Circadian clocks are modulated by compartmentalized oscillating translation.

Terrestrial organisms developed circadian rhythms for adaptation to Earth's quasi-24-h rotation. Achieving precise rhythms requires diurnal oscillation of fundamental biological processes, such as rhythmic shifts in the cellular translational landscape; however, regulatory mechanisms underlying rhythmic translation remain elusive. Here, we identified mammalian ATXN2 and ATXN2L as cooperating master regulators of rhythmic translation, through oscillating phase separation in the suprachiasmatic nucleus along circadian cycles. The spatiotemporal oscillating condensates facilitate sequential initiation of multiple cycling processes, from mRNA processing to protein translation, for selective genes including core clock genes. Depleting ATXN2 or 2L induces opposite alterations to the circadian period, whereas the absence of both disrupts translational activation cycles and weakens circadian rhythmicity in mice. Such cellular defect can be rescued by wild type, but not phase-separation-defective ATXN2. Together, we revealed that oscillating translation is regulated by spatiotemporal condensation of two master regulators to achieve precise circadian rhythm in mammals.

Adult neural stem cell activation in mice is regulated by the day/night cycle and intracellular calcium dynamics.

Neural stem cells (NSCs) in the adult brain transit from the quiescent state to proliferation to produce new neurons. The mechanisms regulating this transition in freely behaving animals are, however, poorly understood. We customized in vivo imaging protocols to follow NSCs for several days up to months, observing their activation kinetics in freely behaving mice. Strikingly, NSC division is more frequent during daylight and is inhibited by darkness-induced melatonin signaling. The inhibition of melatonin receptors affected intracellular Ca2+ dynamics and promoted NSC activation. We further discovered a Ca2+ signature of quiescent versus activated NSCs and showed that several microenvironmental signals converge on intracellular Ca2+ pathways to regulate NSC quiescence and activation. In vivo NSC-specific optogenetic modulation of Ca2+ fluxes to mimic quiescent-state-like Ca2+ dynamics in freely behaving mice blocked NSC activation and maintained their quiescence, pointing to the regulatory mechanisms mediating NSC activation in freely behaving animals.

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.

Multiple Functions and Regulation of Mammalian Peroxiredoxins.

Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (H2O2) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by H2O2, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of H2O2 signaling. Moreover, their high affinity for and sensitivity to oxidation by H2O2 confer on Prxs the ability to serve as sensors and transducers of H2O2 signaling through transfer of their oxidation state to bound effector proteins.

Mutation of the Human Circadian Clock Gene CRY1 in Familial Delayed Sleep Phase Disorder.

Patterns of daily human activity are controlled by an intrinsic circadian clock that promotes ∼24 hr rhythms in many behavioral and physiological processes. This system is altered in delayed sleep phase disorder (DSPD), a common form of insomnia in which sleep episodes are shifted to later times misaligned with the societal norm. Here, we report a hereditary form of DSPD associated with a dominant coding variation in the core circadian clock gene CRY1, which creates a transcriptional inhibitor with enhanced affinity for circadian activator proteins Clock and Bmal1. This gain-of-function CRY1 variant causes reduced expression of key transcriptional targets and lengthens the period of circadian molecular rhythms, providing a mechanistic link to DSPD symptoms. The allele has a frequency of up to 0.6%, and reverse phenotyping of unrelated families corroborates late and/or fragmented sleep patterns in carriers, suggesting that it affects sleep behavior in a sizeable portion of the human population.

Unconventional Roles of Opsins.

Rhodopsin is the classical light sensor. Although rhodopsin has long been known to be important for image formation in the eye, the requirements for opsins in non-image formation and in extraocular light sensation were revealed much later. Most recent is the demonstration that an opsin in the fruit fly, Drosophila melanogaster, is expressed in pacemaker neurons in the brain and functions in light entrainment of circadian rhythms. However, the biggest surprise is that opsins have light-independent roles, countering more than a century of dogma that they function exclusively as light sensors. Through studies in Drosophila, light-independent roles of opsins have emerged in temperature sensation and hearing. Although these findings have been uncovered in the fruit fly, there are hints that opsins have light-independent roles in a wide array of animals, including mammals. Thus, despite the decades of focus on opsins as light detectors, they represent an important new class of polymodal sensory receptor.

