Circadian Clock Gene bmal1 Acts as a Tumor Suppressor Gene in a Mice Model of Human Glioblastoma.

Glioblastomas derived from malignant astrocytes are the most common primary tumors of the central nervous system in humans, exhibiting very bad prognosis. Treatment with surgery, radiotherapy, and chemotherapy (mainly using temozolomide), generates as much one-year survival. The circadian clock controls different aspects of tumor development, and its role in GBM is beginning to be explored. Here, the role of the canonic circadian clock gene bmal1 was studied in vivo in a nude mice model bearing human GBMs from LN229 cells xenografted orthotopically in the dorsal striatum. For that aim, a bmal1 knock-down was generated in LN229 cells by CRISPR/Cas9 gene editing tool, and tumor progression was followed in male mice by measuring survival, tumor growth, cell proliferation and prognosis with CD44 marker, as well as astrocyte activation in the tumor microenvironment with GFAP and nestin markers. Disruption of bmal1 in the tumor decreased survival, increased tumor growth and CD44 expression, worsened motor performance, as well as increased GFAP expression in astrocytes at tumor microenvironment. In addition, survival and tumor progression was not affected in mice bearing LN229 wild type GBM that underwent circadian disruption by constant light, as compared to mice synchronized to 12:12 light-dark cycles. These results consistently demonstrate in an in vivo orthotopic model of human GBM, that bmal1 has a key role as a tumor suppressor gene regulating GBM progression.

Chronic jet lag reduces motivation and affects other mood-related behaviors in male mice.

Introduction: The circadian system regulates various physiological processes such as sleep-wake cycles, hormone secretion, metabolism, and the reaction to both natural and drug-based rewards. Chronic disruption of the circadian system caused by unsteady synchronization with light-dark (LD) schedules, such as advancing chronic jet lag (CJL), leads to adverse physiological effects and pathologies, and is linked with changes in mood and depressive behaviors in humans and rodent models. Methods: C57BL/6J male mice were subjected to circadian disruption through phase advances of 6 h every 2 days (CJL +6/2). Mice under 12:12-h LD cycle were used as controls. After 8 weeks under these conditions, a battery of behavioral tests was performed to assess if mood-related behaviors were affected. Results: Compared to controls under 24 h LD cycles, mice under CJL presented desynchronization of activity-rest rhythms that led to several behavioral impairments, including a decrease in motivation for food reward, and an increase in anxiety, anhedonia, and depressive-like behavior. Conclusion: Chronic circadian disruption, caused by an experimental CJL protocol, affects mood-related and reward-related behaviors in mice. Understanding the importance of the circadian system and its potential role for disruption due to CJL is important for maintaining good health and well-being.

Protein Kinases in the Photic Signaling of the Mammalian Circadian Clock.

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.

Don't just say no: Differential pathways and pharmacological responses to diverse nitric oxide donors.

Nitric oxide (NO) is a gaseous free radical molecule with a short half-life (∼1 s), which can gain or lose an electron into three interchangeable redox-dependent forms, the radical (NO), the nitrosonium cation (NO+), and nitroxyl anion (HNO). NO acts as an intra and extracellular signaling molecule regulating a wide range of functions in the cardiovascular, immune, and nervous system. NO donors are collectively known by their ability to release NOin vitro and in vivo, being proposed as therapeutic pharmacological tools for the treatment of several pathologies, such as cardiovascular disease. The highly reactive NO molecule is easily oxidized under physiological conditions to N-oxides, nitrate/nitrite and nitrogen dioxide. Different cellular responses are triggered depending on: 1) NO concentration [e.g., nanomolar for heme coordination in the allosteric site of guanylate cyclase (sGC) enzyme]; 2) the type of chemical bound to the nitrosated group (i.e., bound to nitrogen, N-nitro, or bound to sulphur atom, S-nitro) determining post-translational cysteine nitrosation; 3) the time-dependent availability of molecular targets. Classic NO donors are: organic nitrates (e.g., nitroglycerin, or glyceryl trinitrate, GTN; isosorbide mononitrate, ISMN), diazeniumdiolates having a diolate group [or NONOates, e.g., 2-(N,N-diethylamino)-diazenolate-2-oxide], S-nitrosothiols (e.g., S-nitroso glutathione, GSNO; S-nitroso-N-acetylpenicillamine, SNAP) or the organic salt sodium nitroprusside (SNP). In addition, nitroxyl (HNO) donors such as Piloty's acid and Angeli's salt can also be considered. The specific NO form released, as well as its differential reactivity to thiols, could act on different molecular targets and should be discussed in the context of: a) the type and amount of NO species determining the sensitivity of molecular targets (e.g., heme coordination, or S-nitrosation); b) the cellular redox state that could gate different effects. Experimental designs should take special care when choosing which NO donors to use, since different outcomes are to be expected. This article will comment recent findings regarding physiological responses involving NO species and their pharmacological modulation with donor drugs, especially in the context of the photic transduction pathways at the hypothalamic circadian clock.

