The NFκB Dif is required for behavioral and molecular correlates of sleep homeostasis in Drosophila.

The nuclear factor binding the κ light chain in B-cells (NFκB) is involved in a wide range of cellular processes including development, growth, innate immunity, and sleep. However, genetic studies of the role of specific NFκB transcription factors in sleep have been limited. Drosophila fruit flies carry three genes encoding NFκB transcription factors, Dorsal, Dorsal Immunity Factor (Dif), and Relish. We previously found that loss of the Relish gene from fat body suppressed daily nighttime sleep, and abolished infection-induced sleep. Here we show that Dif regulates daily sleep and recovery sleep following prolonged wakefulness. Mutants of Dif showed reduced daily sleep and suppressed recovery in response to sleep deprivation. Pan-neuronal knockdown of Dif strongly suppressed daily sleep, indicating that in contrast to Relish, Dif functions from the central nervous system to regulate sleep. Based on the unique expression pattern of a Dif- GAL4 driver, we hypothesized that its effects on sleep were mediated by the pars intercerebralis (PI). While RNAi knock-down of Dif in the PI reduced daily sleep, it had no effect on the recovery response to sleep deprivation. However, recovery sleep was suppressed when RNAi knock-down of Dif was distributed across a wider range of neurons. Induction of the nemuri (nur) antimicrobial peptide by sleep deprivation was reduced in Dif mutants and pan-neuronal over-expression of nur also suppressed the Dif mutant phenotype by significantly increasing sleep and reducing nighttime arousability. Together, these findings indicate that Dif functions from brain to target nemuri and to promote deep sleep.

A neuron-glia lipid metabolic cycle couples daily sleep to mitochondrial homeostasis.

Sleep is thought to be restorative to brain energy homeostasis, but it is not clear how this is achieved. We show here that Drosophila glia exhibit a daily cycle of glial mitochondrial oxidation and lipid accumulation that is dependent on prior wake and requires the Drosophila APOE orthologs NLaz and GLaz, which mediate neuron-glia lipid transfer. In turn, a full night of sleep is required for glial lipid clearance, mitochondrial oxidative recovery and maximal neuronal mitophagy. Knockdown of neuronal NLaz causes oxidative stress to accumulate in neurons, and the neuronal mitochondrial integrity protein, Drp1, is required for daily glial lipid accumulation. These data suggest that neurons avoid accumulation of oxidative mitochondrial damage during wake by using mitophagy and passing damage to glia in the form of lipids. We propose that a mitochondrial lipid metabolic cycle between neurons and glia reflects a fundamental function of sleep relevant for brain energy homeostasis.

Cell state dependent effects of Bmal1 on melanoma immunity and tumorigenicity.

The circadian clock regulator Bmal1 modulates tumorigenesis, but its reported effects are inconsistent. Here, we show that Bmal1 has a context-dependent role in mouse melanoma tumor growth. Loss of Bmal1 in YUMM2.1 or B16-F10 melanoma cells eliminates clock function and diminishes hypoxic gene expression and tumorigenesis, which could be rescued by ectopic expression of HIF1α in YUMM2.1 cells. By contrast, over-expressed wild-type or a transcriptionally inactive mutant Bmal1 non-canonically sequester myosin heavy chain 9 (Myh9) to increase MRTF-SRF activity and AP-1 transcriptional signature, and shift YUMM2.1 cells from a Sox10high to a Sox9high immune resistant, mesenchymal cell state that is found in human melanomas. Our work describes a link between Bmal1, Myh9, mouse melanoma cell plasticity, and tumor immunity. This connection may underlie cancer therapeutic resistance and underpin the link between the circadian clock, MRTF-SRF and the cytoskeleton.

Age-regulated cycling metabolites are relevant for behavior.

Circadian cycles of sleep:wake and gene expression change with age in all organisms examined. Metabolism is also under robust circadian regulation, but little is known about how metabolic cycles change with age and whether these contribute to the regulation of behavioral cycles. To address this gap, we compared cycling of metabolites in young and old Drosophila and found major age-related variations. A significant model separated the young metabolic profiles by circadian timepoint, but could not be defined for the old metabolic profiles due to the greater variation in this dataset. Of the 159 metabolites measured in fly heads, we found 17 that cycle by JTK analysis in young flies and 17 in aged. Only four metabolites overlapped in the two groups, suggesting that cycling metabolites are distinct in young and old animals. Among our top cyclers exclusive to young flies were components of the pentose phosphate pathway (PPP). As the PPP is important for buffering reactive oxygen species, and overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key component of the PPP, was previously shown to extend lifespan in Drosophila, we asked if this manipulation also affects sleep:wake cycles. We found that overexpression in circadian clock neurons decreases sleep in association with an increase in cellular calcium and mitochondrial oxidation, suggesting that altering PPP activity affects neuronal activity. Our findings elucidate the importance of metabolic regulation in maintaining patterns of neural activity, and thereby sleep:wake cycles.

