A role for clock genes in sleep homeostasis.

The timing and quality of both sleep and wakefulness are thought to be regulated by the interaction of two processes. One of these two processes keeps track of the prior sleep-wake history and controls the homeostatic need for sleep while the other sets the time-of-day that sleep preferably occurs. The molecular pathways underlying the latter, circadian process have been studied in detail and their key role in physiological time-keeping has been well established. Analyses of sleep in mice and flies lacking core circadian clock gene proteins have demonstrated, however, that besides disrupting circadian rhythms, also sleep homeostatic processes were affected. Subsequent studies revealed that sleep loss alters both the mRNA levels and the specific DNA-binding of the key circadian transcriptional regulators to their target sequences in the mouse brain. The fact that sleep loss impinges on the very core of the molecular circadian circuitry might explain why both inadequate sleep and disrupted circadian rhythms can similarly lead to metabolic pathology. The evidence for a role for clock genes in sleep homeostasis will be reviewed here.

Uncovering a role for micro-RNAs in sleep homeostasis and energy metabolism

Micro-RNAs (miRNAs) are key, post-transcriptional regulators of gene expression and have been implicated in almost every cellular process investigated thus far. However, their role in sleep, in particular the homeostatic aspect of sleep control, has received little attention. We here assessed the effects of sleep deprivation on the brain miRNA transcriptome in the mouse. Sleep deprivation affected miRNA expression in a brain-region specific manner. The forebrain expression of the miRNA miR-709 was affected the most and in situ analyses confirmed its robust increase throughout the brain, especially in the cerebral cortex and the hippocampus. The hippocampus was a major target of the sleep deprivation affecting 37 miRNAs compared to 52 in the whole forebrain. Moreover, independent from the sleep deprivation condition, miRNA expression was highly region-specific with 45% of all expressed miRNAs showing higher expression in hippocampus and 55% in cortex. Next we demonstrated that down-regulation of miRNAs in Com/c2o-expressing neurons of adult mice, through a conditional and inducible Dicer knockout mice model (cKO), results in an altered homeostatic response after sleep deprivation eight weeks following the tamoxifen-induced recombination. Dicer cKO mice showed a larger increase in the electro-encephalographic (EEG) marker of sleep pressure, EEG delta power, and a reduced Rapid Eye Movement sleep rebound, compared to controls, highlighting a functional role of miRNAs in sleep homeostasis. Beside a sleep phenotype, Dicer cKO mice developed an unexpected, severe obesity phenotype associated with hyperphagia and altered metabolism. Even more surprisingly, after reaching maximum body weight 5 weeks after tamoxifen injection, obese cKO mice spontaneously started losing weight as rapidly as it was gained. Brain transcriptome analyses in obese mice identified several obesity-related pathways (e.g. leptin, somatostatin, and nemo-like kinase signaling), as well as genes involved in feeding and appetite (e.g. Pmch, Neurotensin). A gene cluster with anti-correlated expression in the cerebral cortex of post-obese compared to obese mice was enriched for synaptic plasticity pathways. While other studies have identified a role for miRNAs in obesity, we here present a unique model that allows for the study of processes involved in reversing obesity. Moreover, our study identified the cortex as a brain area important for body weight homeostasis. Together, these observations strongly suggest a role for miRNAs in the maintenance of homeostatic processes in the mouse, and support the hypothesis of a tight relationship between sleep and metabolism at a molecular - Les micro-ARNS (miARNs) sont des régulateurs post-transcriptionnels de l'expression des gènes, impliqués dans la quasi-totalité des processus cellulaires. Cependant, leur rôle dans la régulation du sommeil, et en particulier dans le maintien de l'homéostasie du sommeil, n'a reçu que très peu d'attention jusqu'à présent. Dans cette étude, nous avons étudié les conséquences d'une privation de sommeil sur l'expression cérébrale des miARNs chez la souris, et observé des changements dans l'expression de nombreux miARNs. Dans le cerveau antérieur, miR-709 est le miARN le plus affecté par la perte de sommeil, en particulier dans le cortex cérébral et l'hippocampe. L'hippocampe est la région la plus touchée avec 37 miARNs changés comparés à 52 dans le cerveau entier. Par ailleurs, indépendamment de la privation de sommeil, certains miARNs sont spécifiquement enrichis dans certaines aires cérébrales, 45% des miARNs étant surexprimés dans l'hippocampe contre 55% dans le cortex. Dans une seconde étude, nous avons observé que la délétion de DICER, enzyme essentielle à la biosynthèse des miARNs, et la perte subséquente des miARNs dans les neurones exprimant la protéine CAMK2a altère la réponse homéostatique à une privation de sommeil, 8 semaines après l'induction de la recombinaison génétique par le tamoxifen. Les souris sans Dicer (cKO) ont une plus large augmentation de l'EEG delta power, le principal marqueur électro-encéphalographique du besoin de sommeil, comparée aux contrôles, ainsi qu'un rebond en sommeil paradoxal plus petit. De façon surprenante, les souris Dicer cKO développent une obésité rapide, sévère et transitoire, associée à de l'hyperphagie et une altération de leur métabolisme énergétique. Après avoir atteint un pic maximal d'obésité, les souris cKO entrent spontanément dans une période de perte de poids rapide. L'analyse du transcriptome cérébral des souris obèses nous a permis d'identifier des voies associées à l'obésité (leptine, somatostatine et nemo-like kinase), et à la prise alimentaire (Pmch, Neurotensin), tandis que celui des souris post-obèses a révélé un groupe de gènes liés à la plasticité synaptique. Au-delà des nombreux modèles d'obésité existant chez la souris, notre étude présente un modèle unique permettant d'étudier les mécanismes sous-jacent la perte de poids. De plus, nous avons mis en évidence un rôle important du cortex cérébral dans le maintien de la balance énergétique. En conclusion, toutes ces observations soutiennent l'idée que les miARNs sont des régulateurs cruciaux dans le maintien des processus homéostatiques et confortent l'hypothèse d'une étroite relation moléculaire entre le sommeil et le métabolisme.

