Salt-inducible kinase 3 regulates the mammalian circadian clock by destabilizing PER2 protein.

Salt-inducible kinase 3 (SIK3) plays a crucial role in various aspects of metabolism. In the course of investigating metabolic defects in Sik3-deficient mice (Sik3-/-), we observed that circadian rhythmicity of the metabolisms was phase-delayed. Sik3-/- mice also exhibited other circadian abnormalities, including lengthening of the period, impaired entrainment to the light-dark cycle, phase variation in locomotor activities, and aberrant physiological rhythms. Ex vivo suprachiasmatic nucleus slices from Sik3-/- mice exhibited destabilized and desynchronized molecular rhythms among individual neurons. In cultured cells, Sik3-knockdown resulted in abnormal bioluminescence rhythms. Expression levels of PER2, a clock protein, were elevated in Sik3-knockdown cells but down-regulated in Sik3-overexpressing cells, which could be attributed to a phosphorylation-dependent decrease in PER2 protein stability. This was further confirmed by PER2 accumulation in the Sik3-/- fibroblasts and liver. Collectively, SIK3 plays key roles in circadian rhythms by facilitating phosphorylation-dependent PER2 destabilization, either directly or indirectly.

A new mouse allele of glutamate receptor delta 2 with cerebellar atrophy and progressive ataxia.

Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)-PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants.

In vivo diagnostic imaging using micro-CT: sequential and comparative evaluation of rodent models for hepatic/brain ischemia and stroke.

BACKGROUND: There is an increasing need for animal disease models for pathophysiological research and efficient drug screening. However, one of the technical barriers to the effective use of the models is the difficulty of non-invasive and sequential monitoring of the same animals. Micro-CT is a powerful tool for serial diagnostic imaging of animal models. However, soft tissue contrast resolution, particularly in the brain, is insufficient for detailed analysis, unlike the current applications of CT in the clinical arena. We address the soft tissue contrast resolution issue in this report. METHODOLOGY: We performed contrast-enhanced CT (CECT) on mouse models of experimental cerebral infarction and hepatic ischemia. Pathological changes in each lesion were quantified for two weeks by measuring the lesion volume or the ratio of high attenuation area (%HAA), indicative of increased vascular permeability. We also compared brain images of stroke rats and ischemic mice acquired with micro-CT to those acquired with 11.7-T micro-MRI. Histopathological analysis was performed to confirm the diagnosis by CECT. PRINCIPAL FINDINGS: In the models of cerebral infarction, vascular permeability was increased from three days through one week after surgical initiation, which was also confirmed by Evans blue dye leakage. Measurement of volume and %HAA of the liver lesions demonstrated differences in the recovery process between mice with distinct genetic backgrounds. Comparison of CT and MR images acquired from the same stroke rats or ischemic mice indicated that accuracy of volumetric measurement, as well as spatial and contrast resolutions of CT images, was comparable to that obtained with MRI. The imaging results were also consistent with the histological data. CONCLUSIONS: This study demonstrates that the CECT scanning method is useful in rodents for both quantitative and qualitative evaluations of pathologic lesions in tissues/organs including the brain, and is also suitable for longitudinal observation of the same animals.

Quantitative analysis of phase wave of gene expression in the mammalian central circadian clock network.

BACKGROUND: The suprachiasmatic nucleus (SCN), the master circadian clock, is a heterogeneous oscillator network, yet displays a robust synchronization dynamics. Recent single-cell bioluminescent imaging revealed temporal gradients in circadian clock gene expression in the SCN ex vivo. However, due to technical difficulty in biological approaches to elucidate the entire network structure of the SCN, characteristics of the gradient, which we refer to as phase wave, remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: We implemented new approaches, i.e., quantitative analysis and model simulation to characterize the phase waves in Per2::Luciferase clock reporter gene expression of the rat SCN slice. Our quantitative study demonstrated not only a high degree of synchronization between the neurons and regular occurrence of the phase wave propagation, but also a significant amount of phase fluctuations contained in the wave. In addition, our simulations based on local coupling model suggest that the intercellular coupling strength estimated by the model simulations is significantly higher than the critical value for generating the phase waves. Model simulations also suggest that heterogeneity of the SCN neurons is one of the main factors causing the phase wave fluctuations. Furthermore, robustness of the SCN network against dynamical noise and variation of the natural frequencies inherent in these neurons was quantitatively assessed. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first quantitative evaluation of the phase wave and further characterization of the SCN neuronal network features generating the wave i.e., intercellular synchrony, phase fluctuation, strong local coupling, heterogeneous periodicity and robustness. Our present study provides an approach, which will lead to a comprehensive understanding of mechanistic and/or biological significance of the phase wave in the central circadian oscillatory system.

Attenuated food anticipatory activity and abnormal circadian locomotor rhythms in Rgs16 knockdown mice.

