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Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 ( WFS1 ) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, we aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. We found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, we propose a role for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations.

Most organisms on the earth exhibit circadian rhythms in behavior and physiology, which are driven by endogenous clocks. Phosphorylation plays a central role in timing the clock, but how this contributes to overt rhythms is unclear. Here we conduct phosphoproteomics in conjunction with transcriptomic and proteomic profiling using fly heads. By developing a pipeline for integrating multi-omics data, we identify 789 (~17%) phosphorylation sites with circadian oscillations. We predict 27 potential circadian kinases to participate in phosphorylating these sites, including 7 previously known to function in the clock. We screen the remaining 20 kinases for effects on circadian rhythms and find an additional 3 to be involved in regulating locomotor rhythm. We re-construct a signal web that includes the 10 circadian kinases and identify GASKET as a potentially important regulator. Taken together, we uncover a circadian kinome that potentially shapes the temporal pattern of the entire circadian molecular landscapes. Phosphorylation plays an important role in the regulation of molecular circadian clocks. Here the authors utilize multi-omics data from flies to describe the circadian kinome and identify GASKET as a potentially important regulator within the circadian kinase network.

The circadian rhythm is an evolutionarily conserved mechanism with translational regulation increasingly recognized as pivotal in its modulation. In this study, we found that upstream open reading frames (uORFs) are enriched in Drosophila circadian rhythm genes, with particularly conserved uORFs present in core circadian clock genes. We demonstrate evidence that the uORFs of the core clock gene, Clock ( Clk ), rhythmically and substantially attenuate CLK protein translation in Drosophila , with pronounced suppression occurring during daylight hours. Eliminating Clk uORFs leads to increased CLK protein levels during the day and results in a shortened circadian cycle, along with a broad shift in clock gene expression rhythms. Notably, Clk uORF deletion also augments morning sleep by reducing dopaminergic activity. Beyond daily circadian adjustments, Clk uORFs play a role in modulating sleep patterns in response to seasonal daylight variations. Furthermore, the Clk uORFs act as an important regulator to shape the rhythmic expression of a vast array of genes and influence multifaceted physiological outcomes. Collectively, our research sheds light on the intricate ways uORFs dynamically adjust downstream coding sequences to acclimate to environmental shifts.

Molecular circadian clocks are interconnected via neural networks. In Drosophila, PIGMENT-DISPERSING FACTOR (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF(+) and PDF(-) circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. We demonstrate that genetic inhibition of protein kinase A (PKA) in PDF(-) clock neurons can phenocopy PDF mutants while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, we rescued per in PDF neurons of arrhythmic per⁰¹ mutants. PDF neuron rescue induced high amplitude rhythms in the clock component TIMELESS (TIM) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced TIM levels in non-PDF neurons of per⁰¹ flies. To address how PDF impacts pacemaker neuron output, we focally applied PDF to DN1p neurons and found that it acutely depolarizes and increases firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. We have provided evidence for a signaling mechanism (PKA) and a molecular target (TIM) by which PDF resets and synchronizes clocks and demonstrates an acute direct excitatory effect of PDF on target neurons to control neuronal output. The identification of TIM as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, our results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output.

Adequate sleep is essential for physical and mental health. We previously identified a missense mutation in the human DEC2 gene (BHLHE41) leading to the familial natural short sleep behavioral trait. DEC2 is a transcription factor regulating the circadian clock in mammals, although its role in sleep regulation has been unclear. Here we report that prepro-orexin, also known as hypocretin (Hcrt), gene expression is increased in the mouse model expressing the mutant hDEC2 transgene (hDEC2-P384R). Prepro-orexin encodes a precursor protein of a neuropeptide producing orexin A and B (hcrt1 and hcrt2), which is enriched in the hypothalamus and regulates maintenance of arousal. In cell culture, DEC2 suppressed prepro-orexin promoter-luc (ore-luc) expression through cis-acting E-box elements. The mutant DEC2 has less repressor activity than WT-DEC2, resulting in increased orexin expression. DEC2-binding affinity for the prepro-orexin gene promoter is decreased by the P384R mutation, likely due to weakened interaction with other transcription factors. In vivo, the decreased immobility time of the mutant transgenic mice is attenuated by an orexin receptor antagonist. Our results suggested that DEC2 regulates sleep/wake duration, at least in part, by modulating the neuropeptide hormone orexin.

Sleep, locomotor and social activities are essential animal behaviors, but their reciprocal relationships and underlying mechanisms remain poorly understood. Here, we elicit information from a cutting-edge large-language model (LLM), generative pre-trained transformer (GPT) 3.5, which interprets 10.2–13.8% of Drosophila genes known to regulate the 3 behaviors. We develop an instrument for simultaneous video tracking of multiple moving objects, and conduct a genome-wide screen. We have identified 758 fly genes that regulate sleep and activities, including mre11 which regulates sleep only in the presence of conspecifics, and NELF-B which regulates sleep regardless of whether conspecifics are present. Based on LLM-reasoning, an educated signal web is modeled for understanding of potential relationships between its components, presenting comprehensive molecular signatures that control sleep, locomotor and social activities. This LLM-aided strategy may also be helpful for addressing other complex scientific questions. The knowledge in the large language model (LLM), generative pre-trained transformer (GPT) 3.5, is elicited to facilitate the discovery of MRE11 in regulating sleep in the presence of conspecifics by a multi-object video tracking system.

