Molecular mechanisms and physiological importance of circadian rhythms.

To accommodate daily recurring environmental changes, animals show cyclic variations in behaviour and physiology, which include prominent behavioural states such as sleep-wake cycles but also a host of less conspicuous oscillations in neurological, metabolic, endocrine, cardiovascular and immune functions. Circadian rhythmicity is created endogenously by genetically encoded molecular clocks, whose components cooperate to generate cyclic changes in their own abundance and activity, with a periodicity of about a day. Throughout the body, such molecular clocks convey temporal control to the function of organs and tissues by regulating pertinent downstream programmes. Synchrony between the different circadian oscillators and resonance with the solar day is largely enabled by a neural pacemaker, which is directly responsive to certain environmental cues and able to transmit internal time-of-day representations to the entire body. In this Review, we discuss aspects of the circadian clock in Drosophila melanogaster and mammals, including the components of these molecular oscillators, the function and mechanisms of action of central and peripheral clocks, their synchronization and their relevance to human health.

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.

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.

Chronic social isolation signals starvation and reduces sleep in Drosophila.

Social isolation and loneliness have potent effects on public health1-4. Research in social psychology suggests that compromised sleep quality is a key factor that links persistent loneliness to adverse health conditions5,6. Although experimental manipulations have been widely applied to studying the control of sleep and wakefulness in animal models, how normal sleep is perturbed by social isolation is unknown. Here we report that chronic, but not acute, social isolation reduces sleep in Drosophila. We use quantitative behavioural analysis and transcriptome profiling to differentiate between brain states associated with acute and chronic social isolation. Although the flies had uninterrupted access to food, chronic social isolation altered the expression of metabolic genes and induced a brain state that signals starvation. Chronically isolated animals exhibit sleep loss accompanied by overconsumption of food, which resonates with anecdotal findings of loneliness-associated hyperphagia in humans. Chronic social isolation reduces sleep and promotes feeding through neural activities in the peptidergic fan-shaped body columnar neurons of the fly. Artificial activation of these neurons causes misperception of acute social isolation as chronic social isolation and thereby results in sleep loss and increased feeding. These results present a mechanistic link between chronic social isolation, metabolism, and sleep, addressing a long-standing call for animal models focused on loneliness7.

The Drosophila blood-brain barrier regulates sleep via Moody G protein-coupled receptor signaling.

Sleep is vital for most animals, yet its mechanism and function remain unclear. We found that permeability of the BBB (blood-brain barrier)-the organ required for the maintenance of homeostatic levels of nutrients, ions, and other molecules in the brain-is modulated by sleep deprivation (SD) and can cell-autonomously effect sleep changes. We observed increased BBB permeability in known sleep mutants as well as in acutely sleep-deprived animals. In addition to molecular tracers, SD-induced BBB changes also increased the penetration of drugs used in the treatment of brain pathologies. After chronic/genetic or acute SD, rebound sleep or administration of the sleeping aid gaboxadol normalized BBB permeability, showing that SD effects on the BBB are reversible. Along with BBB permeability, RNA levels of the BBB master regulator moody are modulated by sleep. Conversely, altering BBB permeability alone through glia-specific modulation of moody, gαo, loco, lachesin, or neuroglian-each a well-studied regulator of BBB function-was sufficient to induce robust sleep phenotypes. These studies demonstrate a tight link between BBB permeability and sleep and indicate a unique role for the BBB in the regulation of sleep.

Circadian clock activity of cryptochrome relies on tryptophan-mediated photoreduction.

Cryptochromes (CRYs) entrain the circadian clocks of plants and animals to light. Irradiation of the Drosophila cryptochrome (dCRY) causes reduction of an oxidized flavin cofactor by a chain of conserved tryptophan (Trp) residues. However, it is unclear how redox chemistry within the Trp chain couples to dCRY-mediated signaling. Here, we show that substitutions of four key Trp residues to redox-active tyrosine and redox-inactive phenylalanine tune the light sensitivity of dCRY photoreduction, conformational activation, cellular stability, and targeted degradation of the clock protein timeless (TIM). An essential surface Trp gates electron flow into the flavin cofactor, but can be relocated for enhanced photoactivation. Differential effects of Trp-mediated flavin photoreduction on cellular turnover of TIM and dCRY indicate that these activities are separated in time and space. Overall, the dCRY Trp chain has evolutionary importance for light sensing, and its manipulation has implications for optogenetic applications of CRYs.

Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.

Temperature compensation and temperature sensation in the circadian clock.