Interactive features of proteins composing eukaryotic circadian clocks.

Research into the molecular mechanisms of eukaryotic circadian clocks has proceeded at an electrifying pace. In this review, we discuss advances in our understanding of the structures of central molecular players in the timing oscillators of fungi, insects, and mammals. A series of clock protein structures demonstrate that the PAS (Per/Arnt/Sim) domain has been used with great variation to formulate the transcriptional activators and repressors of the clock. We discuss how posttranslational modifications and external cues, such as light, affect the conformation and function of core clock components. Recent breakthroughs have also revealed novel interactions among clock proteins and new partners that couple the clock to metabolic and developmental pathways. Overall, a picture of clock function has emerged wherein conserved motifs and structural platforms have been elaborated into a highly dynamic collection of interacting molecules that undergo orchestrated changes in chemical structure, conformational state, and partners.

BMAL1 drives muscle repair through control of hypoxic NAD+ regeneration in satellite cells.

The process of tissue regeneration occurs in a developmentally timed manner, yet the role of circadian timing is not understood. Here, we identify a role for the adult muscle stem cell (MuSC)-autonomous clock in the control of muscle regeneration following acute ischemic injury. We observed greater muscle repair capacity following injury during the active/wake period as compared with the inactive/rest period in mice, and loss of Bmal1 within MuSCs leads to impaired muscle regeneration. We demonstrate that Bmal1 loss in MuSCs leads to reduced activated MuSC number at day 3 postinjury, indicating a failure to properly expand the myogenic precursor pool. In cultured primary myoblasts, we observed that loss of Bmal1 impairs cell proliferation in hypoxia (a condition that occurs in the first 1-3 d following tissue injury in vivo), as well as subsequent myofiber differentiation. Loss of Bmal1 in both cultured myoblasts and in vivo activated MuSCs leads to reduced glycolysis and premature activation of prodifferentiation gene transcription and epigenetic remodeling. Finally, hypoxic cell proliferation and myofiber formation in Bmal1-deficient myoblasts are restored by increasing cytosolic NAD+ Together, we identify the MuSC clock as a pivotal regulator of oxygen-dependent myoblast cell fate and muscle repair through the control of the NAD+-driven response to injury.

Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity.

In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, we performed a systematic RNAi screen in human cells and identified protein phosphatase 4 (PPP4) with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila Genetic depletion of PPP4 shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the "kamikaze" properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1δ-mediated inhibition of CLOCK/BMAL1.

BMAL1 dephosphorylation determines the pace of the circadian clock.

In mammals, virtually all body cells harbor cell-autonomous and self-sustained circadian oscillators that rely on delayed negative feedback loops in gene expression. Transcriptional activation and repression play a major role in keeping these clocks ticking, but numerous post-translational mechanisms-and particularly the phosphorylation of core clock components by protein kinases-are also critically involved in setting the pace of these timekeepers. In this issue of Genes & Development, Klemz and colleagues (pp. 1161-1174) now show how dephosphorylation of BMAL1 by protein phosphatase 4 (PPP4) participates in the modulation of circadian timing.

Ion Channels Controlling Circadian Rhythms in Suprachiasmatic Nucleus Excitability.

