A K(ATP) channel gene effect on sleep duration: from genome-wide association studies to function in Drosophila.

Humans sleep approximately a third of their lifetime. The observation that individuals with either long or short sleep duration show associations with metabolic syndrome and psychiatric disorders suggests that the length of sleep is adaptive. Although sleep duration can be influenced by photoperiod (season) and phase of entrainment (chronotype), human familial sleep disorders indicate that there is a strong genetic modulation of sleep. Therefore, we conducted high-density genome-wide association studies for sleep duration in seven European populations (N=4251). We identified an intronic variant (rs11046205; P=3.99 × 10(-8)) in the ABCC9 gene that explains ≈5% of the variation in sleep duration. An influence of season and chronotype on sleep duration was solely observed in the replication sample (N=5949). Meta-analysis of the associations found in a subgroup of the replication sample, chosen for season of entry and chronotype, together with the discovery results showed genome-wide significance. RNA interference knockdown experiments of the conserved ABCC9 homologue in Drosophila neurons renders flies sleepless during the first 3 h of the night. ABCC9 encodes an ATP-sensitive potassium channel subunit (SUR2), serving as a sensor of intracellular energy metabolism.

Cholera toxin discriminates between T helper 1 and 2 cells in T cell receptor-mediated activation: role of cAMP in T cell proliferation.

CD4+ T helper (Th) clones can be divided into interleukin 2 (IL-2)-secreting Th1 and IL-4-secreting Th2 cells. We show in the present report that these two Th subsets have different activation requirements for lymphokine production and proliferation: namely, cholera toxin (CT) as well as forskolin inhibit T cell receptor (TCR)-mediated IL-2 production and proliferation in Th1 cells, while the same reagents fail to block IL-4 production and proliferation in Th2 cells. In addition, CT and forskolin differentially influence the proto-oncogene mRNA expression in Th1 vs. Th2 cells after stimulation with Con A. Since both reagents lead to elevated levels of intracellular cAMP, it is likely that Th1 and Th2 cells differ in their sensitivity to an increase in cAMP. Our results indicate that the two Th subsets use different transmission signal pathways upon TCR-mediated activation.

How temperature changes reset a circadian oscillator.

Circadian rhythms control many physiological activities. The environmental entrainment of rhythms involves the immediate responses of clock components. Levels of the clock protein FRQ were measured in Neurospora at various temperatures; at higher temperatures, the amount of FRQ oscillated around higher levels. Absolute FRQ amounts thus identified different times at different temperatures, so temperature shifts corresponded to shifts in clock time without immediate synthesis or turnover of components. Moderate temperature changes could dominate light-to-dark shifts in the influence of circadian timing. Temperature regulation of clock components could explain temperature resetting of rhythms and how single transitions can initiate rhythmicity from characteristic circadian phases.

Circadian clocks: Omnes viae Romam ducunt.

The circadian clock in all organisms is so intimately linked to light reception that it appears as if evolution has simply wired a timer into the mechanism that processes photic information. Several recent studies have provided new insights into the role of light input pathways in the circadian system of Arabidopsis.

The circadian cycle: is the whole greater than the sum of its parts?

The term 'circadian rhythm' describes an oscillatory behavior in the absence of exogenous environmental cues, with a period of about a day. As yet, we don't fully understand which biological mechanisms join together to supply a stable and self-sustained oscillation with such a long period. By chipping away at the molecular mechanism with genetic approaches, some common features are emerging. In combining molecular analyses and physiological experiments, those features that are crucial for structuring a circadian day could be uncovered.

Sleeping Beauty? Developmental Timing, Sleep, and the Circadian Clock in Caenorhabditis elegans.

The genetics toolkit is pretty successful in drilling down into minutiae. The big challenge is to integrate the information from this specialty as well as those of biochemistry, physiology, behavior, and anatomy to explain how fundamental biological processes really work. Sleep, the circadian clock and development all qualify as overarching processes that encompass levels from molecule to behavior as part of their known mechanisms. They overlap each other, such that understanding the mechanisms of one can lead to insights into one of the others. In this essay, we consider how the experimental approaches and findings relating to Caenorhabditis elegans development and lethargus on one hand, and to the circadian clock and sleep in higher organisms on the other, could complement and enhance one another.