Glucose Challenge Uncovers Temporal Fungibility of Metabolic Homeostasis Throughout the Day.

Rhythmicity is a central feature of behavioral and biological processes including metabolism, however, the mechanisms of metabolite cycling are poorly understood. A robust oscillation in a network of key metabolite pathways downstream of glucose is described in humans, then these pathways mechanistically probed through purpose-built 13C6-glucose isotope tracing in Drosophila every 4h. A temporal peak in biosynthesis was noted by broad labelling of pathways downstream of glucose in wild-type flies shortly following lights on. Krebs cycle labelling was generally increased in a hyperactive mutant (fumin) along with glycolysis labelling primarily observed at dawn. Surprisingly, neither underlying feeding rhythms nor the presence of food explains the rhythmicity of glucose processing across genotypes. These results are consistent with clinical data demonstrating detrimental effects of mis-timed energy intake. This approach provides a window into the dynamic range of metabolic processing ability through the day and mechanistic basis for exploring circadian metabolic homeostasis in disease states.

Altered Metabolism During the Dark Period in Drosophila Short Sleep Mutants.

Sleep is an almost universally required state in biology. Disrupted sleep has been associated with adverse health risks including metabolic perturbations. Sleep is in part regulated via circadian mechanisms, however, metabolic dysfunction at different times of day arising from sleep disruption is unclear. We used targeted liquid chromatography-mass spectrometry to probe metabolic alterations using high-resolution temporal sampling of two Drosophila short sleep mutants, fumin and sleepless, across a circadian day. Discriminant analyses revealed overall distinct metabolic profiles for mutants when compared to a wild type dataset. Altered levels of metabolites involved in nicotinate/nicotinamide, alanine, aspartate, and glutamate, glyoxylate and dicarboxylate metabolism, and the TCA cycle were observed in mutants suggesting increased energetic demands. Furthermore, rhythmicity analyses revealed fewer 24 hr rhythmic metabolites in both mutants. Interestingly, mutants displayed two major peaks in phases while wild type displayed phases that were less concerted. In contrast to 24 hr rhythmic metabolites, an increase in the number of 12 hr rhythmic metabolites was observed in fumin while sleepless displayed a decrease. These results support that decreased sleep alters the overall metabolic profile with short sleep mutants displaying altered metabolite levels associated with a number of pathways in addition to altered neurotransmitter levels.

Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Crosstalk between cellular metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to degenerative disease, including cancer. Here, we investigated whether maintenance of circadian rhythms depends upon specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to overall levels of a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function in an in vitro mouse model of pancreatic adenocarcinoma. Metabolic profiling of a library of congenic tumor cell clones revealed significant differences in levels of lactate, pyruvate, ATP, and other crucial metabolites that we used to identify candidate clones with which to generate circadian reporter lines. Despite the shared genetic background of the clones, we observed diverse circadian profiles among these lines that varied with their metabolic phenotype: the most hypometabolic line had the strongest circadian rhythms while the most hypermetabolic line had the weakest rhythms. Treatment of these tumor cell lines with bezafibrate, a peroxisome proliferator-activated receptor (PPAR) agonist shown to increase OxPhos, decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, treatment with the Complex I antagonist rotenone enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function, and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

The NFκB Dif is required for behavioral and molecular correlates of sleep homeostasis in Drosophila.

The nuclear factor binding the κ light chain in B-cells (NFκB) is involved in a wide range of cellular processes including development, growth, innate immunity, and sleep. However, efforts have been limited toward understanding how specific NFκB transcription factors function in sleep. Drosophila fruit flies carry three genes encoding NFκB transcription factors, Dorsal, Dorsal Immunity Factor (Dif), and Relish. We previously found that loss of the Relish gene from fat body suppressed daily nighttime sleep, and abolished infection-induced sleep. Here we show that Dif regulates daily sleep and recovery sleep following prolonged wakefulness. Mutants of Dif showed reduced daily sleep and suppressed recovery in response to sleep deprivation. Pan-neuronal knockdown of Dif strongly suppressed daily sleep, indicating that in contrast to Relish, Dif functions from the central nervous system to regulate sleep. Based on the distribution of a Dif-associated GAL4 driver, we hypothesized that its effects on sleep were mediated by the pars intercerebralis (PI). While RNAi knock-down of Dif in the PI reduced daily sleep, it had no effect on the recovery response to sleep deprivation. However, recovery sleep was suppressed when RNAi knock-down of Dif was distributed across a wider range of neurons. Induction of the nemuri (nur) antimicrobial peptide by sleep deprivation was suppressed in Dif mutants and pan-neuronal over-expression of nur also suppressed the Dif mutant phenotype. Together, these findings indicate that Dif functions from brain to target nemuri and to promote sleep.

