A role of cryptochrome for magnetic field-dependent improvement of sleep quality, lifespan, and motor function in Drosophila.

Understanding the molecular genetic basis of animal magnet reception has been one of the big challenges in molecular biology. Recently it was discovered that the magnetic sense of Drosophila melanogaster is mediated by the ultraviolet (UV)-A/blue light photoreceptor cryptochrome (Cry). Here, using the fruit fly as a magnet-receptive model organism, we show that the magnetic field exposure (0.4-0.6 mT) extended lifespan under starvation, but not in cryptochrome mutant flies (cryb ). The magnetic field exposure increases motor function in wild type and neurodegenerative disease model flies. Furthermore, the magnetic field exposure improved sleep quality at night-time specific manner, but not in cryb . We also showed that repeated AC magnetic field exposure increased climbing activity in wild-type Drosophila, but not in cryb . The data suggests that magnetic field-dependent improvement of lifespan, sleep quality, and motor function is mediated through a cry-dependent pathway in Drosophila.

Novel hypnotics of Japanese traditional herbal medicines to caffeine-induced insomnia in Drosophila by using Newly-developed automated sleep and rhythm analysis system (AutoCircaS).

Sleep in Drosophila was defined in the year 2000 by using Drosophila Activity Monitor (DAM) system. But DAM is very small tube space and one fly per tube is very limited to analyze for fly social behavior. To overcome such demerits of DAM system, we developed a novel automated sleep and rhythm analysis system (AutoCircaS) which monitors and records any behaviors like social mating, sleep, and circadian rhythm in flies (Drosophila) and small fishes medaka (Oryzias latipes) in free space using the time-lapse (one frame per 10 sec) imaging. AutoCircaS can detect the caffeine-induced insomnia in flies in light-dark (LD) and constant dark (DD) conditions. Thus, using the AutoCircaS, we discovered that Japanese traditional herbal medicines, KyushinKannouGan-ki (KKG), NouKassei (NK) as well as, and Sansoninto, significantly improved caffeine-induced insomnia in flies. The data suggest that AutoCircaS is useful for sleep analysis of small animals and screening of new sedative-hypnotics from many origins.

Effects of an electric field on sleep quality and life span mediated by ultraviolet (UV)-A/blue light photoreceptor CRYPTOCHROME in Drosophila.

Although electric fields (EF) exert beneficial effects on animal wound healing, differentiation, cancers and rheumatoid arthritis, the molecular mechanisms of these effects have remained unclear about a half century. Therefore, we aimed to elucidate the molecular mechanisms underlying EF effects in Drosophila melanogaster as a genetic animal model. Here we show that the sleep quality of wild type (WT) flies was improved by exposure to a 50-Hz (35 kV/m) constant electric field during the day time, but not during the night time. The effect was undetectable in cryptochrome mutant (cryb) flies. Exposure to a 50-Hz electric field under low nutrient conditions elongated the lifespan of male and female WT flies by ~ 18%, but not of several cry mutants and cry RNAi strains. Metabolome analysis indicated that the adenosine triphosphate (ATP) content was higher in intact WT than cry gene mutant strains exposed to an electric field. A putative magnetoreceptor protein and UV-A/blue light photoreceptor, CRYPTOCHROME (CRY) is involved in electric field (EF) receptors in animals. The present findings constitute hitherto unknown genetic evidence of a CRY-based system that is electric field sensitive in animals.

Saffron ameliorated motor symptoms, short life span and retinal degeneration in Parkinson's disease fly models.

Parkinson's disease (PD) is a common neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the brain. α-Synuclein is an aggregation-prone neural protein that plays a role in the pathogenesis of PD. In our previous paper, we found that saffron; the stigma of Crocus sativus Linné (Iridaceae), and its constituents (crocin and crocetin) suppressed aggregation of α-synuclein and promoted the dissociation of α-synuclein fibrils in vitro. In this study, we investigated the effect of dietary saffron and its constituent, crocetin, in vivo on a fly PD model overexpressing several mutant α-synuclein in a tissue-specific manner. Saffron and crocetin significantly suppressed the decrease of climbing ability in the Drosophila overexpressing A30P (A30P fly PD model) or G51D (G51D fly PD model) mutated α-synuclein in neurons. Saffron and crocetin extended the life span in the G51D fly PD model. Saffron suppressed the rough-eyed phenotype and the dispersion of the size histogram of the ocular long axis in the eye of A30P fly PD model. Saffron had a cytoprotective effect on a human neuronal cell line with α-synuclein fibrils. These data showed that saffron and its constituent crocetin have protective effects on the progression of PD disease in animals in vivo and suggest that saffron and crocetin can be used to treat PD.