Molecular circadian oscillators: an alternative hypothesis.

Results from experiments in different organisms have shown that elements of input pathways can themselves be under circadian control and that outputs might feed back into the oscillator. In addition, it has become clear that there might be redundancies in the generation of circadian rhythmicity, even within single cells. In view of these results, it is worth reevaluating our current working hypotheses about the pacemaker's molecular mechanisms and the involvement of single autoregulatory genes. On one hand, redundancies in the generation of circadian rhythmicity might make the approach of defining a discrete circadian oscillator with the help of single gene mutations extremely difficult. On the other hand, many examples show that components of signal transduction pathways can indeed be encoded by single genes. The authors have constructed a model placing an autoregulatory gene and its products on an input pathway feeding into a separate oscillator. The behavior of this model can explain the majority of results of molecular circadian biology published to date. In addition, it shows that different qualities of the circadian system might be associated with different cellular functions that can exist independently and, only if put together, will lead to the known circadian phenotype.

Circadian systems and metabolism.

Circadian systems direct many metabolic parameters and, at the same time, they appear to be exquisitely shielded from metabolic variations. Although the recent decade of circadian research has brought insights into how circadian periodicity may be generated at the molecular level, little is known about the relationship between this molecular feedback loop and metabolism both at the cellular and at the organismic level. In this theoretical paper, we conjecture about the interdependence between circadian rhythmicity and metabolism. A mathematical model based on the chemical reactions of photosynthesis demonstrates that metabolism as such may generate rhythmicity in the circadian range. Two additional models look at the possible function of feedback loops outside of the circadian oscillator. These feedback loops contribute to the robustness and sustainability of circadian oscillations and to compensation for long- and short-term metabolic variations. The specific circadian property of temperature compensation is put into the context of metabolism. As such, it represents a general compensatory mechanism that shields the clock from metabolic variations.

Seasonality and photoperiodism in fungi.

This review gives a retrospective of what is known about photoperiodism in fungi, which is largely based on reports about seasonal spore concentrations. Relatively few species have been investigated under laboratory conditions, so that our knowledge whether seasonal reproduction in fungi is mainly a direct response to environmental conditions or whether it involves a photoperiodic machinery with memory capacities and a relationship to the circadian system is extremely limited. To form a basis for further experimental endeavors into fungal photoperiodism, we review the reports about endogenous rhythms and photobiology. Finally, we will look at the possibilities of using the fungal circadian model system of Neurospora crassa for future work on photoperiodism.

The genetic and molecular dissection of a prototypic circadian system.

A great deal is known about this archetypal circadian system, and it is likely that Neurospora will represent the first circadian system in which it will be possible to provide a complete description of the flow of information from the photoreceptor, through the components of oscillator, out to a terminal aspect of regulation. In Neurospora the strongest case has been made for there being a state variable of clock identified (Hall, 1995), it has now been shown that light resetting of the clock is mediated by the rapid light induction of the gene encoding this state variable, and a number of defined clock-regulated output genes have been identified, in two of which the clock-specific parts of the promoters have been localized. In addition to the importance of these factoids themselves, our efforts towards understanding of this system has allowed the development of tools and paradigms (e.g. Loros et al., 1989; Loros and Dunlap, 1991; Aronson et al., 1994a) that will help to pave the way for proving the identity of clock components in more complex systems, for understanding how clocks are regulated by entraining factors, and for showing how time information eventually is used to regulate the behaviors of clock cells, and of whole organisms.

Circadian entrainment of Neurospora crassa.

The circadian clock evolved under entraining conditions, yet most circadian experiments and much circadian theory are built around free-running rhythms. The interpretation of entrainment experiments is certainly more complex than that of free-running rhythms due to the relationship between exogenous and endogenous cycles. Here, we systematically describe entrainment in the simplest of the traditional eukaryotic model systems in circadian research, Neurospora crassa. This fungus forms a mass of spores (bands of conidia) each day. Over a wide range of photoperiods, these bands begin to appear at midnight, suggesting integration of neither dawn nor dusk signals alone. However, when symmetrical light/dark cycles (T cycles, each with 50% light) are applied, dusk determines the time of conidiation with a uniform, period-dependent delay in phase. This "forced" synchronization appears to be specific for the zeitgeber light because similar experiments, but using temperature, result in systematic entrainment, with bands appearing relatively later in shorter cycles and earlier in longer cycles. We find that the molecular mechanism of entrainment primarily concerns posttranscriptional regulation. Finally, we have used Neurospora to investigate acute effects of zeitgeber stimuli known as "masking."

