RESUMEN
Sleep is regulated by homeostatic sleep drive and the circadian clock. While tremendous progress has been made in elucidating the molecular components of the core circadian oscillator, the output mechanisms by which this robust oscillator generates rhythmic sleep behavior remain poorly understood. At the cellular level, growing evidence suggests that subcircuits in the master circadian pacemaker suprachiasmatic nucleus (SCN) in mammals and in the clock network in Drosophila regulate distinct aspects of sleep. Thus, to identify novel molecules regulating the circadian timing of sleep, we conducted a large-scale screen of mouse SCN-enriched genes in Drosophila Here, we show that Tob (Transducer of ERB-B2) regulates the timing of sleep onset at night in female fruit flies. Knockdown of Tob pan-neuronally, either constitutively or conditionally, advances sleep onset at night. We show that Tob is specifically required in "evening neurons" (the LNds and the fifth s-LNv) of the clock network for proper timing of sleep onset. Tob levels cycle in a clock-dependent manner in these neurons. Silencing of these "evening" clock neurons results in an advanced sleep onset at night, similar to that seen with Tob knockdown. Finally, sharp intracellular recordings demonstrate that the amplitude and kinetics of LNd postsynaptic potentials (PSPs) cycle between day and night, and this cycling is attenuated with Tob knockdown in these cells. Our data suggest that Tob acts as a clock output molecule in a subset of clock neurons to potentiate their activity in the evening and enable the proper timing of sleep onset at night.
Asunto(s)
Ritmo Circadiano , Proteínas de Drosophila , Drosophila , Sueño , Animales , Femenino , Animales Modificados Genéticamente , Ritmo Circadiano/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Neuronas/fisiología , Sueño/fisiología , Núcleo Supraquiasmático/fisiologíaRESUMEN
Sleep is an evolutionarily conserved behavior that is increasingly recognized as important for human health. While its precise function remains controversial, sleep has been suggested to play a key role in a variety of biological phenomena ranging from synaptic plasticity to metabolic clearance. Although it is clear that sleep is regulated by the circadian clock, how this occurs remains enigmatic. Here we examine the genetic mechanisms by which the circadian clock regulates sleep, drawing on recent work in fruit flies, zebrafish, mice, and humans. These studies reveal that central and local clocks utilize diverse mechanisms to regulate different aspects of sleep, and a better understanding of this multilayered regulation may lead to a better understanding of the functions of sleep.
Asunto(s)
Relojes Circadianos/genética , Ritmo Circadiano/genética , Plasticidad Neuronal/genética , Sueño/genética , Animales , Relojes Circadianos/fisiología , Ritmo Circadiano/fisiología , Drosophila/genética , Humanos , Ratones , Plasticidad Neuronal/fisiología , Sueño/fisiología , Pez Cebra/genéticaRESUMEN
BACKGROUND: Circadian clocks control cell cycle factors, and circadian disruption promotes cancer. To address whether enhancing circadian rhythmicity in tumor cells affects cell cycle progression and reduces proliferation, we compared growth and cell cycle events of B16 melanoma cells and tumors with either a functional or dysfunctional clock. RESULTS: We found that clock genes were suppressed in B16 cells and tumors, but treatments inducing circadian rhythmicity, such as dexamethasone, forskolin and heat shock, triggered rhythmic clock and cell cycle gene expression, which resulted in fewer cells in S phase and more in G1 phase. Accordingly, B16 proliferation in vitro and tumor growth in vivo was slowed down. Similar effects were observed in human colon carcinoma HCT-116 cells. Notably, the effects of dexamethasone were not due to an increase in apoptosis nor to an enhancement of immune cell recruitment to the tumor. Knocking down the essential clock gene Bmal1 in B16 tumors prevented the effects of dexamethasone on tumor growth and cell cycle events. CONCLUSIONS: Here we demonstrated that the effects of dexamethasone on cell cycle and tumor growth are mediated by the tumor-intrinsic circadian clock. Thus, our work reveals that enhancing circadian clock function might represent a novel strategy to control cancer progression.
