mPer2 antisense oligonucleotides inhibit mPER2 expression but not circadian rhythms of physiological activity in cultured suprachiasmatic nucleus neurons

Various day-night rhythms, observed at molecular, cellular, and behavioral levels, are governed by an endogenous circadian clock, predominantly functioning in the hypothalamic suprachiasmatic nucleus (SCN). A class of clock genes, mammalian Period (mPer), is known to be rhythmically expressed in SCN neurons, but the correlation between mPER protein levels and autonomous rhythmic activity in SCN neurons is not well understood. Therefore, we blocked mPer translation using antisense phosphothioate oligonucleotides (ODNs) for mPer1 and mPer2 mRNAs and examined the effects on the circadian rhythm of cytosolic Ca2+ concentration and action potentials in SCN slice cultures. Treatment with mPer2 ODNs (20microM for 3 days) but not randomized control ODNs significantly reduced mPER2 immunoreactivity (-63%) in the SCN. Nevertheless, mPer1/2 ODNs treatment inhibited neither action potential firing rhythms nor cytosolic Ca2+ rhythms. These suggest that circadian rhythms in mPER protein levels are not necessarily coupled to autonomous rhythmic activity in SCN neurons.     

Methylation analyses on promoters of mPer1, mPer2, and mCry1 during perinatal development

Recent studies revealed dramatic changes in circadian clock genes’ expression during the perinatal period. In this study, we characterized DNA methylation for three clock genes mPer1, mPer2, and mCry1 at their selected promoter regions during development. Results for the suprachiasmatic nucleus (SCN) and liver (at embryonic day 19, postnatal day 1 and postnatal day 7) were compared to those of sperm. Few methylations were detected for the mPer2 and mCry1 promoters. The 3rd E-box region of the mPer1 promoter exhibited methylation only in sperm. Significant demethylation was observed in the 4th E-box region of the mPer1 promoter between E19 and P1 in the SCN but not in liver tissue. This demethylation state was maintained at P7 for the SCN. Luciferase reporter assays using in vitro methylated promoters revealed an inhibitory effect of promoter methylation on mPer1 expression. The results suggested that epigenetic mechanisms such as DNA methylation might contribute to the developmental expression of clock genes.     

Serine 714 might be implicated in the regulation of the phosphorylation in other areas of mPer1 protein

The phosphorylation of mPer proteins may play important roles in the mechanism of the circadian clock via changes in subcellular localization and degradation. However, the mechanism has remained unclear. Previously, we identified three putative casein kinase (CK)1epsilon phosphorylation motif clusters in mPer1. In this work, we examined the role of the phosphorylation of serine residue, Ser(S)714, in mPer1. mPer1 S[714-726]A mutant, in which potential phosphorylation serine residues replaced by alanine residues, is rapidly phosphorylated compared with wild-type mPer1 by CK1epsilon. Coexpression with S[714]G mutant of mPer1 advanced phase of circadian expression of mPer2-luc expression, which was monitored by in vitro bioluminescence system. This result showed that the mPER1 S[714]G mutation affects circadian core oscillator. Considering these, it seems that Ser 714 might be involved in the regulation of the phosphorylation of other sites in mPer1 by CK1epsilon.     

Identification of mPer1 phosphorylation sites responsible for the nuclear entry

Casein kinase 1 epsilon (CK1 epsilon) is an essential component of the circadian clock in mammals and Drosophila. The phosphorylation of Period (Per) proteins by CK1 epsilon is believed to be implicated in their subcellular localization and degradation, but the precise mechanism by which CK1 epsilon affects Per proteins has not been determined. In this study, three putative CK1 epsilon phosphorylation motif clusters in mouse Per1 (mPer1) were identified, and the phosphorylation status of serine and threonine residues in these clusters was examined. Phosphorylation of residues within a region defined by amino acids 653-663 and in particular of Ser-661 and Ser-663, was identified as responsible for the nuclear translocation of mPer1. Furthermore, phosphorylation of these residues may influence the nuclear translocation of a clock protein complex containing mPer1. These findings indicate that mPer1 phosphorylation is a critical aspect of the circadian clock mechanism.     

Differential effects of two period genes on the physiology and proteomic profiles of mouse anterior tibialis muscles

The molecular components that generate and maintain circadian rhythms of physiology and behavior in mammals are present both in the brain (suprachiasmatic nucleus; SCN) and in peripheral tissues. Examination of mice with targeted disruptions of either mPer1 or mPer2 has shown that these two genes have key roles in the SCN circadian clock. Here we show that loss of the clock gene mPer2 affects forced locomotor performance in mice without altering muscle contractility. A proteomic analysis revealed that the anterior tibialis muscles of the mPer2 knockout mice had higher levels of glycolytic enzymes such as triose phosphate isomerase and enolase than those of either the wild type or mPer1 knockout mice. In addition, the level of expression of HSP90 in the mPer2 mutant mice was also significantly higher than in wildtype mice. These results suggest that the reduced locomotor endurance of the mPer2 knockout mice reflects a greater dependence on anaerobic metabolism under stress conditions, and that the two canonical clock genes, mPer1 and mPer2, play distinct roles in the physiology of skeletal muscle.     

