Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis.

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.

Nipped-A regulates the Drosophila circadian clock via histone deubiquitination.

Psychiatric diseases are often accompanied by circadian disruptions, but the molecular underpinnings remain largely unclear. To address this, we screened genes that have been previously reported to be associated with psychiatric diseases and found that TRRAP, a gene associated with schizophrenia, is involved in circadian rhythm regulation. Knocking down Nipped-A, the Drosophila homolog of human TRRAP, leads to lengthened period of locomotor rhythms in flies. Molecular analysis demonstrates that NIPPED-A sets the pace of the clock by increasing the mRNA and protein levels of core clock genes timeless (tim) and Par domain protein 1ε (Pdp1ε). Furthermore, we found that NIPPED-A promotes the transcription of tim and Pdp1ε possibly by facilitating deubiquitination of histone H2B via the deubiquitination module of the transcription co-activator Spt-Ada-Gcn5 acetyltransferase complex. Taken together, these findings reveal a novel role for NIPPED-A in epigenetic regulation of the clock.

A Novel SPAST Mutation Results in Spastin Accumulation and Defects in Microtubule Dynamics.

BACKGROUND: Haploinsufficiency is widely accepted as the pathogenic mechanism of spastic paraplegia type 4 (SPG4). However, there are some cases that cannot be explained by reduced function of the spastin protein encoded by SPAST. OBJECTIVES: To identify the causative gene of autosomal dominant hereditary spastic paraplegia in three large Chinese families and explore the pathological mechanism of a spastin variant. METHODS: Three large Chinese hereditary spastic paraplegia families with a total of 247 individuals (67 patients) were investigated, of whom 59 members were recruited to the study. Genetic testing was performed to identify the causative gene. Western blotting and immunofluorescence were used to analyze the effects of the mutant proteins in vitro. RESULTS: In the three hereditary spastic paraplegia families, of whom three index cases were misdiagnosed as other types of neurological diseases, a novel c.985dupA (p.Met329Asnfs*3) variant in SPAST was identified and was shown to cosegregate with the phenotype in the three families. The c.985dupA mutation produced two truncated mutants (mutant M1 and M87 isoforms) that accumulated to a higher level than their wild-type counterparts. Furthermore, the mutant M1 isoform heavily decorated the microtubules and rendered them resistant to depolymerization. In contrast, the mutant M87 isoform was diffusely localized in both the nucleus and the cytoplasm, could not decorate microtubules, and was not able to promote microtubule disassembly. CONCLUSIONS: SPAST mutations leading to premature stop codons do not always act through haploinsufficiency. The truncated spastin may damage the corticospinal tracts through an isoform-specific toxic effect.

DEC2 modulates orexin expression and regulates sleep.

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.

The molecular genetics of human sleep.

It has been known for many years that genetic influences account for some of the individual differences in human sleep parameters, but the underlying molecular mechanisms remain unclear. With major advances of molecular biology and the recognition of heritable sleep behaviors in humans over the past 30 years, a number of genetic variants have been identified to be associated with human sleep timing, duration and quality, both in healthy individuals and under pathological conditions. Some of these variants were further validated and characterized in animal models, shedding light on the mechanism of how these variants likely alter sleep in humans, which may provide new insights into developing more effective treatments to improve human sleep.

A single factor dominates the behavior of rhythmic genes in mouse organs.

BACKGROUND: Circadian rhythm, regulated by both internal and external environment of the body, is a multi-scale biological oscillator of great complexity. On the molecular level, thousands of genes exhibit rhythmic transcription, which is both organ- and species-specific, but it remains a mystery whether some common factors could potentially explain their rhythmicity in different organs. In this study we address this question by analyzing the transcriptome data in 12 mouse organs to determine such major impacting factors. RESULTS: We found a strong positive correlation between the transcriptional level and rhythmic amplitude of circadian rhythmic genes in mouse organs. Further, transcriptional level could explain over 70% of the variation in amplitude. In addition, the functionality and tissue specificity were not strong predictors of amplitude, and the expression level of rhythmic genes was linked to the energy consumption associated with transcription. CONCLUSION: Expression level is a single major factor impacts the behavior of rhythmic genes in mouse organs. This single determinant implicates the importance of rhythmic expression itself on the design of the transcriptional system. So, rhythmic regulation of highly expressed genes can effectively reduce the energetic cost of transcription, facilitating the long-term adaptive evolution of the entire genetic system.

CGDB: a database of circadian genes in eukaryotes.