A non-circadian role for clock-genes in sleep homeostasis: a strain comparison.

BACKGROUND: We have previously reported that the expression of circadian clock-genes increases in the cerebral cortex after sleep deprivation (SD) and that the sleep rebound following SD is attenuated in mice deficient for one or more clock-genes. We hypothesized that besides generating circadian rhythms, clock-genes also play a role in the homeostatic regulation of sleep. Here we follow the time course of the forebrain changes in the expression of the clock-genes period (per)-1, per2, and of the clock-controlled gene albumin D-binding protein (dbp) during a 6 h SD and subsequent recovery sleep in three inbred strains of mice for which the homeostatic sleep rebound following SD differs. We reasoned that if clock genes are functionally implicated in sleep homeostasis then the SD-induced changes in gene expression should vary according to the genotypic differences in the sleep rebound. RESULTS: In all three strains per expression was increased when animals were kept awake but the rate of increase during the SD as well as the relative increase in per after 6 h SD were highest in the strain for which the sleep rebound was smallest; i.e., DBA/2J (D2). Moreover, whereas in the other two strains per1 and per2 reverted to control levels with recovery sleep, per2 expression specifically, remained elevated in D2 mice. dbp expression increased during the light period both during baseline and during SD although levels were reduced during the latter condition compared to baseline. In contrast to per2, dbp expression reverted to control levels with recovery sleep in D2 only, whereas in the two other strains expression remained decreased. CONCLUSION: These findings support and extend our previous findings that clock genes in the forebrain are implicated in the homeostatic regulation of sleep and suggest that sustained, high levels of per2 expression may negatively impact recovery sleep.

Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice.

