RESUMEN
BACKGROUND: Sleep deprivation (SD) is a growing global health problem with many deleterious effects, such as cognitive impairment. Microglia activation-induced neuroinflammation may be an essential factor in this. Propofol has been shown to clear sleep debt after SD in rats. This study aims to evaluate the effects of propofol-induced sleep on ameliorating sleep quality impairment and cognitive decline after 48 h SD. METHODS: Almost 8-12-week-old rats were placed in the SD system for 48 h of natural sleep or continuous SD. Afterwards, rats received propofol (20 mg·kg-1·h-1, 6 h) via the tail or slept naturally. The Morris water maze (MWM) and Y-maze test assessed spatial learning and memory abilities. Rat EEG/EMG monitored sleep. The expression of brain and muscle Arnt-like protein 1 (BMAL1), brain-derived neurotrophic factor (BDNF) in the hippocampus and BMAL1 in the hypothalamus were assessed by western blot. Enzyme-linked immunosorbent assay detected IL-6, IL-1ß, arginase 1 (Arg1), and IL-10 levels in the hippocampus. Immunofluorescence was used to determine microglia expression as well as morphological changes. RESULTS: Compared to the control group, the sleep-deprived rats showed poor cognitive performance on both the MWM test and the Y-maze test, accompanied by disturbances in sleep structure, including increased total sleep time, and increased time spent and delta power in non-rapid eye movement sleep. In addition, SD induces abnormal expression of the circadian rhythm protein BMAL1, activates microglia, and causes neuroinflammation and nerve damage. Propofol reversed these changes and saved sleep and cognitive impairment. Furthermore, propofol treatment significantly reduced hippocampal IL-1ß and IL-6 levels, increased BDNF, Arg1, and IL-10 levels, and switched microglia surface markers from the inflammatory M1 type to the anti-inflammatory M2 type. CONCLUSION: Propofol reduces SD-induced cognitive impairment and circadian rhythm disruption, possibly by lowering neuronal inflammation and switching the microglia phenotype from an M1 to an M2 activated state, thus exerting neuroprotective effects.
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Factores de Transcripción ARNTL , Disfunción Cognitiva , Aprendizaje por Laberinto , Microglía , Propofol , Ratas Sprague-Dawley , Privación de Sueño , Animales , Privación de Sueño/complicaciones , Microglía/efectos de los fármacos , Microglía/metabolismo , Factores de Transcripción ARNTL/metabolismo , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/biosíntesis , Masculino , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/metabolismo , Ratas , Propofol/farmacología , Aprendizaje por Laberinto/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Sueño/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismoRESUMEN
The cardiac circadian clock is responsible for the modulation of different myocardial processes, and its dysregulation has been linked to disease development. How this clock machinery is regulated in the heart remains an open question. Because noradrenaline (NE) can act as a zeitgeber in cardiomyocytes, we tested the hypothesis that adrenergic signaling resets cardiac clock gene expression in vivo. In its anti-phase with Clock and Bmal1, cardiac Per1 abundance increased during the dark phase, concurrent with the rise in heart rate and preceded by an increase in NE levels. Sympathetic denervation altered Bmal1 and Clock amplitude, while Per1 was affected in both amplitude and oscillatory pattern. We next treated mice with a ß-adrenergic receptor (ß-AR) blocker. Strikingly, the ß-AR blockade during the day suppressed the nocturnal increase in Per1 mRNA, without altering Clock or Bmal1. In contrast, activating ß-AR with isoproterenol (ISO) promoted an increase in Per1 expression, demonstrating its responsiveness to adrenergic input. Inhibitors of ERK1/2 and CREB attenuated ISO-induced Per1 expression. Upstream of ERK1/2, PI3Kγ mediated ISO induction of Per1 transcription, while activation of ß2-AR, but not ß1-AR induced increases in ERK1/2 phosphorylation and Per1 expression. Consistent with the ß2-induction of Per1 mRNA, ISO failed to activate ERK1/2 and elevate Per1 in the heart of ß2-AR-/- mice, whereas a ß2-AR antagonist attenuated the nocturnal rise in Per1 expression. Our study established a link between NE/ß2-AR signaling and Per1 oscillation via the PI3Ky-ERK1/2-CREB pathway, providing a new framework for understanding the physiological mechanism involved in resetting cardiac clock genes.