Circadian and Metabolic Effects of Light: Implications in Weight Homeostasis and Health.

Daily interactions between the hypothalamic circadian clock at the suprachiasmatic nucleus (SCN) and peripheral circadian oscillators regulate physiology and metabolism to set temporal variations in homeostatic regulation. Phase coherence of these circadian oscillators is achieved by the entrainment of the SCN to the environmental 24-h light:dark (LD) cycle, coupled through downstream neural, neuroendocrine, and autonomic outputs. The SCN coordinate activity and feeding rhythms, thus setting the timing of food intake, energy expenditure, thermogenesis, and active and basal metabolism. In this work, we will discuss evidences exploring the impact of different photic entrainment conditions on energy metabolism. The steady-state interaction between the LD cycle and the SCN is essential for health and wellbeing, as its chronic misalignment disrupts the circadian organization at different levels. For instance, in nocturnal rodents, non-24 h protocols (i.e., LD cycles of different durations, or chronic jet-lag simulations) might generate forced desynchronization of oscillators from the behavioral to the metabolic level. Even seemingly subtle photic manipulations, as the exposure to a "dim light" scotophase, might lead to similar alterations. The daily amount of light integrated by the clock (i.e., the photophase duration) strongly regulates energy metabolism in photoperiodic species. Removing LD cycles under either constant light or darkness, which are routine protocols in chronobiology, can also affect metabolism, and the same happens with disrupted LD cycles (like shiftwork of jetlag) and artificial light at night in humans. A profound knowledge of the photic and metabolic inputs to the clock, as well as its endocrine and autonomic outputs to peripheral oscillators driving energy metabolism, will help us to understand and alleviate circadian health alterations including cardiometabolic diseases, diabetes, and obesity.

Effects of chronic forced circadian desynchronization on body weight and metabolism in male mice.

Metabolic functions are synchronized by the circadian clock setting daily patterns of food intake, nutrient delivery, and behavioral activity. Here, we study the impact of chronic jet-lag (CJL) on metabolism, and test manipulations aimed to overcome potential alterations. We recorded weight gain in C57Bl/6 mice under chronic 6 h advances or delays of the light-dark cycle every 2 days (ChrA and ChrD, respectively). We have previously reported ChrA, but not ChrD, to induce forced desynchronization of locomotor activity rhythms in mice (Casiraghi et al. 2012). Body weight was rapidly increased under ChrA, with animals tripling the mean weight gain observed in controls by day 10, and doubling it by day 30 (6% vs. 2%, and 15% vs. 7%, respectively). Significant increases in retroperitoneal and epidydimal adipose tissue masses (172% and 61%, respectively), adipocytes size (28%), and circulating triglycerides (39%) were also detected. Daily patterns of food and water intake were abolished under ChrA In contrast, ChrD had no effect on body weight. Wheel-running, housing of animals in groups, and restriction of food availability to hours of darkness prevented abnormal increase in body weight under ChrA Our findings suggest that the observed alterations under ChrA may arise either from a direct effect of circadian disruption on metabolism, from desynchronization between feeding and metabolic rhythms, or both. Direction of shifts, timing of feeding episodes, and other reinforcing signals deeply affect the outcome of metabolic function under CJL Such features should be taken into account in further studies of shift working schedules in humans.

N-nitrosomelatonin enhances photic synchronization of mammalian circadian rhythms.

Most physiological processes in mammals are synchronized to the daily light:dark cycle by a circadian clock located in the hypothalamic suprachiasmatic nucleus. Signal transduction of light-induced phase advances of the clock is mediated through a neuronal nitric oxide synthase-guanilyl cyclase pathway. We have employed a novel nitric oxide-donor, N-nitrosomelatonin, to enhance the photic synchronization of circadian rhythms in hamsters. The intraperitoneal administration of this drug before a sub-saturating light pulse at circadian time 18 generated a twofold increase of locomotor rhythm phase-advances, having no effect over saturating light pulses. This potentiation was also obtained even when inhibiting suprachiasmatic nitric oxide synthase activity. However, N-nitrosomelatonin had no effect on light-induced phase delays at circadian time 14. The photic-enhancing effects were correlated with an increased suprachiasmatic immunoreactivity of FBJ murine osteosarcoma viral oncogene and period1. Moreover, in vivo nitric oxide release by N-nitrosomelatonin was verified by measuring nitrate and nitrite levels in suprachiasmatic nuclei homogenates. The compound also accelerated resynchronization to an abrupt 6-h advance in the light:dark cycle (but not resynchronization to a 6-h delay). Here, we demonstrate the chronobiotic properties of N-nitrosomelatonin, emphasizing the importance of nitric oxide-mediated transduction for circadian phase advances.