Entrainment of the human circadian clock.

Humans are an excellent model system for studying entrainment of the circadian clock in the real world. Unlike the situation in laboratory experiments, entrainment under natural conditions is achieved by different external signals as well as by internal signals generated by multiple feedbacks within the system (e.g., behavior-dependent light and temperature changes, melatonin levels, or regular nutrient intake). Signals that by themselves would not be sufficient zeitgebers may contribute to entrainment in conjunction with other self-sufficient zeitgeber signals (e.g., light). The investigation of these complex zeitgeber interactions seems to be problematic in most model systems and strengthens the human system for circadian research. Here, we review our endeavors measuring human entrainment in real life, predominantly with the help of the Munich ChronoType Questionnaire (MCTQ). The large number of participants in our current MCTQ database allows accurate quantification of the human phase of entrainment (chronotype) and how it depends on age or sex. We also present new data showing how chronotype depends on natural light exposure. The results indicate the importance of zeitgeber strength on human entrainment and help in understanding the differences in chronotype, e.g., between urban and rural regions.

Circadian clocks--from genes to complex behaviour.

Circadian clocks control temporal structure in practically all organisms and on all levels of biology, from gene expression to complex behaviour and cognition. Over the last decades, research has begun to unravel the physiological and, more recently, molecular mechanisms that underlie this endogenous temporal programme. The generation of circadian rhythms can be explained, at the molecular level, by a model based upon a set of genes and their products which form an autoregulating negative feedback loop. The elements contributing to this transcriptional feedback appear to be conserved from insects to mammals. Here, we summarize the process of the genetic and molecular research that led to 'closing the molecular loop'. Now that the reductionist approach has led to the description of a detailed clock model at the molecular level, further insights into the circadian system can be provided by combining the extensive knowledge gained from decades of physiological research with molecular tools, thereby reconstructing the clock within the organism and its environment. We describe experiments combining old and new tools and show that they constitute a powerful approach to understanding the mechanisms that lead to temporal structure in complex behaviour.

Circadian systems: different levels of complexity.

After approximately 50 years of circadian research, especially in selected circadian model systems (Drosophila, Neurospora, Gonyaulax and, more recently, cyanobacteria and mammals), we appreciate the enormous complexity of the circadian programme in organisms and cells, as well as in physiological and molecular circuits. Many of our insights into this complexity stem from experimental reductionism that goes as far as testing the interaction of molecular clock components in heterologous systems or in vitro. The results of this enormous endeavour show circadian systems that involve several oscillators, multiple input pathways and feedback loops that contribute to specific circadian qualities but not necessarily to the generation of circadian rhythmicity. For a full appreciation of the circadian programme, the results from different levels of the system eventually have to be put into the context of the organism as a whole and its specific temporal environment. This review summarizes some of the complexities found at the level of organisms, cells and molecules, and highlights similar strategies that apparently solve similar problems at the different levels of the circadian system.

rIL-4 differentially regulates rIL-2-induced murine NK and LAK killing in CD8+ and CD8- precursor cell subsets.

Interleukin 4 (IL-4) and IL-2 have complementary or synergistic roles in many aspects of lymphocyte development. IL-2 supports the induction of cytolytic activity in cytotoxic T lymphocyte (CTL), natural killer (NK), and lymphokine-activated killer (LAK) cells. IL-4 has also been shown to support CTL and LAK in primary murine spleen cell culture. This report demonstrates that IL-4 selectively down-regulates IL-2 inducible murine CD8- precursors of NK cells. For maximal regulatory effect it is necessary to add IL-4 to cultures before 40 h. Enrichment for NK1.1+ cells failed to recover precursor cells which are down-regulated in overnight cultures or can be cultivated in vitro to yield NK cytolytic activity. Furthermore, phenotypic analysis of effector cells demonstrated a marked inhibition of development of NK1.1+ cells in cultures containing IL-4 plus IL-2 versus IL-2 alone. Thus, it appears that IL-4 down-regulates the precursors of murine NK cells by inhibiting proliferation and/or development. In addition, we show that IL-2-induced murine LAK activity mediated by CD8- precursor cells is unaffected by IL-4, while CD8(+)-derived LAK cells are up-regulated by co-culture with IL-4 and IL-2. Analysis of these data relative to reports documenting down-regulation of human LAK by IL-4 suggests that in vitro cultured, IL-2-activated murine NK cells are the correlates to what are commonly described as human LAK cells. The discrepancy may stem from differences in the characteristics of target cells used in the murine versus the human systems. These results clarify the conflicting reports on the effect of IL-4 on killing activity.

