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insomniac links the development and function of a sleep regulatory circuit

Li, Qiuling; Jang, Hyunsoo; Lim, Kayla Y; Lessing, Alexie; Stavropoulos, Nicholas
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, a center for sensory integration, associative learning, and sleep regulation. In inc mutants, mushroom body 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.
PMID: 34908527
ISSN: 2050-084x
CID: 5109722

Cul3 and insomniac are required for rapid ubiquitination of postsynaptic targets and retrograde homeostatic signaling

Kikuma, Koto; Li, Xiling; Perry, Sarah; Li, Qiuling; Goel, Pragya; Chen, Catherine; Kim, Daniel; Stavropoulos, Nicholas; Dickman, Dion
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 Ca2+-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca2+ 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.
PMID: 31278365
ISSN: 2041-1723
CID: 3968422

Elementary sensory-motor transformations underlying olfactory navigation in walking fruit-flies

Álvarez-Salvado, Efrén; Licata, Angela M; Connor, Erin G; McHugh, Margaret K; King, Benjamin Mn; Stavropoulos, Nicholas; Victor, Jonathan D; Crimaldi, John P; Nagel, Katherine I
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.
PMCID:6103744
PMID: 30129438
ISSN: 2050-084x
CID: 3246092

A bidirectional relationship between sleep and oxidative stress in Drosophila

Hill, Vanessa M; O'Connor, Reed M; Sissoko, Gunter B; Irobunda, Ifeoma S; Leong, Stephen; Canman, Julie C; Stavropoulos, Nicholas; Shirasu-Hiza, Mimi
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.
PMCID:6042693
PMID: 30001323
ISSN: 1545-7885
CID: 3192292

Conserved properties of Drosophila insomniac link sleep regulation and synaptic function

Li, Qiuling; Kellner, David A; Hatch, Hayden A M; Yumita, Tomohiro; Sanchez, Sandrine; Machold, Robert P; Frank, C Andrew; Stavropoulos, Nicholas
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.
PMCID:5469494
PMID: 28558011
ISSN: 1553-7404
CID: 2582082

Evaluation of Ligand-Inducible Expression Systems for Conditional Neuronal Manipulations of Sleep in Drosophila

Li, Qiuling; Stavropoulos, Nicholas
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.
PMCID:5068954
PMID: 27558667
ISSN: 2160-1836
CID: 2221572

insomniac and Cullin-3 regulate sleep and wakefulness in Drosophila

Stavropoulos, Nicholas; Young, Michael W
In a forward genetic screen in Drosophila, we have isolated insomniac, a mutant that severely reduces the duration and consolidation of sleep. Anatomically restricted genetic manipulations indicate that insomniac functions within neurons to regulate sleep. insomniac expression does not oscillate in a circadian manner, and conversely, the circadian clock is intact in insomniac mutants, suggesting that insomniac regulates sleep by pathways distinct from the circadian clock. The protein encoded by insomniac is a member of the BTB/POZ superfamily, which includes many proteins that function as adaptors for the Cullin-3 (Cul3) ubiquitin ligase complex. We show that Insomniac can physically associate with Cul3, and that reduction of Cul3 activity in neurons recapitulates the insomniac phenotype. The extensive evolutionary conservation of insomniac and Cul3 suggests that protein degradation pathways may have a general role in governing the sleep and wakefulness of animals.
PMCID:3244879
PMID: 22196332
ISSN: 0896-6273
CID: 176024

Identification of developmentally specific enhancers for Tsix in the regulation of X chromosome inactivation

Stavropoulos, Nicholas; Rowntree, Rebecca K; Lee, Jeannie T
X chromosome inactivation silences one of two X chromosomes in the mammalian female cell and is controlled by a binary switch that involves interactions between Xist and Tsix, a sense-antisense pair of noncoding genes. On the future active X chromosome, Tsix expression suppresses Xist upregulation, while on the future inactive X chromosome, Tsix repression is required for Xist-mediated chromosome silencing. Thus, understanding the binary switch mechanism depends on ascertaining how Tsix expression is regulated. Here we have taken an unbiased approach toward identifying Tsix regulatory elements within the X chromosome inactivation center. First, we defined the major Tsix promoter and found that it cannot fully recapitulate the developmental dynamics of Tsix expression, indicating a requirement for additional regulatory elements. We then delineated two enhancers, one classical enhancer mapping upstream of Tsix and a bipartite enhancer that flanks the major Tsix promoter. These experiments revealed the intergenic transcription element Xite as an enhancer of Tsix and the repeat element DXPas34 as a component of the bipartite enhancer. Each enhancer contains DNase I-hypersensitive sites and appears to confer developmental specificity to Tsix expression. Characterization of these enhancers will facilitate the identification of trans-acting regulatory factors for X chromosome counting and choice.
PMCID:1061649
PMID: 15767680
ISSN: 0270-7306
CID: 176025

A functional role for Tsix transcription in blocking Xist RNA accumulation but not in X-chromosome choice

Stavropoulos, N; Lu, N; Lee, J T
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.
PMCID:56944
PMID: 11481444
ISSN: 0027-8424
CID: 179048

Further examination of the Xist promoter-switch hypothesis in X inactivation: evidence against the existence and function of a P(0) promoter

Warshawsky, D; Stavropoulos, N; Lee, J T
The onset of X inactivation coincides with accumulation of Xist RNA along the future inactive X chromosome. A recent hypothesis proposed that accumulation is initiated by a promoter switch within Xist. In this hypothesis, an upstream promoter (P(0)) produces an unstable transcript, while the known downstream promoter (P(1)) produces a stable RNA. To test this hypothesis, we examined expression and half-life of Xist RNA produced from an Xist transgene lacking P(0) but retaining P(1). We confirm the previous finding that P(0) is dispensable for Xist expression in undifferentiated cells and that P(1) can be used in both undifferentiated and differentiated cells. Herein, we show that Xist RNA initiated at P(1) is unstable and does not accumulate. Further analysis indicates that the transcriptional boundary at P(0) does not represent the 5' end of a distinct Xist isoform. Instead, P(0) is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribosomal protein S12 gene Rps12. Using strand-specific techniques, we find that transcription upstream of P(1) originates from the DNA strand opposite Xist and represents the 3' end of the antisense Tsix RNA. Thus, these data do not support the existence of a P(0) promoter and suggest that mechanisms other than switching of functionally distinct promoters control the up-regulation of Xist.
PMCID:24452
PMID: 10588721
ISSN: 0027-8424
CID: 179049