András D. Nagy

Cell autonomous regulation of herpes and influenza virus infection by the circadian clock

Significance The circadian clock coordinates our physiology. Circadian disruption, as occurs during shift work, increases the risk of chronic diseases. For infectious diseases, circadian regulation of systemic immunity seems to underpin “time-of-day” differences in responses to extracellular pathogens. However, circadian rhythms are cell autonomous, and their interaction with intracellular pathogens, such as viruses, is poorly understood. We demonstrate that time of day of virus infection has a major impact on disease progression, in cellular models as well as in animals, highlighting the key role that cellular clocks play in this phenomenon. Clock disruption leads to increased virus replication and dissemination, indicating that severity of acute infections is influenced by circadian timekeeping. Viruses are intracellular pathogens that hijack host cell machinery and resources to replicate. Rather than being constant, host physiology is rhythmic, undergoing circadian (∼24 h) oscillations in many virus-relevant pathways, but whether daily rhythms impact on viral replication is unknown. We find that the time of day of host infection regulates virus progression in live mice and individual cells. Furthermore, we demonstrate that herpes and influenza A virus infections are enhanced when host circadian rhythms are abolished by disrupting the key clock gene transcription factor Bmal1. Intracellular trafficking, biosynthetic processes, protein synthesis, and chromatin assembly all contribute to circadian regulation of virus infection. Moreover, herpesviruses differentially target components of the molecular circadian clockwork. Our work demonstrates that viruses exploit the clockwork for their own gain and that the clock represents a novel target for modulating viral replication that extends beyond any single family of these ubiquitous pathogens.

The role of PACAP in the control of circadian expression of clock genes in the chicken pineal gland

Several features of the molecular circadian oscillator of the chicken pineal gland show homology with those in the mammalian SCN. Studies have shown the effects of PACAP on the mammalian SCN, but its effects on the expression of clock genes in the avian pineal gland have not yet been demonstrated. Clock and Cry1 expression was analyzed in pineal glands of chicken embryos after exposure to PACAP-38 in vitro. PACAP reduced expression of both clock genes within 2h. Ten hours after exposure, mRNA contents exceeded that of the controls. Our results support the hypothesis that the molecular clock machinery in the chicken pineal gland is also sensitive to PACAP.

Melatonin adjusts the expression pattern of clock genes in the suprachiasmatic nucleus and induces antidepressant-like effect in a mouse model of seasonal affective disorder

Recently, we have shown that C57BL/6J mice exhibit depression-like behavior under short photoperiod and suggested them as an animal model for investigating seasonal affective disorder (SAD). In this study, we tested if manipulations of the circadian clock with melatonin treatment could effectively modify depression-like and anxiety-like behaviors and brain serotonergic system in C57BL/6J mice. Under short photoperiods (8-h light/16-h dark), daily melatonin treatments 2 h before light offset have significantly altered the 24-h patterns of mRNA expression of circadian clock genes (per1, per2, bmal1 and clock) within the suprachiasmatic nuclei (SCN) mostly by increasing amplitude in their expressional rhythms without inducing robust phase shifts in them. Melatonin treatments altered the expression of genes of serotonergic neurotransmission in the dorsal raphe (tph2, sert, vmat2 and 5ht1a) and serotonin contents in the amygdala. Importantly, melatonin treatment reduced the immobility in forced swim test, a depression-like behavior. As a key mechanism of melatonin-induced antidepressant-like effect, the previously proposed phase-advance hypothesis of the circadian clock could not be confirmed under conditions of our experiment. However, our findings of modest adjustments in both the amplitude and phase of the transcriptional oscillators in the SCN as a result of melatonin treatments may be sufficient to associate with the effects seen in the brain serotonergic system and with the improvement in depression-like behavior. Our study confirmed a predictive validity of C57BL/6J mice as a useful model for the molecular analysis of links between the clock and brain serotonergic system, which could greatly accelerate our understanding of the pathogenesis of SAD, as well as the search for new treatments.

Structural and Morphometric Comparison of the Molar Teeth in Pre-eruptive Developmental Stage of PACAP-Deficient and Wild-Type Mice

