All kinds of animals use their sense of smell to find things. Doing this is difficult because odors in air travel as plumes, which meander downwind and break apart. Scientists are interested in learning the rules that animals use to decipher these odor signals and trace them back to their source. For example, do animals use patterns of timing in the odor, differences between smell at the two nostrils, or the direction of the wind? Scientists would also like to know how animal’s brain circuits decipher this information. Tiny fruit flies make a good model for studying the way animals detect odors because scientists have already learned a great deal about how their brains work. There are also many tools available to help scientists study the brain circuits of fruit flies. Now, Álvarez-Salvado et al. show that fruit flies use multiple senses to track odors to their source. In the experiments, fruit flies that were blind and could not fly were placed in tiny wind tunnels and their behavior in response to a smell or no smell in the tunnel was carefully documented. When the flies detected an odor, they turned to face the wind using their antennae to detect wind direction and run toward it. When flies lost track of an odor they began to search for it at the spot where they last smelled it. Next, Álvarez-Salvado et al. created a computer model that recreated the flies’ behavior and was able to find the odor source as well as real flies. The model added together these basic behaviors to successfully recreate the flies’ odor-search strategy. Other animals are often better than humans at finding odor sources. As a result, people use pigs to find truffles and dogs to find lost hikers. The computer model Álvarez-Salvado et al. developed might help design robots that can search for truffles, hikers, or landmines, without risking the lives of animals. It might also be useful for designing autonomous vehicles that must respond to many types of information in changing environments to make decisions.

Mutations in Cullin-3 ( Cul3 ), a conserved gene encoding a ubiquitin ligase, are strongly associated with autism spectrum disorder (ASD). Here, we characterize ASD-related pathologies caused by neuron-specific Cul3 knockdown in Drosophila . We confirmed that neuronal Cul3 knockdown causes short sleep, paralleling sleep disturbances in ASD. Because sleep defects and ASD are linked to metabolic dysregulation, we tested the starvation response of neuronal Cul3 knockdown flies; they starved faster and had lower triacylglyceride levels than controls, suggesting defects in metabolic homeostasis. ASD is also characterized by increased biomarkers of oxidative stress; we found that neuronal Cul3 knockdown increased sensitivity to hyperoxia, an exogenous oxidative stress. Additional hallmarks of ASD are deficits in social interactions and learning. Using a courtship suppression assay that measures social interactions and memory of prior courtship, we found that neuronal Cul3 knockdown reduced courtship and learning compared to controls. Finally, we found that neuronal Cul3 depletion alters the anatomy of the mushroom body, a brain region required for memory and sleep. Taken together, the ASD-related phenotypes of neuronal Cul3 knockdown flies establish these flies as a genetic model to study molecular and cellular mechanisms underlying ASD pathology, including metabolic and oxidative stress dysregulation and neurodevelopment.

Although many genes are known to influence sleep, when and how they impact sleep-regulatory circuits remain ill-defined. Here, we show that insomniac ( inc ), a conserved adaptor for the autism-associated Cul3 ubiquitin ligase, acts in a restricted period of neuronal development to impact sleep in adult Drosophila . The loss of inc causes structural and functional alterations within the mushroom body (MB), a center for sensory integration, associative learning, and sleep regulation. In inc mutants, MB neurons are produced in excess, develop anatomical defects that impede circuit assembly, and are unable to promote sleep when activated in adulthood. Our findings link neurogenesis and postmitotic development of sleep-regulatory neurons to their adult function and suggest that developmental perturbations of circuits that couple sensory inputs and sleep may underlie sleep dysfunction in neurodevelopmental disorders.

Drosophila melanogaster is a powerful model organism for dissecting the molecular mechanisms that regulate sleep, and numerous studies in the fly have identified genes that impact sleep–wake cycles. Conditional genetic analysis is essential to distinguish the mechanisms by which these genes impact sleep: some genes might exert their effects developmentally, for instance by directing the assembly of neuronal circuits that regulate sleep; other genes may regulate sleep in adulthood; and yet other genes might influence sleep by both developmental and adult mechanisms. Here we have assessed two ligand-inducible expression systems, Geneswitch and the Q-system, for conditional and neuronally restricted manipulations of sleep in Drosophila. While adult-specific induction of a neuronally expressed Geneswitch transgene (elav-GS) is compatible with studies of sleep as shown previously, developmental induction of elav-GS strongly and nonspecifically perturbs sleep in adults. The alterations of sleep in elav-GS animals occur at low doses of Geneswitch agonist and in the presence of transgenes unrelated to sleep, such as UAS-CD8-GFP. Furthermore, developmental elav-GS induction is toxic and reduces brood size, indicating multiple adverse effects of neuronal Geneswitch activation. In contrast, the transgenes and ligand of the Q-system do not significantly impact sleep–wake cycles when used for constitutive, developmental, or adult-specific neuronal induction. The nonspecific effects of developmental elav-GS activation on sleep indicate that such manipulations require cautious interpretation, and suggest that the Q-system or other strategies may be more suitable for conditional genetic analysis of sleep and other behaviors in Drosophila.

