Hiroshi Fujishima

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.

Regulation of Prostaglandin Endoperoxide Synthase-2 and IL-6 Expression in Mouse Bone Marrow-Derived Mast Cells by Exogenous But Not Endogenous Prostanoids

Mouse bone marrow-derived mast cells (BMMC), stimulated with stem cell factor, IL-1β, and IL-10, secrete IL-6 and demonstrate a delayed phase of PGD2 generation that is dependent upon the induced expression of PG endoperoxide synthase (PGHS)-2. We have examined the potential for exogenous prostanoids, acting in a paracrine fashion, and endogenous prostanoids, acting in an autocrine fashion, to regulate PGHS-2 induction and IL-6 secretion in mouse BMMC. Exogenous PGE2, which acts through G protein-coupled receptors, and 15-deoxy-Δ12,14-PGJ2, which is a ligand for peroxisome proliferator-activated receptor (PPAR)γ, elicited a 2- to 3-fold amplification of PGHS-2 induction, delayed-phase PGD2 generation, and IL-6 secretion in response to stem cell factor, IL-1β, and IL-10. The effect of PGE2 was reproduced by the E prostanoid (EP)1 receptor agonist 17-trinor-PGE2, and the EP1/EP3 agonist, sulprostone, but not the EP2 receptor agonist, butaprost. Although BMMC express PPARγ, the effects of 15-deoxy-Δ12,14-PGJ2 were not reproduced by the PPARγ agonists, troglitazone and ciglitazone. PGHS-2 induction, but not IL-6 secretion, was impaired in cPLA2-deficient BMMC. However, there was no impairment of PGHS-2 induction in BMMC deficient in hematopoietic PGD synthase or PGHS-1 in the presence or absence of the PGHS-2 inhibitor, NS-398. Thus, although exogenous prostanoids may contribute to amplification of the inflammatory response by augmenting PGD2 generation and IL-6 secretion from mast cells, endogenous prostanoids do not play a role.

Involvement of Ca2+-Dependent Hyperpolarization in Sleep Duration in Mammals

The detailed molecular mechanisms underlying the regulation of sleep duration in mammals are still elusive. To address this challenge, we constructed a simple computational model, which recapitulates the electrophysiological characteristics of the slow-wave sleep and awake states. Comprehensive bifurcation analysis predicted that a Ca(2+)-dependent hyperpolarization pathway may play a role in slow-wave sleep and hence in the regulation of sleep duration. To experimentally validate the prediction, we generate and analyze 21 KO mice. Here we found that impaired Ca(2+)-dependent K(+) channels (Kcnn2 and Kcnn3), voltage-gated Ca(2+) channels (Cacna1g and Cacna1h), or Ca(2+)/calmodulin-dependent kinases (Camk2a and Camk2b) decrease sleep duration, while impaired plasma membrane Ca(2+) ATPase (Atp2b3) increases sleep duration. Pharmacological intervention and whole-brain imaging validated that impaired NMDA receptors reduce sleep duration and directly increase the excitability of cells. Based on these results, we propose a hypothesis that a Ca(2+)-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals.

Mammalian reverse genetics without crossing reveals Nr3a as a short-sleeper gene

The identification of molecular networks at the system level in mammals is accelerated by next-generation mammalian genetics without crossing, which requires both the efficient production of whole-body biallelic knockout (KO) mice in a single generation and high-performance phenotype analyses. Here, we show that the triple targeting of a single gene using the CRISPR/Cas9 system achieves almost perfect KO efficiency (96%-100%). In addition, we developed a respiration-based fully automated non-invasive sleep phenotyping system, the Snappy Sleep Stager (SSS), for high-performance (95.3% accuracy) sleep/wake staging. Using the triple-target CRISPR and SSS in tandem, we reliably obtained sleep/wake phenotypes, even in double-KO mice. By using this system to comprehensively analyze all of the N-methyl-D-aspartate (NMDA) receptor family members, we found Nr3a as a short-sleeper gene, which is verified by an independent set of triple-target CRISPR. These results demonstrate the application of mammalian reverse genetics without crossing to organism-level systems biology in sleep research.

Participation of Cytosolic Phospholipase A2 in Eicosanoid Generation by Mouse Bone Marrow-Derived Mast Cells

Leukotrienes and prostaglandins, derived from the oxidative metabolism of arachidonic acid1’2are potent lipid mediators of tissue inflammation.3’4The first step in the generation of these eicosanoids, is the liberation of esterified arachidonic acid from the sn-2 position of cell membrane glycerophospholipids by the action of phospholipase A2 (PLA2)5. The family of mammalian PLA2enzymes includes the 85-kDa group IV cytosolic PLA2(cPLA2); at least eight low molecular weight, cysteine-rich PLA2enzymes; and calcium-independent species of PLA2.6

Cytosolic phospholipase A2 is essential for both the immediate and the delayed phases of eicosanoid generation in mouse bone marrow-derived mast cells

We have used mice in which the gene for cytosolic phospholipase A2 (cPLA2) has been disrupted to demonstrate the absolute requirement for cPLA2 in both the immediate and the delayed phases of eicosanoid generation by bone marrow-derived mast cells. For the immediate phase, quantitative analysis of the products of the 5-lipoxygenase pathway showed that gene disruption of cPLA2 prevented the provision of arachidonic acid substrate for biosynthesis of proximal intermediates. By analogy, we conclude that arachidonic acid substrate was also not available to prostaglandin endoperoxide synthase 1 in the immediate phase of prostaglandin (PG) D2 generation. These defects occurred with two distinct stimuli, stem cell factor and IgE/antigen, which were, however, sufficient for signal transduction defined by exocytosis of beta-hexosaminidase. Whereas cPLA2 is essential for immediate eicosanoid generation by providing arachidonic acid, its role in delayed-phase PGD2 generation is more complex and involves the activation-dependent induction of prostaglandin endoperoxide synthase 2 and the supply of arachidonic acid for metabolism to PGD2.

Leak potassium channels regulate sleep duration

Significance To address molecular mechanisms regulating sleep duration, a simple computational model of a cortical neuron [simplified averaged neuron (SAN) model], which recapitulates the electrophysiological characteristics of slow-wave sleep (SWS) and wakefulness, is developed in this study. Comprehensive bifurcation and detailed mathematical analyses predicted that leak K+ channels play a role in generating the cortical electrophysiological characteristics of SWS, leading to a hypothesis that leak K+ channels play a role in the regulation of sleep duration. We comprehensively generated and analyzed 14 knockout mice of the leak K+ channel family, which demonstrated that impairment of the leak K+ channel (Kcnk9) decreases sleep duration. The results confirm the validity of the SAN model and suggest a molecular mechanism regulating sleep duration. A primary goal of sleep research is to understand the molecular basis of sleep. Although some sleep/wake-promoting circuits and secreted substances have been identified, the detailed molecular mechanisms underlying the regulation of sleep duration have been elusive. Here, to address these mechanisms, we developed a simple computational model of a cortical neuron with five channels and a pump, which recapitulates the cortical electrophysiological characteristics of slow-wave sleep (SWS) and wakefulness. Comprehensive bifurcation and detailed mathematical analyses predicted that leak K+ channels play a role in generating the electrophysiological characteristics of SWS, leading to a hypothesis that leak K+ channels play a role in the regulation of sleep duration. To test this hypothesis experimentally, we comprehensively generated and analyzed 14 KO mice, and found that impairment of the leak K+ channel (Kcnk9) decreased sleep duration. Based on these results, we hypothesize that leak K+ channels regulate sleep duration in mammals.