We report a database of circadian genes in eukaryotes (CGDB, http://cgdb.biocuckoo.org), containing ∼73 000 circadian-related genes in 68 animals, 39 plants and 41 fungi. Circadian rhythm is ∼24 h rhythm in behavioral and physiological processes that exists in almost all organisms on the earth. Defects in the circadian system are highly associated with a number of diseases such as cancers. Although several databases have been established for rhythmically expressed genes, a comprehensive database of cycling genes across phyla is still lacking. From the literature, we collected 1382 genes of which transcript level oscillations were validated using methods such as RT-PCR, northern blot and in situ hybridization. Given that many genes exhibit different oscillatory patterns in different tissues/cells within an organism, we have included information regarding the phase and amplitude of the oscillation, as well as the tissue/cells in which the oscillation was identified. Using these well characterized cycling genes, we have then conducted an orthologous search and identified ∼45 000 potential cycling genes from 148 eukaryotes. Given that significant effort has been devoted to identifying cycling genes by transcriptome profiling, we have also incorporated these results, a total of over 26 000 genes, into our database.

A PERIOD3 variant causes a circadian phenotype and is associated with a seasonal mood trait.

In humans, the connection between sleep and mood has long been recognized, although direct molecular evidence is lacking. We identified two rare variants in the circadian clock gene PERIOD3 (PER3-P415A/H417R) in humans with familial advanced sleep phase accompanied by higher Beck Depression Inventory and seasonality scores. hPER3-P415A/H417R transgenic mice showed an altered circadian period under constant light and exhibited phase shifts of the sleep-wake cycle in a short light period (photoperiod) paradigm. Molecular characterization revealed that the rare variants destabilized PER3 and failed to stabilize PERIOD1/2 proteins, which play critical roles in circadian timing. Although hPER3-P415A/H417R-Tg mice showed a mild depression-like phenotype, Per3 knockout mice demonstrated consistent depression-like behavior, particularly when studied under a short photoperiod, supporting a possible role for PER3 in mood regulation. These findings suggest that PER3 may be a nexus for sleep and mood regulation while fine-tuning these processes to adapt to seasonal changes.

A PRRT2 variant in a Chinese family with paroxysmal kinesigenic dyskinesia and benign familial infantile seizures results in loss of interaction with STX1B.

OBJECTIVE: To identify the causative gene of autosomal dominant paroxysmal kinesigenic dyskinesia and benign familial infantile seizures (PKD/BFIS) in a large Chinese family and explore the potential pathogenic mechanism of a PRRT2 (proline-rich transmembrane protein 2) variant. METHODS: Genetic testing was performed via whole exome sequencing. Western blotting and immunofluorescence were used to analyze the protein expression level and subcellular localization of the PRRT2 mutant in HeLa cells and N2A cells. Coimmunoprecipitation was conducted to investigate the interaction of the PRRT2 mutant with syntaxin 1B (STX1B). RESULTS: In a large Chinese family with autosomal dominant PKD/BFIS showing wide phenotypic heterogeneity, including patients suffering from PKD, BFIS, or epilepsy and asymptomatic variant carriers, a c.621dupA variant in PRRT2 was identified in the proband and was shown to cosegregate with the phenotype in this family. This variant results in premature termination at codon 224, producing a truncated protein (p.Ser208Ilefs*17) in which the two conserved hydrophobic segments and the cytoplasmic loop are missing. Both the expression and subcellular localization of PRRT2 are strongly affected by the c.621dupA variant. In addition, we found that PRRT2 directly interacts with STX1B, a SNARE protein critical for neurotransmitter release, whereas the truncated variant p.Ser208Ilefs*17 lacking the helix-loop-helix domain fails to bind to STX1B. SIGNIFICANCE: Our findings identified a PRRT2 variant in a family with PKD/BFIS and confirmed STX1B as a new binding partner of PRRT2, which suggested that the loss of the interaction between PRRT2 and STX1B may contribute to the pathogenesis of PKD/BFIS.

Dual PDF signaling pathways reset clocks via TIMELESS and acutely excite target neurons to control circadian behavior.

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 per°¹ 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 per°¹ 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.

MicroRNA-23a promotes myelination in the central nervous system.

Demyelinating disorders including leukodystrophies are devastating conditions that are still in need of better understanding, and both oligodendrocyte differentiation and myelin synthesis pathways are potential avenues for developing treatment. Overexpression of lamin B1 leads to leukodystrophy characterized by demyelination of the central nervous system, and microRNA-23 (miR-23) was found to suppress lamin B1 and enhance oligodendrocyte differentiation in vitro. Here, we demonstrated that miR-23a-overexpressing mice have increased myelin thickness, providing in vivo evidence that miR-23a enhances both oligodendrocyte differentiation and myelin synthesis. Using this mouse model, we explored possible miR-23a targets and revealed that the phosphatase and tensin homologue/phosphatidylinositol trisphosphate kinase/Akt/mammalian target of rapamycin pathway is modulated by miR-23a. Additionally, a long noncoding RNA, 2700046G09Rik, was identified as a miR-23a target and modulates phosphatase and tensin homologue itself in a miR-23a-dependent manner. The data presented here imply a unique role for miR-23a in the coordination of proteins and noncoding RNAs in generating and maintaining healthy myelin.

