RESUMEN
We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the Per2 3'-UTR region: Per2::Luc, which retains the endogenous Per2 3'-UTR and Per2::LucSV, where the endogenous Per2 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of Per2 3'-UTR, we analyzed circadian rhythms of Per2::LucSV mice. Interestingly, Per2::LucSV mice displayed more than threefold stronger amplitude in bioluminescence rhythms than Per2::Luc mice, and also exhibited lengthened free-running periods (â¼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Per2::Luc Analysis of the Per2 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in Per2::LucSV augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, Bmal1 mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in Per2::LucSV mice, suggesting rhythmic overexpression of PER2 enhances expression of Per2 and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of Per2 3'-UTR, miR-24, and PER2 in Per2 expression and core clock function.
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Ritmo Circadiano/fisiología , MicroARNs/genética , Proteínas Circadianas Period/genética , Regiones no Traducidas 3' , Animales , Relojes Circadianos/genética , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Luciferasas/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Circadianas Period/metabolismo , Biosíntesis de Proteínas , TemperaturaRESUMEN
The effects of spherical nucleic acid (SNA) gold nanoparticle conjugates on the activation of macrophages in vitro and release of cytokines in vivo were explored. Herein, we show that G-quadruplexes, the formation of which is enhanced on gold nanoparticle surfaces, elicit an increase in cytokine release from mouse and human macrophages and induce the upregulation of activation receptors as well as NO2 production in vitro. Moreover, these G-rich SNAs can induce cytokine release when injected intravenously, though there were no severe, long-term effects observed. These results further reinforce the notion that nucleic acid sequence and structure play an important role in how SNAs interact in biological milieu and highlight a key design parameter.
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G-Cuádruplex , Oro/química , Activación de Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Nanopartículas del Metal/administración & dosificación , Ácidos Nucleicos/química , Animales , Células Cultivadas , Citocinas/metabolismo , Humanos , Técnicas In Vitro , Macrófagos/efectos de los fármacos , Nanopartículas del Metal/química , Ratones , Ratones Endogámicos C57BL , Dióxido de Nitrógeno/metabolismo , Ácidos Nucleicos/metabolismoRESUMEN
The effect of serum protein adsorption on the biological fate of Spherical Nucleic Acids (SNAs) is investigated. Through a proteomic analysis, it is shown that G-quadruplexes templated on the surface of a gold nanoparticle in the form of SNAs mediate the formation of a protein corona that is rich in complement proteins relative to SNAs composed of poly-thymine (poly-T) DNA. Cellular uptake studies show that complement receptors on macrophage cells recognize the SNA protein corona, facilitating their internalization, and causing G-rich SNAs to accumulate in the liver and spleen more than poly-T SNAs in vivo. These results support the conclusion that nucleic acid sequence and architecture can mediate nanoparticle-biomolecule interactions and alter their cellular uptake and biodistribution properties and illustrate that nucleic acid sequence is an important parameter in the design of SNA therapeutics.
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Endocitosis , Macrófagos/metabolismo , Ácidos Nucleicos/metabolismo , Corona de Proteínas/metabolismo , Animales , Secuencia de Bases , Proteínas Sanguíneas/metabolismo , Línea Celular , G-Cuádruplex , Humanos , Hígado/metabolismo , Ratones Endogámicos C57BL , Receptores de Superficie Celular/metabolismo , Bazo/metabolismo , Distribución TisularRESUMEN
The molecular clock maintains energy constancy by producing circadian oscillations of rate-limiting enzymes involved in tissue metabolism across the day and night. During periods of feeding, pancreatic islets secrete insulin to maintain glucose homeostasis, and although rhythmic control of insulin release is recognized to be dysregulated in humans with diabetes, it is not known how the circadian clock may affect this process. Here we show that pancreatic islets possess self-sustained circadian gene and protein oscillations of the transcription factors CLOCK and BMAL1. The phase of oscillation of islet genes involved in growth, glucose metabolism and insulin signalling is delayed in circadian mutant mice, and both Clock and Bmal1 (also called Arntl) mutants show impaired glucose tolerance, reduced insulin secretion and defects in size and proliferation of pancreatic islets that worsen with age. Clock disruption leads to transcriptome-wide alterations in the expression of islet genes involved in growth, survival and synaptic vesicle assembly. Notably, conditional ablation of the pancreatic clock causes diabetes mellitus due to defective beta-cell function at the very latest stage of stimulus-secretion coupling. These results demonstrate a role for the beta-cell clock in coordinating insulin secretion with the sleep-wake cycle, and reveal that ablation of the pancreatic clock can trigger the onset of diabetes mellitus.
