Understanding circadian rhythmicity in Neurospora crassa: from behavior to genes and back again

Circadian clocks have been described in organisms ranging in complexity from unicells to mammals, in which they function to control daily rhythms in cellular activities and behavior. The significance of a detailed understanding of the clock can be appreciated by its ubiquity and its established involvement in human physiology, including endocrine function, sleep/wake cycles, psychiatric illness, and drug tolerances and effectiveness. Because the clock in all organisms is assembled within the cell and clock mechanisms are evolutionarily conserved, simple eukaryotes provide appropriate experimental systems for dissecting the clock. Significant progress has been made in deciphering the circadian system in Neurospora crassa using both genetic and molecular approaches, and Neurospora has contributed greatly to our understanding of (1) the feedback cycle that comprises a circadian oscillator, (2) the mechanisms by which the clock is kept in synchrony with the environment, and (3) the genes that reside in rhythmic output pathways. Importantly, the lessons learned in Neurospora are relevant to our understanding of clocks in higher eukaryotes.     

Analysis of the molecular pathophysiology of sleep disorders relevant to a disturbed biological clock

Genetic studies have revealed several clock gene variations/mutations involved in the manifestation of sleep disorders or interindividual differences in sleep–wake patterns, but only part of the genetic risk can be explained by the gene variations/mutations identified to date. Recent progress in research into circadian rhythm generation has provided efficient tools for eliciting the molecular basis of clock-relevant sleep disorders, complementing traditional genetic analysis. While the human master clock resides in the suprachiasmatic nucleus of the hypothalamus (central clock), peripheral tissue cells also generate self-sustained circadian oscillations of clock gene expression (peripheral clock), enabling estimation of individual human clock properties through a single collection of skin fibroblasts or venous blood cells. Some of the established cell lines exhibit autonomous circadian oscillations of clock gene expression, and introduction of clock gene variations into these cell lines by gene targeting makes it possible to investigate changes in the circadian phenotype induced by these variations/mutations without the need for generating transgenic animals. Estimation of human clock properties using peripheral tissue cells, in addition to genetic analysis, will facilitate comprehensive explication of the genetic risk of a variety of disorders relevant to biological clock disturbances, including sleep disorders, mood disorders, and metabolic diseases.     

Solving the mystery of human sleep schedules one mutation at a time

Abstract

Sleep behavior remains one of the most enigmatic areas of life. The unanswered questions range from “why do we sleep?” to “how we can improve sleep in today’s society?” Identification of mutations responsible for altered circadian regulation of human sleep lead to unique opportunities for probing these territories. In this review, we summarize causative circadian mutations found from familial genetic studies to date. We also describe how these mutations mechanistically affect circadian function and lead to altered sleep behaviors, including shifted or shortening of sleep patterns. In addition, we discuss how the investigation of mutations can not only expand our understanding of the molecular mechanisms regulating the circadian clock and sleep duration, but also bridge the pathways between clock/sleep and other human physiological conditions and ailments such as metabolic regulation and migraine headaches.     

Neural organization of the circadian system of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae was the first animal in which lesion experiments localized an endogenous circadian clock to a particular brain area, the optic lobe. The neural organization of the circadian system, however, including entrainment pathways, coupling elements of the bilaterally distributed internal clock, and output pathways controlling circadian locomotor rhythms are only recently beginning to be elucidated. As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreac- tive neurons of the accessory medulla of the cockroach are crucial elements of the circadian system. Lesions and transplantation experiments showed that the endogeneous circadian clock of the brain resides in neurons associated with the accessory medulla. The accessory medulla is organized into a nodular core receiving photic input, and into internodular and peripheral neuropil involved in efferent output and coupling input. Photic entrainment of the clock through compound eye photoreceptors appears to occur via parallel, indirect pathways through the medulla. Light-like phase shifts in circadian locomotor activity after injections of γ-aminobutyric acid (GABA)- or Mas-allatotropin into the vicinity of the accessory medulla suggest that both substances are involved in photic entrainment. Extraocular, cryptochrome-based photoreceptors appear to be present in the optic lobe, but their role in photic entrainment has not been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent output from the accessory medulla to several brain areas and to the peripheral visual system. Pigment-dispersing hormone-immunoreactive neurons, and additional heterolateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers. The neuronal organization, as well as the prominent involvement of GABA and neuropeptides, shows striking similarities to the organization of the suprachiasmatic nucleus, the circadian clock of the mammalian brain.     