All known circadian clocks have an endogenous period that is remarkably insensitive to temperature, a property known as temperature compensation, while at the same time being readily entrained by a diurnal temperature oscillation. Although temperature compensation and entrainment are defining features of circadian clocks, their mechanisms remain poorly understood. Most models presume that multiple steps in the circadian cycle are temperature-dependent, thus facilitating temperature entrainment, but then insist that the effect of changes around the cycle sums to zero to enforce temperature compensation. An alternative theory proposes that the circadian oscillator evolved from an adaptive temperature sensor: a gene circuit that responds only to temperature changes. This theory implies that temperature changes should linearly rescale the amplitudes of clock component oscillations but leave phase relationships and shapes unchanged. We show using timeless luciferase reporter measurements and Western blots against TIMELESS protein that this prediction is satisfied by the Drosophila circadian clock. We also review evidence for pathways that couple temperature to the circadian clock, and show previously unidentified evidence for coupling between the Drosophila clock and the heat-shock pathway.

Drosophila TIM binds importin α1, and acts as an adapter to transport PER to the nucleus.

Regulated nuclear entry of clock proteins is a conserved feature of eukaryotic circadian clocks and serves to separate the phase of mRNA activation from mRNA repression in the molecular feedback loop. In Drosophila, nuclear entry of the clock proteins, PERIOD (PER) and TIMELESS (TIM), is tightly controlled, and impairments of this process produce profound behavioral phenotypes. We report here that nuclear entry of PER-TIM in clock cells, and consequently behavioral rhythms, require a specific member of a classic nuclear import pathway, Importin α1 (IMPα1). In addition to IMPα1, rhythmic behavior and nuclear expression of PER-TIM require a specific nuclear pore protein, Nup153, and Ran-GTPase. IMPα1 can also drive rapid and efficient nuclear expression of TIM and PER in cultured cells, although the effect on PER is mediated by TIM. Mapping of interaction domains between IMPα1 and TIM/PER suggests that TIM is the primary cargo for the importin machinery. This is supported by attenuated interaction of IMPα1 with TIM carrying a mutation previously shown to prevent nuclear entry of TIM and PER. TIM is detected at the nuclear envelope, and computational modeling suggests that it contains HEAT-ARM repeats typically found in karyopherins, consistent with its role as a co-transporter for PER. These findings suggest that although PER is the major timekeeper of the clock, TIM is the primary target of nuclear import mechanisms. Thus, the circadian clock uses specific components of the importin pathway with a novel twist in that TIM serves a karyopherin-like role for PER.

Time Travels: A 40-Year Journey from Drosophila's Clock Mutants to Human Circadian Disorders (Nobel Lecture).

Living organisms have a biological clock that helps to prepare our physiology for the fluctuations of the day. Key research to elucidate the biological mechanisms of this regular adaptation, referred to as the circadian rhythm, is described by M. W. Young in his Nobel lecture.

miR-124 Regulates Diverse Aspects of Rhythmic Behavior in Drosophila.

Circadian clocks enable organisms to anticipate and adapt to fluctuating environmental conditions. Despite substantial knowledge of central clock machineries, we have less understanding of how the central clock's behavioral outputs are regulated. Here, we identify Drosophila miR-124 as a critical regulator of diurnal activity. During normal light/dark cycles, mir-124 mutants exhibit profoundly abnormal locomotor activity profiles, including loss of anticipatory capacities at morning and evening transitions. Moreover,mir-124 mutants exhibited striking behavioral alterations in constant darkness (DD), including a temporal advance in peak activity. Nevertheless, anatomical and functional tests demonstrate a normal circadian pacemaker in mir-124 mutants, indicating this miRNA regulates clock output. Among the extensive miR-124 target network, heterozygosity for targets in the BMP pathway substantially corrected the evening activity phase shift in DD. Thus, excess BMP signaling drives specific circadian behavioral output defects in mir-124 knock-outs. SIGNIFICANCE STATEMENT: Circadian clocks control rhythmic behaviors of most life-forms. Despite extensive knowledge of the central clock, there is less understanding of how its behavioral outputs are regulated. Here, we identify a conserved neural microRNA as a critical regulator of diurnal behavior. We find Drosophila mir-124 mutants exhibit robust activity abnormalities during normal light/dark cycles and during constant darkness. Nevertheless, as the central pacemaker is functional in these mutants, miR-124 regulates clock output. We provide mechanistic insight by showing deregulation of miR-124 targets in BMP signaling drives specific mir-124 defects. In summary,Drosophila mir-124 mutants reveal post-transcriptional control of circadian activities, and impact of BMP signaling in behavioral output.