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Factores de Transcripción ARNTL/genética , Proteínas CLOCK/genética , Ritmo Circadiano/fisiología , Diabetes Mellitus/metabolismo , Insulina/sangre , Islotes Pancreáticos/metabolismo , Factores de Transcripción ARNTL/deficiencia , Factores de Transcripción ARNTL/metabolismo , Envejecimiento/genética , Envejecimiento/patología , Animales , Glucemia/análisis , Glucemia/metabolismo , Proteínas CLOCK/deficiencia , Proteínas CLOCK/metabolismo , Proliferación Celular , Tamaño de la Célula , Supervivencia Celular , Ritmo Circadiano/genética , Diabetes Mellitus/genética , Perfilación de la Expresión Génica , Intolerancia a la Glucosa/genética , Prueba de Tolerancia a la Glucosa , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/patología , Ratones , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fenotipo , Sueño/genética , Sueño/fisiología , Vesículas Sinápticas/metabolismo , Vigilia/genética , Vigilia/fisiologíaRESUMEN
Circadian cycles affect a variety of physiological processes, and disruptions of normal circadian biology therefore have the potential to influence a range of disease-related pathways. The genetic basis of circadian rhythms is well studied in model organisms and, more recently, studies of the genetic basis of circadian disorders has confirmed the conservation of key players in circadian biology from invertebrates to humans. In addition, important advances have been made in understanding how these molecules influence physiological functions in tissues throughout the body. Together, these studies set the scene for applying our knowledge of circadian biology to the understanding and treatment of a range of human diseases, including cancer and metabolic and behavioural disorders.
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Trastornos Cronobiológicos/genética , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Enfermedad/etiología , Animales , Relojes Biológicos/genética , Encéfalo/fisiología , Trastornos Cronobiológicos/complicaciones , Esquema de Medicación , Quimioterapia/métodos , Retroalimentación Fisiológica/genética , Redes Reguladoras de Genes/fisiología , Humanos , Modelos Biológicos , Trastornos del Humor/etiología , Trastornos del Humor/genética , Especificidad de Órganos/genéticaRESUMEN
Hepatorenal syndrome (HRS) is a life-threatening complication of end-stage liver disease first reported over a century ago, but its management still poses an unmet challenge. A therapeutic agent found to stabilize the condition is a short cyclic peptide, vasopressin analogue, terlipressin (TP). While TP is commonly prescribed for HRS patients in most parts of the world, it was only recently approved for use in the United States. TP exhibits short circulation half-lives and adverse side effects associated with the dose required. Herein, we present a 1,18-octadecanedioic acid (ODDA) conjugate of the cyclic peptide (ODDA-TP), which enables noncovalent binding to serum albumin via native fatty acid binding modes. ODDA-TP is demonstrated to outperform TP alone in studies including in vitro cellular receptor activation, stability in plasma, pharmacokinetics, and performance in vivo in rats. Specifically, ODDA-TP had an elimination half-life 20 times that of TP alone while exhibiting a superior safety profile.
RESUMEN
Bmal1 is an essential transcriptional activator within the mammalian circadian clock. We report here that the suprachiasmatic nucleus (SCN) of Bmal1-null mutant mice, unexpectedly, generates stochastic oscillations with periods that overlap the circadian range. Dissociated SCN neurons expressed fluctuating levels of PER2 detected by bioluminescence imaging but could not generate circadian oscillations intrinsically. Inhibition of intercellular communication or cyclic-AMP signaling in SCN slices, which provide a positive feed-forward signal to drive the intracellular negative feedback loop, abolished the stochastic oscillations. Propagation of this feed-forward signal between SCN neurons then promotes quasi-circadian oscillations that arise as an emergent property of the SCN network. Experimental analysis and mathematical modeling argue that both intercellular coupling and molecular noise are required for the stochastic rhythms, providing a novel biological example of noise-induced oscillations. The emergence of stochastic circadian oscillations from the SCN network in the absence of cell-autonomous circadian oscillatory function highlights a previously unrecognized level of circadian organization.
