Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Here we show that mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557-phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by ϳ4 h in both light-dark cycle and constant dark conditions. The Ser-557-phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3␤ (GSK-3␤), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3␤ at Ser-9, which negatively regulates the kinase activity, GSK-3␤ exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein.The physiology and behavior of living organisms from bacteria to humans show daily fluctuations, and those controlled by autonomous clocks are termed circadian rhythms (1, 2). These rhythms are synchronized with (entrained to) environmental time cues such as light, and the rhythms are sustained with a period of ϳ24 h even in the absence of the time cues. In mammals, the suprachiasmatic nucleus in the anterior hypothalamus serves as the central clock of the circadian timing system (3-5). Peripheral tissues throughout the body also have circadian clocks, and both the central and peripheral clocks generate the 24-h rhythm with molecular machinery very similar to each other (6 -8).The molecular mechanism of the circadian oscillator has been investigated extensively by genetic and molecular studies on Drosophila and mice. In the mouse molecular clock, a heterodimer of the two transcription factors, CLOCK and BMAL1, activates E-box-dependent transcription of two cryptochrome genes, mCry1 1 and mCry2, and three period genes, mPer1, mPer2, and mPer3 (9, 10). Translated mCRY and mPER proteins translocate to the nucleus where mCRY proteins act as predominant negative regulators by interacting directly with CLOCK/BMAL1 heterodimer to inhibit the transactivation from the E-box (10, 11). The negative regulation in turn results in decrease of the protein levels of mCRYs and mPERs and allows the molecular cycle to start again with the activation of the E-box-dependent transcription. Importantly, mice lacking both mCry1 and mCry2 exhibit arrhythmic behavior immediately after being placed in constant darkness (12), indicating their c...

Circadian molecular oscillation is generated by a transcription/translation-based feedback loop in which CRY proteins play critical roles as potent inhibitors for E-box-dependent clock gene expression. Although CRY2 undergoes rhythmic phosphorylation in its C-terminal tail, structurally distinct from the CRY1 tail, little is understood about how protein kinase(s) controls the CRY2-specific phosphorylation and contributes to the molecular clockwork. Here we found that Ser557 in the C-terminal tail of CRY2 is phosphorylated by DYRK1A as a priming kinase for subsequent GSK-3␤ (glycogen synthase kinase 3␤)-mediated phosphorylation of Ser553, which leads to proteasomal degradation of CRY2. In the mouse liver, DYRK1A kinase activity toward Ser557 of CRY2 showed circadian variation, with its peak in the accumulating phase of CRY2 protein.Knockdown of Dyrk1a caused abnormal accumulation of cytosolic CRY2, advancing the timing of a nuclear increase of CRY2, and shortened the period length of the cellular circadian rhythm. Expression of an S557A/S553A mutant of CRY2 phenocopied the effect of Dyrk1a knockdown in terms of the circadian period length of the cellular clock. DYRK1A is a novel clock component cooperating with GSK-3␤ and governs the Ser557 phosphorylation-triggered degradation of CRY2.

The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day-dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by >2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF)1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7α-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7α-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-daydependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland.

Down's syndrome (DS), a major genetic cause of mental retardation, arises from triplication of genes on human chromosome 21. Here we show that DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) and DSCR1 (DS critical region 1), two genes lying within human chromosome 21 and encoding for a serine/ threonine kinase and calcineurin regulator, respectively, are expressed in neural progenitors in the mouse developing neocortex. Increasing the dosage of both proteins in neural progenitors leads to a delay in neuronal differentiation, resulting ultimately in alteration of their laminar fate. This defect is mediated by the cooperative actions of DYRK1A and DSCR1 in suppressing the activity of the transcription factor NFATc. In Ts1Cje mice, a DS mouse model, dysregulation of NFATc in conjunction with increased levels of DYRK1A and DSCR1 was observed. Furthermore, counteracting the dysregulated pathway ameliorates the delayed neuronal differentiation observed in Ts1Cje mice. In sum, our findings suggest that dosage of DYRK1A and DSCR1 is critical for proper neurogenesis through NFATc and provide a potential mechanism to explain the neurodevelopmental defects in DS.

Down syndrome (DS) arises from triplication of genes on human chromosome 21 and is associated with anomalies in brain development such as reduced production of neurons and increased generation of astrocytes. Here, we show that differentiation of cortical progenitor cells into astrocytes is promoted by DYRK1A, a Ser/Thr kinase encoded on human chromosome 21. In the Ts1Cje mouse model of DS, increased dosage of DYRK1A augments the propensity of progenitors to differentiate into astrocytes. This tendency is associated with enhanced astrogliogenesis in the developing neocortex. We also find that overexpression of DYRK1A upregulates the activity of the astrogliogenic transcription factor STAT in wildtype progenitors. Ts1Cje progenitors exhibit elevated STAT activity, and depletion of DYRK1A in these cells reverses the deregulation of STAT. In sum, our findings indicate that potentiation of the DYRK1A-STAT pathway in progenitors contributes to aberrant astrogliogenesis in DS.