Structure of full-length Drosophila cryptochrome.

The cryptochrome/photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to ultraviolet and blue light exposure in all kingdoms of life. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs) and 6-4 photolesions caused by ultraviolet radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalysed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable carboxy-terminal tail that appends the conserved PL homology domain (PHD) and is important for function. Here, we report a 2.3-Å resolution crystal structure of Drosophila CRY with an intact C terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp 536 juts into the CRY catalytic centre to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture and photochemistry can be elaborated into a range of light-driven functions.

Flavin reduction activates Drosophila cryptochrome.

Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation.

Application of an ex vivo cellular model of circadian variation for bipolar disorder research: a proof of concept study.

OBJECTIVES: Disruption of circadian function has been observed in several human disorders, including bipolar disorder (BD). Research into these disorders can be facilitated by human cellular models that evaluate external factors (zeitgebers) that impact circadian pacemaker activity. Incorporating a firefly luciferase reporter system into human fibroblasts provides a facile, bioluminescent readout that estimates circadian phase, while leaving the cells intact. We evaluated whether this system can be adapted to clinical BD research and whether it can incorporate zeitgeber challenge paradigms. METHODS: Fibroblasts from patients with bipolar I disorder (BD-I) (n = 13) and controls (n = 12) were infected ex vivo with a lentiviral reporter incorporating the promoter sequences for Bmal1, a circadian gene to drive expression of the firefly luciferase gene. Following synchronization, the bioluminescence was used to estimate period length. Phase response curves (PRCs) were also generated following forskolin challenge and the phase response patterns were characterized. RESULTS: Period length and PRCs could be estimated reliably from the constructs. There were no significant case-control differences in period length, with a nonsignificant trend for differences in PRCs following the phase-setting experiments. CONCLUSIONS: An ex vivo cellular fibroblast-based model can be used to investigate circadian function in BD-I. It can be generated from specific individuals and this could usefully complement ongoing circadian clinical research.

The importance of sleep regularity: a consensus statement of the National Sleep Foundation sleep timing and variability panel.

OBJECTIVE: To develop and present consensus findings of the National Sleep Foundation sleep timing and variability panel regarding the impact of sleep timing variability on health and performance. METHODS: The National Sleep Foundation assembled a panel of sleep and circadian experts to evaluate the scientific evidence and conduct a formal consensus and voting procedure. A systematic literature review was conducted using the NIH National Library of Medicine PubMed database, and panelists voted on the appropriateness of 3 questions using a modified Delphi RAND/UCLA Appropriateness Method with 2 rounds of voting. RESULTS: The literature search and panel review identified 63 full text publications to inform consensus voting. Panelists achieved consensus on each question: (1) is daily regularity in sleep timing important for (a) health or (b) performance? and (2) when sleep is of insufficient duration during the week (or work days), is catch-up sleep on weekends (or non-work days) important for health? Based on the evidence currently available, panelists agreed to an affirmative response to all 3 questions. CONCLUSIONS: Consistency of sleep onset and offset timing is important for health, safety, and performance. Nonetheless, when insufficient sleep is obtained during the week/work days, weekend/non-work day catch-up sleep may be beneficial.

Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein.

Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT(S)), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT(S) in cell culture resembles that with wild-type DBT and posits that, in flies DBT(S) likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation.

Chronic sleep loss disrupts rhythmic gene expression in Drosophila.

Genome-wide profiling of rhythmic gene expression has offered new avenues for studying the contribution of circadian clock to diverse biological processes. Sleep has been considered one of the most important physiological processes that are regulated by the circadian clock, however, the effects of chronic sleep loss on rhythmic gene expression remain poorly understood. In the present study, we exploited Drosophila sleep mutants insomniac 1 (inc 1 ) and wide awake D2 (wake D2 ) as models for chronic sleep loss. We profiled the transcriptomes of head tissues collected from 4-week-old wild type flies, inc 1 and wake D2 at timepoints around the clock. Analysis of gene oscillation revealed a substantial loss of rhythmicity in inc 1 and wake D2 compared to wild type flies, with most of the affected genes common to both mutants. The disruption of gene oscillation was not due to changes in average gene expression levels. We also identified a subset of genes whose loss of rhythmicity was shared among animals with chronic sleep loss and old flies, suggesting a contribution of aging to chronic, sleep-loss-induced disruption of gene oscillation.

Activating PER repressor through a DBT-directed phosphorylation switch.

Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT), a Drosophila ortholog of human casein kinase I (CKI)epsilon/delta. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER). DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The per(S) mutation, which is associated with short-period (19-h) circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function.