Asunto(s)
Regulación de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Miocardio/metabolismo , Proteínas Circadianas Period/biosíntesis , Receptores Adrenérgicos beta 2/metabolismo , Factores de Transcripción ARNTL/biosíntesis , Factores de Transcripción ARNTL/genética , Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Animales , Proteínas CLOCK/biosíntesis , Isoproterenol/farmacología , Masculino , Ratones , Ratones Noqueados , Proteínas Circadianas Period/genética , Receptores Adrenérgicos beta 2/genéticaRESUMEN
INTRODUCTION: The occurrence of circadian rhythm disorder in patients with Alzheimer's disease (AD) is closely related to the abnormal deposition of amyloid-ß (Aß), and d-Ser2-oxyntomodulin (Oxy) is a protease-resistant oxyntomodulin analogue that has been shown to exert neuroprotective effects. AIMS: This study aimed to explore whether Oxy, a new GLP-1R/GCGR dual receptor agonist, can improve the Aß-induced disrupted circadian rhythm and the role of GLP-1R. METHODS: A mouse wheel-running experiment was performed to explore the circadian rhythm, and western blotting and real-time PCR were performed to assess the expression of the circadian clock genes Bmal1 and Per2. Furthermore, a lentivirus encoding an shGLP-1R-GFP-PURO was used to interfere with GLP-1R gene expression and so explore the role of GLP-1R. RESULTS: The present study has confirmed that Oxy could restore Aß31-35-induced circadian rhythm disorders and improve the abnormal expression of Bmal1 and Per2. After interfering the GLP-1R gene, we found that Oxy could not improve the Aß31-35-induced circadian rhythm disorder and abnormal expression of clock genes. CONCLUSION: This study demonstrated that Oxy could improve Aß31-35-induced circadian rhythm disorders, and GLP-1R plays a critical role. This study thus describes a novel target that may be potentially used in the treatment of AD.
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Péptidos beta-Amiloides/toxicidad , Trastornos Cronobiológicos/inducido químicamente , Trastornos Cronobiológicos/tratamiento farmacológico , Oxintomodulina/uso terapéutico , Fragmentos de Péptidos/toxicidad , Factores de Transcripción ARNTL/biosíntesis , Animales , Línea Celular , Trastornos Cronobiológicos/metabolismo , Receptor del Péptido 1 Similar al Glucagón/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Circadianas Period/biosíntesisRESUMEN
Trimethylamine N-oxide (TMAO) promotes atherosclerosis in association with the functions of endothelial cells. Clock and Bmal1, as two main components of molecular circadian clock, play important regulatory roles during progression of atherogenesis. However, whether Clock and Bmal1 are involved in the regulation of endothelial proliferation disturbed by TMAO are unclear. We observed that cell proliferation of human umbilical vein endothelial cells (HUVECs) was inhibited after exposed to TMAO for 24â¯h. Besides, TMAO caused increased expression of lncRNA-NEAT1, Clock and Bmal1, and inhibited MAPK pathways. While MAPK pathways were blocked, the expression of Clock and Bmal1 was elevated. NEAT1 showed a circadian rhythmic expression in HUVECs, and its overexpression reduced cell proliferation. Knockdown or overexpression of NEAT1 might decrease or increase the expression of Clock and Bmal1 respectively, while raised or suppressed the expression of MAPK pathways correspondingly. Asparagus extract (AE) was found to improve the TMAO-reduced HUVECs proliferation. Moreover, it ameliorated the disorders of NEAT1, Clock, Bmal1, and MAPK signaling pathways induced by TMAO. Therefore, our findings indicated that NEAT1 regulating Clock-Bmal1 via MAPK pathways was involved in TMAO-repressed HUVECs proliferation, and AE improved endothelial proliferation by TMAO, proposing a novel mechanism for cardiovascular disease prevention.
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Asparagaceae/química , Ritmo Circadiano/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Metilaminas/toxicidad , Extractos Vegetales/farmacología , ARN Largo no Codificante/fisiología , Factores de Transcripción ARNTL/antagonistas & inhibidores , Factores de Transcripción ARNTL/biosíntesis , Factores de Transcripción ARNTL/genética , Aterosclerosis/genética , Aterosclerosis/fisiopatología , Proteínas CLOCK/biosíntesis , Proteínas CLOCK/genética , División Celular/efectos de los fármacos , Ritmo Circadiano/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Metilaminas/farmacología , Tallos de la Planta/química , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacologíaRESUMEN
Purpose: Diabetes leads to the downregulation of the retinal Kir4.1 channels and Müller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Müller cells. Circadian rhythms play an integral role in normal physiology; however, diabetes leads to a circadian dysrhythmia. We hypothesize that diabetes will result in a circadian dysrhythmia of IRS-1 and Kir4.1 and disturbed clock gene function will have a critical role in regulating Kir4.1 channels. Methods: We assessed a diurnal rhythm of retinal IRS-1 and Kir4.1 in db/db mice. The Kir4.1 function was evaluated using a whole-cell recording of Müller cells. The rat Müller cells (rMC-1) were used to undertake in vitro studies using a siRNA. Results: The IRS-1 exhibited a diurnal rhythm in control mice; however, with diabetes, this natural rhythm was lost. The Kir4.1 levels peaked and troughed at times similar to the IRS-1 rhythm. The IRS-1 silencing in the rMC-1 led to a decrease in Kir4.1 and BMAL1. The insulin treatment of retinal explants upregulated Kir4.1 possibly via upregulation of BMAL1 and phosphorylation of IRS-1 and Akt-1. Conclusions: Our studies highlight that IRS-1, by regulating BMAL1, is an important regulator of Kir4.1 in Müller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1.