Light influences sleep and alertness either indirectly through a well-characterized circadian pathway or directly through yet poorly understood mechanisms. Melanopsin (Opn4) is a retinal photopigment crucial for conveying nonvisual light information to the brain. Through extensive characterization of sleep and the electrocorticogram (ECoG) in melanopsin-deficient (Opn4(-/-)) mice under various light-dark (LD) schedules, we assessed the role of melanopsin in mediating the effects of light on sleep and ECoG activity. In control mice, a light pulse given during the habitual dark period readily induced sleep, whereas a dark pulse given during the habitual light period induced waking with pronounced theta (7-10 Hz) and gamma (40-70 Hz) activity, the ECoG correlates of alertness. In contrast, light failed to induce sleep in Opn4(-/-) mice, and the dark-pulse-induced increase in theta and gamma activity was delayed. A 24-h recording under a LD 1-hratio1-h schedule revealed that the failure to respond to light in Opn4(-/-) mice was restricted to the subjective dark period. Light induced c-Fos immunoreactivity in the suprachiasmatic nuclei (SCN) and in sleep-active ventrolateral preoptic (VLPO) neurons was importantly reduced in Opn4(-/-) mice, implicating both sleep-regulatory structures in the melanopsin-mediated effects of light. In addition to these acute light effects, Opn4(-/-) mice slept 1 h less during the 12-h light period of a LD 12ratio12 schedule owing to a lengthening of waking bouts. Despite this reduction in sleep time, ECoG delta power, a marker of sleep need, was decreased in Opn4(-/-) mice for most of the (subjective) dark period. Delta power reached after a 6-h sleep deprivation was similarly reduced in Opn4(-/-) mice. In mice, melanopsin's contribution to the direct effects of light on sleep is limited to the dark or active period, suggesting that at this circadian phase, melanopsin compensates for circadian variations in the photo sensitivity of other light-encoding pathways such as rod and cones. Our study, furthermore, demonstrates that lack of melanopsin alters sleep homeostasis. These findings call for a reevaluation of the role of light on mammalian physiology and behavior.

Separating the contribution of glucocorticoids and wakefulness to the molecular and electrophysiological correlates of sleep homeostasis.

Study Objectives: The sleep-deprivation-induced changes in delta power, an electroencephalographical correlate of sleep need, and brain transcriptome profiles have importantly contributed to current hypotheses on sleep function. Because sleep deprivation also induces stress, we here determined the contribution of the corticosterone component of the stress response to the electrophysiological and molecular markers of sleep need in mice. Design: N/A Settings: Mouse sleep facility. Participants: C57BL/6J, AKR/J, DBA/2J mice. Interventions: Sleep deprivation, adrenalectomy (ADX). Measurements and Results: Sleep deprivation elevated corticosterone levels in 3 inbred strains, but this increase was larger in DBA/2J mice; i.e., the strain for which the rebound in delta power after sleep deprivation failed to reach significance. Elimination of the sleep-deprivation-associated corticosterone surge through ADX in DBA/2J mice did not, however, rescue the delta power rebound but did greatly reduce the number of transcripts affected by sleep deprivation. Genes no longer affected by sleep deprivation cover pathways previously implicated in sleep homeostasis, such as lipid, cholesterol (e.g., Ldlr, Hmgcs1, Dhcr7, -24, Fkbp5), energy and carbohydrate metabolism (e.g., Eno3, G6pc3, Mpdu1, Ugdh, Man1b1), protein biosynthesis (e.g., Sgk1, Alad, Fads3, Eif2c2, -3, Mat2a), and some circadian genes (Per1, -3), whereas others, such as Homer1a, remained unchanged. Moreover, several microRNAs were affected both by sleep deprivation and ADX. Conclusions: Our findings indicate that corticosterone contributes to the sleep-deprivation-induced changes in brain transcriptome that have been attributed to wakefulness per se. The study identified 78 transcripts that respond to sleep loss independent of corticosterone and time of day, among which genes involved in neuroprotection prominently feature, pointing to a molecular pathway directly relevant for sleep function.

Circadian clock genes and sleep homeostasis.

Circadian and sleep-homeostatic processes both contribute to sleep timing and sleep structure. Elimination of circadian rhythms through lesions of the suprachiasmatic nuclei (SCN), the master circadian pacemaker, leads to fragmentation of wakefulness and sleep but does not eliminate the homeostatic response to sleep loss as indexed by the increase in EEG delta power. In humans, EEG delta power declines during sleep episodes nearly independently of circadian phase. Such observations have contributed to the prevailing notion that circadian and homeostatic processes are separate but recent data imply that this segregation may not extend to the molecular level. Here we summarize the criteria and evidence for a role for clock genes in sleep homeostasis. Studies in mice with targeted disruption for core circadian clock genes have revealed alterations in circadian rhythmicity as well as changes in sleep duration, sleep structure and EEG delta power. Clock-gene expression in brain areas outside the SCN, in particular the cerebral cortex, depends to a large extent on prior sleep-wake history. Evidence for effects of clock genes on sleep homeostasis has also been obtained in Drosophila and humans, pointing to a phylogenetically preserved pathway. These findings suggest that, while within the SCN clock genes are utilized to set internal time-of-day, in the forebrain the same feedback circuitry may be utilized to track time spent awake and asleep. The mechanisms by which clock-gene expression is coupled to the sleep-wake distribution could be through cellular energy charge whereby clock genes act as energy sensors. The data underscore the interrelationships between energy metabolism, circadian rhythmicity, and sleep regulation.

