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1.
EMBO J ; 40(20): e108614, 2021 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-34487375

RESUMO

Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN.

2.
J Neurosci ; 41(41): 8562-8576, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34446572

RESUMO

The timing and quality of sleep-wake cycles are regulated by interacting circadian and homeostatic mechanisms. Although the suprachiasmatic nucleus (SCN) is the principal clock, circadian clocks are active across the brain and the respective sleep-regulatory roles of SCN and local clocks are unclear. To determine the specific contribution(s) of the SCN, we used virally mediated genetic complementation, expressing Cryptochrome1 (Cry1) to establish circadian molecular competence in the suprachiasmatic hypothalamus of globally clockless, arrhythmic male Cry1/Cry2-null mice. Under free-running conditions, the rest/activity behavior of Cry1/Cry2-null controls expressing EGFP (SCNCon) was arrhythmic, whereas Cry1-complemented mice (SCNCry1) had coherent circadian behavior, comparable to that of Cry1,2-competent wild types (WTs). In SCNCon mice, sleep-wakefulness, assessed by electroencephalography (EEG)/electromyography (EMG), lacked circadian organization. In SCNCry1 mice, however, it matched WTs, with consolidated vigilance states [wake, rapid eye movement sleep (REMS) and non-REMS (NREMS)] and rhythms in NREMS δ power and expression of REMS within total sleep (TS). Wakefulness in SCNCon mice was more fragmented than in WTs, with more wake-NREMS-wake transitions. This disruption was reversed in SCNCry1 mice. Following sleep deprivation (SD), all mice showed a homeostatic increase in NREMS δ power, although the SCNCon mice had reduced NREMS during the inactive (light) phase of recovery. In contrast, the dynamics of homeostatic responses in the SCNCry1 mice were comparable to WTs. Finally, SCNCon mice exhibited poor sleep-dependent memory but this was corrected in SCNCry1mice. In clockless mice, circadian molecular competence focused solely on the SCN rescued the architecture and consolidation of sleep-wake and sleep-dependent memory, highlighting its dominant role in timing sleep.SIGNIFICANCE STATEMENT The circadian timing system regulates sleep-wake cycles. The hypothalamic suprachiasmatic nucleus (SCN) is the principal circadian clock, but the presence of multiple local brain and peripheral clocks mean the respective roles of SCN and other clocks in regulating sleep are unclear. We therefore used virally mediated genetic complementation to restore molecular circadian functions in the suprachiasmatic hypothalamus, focusing on the SCN, in otherwise genetically clockless, arrhythmic mice. This initiated circadian activity-rest cycles, and circadian sleep-wake cycles, circadian patterning to the intensity of non-rapid eye movement sleep (NREMS) and circadian control of REMS as a proportion of total sleep (TS). Consolidation of sleep-wake established normal dynamics of sleep homeostasis and enhanced sleep-dependent memory. Thus, the suprachiasmatic hypothalamus, alone, can direct circadian regulation of sleep-wake.


Assuntos
Ritmo Circadiano/fisiologia , Criptocromos/biossíntese , Sono/fisiologia , Núcleo Supraquiasmático/metabolismo , Vigília/fisiologia , Animais , Relógios Circadianos/fisiologia , Criptocromos/genética , Eletroencefalografia/métodos , Eletromiografia/métodos , Masculino , Transtornos da Memória , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
3.
EMBO J ; 40(7): e106745, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33491228

RESUMO

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ε activity to be maintained. In the absence of CRY-mediated feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational.