Behavioral and Serotonergic Regulation of Circadian Rhythms

Endogenous depression is often accompanied by alterations in core parameters of circadian rhythms, and antidepressant treatments, including serotonergic drugs, sleep deprivation and exercise, alter circadian phase or period in humans or animal models. Antidepressants may act in part through the circadian system, and behavioral antidepressants through a common serotonergic path to the clock. This review evaluates the evidence from animal models that serotonin (5-HT) mediates phase-shifting effects of behavioral stimuli on circadian rhythms. In rodents, 'exercise' stimulated during the rest phase of the rest-activity cycle induces large phase shifts of circadian rhythms. These shifts can be mimicked by short-term sleep deprivation without intense activity. During wheel running or sleep deprivation, 5-HT release in the suprachiasmatic nucleus (SCN) circadian clock is significantly elevated. Lesions of 5-HT afferents to the SCN attenuate phase shifts or entrainment induced by activity in response to some stimuli (e.g., triazolam injections in hamsters, treadmill running in mice) but not others (e.g., novel wheel confinement in hamsters). Antagonists selective to 5HT_{1, 2} or ₇ receptors do not attenuate shifts induced by wheel running, although 5-HT_2/7 antagonists do partially block shifts to saline injections. 5-HT agonists (e.g., 8-OH-DPAT) induce large shifts in vitro, but much smaller shifts in vivo, particularly if administered directly to the SCN. Procedures for inducing 5-HT supersensitivity in vivo result in larger shifts to 8-OH-DPAT. 5-HT stimuli may affect the clock by direct and indirect pathways, particularly through the thalamic intergeniculate leaflet, and the role of these pathways may differ across species. At the level of the SCN, 5-HT likely acts through 5-HT₇ receptors on neurons and possibly also glial cells. These receptors may be useful targets for the development of antidepressant drugs. In aggregate, the literature provides mixed support for the hypothesis that exercise or behavioral arousal shift the circadian clock by a 5-HT pathway; the role of indirect pathways, interactions with other transmitters, cellular adaptations to denervation, glial cells, and species differences remain to be more fully clarified. Serotonergic and behavioral stimuli provide an intriguing route to elucidate the circadian clockworks and their possible role in depression.     

Drosophila lacking dfmr1 activity show defects in circadian output and fail to maintain courtship interest

Fragile X mental retardation is a prominent genetic disorder caused by the lack of the FMR1 gene product, a known RNA binding protein. Specific physiologic pathways regulated by FMR1 function have yet to be identified. Adult dfmr1 (also called dfxr) mutant flies display arrhythmic circadian activity and have erratic patterns of locomotor activity, whereas overexpression of dFMR1 leads to a lengthened period. dfmr1 mutant males also display reduced courtship activity which appears to result from their inability to maintain courtship interest. Molecular analysis fails to reveal any defects in the expression of clock components; however, the CREB output is affected. Morphological analysis of neurons required for normal circadian behavior reveals subtle abnormalities, suggesting that defects in axonal pathfinding or synapse formation may cause the observed behavioral defects.     

Cul3 and the BTB Adaptor Insomniac Are Key Regulators of Sleep Homeostasis and a Dopamine Arousal Pathway in Drosophila

Sleep is homeostatically regulated, such that sleep drive reflects the duration of prior wakefulness. However, despite the discovery of genes important for sleep, a coherent molecular model for sleep homeostasis has yet to emerge. To better understand the function and regulation of sleep, we employed a reverse-genetics approach in Drosophila. An insertion in the BTB domain protein CG32810/insomniac (inc) exhibited one of the strongest baseline sleep phenotypes thus far observed, a ∼10 h sleep reduction. Importantly, this is coupled to a reduced homeostatic response to sleep deprivation, consistent with a disrupted sleep homeostat. Knockdown of the INC-interacting protein, the E3 ubiquitin ligase Cul3, results in reduced sleep duration, consolidation, and homeostasis, suggesting an important role for protein turnover in mediating INC effects. Interestingly, inc and Cul3 expression in post-mitotic neurons during development contributes to their adult sleep functions. Similar to flies with increased dopaminergic signaling, loss of inc and Cul3 result in hyper-arousability to a mechanical stimulus in adult flies. Furthermore, the inc sleep duration phenotype can be rescued by pharmacological inhibition of tyrosine hydroxylase, the rate-limiting enzyme for dopamine biosynthesis. Taken together, these results establish inc and Cul3 as important new players in setting the sleep homeostat and a dopaminergic arousal pathway in Drosophila.     