Effects of Kamikihito and Unkei-to on Sleep Behavior of Wild Type and Parkinson Model in Drosophila.

Parkinson's disease (PD) is the second most common neurodegenerative disease, and it is associated with sleep behavior disorders. In Drosophila melanogaster disease model, human α-synuclein A30P overexpressing flies (A30P PD model) have been shown for levy body aggregation and movement disorders. We measured sleep rhythms in the A30P PD model flies using the Drosophila Activity Monitoring system and found that they develop sleep defects at 20 days after eclosion. Furthermore, the total amount of sleep is significantly reduced in middle-aged PD model flies and the reduction has been attributed to nighttime sleep. The number and length of sleep bouts also decreased in middle-aged A30P PD model flies. Feeding of the oriental traditional herbal medicines (Kampo), Kamikihito and Unkei-to significantly ameliorate the level of sleep defects in A30P PD model flies. The Kamikihito and Unkei-to recovered 60-min sleep bouts number in the A30P PD model flies to the level of young (5 days after eclosion) flies. Kamikihito recovered sleep both in wild-type and PD model flies. Unkei-to ameliorates not only sleep but also motor function in PD model flies. The data suggest that Kamikihito and Unkei-to might be useful for the sleep defects in human PD patients as well as healthy human.

Minos-insertion mutant of the Drosophila GBA gene homologue showed abnormal phenotypes of climbing ability, sleep and life span with accumulation of hydroxy-glucocerebroside.

Gaucher's disease in humans is considered a deficiency of glucocerebrosidase (GlcCerase) that result in the accumulation of its substrate, glucocerebroside (GlcCer). Although mouse models of Gaucher's disease have been reported from several laboratories, these models are limited due to the perinatal lethality of GlcCerase gene. Here, we examined phenotypes of Drosophila melanogaster homologues genes of the human Gaucher's disease gene by using Minos insertion. One of two Minos insertion mutants to unknown function gene (CG31414) accumulates the hydroxy-GlcCer in whole body of Drosophila melanogaster. This mutant showed abnormal phenotypes of climbing ability and sleep, and short lifespan. These abnormal phenotypes are very similar to that of Gaucher's disease in human. In contrast, another Minos insertion mutant (CG31148) and its RNAi line did not show such severe phenotype as observed in CG31414 gene mutation. The data suggests that Drosophila CG31414 gene mutation might be useful for unraveling the molecular mechanism of Gaucher's disease.

Inositols affect the mating circadian rhythm of Drosophila melanogaster.

Accumulating evidence indicates that the molecular circadian clock underlies the mating behavior of Drosophila melanogaster. However, information about which food components affect circadian mating behavior is scant. The ice plant, Mesembryanthemum crystallinum has recently become a popular functional food. Here, we showed that the close-proximity (CP) rhythm of D. melanogaster courtship behavior was damped under low-nutrient conditions, but significantly enhanced by feeding the flies with powdered ice plant. Among various components of ice plants, we found that myo-inositol increased the amplitude and slightly shortened the period of the CP rhythm. Real-time reporter assays showed that myo-inositol and D-pinitol shortened the period of the circadian reporter gene Per2-luc in NIH 3T3 cells. These data suggest that the ice plant is a useful functional food and that the ability of inositols to shorten rhythms is a general phenomenon in insects as well as mammals.

Effect of feeding behavior on circadian regulation of endothelin expression in mouse colon.