Assuntos
Ritmo Circadiano , Criptocromos/metabolismo , Animais , Células Cultivadas , Criptocromos/deficiência , Criptocromos/genética , Drosophila melanogaster , Feminino , Locomoção , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo
4.
J Neurosci ; 41(3): 502-512, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33234609

RESUMO

Circadian (approximately daily) rhythms pervade mammalian behavior. They are generated by cell-autonomous, transcriptional/translational feedback loops (TTFLs), active in all tissues. This distributed clock network is coordinated by the principal circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). Its robust and accurate time-keeping arises from circuit-level interactions that bind its individual cellular clocks into a coherent time-keeper. Cells that express the neuropeptide vasoactive intestinal peptide (VIP) mediate retinal entrainment of the SCN; and in the absence of VIP, or its cognate receptor VPAC2, circadian behavior is compromised because SCN cells cannot synchronize. The contributions to pace-making of other cell types, including VPAC2-expressing target cells of VIP, are, however, not understood. We therefore used intersectional genetics to manipulate the cell-autonomous TTFLs of VPAC2-expressing cells. Measuring circadian behavioral and SCN rhythmicity in these temporally chimeric male mice thus enabled us to determine the contribution of VPAC2-expressing cells (∼35% of SCN cells) to SCN time-keeping. Lengthening of the intrinsic TTFL period of VPAC2 cells by deletion of the CK1εTau allele concomitantly lengthened the period of circadian behavioral rhythms. It also increased the variability of the circadian period of bioluminescent TTFL rhythms in SCN slices recorded ex vivo Abrogation of circadian competence in VPAC2 cells by deletion of Bmal1 severely disrupted circadian behavioral rhythms and compromised TTFL time-keeping in the corresponding SCN slices. Thus, VPAC2-expressing cells are a distinct, functionally powerful subset of the SCN circuit, contributing to computation of ensemble period and maintenance of circadian robustness. These findings extend our understanding of SCN circuit topology.


Assuntos
Comportamento Animal/fisiologia , Ritmo Circadiano/fisiologia , Periodicidade , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/fisiologia , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/fisiologia , Animais , Ritmo Circadiano/genética , Retroalimentação Fisiológica , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Proteínas Mutantes Quiméricas/genética , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Núcleo Supraquiasmático/fisiologia
5.
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
6.
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
7.
Cell ; 177(4): 896-909.e20, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31030999

RESUMO

In mammals, endogenous circadian clocks sense and respond to daily feeding and lighting cues, adjusting internal ∼24 h rhythms to resonate with, and anticipate, external cycles of day and night. The mechanism underlying circadian entrainment to feeding time is critical for understanding why mistimed feeding, as occurs during shift work, disrupts circadian physiology, a state that is associated with increased incidence of chronic diseases such as type 2 (T2) diabetes. We show that feeding-regulated hormones insulin and insulin-like growth factor 1 (IGF-1) reset circadian clocks in vivo and in vitro by induction of PERIOD proteins, and mistimed insulin signaling disrupts circadian organization of mouse behavior and clock gene expression. Insulin and IGF-1 receptor signaling is sufficient to determine essential circadian parameters, principally via increased PERIOD protein synthesis. This requires coincident mechanistic target of rapamycin (mTOR) activation, increased phosphoinositide signaling, and microRNA downregulation. Besides its well-known homeostatic functions, we propose insulin and IGF-1 are primary signals of feeding time to cellular clocks throughout the body.


Assuntos
Relógios Circadianos/fisiologia , Comportamento Alimentar/fisiologia , Proteínas Circadianas Period/metabolismo , Animais , Ritmo Circadiano/fisiologia , Feminino , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais
8.
Nat Commun ; 10(1): 542, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30710088

RESUMO

The suprachiasmatic nucleus (SCN) co-ordinates circadian behaviour and physiology in mammals. Its cell-autonomous circadian oscillations pivot around a well characterised transcriptional/translational feedback loop (TTFL), whilst the SCN circuit as a whole is synchronised to solar time by its retinorecipient cells that express and release vasoactive intestinal peptide (VIP). The cell-autonomous and circuit-level mechanisms whereby VIP synchronises the SCN are poorly understood. We show that SCN slices in organotypic culture demonstrate rapid and sustained circuit-level circadian responses to VIP that are mediated at a cell-autonomous level. This is accompanied by changes across a broad transcriptional network and by significant VIP-directed plasticity in the internal phasing of the cell-autonomous TTFL. Signalling via ERK1/2 and tuning by its negative regulator DUSP4 are critical elements of the VIP-directed circadian re-programming. In summary, we provide detailed mechanistic insight into VIP signal transduction in the SCN at the level of genes, cells and neural circuit.