Ecological segregation in space, time and trophic niche of sympatric planktivorous petrels.

The principle of competitive exclusion postulates that ecologically-similar species are expected to partition their use of resources, leading to niche divergence. The most likely mechanisms allowing such coexistence are considered to be segregation in a horizontal, vertical or temporal dimension, or, where these overlap, a difference in trophic niche. Here, by combining information obtained from tracking devices (geolocator-immersion and time depth recorders), stable isotope analyses of blood, and conventional morphometry, we provide a detailed investigation of the ecological mechanisms that explain the coexistence of four species of abundant, zooplanktivorous seabirds in Southern Ocean ecosystems (blue petrel Halobaena caerulea, Antarctic prion Pachyptila desolata, common diving petrel Pelecanoides urinatrix and South Georgian diving petrel P. georgicus). The results revealed a combination of horizontal, vertical and temporal foraging segregation during the breeding season. The stable isotope and morphological analyses reinforced this conclusion, indicating that each species occupied a distinct trophic space, and that this appears to reflect adaptations in terms of flight performance. In conclusion, the present study indicated that although there was a degree of overlap in some measures of foraging behaviour, overall the four taxa operated in very different ecological space despite breeding in close proximity. We therefore provide important insight into the mechanisms allowing these very large populations of ecologically-similar predators to coexist.

The times they're a-changing: effects of circadian desynchronization on physiology and disease.

Circadian rhythms are endogenous and need to be continuously entrained (synchronized) with the environment. Entrainment includes both coupling internal oscillators to external periodic changes as well as synchrony between the central clock and peripheral oscillators, which have been shown to exhibit different phases and resynchronization speed. Temporal desynchronization induces diverse physiological alterations that ultimately decrease quality of life and induces pathological situations. Indeed, there is a considerable amount of evidence regarding the deleterious effect of circadian dysfunction on overall health or on disease onset and progression, both in human studies and in animal models. In this review we discuss the general features of circadian entrainment and introduce diverse experimental models of desynchronization. In addition, we focus on metabolic, immune and cognitive alterations under situations of acute or chronic circadian desynchronization, as exemplified by jet-lag and shiftwork schedules. Moreover, such situations might lead to an enhanced susceptibility to diverse cancer types. Possible interventions (including light exposure, scheduled timing for meals and use of chronobiotics) are also discussed.

Forced desynchronization of activity rhythms in a model of chronic jet lag in mice.

We studied locomotor activity rhythms of C57/Bl6 mice under a chronic jet lag (CJL) protocol (ChrA(6/2) ), which consisted of 6-hour phase advances of the light-dark schedule (LD) every 2 days. Through periodogram analysis, we found 2 components of the activity rhythm: a short-period component (21.01 ± 0.04 h) that was entrained by the LD schedule and a long-period component (24.68 ± 0.26 h). We developed a mathematical model comprising 2 coupled circadian oscillators that was tested experimentally with different CJL schedules. Our simulations suggested that under CJL, the system behaves as if it were under a zeitgeber with a period determined by (24 - [phase shift size/days between shifts]). Desynchronization within the system arises according to whether this effective zeitgeber is inside or outside the range of entrainment of the oscillators. In this sense, ChrA(6/2) is interpreted as a (24 - 6/2 = 21 h) zeitgeber, and simulations predicted the behavior of mice under other CJL schedules with an effective 21-hour zeitgeber. Animals studied under an asymmetric T = 21 h zeitgeber (carried out by a 3-hour shortening of every dark phase) showed 2 activity components as observed under ChrA(6/2): an entrained short-period (21.01 ± 0.03 h) and a long-period component (23.93 ± 0.31 h). Internal desynchronization was lost when mice were subjected to 9-hour advances every 3 days, a possibility also contemplated by the simulations. Simulations also predicted that desynchronization should be less prevalent under delaying than under advancing CJL. Indeed, most mice subjected to 6-hour delay shifts every 2 days (an effective 27-hour zeitgeber) displayed a single entrained activity component (26.92 ± 0.11 h). Our results demonstrate that the disruption provoked by CJL schedules is not dependent on the phase-shift magnitude or the frequency of the shifts separately but on the combination of both, through its ratio and additionally on their absolute values. In this study, we present a novel model of forced desynchronization in mice under a specific CJL schedule; in addition, our model provides theoretical tools for the evaluation of circadian disruption under CJL conditions that are currently used in circadian research.