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Relojes Circadianos/fisiología , Ritmo Circadiano/fisiología , Núcleo Supraquiasmático/fisiología , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Animales , Comunicación Celular/fisiología , AMP Cíclico/metabolismo , Ratones , Ratones Noqueados , Neuronas/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Procesos Estocásticos , Núcleo Supraquiasmático/citología , Técnicas de Cultivo de TejidosRESUMEN
Glioblastoma (GBM) is one of the most difficult cancers to effectively treat, in part because of the lack of precision therapies and limited therapeutic access to intracranial tumor sites due to the presence of the blood-brain and blood-tumor barriers. We have developed a precision medicine approach for GBM treatment that involves the use of brain-penetrant RNA interference-based spherical nucleic acids (SNAs), which consist of gold nanoparticle cores covalently conjugated with radially oriented and densely packed small interfering RNA (siRNA) oligonucleotides. On the basis of previous preclinical evaluation, we conducted toxicology and toxicokinetic studies in nonhuman primates and a single-arm, open-label phase 0 first-in-human trial (NCT03020017) to determine safety, pharmacokinetics, intratumoral accumulation and gene-suppressive activity of systemically administered SNAs carrying siRNA specific for the GBM oncogene Bcl2Like12 (Bcl2L12). Patients with recurrent GBM were treated with intravenous administration of siBcl2L12-SNAs (drug moniker: NU-0129), at a dose corresponding to 1/50th of the no-observed-adverse-event level, followed by tumor resection. Safety assessment revealed no grade 4 or 5 treatment-related toxicities. Inductively coupled plasma mass spectrometry, x-ray fluorescence microscopy, and silver staining of resected GBM tissue demonstrated that intravenously administered SNAs reached patient tumors, with gold enrichment observed in the tumor-associated endothelium, macrophages, and tumor cells. NU-0129 uptake into glioma cells correlated with a reduction in tumor-associated Bcl2L12 protein expression, as indicated by comparison of matched primary tumor and NU-0129-treated recurrent tumor. Our results establish SNA nanoconjugates as a potential brain-penetrant precision medicine approach for the systemic treatment of GBM.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Nanopartículas del Metal , Ácidos Nucleicos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioblastoma/genética , Glioblastoma/terapia , Oro , Humanos , Proteínas Musculares/metabolismo , Recurrencia Local de Neoplasia , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARNRESUMEN
The mechanism of circadian oscillations in mammals is cell autonomous and is generated by a set of genes that form a transcriptional autoregulatory feedback loop. While these "clock genes" are well conserved among animals, their specific functions remain to be fully understood and their roles in central versus peripheral circadian oscillators remain to be defined. We utilized the in vivo inducible tetracycline-controlled transactivator (tTA) system to regulate Clock gene expression conditionally in a tissue-specific and temporally controlled manner. Through the use of Secretogranin II to drive tTA expression, suprachiasmatic nucleus- and brain-directed expression of a tetO::Clock(Delta19) dominant-negative transgene lengthened the period of circadian locomotor rhythms in mice, whereas overexpression of a tetO::Clock(wt) wild-type transgene shortened the period. Low doses (10 mug/ml) of doxycycline (Dox) in the drinking water efficiently inactivated the tTA protein to silence the tetO transgenes and caused the circadian periodicity to return to a wild-type state. Importantly, low, but not high, doses of Dox were completely reversible and led to a rapid reactivation of the tetO transgenes. The rapid time course of tTA-regulated transgene expression demonstrates that the CLOCK protein is an excellent indicator for the kinetics of Dox-dependent induction/repression in the brain. Interestingly, the daily readout of circadian period in this system provides a real-time readout of the tTA transactivation state in vivo. In summary, the tTA system can manipulate circadian clock gene expression in a tissue-specific, conditional, and reversible manner in the central nervous system. The specific methods developed here should have general applicability for the study of brain and behavior in the mouse.
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Conducta Animal , Encéfalo/metabolismo , Ritmo Circadiano , Vectores Genéticos , Transactivadores/metabolismo , Animales , Proteínas CLOCK , Ritmo Circadiano/genética , Doxiciclina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Luz , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Datos de Secuencia Molecular , Actividad Motora , Transactivadores/genética , TransgenesRESUMEN
Rats with hippocampal or sham lesions were trained on a stimulus-response task developed for the 8-arm radial maze. After reaching a stringent learning criterion, different context manipulations were performed. In Experiment I, the different groups were transferred to an identical radial maze in a different room to determine the context specificity of the discrimination learning. Experiment I revealed that although rats with hippocampal lesions did not show a normal context detection effect, the expression of the discrimination was not context dependent for either the lesion or sham groups. In Experiment II, animals were trained to criterion on the discrimination task and then both groups were divided into sub-groups based on whether they would experience reversal training in the same or different context from original training. Experiment II indicated that animals with hippocampal lesions and shams reversed in a different context were significantly enhanced in reaching the learning criterion compared to either counterparts that were reversed in the same context. Reversal learning in rats with hippocampal lesions was faster than sham animals in the same context suggesting that the context-specific inhibition effect was hippocampal-based. After learning the reversal task, the groups of animals trained and reversed in different contexts were brought back into the original training context to test for competitive effects. Animals with hippocampal lesions that were reversed in the different context, did not show a competition between the most recently acquired discrimination and a context-specific association acquired during original training whereas sham animals in the same condition did. Taken together these results suggest that rats with hippocampal lesions do not acquire normal context-specific inhibition during discrimination learning.