Assuntos
Relógios Circadianos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Tirosina Fosfatases/metabolismo , Núcleo Supraquiasmático/fisiologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Sistemas CRISPR-Cas , Relógios Circadianos/genética , Relógios Circadianos/efeitos da radiação , AMP Cíclico/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos da radiação , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos da radiação , Luz , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Camundongos Knockout , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Elementos de Resposta/genética , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/efeitos da radiação , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/efeitos da radiação
9.
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
10.
Proc Natl Acad Sci U S A ; 115(52): E12388-E12397, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30487216

RESUMO

The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional-translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.


Assuntos
Transtornos Cronobiológicos/genética , Criptocromos/genética , Criptocromos/metabolismo , Animais , Transtornos Cronobiológicos/fisiopatologia , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Circadianas Period/metabolismo , Biossíntese de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional , Núcleo Supraquiasmático/metabolismo , Fatores de Transcrição/metabolismo
11.
Sci Transl Med ; 9(415)2017 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-29118260

RESUMO

Fibroblasts are primary cellular protagonists of wound healing. They also exhibit circadian timekeeping, which imparts an approximately 24-hour rhythm to their biological function. We interrogated the functional consequences of the cell-autonomous clockwork in fibroblasts using a proteome-wide screen for rhythmically expressed proteins. We observed temporal coordination of actin regulators that drives cell-intrinsic rhythms in actin dynamics. In consequence, the cellular clock modulates the efficiency of actin-dependent processes such as cell migration and adhesion, which ultimately affect the efficacy of wound healing. Accordingly, skin wounds incurred during a mouse's active phase exhibited increased fibroblast invasion in vivo and ex vivo, as well as in cultured fibroblasts and keratinocytes. Our experimental results correlate with the observation that the time of injury significantly affects healing after burns in humans, with daytime wounds healing ~60% faster than nighttime wounds. We suggest that circadian regulation of the cytoskeleton influences wound-healing efficacy from the cellular to the organismal scale.


Assuntos
Actinas/metabolismo , Ritmo Circadiano , Fibroblastos/metabolismo , Fibroblastos/patologia , Cicatrização , Queimaduras/patologia , Relógios Circadianos , Humanos , Queratinócitos/patologia , Polimerização , Proteoma/metabolismo
12.
Neuron ; 93(6): 1420-1435.e5, 2017 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-28285822

RESUMO

The suprachiasmatic nucleus (SCN) of the hypothalamus orchestrates daily rhythms of physiology and behavior in mammals. Its circadian (∼24 hr) oscillations of gene expression and electrical activity are generated intrinsically and can persist indefinitely in temporal isolation. This robust and resilient timekeeping is generally regarded as a product of the intrinsic connectivity of its neurons. Here we show that neurons constitute only one "half" of the SCN clock, the one metabolically active during circadian daytime. In contrast, SCN astrocytes are active during circadian nighttime, when they suppress the activity of SCN neurons by regulating extracellular glutamate levels. This glutamatergic gliotransmission is sensed by neurons of the dorsal SCN via specific pre-synaptic NMDA receptor assemblies containing NR2C subunits. Remarkably, somatic genetic re-programming of intracellular clocks in SCN astrocytes was capable of remodeling circadian behavioral rhythms in adult mice. Thus, SCN circuit-level timekeeping arises from interdependent and mutually supportive astrocytic-neuronal signaling.


