RESUMO
The circadian system is a conserved time-keeping machinery that regulates a wide range of processes such as sleep/wake, feeding/fasting, and activity/rest cycles to coordinate behavior and physiology. Circadian disruption can be a contributing factor in the development of metabolic diseases, inflammatory disorders, and higher risk of cancer. Glioblastoma (GBM) is a highly aggressive grade 4 brain tumor that is resistant to conventional therapies and has a poor prognosis after diagnosis, with a median survival of only 12-15 months. GBM cells kept in culture were shown to contain a functional circadian oscillator. In seeking more efficient therapies with lower side effects, we evaluated the pharmacological modulation of the circadian clock by targeting the cytosolic kinases glycogen synthase kinase-3 (GSK-3) and casein kinase 1 ε/δ (CK1ε/δ) with specific inhibitors (CHIR99021 and PF670462, respectively), the cryptochrome protein stabilizer (KL001), or circadian disruption after Per2 knockdown expression in GBM-derived cells. CHIR99021-treated cells had a significant effect on cell viability, clock protein expression, migration, and cell cycle distribution. Moreover, cultures exhibited higher levels of reactive oxygen species and alterations in lipid droplet content after GSK-3 inhibition compared to control cells. The combined treatment of CHIR99021 with temozolomide was found to improve the effect on cell viability compared to temozolomide therapy alone. Per2 disruption affected both GBM migration and cell cycle progression. Overall, our results suggest that pharmacological modulation or molecular clock disruption severely affects GBM cell biology.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioblastoma/patologia , Glioblastoma/metabolismo , Glioblastoma/tratamento farmacológico , Humanos , Linhagem Celular Tumoral , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Piridinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Citosol/metabolismo , Citosol/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Pirimidinas/farmacologia , Movimento Celular/efeitos dos fármacos , Relógios Circadianos/efeitos dos fármacos , Relógios Circadianos/fisiologia , Proteínas CLOCK/metabolismo , Proteínas CLOCK/genética , Proteínas Circadianas Period/metabolismo , Proteínas Circadianas Period/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Aging impairs both circadian rhythms and memory, though the relationship between these impairments is not fully understood. Circadian rhythms are largely dictated by clock genes within the body's central pacemaker, the suprachiasmatic nucleus (SCN), though these genes are also expressed in local clocks throughout the body. As circadian rhythms can directly affect memory performance, one possibility is that memory deficits observed with age are downstream of global circadian rhythm disruptions stemming from the SCN. Here, we demonstrate that expression of clock gene Period1 within a memory-relevant cortical structure, the retrosplenial cortex (RSC), is necessary for incidental learning, and that age-related disruption of Period1 within the RSC-but not necessarily the SCN-contributes to cognitive decline. These data expand the known functions of clock genes beyond maintaining circadian rhythms and suggests that age-associated changes in clock gene expression modulates circadian rhythms and memory performance in a brain region-dependent manner.
Assuntos
Relógios Circadianos , Giro do Cíngulo , Camundongos , Animais , Masculino , Giro do Cíngulo/metabolismo , Núcleo Supraquiasmático/metabolismo , Ritmo Circadiano/genética , Encéfalo/metabolismo , Fatores de Transcrição/metabolismo , Envelhecimento/genética , Relógios Circadianos/genética , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismoRESUMO
The molecular circadian clock is based on a transcriptional/translational feedback loop in which the stability and half-life of circadian proteins is of importance. Cysteine residues of proteins are subject to several redox reactions leading to S-thiolation and disulfide bond formation, altering protein stability and function. In this work, the ability of the circadian protein period 2 (PER2) to undergo oxidation of cysteine thiols was investigated in HEK-293T cells. PER2 includes accessible cysteines susceptible to oxidation by nitroso cysteine (CysNO), altering its stability by decreasing its monomer form and subsequently increasing PER2 homodimers and multimers. These changes were reversed by treatment with 2-mercaptoethanol and partially mimicked by hydrogen peroxide. These results suggest that cysteine oxidation can prompt PER2 homodimer and multimer formation in vitro, likely by S-nitrosation and disulphide bond formation. These kinds of post-translational modifications of PER2 could be part of the redox regulation of the molecular circadian clock.