CD40 activation induces NREM sleep and modulates genes associated with sleep homeostasis.

The T-cell derived cytokine CD40 ligand is overexpressed in patients with autoimmune diseases. Through activation of its receptor, CD40 ligand leads to a tumor necrosis factor (TNF) receptor 1 (TNFR1) dependent impairment of locomotor activity in mice. Here we report that this effect is explained through a promotion of sleep, which was specific to non-rapid eye movement (NREM) sleep while REM sleep was suppressed. The increase in NREM sleep was accompanied by a decrease in EEG delta power during NREM sleep and by a decrease in the expression of transcripts in the cerebral cortex known to be associated with homeostatic sleep drive, such as Homer1a, Early growth response 2, Neuronal pentraxin 2, and Fos-like antigen 2. The effect of CD40 activation was mimicked by peripheral TNF injection and prevented by the TNF blocker etanercept. Our study indicates that sleep-wake dysregulation in autoimmune diseases may result from CD40 induced TNF:TNFR1 mediated alterations of molecular pathways, which regulate sleep-wake behavior.

Sustained sleep fragmentation induces sleep homeostasis in mice.

STUDY OBJECTIVES: Sleep fragmentation (SF) is an integral feature of sleep apnea and other prevalent sleep disorders. Although the effect of repetitive arousals on cognitive performance is well documented, the effects of long-term SF on electroencephalography (EEG) and molecular markers of sleep homeostasis remain poorly investigated. To address this question, we developed a mouse model of chronic SF and characterized its effect on EEG spectral frequencies and the expression of genes previously linked to sleep homeostasis including clock genes, heat shock proteins, and plasticity-related genes. DESIGN: N/A. SETTING: Animal sleep research laboratory. PARTICIPANTS: Sixty-six C57BL6/J adult mice. INTERVENTIONS: Instrumental sleep disruption at a rate of 60/h during 14 days. MEASUREMENTS AND RESULTS: Locomotor activity and EEG were recorded during 14 days of SF followed by recovery for 2 days. Despite a dramatic number of arousals and decreased sleep bout duration, SF minimally reduced total quantity of sleep and did not significantly alter its circadian distribution. Spectral analysis during SF revealed a homeostatic drive for slow wave activity (SWA; 1-4 Hz) and other frequencies as well (4-40 Hz). Recordings during recovery revealed slow wave sleep consolidation and a transient rebound in SWA, and paradoxical sleep duration. The expression of selected genes was not induced following chronic SF. CONCLUSIONS: Chronic SF increased sleep pressure confirming that altered quality with preserved quantity triggers core sleep homeostasis mechanisms. However, it did not induce the expression of genes induced by sleep loss, suggesting that these molecular pathways are not sustainably activated in chronic diseases involving SF.

Genetic dissection of sleep homeostasis.

Sleep is a complex behavior both in its manifestation and regulation, that is common to almost all animal species studied thus far. Sleep is not a unitary behavior and has many different aspects, each of which is tightly regulated and influenced by both genetic and environmental factors. Despite its essential role for performance, health, and well-being, genetic mechanisms underlying this complex behavior remain poorly understood. One important aspect of sleep concerns its homeostatic regulation, which ensures that levels of sleep need are kept within a range still allowing optimal functioning during wakefulness. Uncovering the genetic pathways underlying the homeostatic aspect of sleep is of particular importance because it could lead to insights concerning sleep's still elusive function and is therefore a main focus of current sleep research. In this chapter, we first give a definition of sleep homeostasis and describe the molecular genetics techniques that are used to examine it. We then provide a conceptual discussion on the problem of assessing a sleep homeostatic phenotype in various animal models. We finally highlight some of the studies with a focus on clock genes and adenosine signaling molecules.

Altered Sleep Homeostasis in Rev-erbα Knockout Mice.

STUDY OBJECTIVES: The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep. METHODS: EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline. RESULTS: Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1-4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain. CONCLUSIONS: Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context.