Evidence for a role of human blood-borne factors in mediating age-associated changes in molecular circadian rhythms.

Aging is associated with a number of physiologic changes including perturbed circadian rhythms; however, mechanisms by which rhythms are altered remain unknown. To test the idea that circulating factors mediate age-dependent changes in peripheral rhythms, we compared the ability of human serum from young and old individuals to synchronize circadian rhythms in culture. We collected blood from apparently healthy young (age 25-30) and old (age 70-76) individuals and used the serum to synchronize cultured fibroblasts. We found that young and old sera are equally competent at driving robust ~24h oscillations of a luciferase reporter driven by clock gene promoter. However, cyclic gene expression is affected, such that young and old sera drive cycling of different genes. While genes involved in the cell cycle and transcription/translation remain rhythmic in both conditions, genes identified by STRING and IPA analyses as associated with oxidative phosphorylation and Alzheimer's Disease lose rhythmicity in the aged condition. Also, the expression of cycling genes associated with cholesterol biosynthesis increases in the cells entrained with old serum. We did not observe a global difference in the distribution of phase between groups, but find that peak expression of several clock controlled genes (PER3, NR1D1, NR1D2, CRY1, CRY2, and TEF) lags in the cells synchronized with old serum. Taken together, these findings demonstrate that age-dependent blood-borne factors affect peripheral circadian rhythms in cells and have the potential to impact health and disease via maintaining or disrupting rhythms respectively.

Sleepiness, not total sleep amount, increases seizure risk.

Sleep loss has been associated with increased seizure risk since antiquity. Despite this observation standing the test of time, how poor sleep drives susceptibility to seizures remains unclear. To identify underlying mechanisms, we restricted sleep in Drosophila epilepsy models and developed a method to identify spontaneous seizures using quantitative video tracking. Here we find that sleep loss exacerbates seizures but only when flies experience increased sleep need, or sleepiness , and not necessarily with reduced sleep quantity. This is supported by the paradoxical finding that acute activation of sleep-promoting circuits worsens seizures, because it increases sleep need without changing sleep amount. Sleep-promoting circuits become hyperactive after sleep loss and are associated with increased whole-brain activity. During sleep restriction, optogenetic inhibition of sleep-promoting circuits to reduce sleepiness protects against seizures. Downregulation of the 5HT1A serotonin receptor in sleep-promoting cells mediates the effect of sleep need on seizures, and we identify an FDA-approved 5HT1A agonist to mitigate seizures. Our findings demonstrate that while homeostatic sleep is needed to recoup lost sleep, it comes at the cost of increasing seizure susceptibility. We provide an unexpected perspective on interactions between sleep and seizures, and surprisingly implicate sleep- promoting circuits as a therapeutic target for seizure control.

Circadian regulation of lung repair and regeneration.

Optimal lung repair and regeneration are essential for recovery from viral infections, including influenza A virus (IAV). We have previously demonstrated that acute inflammation and mortality induced by IAV is under circadian control. However, it is not known whether the influence of the circadian clock persists beyond the acute outcomes. Here, we utilize the UK Biobank to demonstrate an association between poor circadian rhythms and morbidity from lower respiratory tract infections, including the need for hospitalization and mortality after discharge; this persists even after adjusting for common confounding factors. Furthermore, we use a combination of lung organoid assays, single-cell RNA sequencing, and IAV infection in different models of clock disruption to investigate the role of the circadian clock in lung repair and regeneration. We show that lung organoids have a functional circadian clock and the disruption of this clock impairs regenerative capacity. Finally, we find that the circadian clock acts through distinct pathways in mediating lung regeneration - in tracheal cells via the Wnt/ß-catenin pathway and through IL-1ß in alveolar epithelial cells. We speculate that adding a circadian dimension to the critical process of lung repair and regeneration will lead to novel therapies and improve outcomes.

Modulation of sleep by trafficking of lipids through the Drosophila blood-brain barrier.