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Atención/fisiología , Hipocampo/fisiología , Aprendizaje Inverso/fisiología , Animales , Atropina/toxicidad , Conducta Competitiva/efectos de los fármacos , Conducta Competitiva/fisiología , Condicionamiento Operante/efectos de los fármacos , Aprendizaje Discriminativo/fisiología , Hipocampo/anatomía & histología , Hipocampo/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Antagonistas Muscarínicos/toxicidad , Ratas , Ratas Long-EvansRESUMEN
In humans and animal models, circadian modulation of learning has been demonstrated on numerous tests. However, it is unclear which aspects of the cognitive process are rhythmically regulated. In these experiments, we used a conditioned place preference task in hamsters to ask whether memory acquisition (hypothesis 1) or memory recall and performance (hypothesis 2) were subject to circadian modulation. In golden hamsters, access to a running wheel has been used as a reward to condition a place preference, but when given unrestricted access to a wheel, animals perform most of their spontaneous running within a few hours each day or circadian cycle. This suggested that either the perceived reward value of the wheel changes through the day or that the response to this reward is temporally restricted. Contrary to the hypotheses, we found that learning was not tied to the time of training nor to the time of testing, but rather animals showed a preference for a reward-paired context only at the circadian time that training had taken place. Timing is not an explicit discriminative cue in these experiments. Hence, the learning mechanism must be predisposed to register circadian time as an attribute during context learning.
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Ritmo Circadiano/fisiología , Condicionamiento Operante/fisiología , Recompensa , Animales , Cricetinae , Señales (Psicología) , Aprendizaje Discriminativo/fisiología , Iluminación , Masculino , Mesocricetus , Desempeño Psicomotor/fisiologíaRESUMEN
We report that the neural representation of the time of day (time memory) in golden hamsters involves the setting of a 24-h oscillator that is functionally and anatomically distinct from the circadian clock in the suprachiasmatic nucleus (SCN), but is entrained by the SCN acting as a weak zeitgeber. In hamsters, peak conditioned place avoidance (CPA) was expressed only near the time of day of the learning experience (± 2 h) for the first days after conditioning. On a 14:10 light:dark cycle, with conditioning at the end of the light period (zeitgeber time 11 [ZT11]), CPA behavior, including time of day memory, was retained for more than 18 d. With conditioning in the early day (zeitgeber time 03 [ZT03]), CPA was completely lost after 5 d but reemerged after an additional 6 d, with the peak avoidance time shifted to ZT11. When the entraining light cycle was shifted immediately following learning at either ZT11 or ZT03, with no additional experience in the training apparatus, peak CPA 18 d later was always found at ZT11 on the shifted light cycles. When conditioned at ZT03, then placed into constant dark for 18 cycles, the peak shifted to subjective circadian time 11 (CT11). In all experiments, the peak CPA time was set initially to the time of experience, and was reset subsequently to the end of the subjective day, without memory loss for other context associations. In the absence of an SCN, peak avoidance was not reset. Therefore, time memory is distinct from other context memories, and involves the setting of a non-SCN circadian oscillator. We suggest that circadian oscillators underlying time memory work in concert with the SCN to enable anticipation of critical conditions according to both immediate- and long-term probabilities of where and when important conditions could be encountered again.
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Relojes Circadianos/fisiología , Memoria/fisiología , Percepción del Tiempo/fisiología , Animales , Cricetinae , Masculino , FotoperiodoRESUMEN
Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many "undruggable" oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)-based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)--an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas--were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.