Behavioral and Serotonergic Regulation of Circadian Rhythms

Endogenous depression is often accompanied by alterations in core parameters of circadian rhythms, and antidepressant treatments, including serotonergic drugs, sleep deprivation and exercise, alter circadian phase or period in humans or animal models. Antidepressants may act in part through the circadian system, and behavioral antidepressants through a common serotonergic path to the clock. This review evaluates the evidence from animal models that serotonin (5-HT) mediates phase-shifting effects of behavioral stimuli on circadian rhythms. In rodents, 'exercise' stimulated during the rest phase of the rest-activity cycle induces large phase shifts of circadian rhythms. These shifts can be mimicked by short-term sleep deprivation without intense activity. During wheel running or sleep deprivation, 5-HT release in the suprachiasmatic nucleus (SCN) circadian clock is significantly elevated. Lesions of 5-HT afferents to the SCN attenuate phase shifts or entrainment induced by activity in response to some stimuli (e.g., triazolam injections in hamsters, treadmill running in mice) but not others (e.g., novel wheel confinement in hamsters). Antagonists selective to 5HT_{1, 2} or ₇ receptors do not attenuate shifts induced by wheel running, although 5-HT_2/7 antagonists do partially block shifts to saline injections. 5-HT agonists (e.g., 8-OH-DPAT) induce large shifts in vitro, but much smaller shifts in vivo, particularly if administered directly to the SCN. Procedures for inducing 5-HT supersensitivity in vivo result in larger shifts to 8-OH-DPAT. 5-HT stimuli may affect the clock by direct and indirect pathways, particularly through the thalamic intergeniculate leaflet, and the role of these pathways may differ across species. At the level of the SCN, 5-HT likely acts through 5-HT₇ receptors on neurons and possibly also glial cells. These receptors may be useful targets for the development of antidepressant drugs. In aggregate, the literature provides mixed support for the hypothesis that exercise or behavioral arousal shift the circadian clock by a 5-HT pathway; the role of indirect pathways, interactions with other transmitters, cellular adaptations to denervation, glial cells, and species differences remain to be more fully clarified. Serotonergic and behavioral stimuli provide an intriguing route to elucidate the circadian clockworks and their possible role in depression.     

An emerging role for Cullin-3 mediated ubiquitination in sleep and circadian rhythm

Although the neurophysiological correlates of sleep have been thoroughly described, genetic mechanisms that control sleep architecture, long surmised from ethological studies, family histories and clinical observations, have only been investigated during the past decade. Key contributions to the molecular understanding of sleep have come from studies in Drosophila, benefitting from a strong history of circadian rhythm research. For instance, a number of recent papers have highlighted the role of the E3 ubiquitin ligase Cullin-3 in the regulation of circadian rhythm and sleep. We propose that different Cullin-3 substrate adaptors may affect specific molecular pathways and diverse aspects of circadian rhythm and sleep. We have previously shown that mutations in BTBD9, a risk factor for Restless Legs Syndrome (RLS) encoding a Cullin-3 substrate adaptor, lead to reduced dopamine, increased locomotion and sleep fragmentation. Here, we propose that Cullin-3 acts together with BTBD9 to limit the accumulation of iron regulatory proteins in conditions of iron deficiency. Our model is consistent with clinical observations implicating iron homeostasis in the pathophysiology of RLS and predicts that lack of BTBD9 leads to misregulation of cellular iron storage, inactivating the critical biosynthetic enzyme Tyrosine Hydroxylase in dopaminergic neurons, with consequent phenotypic effects on sleep.     

