A Circadian Clock in the Blood-Brain Barrier Regulates Xenobiotic Efflux.

Endogenous circadian rhythms are thought to modulate responses to external factors, but mechanisms that confer time-of-day differences in organismal responses to environmental insults/therapeutic treatments are poorly understood. Using a xenobiotic, we find that permeability of the Drosophila "blood"-brain barrier (BBB) is higher at night. The permeability rhythm is driven by circadian regulation of efflux and depends on a molecular clock in the perineurial glia of the BBB, although efflux transporters are restricted to subperineurial glia (SPG). We show that transmission of circadian signals across the layers requires cyclically expressed gap junctions. Specifically, during nighttime, gap junctions reduce intracellular magnesium ([Mg2+]i), a positive regulator of efflux, in SPG. Consistent with lower nighttime efflux, nighttime administration of the anti-epileptic phenytoin is more effective at treating a Drosophila seizure model. These findings identify a novel mechanism of circadian regulation and have therapeutic implications for drugs targeted to the central nervous system.

Senescent glia link mitochondrial dysfunction and lipid accumulation.

Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species1,2. Acutely, senescent cells promote wound healing3,4 and prevent tumour formation5; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy6-11, why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear12,13. Here we identify naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence14,15, we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation.

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

Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos 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.

Circadian Rhythms of the Blood-Brain Barrier and Drug Delivery.

The blood-brain barrier (BBB) is a critical interface separating the central nervous system from the peripheral circulation, ensuring brain homeostasis and function. Recent research has unveiled a profound connection between the BBB and circadian rhythms, the endogenous oscillations synchronizing biological processes with the 24-hour light-dark cycle. This review explores the significance of circadian rhythms in the context of BBB functions, with an emphasis on substrate passage through the BBB. Our discussion includes efflux transporters and the molecular timing mechanisms that regulate their activities. A significant focus of this review is the potential implications of chronotherapy, leveraging our knowledge of circadian rhythms for improving drug delivery to the brain. Understanding the temporal changes in BBB can lead to optimized timing of drug administration, to enhance therapeutic efficacy for neurological disorders while reducing side effects. By elucidating the interplay between circadian rhythms and drug transport across the BBB, this review offers insights into innovative therapeutic interventions.

Losing TREC with age.

In this issue of Immunity, den Braber et al. (2012) highlight differences in naive T cell lifespan between mice and humans. Their data suggest that mechanisms of naive T cell maintenance may differ between mice and men.

Human and rat gut microbiome composition is maintained following sleep restriction.

Insufficient sleep increasingly characterizes modern society, contributing to a host of serious medical problems. Loss of sleep is associated with metabolic diseases such as obesity and diabetes, cardiovascular disorders, and neurological and cognitive impairments. Shifts in gut microbiome composition have also been associated with the same pathologies; therefore, we hypothesized that sleep restriction may perturb the gut microbiome to contribute to a disease state. In this study, we examined the fecal microbiome by using a cross-species approach in both rat and human studies of sleep restriction. We used DNA from hypervariable regions (V1-V2) of 16S bacteria rRNA to define operational taxonomic units (OTUs) of the microbiome. Although the OTU richness of the microbiome is decreased by sleep restriction in rats, major microbial populations are not altered. Only a single OTU, TM7-3a, was found to increase with sleep restriction of rats. In the human microbiome, we find no overt changes in the richness or composition induced by sleep restriction. Together, these results suggest that the microbiome is largely resistant to changes during sleep restriction.

Chemokine treatment rescues profound T-lineage progenitor homing defect after bone marrow transplant conditioning in mice.

Development of T cells in the thymus requires continuous importation of T-lineage progenitors from the bone marrow via the circulation. Following bone marrow transplant, recovery of a normal peripheral T-cell pool depends on production of naïve T cells in the thymus; however, delivery of progenitors to the thymus limits T-lineage reconstitution. Here, we examine homing of intravenously delivered progenitors to the thymus following irradiation and bone marrow reconstitution. Surprisingly, following host conditioning by irradiation, we find that homing of lymphoid-primed multipotent progenitors and common lymphoid progenitors to the thymus decreases more than 10-fold relative to unirradiated mice. The reduction in thymic homing in irradiated mice is accompanied by a significant reduction in CCL25, an important chemokine ligand for thymic homing. We show that pretreatment of bone marrow progenitors with CCL25 and CCL21 corrects the defect in thymic homing after irradiation and promotes thymic reconstitution. These data suggest new therapeutic approaches to promote T-cell regeneration.