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Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Nanopartículas/química , Ácidos Nucleicos/química , Interferencia de ARN , Animales , Apoptosis , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Ratones SCID , Proteínas Musculares/metabolismo , Ácidos Nucleicos/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Atherosclerosis is the disease mechanism responsible for coronary heart disease (CHD), the leading cause of death worldwide. One strategy to combat atherosclerosis is to increase the amount of circulating high-density lipoproteins (HDL), which transport cholesterol from peripheral tissues to the liver for excretion. The process, known as reverse cholesterol transport, is thought to be one of the main reasons for the significant inverse correlation observed between HDL blood levels and the development of CHD. This article highlights the most common strategies for treating atherosclerosis using HDL. We further detail potential treatment opportunities that utilize nanotechnology to increase the amount of HDL in circulation. The synthesis of biomimetic HDL nanostructures that replicate the chemical and physical properties of natural HDL provides novel materials for investigating the structure-function relationships of HDL and for potential new therapeutics to combat CHD.
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Lipoproteínas HDL/síntesis química , Nanotecnología , Animales , Enfermedad Coronaria/sangre , Enfermedad Coronaria/tratamiento farmacológico , Humanos , Lipoproteínas HDL/sangre , Lipoproteínas HDL/uso terapéutico , Nanopartículas del Metal/químicaRESUMEN
Circadian activity rhythms in mammals are controlled by the expression and transcriptional regulation of clock genes in the suprachiasmatic nucleus (SCN). The circadian cycle length in hamsters is regulated in part by casein kinase I epsilon (CKIepsilon). A semidominant mutation (C-->T, R178C, CKIepsilon(tau)) appears to act as a dominant-negative allele to shorten the period of circadian rhythms. We tested this hypothesis in vivo by expressing wild-type CKIepsilon gene in homozygous tau mutant hamsters. High-level CKIepsilon(+/+) gene transfer and expression (as indicated by green fluorescent protein) were obtained by injecting CKIepsilon-containing plasmids bilaterally near the SCN, followed by in vivo electroporation. Rhythmicity reappeared 5-7 days after electroporation, with a gradual increase in circadian period over the next 10 days. The circadian period returned to the baseline over the next 20 days. For the five hamsters with clearest gene expression in the SCN, the mean lengthening time was 39.6 min. Period change was not observed in either control tau mutant hamsters electroporated with plasmids lacking the CKIepsilon gene or in wild-type hamsters with plasmids containing the wild-type CKIepsilon gene. Therefore, normal periodicity in homozygous CKIepsilon(tau) hamsters was partially rescued by expression of the wild-type CKIepsilon gene in the SCN, supporting a competitive and dominant-negative action of the mutant allele. This study shows that electroporation of wild-type CKIepsilon gene into the SCN is sufficient for lengthening the shorter circadian period of tau mutant hamsters in a time-dependent way and supports the conclusion that CKIepsilon(tau) is the cause of the shorter period.
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Caseína Cinasa 1 épsilon/genética , Caseína Cinasa 1 épsilon/metabolismo , Ritmo Circadiano/genética , Mutación , Núcleo Supraquiasmático/fisiología , Proteínas tau/genética , Animales , Animales Modificados Genéticamente , Cricetinae , Electroporación/métodos , Masculino , Factores de Tiempo , Transcripción Genética/fisiologíaRESUMEN
Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.
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Relojes Biológicos/fisiología , Proteínas de Ciclo Celular/fisiología , Ritmo Circadiano/fisiología , Flavoproteínas/fisiología , Proteínas Nucleares/fisiología , Núcleo Supraquiasmático/fisiología , Factores de Transcripción/fisiología , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Criptocromos , Fibroblastos , Flavoproteínas/genética , Ratones , Actividad Motora , Mutación , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Circadianas Period , Núcleo Supraquiasmático/citología , Factores de Transcripción/genéticaRESUMEN
Circadian rhythms are approximately 24-h oscillations in behavior and physiology, which are internally generated and function to anticipate the environmental changes associated with the solar day. A conserved transcriptional-translational autoregulatory loop generates molecular oscillations of 'clock genes' at the cellular level. In mammals, the circadian system is organized in a hierarchical manner, in which a master pacemaker in the suprachiasmatic nucleus (SCN) regulates downstream oscillators in peripheral tissues. Recent findings have revealed that the clock is cell-autonomous and self-sustained not only in a central pacemaker, the SCN, but also in peripheral tissues and in dissociated cultured cells. It is becoming evident that specific contribution of each clock component and interactions among the components vary in a tissue-specific manner. Here, we review the general mechanisms of the circadian clockwork, describe recent findings that elucidate tissue-specific expression patterns of the clock genes and address the importance of circadian regulation in peripheral tissues for an organism's overall well-being.