Assuntos
Astrócitos/fisiologia , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Ácido Glutâmico/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora/fisiologia , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/genética
13.
J Neurosci ; 36(36): 9326-41, 2016 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-27605609

RESUMO

UNLABELLED: The suprachiasmatic nucleus (SCN) is the master circadian oscillator encoding time-of-day information. SCN timekeeping is sustained by a cell-autonomous transcriptional-translational feedback loop, whereby expression of the Period and Cryptochrome genes is negatively regulated by their protein products. This loop in turn drives circadian oscillations in gene expression that direct SCN electrical activity and thence behavior. The robustness of SCN timekeeping is further enhanced by interneuronal, circuit-level coupling. The aim of this study was to combine pharmacological and genetic manipulations to push the SCN clockwork toward its limits and, by doing so, probe cell-autonomous and emergent, circuit-level properties. Circadian oscillation of mouse SCN organotypic slice cultures was monitored as PER2::LUC bioluminescence. SCN of three genetic backgrounds-wild-type, short-period CK1ε(Tau/Tau) mutant, and long-period Fbxl3(Afh/Afh) mutant-all responded reversibly to pharmacological manipulation with period-altering compounds: picrotoxin, PF-670462 (4-[1-Cyclohexyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-2-pyrimidinamine dihydrochloride), and KNK437 (N-Formyl-3,4-methylenedioxy-benzylidine-gamma-butyrolactam). This revealed a remarkably wide operating range of sustained periods extending across 25 h, from ≤17 h to >42 h. Moreover, this range was maintained at network and single-cell levels. Development of a new technique for formal analysis of circadian waveform, first derivative analysis (FDA), revealed internal phase patterning to the circadian oscillation at these extreme periods and differential phase sensitivity of the SCN to genetic and pharmacological manipulations. For example, FDA of the CK1ε(Tau/Tau) mutant SCN treated with the CK1ε-specific inhibitor PF-4800567 (3-[(3-Chlorophenoxy)methyl]-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine hydrochloride) revealed that period acceleration in the mutant is due to inappropriately phased activity of the CK1ε isoform. In conclusion, extreme period manipulation reveals unprecedented elasticity and temporal structure of the SCN circadian oscillation. SIGNIFICANCE STATEMENT: The master circadian clock of the suprachiasmatic nucleus (SCN) encodes time-of-day information that allows mammals to predict and thereby adapt to daily environmental cycles. Using combined genetic and pharmacological interventions, we assessed the temporal elasticity of the SCN network. Despite having evolved to generate a 24 h circadian period, we show that the molecular clock is surprisingly elastic, able to reversibly sustain coherent periods between ≤17 and >42 h at the levels of individual cells and the overall circuit. Using quantitative techniques to analyze these extreme periodicities, we reveal that the oscillator progresses as a sequence of distinct stages. These findings reveal new properties of how the SCN functions as a network and should inform biological and mathematical analyses of circadian timekeeping.


Assuntos
Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/genética , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/fisiologia , Proteínas tau/genética , Animais , Animais Recém-Nascidos , Compostos Benzidrílicos/farmacologia , Inibidores Enzimáticos/farmacologia , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/genética , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Antagonistas GABAérgicos/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Proteínas Circadianas Period/genética , Pirimidinas/farmacologia , Pirrolidinonas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Núcleo Supraquiasmático/citologia , Tetrodotoxina/farmacologia , Fatores de Tempo
14.
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
15.
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
16.
Proc Natl Acad Sci U S A ; 113(10): 2756-61, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26903623

RESUMO

The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1-CLOCK complexes is suppressed by PER-CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2(Edo/Edo) mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2(Edo) complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2(Edo/Edo); Csnk1e(Tau/Tau) mice and the SCN. These periods are unprecedented in mice. Thus, Per2(Edo) reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.


Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Mutação de Sentido Incorreto , Proteínas Circadianas Period/genética , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Caseína Quinase Iépsilon/genética , Caseína Quinase Iépsilon/metabolismo , Chlorocebus aethiops , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Atividade Motora/genética , Atividade Motora/fisiologia , Proteínas Circadianas Period/química , Proteínas Circadianas Period/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiopatologia
17.
Proc Natl Acad Sci U S A ; 113(10): 2732-7, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26903624

RESUMO

Circadian rhythms in mammals are coordinated by the suprachiasmatic nucleus (SCN). SCN neurons define circadian time using transcriptional/posttranslational feedback loops (TTFL) in which expression of Cryptochrome (Cry) and Period (Per) genes is inhibited by their protein products. Loss of Cry1 and Cry2 stops the SCN clock, whereas individual deletions accelerate and decelerate it, respectively. At the circuit level, neuronal interactions synchronize cellular TTFLs, creating a spatiotemporal wave of gene expression across the SCN that is lost in Cry1/2-deficient SCN. To interrogate the properties of CRY proteins required for circadian function, we expressed CRY in SCN of Cry-deficient mice using adeno-associated virus (AAV). Expression of CRY1::EGFP or CRY2::EGFP under a minimal Cry1 promoter was circadian and rapidly induced PER2-dependent bioluminescence rhythms in previously arrhythmic Cry1/2-deficient SCN, with periods appropriate to each isoform. CRY1::EGFP appropriately lengthened the behavioral period in Cry1-deficient mice. Thus, determination of specific circadian periods reflects properties of the respective proteins, independently of their phase of expression. Phase of CRY1::EGFP expression was critical, however, because constitutive or phase-delayed promoters failed to sustain coherent rhythms. At the circuit level, CRY1::EGFP induced the spatiotemporal wave of PER2 expression in Cry1/2-deficient SCN. This was dependent on the neuropeptide arginine vasopressin (AVP) because it was prevented by pharmacological blockade of AVP receptors. Thus, our genetic complementation assay reveals acute, protein-specific induction of cell-autonomous and network-level circadian rhythmicity in SCN never previously exposed to CRY. Specifically, Cry expression must be circadian and appropriately phased to support rhythms, and AVP receptor signaling is required to impose circuit-level circadian function.


Assuntos
Criptocromos/metabolismo , Receptores de Vasopressinas/metabolismo , Transdução de Sinais , Núcleo Supraquiasmático/metabolismo , Animais , Arritmias Cardíacas/fisiopatologia , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Criptocromos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Núcleo Supraquiasmático/fisiopatologia , Fatores de Tempo
18.
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
19.
Curr Biol ; 24(23): 2838-44, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25454592

RESUMO

Circadian clocks allow anticipation of daily environmental changes. The suprachiasmatic nucleus (SCN) houses the master clock, but clocks are also widely expressed elsewhere in the body. Although some peripheral clocks have established roles, it is unclear what local brain clocks do. We tested the contribution of one putative local clock in mouse histaminergic neurons in the tuberomamillary nucleus to the regulation of the sleep-wake cycle. Histaminergic neurons are silent during sleep, and start firing after wake onset; the released histamine, made by the enzyme histidine decarboxylase (HDC), enhances wakefulness. We found that hdc gene expression varies with time of day. Selectively deleting the Bmal1 (also known as Arntl or Mop3) clock gene from histaminergic cells removes this variation, producing higher HDC expression and brain histamine levels during the day. The consequences include more fragmented sleep, prolonged wake at night, shallower sleep depth (lower nonrapid eye movement [NREM] δ power), increased NREM-to-REM transitions, hindered recovery sleep after sleep deprivation, and impaired memory. Removing BMAL1 from histaminergic neurons does not, however, affect circadian rhythms. We propose that for mammals with polyphasic/nonwake consolidating sleep, the local BMAL1-dependent clock directs appropriately timed declines and increases in histamine biosynthesis to produce an appropriate balance of wake and sleep within the overall daily cycle of rest and activity specified by the SCN.


Assuntos
Fatores de Transcrição ARNTL/fisiologia , Neurônios/metabolismo , Sono/fisiologia , Animais , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica , Histamina/metabolismo , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Privação do Sono , Núcleo Supraquiasmático/fisiologia
20.
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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