Animals synchronize to the environmental day-night cycle by means of an internal circadian clock in the brain. In mammals, this timekeeping mechanism is housed in the suprachiasmatic nucleus (SCN) of the hypothalamus and is entrained by light input from the retina. One output of the SCN is a neural code for circadian time, which arises from the collective activity of neurons within the SCN circuit and comprises two fundamental components: 1) periodic alterations in the spontaneous excitability of individual neurons that result in higher firing rates during the day and lower firing rates at night, and 2) synchronization of these cellular oscillations throughout the SCN. In this review, we summarize current evidence for the identity of ion channels in SCN neurons and the mechanisms by which they set the rhythmic parameters of the time code. During the day, voltage-dependent and independent Na+ and Ca2+ currents, as well as several K+ currents, contribute to increased membrane excitability and therefore higher firing frequency. At night, an increase in different K+ currents, including Ca2+-activated BK currents, contribute to membrane hyperpolarization and decreased firing. Layered on top of these intrinsically regulated changes in membrane excitability, more than a dozen neuromodulators influence action potential activity and rhythmicity in SCN neurons, facilitating both synchronization and plasticity of the neural code.

Circadian clock gene polymorphisms implicated in human pathologies.

Circadian rhythms, ~24 h cycles of physiological and behavioral processes, can be synchronized by external signals (e.g., light) and persist even in their absence. Consequently, dysregulation of circadian rhythms adversely affects the well-being of the organism. This timekeeping system is generated and sustained by a genetically encoded endogenous mechanism composed of interlocking transcriptional/translational feedback loops that generate rhythmic expression of core clock genes. Genome-wide association studies (GWAS) and forward genetic studies show that SNPs in clock genes influence gene regulation and correlate with the risk of developing various conditions. We discuss genetic variations in core clock genes that are associated with various phenotypes, their implications for human health, and stress the need for thorough studies in this domain of circadian regulation.

Altered circadian rhythm, sleep, and rhodopsin 7-dependent shade preference during diapause in Drosophila melanogaster.

To survive adverse environments, many animals enter a dormant state such as hibernation, dauer, or diapause. Various Drosophila species undergo adult reproductive diapause in response to cool temperatures and/or short day-length. While flies are less active during diapause, it is unclear how adverse environmental conditions affect circadian rhythms and sleep. Here we show that in diapause-inducing cool temperatures, Drosophila melanogaster exhibit altered circadian activity profiles, including severely reduced morning activity and an advanced evening activity peak. Consequently, the flies have a single activity peak at a time similar to when nondiapausing flies take a siesta. Temperatures ≤15 °C, rather than photoperiod, primarily drive this behavior. At cool temperatures, flies rapidly enter a deep-sleep state that lacks the sleep cycles of flies at higher temperatures and require high levels of stimulation for arousal. Furthermore, we show that at 25 °C, flies prefer to siesta in the shade, a preference that is virtually eliminated at 10 °C. Resting in the shade is driven by an aversion to blue light that is sensed by Rhodopsin 7 outside of the eyes. Flies at 10 °C show neuronal markers of elevated sleep pressure, including increased expression of Bruchpilot and elevated Ca2+ in the R5 ellipsoid body neurons. Therefore, sleep pressure might overcome blue light aversion. Thus, at the same temperatures that cause reproductive arrest, preserve germline stem cells, and extend lifespan, D. melanogaster are prone to deep sleep and exhibit dramatically altered, yet rhythmic, daily activity patterns.

Phosphorylation of DNA-binding domains of CLOCK-BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells.

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including Period (Per). Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.

Coordination between circadian neural circuit and intracellular molecular clock ensures rhythmic activation of adult neural stem cells.

The circadian clock throughout the day organizes the activity of neural stem cells (NSCs) in the dentate gyrus (DG) of adult hippocampus temporally. However, it is still unclear whether and how circadian signals from the niches contribute to daily rhythmic variation of NSC activation. Here, we show that norepinephrinergic (NEergic) projections from the locus coeruleus (LC), a brain arousal system, innervate into adult DG, where daily rhythmic release of norepinephrine (NE) from the LC NEergic neurons controlled circadian variation of NSC activation through ß3-adrenoceptors. Disrupted circadian rhythmicity by acute sleep deprivation leads to transient NSC overactivation and NSC pool exhaustion over time, which is effectively ameliorated by the inhibition of the LC NEergic neuronal activity or ß3-adrenoceptors-mediated signaling. Finally, we demonstrate that NE/ß3-adrenoceptors-mediated signaling regulates NSC activation through molecular clock BMAL1. Therefore, our study unravels that adult NSCs precisely coordinate circadian neural circuit and intrinsic molecular circadian clock to adapt their cellular behavior across the day.