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Factores de Transcripción ARNTL/genética , Ritmo Circadiano/fisiología , Retinopatía Diabética/genética , Células Ependimogliales/metabolismo , Regulación de la Expresión Génica , Proteínas Sustrato del Receptor de Insulina/genética , Canales de Potasio de Rectificación Interna/genética , Factores de Transcripción ARNTL/biosíntesis , Animales , Western Blotting , Células Cultivadas , Diabetes Mellitus Experimental , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Células Ependimogliales/patología , Humanos , Proteínas Sustrato del Receptor de Insulina/biosíntesis , Ratones , Reacción en Cadena de la Polimerasa , Canales de Potasio de Rectificación Interna/biosíntesis , ARN/genética , RatasRESUMEN
Most living creatures have a circadian rhythm that is generated by a precisely regulated transcriptional-translational feedback loop of clock genes. Brain and muscle ARNT-like 1 (BMAL1) is one of the core clock genes and transcription factors that represents a positive arm of this autoregulatory circadian clock system. Despite the indispensable role of BMAL1 in the circadian rhythm, the molecular mechanisms underlying translational control of BMAL1 are largely unknown. Here, using murine NIH-3T3 cells, gene constructs, and a variety of biochemical approaches, including RNAi- and luciferase reporter gene-based assays, along with immunoblotting, in vitro transcription, quantitative real-time PCR, and real-time bioluminescence experiments, we show that translation of Bmal1 is negatively regulated by an RNA-binding protein, heterogeneous nuclear ribonucleoprotein Q (hnRNP Q). Interestingly, we found that hnRNP Q rhythmically binds to a specific region of the Bmal1 mRNA 5' UTR and controls its time-dependent expression. Moreover, we demonstrate that knockdown of hnRNP Q modulates BMAL1 protein oscillation amplitude without affecting mRNA rhythmic patterns. Furthermore, hnRNP Q depletion increases the mRNA oscillation amplitudes of BMAL1-regulated target genes. Together, our results suggest that hnRNP Q plays a pivotal role in both Bmal1 translation and BMAL1-regulated gene expression.
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Regiones no Traducidas 5' , Factores de Transcripción ARNTL/biosíntesis , Regulación de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Factores de Transcripción ARNTL/genética , Animales , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ratones , Células 3T3 NIH , Transporte de Proteínas/genética , ARN Mensajero/genéticaRESUMEN
OBJECTIVE(S): To determine whether variation in cell-associated unspliced (CA-US) HIV RNA in HIV-infected individuals on antiretroviral therapy (ART) has a circadian basis. METHODS: Prospective observational study of HIV-infected individuals on ART. Blood was collected on three occasions and CA-US HIV RNA and mRNA of the circadian-locomotor-output-cycles-kaput (CLOCK)-associated genes quantified by real time PCR. CLOCK-associated proteins were over-expressed in a cell line stably transfected with an HIV long-terminal repeat (LTR) luciferase reporter. RESULTS: Using a mixed effects model, there was a significant increase in log-CA-US RNA at the third visit compared with the first visit (effect size of 0.619 with standard error (SE) of 0.098, Pâ<â0.001) and an independent effect of time of blood draw (effect size 0.051 (SE 0.025), Pâ=â0.040). The CLOCK-associated gene, brain-and-muscle-ARNT-like-1 (BMAL-1) had a significant relationship with log CA-US HIV RNA (effect size 8.508 (SE 3.777), Pâ=â0.028) and also with time (Pâ=â0.045). Over expression of BMAL-1 and CLOCK in a cell line stably transfected with an HIV-LTR luciferase reporter resulted in an increase in luciferase expression and this was reduced following mutation of the second E-box in the HIV-LTR. CONCLUSION: The basal level of HIV transcription on ART can vary significantly and is modulated by the circadian regulator BMAL-1, amongst other factors.