AIMS: The function, regulation and gene expression of the endothelin (ET) system in the intestine is not well understood. We investigated the dependence on feeding schedule and biological clock of the regulation of ET-1 gene expression in mouse colon. MAIN METHODS: Mice were fed freely, fasted for 48 h and re-fed after fasting. KEY FINDINGS: Where indicated ET-1 gene expression was highest in the colon compared with other tissues examined in fasted mice. Fasting increased the level, while maintaining the rhythmicity, of ET-1 gene expression in epithelial colonic tissue. Re-feeding, however, decreased ET-1 gene expression and suppressed rhythmic oscillation, and the rhythmicity also changed for gene expression for circadian clocks, period-1 and period-2 (Per1 and Per2). Furthermore, the decrease in ET-1 gene expression induced by re-feeding was blocked by pre-treatment with hexamethonium and atropine. The daily change in ET-1 gene expression in colon, which depends on feeding schedule via the autonomic nervous system, is synchronized with peripheral circadian oscillators under conditions of free feeding and fasting but not re-feeding. The decrease in ET-1 gene expression in the proximal colon induced by re-feeding occurs via the nervous system. SIGNIFICANCE: ET-1 plays an important physiological role, which is dependent on feeding behavior.

Role of p53 in the entrainment of mammalian circadian behavior rhythms.

p53 protein plays a role for control of cell proliferation and the induction of apoptosis in normal cells. However, its role in the circadian rhythms that control many physiological functions including locomotor behavior remains unknown. The present study examined the locomotors activity rhythms of mice which have homozygous mutations of p53 gene. The period of drinking activity rhythms in p53 knockout (p53 KO) mice became unstable under constant dark. Light pulse causes a big phase shifts at CT15.5-17, when p53 mRNA expression peaks in the suprachiasmatic nucleus (SCN). Furthermore, photic entrainment of p53 KO mice is unusual under light-dark conditions. These findings suggest that p53 is involved in entrainment of the circadian behavioral rhythm.

Directed evolution of three-finger toxin to produce serine protease inhibitors.

Directed evolution is a very popular strategy for improving biophysical properties and even for generating proteins with novel functions. Recent advances in combinatorial protein engineering mean it is now possible to develop protein scaffolds that could substitute for whole antibody-associated properties as emerging therapeutic proteins. In particular, disulfide-rich proteins are attractive templates for directed evolution in the search for novel molecules because they can regulate the activities of receptors, enzymes, and other molecules. Previously, we demonstrated that functional regulatory molecules against interleukin-6 receptor (IL-6R) could be obtained by directed evolution of the three-finger toxin (3F) scaffold. In the present study, trypsin was selected as a target for directed evolution to further explore the potential use of the 3F cDNA display library. After seven rounds of selection, the DNA sequences converged. The recombinant proteins produced by the selected candidates had inhibitory activity against trypsin (Ki of 33-450 nM). Three of the six groups had Ki values that were comparable to bovine pancreatic trypsin inhibitor and soybean trypsin inhibitor. Two of the candidates also had inhibitory effects against chymotrypsin and kallikrein. This study suggests that 3F protein is suitable for the preparation of high-diversity libraries that can be utilized to obtain protease inhibitors. In addition to our previous successful targeting of IL-6R, the technique developed in our studies may have wide applications in the generation of regulatory molecules for targets of interest, such as receptors, enzymes for research, diagnostic applications, and therapeutic uses.

Expression of human Gaucher disease gene GBA generates neurodevelopmental defects and ER stress in Drosophila eye.

Gaucher disease (GD) is the most common of the lysosomal storage disorders and is caused by defects in the GBA gene encoding glucocerebrosidase (GlcCerase). The accumulation of its substrate, glucocylceramide (GlcCer) is considered the main cause of GD. We found here that the expression of human mutated GlcCerase gene (hGBA) that is associated with neuronopathy in GD patients causes neurodevelopmental defects in Drosophila eyes. The data indicate that endoplasmic reticulum (ER) stress was elevated in Drosophila eye carrying mutated hGBAs by using of the ER stress markers dXBP1 and dBiP. We also found that Ambroxol, a potential pharmacological chaperone for mutated hGBAs, can alleviate the neuronopathic phenotype through reducing ER stress. We demonstrate a novel mechanism of neurodevelopmental defects mediated by ER stress through expression of mutants of human GBA gene in the eye of Drosophila.

The circadian binding of CLOCK protein to the promoter of C/ebpα gene in mouse cells.