Shift work is known to be associated with an increased risk of neurological and psychiatric diseases, but how it contributes to the development of these diseases remains unclear. Chronic jet lag (CJL) induced by shifting light-dark cycles repeatedly is a commonly used protocol to mimic the environmental light/dark changes encountered by shift workers. Here we subjected wildtype mice to CJL and performed positron emission tomography imaging of glucose metabolism to monitor brain activities. We also conducted RNA sequencing using prefrontal cortex and nucleus accumbens tissues from these animals, which are brain regions strongly implicated in the pathology of various neurological and psychiatric conditions. Our results reveal the alterations of brain activities and systematic reprogramming of gene expression in brain tissues under CJL, building hypothesis for how CJL increases the susceptibility to neurological and psychiatric diseases.Measurement(s)brain measurement • sleep duration • sleep quality • fragments per kilobase of exon per million mapped readsTechnology Type(s)positron emission tomography • animal activity monitoring system • RNA sequencingFactor Type(s)chronic jet lagSample Characteristic - OrganismMus musculusSample Characteristic - Environmentlaboratory environmentMachine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12924248

Proteostasis is fundamental for maintaining organismal health. However, the mechanisms underlying its dynamic regulation and how its disruptions lead to diseases are largely unclear. Here, we conduct in-depth propionylomic profiling in Drosophila , and develop a small-sample learning framework to prioritize the propionylation at lysine 17 of H2B (H2BK17pr) to be functionally important. Mutating H2BK17 which eliminates propionylation leads to elevated total protein level in vivo. Further analyses reveal that H2BK17pr modulates the expression of 14.7–16.3% of genes in the proteostasis network, and determines global protein level by regulating the expression of genes involved in the ubiquitin-proteasome system. In addition, H2BK17pr exhibits daily oscillation, mediating the influences of feeding/fasting cycles to drive rhythmic expression of proteasomal genes. Our study not only reveals a role of lysine propionylation in regulating proteostasis, but also implements a generally applicable method which can be extended to other issues with little prior knowledge. The development of a new smallsample learning framework, KprFunc, leads to the discovery of an important role for lysine propionylation in determining global protein homeostasis, mediated by a critical propionylation site on histone H2B, H2BK17pr.

The occurrence of aging is intricately associated with alterations in circadian rhythms that coincide with stem cell exhaustion. Nonetheless, the extent to which the circadian system governs skeletal aging remains inadequately understood. Here, we noticed that skeletal aging in male mice was accompanied by a decline in a core circadian protein, BMAL1, especially in bone marrow endothelial cells (ECs). Using male mice with endothelial KO of aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), we ascertained that endothelial BMAL1 in bone played a crucial role in ensuring the stability of an extracellular structural component, fibrillin-1 (FBN1), through regulation of the equilibrium between the extracellular matrix (ECM) proteases thrombospondin type 1 domain-containing protein 4 (THSD4) and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), which promote FBN1 assembly and breakdown, respectively. The decline of endothelial BMAL1 during aging prompted excessive breakdown of FBN1, leading to persistent activation of TGF-β/SMAD3 signaling and exhaustion of bone marrow mesenchymal stem cells. Meanwhile, the free TGF-β could promote osteoclast formation. Further analysis revealed that activation of ADAMTS4 in ECs lacking BMAL1 was stimulated by TGF-β/SMAD3 signaling through an ECM-positive feedback mechanism, whereas THSD4 was under direct transcriptional control by endothelial BMAL1. Our investigation has elucidated the etiology of bone aging in male mice by defining the role of ECs in upholding the equilibrium within the ECM, consequently coordinating osteogenic and osteoclastic activities and retarding skeletal aging.

Neuropathic pain causes enduring physical discomfort and emotional distress. Conventional pharmacological treatments often provide restricted relief and may result in undesirable side effects, posing a substantial clinical challenge. Peripheral and spinal redox homeostasis plays an important role in pain processing and perception. However, the roles of oxidative stress and antioxidants in pain and analgesia on the cortical region during chronic pain remains obscure. Here we focus on the ventrolateral orbital cortex (VLO), a brain region associated with pain severity and involved in pain inhibition. Using a spared nerve injury mouse model, we observed the notable reactive oxygen species (ROS)-mediated suppression of the excitability of pyramidal cells (PYR VLO ) in the VLO. Nasal application or microinjection of the natural antioxidants proanthocyanidins (PACs) to the VLO specifically increased the activity of PYR VLO and induced a significant analgesic effect. Mechanistically, PACs activate PYR VLO by inhibiting distinct potassium channels in different ways: (1) by scavenging ROS to reduce ROS-sensitive voltage-gated potassium currents and (2) by acting as a channel blocker through direct binding to the cap structure of KCNK3 to inhibit the leak potassium current ( I leak ). These results reveal the role of cortical oxidative stress in central hyperalgesia and elucidate the mechanism and potential translational significance of PACs in central analgesia. These findings suggest that the effects of PACs extend beyond their commonly assumed antioxidant or anti-inflammatory effects. Proanthocyanidins enhance pain relief in brain cortex Neuropathic pain is a challenging condition that affects the nervous system, causing abnormal sensations and heightened sensitivity. Researchers explored the potential of proanthocyanidins, natural antioxidants found in plants, to alleviate neuropathic pain. The study involves experiments on mice with nerve injuries to mimic neuropathic pain. Researchers administered proanthocyanidins to the ventrolateral orbitofrontal cortex, a key brain region for pain regulation. They measured pain responses and examined brain activity using various techniques, including electrode recordings and molecular analysis. Results showed that proanthocyanidins decreased pain sensitivity in mice by lowering oxidative stress and modulating specific potassium channels that help control nerve cell activity. This dual mechanism—antioxidant effects and potassium channel regulation—highlights proanthocyanidins as an alternative to conventional pain treatments. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.