Regulators of G protein signaling (RGS) are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs) of G protein α-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN), the master circadian light-entrainable oscillator (LEO) of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA) targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO)-driven elevated food-anticipatory activity (FAA) observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s).

Differential contribution of rod and cone circadian clocks in driving retinal melatonin rhythms in Xenopus.

BACKGROUND: Although an endogenous circadian clock located in the retinal photoreceptor layer governs various physiological events including melatonin rhythms in Xenopus laevis, it remains unknown which of the photoreceptors, rod and/or cone, is responsible for the circadian regulation of melatonin release. METHODOLOGY/PRINCIPAL FINDINGS: We selectively disrupted circadian clock function in either the rod or cone photoreceptor cells by generating transgenic Xenopus tadpoles expressing a dominant-negative CLOCK (XCLΔQ) under the control of a rod or cone-specific promoter. Eyecup culture and continuous melatonin measurement revealed that circadian rhythms of melatonin release were abolished in a majority of the rod-specific XCLΔQ transgenic tadpoles, although the percentage of arrhythmia was lower than that of transgenic tadpole eyes expressing XCLΔQ in both rods and cones. In contrast, whereas a higher percentage of arrhythmia was observed in the eyes of the cone-specific XCLΔQ transgenic tadpoles compare to wild-type counterparts, the rate was significantly lower than in rod-specific transgenics. The levels of the transgene expression were comparable between these two different types of transgenics. In addition, the average overall melatonin levels were not changed in the arrhythmic eyes, suggesting that CLOCK does not affect absolute levels of melatonin, only its temporal expression pattern. CONCLUSIONS/SIGNIFICANCE: These results suggest that although the Xenopus retina is made up of approximately equal numbers of rods and cones, the circadian clocks in the rod cells play a dominant role in driving circadian melatonin rhythmicity in the Xenopus retina, although some contribution of the clock in cone cells cannot be excluded.

Mitogen-activated protein kinase is a functional component of the autonomous circadian system in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker driving behavioral and physiological rhythms in mammals. Circadian activation of mitogen-activated protein kinase [MAPK; also known as ERK (extracellular signal-regulated kinase)] is observed in vivo in the SCN under constant darkness, although the biological significance of this remains unclear. To elucidate this question, we first examined whether MAPK was autonomously activated in ex vivo SCN slices. Moreover, we investigated the effect of MAPK inhibition on circadian clock gene expression and neuronal firing rhythms using SCN-slice culture systems. We show herein that MAPK is autonomously activated in the SCN, and our data demonstrate that inhibition of the MAPK activity results in dampened rhythms and reduced basal levels in circadian clock gene expression at the SCN single-neuron level. Furthermore, MAPK inhibition attenuates autonomous circadian neuronal firing rhythms in the SCN. Thus, our data suggest that light-independent MAPK activity contributes to the robustness of the SCN autonomous circadian system.

Modulatory effects of 5-fluorouracil on the rhythmic expression of circadian clock genes: a possible mechanism of chemotherapy-induced circadian rhythm disturbances.

The circadian clock system is necessary to adapt endogenous physiological functions to daily variations in environmental conditions. Abnormality in circadian rhythms, such as the sleep-wake cycle and the timing of hormonal secretions, is implicated in various physiological and psychiatrical disorders. Recent molecular studies have revealed that oscillation in the transcription of specific clock genes plays a central role in the generation of 24h cycles of physiology and behavior. It has been noticed that patients receiving chemotherapeutic agents experience disturbances in their behavioral and physical performances, including circadian rhythms. To explore the underlying mechanism of chemotherapeutic agent-induced disturbance of these rhythms, we investigated the influence of 5-fluorouracil (5-FU), one of the most widely used chemotherapeutic agents for the treatment of cancers, on the expression of clock genes. Treatment of cultured NIH3T3 cells with 5-FU for 48 h resulted in a significant reduction of mRNA levels of Period1 (Per1) and Period2 (Per2) without affecting cell viability; however, treatment with the same amount of uracil, a structural analog of 5-FU, had little effect on the expression of clock genes. Consistent with its inhibitory actions, continuous administration of 5-FU (2 mg/kg/h) to mice attenuated the oscillation in the expressions of Per1 and Per2 in the liver and suprachiasmatic nuclei, the center of the mammalian circadian clock. These results reveal a possible pharmacological action by the chemotherapeutic agent 5-FU on the circadian clock mechanism, which is the underlying cause of its adverse effects on 24-h rhythms of physiology and behavior.

Gq/11-induced intracellular calcium mobilization mediates Per2 acute induction in Rat-1 fibroblasts.