Assuntos
Relógios Circadianos , Proteínas Circadianas Period , Ritmo Circadiano/fisiologia , Cisteína/metabolismo , Dimerização , Oxirredução , Proteínas Circadianas Period/química , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Proteínas/metabolismoRESUMO
The important involvement of the suprachiasmatic nucleus (SCN) and the activity of vasopressinergic neurons in maintaining the rhythmicity of the female reproductive system depends on the mRNA transcription-translation feedback loops. Therefore, circadian clock function, like most physiological processes, is involved in the events that determine reproductive aging. This study describes the change of mRNA expression of clock genes, Per2, Bmal1, and Rev-erbα, in the hypothalamus-pituitary-gonadal axis (HPG) of female rats with regular cycle (RC) and irregular cycle (IC), and the vasopressinergic neurons activity in the SCN and kisspeptin neurons in the arcuate nucleus (ARC) of these animals. Results for gonadotropins and the cFos/AVP-ir neurons in the SCN of IC were higher, but kisspeptin-ir was minor. Change in the temporal synchrony of the clock system in the HPG axis, during the period prior to the cessation of ovulatory cycles, was identified. The analysis of mRNA for Per2, Bmal1, and Rev-erbα in the reproductive axis of adult female rodents shows that the regularity of the estrous cycle is guaranteed by alternation in the amount of expression of Bmal1 and Per2, and Rev-erbα and Bmal1 between light and dark phases, which ceases to occur and contributes to determining reproductive senescence. These results showed that the desynchronization between the central and peripheral circadian clocks contributes to the irregularity of reproductive events. We suggest that the feedback loops of clock genes on the HPG axis modulate the spontaneous transition from regular to irregular cycle and to acyclicity in female rodents.
Assuntos
Envelhecimento , Ritmo Circadiano , Gônadas/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , RNA Mensageiro/metabolismo , Núcleo Supraquiasmático/metabolismo , Vasopressinas/farmacologia , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Relógios Circadianos , Feminino , Gônadas/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Wistar , Núcleo Supraquiasmático/efeitos dos fármacosRESUMO
BACKGROUND: Obesity has reached epidemic proportions worldwide, affecting life quality and span. Susceptibility to obesity is partly mediated by genetic differences. Indeed, several genes from the clock gene family have already been shown to be intimately associated with obesity in diverse ethnic groups. In the present study, an association between BMI and the rs707467, rs228697 and rs228729 PER3 (Period Circadian Clock 3) polymorphisms in subjects with class II (BMI ≥ 35.0-39.9 kg/m2) and class III obesity (>40 kg/m2, extreme obesity) were carried out using TaqMan real-time PCR. Overall, 259 Brazilian adults were genotyped, of whom 122 had class II or III obesity (BMI ≥ 35.0 kg/m2) and 137 were controls having normal weight (BMI > 18.5 and <24.9 kg/m2). RESULTS: PER3 tag SNP (rs228729) shows a significant association with extreme obesity (1000 permutation p = 0.03 and p = 0.04), for genotype and allele frequency respectively) and a haplotype among the three assessed SNPs (alleles G/T/A, rs228697, rs228729, and rs707467, respectively, 1000 permutation p = 0.03) was significantly more prevalent in the group with obesity. CONCLUSION: This exploratory association study suggests that PER3 rs228729 may be associated with extreme obesity in Brazilian adults, however, replication is needed.
