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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.