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Factores de Transcripción ARNTL/biosíntesis , Antirretrovirales/uso terapéutico , Células Sanguíneas/virología , Infecciones por VIH/virología , VIH/crecimiento & desarrollo , ARN Viral/análisis , Transcripción Genética , Factores de Transcripción ARNTL/genética , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Infecciones por VIH/tratamiento farmacológico , Interacciones Huésped-Patógeno , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Many mammalian genes are transcribed during short bursts of variable frequencies and sizes that substantially contribute to cell-to-cell variability. However, which molecular mechanisms determine bursting properties remains unclear. To probe putative mechanisms, we combined temporal analysis of transcription along the circadian cycle with multiple genomic reporter integrations, using both short-lived luciferase live microscopy and single-molecule RNA-FISH. Using the Bmal1 circadian promoter as our model, we observed that rhythmic transcription resulted predominantly from variations in burst frequency, while the genomic position changed the burst size. Thus, burst frequency and size independently modulated Bmal1 transcription. We then found that promoter histone-acetylation level covaried with burst frequency, being greatest at peak expression and lowest at trough expression, while remaining unaffected by the genomic location. In addition, specific deletions of ROR-responsive elements led to constitutively elevated histone acetylation and burst frequency. We then investigated the suggested link between histone acetylation and burst frequency by dCas9p300-targeted modulation of histone acetylation, revealing that acetylation levels influence burst frequency more than burst size. The correlation between acetylation levels at the promoter and burst frequency was also observed in endogenous circadian genes and in embryonic stem cell fate genes. Thus, our data suggest that histone acetylation-mediated control of transcription burst frequency is a common mechanism to control mammalian gene expression.
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Factores de Transcripción ARNTL/biosíntesis , Ritmo Circadiano/fisiología , Regulación de la Expresión Génica/fisiología , Histonas/metabolismo , Modelos Biológicos , Regiones Promotoras Genéticas/fisiología , Transcripción Genética/fisiología , Factores de Transcripción ARNTL/genética , Acetilación , Animales , Ratones , Células 3T3 NIHRESUMEN
Occurrence of stroke cases displays a time-of-day variation in human. However, the mechanism linking circadian rhythm to the internal response mechanisms against pathophysiological events after ischemic stroke remained largely unknown. To this end, temporal changes in the susceptibility to ischemia/reperfusion (I/R) injury were investigated in mice in which the ischemic stroke induced at four different Zeitgeber time points with 6-h intervals (ZT0, ZT6, ZT12, and ZT18). Besides infarct volume and brain swelling, neuronal survival, apoptosis, ischemia, and circadian rhythm related proteins were examined using immunohistochemistry, Western blot, planar surface immune assay, and liquid chromatography-mass spectrometry tools. Here, we present evidence that midnight (ZT18; 24:00) I/R injury in mice resulted in significantly improved infarct volume, brain swelling, neurological deficit score, neuronal survival, and decreased apoptotic cell death compared with ischemia induced at other time points, which were associated with increased expressions of circadian proteins Bmal1, PerI, and Clock proteins and survival kinases AKT and Erk-1/2. Moreover, ribosomal protein S6, mTOR, and Bad were also significantly increased, while the levels of PRAS40, negative regulator of AKT and mTOR, and phosphorylated p53 were decreased at this time point compared to ZT0 (06:00). Furthermore, detailed proteomic analysis revealed significantly decreased CSKP, HBB-1/2, and HBA levels, while increased GNAZ, NEGR1, IMPCT, and PDE1B at midnight as compared with early morning. Our results indicate that nighttime I/R injury results in less severe neuronal damage, with increased neuronal survival, increased levels of survival kinases and circadian clock proteins, and also alters the circadian-related proteins.