Asunto(s)
Relojes Circadianos , Melanoma Experimental/genética , Melanoma Experimental/patología , Factores de Transcripción ARNTL/metabolismo , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relojes Circadianos/efectos de los fármacos , Relojes Circadianos/genética , Ritmo Circadiano/efectos de los fármacos , Colforsina/farmacología , Dexametasona/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HCT116 , Respuesta al Choque Térmico/efectos de los fármacos , Humanos , Ratones Endogámicos C57BL , Modelos BiológicosRESUMEN
Noradrenaline (NE) plays an integral role in shaping behavioral outcomes including anxiety/depression, fear, learning and memory, attention and shifting behavior, sleep-wake state, pain, and addiction. However, it is unclear whether dysregulation of NE release is a cause or a consequence of maladaptive orientations of these behaviors, many of which associated with psychiatric disorders. To address this question, we used a unique genetic model in which the brain-specific vesicular monoamine transporter-2 (VMAT2) gene expression was removed in NE-positive neurons disabling NE release in the entire brain. We engineered VMAT2 gene splicing and NE depletion by crossing floxed VMAT2 mice with mice expressing the Cre-recombinase under the dopamine ß-hydroxylase (DBH) gene promotor. In this study, we performed a comprehensive behavioral and transcriptomic characterization of the VMAT2DBHcre KO mice to evaluate the role of central NE in behavioral modulations. We demonstrated that NE depletion induces anxiolytic and antidepressant-like effects, improves contextual fear memory, alters shifting behavior, decreases the locomotor response to amphetamine, and induces deeper sleep during the non-rapid eye movement (NREM) phase. In contrast, NE depletion did not affect spatial learning and memory, working memory, response to cocaine, and the architecture of the sleep-wake cycle. Finally, we used this model to identify genes that could be up- or down-regulated in the absence of NE release. We found an up-regulation of the synaptic vesicle glycoprotein 2c (SV2c) gene expression in several brain regions, including the locus coeruleus (LC), and were able to validate this up-regulation as a marker of vulnerability to chronic social defeat. The NE system is a complex and challenging system involved in many behavioral orientations given it brain wide distribution. In our study, we unraveled specific role of NE neurotransmission in multiple behavior and link it to molecular underpinning, opening future direction to understand NE role in health and disease.
Asunto(s)
Encéfalo , Transcriptoma , Ratones , Animales , Encéfalo/metabolismo , Norepinefrina/metabolismo , Depresión/metabolismo , Antidepresivos/farmacologíaRESUMEN
Circadian clocks generate rhythms of arousal, but the underlying molecular and cellular mechanisms remain unclear. In Drosophila, the clock output molecule WIDE AWAKE (WAKE) labels rhythmic neural networks and cyclically regulates sleep and arousal. Here, we show, in a male mouse model, that mWAKE/ANKFN1 labels a subpopulation of dorsomedial hypothalamus (DMH) neurons involved in rhythmic arousal and acts in the DMH to reduce arousal at night. In vivo Ca2+ imaging reveals elevated DMHmWAKE activity during wakefulness and rapid eye movement (REM) sleep, while patch-clamp recordings show that DMHmWAKE neurons fire more frequently at night. Chemogenetic manipulations demonstrate that DMHmWAKE neurons are necessary and sufficient for arousal. Single-cell profiling coupled with optogenetic activation experiments suggest that GABAergic DMHmWAKE neurons promote arousal. Surprisingly, our data suggest that mWAKE acts as a clock-dependent brake on arousal during the night, when mice are normally active. mWAKE levels peak at night under clock control, and loss of mWAKE leads to hyperarousal and greater DMHmWAKE neuronal excitability specifically at night. These results suggest that the clock does not solely promote arousal during an animal's active period, but instead uses opposing processes to produce appropriate levels of arousal in a time-dependent manner.
Asunto(s)
Relojes Circadianos , Sueño , Ratones , Animales , Masculino , Nivel de Alerta/fisiología , Neuronas/fisiología , Hipotálamo/fisiología , Ritmo Circadiano/fisiologíaRESUMEN
Sleep is under homeostatic control, whereby increasing wakefulness generates sleep need and triggers sleep drive. However, the molecular and cellular pathways by which sleep need is encoded are poorly understood. In addition, the mechanisms underlying both how and when sleep need is transformed to sleep drive are unknown. Here, using ex vivo and in vivo imaging, we show in Drosophila that astroglial Ca2+ signaling increases with sleep need. We demonstrate that this signaling is dependent on a specific L-type Ca2+ channel and is necessary for homeostatic sleep rebound. Thermogenetically increasing Ca2+ in astrocytes induces persistent sleep behavior, and we exploit this phenotype to conduct a genetic screen for genes required for the homeostatic regulation of sleep. From this large-scale screen, we identify TyrRII, a monoaminergic receptor required in astrocytes for sleep homeostasis. TyrRII levels rise following sleep deprivation in a Ca2+-dependent manner, promoting further increases in astrocytic Ca2+ and resulting in a positive-feedback loop. Moreover, our findings suggest that astrocytes then transmit this sleep need to a sleep drive circuit by upregulating and releasing the interleukin-1 analog Spätzle, which then acts on Toll receptors on R5 neurons. These findings define astroglial Ca2+ signaling mechanisms encoding sleep need and reveal dynamic properties of the sleep homeostatic control system.
Asunto(s)
Astrocitos/metabolismo , Señalización del Calcio/fisiología , Sueño/fisiología , Animales , Animales Modificados Genéticamente , Calcio/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Retroalimentación Fisiológica , Femenino , Técnicas de Silenciamiento del Gen , Microscopía Intravital , Canales Iónicos/genética , Canales Iónicos/metabolismo , Neuronas/metabolismo , Receptores de Amina Biogénica/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.
