PACAP-deficient mice exhibit light parameter-dependent abnormalities on nonvisual photoreception and early activity onset.

BACKGROUND: The photopigment melanopsin has been suggested to act as a dominant photoreceptor in nonvisual photoreception including resetting of the circadian clock (entrainment), direct tuning or masking of vital status (activity, sleep/wake cycles, etc.), and the pupillary light reflex (PLR). Pituitary adenylate cyclase-activating polypeptide (PACAP) is exclusively coexpressed with melanopsin in a small subset of retinal ganglion cells and is predicted to be involved extensively in these responses; however, there were inconsistencies in the previous reports, and its functional role has not been well understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that PACAP-deficient mice exhibited severe dysfunctions of entrainment in a time-dependent manner. The abnormalities in the mutant mice were intensity-dependent in phase delay and duration-dependent in phase advance. The knockout mice also displayed blunted masking, which was dependent on lighting conditions, but not completely lost. The dysfunctions of masking in the mutant mice were recovered by infusion of PACAP-38. By contrast, these mutant mice show a normal PLR. We examined the retinal morphology and innervations in the mutant mice, and no apparent changes were observed in melanopsin-immunoreactive cells. These data suggest that the dysfunctions of entrainment and masking were caused by the loss of PACAP, not by the loss of light input itself. Moreover, PACAP-deficient mice express an unusually early onset of activities, from approximately four hours before the dark period, without influencing the phase of the endogenous circadian clock. CONCLUSIONS/SIGNIFICANCE: Although some groups including us reported the abnormalities in photic entrainments in PACAP- and PAC(1)-knockout mice, there were inconsistencies in their results. The time-dependent dysfunctions of photic entrainment in the PACAP-knockout mice described in this paper can integrate the incompatible data in previous reports. The recovery of impaired masking by infusion of PACAP-38 in the mutant mice is the first direct evidence of the relationship between PACAP and masking. These results indicate that PACAP regulates particular nonvisual light responses by conveying parametric light information--that is, intensity and duration. The "early-bird" phenotype in the mutant mice originally reported in this paper supposed that PACAP also has a critical role in daily behavioral patterns, especially during the light-to-dark transition period.

CKIepsilon/delta-dependent phosphorylation is a temperature-insensitive, period-determining process in the mammalian circadian clock.

A striking feature of the circadian clock is its flexible yet robust response to various environmental conditions. To analyze the biochemical processes underlying this flexible-yet-robust characteristic, we examined the effects of 1,260 pharmacologically active compounds in mouse and human clock cell lines. Compounds that markedly (>10 s.d.) lengthened the period in both cell lines, also lengthened it in central clock tissues and peripheral clock cells. Most compounds inhibited casein kinase Iepsilon (CKIepsilon) or CKIdelta phosphorylation of the PER2 protein. Manipulation of CKIepsilon/delta-dependent phosphorylation by these compounds lengthened the period of the mammalian clock from circadian (24 h) to circabidian (48 h), revealing its high sensitivity to chemical perturbation. The degradation rate of PER2, which is regulated by CKIepsilon/delta-dependent phosphorylation, was temperature-insensitive in living clock cells, yet sensitive to chemical perturbations. This temperature-insensitivity was preserved in the CKIepsilon/delta-dependent phosphorylation of a synthetic peptide in vitro. Thus, CKIepsilon/delta-dependent phosphorylation is likely a temperature-insensitive period-determining process in the mammalian circadian clock.

CK2alpha phosphorylates BMAL1 to regulate the mammalian clock.

Clock proteins govern circadian physiology and their function is regulated by various mechanisms. Here we demonstrate that Casein kinase (CK)-2alpha phosphorylates the core circadian regulator BMAL1. Gene silencing of CK2alpha or mutation of the highly conserved CK2-phosphorylation site in BMAL1, Ser90, result in impaired nuclear BMAL1 accumulation and disruption of clock function. Notably, phosphorylation at Ser90 follows a rhythmic pattern. These findings reveal that CK2 is an essential regulator of the mammalian circadian system.

Lack of light-induced elevation of renal sympathetic nerve activity and plasma corticosterone levels in PACAP-deficient mice.

PACAP is a neurotransmitter involved in the signal transduction of light stimulation in the suprachiasmatic nucleus (SCN). Light stimulation affects autonomic nerve activity via the SCN, and here we tested whether PACAP participates in light-induced regulation of sympatho-adrenal activity by using PACAP-deficient (Adcyap1(-/-)) mice. Light stimulation (100 lux, 30 min) significantly elevated both renal sympathetic nerve activity (RSNA), which was monitored on a digital oscilloscope, and plasma corticosterone levels in wild-type mice, but both responses were almost abolished in Adcyap1(-/-) mice. Although light-induced c-Fos expression in the SCN was observed in both genotypes, the numbers of c-Fos positive cells were significantly decreased in Adcyap1(-/-) mice. These data suggest that PACAP signaling pathway is involved in light-induced stimulation of RSNA and plasma corticosterone release through SCN of brain.

