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1.
Nat Commun ; 11(1): 3394, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636383

RESUMO

The hypothalamic suprachiasmatic nuclei (SCN) are the principal mammalian circadian timekeeper, co-ordinating organism-wide daily and seasonal rhythms. To achieve this, cell-autonomous circadian timing by the ~20,000 SCN cells is welded into a tight circuit-wide ensemble oscillation. This creates essential, network-level emergent properties of precise, high-amplitude oscillation with tightly defined ensemble period and phase. Although synchronised, regional cell groups exhibit differentially phased activity, creating stereotypical spatiotemporal circadian waves of cellular activation across the circuit. The cellular circuit pacemaking components that generate these critical emergent properties are unknown. Using intersectional genetics and real-time imaging, we show that SCN cells expressing vasoactive intestinal polypeptide (VIP) or its cognate receptor, VPAC2, are neurochemically and electrophysiologically distinct, but together they control de novo rhythmicity, setting ensemble period and phase with circuit-level spatiotemporal complexity. The VIP/VPAC2 cellular axis is therefore a neurochemically and topologically specific pacemaker hub that determines the emergent properties of the SCN timekeeper.


Assuntos
Ritmo Circadiano , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Núcleo Supraquiasmático/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Relógios Circadianos , Criptocromos/genética , Feminino , Genes Reporter , Teste de Complementação Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Optogenética , Oscilometria , Transdução de Sinais , Núcleo Supraquiasmático/citologia
2.
PLoS Genet ; 16(4): e1008729, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32352975

RESUMO

Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.


Assuntos
Fatores de Transcrição ARNTL/genética , Envelhecimento/genética , Ritmo Circadiano , Fatores de Transcrição ARNTL/metabolismo , Envelhecimento/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Encéfalo/embriologia , Células Cultivadas , Retroalimentação Fisiológica , Fígado/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Músculo Esquelético/metabolismo , Biossíntese de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Célula Única/métodos
3.
J Mol Biol ; 432(12): 3639-3660, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996314

RESUMO

Circadian (approximately daily) rhythms of physiology and behaviour adapt organisms to the alternating environments of day and night. The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian timekeeper of mammals. The mammalian cell-autonomous circadian clock is built around a self-sustaining transcriptional-translational negative feedback loop (TTFL) in which the negative regulators Per and Cry suppress their own expression, which is driven by the positive regulators Clock and Bmal1. Importantly, such TTFL-based clocks are present in all major tissues across the organism, and the SCN is their central co-ordinator. First, we analyse SCN timekeeping at the cell-autonomous and the circuit-based levels of organisation. We consider how molecular-genetic manipulations have been used to probe cell-autonomous timing in the SCN, identifying the integral components of the clock. Second, we consider new approaches that enable real-time monitoring of the activity of these clock components and clock-driven cellular outputs. Finally, we review how intersectional genetic manipulations of the cell-autonomous clockwork can be used to determine how SCN cells interact to generate an ensemble circadian signal. Critically, it is these network-level interactions that confer on the SCN its emergent properties of robustness, light-entrained phase and precision- properties that are essential for its role as the central co-ordinator. Remaining gaps in knowledge include an understanding of how the TTFL proteins behave individually and in complexes: whether particular SCN neuronal populations act as pacemakers, and if so, by which signalling mechanisms, and finally the nature of the recently discovered role of astrocytes within the SCN network.


Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Neurônios/metabolismo , Núcleo Supraquiasmático/metabolismo , Animais , Astrócitos/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/genética , Transdução de Sinais/genética
4.
Science ; 363(6423): 187-192, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30630934

RESUMO

Circadian (~24-hour) rhythms depend on intracellular transcription-translation negative feedback loops (TTFLs). How these self-sustained cellular clocks achieve multicellular integration and thereby direct daily rhythms of behavior in animals is largely obscure. The suprachiasmatic nucleus (SCN) is the fulcrum of this pathway from gene to cell to circuit to behavior in mammals. We describe cell type-specific, functionally distinct TTFLs in neurons and astrocytes of the SCN and show that, in the absence of other cellular clocks, the cell-autonomous astrocytic TTFL alone can drive molecular oscillations in the SCN and circadian behavior in mice. Astrocytic clocks achieve this by reinstating clock gene expression and circadian function of SCN neurons via glutamatergic signals. Our results demonstrate that astrocytes can autonomously initiate and sustain complex mammalian behavior.


Assuntos
Astrócitos/fisiologia , Relógios Circadianos , Ritmo Circadiano , Núcleo Supraquiasmático/fisiologia , Animais , Criptocromos/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia
5.
FASEB J ; 32(8): 4302-4314, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29561690

RESUMO

Cryptochromes 1 and 2 (CRY1/2) are key components of the negative limb of the mammalian circadian clock. Like many peripheral tissues, Cry1 and -2 are expressed in the retina, where they are thought to play a role in regulating rhythmic physiology. However, studies differ in consensus as to their localization and function, and CRY1 immunostaining has not been convincingly demonstrated in the retina. Here we describe the expression and function of CRY1 and -2 in the mouse retina in both sexes. Unexpectedly, we show that CRY1 is expressed throughout all retinal layers, whereas CRY2 is restricted to the photoreceptor layer. Retinal period 2::luciferase recordings from CRY1-deficient mice show reduced clock robustness and stability, while those from CRY2-deficient mice show normal, albeit long-period, rhythms. In functional studies, we then investigated well-defined rhythms in retinal physiology. Rhythms in the photopic electroretinogram, contrast sensitivity, and pupillary light response were all severely attenuated or abolished in CRY1-deficient mice. In contrast, these physiological rhythms are largely unaffected in mice lacking CRY2, and only photopic electroretinogram rhythms are affected. Together, our data suggest that CRY1 is an essential component of the mammalian retinal clock, whereas CRY2 has a more limited role.-Wong, J. C. Y., Smyllie, N. J., Banks, G. T., Pothecary, C. A., Barnard, A. R., Maywood, E. S., Jagannath, A., Hughes, S., van der Horst, G. T. J., MacLaren, R. E., Hankins, M. W., Hastings, M. H., Nolan, P. M., Foster, R. G., Peirson, S. N. Differential roles for cryptochromes in the mammalian retinal clock.


