Yasushi Isojima

CKIε/δ-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 Iε (CKIε) or CKIδ phosphorylation of the PER2 protein. Manipulation of CKIε/δ-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 CKIε/δ-dependent phosphorylation, was temperature-insensitive in living clock cells, yet sensitive to chemical perturbations. This temperature-insensitivity was preserved in the CKIε/δ-dependent phosphorylation of a synthetic peptide in vitro. Thus, CKIε/δ-dependent phosphorylation is likely a temperature-insensitive period-determining process in the mammalian circadian clock.

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 (CKIɛ), which phosphorylates clock proteins and plays a pivotal role in the circadian clock. To determine whether variations in CKIɛ 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 CKIɛ 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 CKIɛ. 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 CKIɛ with the S408N variation was ∼1.8-fold more active than wild-type CKIɛ. These results indicate that the N408 allele in CKIɛ plays a protective role in the development of DSPS and N-24 through alteration of the enzyme activity.

CK2|[alpha]| 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)-2α phosphorylates the core circadian regulator BMAL1. Gene silencing of CK2α 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.

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 ∼24 h‐periodicity remain unclear.

Ultraweak biochemiluminescence detected from rat hippocampal slices

From any kind of living organisms, very weak spontaneous light emission without excitation light (ultraweak biochemiluminescence) is observed. We succeeded to detect ultraweak biochemiluminescence from rat hippocampal slice preparation at the order of 10(-19) W/mm2 by a single-photon detector using a silicon avalanche photodiode. It was shown that depolarization induced by the high concentration of potassium caused an increase in the intensity of biochemiluminescence from the rat hippocampal slice, and that suppression of neural activity by tetrodotoxin elicited a decrease of its luminescence. These findings suggest that correlation between the intensity of ultraweak biochemiluminescence and neural metabolic activity.

Ultrahigh sensitivity single‐photon detector using a Si avalanche photodiode for the measurement of ultraweak biochemiluminescence

A single‐photon detector using a Si avalanche photodiode (APD) has been constructed. A trans‐impedance type front‐end circuit was employed in order to achieve a high signal‐to‐noise ratio. The APD and front‐end circuit were cooled in liquid nitrogen to reduce dark count rate and circuit noise. The system performances were measured, and the achieved dark count rate and total quantum efficiency were 0.08 count/s and 5%, respectively. The sensitivity was evaluated to be of the order of 10−19 W. The APD single‐photon detector was demonstrated to be available for measurement of the ultraweak biochemiluminescence at the order of 10−19 W/mm2 from brain slices, i.e., hippocampal slices. It was shown that the addition of tetrodotoxin to the hippocampal slice caused a decrease in the intensity of biochemiluminescence.

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.

Phorbol esters promote postsynaptic accumulation of Vesl-1S/Homer-1a protein: Phorbol esters promote postsynaptic accumulation of Vesl-1S

We examined effects of phorbol esters on the amount and the subcellular distribution of the activity‐regulated protein Vesl‐1S/Homer‐1a in cultured hippocampal neurons. Major Vesl‐1S immunoreactivity (IR) was detected throughout neuronal somata under control conditions. Bath application of phorbol esters, PMA and PDBu resulted in the increase in the amount of Vesl‐1S proteins and promoted punctate distribution of Vesl‐1S IR at the cortical regions of the neuronal somata. Immunofluorescent observations using antisynaptophysin and anti‐Vesl‐1S antibodies, and electron microscopic observations, revealed that Vesl‐1S accumulated at postsynaptic regions following PMA application. Membrane depolarization with high concentrations of external potassium also promoted the punctate distribution of Vesl‐1S IR. These results demonstrate that phorbol‐triggered reaction cascades result in the accumulation of Vesl‐1S protein at postsynaptic regions, and suggest that these phorbol effects may mimic those caused by synaptic activities.

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 PAC1-knockout mice, there were inconsistencies in their results [1], [2], [3], [4]. 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.

Changes in circadian period and morphology of the hypothalamic suprachiasmatic nucleus in fyn kinase-deficient mice.

Protein tyrosine phosphorylation is involved in intracellular signal transduction and plays important roles in various physiological events. To understand the role of Fyn, a non-receptor type tyrosine kinase of Src family kinases, in the mechanism of circadian rhythms, we analyzed the circadian locomotor behavior and morphology of the hypothalamic suprachiasmatic nucleus (SCN), a master circadian oscillator in Fyn mutant mice, because Fyn-like immunoreactive substance was observed in the SCN. Under constant dark (DD) condition the Fyn (-/-) mutant mice showed a free-running circadian rhythm, and the period of the circadian rhythm of the locomotor activity was significantly longer than that of the control mice. Fyn (-/-) mutant mice had abnormal distribution of neurons containing vasoactive intestinal polypeptide (VIP)-like immunoreactive substance in the SCN. These findings suggest that Fyn is involved in the mechanism of circadian oscillation and morphological formation of the SCN. The mechanism of the implication of Fyn discussed with the Fyns roles in neural network formation and cellular signal transduction pathway.