Uncoupling protein 2 negatively regulates neurite extensions in PC12h cells.

Uncoupling protein 2 (UCP2) distributes in many organs including the brain. Though recent reports suggest that UCP2 is involved in the neuroprotection and the regulation of neurosecretion, the roles of UCP2 in the central nervous systems remain largely unclear. In order to clarify the significance of UCP2 in the brain especially at developmental stage, subcellular localizations of rat UCP2 (rUCP2) in the developing cerebellar Purkinje cells were immunochemically examined. The rUCP2-like immunoreactivities observed axon or its terminal during axonal maturation. This result implies that rUCP2 contributes to the neurite development. In the PC12h cells overexpressing rUCP2 or active mutant of rUCP2, the neurite outgrowth was significantly inhibited along with a reduction of cellular ATP level. These findings suggest a possibility that UCP2 is involved in negative regulation of neurite extensions through repression of the energy supply.

Role of Per1-interacting protein of the suprachiasmatic nucleus in NGF mediated neuronal survival.

We previously identified Per1-interacting protein of the suprachiasmatic nucleus (PIPS) in rats. To reveal its role, its tissue distribution was examined by immunoblotting. PIPS-like immunoreactive substance (PIPSLS) was observed in the brain, adrenal gland, and PC12 cells. Since PIPS, which has no nuclear localization signal (NLS), is translocated into nuclei of COS-7 cells in the presence of mPer1, the effect of NGF on nuclear localization of PIPS was examined using PC12 cells. NGF caused nuclear translocation of either PIPSLS or GFP-PIPS. NGF mediated nuclear translocation of PIPSLS was blocked by K252a, a TrkA-inhibitor, or wortmannin, a PI3K-inhibitor. Gab1, which is implicated in TrkA signaling and has NLS, co-immunoprecipitated with PIPSLS from PC12 cells using an anti-PIPS antibody. Inhibition of PIPS expression by RNAi increased levels of apoptosis in PC12 cells. These findings suggest that nuclear translocation of PIPS is involved in NGF mediated neuronal survival via TrkA, PI3K, and Gab1 signaling pathway.

Analysis of the expression, localization and activity of rat casein kinase 1epsilon-3.

Casein kinase 1epsilon (CK1epsilon) is a serine/threonine protein kinase that has been suggested to participate in the regulation of various signaling pathways. In this report, we examined the tissue distributions of three putative alternatively spliced forms of rCk1epsilon by RT-PCR. This analysis confirmed that all three isoforms are expressed in rat tissues with different tissue-specific expression patterns. Immunohistochemical analysis showed that the intracellular distribution of rCK1epsilon-3 in neurons was broader than that of rCK1epsilon-1. Moreover, the kinase activity of the rCK1epsilon-3 protein differed from that of rCK1epsilon-1. These data suggest that rCK1epsilon-1 and rCK1epsilon-3 may play different functional roles.

A missense variation in human casein kinase I epsilon gene that induces functional alteration and shows an inverse association with circadian rhythm sleep disorders.

Recent studies have shown that functional variations in clock genes, which generate circadian rhythms through interactive positive/negative feedback loops, contribute to the development of circadian rhythm sleep disorders in humans. Another potential candidate for rhythm disorder susceptibility is casein kinase I epsilon (CKIepsilon), which phosphorylates clock proteins and plays a pivotal role in the circadian clock. To determine whether variations in CKIepsilon induce vulnerability to human circadian rhythm sleep disorders, such as delayed sleep phase syndrome (DSPS) and non-24-h sleep-wake syndrome (N-24), we analyzed all of the coding exons of the human CKIepsilon gene. One of the variants identified encoded an amino-acid substitution S408N, eliminating one of the putative autophosphorylation sites in the carboxyl-terminal extension of CKIepsilon. The N408 allele was less common in both DSPS (p = 0.028) and N-24 patients (p = 0.035) compared to controls. When DSPS and N-24 subjects were combined, based on an a priori prediction of a common mechanism underlying both DSPS and N-24, the inverse association between the N408 allele and rhythm disorders was highly significant (p = 0.0067, odds ratio = 0.42, 95% confidence interval: 0.22-0.79). In vitro kinase assay revealed that CKIepsilon with the S408N variation was approximately 1.8-fold more active than wild-type CKIepsilon. These results indicate that the N408 allele in CKIepsilon plays a protective role in the development of DSPS and N-24 through alteration of the enzyme activity.

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.