C/EBPα plays important roles in metabolism as well as in the maintenance of energy homeostasis. Here we describe loss of the circadian oscillation of C/ebpα expression in liver of Clock mutant mice. Reporter assays indicate Clock and Bmal significantly induced C/ebpα gene expression whereas Cry suppressed. Real time reporter assays showed that two mutated E-boxes disrupted C/ebpα promoter dependent-oscillation. Chromatin immunoprecipitation suggests Clock can bind to two E-boxes in the C/ebpα promoter with a circadian manner in vivo. Thus, C/ebpα gene transcription is under circadian control of a core clock component, Clock. The data suggests that circadian disturbances may affect metabolic abnormalities through the C/ebpα pathway in liver.

Chronic stress affects PERIOD2 expression through glycogen synthase kinase-3ß phosphorylation in the central clock.

Mood disorders are considered to be associated with altered circadian rhythms, but the correlation between them has remained obscure. The mood stabilizer, lithium, is an inhibitor of glycogen synthase kinase-3ß (GSK-3ß), which is a modulator of the circadian clock system. Here, we show that chronic restraint (CR) stress diminishes behavioral activity and rhythmicity in mice. CR stress elevated GSK-3ß phosphorylation and blunted the rhythmic expression of PERIOD2 (PER2) in the brain. Moreover, lithium, when administered to the stress-imposed mice, reduced GSK-3ß phosphorylation and restored PER2 expression in the suprachiasmatic nucleus in a nighttime-specific manner. These data suggest that CR stress altered the circadian behavioral rhythm through a change in circadian gene expression of PER2 and GSK-3ß phosphorylation in the suprachiasmatic nucleus.

Feeding cues and injected nutrients induce acute expression of multiple clock genes in the mouse liver.

The circadian clock is closely associated with energy metabolism. The liver clock can rapidly adapt to a new feeding cycle within a few days, whereas the lung clock is gradually entrained over one week. However, the mechanism underlying tissue-specific clock resetting is not fully understood. To characterize the rapid response to feeding cues in the liver clock, we examined the effects of a single time-delayed feeding on circadian rhythms in the liver and lungs of Per2::Luc reporter knockin mice. After adapting to a night-time restricted feeding schedule, the mice were fed according to a 4, 8, or 13 h delayed schedule on the last day. The phase of the liver clock was delayed in all groups with delayed feeding, whereas the lung clock remained unaffected. We then examined the acute response of clock and metabolism-related genes in the liver using focused DNA-microarrays. Clock mutant mice were bred under constant light to attenuate the endogenous circadian rhythm, and gene expression profiles were determined during 24 h of fasting followed by 8 h of feeding. Per2 and Dec1 were significantly increased within 1 h of feeding. Real-time RT-PCR analysis revealed a similarly acute response in hepatic clock gene expression caused by feeding wild type mice after an overnight fast. In addition to Per2 and Dec1, the expression of Per1 increased, and that of Rev-erbα decreased in the liver within 1 h of feeding after fasting, whereas none of these clock genes were affected in the lung. Moreover, an intraperitoneal injection of glucose combined with amino acids, but not either alone, reproduced a similar hepatic response. Our findings show that multiple clock genes respond to nutritional cues within 1 h in the liver but not in the lung.

Caffeine lengthens circadian rhythms in mice.

Although caffeine alters sleep in many animals, whether or not it affects mammalian circadian clocks remains unknown. Here, we found that incubating cultured mammalian cell lines, human osteosarcoma U2OS cells and mouse fibroblast NIH3T3 cells, with caffeine lengthened the period of circadian rhythms. Adding caffeine to ex vivo cultures also lengthened the circadian period in mouse liver explants from Per2::Luciferase reporter gene knockin mice, and caused a phase delay in brain slices containing the suprachiasmatic nucleus (SCN), where the central circadian clock in mammals is located. Furthermore, chronic caffeine consumption ad libitum for a week delayed the phase of the mouse liver clock in vivo under 12 h light-dark conditions and lengthened the period of circadian locomotor rhythms in mice under constant darkness. Our results showed that caffeine alters circadian clocks in mammalian cells in vitro and in the mouse ex vivo and in vivo.

Evening circadian oscillator as the primary determinant of rhythmic motivation for Drosophila courtship behavior.