Both retention and recirculation contribute to long-lived regulatory T-cell accumulation in the thymus.

Natural Treg cells acquire their lineage-determining transcription factor Foxp3 during development in the thymus and are important in maintaining immunologic tolerance. Here, we analyzed the composition of the thymic Treg-cell pool using RAG2-GFP/FoxP3-RFP dual reporter mice and found that a population of long-lived GFP(-) Treg cells exists in the thymus. These long-lived Treg cells substantially increased with age, to a point where they represent >90% of the total thymic Treg-cell pool at 6 months of age. In contrast, long-lived conventional T cells remained at ∼ 15% of the total thymic pool at 6 months of age. Consistent with these studies, we noticed that host-derived Treg cells represented a large fraction (∼ 10%) of the total thymic Treg-cell pool in bone marrow chimeras, suggesting that long-lived Treg cells also reside in the thymus of these mice. The pool of long-lived Treg cells in the thymus was sustained by retention of Treg cells in the thymus and by recirculation of peripheral Treg cells back into the thymus. These long-lived thymic Treg cells suppressed T-cell proliferation to an equivalent extent to splenic Treg cells. Together, these data demonstrate that long-lived Treg cells accumulate in the thymus by both retention and recirculation.

Trafficking to the thymus.

The continuous production of T lymphocytes requires that hematopoietic progenitors developing in the bone marrow migrate to the thymus. Rare progenitors egress from the bone marrow into the circulation, then traffic via the blood to the thymus. It is now evident that thymic settling is tightly regulated by selectin ligands, chemokine receptors, and integrins, among other factors. Identification of these signals has enabled progress in identifying specific populations of hematopoietic progenitors that can settle the thymus. Understanding the nature of progenitor cells and the molecular mechanisms involved in thymic settling may allow for therapeutic manipulation of this process, and improve regeneration of the T lineage in patients with impaired T cell numbers.

Expression of functional P-selectin glycoprotein ligand 1 on hematopoietic progenitors is developmentally regulated.

T cell development requires periodic importation of hematopoietic progenitors into the thymus. The receptor-ligand pair P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) are critically involved in this process. In this study, we examined the expression of functional PSGL-1 on bone marrow hematopoietic progenitors. We demonstrate that functional PSGL-1 is expressed at low levels on hematopoietic stem cells, but upregulated on the cell surface of progenitors that bear other homing molecules known to be important for thymic settling. We found that progenitors able to home to the thymus expressed high levels of PSGL-1 transcripts compared with hematopoietic stem cells. We further demonstrate that hematopoietic progenitors lacking fucosyltransferase 4 and 7 do not express functional PSGL-1, and do not home efficiently to the thymus. These studies provide insight into the developmentally regulated expression of a critical determinant involved in progenitor homing to the thymus.

A circadian clock regulates the blood-brain barrier across phylogeny.

As the central regulatory system of an organism, the brain is responsible for overseeing a wide variety of physiological processes essential for an organism's survival. To maintain the environment necessary for neurons to function, the brain requires highly selective uptake and elimination of specific molecules through the blood-brain barrier (BBB). As an organism's activities vary throughout the day, how does the BBB adapt to meet the changing needs of the brain? A mechanism is through temporal regulation of BBB permeability via its circadian clock, which will be the focal point of this chapter. To comprehend the circadian clock's role within the BBB, we will first examine the anatomy of the BBB and the transport mechanisms enabling it to fulfill its role as a restrictive barrier. Next, we will define the circadian clock, and the discussion will encompass an introduction to circadian rhythms, the Transcription-Translation Feedback Loop (TTFL) as the mechanistic basis of circadian timekeeping, and the organization of tissue clocks found in organisms. Then, we will cover the role of the circadian rhythms in regulating the cellular mechanisms and functions of the BBB. We discuss the implications of this regulation in influencing sleep behavior, the progression of neurodegenerative diseases, and finally drug delivery for treatment of neurological diseases.