SUMMARYNeural progenitor cells in the developing brain give rise to neurons and glia. Multiple extrinsic signalling molecules and their cognate membrane receptors have been identified to control neural progenitor fate. However, a role for G protein-coupled receptors in cell fate decisions in the brain remains largely putative. Here we show that GPRC5B, which encodes an orphan G protein-coupled receptor, is present in the ventricular surface of cortical progenitors in the mouse developing neocortex and is required for their neuronal differentiation. GPRC5B-depleted progenitors fail to adopt a neuronal fate and ultimately become astrocytes. Furthermore, GPRC5B-mediated signalling is associated with the proper regulation of β-catenin signalling, a pathway crucial for progenitor fate decision. Our study uncovers G protein-coupled receptor signalling in the neuronal fate determination of cortical progenitors.

The circadian clock is finely regulated by posttranslational modifications of clock components. Mouse CRY2, a critical player in the mammalian clock, is phosphorylated at Ser557 for proteasome-mediated degradation, but its in vivo role in circadian organization was not revealed. Here, we generated CRY2(S557A) mutant mice, in which Ser557 phosphorylation is specifically abolished. The mutation lengthened free-running periods of the behavioral rhythms and PER2::LUC bioluminescence rhythms of cultured liver. In livers from mutant mice, the nuclear CRY2 level was elevated, with enhanced PER2 nuclear occupancy and suppression of E-box-regulated genes. Thus, Ser557 phosphorylation-dependent regulation of CRY2 is essential for proper clock oscillation in vivo.T ranscription-and translation-based negative-feedback loops play an important role in circadian clock regulation (1, 2). In mammals, a heterodimer of positive factors, CLOCK and BMAL1, activates the transcription of genes encoding negative factors such as PERIOD (PER1 to -3) and cryptochrome (CRY1 and -2) through binding to E-box enhancer elements in their promoter regions (3). Translated PERs and CRYs associate with each other, enter into cell nuclei, and inhibit their own transcription by interacting with the CLOCK-BMAL1 heterodimer (4). In addition to transcriptional and translational regulation, posttranslational modifications of the clock proteins play critical roles in the clockwork (5-8).Among the negative factors, CRY1 and CRY2 play major roles for repression of E-box-dependent gene expression (9). They share highly conserved N-terminal and central regions while having unique C-terminal tails. Although the roles of the diverged C-terminal regions remain unclear, both CRY1 and CRY2 have strong repressor activities (9), and therefore, their accumulation and decline are the major period-determining steps for circadian molecular oscillation. Previous studies reported that FBXL3, an F-box-type E3 ubiquitin ligase, enhances proteasomal degradation of . Recently, we and other groups demonstrated that ubiquitination of CRY1 and CRY2 by FBXL21, the closest paralog of FBXL3, stabilizes CRYs (13,14). It has been reported that FBXL3-dependent CRY1 degradation is regulated by CRY1 phosphorylation (15, 16). In contrast, we previously demonstrated that priming phosphorylation of CRY2 at Ser557 in the C-terminal region by DYRK1A is required for secondary phosphorylation at Ser553 by glycogen synthase kinase 3␤ (GSK-3␤) (17, 18). The dually phosphorylated CRY2 is led to proteasomal degradation (18), which is independent of FBXL3 action (17, 18). So far, the functions of CRY regulators, such as protein kinases and ubiquitin ligases, have been examined by using their inhibitors and/or gene knockout/knockdown (17-19). On the other hand, site-directed mutagenesis in CRY proteins will be the most specific strategy because the upstream regulators would have multiple targets in the clockwork. In the present study, we generated knock-in mice carrying a mutation at the priming pho...

The ability of radial glial progenitors (RGPs) to generate cortical neurons is determined by local extracellular factors and signaling pathways intrinsic to RGPs. Here we find that GPR157, an orphan G protein-coupled receptor, localizes to RGPs’ primary cilia exposed to the cerebrospinal fluid (CSF). GPR157 couples with Gq-class of the heterotrimeric G-proteins and signals through IP3-mediated Ca2+ cascade. Activation of GPR157-Gq signaling enhances neuronal differentiation of RGPs whereas interfering with GPR157-Gq-IP3 cascade in RGPs suppresses neurogenesis. We also detect the presence of putative ligand(s) for GPR157 in the CSF, and demonstrate the increased ability of the CSF to activate GPR157 at neurogenic phase. Thus, GPR157-Gq signaling at the primary cilia of RGPs is activated by the CSF and contributes to neurogenesis.