Circadian entrainment to light-dark cycles involves extracellular nitric oxide communication within the suprachiasmatic nuclei.

The ability to synchronize to light-dark (LD) cycles is an essential property of the circadian clock, located in mammals within the hypothalamic suprachiasmatic nuclei (SCN). Single light pulses activate nitric oxide (NO) intracellular signaling, leading to circadian phase-shifts required for synchronization. In addition, extracellular NO has a role in the SCN paracrine communication of photic phase advances. In this work, the extracellular nitrergic transmission was assessed in steady-state synchronization to LD cycles of locomotor rhythms in the golden hamster (Mesocricetus auratus). Extracellular NO levels were pharmacologically decreased in vivo with the specific scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Hamsters were subjected to LD cycles different from normal 24 h (LD 14 : 10) cycles (i.e. T-cycles), with a single 30-min light pulse presented either every 23 h (T23 cycles), or every 25 h (T25 cycles), thus allowing synchronization by advances or delays, respectively. Acute PTIO intracerebroventricular microinjections, delivered 30 min previous to the light pulse, inhibited synchronization by phase advances to T23 cycles, but did not alter phase delays under T25 cycles. In addition, NO scavenging inhibited light-induced expression of PERIOD1 protein at circadian time 18 (i.e. the time for light-induced phase advances). These findings demonstrate the role of extracellular NO communication within the SCN in the steady-state synchronization to LD cycles.

History-dependent changes in entrainment of the activity rhythm in the Syrian hamster (Mesocricetus auratus).

The authors have studied the activity rhythm of Syrian hamsters exposed to square LD cycles with a 22-h period (T22) with the aim of testing the effects of the previous history on the rhythmic pattern. To do so, sequential changes of different lighting environments were established, followed by the same LD condition. Also, the protocol included T22 cycles with varying lighting contrasts to test the extent to which a computational model predicts experimental outcomes. At the beginning of the experiment, exposure to T22 with 300 lux and dim red light occurring respectively at photophase and scotophase (LD300/dim red) mainly generated relative coordination. Subsequent transfer to cycles with approximately 0.1-lux dim light during the scotophase (LD300/0.1) promoted entrainment to T22. However, a further reduction in light intensity to 10 lux during the photophase (LD10/0.1) generated weak and unstable T22 rhythms. When, after that, animals were transferred again to the initial LD300/dim red cycles, the amplitude of the rhythm still remained very low, and the phases were very unstable. Exposure to constant darkness partially restored the activity rhythm, and when, afterwards, the animals were submitted again to LD300/dim red cycles, a robust T22 rhythm appeared. The results demonstrate history-dependent changes in the hamster circadian system because the locomotor activity pattern under the same T22 cycle can show relative coordination or unstable or robust entrainment depending on the prior lighting condition. This suggests that the circadian system responds to environmental stimuli depending on its previous history. Moreover, computer simulations allow the authors to predict entrainment under LD300/0.1 cycles and indicate that most of the patterns observed in the animals due to the light in the scotophase can be explained by different degrees of coupling among the oscillators of the circadian system.

Activity rhythm of golden hamster (Mesocricetus auratus) can be entrained to a 19-h light-dark cycle.

Both temporary access to a running wheel and temporary exposure to light systematically influence the phase producing entrainment of the circadian activity rhythm in the golden hamster (Mesocricetus auratus). However, precise determination of entrainment limits remains methodologically difficult, because such calculations may be influenced by varying experimental paradigms. In this study, effects on the entrainment of the activity pattern during successive light-dark (LD) cycles of stepwise decreasing periods, as well as wheel running activity, were investigated. In particular, the hamster activity rhythm under LD cycles with a period (T) shorter than 22 h was studied, i.e., when the LD cycle itself had been shown to be an insufficiently strong zeitgeber to synchronize activity rhythms. Indeed, it was confirmed that animals without a wheel do not entrain under 11:11-h LD cycles (T = 22 h). Subsequently providing hamsters continuous access to a running wheel established entrainment to T = 22 h. Moreover, this paradigm underwent further reductions of the T period to T = 19.6 h without loss of entrainment. Furthermore, restricting access to the wheel did not result in loss of entrainment, while even entrainment to T = 19 h was observed. To explain this observed shift in the lower entrainment limit, our speculation centers on changes in pacemaker response facilitated by stepwise changes of T spaced very far apart, thus allowing time for adaptation.