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Factores de Transcripción ARNTL/biosíntesis , Isquemia Encefálica/metabolismo , Ritmo Circadiano/fisiología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Accidente Cerebrovascular/metabolismo , Animales , Isquemia Encefálica/patología , Supervivencia Celular/fisiología , Relojes Circadianos/fisiología , Fragmentación del ADN , Masculino , Ratones , Ratones Endogámicos BALB C , Neuronas/patología , Accidente Cerebrovascular/patologíaRESUMEN
The temporal order of physiology and behavior in mammals is primarily regulated by the circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Taking advantage of bioluminescence reporters, we monitored the circadian rhythms of the expression of clock genes Per1 and Bmal1 in the SCN of freely moving mice and found that the rate of phase shifts induced by a single light pulse was different in the two rhythms. The Per1-luc rhythm was phase-delayed instantaneously by the light presented at the subjective evening in parallel with the activity onset of behavioral rhythm, whereas the Bmal1-ELuc rhythm was phase-delayed gradually, similar to the activity offset. The dissociation was confirmed in cultured SCN slices of mice carrying both Per1-luc and Bmal1-ELuc reporters. The two rhythms in a single SCN slice showed significantly different periods in a long-term (3 wk) culture and were internally desynchronized. Regional specificity in the SCN was not detected for the period of Per1-luc and Bmal1-ELuc rhythms. Furthermore, neither is synchronized with circadian intracellular Ca2+ rhythms monitored by a calcium indicator, GCaMP6s, or with firing rhythms monitored on a multielectrode array dish, although the coupling between the circadian firing and Ca2+ rhythms persisted during culture. These findings indicate that the expressions of two key clock genes, Per1 and Bmal1, in the SCN are regulated in such a way that they may adopt different phases and free-running periods relative to each other and are respectively associated with the expression of activity onset and offset.
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Factores de Transcripción ARNTL/biosíntesis , Conducta Animal , Señalización del Calcio , Ritmo Circadiano , Proteínas Circadianas Period/biosíntesis , Núcleo Supraquiasmático/metabolismo , Factores de Transcripción ARNTL/genética , Animales , Calcio/metabolismo , Femenino , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Proteínas Circadianas Period/genéticaRESUMEN
Sleep is governed by homeostasis and the circadian clock. Clock genes play an important role in the generation and maintenance of circadian rhythms but are also involved in regulating sleep homeostasis. The lateral habenular nucleus (LHb) has been implicated in sleep-wake regulation, since LHb gene expression demonstrates circadian oscillation characteristics. This study focuses on the participation of LHb clock genes in regulating sleep homeostasis, as the nature of their involvement is unclear. In this study, we observed changes in sleep pattern following sleep deprivation in LHb-lesioned rats using EEG recording techniques. And then the changes of clock gene expression (Per1, Per2, and Bmal1) in the LHb after 6 hours of sleep deprivation were detected by using real-time quantitative PCR (qPCR). We found that sleep deprivation increased the length of Non-Rapid Eye Movement Sleep (NREMS) and decreased wakefulness. LHb-lesioning decreased the amplitude of reduced wake time and increased NREMS following sleep deprivation in rats. qPCR results demonstrated that Per2 expression was elevated after sleep deprivation, while the other two genes were unaffected. Following sleep recovery, Per2 expression was comparable to the control group. This study provides the basis for further research on the role of LHb Per2 gene in the regulation of sleep homeostasis.
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Ritmo Circadiano/genética , Habénula/fisiología , Privación de Sueño/genética , Factores de Transcripción ARNTL/biosíntesis , Factores de Transcripción ARNTL/genética , Animales , Expresión Génica , Habénula/metabolismo , Habénula/patología , Masculino , Proteínas Circadianas Period/biosíntesis , Proteínas Circadianas Period/genética , Ratas , Ratas Wistar , Sueño/fisiología , Privación de Sueño/metabolismo , Vigilia/fisiologíaRESUMEN
Vasoactive intestinal peptide (VIP) signaling via cyclic adenosine monophosphate (cAMP) is reported to stimulate steroidogenesis in ovarian granulosa cells from a variety of vertebrate species, including the domestic hen. Prior to follicle selection in the hen ovary (i.e., cyclic recruitment) follicle-stimulating-hormone (FSH)-induced cAMP signaling is absent within the granulosa layer until immediately following follicle selection. As a consequence, granulosa cells remain in an undifferentiated state and are unable to initiate FSH-induced steroidogenesis. VIP receptors (VPAC1 and VPAC2), like the FSH receptor, are G protein-coupled receptors, so we predicted that VIP signaling in granulosa cells is also absent in follicles that have not yet been selected into the preovulatory hierarchy. Initial studies established that mRNA encoding VPAC1 and VPAC2 are expressed within the granulosa cells throughout follicle development. Nevertheless, undifferentiated granulosa cells from prehierarchal (6-8 mm) follicles do not accumulate cAMP in response to a 4-hr incubation with chicken VIP; the capacity for such receptor signaling is attained only following selection within actively differentiating granulosa cells. VIP treatment did, however, increase expression of mRNA encoding the Gallus circadian clock protein, BMAL1-but only in granulosa cells collected from selected follicles. These findings provide evidence that, at follicle selection, the acquisition of VIP-induced cAMP cell signaling helps initiate and promote the differentiation of of granulosa cells. Furthermore, we propose that VIP signaling may regulate BMAL1 expression and, thus, a daily rhythmicity within granulosa cells of preovulatory follicles. Mol. Reprod. Dev. 83: 455-463, 2016. © 2016 Wiley Periodicals, Inc.