Asunto(s)
Envejecimiento/fisiología , Evolución Biológica , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vertebrados/fisiología , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas/genética , Señalización del Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Femenino , Edición Génica , Técnicas de Sustitución del Gen , Masculino , Ratones , Modelos Animales , Oxidación-Reducción , Filogenia , Aptitud Física/fisiología , Mutación PuntualRESUMEN
Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell-derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Microfilamentos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Mutación Missense , Miocardio/enzimología , Miocitos Cardíacos/enzimología , Taquicardia Ventricular/enzimología , Sustitución de Aminoácidos , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Oxigenasas de Función Mixta/genética , Miocardio/patología , Miocitos Cardíacos/patología , Oxidación-Reducción , Taquicardia Ventricular/genética , Taquicardia Ventricular/patologíaRESUMEN
Slow-wave sleep is a marker of sleep need, but its presence and function in non-mammalian species have been controversial. A new study finds sleep-dependent slow wave oscillations in the fruit fly, which act to inhibit sensory input during sleep.
Asunto(s)
Drosophila , Electroencefalografía , Animales , Proteínas de Drosophila , Sueño , Ubiquitina-Proteína LigasasRESUMEN
The circadian (â¼24 h) clock is continuously entrained (reset) by ambient light so that endogenous rhythms are synchronized with daily changes in the environment. Light-induced gene expression is thought to be the molecular mechanism underlying clock entrainment. mRNA translation is a key step of gene expression, but the manner in which clock entrainment is controlled at the level of mRNA translation is not well understood. We found that a light- and circadian clock-regulated MAPK/MNK pathway led to phosphorylation of the cap-binding protein eIF4E in the mouse suprachiasmatic nucleus of the hypothalamus, the locus of the master circadian clock in mammals. Phosphorylation of eIF4E specifically promoted translation of Period 1 (Per1) and Period 2 (Per2) mRNAs and increased the abundance of basal and inducible PER proteins, which facilitated circadian clock resetting and precise timekeeping. Together, these results highlight a critical role for light-regulated translational control in the physiology of the circadian clock.
Asunto(s)
Conducta Animal/fisiología , Ritmo Circadiano/fisiología , Factor 4E Eucariótico de Iniciación/fisiología , Animales , Conducta Animal/efectos de la radiación , Química Encefálica/genética , Química Encefálica/fisiología , Ritmo Circadiano/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Luz , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/fisiología , Fosforilación , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiologíaRESUMEN
Ultradian (~4 hr) rhythms in locomotor activity that do not depend on the master circadian pacemaker in the suprachiasmatic nucleus have been observed across mammalian species, however, the underlying mechanisms driving these rhythms are unknown. We show that disruption of the dopamine transporter gene lengthens the period of ultradian locomotor rhythms in mice. Period lengthening also results from chemogenetic activation of midbrain dopamine neurons and psychostimulant treatment, while the antipsychotic haloperidol has the opposite effect. We further reveal that striatal dopamine levels fluctuate in synchrony with ultradian activity cycles and that dopaminergic tone strongly predicts ultradian period. Our data indicate that an arousal regulating, dopaminergic ultradian oscillator (DUO) operates in the mammalian brain, which normally cycles in harmony with the circadian clock, but can desynchronize when dopamine tone is elevated, thereby producing aberrant patterns of arousal which are strikingly similar to perturbed sleep-wake cycles comorbid with psychopathology.
Asunto(s)
Ciclos de Actividad/fisiología , Nivel de Alerta/fisiología , Conducta Animal , Dopamina/fisiología , Ciclos de Actividad/efectos de los fármacos , Animales , Haloperidol/farmacología , Locomoción , Metanfetamina/farmacología , Ratones , Ratones Noqueados , Núcleo Supraquiasmático/efectos de los fármacos , Núcleo Supraquiasmático/fisiologíaRESUMEN
Although there is evidence for a circadian regulation of the preovulatory LH surge, the contributions of individual tissue clocks to this process remain unclear. We studied female mice deficient in the Bmal1 gene (Bmal1(-/-)), which is essential for circadian clock function, and found that they lack the proestrous LH surge. However, spontaneous ovulation on the day of estrus was unaffected in these animals. Bmal1(-/-) females were also deficient in the proestrous FSH surge, which, like the LH surge, is GnRH-dependent. In the absence of circadian or external timing cues, Bmal1(-/-) females continued to cycle in constant darkness albeit with increased cycle length and time spent in estrus. Because pituitary gonadotropes are the source of circulating LH and FSH, we assessed hypophyseal circadian clock function and found that female pituitaries rhythmically express clock components throughout all cycle stages. To determine the role of the gonadotrope clock in the preovulatory LH and FSH surge process, we generated mice that specifically lack BMAL1 in gonadotropes (GBmal1KO). GBmal1KO females exhibited a modest elevation in both proestrous and baseline LH levels across all estrous stages. BMAL1 elimination from gonadotropes also led to increased variability in estrous cycle length, yet GBmal1KO animals were otherwise reproductively normal. Together our data suggest that the intrinsic clock in gonadotropes is dispensable for LH surge regulation but contributes to estrous cycle robustness. Thus, clocks in the suprachiasmatic nucleus or elsewhere must be involved in the generation of the LH surge, which, surprisingly, is not required for spontaneous ovulation.