Endocytosis through Drosophila glia is a significant determinant of sleep amount and occurs preferentially during sleep in glia of the blood-brain barrier (BBB). To identify metabolites whose trafficking is mediated by sleep-dependent endocytosis, we conducted metabolomic analysis of flies that have increased sleep due to a block in glial endocytosis. We report that acylcarnitines, fatty acids conjugated to carnitine to promote their transport, accumulate in heads of these animals. In parallel, to identify transporters and receptors whose loss contributes to the sleep phenotype caused by blocked endocytosis, we screened genes enriched in barrier glia for effects on sleep. We find that knockdown of lipid transporters LRP1&2 or of carnitine transporters ORCT1&2 increases sleep. In support of the idea that the block in endocytosis affects trafficking through specific transporters, knockdown of LRP or ORCT transporters also increases acylcarnitines in heads. We propose that lipid species, such as acylcarnitines, are trafficked through the BBB via sleep-dependent endocytosis, and their accumulation reflects an increased need for sleep.

Variant-to-gene, gene-to-trait: finding new sleep genes through GWAS.

Distilling insomnia genome-wide association study (GWAS) variants, Palermo and colleagues identified several genes that participate in sleep regulation in two different model organisms. This workflow sets off an innovative strategy to extract biological relevance from large human genomic databases.

Chronic sleep loss sensitizes Drosophila melanogaster to nitrogen stress.

Chronic sleep loss profoundly impacts metabolic health and shortens lifespan, but studies of the mechanisms involved have focused largely on acute sleep deprivation.1,2 To identify metabolic consequences of chronically reduced sleep, we conducted unbiased metabolomics on heads of three adult Drosophila short-sleeping mutants with very different mechanisms of sleep loss: fumin (fmn), redeye (rye), and sleepless (sss).3,4,5,6,7 Common features included elevated ornithine and polyamines, with lipid, acyl-carnitine, and TCA cycle changes suggesting mitochondrial dysfunction. Studies of excretion demonstrate inefficient nitrogen elimination in adult sleep mutants, likely contributing to their polyamine accumulation. Increasing levels of polyamines, particularly putrescine, promote sleep in control flies but poison sleep mutants. This parallels the broadly enhanced toxicity of high dietary nitrogen load from protein in chronically sleep-restricted Drosophila, including both sleep mutants and flies with hyper-activated wake-promoting neurons. Together, our results implicate nitrogen stress as a novel mechanism linking chronic sleep loss to adverse health outcomes-and perhaps for linking food and sleep homeostasis at the cellular level in healthy organisms.

The microbiome stabilizes circadian rhythms in the gut.

The gut microbiome is well known to impact host physiology and health. Given widespread control of physiology by circadian clocks, we asked how the microbiome interacts with circadian rhythms in the Drosophila gut. The microbiome did not cycle in flies fed ad libitum, and timed feeding (TF) drove limited cycling only in clockless per01 flies. However, TF and loss of the microbiome influenced the composition of the gut cycling transcriptome, independently and together. Moreover, both interventions increased the amplitude of rhythmic gene expression, with effects of TF at least partly due to changes in histone acetylation. Contrary to expectations, timed feeding rendered animals more sensitive to stress. Analysis of microbiome function in circadian physiology revealed that germ-free flies reset more rapidly with shifts in the light:dark cycle. We propose that the microbiome stabilizes cycling in the host gut to prevent rapid fluctuations with changing environmental conditions.

Ecdysone acts through cortex glia to regulate sleep in Drosophila.

Steroid hormones are attractive candidates for transmitting long-range signals to affect behavior. These lipid-soluble molecules derived from dietary cholesterol easily penetrate the brain and act through nuclear hormone receptors (NHRs) that function as transcription factors. To determine the extent to which NHRs affect sleep:wake cycles, we knocked down each of the 18 highly conserved NHRs found in Drosophila adults and report that the ecdysone receptor (EcR) and its direct downstream NHR Eip75B (E75) act in glia to regulate the rhythm and amount of sleep. Given that ecdysone synthesis genes have little to no expression in the fly brain, ecdysone appears to act as a long-distance signal and our data suggest that it enters the brain more at night. Anti-EcR staining localizes to the cortex glia in the brain and functional screening of glial subtypes revealed that EcR functions in adult cortex glia to affect sleep. Cortex glia are implicated in lipid metabolism, which appears to be relevant for actions of ecdysone as ecdysone treatment mobilizes lipid droplets (LDs), and knockdown of glial EcR results in more LDs. In addition, sleep-promoting effects of exogenous ecdysone are diminished in lsd-2 mutant flies, which are lean and deficient in lipid accumulation. We propose that ecdysone is a systemic secreted factor that modulates sleep by stimulating lipid metabolism in cortex glia.