Circadian clocks of Drosophila melanogaster motivate males to court females at a specific time of day. However, clock neurons involved in courtship rhythms in the brain of Drosophila remain totally unknown. The circadian locomotor behavior of Drosophila is controlled by morning (M cells) and evening (E cells) cells in the brain, which regulate morning and evening activities, respectively. Here, we identified the brain clock neurons that are responsible for the circadian rhythms of the close-proximity (CP) behavior that reflects male courtship motivation. Interestingly, the ablation or functional molecular clock disruption of E cells caused arrhythmic CP behavior, but that of M cells resulted in sustained CP rhythms even in constant darkness. In addition, the ablation of some dorsal lateral neurons (LNd) of E cells using neuropeptide-F (NPF)-GAL4 did not impair CP rhythms. These findings suggested that the NPF-negative LNds and DN1s of E cells include cells essential for circadian CP behavior in Drosophila.

High-salt diet advances molecular circadian rhythms in mouse peripheral tissues.

Dietary compounds influence the expression of various genes and play a major role in changing physiological and metabolic states. However, little is known about the role of food ingredients in the regulation of circadian gene expression. Here, we show that feeding mice with a high-salt (HS) diet ad libitum for over 2weeks advanced the phase of clock gene expression by about 3h in the liver, kidney, and lung, but did not change circadian feeding, drinking, and locomotor rhythms. Focused DNA microarray analysis showed that the expression phase of many genes related to metabolism in the liver was also advanced. Immediately before phase advancement in peripheral tissues, the mRNA expression of sodium-glucose cotransporter 1 (Sglt1) and glucose transporter 2 (Glut2), that are responsible for glucose absorption, was increased in the jejunum. Furthermore, blood glucose uptake increased more rapidly after consuming the HS diet than the control diet. Moreover, phloridzin, a specific inhibitor of SGLT1, prevented the increased glucose transporter expression in the jejunum and phase advancement in the livers of mice on the HS diet. These results suggest that increased glucose absorption induced by dietary HS alters the food entrainment of peripheral molecular circadian rhythms.

CLOCK regulates circadian rhythms of hepatic glycogen synthesis through transcriptional activation of Gys2.

Hepatic glycogen content is important for glucose homeostasis and exhibits robust circadian rhythms that peak at the end of the active phase in mammals. The activities of the rate-limiting enzymes for glycogenesis and glycogenolysis also show circadian rhythms, and the balance between them forms the circadian rhythm of the hepatic glycogen content. However, no direct evidence has yet implicated the circadian clock in the regulation of glycogen metabolism at the molecular level. We show here that a Clock gene mutation damps the circadian rhythm of the hepatic glycogen content, as well as the circadian mRNA and protein expression of Gys2 (glycogen synthase 2), which is the rate-limiting enzyme of glycogenesis in the liver. Transient reporter assays revealed that CLOCK drives the transcriptional activation of Gys2 via two tandemly located E-boxes. Chromatin immunoprecipitation assays of liver tissues revealed that CLOCK binds to these E-box elements in vivo, and real time reporter assays showed that these elements are sufficient for circadian Gys2 expression in vitro. Thus, CLOCK regulates the circadian rhythms of hepatic glycogen synthesis through transcriptional activation of Gys2.

Tumor growth suppression in vivo by overexpression of the circadian component, PER2.

Some reports have indicated that the core clock gene, Per2 regulates the cell cycle, immune system and neural functions. To understand the effects of PER2 on tumor growth in vivo, stable transformants of murine sarcoma 180 (S-180) cell lines expressing different levels of PER2 were established. The growth of stable PER2 transformants in vivo was significantly and dose-dependently suppressed according to the amount of PER2 expressed, indicating that PER2 plays a role in the growth suppression of sarcoma cells. The anchorage-dependent and -independent growth in vitro and expression of the clock controlled cell-cycle related genes, wee1, myc, and VEGF were not altered in stable PER2 transformants. In contrast, susceptibility to murine natural killer (NK) cell cytolytic activity was enhanced in PER2 transformants. Furthermore, PER2 transformants suppressed cell motility and reduced fibronectin expression, but the expression of integrin receptors was not affected. These results suggest that sarcoma cells overexpressing PER2 suppress tumors in vivo by changing the nature of tumor cell adhesion.