Assuntos
Obesidade Mórbida/genética , Proteínas Circadianas Period , Polimorfismo de Nucleotídeo Único , Adulto , Alelos , Proteínas CLOCK/genética , Ritmo Circadiano , Frequência do Gene , Genótipo , Humanos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismoRESUMO
Behavioral outputs arise as a result of highly regulated yet flexible communication among neurons. The Drosophila circadian network includes 150 neurons that dictate the temporal organization of locomotor activity; under light-dark (LD) conditions, flies display a robust bimodal pattern. The pigment-dispersing factor (PDF)-positive small ventral lateral neurons (sLNv) have been linked to the generation of the morning activity peak (the "M cells"), whereas the Cryptochrome (CRY)-positive dorsal lateral neurons (LNds) and the PDF-negative sLNv are necessary for the evening activity peak (the "E cells") [1, 2]. While each group directly controls locomotor output pathways [3], an interplay between them along with a third dorsal cluster (the DN1ps) is necessary for the correct timing of each peak and for adjusting behavior to changes in the environment [4-7]. M cells set the phase of roughly half of the circadian neurons (including the E cells) through PDF [5, 8-10]. Here, we show the existence of synaptic input provided by the evening oscillator onto the M cells. Both structural and functional approaches revealed that E-to-M cell connectivity changes across the day, with higher excitatory input taking place before the day-to-night transition. We identified two different neurotransmitters, acetylcholine and glutamate, released by E cells that are relevant for robust circadian output. Indeed, we show that acetylcholine is responsible for the excitatory input from E cells to M cells, which show preferential responsiveness to acetylcholine during the evening. Our findings provide evidence of an excitatory feedback between circadian clusters and unveil an important plastic remodeling of the E cells' synaptic connections.
Assuntos
Relógios Biológicos/fisiologia , Drosophila melanogaster/fisiologia , Locomoção/fisiologia , Terminações Pré-Sinápticas/metabolismo , Acetilcolina/metabolismo , Animais , Animais Geneticamente Modificados , Ritmo Circadiano/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Retroalimentação Fisiológica , Ácido Glutâmico/metabolismo , Masculino , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , FotoperíodoRESUMO
Circadian (~24 h) rhythms in behavior and physiological functions are under control of an endogenous circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN directly drives some of these rhythms or serves as a coordinator of peripheral oscillators residing in other tissues and organs. Disruption of the circadian organization may contribute to disease, including stress-related disorders. Previous research indicates that the master clock in the SCN is resistant to stress, although it is unclear whether stress affects rhythmicity in other tissues, possibly mediated by glucocorticoids, released in stressful situations. In the present study, we examined the effect of uncontrollable social defeat stress and glucocorticoid hormones on the central and peripheral clocks, respectively in the SCN and liver. Transgenic PERIOD2::LUCIFERASE knock-in mice were used to assess the rhythm of the clock protein PERIOD2 (PER2) in SCN slices and liver tissue collected after 10 consecutive days of social defeat stress. The rhythmicity of PER2 expression in the SCN was not affected by stress exposure, whereas in the liver the expression showed a delayed phase in defeated compared to non-defeated control mice. In a second experiment, brain slices and liver samples were collected from transgenic mice and exposed to different doses of corticosterone. Corticosterone did not affect PER2 rhythm of the SCN samples, but caused a phase shift in PER2 expression in liver samples. This study confirms earlier findings that the SCN is resistant to stress and shows that clocks in the liver are affected by social stress, which might be due to the direct influence of glucocorticoids released from the adrenal gland.