CD40 activation induces NREM sleep and modulates genes associated with sleep homeostasis

The T-cell derived cytokine CD40 ligand is overexpressed in patients with autoimmune diseases. Through activation of its receptor, CD40 ligand leads to a tumor necrosis factor (TNF) receptor 1 (TNFR1) dependent impairment of locomotor activity in mice. Here we report that this effect is explained through a promotion of sleep, which was specific to non-rapid eye movement (NREM) sleep while REM sleep was suppressed. The increase in NREM sleep was accompanied by a decrease in EEG delta power during NREM sleep and by a decrease in the expression of transcripts in the cerebral cortex known to be associated with homeostatic sleep drive, such as Homer1a, Early growth response 2, Neuronal pentraxin 2, and Fos-like antigen 2. The effect of CD40 activation was mimicked by peripheral TNF injection and prevented by the TNF blocker etanercept. Our study indicates that sleep-wake dysregulation in autoimmune diseases may result from CD40 induced TNF:TNFR1 mediated alterations of molecular pathways, which regulate sleep-wake behavior.

Sleep loss reduces the DNA-binding of BMAL1, CLOCK, and NPAS2 to specific clock genes in the mouse cerebral cortex.

We have previously demonstrated that clock genes contribute to the homeostatic aspect of sleep regulation. Indeed, mutations in some clock genes modify the markers of sleep homeostasis and an increase in homeostatic sleep drive alters clock gene expression in the forebrain. Here, we investigate a possible mechanism by which sleep deprivation (SD) could alter clock gene expression by quantifying DNA-binding of the core-clock transcription factors CLOCK, NPAS2, and BMAL1 to the cis-regulatory sequences of target clock genes in mice. Using chromatin immunoprecipitation (ChIP), we first showed that, as reported for the liver, DNA-binding of CLOCK and BMAL1 to target clock genes changes in function of time-of-day in the cerebral cortex. Tissue extracts were collected at ZT0 (light onset), -6, -12, and -18, and DNA enrichment of E-box or E'-box containing sequences was measured by qPCR. CLOCK and BMAL1 binding to Cry1, Dbp, Per1, and Per2 depended on time-of-day, with maximum values reached at around ZT6. We then observed that SD, performed between ZT0 and -6, significantly decreased DNA-binding of CLOCK and BMAL1 to Dbp, consistent with the observed decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was also decreased by SD, although SD is known to increase Per2 expression in the cortex. DNA-binding to Per1 and Cry1 was not affected by SD. Our results show that the sleep-wake history can affect the clock molecular machinery directly at the level of chromatin binding thereby altering the cortical expression of Dbp and Per2 and likely other targets. Although the precise dynamics of the relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive, the results also suggest that part of the reported circadian changes in DNA-binding of core clock components in tissues peripheral to the suprachiasmatic nuclei could, in fact, be sleep-wake driven.

Sleep deprivation effects on circadian clock gene expression in the cerebral cortex parallel electroencephalographic differences among mouse strains.

Sleep deprivation (SD) results in increased electroencephalographic (EEG) delta power during subsequent non-rapid eye movement sleep (NREMS) and is associated with changes in the expression of circadian clock-related genes in the cerebral cortex. The increase of NREMS delta power as a function of previous wake duration varies among inbred mouse strains. We sought to determine whether SD-dependent changes in circadian clock gene expression parallel this strain difference described previously at the EEG level. The effects of enforced wakefulness of incremental durations of up to 6 h on the expression of circadian clock genes (bmal1, clock, cry1, cry2, csnk1epsilon, npas2, per1, and per2) were assessed in AKR/J, C57BL/6J, and DBA/2J mice, three strains that exhibit distinct EEG responses to SD. Cortical expression of clock genes subsequent to SD was proportional to the increase in delta power that occurs in inbred strains: the strain that exhibits the most robust EEG response to SD (AKR/J) exhibited dramatic increases in expression of bmal1, clock, cry2, csnkIepsilon, and npas2, whereas the strain with the least robust response to SD (DBA/2) exhibited either no change or a decrease in expression of these genes and cry1. The effect of SD on circadian clock gene expression was maintained in mice in which both of the cryptochrome genes were genetically inactivated. cry1 and cry2 appear to be redundant in sleep regulation as elimination of either of these genes did not result in a significant deficit in sleep homeostasis. These data demonstrate transcriptional regulatory correlates to previously described strain differences at the EEG level and raise the possibility that genetic differences underlying circadian clock gene expression may drive the EEG differences among these strains.