Sleep is an ancient animal behavior that is regulated similarly in species ranging from flies to humans. Various genes that regulate sleep have been identified in invertebrates, but whether the functions of these genes are conserved in mammals remains poorly explored. Drosophila insomniac (inc) mutants exhibit severely shortened and fragmented sleep. Inc protein physically associates with the Cullin-3 (Cul3) ubiquitin ligase, and neuronal depletion of Inc or Cul3 strongly curtails sleep, suggesting that Inc is a Cul3 adaptor that directs the ubiquitination of neuronal substrates that impact sleep. Three proteins similar to Inc exist in vertebrates-KCTD2, KCTD5, and KCTD17-but are uncharacterized within the nervous system and their functional conservation with Inc has not been addressed. Here we show that Inc and its mouse orthologs exhibit striking biochemical and functional interchangeability within Cul3 complexes. Remarkably, KCTD2 and KCTD5 restore sleep to inc mutants, indicating that they can substitute for Inc in vivo and engage its neuronal targets relevant to sleep. Inc and its orthologs localize similarly within fly and mammalian neurons and can traffic to synapses, suggesting that their substrates may include synaptic proteins. Consistent with such a mechanism, inc mutants exhibit defects in synaptic structure and physiology, indicating that Inc is essential for both sleep and synaptic function. Our findings reveal that molecular functions of Inc are conserved through ~600 million years of evolution and support the hypothesis that Inc and its orthologs participate in an evolutionarily conserved ubiquitination pathway that links synaptic function and sleep regulation.

In female mammals, up-regulation of Xist triggers X-chromosome inactivation in cis. Up-regulation is inhibited by sequences 3′ to Xist contained within the antisense locus, Tsix. Inhibition could depend on transcription of Tsix and/or on DNA elements therein. Here we test the role of Tsix transcription by augmenting the duration and strength of Tsix expression. We find that Tsix hypertranscription is sufficient to block Xist RNA accumulation in a cis-limited manner. We propose that Tsix transcription is necessary to restrict Xist activity on the future active X and, conversely, that Tsix repression is required for Xist RNA accumulation on the future inactive X. We also find that Tsix hypertranscription does not affect X-chromosome choice. Thus, choice is mediated by elements within Tsix that are independent of promoter activity.

AimTo systematically review clinical presentation, diagnosis, and therapy of achalasia, focusing on recent developments in high-resolution esophageal manometry (HREM) for diagnosis and peroral endoscopic myotomy (POEM) for therapy.MethodsSystematic review of achalasia using computerized literature search via PubMed and Ovid of articles published since 2005 with keywords (“achalasia”) AND (“high resolution” or “HREM” or “peroral endoscopic myotomy” or “POEM”). Two authors independently performed literature searches and incorporated articles into this review by consensus according to prospectively determined criteria.ResultsAchalasia is an uncommon esophageal motility disorder, usually manifested by dysphagia to solids and liquids, and sometimes manifested by chest pain, regurgitation, and weight loss. Symptoms often suggest more common disorders, such as gastroesophageal reflux disease (GERD), thus often delaying diagnosis. Achalasia is a predominantly idiopathic chronic disease. Diagnosis is typically suggested by barium swallow showing esophageal dilation; absent distal esophageal peristalsis; smoothly tapered narrowing (“bird’s beak”) at esophagogastric junction; and delayed passage of contrast into stomach. Diagnostic findings at high-resolution esophageal manometry (HREM) include: distal esophageal aperistalsis and integrated relaxation pressure (trough LES pressure during 4 s) > 15 mmHg. Achalasia is classified by HREM into: type 1 classic; type 2 compartmentalized high pressure in esophageal body, and type 3 spastic. This classification impacts therapeutic decisions. Esophagogastroduodenoscopy is required before therapy to assess esophagus and esophagogastric junction and to exclude distal esophageal malignancy. POEM is a revolutionizing achalasia therapy. POEM creates a myotomy via interventional endoscopy. Numerous studies demonstrate that POEM produces comparable, if not superior, results compared to standard laparoscopic Heller myotomy (LHM), as determined by LES pressure, dysphagia frequency, Eckardt score, hospital length of stay, therapy durability, and incidence of GERD. Other therapies, including botulinum toxin injection and pneumatic dilation, have moderately less efficacy and much less durability than POEM.ConclusionThis comprehensive review suggests that POEM is equivalent or perhaps superior to LHM for achalasia in terms of cost efficiency, hospital length of stay, and relief of dysphagia, with comparable side effects. The data are, however, not conclusive due to sparse long-term follow-up and lack of randomized comparative clinical trials. POEM therapy is currently limited by a shortage of trained endoscopists.