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Factores de Transcripción ARNTL/biosíntesis , Proteínas Aviares/biosíntesis , Diferenciación Celular/fisiología , Pollos/metabolismo , Regulación de la Expresión Génica/fisiología , Células de la Granulosa/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Animales , AMP Cíclico/metabolismo , Femenino , Células de la Granulosa/citología , Sistemas de Mensajero Secundario/fisiologíaRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disease often accompanied with disruption of sleep-wake cycle. The sleep-wake cycle is controlled by mechanisms involving internal timekeeping (circadian) regulation. The aim of our present pilot study was to assess the circadian system in patients with mild form of AD in their home environment. In the study, 13 elderly AD patients and 13 age-matched healthy control subjects (the patient's spouses) were enrolled. Sleep was recorded for 21 days by sleep diaries in all participants and checked by actigraphy in 4 of the AD patient/control couples. The samples of saliva and buccal mucosa were collected every 4 hours during the same 24 h-interval to detect melatonin and clock gene (PER1 and BMAL1) mRNA levels, respectively. The AD patients exhibited significantly longer inactivity interval during the 24 h and significantly higher number of daytime naps than controls. Daily profiles of melatonin levels exhibited circadian rhythms in both groups. Compared with controls, decline in amplitude of the melatonin rhythm in AD patients was not significant, however, in AD patients more melatonin profiles were dampened or had atypical waveforms. The clock genes PER1 and BMAL1 were expressed rhythmically with high amplitudes in both groups and no significant differences in phases between both groups were detected. Our results suggest moderate differences in functional state of the circadian system in patients with mild form of AD compared with healthy controls which are present in conditions of their home dwelling.
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Enfermedad de Alzheimer/fisiopatología , Ritmo Circadiano/fisiología , Factores de Transcripción ARNTL/biosíntesis , Factores de Transcripción ARNTL/genética , Actigrafía , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/complicaciones , Estudios de Casos y Controles , Ambiente , Femenino , Regulación de la Expresión Génica , Vivienda , Humanos , Masculino , Registros Médicos , Melatonina/análisis , Mucosa Bucal/química , Proteínas Circadianas Period/biosíntesis , Proteínas Circadianas Period/genética , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Saliva/química , Trastornos Intrínsecos del Sueño/complicaciones , Trastornos Intrínsecos del Sueño/fisiopatologíaRESUMEN
Circadian rhythm disturbance promotes of carcinogenesis and is associated with changes in clock genes and proteins expression. In the current study we observed that continu- ous light exposure potentiated skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene and 12-0-tetradecanoylphor- bol-13-acetate while melatonin had opposite effect. Carcinogenic exposure decreased BMAL1 and CRYI- expression in the skin, CLOCK expression was upregulated and CRYl downregulated in tumor compared to normal skin. BMAL1 expression increased under disrupted light regimen; melatonin treatment affected CLOCK expression in tumors and CRYI in skin at the carcinogens application sites.
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Proteínas CLOCK/biosíntesis , Transformación Celular Neoplásica/metabolismo , Regulación Neoplásica de la Expresión Génica , Iluminación , Proteínas de Neoplasias/biosíntesis , Neoplasias Cutáneas/metabolismo , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Factores de Transcripción ARNTL/biosíntesis , Animales , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/patología , Criptocromos/biosíntesis , Masculino , Ratones , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol/toxicidadRESUMEN
The circadian clock is driven by transcriptional oscillation of clock genes in almost all body cells. To investigate the effect of cell type-specific intracellular environment on the circadian machinery, we examined gene expression profiles in five peripheral tissues. As expected, the phase relationship between expression rhythms of nine clock genes was similar in all tissues examined. We also compared relative expression levels of clock genes among tissues, and unexpectedly found that quantitative variation remained within an approximately three-fold range, which was substantially smaller than that of metabolic housekeeping genes. Interestingly, circadian gene expression was little affected even when fibroblasts were cultured with different concentrations of serum. Together, these findings support a hypothesis that expression levels of clock genes are quantitatively compensated for the intracellular environment, such as redox potential and metabolite composition. However, more comprehensive studies are required to reach definitive conclusions.