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with widespread distribution. It plays pivotal role in neuronal development. PACAP-immunoreactive fibers have been found in the tooth pulp, and recently, it has been shown that PACAP may also play a role in the regeneration of the periodontium after luxation injuries. However, there is no data about the effect of endogenous PACAP on tooth development. Ectodermal organogenesis including tooth development is regulated by different members of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH), and Wnt families. There is also a growing evidence to support the hypothesis that PACAP interacts with sonic hedgehog (SHH) receptor (PTCH1) and its downstream target (Gli1) suggesting its role in tooth development. Therefore, our aim was to study molar tooth development in mice lacking endogenous PACAP. In this study morphometric, immunohistochemical and structural comparison of molar teeth in pre-eruptive developmental stage was performed on histological sections of 7-day-old wild-type and PACAP-deficient mice. Further structural analysis was carried out with Raman microscope. The morphometric comparison of the 7-day-old samples revealed that the dentin was significantly thinner in the molars of PACAP-deficient mice compared to wild-type animals. Raman spectra of the enamel in wild-type mice demonstrated higher diversity in secondary structure of enamel proteins. In the dentin of PACAP-deficient mice higher intracrystalline disordering in the hydroxyapatite molecular structure was found. We also obtained altered SHH, PTCH1 and Gli1 expression level in secretory ameloblasts of PACAP-deficient mice compared to wild-type littermates suggesting that PACAP might play an important role in molar tooth development and matrix mineralization involving influence on SHH signaling cascade.

Circadian clocks and tumor biology: What is to learn from human skin biopsies?

Some of the components of the circadian molecular clock have been shown to link directly to tumor suppression. Most studies on human tumorous biopsies with consistently down-regulated clock gene expression suggested a protective role for these genes against cancer formation. To highlight some limitations of this hypothesis we review these data in light of recent evidences from animal research, epidemiologic studies, and clinical data on skin tumors. We emphasize the role of circadian rhythmic orchestration in cellular metabolism with a potential in cancer development.

Expression pattern of clock under acute phase-delay of the light/dark cycle in the chicken pineal model

Shift workers have a higher risk of metabolic syndrome, a condition that also develops in mice carrying mutation in their circadian clock gene clock. To collect more data on the transcriptional changes of clock under phase-shifted light/dark LD conditions, we examined the 24h patterns of clock mRNA expression in vivo and in vitro in chickens exposed acutely to a reversed LD (DL) cycle. Under controlled LD conditions (lights on at 6:00, lights off at 20:00), clock mRNA expression peaked in vivo at 2:00 (Zeitgeber Time 20, ZT20) and in vitro at 22:00 (ZT16). Even higher mRNA contents were measured in the first cycle of in vivo DL conditions between 22:00 and 6:00 (lights at night), but in the second cycle by 2:00, lower mRNA contents were detected than the control peak values seen at this time point. Furthermore, no alterations were found in vitro in clock mRNA content during the first 12h of DL conditions (lights at night). The differences seen between the first and the second DL cycles in vivo and between the in vivo and in vitro data for the first DL cycle support the idea that neurohumoral signals perturbed by a phase-delayed light-dark cycle may also play a role in the in vivo rapid transcriptional resetting of the circadian clock in the chicken pineal model.

Expression of Cry2 in the chicken pineal gland: Effects of changes in light-dark conditions

Pineal expression of Cry2 mRNA has been examined in chickens under normal (LD) and reversed (DL) light–dark conditions. In vivo the peak of Cry2 mRNA content at late subjective day under LD diminished after switching to a DL schedule. In vitro, Cry2 mRNA levels showed a steady decrease during light exposure at subjective night. Our data show that light‐sensitive clock components in the pinealocytes may be involved in the repression of Cry2 transcription at night, which may contribute to resetting the phase of the clock within 24 h.

Circadian Expression of Clock Genes Clock and Cry1 in the Embryonic Chicken Pineal Gland

Clock and Cry1 expression were examined in the pineal gland of chicken embryos incubated under constant darkness from embryonic day (ED) 0. From ED13, Clock and Cry1 mRNA levels showed episodic alterations. After ED17, circadian pattern of clock gene expression was seen both in vivo and in vitro. Our results support the idea that rhythmic environmental factors are not necessary for the generation of circadian patterns of clock gene expression during development.

The embryonic pineal gland of the chicken as a model for experimental jet lag.

The circadian clock in the chicken pineal model develops before hatching, at around the 17th embryonic day (ED17). By this stage, it runs in synchrony with environmental cues. To address if phase resetting mechanisms are comparable to those of post-hatched chicken, we investigated ED19 stage chicken embryos under 12h light:12h dark (LD), under constant darkness (DD), or under acute 4h phase delay of the LD condition (LD+4). The 24h mRNA-expression patterns of clock gene clock and of clock controlled genes Aanat and hiomt were analyzed with qRT-PCR. Under DD the rhythm of Aanat did not change significantly, however the 24h pattern of hiomt was altered. Clock shows a delayed response to DD with a phase-shift in its rhythm. After the first cycle under LD+4 conditions, the 24h patterns of Aa-nat and hiomt mRNA-s were phase delayed. Clock showed both acute and delayed changes in response to LD+4. These results show that the embryonic chicken pineal gland has a fully functioning clock mechanism, and that it is a good model for phase-change experiments. In addition it demonstrates that only one cycle of altered light schedule is sufficient to trigger changes within the molecular clock mechanisms of the chicken embryonic pineal model.