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Ritmo Circadiano/genética , Animales , Ritmo Circadiano/fisiología , Retroalimentación Fisiológica , Expresión Génica , Humanos , Ratones , Modelos Genéticos , Mutación , Fenotipo , Núcleo Supraquiasmático/fisiología , Distribución TisularRESUMEN
The mouse Clock gene encodes a basic helix-loop-helix-PAS transcription factor, CLOCK, that acts in concert with BMAL1 to form the positive elements of the circadian clock mechanism in mammals. The original Clock mutant allele is a dominant negative (antimorphic) mutation that deletes exon 19 and causes an internal deletion of 51 aa in the C-terminal activation domain of the CLOCK protein. Here we report that heterozygous Clock/+ mice exhibit high-amplitude phase-resetting responses to 6-h light pulses (Type 0 resetting) as compared with wild-type mice that have low amplitude (Type 1) phase resetting. The magnitude and time course of acute light induction in the suprachiasmatic nuclei of the only known light-induced core clock genes, Per1 and Per2, are not affected by the Clock/+ mutation. However, the amplitude of the circadian rhythms of Per gene expression are significantly reduced in Clock homozygous and heterozygous mutants. Rhythms of PER2::LUCIFERASE expression in suprachiasmatic nuclei explant cultures also are reduced in amplitude in Clock heterozygotes. The phase-response curves to changes in culture medium are Type 0 in Clock heterozygotes, but Type 1 in wild types, similar to that seen for light in vivo. The increased efficacy of resetting stimuli and decreased PER expression amplitude can be explained in a unified manner by a model in which the Clock mutation reduces circadian pacemaker amplitude in the suprachiasmatic nuclei.
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Relojes Biológicos/fisiología , Ritmo Circadiano/fisiología , Mutación , Transactivadores/metabolismo , Animales , Proteínas CLOCK , Proteínas de Ciclo Celular , Regulación de la Expresión Génica , Luz , Ratones , Ratones Endogámicos , Actividad Motora/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Fotoperiodo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Núcleo Supraquiasmático/fisiología , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) domain transcription factor BMAL1 is an essential component of the mammalian circadian pacemaker. Bmal1-/- mice lose circadian rhythmicity but also display tendon calcification and decreased activity, body weight, and longevity. To investigate whether these diverse functions of BMAL1 are tissue-specific, we produced transgenic mice that constitutively express Bmal1 in brain or muscle and examined the effects of rescued gene expression in Bmal1-/- mice. Circadian rhythms of wheel-running activity were restored in brain-rescued Bmal1-/- mice in a conditional manner; however, activity levels and body weight were lower than those of wild-type mice. In contrast, muscle-rescued Bmal1-/- mice exhibited normal activity levels and body weight yet remained behaviorally arrhythmic. Thus, Bmal1 has distinct tissue-specific functions that regulate integrative physiology.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Encéfalo/metabolismo , Ritmo Circadiano , Actividad Motora , Músculo Esquelético/metabolismo , Factores de Transcripción ARNTL , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Peso Corporal , Calcinosis , Proteínas de Ciclo Celular/genética , Cromosomas Artificiales Bacterianos , Expresión Génica , Longevidad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/genética , Especificidad de Órganos , Proteínas Circadianas Period , Núcleo Supraquiasmático/metabolismo , Tendones/patología , Factores de Transcripción/genéticaRESUMEN
The mouse Period2 (mPer2) locus is an essential negative-feedback element of the mammalian circadian-clock mechanism. Recent work has shown that mPer2 circadian gene expression persists in both central and peripheral tissues. Here, we analyze the mouse mPer2 promoter and identify a circadian enhancer (E2) with a noncanonical 5'-CACGTT-3' E-box located 20 bp upstream of the mPer2 transcription start site. The E2 enhancer accounts for most circadian transcriptional drive of the mPer2 locus by CLOCK:BMAL1, is a major site of DNaseI hypersensitivity in this region, and is constitutively bound by a transcriptional complex containing the CLOCK protein. Importantly, the E2 enhancer is sufficient to drive self-sustained circadian rhythms of luciferase activity in central and peripheral tissues from mPer2-E2::Luciferase transgenic mice with tissue-specific phase and period characteristics. Last, genetic analysis with mutations in Clock and Bmal1 shows that the E2 enhancer is a target of CLOCK and BMAL1 in vivo.