PKCγ participates in food entrainment by regulating BMAL1.

Temporally restricted feeding (RF) can phase reset the circadian clocks in numerous tissues in mammals, contributing to altered timing of behavioral and physiological rhythms. However, little is known regarding the underlying molecular mechanism. Here we demonstrate a role for the gamma isotype of protein kinase C (PKCγ) in food-mediated entrainment of behavior and the molecular clock. We found that daytime RF reduced late-night activity in wild-type mice but not mice homozygous for a null mutation of PKCγ (PKCγ(-/-)). Molecular analysis revealed that PKCγ exhibited RF-induced changes in activation patterns in the cerebral cortex and that RF failed to substantially phase shift the oscillation of clock gene transcripts in the absence of PKCγ. PKCγ exerts effects on the clock, at least in part, by stabilizing the core clock component brain and muscle aryl hydrocarbon receptor nuclear translocator like 1 (BMAL1) and reducing its ubiquitylation in a deubiquitination-dependent manner. Taken together, these results suggest that PKCγ plays a role in food entrainment by regulating BMAL1 stability.

p75 neurotrophin receptor is a clock gene that regulates oscillatory components of circadian and metabolic networks.

The p75 neurotrophin receptor (p75(NTR)) is a member of the tumor necrosis factor receptor superfamily with a widespread pattern of expression in tissues such as the brain, liver, lung, and muscle. The mechanisms that regulate p75(NTR) transcription in the nervous system and its expression in other tissues remain largely unknown. Here we show that p75(NTR) is an oscillating gene regulated by the helix-loop-helix transcription factors CLOCK and BMAL1. The p75(NTR) promoter contains evolutionarily conserved noncanonical E-box enhancers. Deletion mutagenesis of the p75(NTR)-luciferase reporter identified the -1039 conserved E-box necessary for the regulation of p75(NTR) by CLOCK and BMAL1. Accordingly, gel-shift assays confirmed the binding of CLOCK and BMAL1 to the p75(NTR-)1039 E-box. Studies in mice revealed that p75(NTR) transcription oscillates during dark and light cycles not only in the suprachiasmatic nucleus (SCN), but also in peripheral tissues including the liver. Oscillation of p75(NTR) is disrupted in Clock-deficient and mutant mice, is E-box dependent, and is in phase with clock genes, such as Per1 and Per2. Intriguingly, p75(NTR) is required for circadian clock oscillation, since loss of p75(NTR) alters the circadian oscillation of clock genes in the SCN, liver, and fibroblasts. Consistent with this, Per2::Luc/p75(NTR-/-) liver explants showed reduced circadian oscillation amplitude compared with those of Per2::Luc/p75(NTR+/+). Moreover, deletion of p75(NTR) also alters the circadian oscillation of glucose and lipid homeostasis genes. Overall, our findings reveal that the transcriptional activation of p75(NTR) is under circadian regulation in the nervous system and peripheral tissues, and plays an important role in the maintenance of clock and metabolic gene oscillation.

Time of exercise differentially impacts bone growth in mice.

Although physical training has been shown to improve bone mass, the time of day to exercise for optimal bone growth remains uncertain. Here we show that engaging in physical activity during the early active phase, as opposed to the subsequent active or rest phase, results in a more substantial increase in bone length of male and female mice. Transcriptomic and metabolomic methodologies identify that exercise during the early active phase significantly upregulates genes associated with bone development and metabolism. Notably, oxidative phosphorylation-related genes show a rhythmic expression in the chondrification centre, with a peak at the early active phase, when more rhythmic genes in bone metabolism are expressed and bone growth is synergistically promoted by affecting oxidative phosphorylation, which is confirmed by subsequent pharmacological investigations. Finally, we construct a signalling network to predict the impact of exercise on bone growth. Collectively, our research sheds light on the intricacies of human exercise physiology, offering valuable implications for interventions.

Large-language models facilitate discovery of the molecular signatures regulating sleep and activity.

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.

Phosphorylation Promotes the Accumulation of PERIOD Protein Foci.