Assuntos
Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/genética , Glucocorticoides/farmacologia , Fígado/metabolismo , Proteínas Circadianas Period/genética , Estresse Psicológico , Núcleo Supraquiasmático/metabolismo , Glândulas Suprarrenais/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ritmo Circadiano/fisiologia , Corticosterona/metabolismo , Dominação-Subordinação , Técnicas de Introdução de Genes , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Circadianas Period/metabolismo , Comportamento Social , Estresse Psicológico/genética , Estresse Psicológico/metabolismo , Estresse Psicológico/fisiopatologia , Núcleo Supraquiasmático/efeitos dos fármacosRESUMO
Organisms have evolved in a daily cyclic environment, developing circadian cell-autonomous clocks that temporally organize a wide range of biological processes. Translation is a highly regulated process mainly associated with the activity of microRNAs (miRNAs) at the translation initiation step that impacts on the molecular circadian clock dynamics. Recently, a molecular titration mechanism was proposed to explain the interactions between some miRNAs and their target mRNAs; new evidence also indicates that regulation by miRNA is a nonlinear process such that there is a threshold level of target mRNA below which protein production is drastically repressed. These observations led us to use a theoretical model of the circadian molecular clock to study the effect of miRNA-mediated translational thresholds on the molecular clock dynamics. We model the translational threshold by introducing a phenomenological Hill equation for the kinetics of PER translation and show how the parameters associated with translation kinetics affect the period, amplitude, and time delays between clock mRNA and clock protein expression. We show that our results are useful for analyzing experiments related to the translational regulation of negative elements of transcriptional-translational feedback loops. We also provide new elements for thinking about the translational threshold as a mechanism that favors the emergence of circadian rhythmicity, the tuning of the period-delay relationship and the cell capacity to control the protein oscillation amplitude with almost negligible changes in the mRNA amplitudes.
Assuntos
Relógios Circadianos/genética , Modelos Genéticos , Biossíntese de Proteínas , Cinética , Proteínas Circadianas Period/metabolismo , RNA Mensageiro/genéticaRESUMO
Circadian clocks drive biological rhythms in physiology and behavior, providing a selective advantage by enabling organisms to synchronize to the 24 h environmental day. This process depends on light-dark transitions as the main signal that shifts the phase of the clock. In mammals, the light input reaches the master circadian clock in the hypothalamic suprachiasmatic nucleus through glutamatergic afferents from the retina, resulting in phase-shifts of the overt rhythms which depend on the time of the day at which light is applied, leading to changes in the activity of circadian core clock genes (i.e., Per1). This circadian gating of the synchronizing effect of light is dependent on the specific activation of signal transduction pathways involving several kinases acting on protein effectors. Protein phosphorylation is also an important regulatory mechanism essential for the generation and maintenance of circadian rhythms and plays a crucial role in the degradation and the appropriate turnover of PER proteins. In this work, we review the role of the main kinases implicated in the function of the master clock, with emphasis in those involved in circadian photic entrainment.
Assuntos
Relógios Circadianos/fisiologia , Transdução de Sinal Luminoso/fisiologia , Mamíferos/fisiologia , Proteínas Quinases/metabolismo , Animais , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Luz , Mamíferos/genética , Mamíferos/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fosforilação/efeitos da radiaçãoRESUMO
Shift-work and exposure to light at night lead to circadian disruption, which favors the use of alcohol and may be a risk factor for development of addictive behavior. This study evaluated in two experimental models of circadian disruption behavioral indicators of elevated alcohol intake and looked for ΔFosB, which is a transcription factor for neuronal plasticity in corticolimbic structures. Male Wistar rats were exposed to experimental shift-work (AR) or to constant light (LL) and were compared with a control group (LD). After 4 weeks in their corresponding conditions, control LD rats remained rhythmic, AR rats exhibited a loss of day-night patterns in the brain and the LL rats showed arrhythmicity in general activity and day-night PER1 patterns in corticolimbic structures. During 12 days of exposure to 10 percent alcohol solution, the AR group showed daily increased alcohol intake while LD and LL rats ingested similar amounts. After 72 h of alcohol deprivation, AR and LL rats increased alcohol intake in a binge-like test; this could be due not only to circadian disruption but also to stress and/or anxiety developed from the AR and LL manipulations. Associated to the increased alcohol intake, the AR and LL rats had significant accumulation of ΔFosB in the nucleus accumbens shell and decreased ΔFosB in the infralimbic cortex. Data here reported confirm that the disruption of temporal patterns favors the increased alcohol consumption and that this is associated with a differential accumulation of ΔFosB which may favor the development of addictive behavior.