Iori Yamanaka

Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro

The molecular oscillations underlying the generation of circadian rhythmicity in mammals develop gradually during ontogenesis. However, the developmental process of mammalian cellular circadian oscillator formation remains unknown. In differentiated somatic cells, the transcriptional–translational feedback loops (TTFL) consisting of clock genes elicit the molecular circadian oscillation. Using a bioluminescence imaging system to monitor clock gene expression, we show here that the circadian bioluminescence rhythm is not detected in the mouse embryonic stem (ES) cells, and that the ES cells likely lack TTFL regulation for clock gene expression. The circadian clock oscillation was induced during the differentiation culture of mouse ES cells without maternal factors. In addition, reprogramming of the differentiated cells by expression of Sox2, Klf4, Oct3/4, and c-Myc genes, which were factors to generate induced pluripotent stem (iPS) cells, resulted in the re-disappearance of circadian oscillation. These results demonstrate that an intrinsic program controls the formation of the circadian oscillator during the differentiation process of ES cells in vitro. The cellular differentiation and reprogramming system using cultured ES cells allows us to observe the circadian clock formation process and may help design new strategies to understand the key mechanisms responsible for the organization of the molecular oscillator in mammals.

Rhythmic post-transcriptional regulation of the circadian clock protein mPER2 in mammalian cells: A real-time analysis

Post-transcriptional/translational mechanisms regulate the circadian clock system of many organisms, including mammals. The level of the essential clock protein mPER2 daily oscillates in peripheral cells as well as in neurons of the master oscillator in the suprachiasmatic nucleus (SCN). Post-translational modifications of mPER2, such as phosphorylation and ubiquitination, are likely involved in the regulation of its stability and intracellular accumulation rhythms, which in turn create an approximately 2-4 h delay from the rhythm of mPer2 mRNA. However, there are no direct evidences linking the above biochemical processes to the generation of the mPER2 protein cycle itself. Here, we show that multiple circadian waves of bioluminescence are detectable in cells constitutively expressing an mPer2-luciferase fusion mRNA. This suggests that a post-transcriptional/translational mechanism itself is capable of generating the circadian mPER2 accumulation cycle, and thus this type of regulation may function in the circadian clock system in mammals.

Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo) mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR) β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho) mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch-once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch-once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs.

Mini Screening of Kinase Inhibitors Affecting Period-length of Mammalian Cellular Circadian Clock

In mammalian circadian rhythms, the transcriptional-translational feedback loop (TTFL) consisting of a set of clock genes is believed to elicit the circadian clock oscillation. The TTFL model explains that the accumulation and degradation of mPER and mCRY proteins control the period-length (tau) of the circadian clock. Although recent studies revealed that the Casein Kinase Iεδ (CKIεδ) regurates the phosphorylation of mPER proteins and the circadian period-length, other kinases are also likely to contribute the phosphorylation of mPER. Here, we performed small scale screening using 84 chemical compounds known as kinase inhibitors to identify candidates possibly affecting the circadian period-length in mammalian cells. Screening by this high-throughput real-time bioluminescence monitoring system revealed that the several chemical compounds apparently lengthened the cellular circadian clock oscillation. These compounds are known as inhibitors against kinases such as Casein Kinase II (CKII), PI3-kinase (PI3K) and c-Jun N-terminal Kinase (JNK) in addition to CKIεδ. Although these kinase inhibitors may have some non-specific effects on other factors, our mini screening identified new candidates contributing to period-length control in mammalian cells.

CH Activation Generates Period‐Shortening Molecules That Target Cryptochrome in the Mammalian Circadian Clock

The synthesis and functional analysis of KL001 derivatives, which are modulators of the mammalian circadian clock, are described. By using cutting-edge C-H activation chemistry, a focused library of KL001 derivatives was rapidly constructed, which enabled the identification of the critical sites on KL001 derivatives that induce a rhythm-changing activity along with the components that trigger opposite modes of action. The first period-shortening molecules that target the cryptochrome (CRY) were thus discovered. Detailed studies on the effects of these compounds on CRY stability implicate the existence of an as yet undiscovered regulatory mechanism.