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Relojes Circadianos/genética , Ritmo Circadiano/genética , Regulación de la Expresión Génica/genética , Factores de Transcripción ARNTL/biosíntesis , Actinas/biosíntesis , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Criptocromos/biosíntesis , Expresión Génica , Perfilación de la Expresión Génica , Células HEK293 , Células Hep G2 , Humanos , Ratones , Células 3T3 NIH , Proteínas Circadianas Period/biosíntesis , Proteínas Circadianas Period/genética , ARN Ribosómico 18S/biosíntesisRESUMEN
The subgranular zone (SGZ) of the adult hippocampus contains a pool of quiescent neural progenitor cells (QNPs) that are capable of entering the cell cycle and producing newborn neurons. The mechanisms that control the timing and extent of adult neurogenesis are not well understood. Here, we show that QNPs of the adult SGZ express molecular-clock components and proliferate in a rhythmic fashion. The clock proteins PERIOD2 and BMAL1 are critical for proper control of neurogenesis. The absence of PERIOD2 abolishes the gating of cell-cycle entrance of QNPs, whereas genetic ablation of bmal1 results in constitutively high levels of proliferation and delayed cell-cycle exit. We use mathematical model simulations to show that these observations may arise from clock-driven expression of a cell-cycle inhibitor that targets the cyclin D/Cdk4-6 complex. Our findings may have broad implications for the circadian clock in timing cell-cycle events of other stem cell populations throughout the body.
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Factores de Transcripción ARNTL/genética , Relojes Circadianos/genética , Hipocampo/crecimiento & desarrollo , Neurogénesis/genética , Proteínas Circadianas Period/genética , Factores de Transcripción ARNTL/biosíntesis , Animales , Ciclo Celular/genética , Proliferación Celular , Ciclina D/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células-Madre Neurales , Neuronas/citología , Proteínas Circadianas Period/biosíntesis , Regiones Promotoras GenéticasRESUMEN
The circadian clock enables organisms to adjust to daily environmental changes and synchronize multiple molecular, biochemical, physiological, and behavioral processes accordingly. In mammalian clock work, Bmal1 is the most important core clock gene, which works with another core clock gene Clock to drive the expression of other clock genes and clock-controlled genes. However, the regulation of Bmal1 has not been fully understood. This work was aimed at identifying the positive regulator(s) of Bmal1 transcription. A series of 5' deletion reporter constructs was generated, and binding site mutations of mouse Bmal1 promoter fragments were cloned into pGL3-basic and pGL3(R2.1)-basic plasmids and transfected into NIH 3T3 cells. Luciferase activity was either measured 48 h after transfection or recorded for 4 days after serum shock. DNA affinity precipitation assay was used to detect the transcription factors binding to Bmal1 promoter. Small interfering RNA against nuclear factor Y, subunit A (NF-YA) and dominant negative NF-YA were employed to study the role of NF-Y in Bmal1 transcription regulation. Deletion and mutation analyses identified two clusters of CCAAT/GC-boxes at the proximal region of Bmal1 promoter as the activating cis-elements. Bmal1 promoter activity was up-regulated by NF-Y and/or Sp1 and repressed by dominant negative NF-YA or siRNA against NF-YA. The activation of Bmal1 promoter activity by NF-Y and Sp1 was inhibited by Rev-Erbα. DNA affinity precipitation assay showed that NF-Y and Sp1 bound to the two CCAAT/GC clusters of Bmal1 promoter. These results indicate that NF-Y is a functional activator of Bmal1 transcription and it cooperates with Sp1 and Rev-Erbα to generate the daily cycle of Bmal1 expression.
