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Exposure to the space environment induces a number of pathophysiological outcomes in astronauts, including bone demineralization, sleep disorders, circadian clock dysregulation, cardiovascular and metabolic dysfunction, and reduced immune system function. A recent report describing experiments aboard the Space Shuttle mission, STS-132, showed that the level of melatonin, a hormone that provides the biochemical signal of darkness, was decreased during microgravity in an in vitro culture model. Additionally, abnormal lighting conditions in outer space, such as low light intensity in orbital spacecraft and the altered 24-h light-dark cycles, may result in the dysregulation of melatonin rhythms and the misalignment of the circadian clock from sleep and work schedules in astronauts. Studies on Earth have demonstrated that melatonin regulates various physiological functions including bone metabolism. These data suggest that the abnormal regulation of melatonin in outer space may contribute to pathophysiological conditions of astronauts. In addition, experiments with high-linear energy transfer radiation, a ground-based model of space radiation, showed that melatonin may serve as a protectant against space radiation. Gene expression profiling using an in vitro culture model exposed to space flight during the STS-132 mission, showed that space radiation alters the expression of DNA repair and oxidative stress response genes, indicating that melatonin counteracts the expression of these genes responsive to space radiation to promote cell survival. These findings implicate the use of exogenous melatonin and the regulation of endogenous melatonin as countermeasures for the physiological consequences of space flight., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The circadian clock is a highly conserved 24 h biological oscillation mechanism and is affected by environmental stimuli such as light, food and temperature. Disruption of the circadian clock results in disorders of diverse biological processes, including the sleep-wake cycle and metabolism. Although we previously identified several components of the circadian clock in zebrafish, our understanding of the relationship between light-inducible clock genes and metabolism remains incomplete. To investigate how light-inducible clock genes regulate metabolism, we performed transcriptomic and metabolomic analyses of the light-inducible clock genes zPer2, zCry1a, and zCry2a in zebrafish. Transcriptomic analysis of zPer2/zCry1a double knockout (DKO) and zPer2/zCry1a/zCry2a triple knockout (TKO) mutants showed that their gene expression profiles differed from that of wild type (WT) zebrafish. In particular, mRNA levels of zKeap1a, which encodes an oxidative stress sensor, were increased in DKO and TKO mutants. Metabolomic analysis showed genotype-dependent alteration of metabolomic profiles. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) showed the alteration of cysteine/methionine metabolism and glutathione metabolism. Specifically, cysteine and glutathione were decreased but methionine sulfoxide was increased in TKO zebrafish. These results indicate that the light-inducible genes zPer2, zCry1a, and zCry2a are involved in regulating the oxidative status of zebrafish.

Circadian clocks are intrinsic, time-tracking processes that confer a survival advantage on an organism. Under natural conditions, they follow approximately a 24-h day, modulated by environmental time cues, such as light, to maximize an organism's physiological efficiency. The exact timing of this rhythm is established by cell-autonomous oscillators called cellular clocks, which are controlled by transcription-translation negative feedback loops. Studies of cell-based systems and wholeanimal models have utilized a pharmacological approach in which chemical compounds are used to identify molecular mechanisms capable of establishing and maintaining cellular clocks, such as posttranslational modifications of cellular clock regulators, chromatin remodeling of cellular clock target genes' promoters, and stability control of cellular clock components. In addition, studies with chemical compounds have contributed to the characterization of light-signaling pathways and their impact on the cellular clock. Here, the use of chemical compounds to study the molecular, cellular, and behavioral aspects of the vertebrate circadian clock system is described., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The circadian clock generates behavioral rhythms to maximize an organism's physiological efficiency. Light induces the formation of these rhythms by synchronizing cellular clocks. In zebrafish, the circadian clock components Period2 (zPER2) and Cryptochrome1a (zCRY1a) are light-inducible, however their physiological functions are unclear. Here, we investigated the roles of zPER2 and zCRY1a in regulating locomotor activity and behavioral rhythms. zPer2/zCry1a double knockout (DKO) zebrafish displayed defects in total locomotor activity and in forming behavioral rhythms when briefly exposed to light for 3-h. Exposing DKO zebrafish to 12-h light improved behavioral rhythm formation, but not total activity. Our data suggest that the light-inducible circadian clock regulator zCRY2a supports rhythmicity in DKO animals exposed to 12-h light. Single cell imaging analysis revealed that zPER2, zCRY1a, and zCRY2a function in synchronizing cellular clocks. Furthermore, microarray analysis of DKO zebrafish showed aberrant expression of genes involved regulating cellular metabolism, including ATP production. Overall, our results suggest that zPER2, zCRY1a and zCRY2a help to synchronize cellular clocks in a light-dependent manner, thus contributing to behavioral rhythm formation in zebrafish. Further, zPER2 and zCRY1a regulate total physical activity, likely via regulating cellular energy metabolism. Therefore, these circadian clock components regulate the rhythmicity and amount of locomotor behavior.