Real-time monitoring of circadian clock oscillations in primary cultures of mammalian cells using Tol2 transposon-mediated gene transfer strategy

BackgroundThe circadian rhythm in mammals is orchestrated by a central pacemaker in the brain, but most peripheral tissues contain their own intrinsic circadian oscillators. The circadian rhythm is a fundamental biological system in mammals involved in the regulation of various physiological functions such as behavior, cardiovascular functions and energy metabolism. Thus, it is important to understand the correlation between circadian oscillator and physiological functions in peripheral tissues. However, it is still difficult to investigate the molecular oscillator in primary culture cells.ResultsIn this study, we used a novel Tol2 transposon based Dbp promoter or Bmal1 promoter driven luciferase reporter vector system to detect and analyze the intrinsic molecular oscillator in primary culture cells (mouse embryonic fibroblasts, fetal bovine heart endothelial cells and rat astrocytes). The results showed circadian molecular oscillations in all examined primary culture cells. Moreover, the phase relationship between Dbp promoter driven and Bmal1 promoter driven molecular rhythms were almost anti-phase, which suggested that these reporters appropriately read-out the intrinsic cellular circadian clock.ConclusionsOur results indicate that gene transfer strategy using the Tol2 transposon system of a useful and safe non-viral vector is a powerful tool for investigating circadian rhythms in peripheral tissues.

Presence of robust circadian clock oscillation under constitutive over-expression of mCry1 in rat-1 fibroblasts

In mammals, mCRY proteins are essential and are major negative elements in circadian feedback loops. In this study, robust circadian clock oscillation was present even under conditions with constitutive over‐expression of mCry1 in rat‐1 cells. Rat‐1 cells were produced to stably express mPer2 promoter‐driven luciferase reporter, in which mCry1 was overexpressed under a tetracycline‐dependent gene expression (Tet‐On) system. Using these cells, we show that circadian clock oscillations in rat‐1 fibroblasts persist when the mCRY1 protein constitutively accumulates in the nuclei.

Glycinergic transmission and postsynaptic activation of CaMKII are required for glycine receptor clustering in vivo

Synaptic transmission‐dependent regulation of neurotransmitter receptor accumulation at postsynaptic sites underlies the formation, maintenance and maturation of synaptic function. Previous in vitro studies showed that glycine receptor (GlyR) clustering requires synaptic inputs. However, in vivo GlyR regulation by synaptic transmission is not fully understood. Here, we established a model system using developing zebrafish, in which GlyRs are expressed in Mauthner cells (M‐cells), a pair of giant, reticulospinal, hindbrain neurons, thereby enabling analysis of GlyR clusters over time in identifiable cells. Bath application of a glycinergic blocker, strychnine, to developing zebrafish prevented postsynaptic GlyR cluster formation in the M‐cells. After strychnine removal, the GlyR clusters appeared in the M‐cells. At a later stage, glycinergic transmission blockade impaired maintenance of GlyR clusters. We also found that pharmacological blockade of either L‐type Ca2+ channels or calcium‐/calmodulin‐dependent protein kinase II (CaMKII) disturbed GlyR clustering. In addition, the M‐cell‐specific CaMKII inactivation using the Gal4‐UAS system significantly impaired GlyR clustering in the M‐cells. Thus, the formation and maintenance of GlyR clusters in the M‐cells in the developing animals are regulated in a synaptic transmission‐dependent manner, and CaMKII activation at the postsynapse is essential for GlyR clustering. This is the first demonstration of synaptic transmission‐dependent modulation of synaptic GlyRs in vivo.

Formation of glycinergic synapses in zebrafish Mauthner cell

The maintenance of synaptic functions is essential for reliable information transfer and storage. However cellular mechanisms underlying synaptic maintenance in the adult brain are not fully understood. In this study, we evaluated the involvement of inositol 1,4,5-trisphosphate (IP3) signaling in synaptic maintenance in the cerebral cortex. Metabotropic glutamate receptor (mGluR) or IP3 signaling in the neocortical pyramidal neurons was chronically inhibited in vivo by intraperitoneal injection of mGluR antagonists or expressing in these neurons exogenous IP3 5-phosphatase, which selectively hydrolyzes IP3. This chronic inhibition in postsynaptic pyramidal neurons significantly increased the value of the paired pulse ratio at glutamatergic synapses, indicating the reduction of presynaptic release probability. In contrast, the chronic inhibition of mGluR-IP3 signaling did not alter the amplitude of quantal synaptic responses, indicating that postsynaptic responsiveness was unchanged. These results suggest that an IP3-dependent retrograde signaling mechanism is involved in the maintenance of excitatory synapses in the cerebral cortex. Recently, we reported a similar IP3-dependent maintenance mechanism at parallel fiber-Purkinje cell synapses in the cerebellum. Thus, these studies provide new insights into the signaling mechanism underlying synaptic maintenance in the adult brain.