Clearance phagocytosis by the retinal pigment epithelial during photoreceptor outer segment renewal: Molecular mechanisms and relation to retinal inflammation.

Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal pigment epithelial (RPE). It is a strictly rhythmic, synchronized process that underlies in part circadian regulation. We highlight findings from recently developed methods that quantify distinct phases of outer segment renewal in retinal tissue. At light onset, outer segments expose the conserved "eat-me" signal phosphatidylserine exclusively at their distal, most aged tip. A coordinated two-receptor efferocytosis process follows, in which ligands bridge outer segment phosphatidylserine with the RPE receptors αvß5 integrin, inducing cytosolic signaling toward Rac1 and focal adhesion kinase/MERTK, and with MERTK directly, additionally inhibiting RhoA/ROCK and thus enabling F-actin dynamics favoring outer segment fragment engulfment. Photoreceptors and RPE persist for life with each RPE cell in the eye servicing dozens of overlying photoreceptors. Thus, RPE cells phagocytose more often and process more material than any other cell type. Mutant mice with impaired outer segment renewal largely retain functional photoreceptors and retinal integrity. However, when anti-inflammatory signaling in the RPE via MERTK or the related TYRO3 is lacking, catastrophic inflammation leads to immune cell infiltration that swiftly destroys the retina causing blindness.

CRYPTOCHROMES promote daily protein homeostasis.

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild-type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild-type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRY-deficient mice. Rather than generating large-scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation.

Lymphocyte Circadian Clocks Control Lymph Node Trafficking and Adaptive Immune Responses.

Lymphocytes circulate through lymph nodes (LN) in search for antigen in what is believed to be a continuous process. Here, we show that lymphocyte migration through lymph nodes and lymph occurred in a non-continuous, circadian manner. Lymphocyte homing to lymph nodes peaked at night onset, with cells leaving the tissue during the day. This resulted in strong oscillations in lymphocyte cellularity in lymph nodes and efferent lymphatic fluid. Using lineage-specific genetic ablation of circadian clock function, we demonstrated this to be dependent on rhythmic expression of promigratory factors on lymphocytes. Dendritic cell numbers peaked in phase with lymphocytes, with diurnal oscillations being present in disease severity after immunization to induce experimental autoimmune encephalomyelitis (EAE). These rhythms were abolished by genetic disruption of T cell clocks, demonstrating a circadian regulation of lymphocyte migration through lymph nodes with time-of-day of immunization being critical for adaptive immune responses weeks later.

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.

Stroke in the Time of Circadian Medicine.

Time-of-day significantly influences the severity and incidence of stroke. Evidence has emerged not only for circadian governance over stroke risk factors, but also for important determinants of clinical outcome. In this review, we provide a comprehensive overview of the interplay between chronobiology and cerebrovascular disease. We discuss circadian regulation of pathophysiological mechanisms underlying stroke onset or tolerance as well as in vascular dementia. This includes cell death mechanisms, metabolism, mitochondrial function, and inflammation/immunity. Furthermore, we present clinical evidence supporting the link between disrupted circadian rhythms and increased susceptibility to stroke and dementia. We propose that circadian regulation of biochemical and physiological pathways in the brain increase susceptibility to damage after stroke in sleep and attenuate treatment effectiveness during the active phase. This review underscores the importance of considering circadian biology for understanding the pathology and treatment choice for stroke and vascular dementia and speculates that considering a patient's chronotype may be an important factor in developing precision treatment following stroke.