The Kinetics and (Dys)kinetics of Cancer Chronotherapy.

Circadian rhythms are the daily cycles that time almost all aspects of physiology, but treatments of the clock or by the clock are rarely tested in the clinic. We develop a framework for identifying interventions that may benefit from administration at the appropriate time of day (chronotherapy). Typically, pharmacokinetics is an important consideration for chronotherapy, with short half-life drugs deemed optimal for such treatments. However, recent data suggest long-lived antibodies can show time-of-day specific effects. Examples include both tumor-targeted antibodies as well as immunotherapies with antibodies that activate T cells. Clues to the immunotherapy mechanism come from animal vaccination studies, which demonstrate circadian responses of T cells to a single dose that leads to long-lasting T-cell activation. Conversely, some studies have challenged the efficacy of chronotherapy, underscoring the need to rigorously investigate its application for each drug and tumor type.

Consolidation of Sleep-Dependent Appetitive Memory Is Mediated by a Sweet-Sensing Circuit.

Sleep is a universally conserved physiological state which contributes toward basic organismal functions, including cognitive operations such as learning and memory. Intriguingly, organisms can sometimes form memory even without sleep, such that Drosophila display sleep-dependent and sleep-independent memory in an olfactory appetitive training paradigm. Sleep-dependent memory can be elicited by the perception of sweet taste, and we now show that a mixed-sex population of flies maintained on sorbitol, a tasteless but nutritive substance, do not require sleep for memory consolidation. Consistent with this, silencing sugar-sensing gustatory receptor neurons in fed flies triggers a switch to sleep-independent memory consolidation, whereas activating sugar-sensing gustatory receptor neurons results in the formation of sleep-dependent memory in starved flies. Sleep-dependent and sleep-independent memory relies on distinct subsets of reward signaling protocerebral anterior medial dopaminergic neurons (PAM DANs) such that PAM-ß'2mp DANs mediate memory in fed flies whereas PAM-α1 DANs are required in starved flies. Correspondingly, we observed a feeding-dependent calcium increase in PAM-ß'2mp DANs, but not in PAM-α1 DANs. Following training, the presence of sweet sugars recruits PAM-ß'2mp DANs, whereas tasteless medium increases calcium in PAM-α1 DANs. Together, this work identifies mechanistic underpinnings of sleep-dependent memory consolidation, in particular demonstrating a role for the processing of sweet taste reward signals.SIGNIFICANCE STATEMENT Sleep is essential for encoding and consolidating memories, but animals must often suppress sleep for survival. Consequently, Drosophila have evolved sleep-independent consolidation that allows retention of essential information without sleep. In the presence of food, sleep is required for memory, but mechanisms that transmit signals from food cues to regulate the need for sleep in memory are largely unknown. We found that sweet-sensing neurons drive the recruitment of specific reward signaling dopaminergic neurons to establish sleep-dependent memory. Conversely, in the absence of a sweet stimulus, different neurons are activated within the same dopaminergic cluster for sleep-independent memory consolidation. Therefore, the processing of sleep-dependent memory relies on the presence of sweet sugars that signal through reward circuitry.

Hugin+ neurons provide a link between sleep homeostat and circadian clock neurons.

Sleep is controlled by homeostatic mechanisms, which drive sleep after wakefulness, and a circadian clock, which confers the 24-h rhythm of sleep. These processes interact with each other to control the timing of sleep in a daily cycle as well as following sleep deprivation. However, the mechanisms by which they interact are poorly understood. We show here that hugin+ neurons, previously identified as neurons that function downstream of the clock to regulate rhythms of locomotor activity, are also targets of the sleep homeostat. Sleep deprivation decreases activity of hugin+ neurons, likely to suppress circadian-driven activity during recovery sleep, and ablation of hugin+ neurons promotes sleep increases generated by activation of the homeostatic sleep locus, the dorsal fan-shaped body (dFB). Also, mutations in peptides produced by the hugin+ locus increase recovery sleep following deprivation. Transsynaptic mapping reveals that hugin+ neurons feed back onto central clock neurons, which also show decreased activity upon sleep loss, in a Hugin peptide-dependent fashion. We propose that hugin+ neurons integrate circadian and sleep signals to modulate circadian circuitry and regulate the timing of sleep.