Assuntos
Criptocromos/metabolismo , Mamíferos/metabolismo , Mamíferos/fisiologia , Retina/metabolismo , Retina/fisiologia , Animais , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Eletrorretinografia/métodos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/fisiologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-28049647

RESUMO

The suprachiasmatic nucleus (SCN) is the principal circadian clock of the brain, directing daily cycles of behavior and physiology. SCN neurons contain a cell-autonomous transcription-based clockwork but, in turn, circuit-level interactions synchronize the 20,000 or so SCN neurons into a robust and coherent daily timer. Synchronization requires neuropeptide signaling, regulated by a reciprocal interdependence between the molecular clockwork and rhythmic electrical activity, which in turn depends on a daytime Na+ drive and nighttime K+ drag. Recent studies exploiting intersectional genetics have started to identify the pacemaking roles of particular neuronal groups in the SCN. They support the idea that timekeeping involves nonlinear and hierarchical computations that create and incorporate timing information through the interactions between key groups of neurons within the SCN circuit. The field is now poised to elucidate these computations, their underlying cellular mechanisms, and how the SCN clock interacts with subordinate circadian clocks across the brain to determine the timing and efficiency of the sleep-wake cycle, and how perturbations of this coherence contribute to neurological and psychiatric illness.


Assuntos
Relógios Circadianos , Núcleo Supraquiasmático/fisiologia , Animais , Humanos , Neurônios/fisiologia , Núcleo Supraquiasmático/citologia
7.
Curr Biol ; 26(14): 1880-6, 2016 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-27374340

RESUMO

Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5-7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock.


Assuntos
Relógios Circadianos/genética , Camundongos/genética , Proteínas Circadianas Period/genética , Núcleo Supraquiasmático/metabolismo , Animais , Proteínas Circadianas Period/metabolismo
8.
Proc Natl Acad Sci U S A ; 113(13): 3657-62, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26966234

RESUMO

The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside non-Drd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells.


Assuntos
Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Camundongos , Camundongos Transgênicos , Atividade Motora/genética , Atividade Motora/fisiologia , Plasticidade Neuronal , Neurônios/fisiologia , Fotoperíodo , Receptores de Dopamina D1/deficiência , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/fisiologia , Transdução de Sinais , Núcleo Supraquiasmático/citologia
9.
Cell ; 162(3): 607-21, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26232227

RESUMO

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.


Assuntos
Ritmo Circadiano , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Neuropeptídeos/genética , Núcleo Supraquiasmático/metabolismo , Sequência de Aminoácidos , Animais , Regulação para Baixo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Alinhamento de Sequência , Transcrição Genética
10.
Proc Natl Acad Sci U S A ; 110(23): 9547-52, 2013 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-23690615

RESUMO

The suprachiasmatic nucleus (SCN) coordinates circadian rhythms that adapt the individual to solar time. SCN pacemaking revolves around feedback loops in which expression of Period (Per) and Cryptochrome (Cry) genes is periodically suppressed by their protein products. Specifically, PER/CRY complexes act at E-box sequences in Per and Cry to inhibit their transactivation by CLOCK/BMAL1 heterodimers. To function effectively, these closed intracellular loops need to be synchronized between SCN cells and to the light/dark cycle. For Per expression, this is mediated by neuropeptidergic and glutamatergic extracellular cues acting via cAMP/calcium-responsive elements (CREs) in Per genes. Cry genes, however, carry no CREs, and how CRY-dependent SCN pacemaking is synchronized remains unclear. Furthermore, whereas reporter lines are available to explore Per circadian expression in real time, no Cry equivalent exists. We therefore created a mouse, B6.Cg-Tg(Cry1-luc)01Ld, carrying a transgene (mCry1-luc) consisting of mCry1 elements containing an E-box and E'-box driving firefly luciferase. mCry1-luc organotypic SCN slices exhibited stable circadian bioluminescence rhythms with appropriate phase, period, profile, and spatial organization. In SCN lacking vasoactive intestinal peptide or its receptor, mCry1 expression was damped and desynchronized between cells. Despite the absence of CREs, mCry1-luc expression was nevertheless (indirectly) sensitive to manipulation of cAMP-dependent signaling. In mPer1/2-null SCN, mCry1-luc bioluminescence was arrhythmic and no longer suppressed by elevation of cAMP. Finally, an SCN graft procedure showed that PER-independent as well as PER-dependent mechanisms could sustain circadian expression of mCry1. The mCry1-luc mouse therefore reports circadian mCry1 expression and its interactions with vasoactive intestinal peptide, cAMP, and PER at the heart of the SCN pacemaker.


Assuntos
Ritmo Circadiano/fisiologia , Criptocromos/metabolismo , Retroalimentação Fisiológica/fisiologia , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/fisiologia , Animais , AMP Cíclico/metabolismo , Primers do DNA/genética , Luciferases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
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