Assignment of circadian function for the Neurospora clock gene frequency.

Circadian clocks consist of three elements: entrainment pathways (inputs), the mechanism generating the rhythmicity (oscillator), and the output pathways that control the circadian rhythms. It is difficult to assign molecular clock components to any one of these elements. Experiments show that inputs can be circadianly regulated and outputs can feed back on the oscillator. Mathematical simulations indicate that under- or overexpression of a gene product can result in arrhythmicity, whether the protein is part of the oscillator or substantially part of a rhythmically expressed input pathway. To distinguish between these two possibilities, we used traditional circadian entrainment protocols on a genetic model system, Neurospora crassa.

Intergeneric complementation of a circadian rhythmicity defect: phylogenetic conservation of structure and function of the clock gene frequency.

The Neurospora crassa frequency locus encodes a 989 amino acid protein that is a central component, a state variable, of the circadian biological clock. We have determined the sequence of all or part of this protein and surrounding regulatory regions from additional fungi representing three genera and report that there is distinct, preferential conservation of the frequency open reading frame (ORF) as compared with non-coding sequences. Within the coding region, many of the domain hallmarks of the N. crassa protein are highly conserved, especially an internal region bearing the causative mutations in frq1 and frq7, the most extreme alleles in the frequency allelic series. Despite considerable diversity among the strains analyzed in terms of morphology, growth, circadian clock output and frq sequence, the ORF from the most distantly related fungus included in this study (Sordaria fimicola) rescues rhythmicity in a N.crassa frequency null strain. Both sequence conservation, and the ability of frequency from a genus displaying one developmental program to complement circadian defects in a separate genus with a distinct, clock-regulated developmental program, are consistent with a central role of the frequency gene product in a general circadian oscillator capable of controlling diverse outputs in a variety of systems.

Dissection of a circadian oscillation into discrete domains.

The circadian oscillator in Neurospora is a negative feedback loop involving as principal players the products of the frequency (frq) locus. frq encodes multiple forms of its protein product FRQ, which act to depress the amounts of frq transcript. In this scheme there are two discrete and separable steps to the circadian cycle, negative feedback itself (repression) in which FRQ acts to decrease the levels of its own transcript, and recovery from repression (derepression) in which frq transcript levels return to peak amounts. By introducing an exogenously regulatable frq transgene into a frq loss-of-function strain (frq9), we created an artificial system in which the two separate steps in the circadian cycle can be initiated and followed separately for purposes of observing their kinetics. Under these conditions the frq-FRQ cycle occupies the time scale of a full circadian cycle. During this time, the process of negative feedback of FRQ on frq transcript levels is rapid and efficient; it requires only 3 to 6 h and can be mediated by on the order of 10 molecules of FRQ per nucleus, a level even less than that seen in the normal oscillation. In contrast, recovery from negative feedback requires 14 to 18 h, most of the circadian cycle, during which time de novo FRQ synthesis has stopped, and existing FRQ is progressively posttranslationally modified. Altogether the time required to complete both of these steps is in good agreement with the 22-h observed period length of the normal circadian cycle.

A fungus among us: the Neurospora crassa circadian system.

Neurospora crassa is the only molecular genetic model system for circadian rhythms research in the fungi. Its strengths as a model organism lie in its relative simplicity--compared to photosynthesizing and vertebrate organisms, it is a stripped-down version of life. It forms syncitial hyphae, propagates and reproduces, and the circadian clock is manifest in numerous processes therein. As with other model circadian systems, Neurospora features a transcription/translation feedback loop that is fundamental to an intact circadian system. The molecular components of this loop converge with those of blue light photoreception, thus bringing the clock and one of its input pathways together.