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Factores de Transcripción ARNTL/biosíntesis , Factor de Unión a CCAAT/metabolismo , Relojes Circadianos/fisiología , Regulación de la Expresión Génica/fisiología , Elementos de Respuesta/fisiología , Transcripción Genética/fisiología , Factores de Transcripción ARNTL/genética , Animales , Factor de Unión a CCAAT/genética , Ratones , Células 3T3 NIH , Proteínas Oncogénicas v-erbA/genética , Proteínas Oncogénicas v-erbA/metabolismo , ARN Interferente Pequeño/genética , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismoRESUMEN
The p75 neurotrophin receptor (p75(NTR)) is a member of the tumor necrosis factor receptor superfamily with a widespread pattern of expression in tissues such as the brain, liver, lung, and muscle. The mechanisms that regulate p75(NTR) transcription in the nervous system and its expression in other tissues remain largely unknown. Here we show that p75(NTR) is an oscillating gene regulated by the helix-loop-helix transcription factors CLOCK and BMAL1. The p75(NTR) promoter contains evolutionarily conserved noncanonical E-box enhancers. Deletion mutagenesis of the p75(NTR)-luciferase reporter identified the -1039 conserved E-box necessary for the regulation of p75(NTR) by CLOCK and BMAL1. Accordingly, gel-shift assays confirmed the binding of CLOCK and BMAL1 to the p75(NTR-)1039 E-box. Studies in mice revealed that p75(NTR) transcription oscillates during dark and light cycles not only in the suprachiasmatic nucleus (SCN), but also in peripheral tissues including the liver. Oscillation of p75(NTR) is disrupted in Clock-deficient and mutant mice, is E-box dependent, and is in phase with clock genes, such as Per1 and Per2. Intriguingly, p75(NTR) is required for circadian clock oscillation, since loss of p75(NTR) alters the circadian oscillation of clock genes in the SCN, liver, and fibroblasts. Consistent with this, Per2::Luc/p75(NTR-/-) liver explants showed reduced circadian oscillation amplitude compared with those of Per2::Luc/p75(NTR+/+). Moreover, deletion of p75(NTR) also alters the circadian oscillation of glucose and lipid homeostasis genes. Overall, our findings reveal that the transcriptional activation of p75(NTR) is under circadian regulation in the nervous system and peripheral tissues, and plays an important role in the maintenance of clock and metabolic gene oscillation.
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Proteínas CLOCK/fisiología , Ritmo Circadiano/fisiología , Metabolismo/fisiología , Receptor de Factor de Crecimiento Nervioso/fisiología , Factores de Transcripción ARNTL/biosíntesis , Factores de Transcripción ARNTL/genética , Animales , Glucemia/metabolismo , Proteínas CLOCK/genética , Ritmo Circadiano/genética , ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Células HEK293 , Homeostasis/genética , Humanos , Hígado/metabolismo , Luciferasas/genética , Metabolismo/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Factor de Crecimiento Nervioso/genética , Choque Séptico/fisiopatología , Núcleo Supraquiasmático/fisiología , TransfecciónRESUMEN
The circadian clock can regulate the metabolic process of xenobiotics, but little is known as to circadian rhythms can be perturbed by xenobiotics. Styrene is a organic chemical widely used in occupational settings. The effects of styrene on the circadian genes of HuDE cells were evaluated after serum-shocking synchronization. A subtoxic dose of 100 µM of styrene altered the expression of clock genes BMAL1, PER2, PER3, CRY1, CRY2, and REV-ERB-α.
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Ritmo Circadiano/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Estireno/administración & dosificación , Factores de Transcripción ARNTL/biosíntesis , Células Cultivadas , Ritmo Circadiano/genética , Criptocromos/biosíntesis , Humanos , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Proteínas Circadianas Period/biosíntesis , Xenobióticos/administración & dosificaciónRESUMEN
Ovarian circadian oscillators have been implicated in the reproductive processes of mammals. However, there are few reports regarding the detection of ovarian clock-controlled genes (CCGs). The present study was designed to unravel the mechanisms through which CCG ovarian circadian oscillators regulate fertility, primarily using quantitative RT-PCR and RNA interference against Bmal1 in rat granulosa cells. Mature granulosa cells were prepared from mouse Per2-destabilized luciferase (dLuc) reporter gene transgenic rats. A real-time monitoring system of Per2 promoter activity was employed to detect Per2-dLuc oscillations. The cells exposed to luteinizing hormone (LH) displayed clear Per2-dLuc oscillations and a rhythmic expression of clock genes (Bmal1, Per1, Per2, Rev-erbα, and Dbp). Meanwhile, the examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Lhcgr, and p53) showed rhythmic transcript profiles except for Hsd3b2, indicating that these rhythmic expression genes may be CCGs. Notably, Bmal1 small interfering (si)RNA treatment significantly decreased both the amplitude of Per2-dLuc oscillations and Bmal1 mRNA levels compared with nonsilencing RNA treatment in luteinizing granulosa cells. Depletion of Bmal1 by siRNA decreased the transcript levels of clock genes (Per1, Per2, Rev-erbα, and Dbp) and examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Hsd3b2, and Lhcgr). Accordingly, knockdown of Bmal1 also inhibited the synthesis of progesterone and prostaglandin E2, which are associated with crucial reproductive processes. Collectively, these data suggest that ovarian circadian oscillators regulate the synthesis of steroid hormones and prostaglandins through ovarian-specific CCGs in response to LH stimuli. The present study provides new insights into the physiologic significance of Bmal1 related to fertility in ovarian circadian oscillators.