Sleep rhythmicity and homeostasis in mice with targeted disruption of mPeriod genes

In mammals, sleep is regulated by circadian and homeostatic mechanisms. The circadian component, residing in the suprachiasmatic nucleus (SCN), regulates the timing of sleep, whereas homeostatic factors determine the amount of sleep. It is believed that these two processes regulating sleep are independent because sleep amount is unchanged after SCN lesions. However, because such lesions necessarily damage neuronal connectivity, it is preferable to investigate this question in a genetic model that overcomes the confounding influence of circadian rhythmicity. Mice with disruption of both mouse Period genes (mPer)1 and mPer2 have a robust diurnal sleep-wake rhythm in an entrained light-dark cycle but lose rhythmicity in a free-run condition. Here, we examine the role of the mPer genes on the rhythmic and homeostatic regulation of sleep. In entrained conditions, when averaged over the 24-h period, there were no significant differences in waking, slow-wave sleep (SWS), or rapid eye movement (REM) sleep between mPer1, mPer2, mPer3, mPer1-mPer2 double-mutant, and wild-type mice. The mice were then kept awake for 6 h (light period 6-12), and the mPer mutants exhibited increased sleep drive, indicating an intact sleep homeostatic response in the absence of the mPer genes. In free-run conditions (constant darkness), the mPer1-mPer2 double mutants became arrhythmic, but they continued to maintain their sleep levels even after 36 days in free-running conditions. Although mPer1 and mPer2 represent key elements of the molecular clock in the SCN, they are not required for homeostatic regulation of the daily amounts of waking, SWS, or REM sleep.     

Casein Kinase 1 Delta (CK1δ) Regulates Period Length of the Mouse Suprachiasmatic Circadian Clock In Vitro

Background

Casein kinase 1 delta (CK1δ) plays a more prominent role in the regulation of circadian cycle length than its homologue casein kinase 1 epsilon (CK1ε) in peripheral tissues such as liver and embryonic fibroblasts. Mice lacking CK1δ die shortly after birth, so it has not been possible to assess the impact of loss of CK1δ on behavioral rhythms controlled by the master circadian oscillator in the suprachiasmatic nuclei (SCN).

Methodology/Principal Findings

In the present study, mPER2::LUCIFERASE bioluminescence rhythms were monitored from SCN explants collected from neonatal mice. The data demonstrate that SCN explants from neonatal CK1δ-deficient mice oscillate, but with a longer circadian period than littermate controls. The cycle length of rhythms recorded from neonatal SCN explants of CK1ε-deficient mice did not differ from control explants.

Conclusions/Significance

The results indicate that CK1δ plays a more prominent role than CK1ε in the maintenance of 24-hour rhythms in the master circadian oscillator.     

Postmortem stability of melatonin receptor binding and clock-relevant mRNAs in mouse suprachiasmatic nucleus.

The stability of receptor proteins and mRNAs in brain tissue is variable after death. As a prelude to quantitative studies of melatonin receptor density and clock gene expression in the human brain, the stability of these macromolecules was examined in the mouse brain under simulated postmortem conditions using the model of Spokes and Koch. In the mouse suprachiasmatic nucleus (SCN), melatonin receptor binding was significantly reduced after 18 to 24 h under postmortem conditions. Two mRNAs that are rhythmically expressed in the SCN, mPer1 and prepropressophysin (AVP), also decreased significantly over the interval studied, and mPer1 declined more rapidly than AVP. Both mPer1 and AVP mRNA levels in the SCN declined more rapidly in vivo than under postmortem conditions, suggesting that the degradation of these mRNAs is an active process. The results indicate that quantitative studies of melatonin receptor density on human postmortem material are feasible and that detection of rhythmic gene expression in the human SCN will likely require collection of specimens with a rather short (< 8 h) interval from death to tissue collection. The relative stability of melatonin receptor binding in the SCN also suggests that receptor binding may be a reliable marker for the location of the SCN in studies assessing clock gene expression in postmortem material.     