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in tumorgenesis. Interactions between both pathways have repeatedly been reported for mammals during the last decade, the molecular basis, though, has not been identified so far. Expression levels of oxygen-regulated and circadian clock genes in zebrafish larvae (Danio rerio) and zebrafish cell lines were significantly altered under hypoxic conditions. Thus, long-term hypoxic incubation of larvae resulted in a dampening of the diurnal oscillation amplitude of the period1 gene expression starting only several hours after start of the hypoxic incubation. A significant decrease in the amplitude of the period1 circadian oscillation in response to hypoxia and in response to the hypoxic mimic CoCl2 was also observed using a zebrafish luciferase reporter cell line in constant darkness. In addition, activity measurements of zebrafish larvae using an infrared-sensitive camera demonstrated the loss of their usual circadian activity pattern under hypoxic conditions. To explore the functional basis of the observed cross-talk between both signaling pathways ChIP assays were performed. Increasing with the duration of hypoxia, a nearly 4-fold occupancy of hypoxia-inducible factor 1 (Hif-1α) at two specific E-box binding sites located in the period1 gene control region was shown, demonstrating therewith the transcriptional co-regulation of the core clock gene by the major transcription factor of the hypoxic pathway. On the other hand, circadian transgenic zebrafish cells, simulating a repressed or an overstimulated circadian clock, modified gene transcription levels of oxygen-regulated genes such as erythropoietin and vascular endothelial growth factor 165 and altered the hypoxia-induced increase in Hif-1α protein concentration. In addition, the amount of Hif-1α protein accumulated during the hypoxic response was shown to depend on the time of the day, with one maximum during the light phase and a second one during the dark phase. The direct binding of Hif-1α to the period1 gene control region provides a mechanistic explanation for the repeatedly observed interaction between hypoxia and the circadian clock. The cross-talk between both major signaling pathways was shown for the first time to be bidirectional and may provide the advantage of orchestrating a broad range of genes and metabolic pathways to cope with altered oxygen availabilities.

The stress kinase mitogen-activated protein kinase kinase 7 (MKK7) is a specific activator of c-Jun N-terminal kinase (JNK), which controls various physiological processes, such as cell proliferation, apoptosis, differentiation, and migration. Here we show that genetic inactivation of MKK7 resulted in an extended period of oscillation in circadian gene expression in mouse embryonic fibroblasts. Exogenous expression in cultured mammalian cells of an MKK7-JNK fusion protein that functions as a constitutively active form of JNK induced phosphorylation of PER2, an essential circadian component. Furthermore, JNK interacted with PER2 at both the exogenous and endogenous levels, and MKK7-mediated JNK activation increased the half-life of PER2 protein by inhibiting its ubiquitination. Notably, the PER2 protein stabilization induced by MKK7-JNK fusion protein reduced the degradation of PER2 induced by casein kinase 1ε. Taken together, our results support a novel function for the stress kinase MKK7 as a regulator of the circadian clock in mammalian cells at steady state.

In most species, solar light is both a DNA-damaging agent and the key entraining stimulus for the endogenous circadian clock. The zebrafish is an attractive vertebrate system in which to study the influence of light on gene expression because the DNA repair proteins and circadian oscillators in this species are light-responsive. At the molecular level, light treatment of zebrafish cells induces the production of reactive oxygen species (ROS). ROS both alters the reduction-oxidation (redox) state of these cells and stimulates intracellular extracellular signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) cascades that transduce photic signals activating the transcription of particular light-responsive genes, including some clock genes and some DNA repair genes involved in photoreactivation. To date, however, the phototransducing molecules responsible for light-dependent ROS production have not been identified. Flavin-containing oxidases, such as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, are versatile flavoenzymes that catalyze molecular oxidation in numerous metabolic pathways. Importantly, light induces the photoreduction of the flavin adenine dinucleotide (FAD) moiety in these oxidases, leading to ROS production. Here, we show in cultured zebrafish cells that diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase, both suppresses ERK/MAPK activation and efficiently reduces light-dependent expression of clock and photoreactivation genes. Our results suggest that flavin-containing oxidases may be responsible for light-dependent ROS production and thus light-dependent gene expression in zebrafish. Our findings also support the existence of a regulatory link between photoreactivation and the circadian clock in this species.