Old flies have a robust central oscillator but weaker behavioral rhythms that can be improved by genetic and environmental manipulations

Sleep-wake cycles break down with age, but the causes of this degeneration are not clear. Using a Drosophila model, we addressed the contribution of circadian mechanisms to this age-induced deterioration. We found that in old flies, free-running circadian rhythms (behavioral rhythms assayed in constant darkness) have a longer period and an unstable phase before they eventually degenerate. Surprisingly, rhythms are weaker in light-dark cycles and the circadian-regulated morning peak of activity is diminished under these conditions. On a molecular level, aging results in reduced amplitude of circadian clock gene expression in peripheral tissues. However, oscillations of the clock protein PERIOD (PER) are robust and synchronized among different clock neurons, even in very old, arrhythmic flies. To improve rhythms in old flies, we manipulated environmental conditions, which can have direct effects on behavior, and also tested a role for molecules that act downstream of the clock. Coupling temperature cycles with a light-dark schedule or reducing expression of protein kinase A (PKA) improved behavioral rhythms and consolidated sleep. Our data demonstrate that a robust molecular timekeeping mechanism persists in the central pacemaker of aged flies, and reducing PKA can strengthen behavioral rhythms.     

Allatostatin A Signalling in Drosophila Regulates Feeding and Sleep and Is Modulated by PDF

Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. We show that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing PLP neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. We further identify the PLP neurons as a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, our results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF.     

昆虫生物钟分子调控研究进展

昆虫生物钟节律的研究是人类了解生物节律的重要途径。昆虫在生理和行为上具有广泛的节律活动,如运动、睡眠、学习记忆、交配、嗅觉等节律活动,其中昼夜活动行为节律的研究广泛而深入。昆虫乃至高等动物普遍具有保守的昼夜节律系统,昼夜生物钟节律主要包括输入系统:用于接受外界光和温度等环境信号并传入核心振荡器,使得生物时钟与环境同步;核心时钟系统:自我维持的昼夜振荡器;输出系统:将生物钟产生的信号传递出去而控制生物行为和生理的节律变化。早期分子和遗传学研究主要关注昼夜节律振荡器的分子机制及神经生物学,阐明了昼夜生物钟节律的主要分子机制及相关神经网络。最近更多的研究关注生物钟信号是如何输入和输出。本文以果蝇运动节律的相关研究为主要内容,围绕生物钟输入系统、振荡器、输出系统这3个组成部分对昆虫生物钟研究进展进行总结。     

Social jetlag and obesity

Obesity has reached crisis proportions in industrialized societies. Many factors converge to yield increased body mass index (BMI). Among these is sleep duration. The circadian clock controls sleep timing through the process of entrainment. Chronotype describes individual differences in sleep timing, and it is determined by genetic background, age, sex, and environment (e.g., light exposure). Social jetlag quantifies the discrepancy that often arises between circadian and social clocks, which results in chronic sleep loss. The circadian clock also regulates energy homeostasis, and its disruption-as with social jetlag-may contribute to weight-related pathologies. Here, we report the results from a large-scale epidemiological study, showing that, beyond sleep duration, social jetlag is associated with increased BMI. Our results demonstrate that living "against the clock" may be a factor contributing to the epidemic of obesity. This is of key importance in pending discussions on the implementation of Daylight Saving Time and on work or school times, which all contribute to the amount of social jetlag accrued by an individual. Our data suggest that improving the correspondence between biological and social clocks will contribute to the management of obesity.     

Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development

The visual system is one of the input pathways for light into the circadian clock of the Drosophila brain. In particular, extra-retinal visual structures have been proposed to play a role in both larval and adult circadian photoreception. We have analyzed the interactions between extra-retinal structures of the visual system and the clock neurons during brain development. We first show that the larval optic nerve, or Bolwig nerve, already contacts clock cells (the lateral neurons) in the embryonic brain. Analysis of visual system-defective genotypes showed that the absence of the afferent Bolwig nerve resulted in a severe reduction of the lateral neurons dendritic arborization, and that the inhibition of nerve activity induced alterations of the dendritic morphology. During wild-type development, the loss of a functional Bolwig nerve in the early pupa was also accompanied by remodeling of the arborization of the lateral neurons. Approximately 1.5 days later, visual fibers that came from the Hofbauer-Buchner eyelet, a putative photoreceptive organ for the adult circadian clock, were seen contacting the lateral neurons. Both types of extra-retinal photoreceptors expressed rhodopsins RH5 and RH6, as well as the norpA-encoded phospholipase C. These data strongly suggest a role for RH5 and RH6, as well as NORPA, signaling in both larval and adult extra-retinal circadian photoreception. The Hofbauer-Buchner eyelet therefore does not appear to account for the previously described norpA-independent light input to the adult clock. This supports the existence of yet uncharacterized photoreceptive structures in Drosophila.