Phase resetting is one of the essential properties of circadian clocks that is required for the adjustment to a particular environment and the induction of Per1 and Per2 clock genes is believed to be a primary molecular event during this process. Although the intracellular signal transduction pathway underlying Per1 gene activation has been well characterized, the mechanisms that control Per2 up-regulation have not yet been elucidated. In our present study, we demonstrate that Gq/11 coupled receptors mediate serum-induced immediate rat Per2 (rPer2) transactivation in Rat-1 fibroblasts via intracellular Ca2+ mobilization. Stimulation of these cells with a high concentration of serum was found to rapidly increase the intracellular Ca2+ levels and strongly up-regulated rPer2 gene. rPer2 induction by serum stimulation was abrogated by intracellular Ca2+ chelation and depletion of intracellular Ca2+ store, which suggests that the calcium mobilization is necessary for the up-regulation of rPer2 gene. In addition, suppression of Gq/11 function was observed to inhibit both Ca2+ mobilization and rPer2 induction. Further, we demonstrated that endothelin-induced acute rPer2 transactivation via Gq/11-coupled endothelin receptors is also suppressed by a Gq/11 specific inhibitor. These findings together suggest that serum and endothelin utilize a common Gq/11-PLC mediated pathway for the transactivation of rPer2, which involves the mobilization of calcium from the intracellular calcium store.

Distinct localization of prokineticin 2 and prokineticin receptor 2 mRNAs in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian clock that regulates physiological and behavioral circadian rhythms in mammals. Prokineticin 2 (PK2) is highly expressed in the SCN, and its involvement in the generation of circadian locomotor activity has been reported previously. In the present study, using in situ hybridization methods, we investigated the localization of PK2 and prokineticin receptor 2 (PKR2), a specific receptor for PK2, in the rat SCN. In steady light : dark (L : D = 12 : 12 h) and constant dark conditions, rPK2 mRNA displayed a robust circadian oscillation with a peak occurring during the day. Moreover, during peak expression, the rPK2 mRNA-positive neurons were scattered in both the dorsomedial and ventrolateral SCN, which are two functionally and morphologically distinct subregions. Furthermore, double-labeling in situ hybridization experiments revealed that greater than 50% of the rPK2 mRNA-containing neurons co-expressed either vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP) or arginine vasopressin (AVP) in the SCN. In contrast, the rPKR2 mRNA levels did not show significant diurnal alterations. rPKR2 mRNA-containing neurons were also clustered in the dorsolateral part of the SCN, which shows negligible labeling of either rAVP, rVIP, rGRP or rPK2 transcripts. In addition, this region exhibited a delayed cycling of the rPer1 gene. These results suggest an intrinsic PK2 neurotransmission and functionally distinct roles for PKR2-expressing neurons in the SCN.

Genetic manipulation of circadian rhythms in Xenopus.

Xenopus laevis retina is an important experimental model system for the study of circadian oscillator mechanisms, as light input pathways, central oscillator mechanisms, and multiple output pathways are all contained within this tissue. These retinas continue to exhibit robust circadian rhythms even after being maintained in culture for many days. The usefulness of this system has been improved even further by the development of a technique for simple genetic manipulation of these animals, which is complemented by expanded genomics resources (Xenopus genome project, microarray, etc.). By taking advantage of the transgenic technique in Xenopus described in this article, many types of analysis can be done on the primary transgenic animals within a couple of weeks after transgenesis. The availability of many cell-type-specific promoters and well-characterized cell types within the Xenopus retina provides the advantage of cell-specific modification of clock function using this method; in other words, contributions of different cell types within the circadian system can be analyzed independently by "molecular dissociation" of these cells. This article describes both how this transgenic technique is useful and various considerations that should be taken into account when these types of experiments are planned and interpreted. Application of these new techniques to studies of clock function provide an opportunity to rapidly assess gene expression and?or function in the context of the intact retina.

In vivo disruption of Xenopus CLOCK in the retinal photoreceptor cells abolishes circadian melatonin rhythmicity without affecting its production levels.

Xenopus laevis retinas, like retinas from all vertebrate classes, have endogenous circadian clocks that control many aspects of normal retinal physiology occurring in cells throughout all layers of the retina. The localization of the clock(s) that controls these various rhythms remains unclear. One of the best studied rhythmic events is the nocturnal release of melatonin. Photoreceptor layers can synthesize rhythmic melatonin when these cells are in isolation. However, within the intact retina, melatonin is controlled in a complex way, indicating that signals from many parts of the retina may contribute to the production of melatonin rhythmicity. To test this hypothesis, we generated transgenic tadpoles that express different levels of a dominant negative Xenopus CLOCK specifically in the retinal photoreceptors. Eyes from these tadpoles continued to produce melatonin at normal levels, but with greatly disrupted rhythmicity, the severity of which correlated with the transgene expression level. These results demonstrate that although many things contribute to melatonin production in vivo, the circadian clock localized in the retinal photoreceptors is necessary for its rhythmicity. Furthermore, these data show that the control of the level of melatonin synthesis is separable from the control of its rhythmicity and may be controlled by different molecular machinery. This type of specific "molecular lesion" allows perturbation of the clock in intact tissues and is valuable for dissection of clock control of tissue-level processes in this and other complex systems.