Reorganization of the suprachiasmatic nucleus coding for day length

In mammals, the suprachiasmatic nucleus (SCN), the circadian pacemaker, receives light information via the retina and functions in the entrainment of circadian rhythms and in phasing the seasonal responses of behavioral and physiological functions. To better understand photoperiod-related alterations in the SCN physiology, we analyzed the clock gene expression in the mouse SCN by performing in situ hybridization and real-time monitoring of the mPer1::luc bioluminescence. Under long photoperiod (LP) conditions, the expression rhythms of mPer1 and Bmal1 in the caudal SCN phase-led those in the rostral SCN; further, within the middle SCN, the rhythms in the ventrolateral (VL)-like subdivision advanced compared with those in the dorsomedial (DM)-like subdivision. The mPer1::luc rhythms in the entire coronal slice obtained from the middle SCN exhibited 2 peaks with a wide peak width under LP conditions. Imaging analysis of the mPer1::luc rhythms in several subdivisions of the rostral, middle, caudal, and horizontal SCN revealed wide regional variations in the peak time in the rostral half of the SCN under LP conditions. These variations were not due to alterations in the waveform of a single SCN neuronal rhythm. Our results indicate that LP conditions induce phase changes in the rhythms in multiple regions in the rostral half of the SCN; this leads to different circadian waveforms in the entire SCN, coding for day length.     

The VPAC(2) receptor is essential for circadian function in the mouse suprachiasmatic nuclei

The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are implicated in the photic entrainment of circadian rhythms in the suprachiasmatic nuclei (SCN). We now report that mice carrying a null mutation of the VPAC(2) receptor for VIP and PACAP (Vipr2(-/-)) are incapable of sustaining normal circadian rhythms of rest/activity behavior. These mice also fail to exhibit circadian expression of the core clock genes mPer1, mPer2, and mCry1 and the clock-controlled gene arginine vasopressin (AVP) in the SCN. Moreover, the mutants fail to show acute induction of mPer1 and mPer2 by nocturnal illumination. This study highlights the role of intercellular neuropeptidergic signaling in maintenance of circadian function within the SCN.     

Acamprosate-responsive brain sites for suppression of ethanol intake and preference

Acamprosate suppresses alcohol intake and craving in recovering alcoholics; however, the central sites of its action are unclear. To approach this question, brain regions responsive to acamprosate were mapped using acamprosate microimplants targeted to brain reward and circadian areas implicated in alcohol dependence. mPer2 mutant mice with nonfunctional mPer2, a circadian clock gene that gates endogenous timekeeping, were included, owing to their high levels of ethanol intake and preference. Male wild-type (WT) and mPer2 mutant mice received free-choice (15%) ethanol/water for 3 wk. The ethanol was withdrawn for 3 wk and then reintroduced to facilitate relapse. Four days before ethanol reintroduction, mice received bilateral blank or acamprosate-containing microimplants releasing ～50 ng/day into reward [ventral tegmental (VTA), peduculopontine tegmentum (PPT), and nucleus accumbens (NA)] and circadian [intergeniculate leaflet (IGL) and suprachiasmatic nucleus (SCN)] areas. The hippocampus was also targeted. Circadian locomotor activity was measured throughout. Ethanol intake and preference were greater in mPer2 mutants than in wild-type (WT) mice (27 g·kg(-1)·day(-1) vs. 13 g·kg(-1)·day(-1) and 70% vs. 50%, respectively; both, P < 0.05). In WTs, acamprosate in all areas, except hippocampus, suppressed ethanol intake and preference (by 40-60%) during ethanol reintroduction. In mPer2 mutants, acamprosate in the VTA, PPT, and SCN suppressed ethanol intake and preference by 20-30%. These data are evidence that acamprosate's suppression of ethanol intake and preference are manifest through actions within major reward and circadian sites.     

Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.     

Daily restricted feeding resets the circadian clock in the suprachiasmatic nucleus of CS mice

Circadian rhythms in clock gene expressions in the suprachiasmatic nucleus (SCN) of CS mice and C57BL/6J mice were measured under a daily restricted feeding (RF) schedule in continuous darkness (DD), and entrainment of the SCN circadian pacemaker to RF was examined. After 2-3 wk under a light-dark cycle with free access to food, animals were released into DD and fed for 3 h at a fixed time of day for 3-4 wk. Subsequently, they returned to having free access to food for 2-3 wk. In CS mice, wheel-running rhythms entrained to RF with a stable phase relationship between the activity onset and feeding time, and the rhythms started to free run from the feeding time after the termination of RF. mPer1, mPer2, and mBMAL1 mRNA rhythms in the SCN showed a fixed phase relationship with feeding time, indicating that the circadian pacemaker in the SCN entrained to RF. On the other hand, in C57BL/6J mice, wheel-running rhythms free ran under RF, and clock gene expression rhythms in the SCN showed a stable phase relation not to feeding time but to the behavioral rhythms, indicating that the circadian pacemaker in the SCN did not entrain. These results indicate that the SCN circadian pacemaker of CS mice is entrainable to RF under DD and suggest that CS mice have a circadian clock system that can be reset by a signal associated with feeding time.