Circadian clock drives the 24-h rhythm in our behavior and physiology. The molecular clock consists of a series of transcriptional/translational feedback loops operated by a number of clock genes. A very recent study reported that the clock protein PERIOD (PER) is organized into discrete foci at the nuclear envelope in fly circadian neurons, which is believed to be important for controlling the subcellular localization of clock genes. Loss of inner nuclear membrane protein lamin B receptor (LBR) leads to disruption of these foci, but how they are regulated is yet unknown. Here, we found that PER foci are likely phase-separated condensates, the formation of which is mediated by intrinsically disordered region in PER. Phosphorylation promotes the accumulation of these foci. Protein phosphatase 2A, which is known to dephosphorylate PER, hampers the accumulation of the foci. On the other hand, the circadian kinase DOUBLETIME (DBT) which phosphorylates PER enhances the accumulation of the foci. LBR likely facilitates PER foci accumulation by destabilizing the catalytic subunit of protein phosphatase 2A, MICROTUBULE STAR (MTS). In conclusion, here, we demonstrate a key role for phosphorylation in promoting the accumulation of PER foci, while LBR modulates this process by impinging on the circadian phosphatase MTS.

Time-restricted feeding is an intervention against excessive dark-phase sleepiness induced by obesogenic diet.

High-fat diet (HFD)-induced obesity is a growing epidemic and major health concern. While excessive daytime sleepiness (EDS) is a common symptom of HFD-induced obesity, preliminary findings suggest that reduced wakefulness could be improved with time-restricted feeding (TRF). At present, however, the underlying neural mechanisms remain largely unknown. The paraventricular thalamic nucleus (PVT) plays a role in maintaining wakefulness. We found that chronic HFD impaired the activity of PVT neurons. Notably, inactivation of the PVT was sufficient to reduce and fragment wakefulness during the active phase in lean mice, similar to the sleep-wake alterations observed in obese mice with HFD-induced obesity. On the other hand, enhancing PVT neuronal activity consolidated wakefulness in mice with HFD-induced obesity. We observed that the fragmented wakefulness could be eliminated and reversed by TRF. Furthermore, TRF prevented the HFD-induced disruptions on synaptic transmission in the PVT, in a feeding duration-dependent manner. Collectively, our findings demonstrate that ad libitum access to a HFD results in inactivation of the PVT, which is critical to impaired nocturnal wakefulness and increased sleep, while TRF can prevent and reverse diet-induced PVT dysfunction and excessive sleepiness. We establish a link between TRF and neural activity, through which TRF can potentially serve as a lifestyle intervention against diet/obesity-related EDS.

Gut AstA mediates sleep deprivation-induced energy wasting in Drosophila.

Severe sleep deprivation (SD) has been highly associated with systemic energy wasting, such as lipid loss and glycogen depletion. Despite immune dysregulation and neurotoxicity observed in SD animals, whether and how the gut-secreted hormones participate in SD-induced disruption of energy homeostasis remains largely unknown. Using Drosophila as a conserved model organism, we characterize that production of intestinal Allatostatin A (AstA), a major gut-peptide hormone, is robustly increased in adult flies bearing severe SD. Interestingly, the removal of AstA production in the gut using specific drivers significantly improves lipid loss and glycogen depletion in SD flies without affecting sleep homeostasis. We reveal the molecular mechanisms whereby gut AstA promotes the release of an adipokinetic hormone (Akh), an insulin counter-regulatory hormone functionally equivalent to mammalian glucagon, to mobilize systemic energy reserves by remotely targeting its receptor AstA-R2 in Akh-producing cells. Similar regulation of glucagon secretion and energy wasting by AstA/galanin is also observed in SD mice. Further, integrating single-cell RNA sequencing and genetic validation, we uncover that severe SD results in ROS accumulation in the gut to augment AstA production via TrpA1. Altogether, our results demonstrate the essential roles of the gut-peptide hormone AstA in mediating SD-associated energy wasting.

Proteome and Ubiquitylome Analyses of Maize Endoplasmic Reticulum under Heat Stress.

High temperatures severely affect plant growth and pose a threat to global crop production. Heat causes the accumulation of misfolded proteins in the endoplasmic reticulum(ER), as well as triggering the heat-shock response (HSR) in the cytosol and the unfolded protein response (UPR) in the ER. Excessive misfolded proteins undergo further degradation through ER-associated degradation (ERAD). Although much research on the plant heat stress response has been conducted, the regulation of ER-localized proteins has not been well-studied thus far. We isolated the microsome fraction from heat-treated and untreated maize seedlings and performed proteome and ubiquitylome analyses. Of the 8306 total proteins detected in the proteomics analysis, 1675 proteins were significantly up-regulated and 708 proteins were significantly down-regulated. Global ubiquitination analysis revealed 1780 proteins with at least one ubiquitination site. Motif analysis revealed that alanine and glycine are the preferred amino acids upstream and downstream of ubiquitinated lysine sites. ERAD components were found to be hyper-ubiquitinated after heat treatment, implying the feedback regulation of ERAD activity through protein degradation.

Small-sample learning reveals propionylation in determining global protein homeostasis.

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.