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.

Delivery of progenitors to the thymus limits T-lineage reconstitution after bone marrow transplantation.

T-cell production depends on the recruitment of hematopoietic progenitors into the thymus. T cells are among the last of the hematopoietic lineages to recover after bone marrow transplantation (BMT), but the reasons for this delay are not well understood. Under normal physiologic conditions, thymic settling is selective and either CCR7 or CCR9 is required for progenitor access into the thymus. The mechanisms of early thymic reconstitution after BMT, however, are unknown. Here we report that thymic settling is briefly CCR7/CCR9-independent after BMT but continues to rely on the selectin ligand PSGL-1. The CCR7/CCR9 independence is transient, and by 3 weeks after BMT these receptors are again strictly required. Despite the normalization of thymic settling signals, the rare bone marrow progenitors that can efficiently repopulate the thymus are poorly reconstituted for at least 4 weeks after BMT. Consistent with reduced progenitor input to the thymus, intrathymic progenitor niches remain unsaturated for at least 10 weeks after BMT. Finally, we show that thymic recovery is limited by the number of progenitors entering the thymus after BMT. Hence, T-lineage reconstitution after BMT is limited by progenitor supply to the thymus.

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.

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.

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.

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.

A circadian clock regulates efflux by the blood-brain barrier in mice and human cells.

The blood-brain barrier (BBB) is critical for neural function. We report here circadian regulation of the BBB in mammals. Efflux of xenobiotics by the BBB oscillates in mice, with highest levels during the active phase and lowest during the resting phase. This oscillation is abrogated in circadian clock mutants. To elucidate mechanisms of circadian regulation, we profiled the transcriptome of brain endothelial cells; interestingly, we detected limited circadian regulation of transcription, with no evident oscillations in efflux transporters. We recapitulated the cycling of xenobiotic efflux using a human microvascular endothelial cell line to find that the molecular clock drives cycling of intracellular magnesium through transcriptional regulation of TRPM7, which appears to contribute to the rhythm in efflux. Our findings suggest that considering circadian regulation may be important when therapeutically targeting efflux transporter substrates to the CNS.

Time-of-day specificity of anticancer drugs may be mediated by circadian regulation of the cell cycle.

Circadian rhythms are an integral part of physiology, underscoring their relevance for the treatment of disease. We conducted cell-based high-throughput screening to investigate time-of-day influences on the activity of known antitumor agents and found that many compounds exhibit daily rhythms of cytotoxicity concomitant with previously reported oscillations of target genes. Rhythmic action of HSP90 inhibitors was mediated by specific isoforms of HSP90, genetic perturbation of which affected the cell cycle. Furthermore, clock mutants affected the cell cycle in parallel with abrogating rhythms of cytotoxicity, and pharmacological inhibition of the cell cycle also eliminated rhythmic drug effects. An HSP90 inhibitor reduced growth rate of a mouse melanoma in a time-of-day-specific manner, but efficacy was impaired in clock-deficient tumors. These results provide a powerful rationale for appropriate daily timing of anticancer drugs and suggest circadian regulation of the cell cycle within the tumor as an underlying mechanism.

Regulation of the Blood-Brain Barrier by Circadian Rhythms and Sleep.

The blood-brain barrier (BBB) is an evolutionarily conserved, structural, and functional separation between circulating blood and the central nervous system (CNS). By controlling permeability into and out of the nervous system, the BBB has a critical role in the precise regulation of neural processes. Here, we review recent studies demonstrating that permeability at the BBB is dynamically controlled by circadian rhythms and sleep. An endogenous circadian rhythm in the BBB controls transporter function, regulating permeability across the BBB. In addition, sleep promotes the clearance of metabolites along the BBB, as well as endocytosis across the BBB. Finally, we highlight the implications of this regulation for diseases, including epilepsy.