Changes in light-induced phase shift of circadian rhythm in mice lacking PACAP.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is one of the neurotransmitters that transfers light signals from the retina to the hypothalamic suprachiasmatic nucleus (SCN) where the master clock of mammalian circadian rhythm locates, and is suggested to be implicated in the mechanism of light-induced phase shift of the circadian clock. Here, we examined changes in the phase shift of circadian rhythm in behavioral activity in mice lacking PACAP (PACAP(-/-)). The phase advance in PACAP(-/-) mice by a light stimulation at late subjective night was significantly attenuated, but the phase delay due to the illumination at the early subjective night slightly diminished. In contrast, the induction of c-Fos in the SCN by the illumination at the early subjective night but not that at the late subjective night was significantly blunted in PACAP(-/-) mice. These data provide new aspects about the roles of PACAP in light-induced phase shift of the circadian clock.

Uncoupling protein 2 influences dopamine secretion in PC12h cells.

Uncoupling protein 2 (UCP2) belongs to the UCP family, and is distributed in many organs including the brain. Although UCP2 is known to be related to many functions such as the regulation of insulin secretion or the scavenging of the radicals, the role of UCP2 in the central nervous system remains unclear. In this report, rat UCP2 (rUCP2) and its mutants were overexpressed in the PC12h cells to determine the physiological roles played by UCP2 in neural cells and to elucidate the mechanisms that regulate these functions. It was found that rUCP2 was activated by the stimulation of the cAMP-protein kinase A (PKA) cascade. Moreover, the activation of rUCP2 suppressed intracellular ATP levels and inhibited the cAMP-dependent increase of dopamine secretion. Thus, UCP2 appears to be regulated by the excitatory stimulus via the cAMP-PKA cascade and serves to negatively control the synaptic output by reducing intracellular ATP levels.

Circadian expression of hnRNP U, a nuclear multi-potent regulatory protein, in the murine suprachiasmatic nucleus.

Heterogeneous ribonuclear protein U (hnRNP U/SAF-A) is a nuclear multi-potent regulatory protein. We investigated whether hnRNP U protein and transcript levels undergo circadian changes by immunoblot and quantitative RT-PCR analyses. In the suprachiasmatic nucleus (SCN), hnRNP U immunoreactivity (ir) changed in a robust circadian pattern as it showed a peak at late nighttime in both light/dark and constant dark conditions. hnRNP U transcript levels in the SCN changed in a similar circadian pattern. In the hippocampus, hnRNP transcript levels also showed a peak at late nighttime but hnRNP U-ir showed an opposite pattern as it peaked at late daytime. These findings suggest that hnRNP U participates in nuclear regulatory events that are involved in mammalian central and peripheral circadian clocks.

Molecular mechanism of mammalian circadian clock.

Circadian rhythms in behaviors and physiological phenomena of plants and animals have long been well known, but the frameworks of the molecular mechanism of circadian clocks have become clearer only within the last decade. A transcription-translation feedback loop has been shown to be an essential component of the clock, and this mechanism seems to be conserved over a wide range of species. The transcriptional activation by a Clock:Bmal1 heterodimer and the inhibition by Cryptochrome and Period are believed to provide the framework of the feedback loop in mammals. Posttranslational modifications such as phosphorylation, nuclear entry and degradation have also been demonstrated to be necessary for the oscillation. Complex auxiliary loops have also been found, and these are thought to contribute to the stabilization of the feedback loop. The molecular mechanisms by which the circadian clock is adjusted to external conditions such as daily light-dark cycles, and by which the oscillation of the feedback loop is transferred to the peripheral organs are also discussed.

Nucleocytoplasmic shuttling and phosphorylation of BMAL1 are regulated by circadian clock in cultured fibroblasts.

BACKGROUND: Recent discoveries of clock proteins have unveiled an important part of the mammalian circadian clock mechanism. However, the molecular clockwork that cause these fundamental feedback loops to stably oscillate with a approximately 24 h-periodicity remain unclear. RESULTS: Serum-shocked fibroblasts were used as a cellular clock model. Circadian changes in the subcellular localization and phosphorylation of BMAL1 protein in these cells were assessed by immunocytochemistry and immunoblotting. A significant time lag between Bmal1 transcription and the cytoplasmic/nuclear accumulation of BMAL1 was observed. After its nuclear accumulation, BMAL1 accumulated in the cytoplasm again, mainly by nucleoexport, before the increase of Bmal1 transcripts. Nuclear accumulation of BMAL1 matched nuclear accumulation of CLOCK and the peak of Per1 transcription. Nuclear BMAL1 was gradually phosphorylated and then dephosphorylated in a temporally regulated manner, although cytoplasmic BMAL1 was not. In serum-shocked mCry1/mCry2 (CRY)-deficient fibroblasts, which lack a functional clock, both the cytoplasmic and nuclear BMAL1 were only present as hyperphosphorylated forms and their circadian nucleocytoplasmic shuttling was absent. CONCLUSIONS: We propose that the nucleocytoplasmic shuttling and phosphorylation states of BMAL1 are regulated by circadian clock, and that this temporally regulated and time-delayed nuclear entry of BMAL1 is important in the maintenance of a stably oscillating clock.