Single-Cell Imaging

the timing of physiological events is carefully controlled so that incompatible processes do not compete and complementary functions coincide. Given the pivotal role of the hypothalamus in homeostatic regulation, the discovery that a master circadian pacemaker resides in this region was not

The influence of circadian rhythms on memory has long been studied; however, the molecular prerequisites for their interaction remain elusive. The hippocampus, which is a region of the brain important for long-term memory formation and temporary maintenance, shows circadian rhythmicity in pathways central to the memory-consolidation process. As neuronal plasticity is the translation of numerous inputs, illuminating the direct molecular links between circadian rhythms and memory consolidation remains a daunting task. However, the elucidation of how clock genes contribute to synaptic plasticity could provide such a link. Furthermore, the idea that memory training could actually function as a zeitgeber for hippocampal neurons is worth consideration, based on our knowledge of the entrainment of the circadian clock system. The integration of many inputs in the hippocampus affects memory consolidation at both the cellular and the systems level, leaving the molecular connections between circadian rhythmicity and memory relatively obscure but ripe for investigation.

The photoneuroendocrine circadian system of the brain consists of (a) specialized photoreceptors in the retina, (b) a circadian generator located in the forebrain that contains "clock genes," (c) specialized nuclei in the forebrain involved in neuroendocrine secretion, and (d) the pineal gland. The circadian generator is a nucleus, called the suprachiasmatic nucleus (SCN). The neurons of this nucleus contain "clock genes," the transcription of which exhibits a circadian rhythm. Most circadian rhythms are generated by the neurons of this nucleus and, via neuronal and humoral connections, the SCN controls circadian activity of the brain and peripheral tissues. The endogenous oscillator of the SCN is each day entrained to the length of the daily photoperiod by light that reach the retina, and specialized photoreceptors transmit impulses to the SCN via the optic nerves. Mass screening for day/night variations in gene expression in the circadian system as well as in the whole brain and peripheral tissues have, during the last decade, been performed. However, studies of circadian changes in the proteome have been less investigated. In this survey, the anatomy and function of the circadian-generating system in mammals is described, and recent proteomic studies that investigate day/night changes in the retina, SCN, and pineal gland are reviewed. Further circadian changes controlled by the SCN in gene and protein expression in the liver are discussed.

Drosophila Ebony Activity Is Required in Glia for the Circadian Regulation of Locomotor Activity

Neurons of the cerebellar cortex contain a circadian oscillator, with circadian expression of clock genes being controlled by the master clock of the suprachiasmatic nucleus (SCN). However, the signaling pathway connecting the SCN to the cerebellum is unknown. Glucocorticoids exhibit a prominent SCN-dependent circadian rhythm, and high levels of the glucocorticoid receptor have been reported in the cerebellar cortex; we therefore hypothesized that glucocorticoids may control the rhythmic expression of clock genes in the cerebellar cortex. We here applied a novel methodology by combining the electrolytic lesion of the SCN with implantation of a micropump programmed to release corticosterone in a circadian manner mimicking the endogenous hormone profile. By use of this approach, we were able to restore the corticosterone rhythm in SCN-lesioned male rats. Clock gene expression in the cerebellum was abolished in rats with a lesioned SCN, but exogenous corticosterone restored the daily rhythm in clock gene expression in the cerebellar cortex, as revealed by quantitative real-time PCR and radiochemical in situ hybridization for the detection of the core clock genes Per1, Per2, and Arntl. On the contrary, exogenous hormone did not restore circadian rhythms in body temperature and running activity. RNAscope in situ hybridization further revealed that the glucocorticoid receptor colocalizes with clock gene products in cells of the cerebellar cortex, suggesting that corticosterone exerts its actions by binding directly to receptors in neurons of the cerebellum. However, rhythmic clock gene expression in the cerebellum was also detectable in adrenalectomized rats, indicating that additional control mechanisms exist. These data show that the cerebellar circadian oscillator is influenced by SCN-dependent rhythmic release of corticosterone.

The clock gene Per1 is necessary in the retrosplenial cortex—but not in the suprachiasmatic nucleus—for incidental learning in young and aging male mice

Virtually all light-sensitive organisms, ranging from bacteria to human, anticipate and adapt to daily changes in luminosity and/or temperature caused by the rotation of the earth around its own axis. Endogenous circadian clocks synchronize to such external cues and regulate many aspects of behavior and physiology. In mammals, the circadian timing system comprises the master clock in the suprachiasmatic nuclei (SCN) in the brain and subsidiary clocks in most cells of the body. The SCN is reset by light in a daily fashion and in turn ensures phase coherence of subsidiary oscillators in the periphery. The master clock synchronizes peripheral oscillators by diverse means. These include hormone secretion, neuronal cues, rest-activity and feeding-fasting cycles of the animal as well as circadian fluctuations in body temperature. In order to gain insights into how SCN-emitted cues are integrated into peripheral oscillators, we attempted to characterize the function of Cold-inducible RNA-binding protein (CIRP) and to examine its effect on circadian rhythms. CIRP belongs to a group of genes, which are expressed in a circadian manner in mouse liver irrespective of whether local hepatocyte clocks are functional or not. These genes must therefore be regulated by oscillating systemic signals under the direct control of the master clock in the SCN. Conversely, if systemically driven genes, such as Cirp, were involved in the regulation of peripheral core clock components, this would provide a plausible mechanism to relay cyclic SCN signals to the circadian clock circuitry. Indeed, loss-of-function experiments demonstrated that CIRP is required for high-amplitude circadian gene expression and thereby confers robustness to oscillators. We aimed at identifying the molecular interactions between CIRP and circadian clocks. For this purpose, we, purified CIRP–RNA complexes and analyzed CIRP-interacting RNAs through high-throughput sequencing (CLIP-seq). We furthermore deep-sequenced whole transcriptomes (RNA-seq) from control and CIRP-depleted cells, respectively. We thereby identified transcripts associated with and regulated by CIRP. These experiments revealed Clock and several additional core clock components as targets of CIRP and therefore established a molecular link between CIRP and the core clock. Using cultured fibroblasts, a clock model devoid of complex SCN signals, we were able to reconstitute the rhythmic expression of CIRP by imposed circadian temperature fluctuations, suggesting that body temperature cycles act as the systemic signal that regulates CIRP abundance. Thus, CIRP might participate in a pathway, which relays temperature cues to circadian oscillators.

EGFP Is a Useful Long-Term Expression Tracer for Hematopoietic Stem Cells While DsRed Fluorescent Protein Is Not.

Annual Meeting, Winnipeg, Manitoba, 2009

Nutrient and energy metabolism in organisms oscillates in a time-of the-day-dependent manner under the control of an endogenous timing mechanism called the circadian clock. This is a cell autonomous, self-sustained molecular mechanism, which is synchronized by a key environmental signals, notably light and food availability. There is a wealth of evidence showing a bidirectional interaction between food-regulated clocks and the rhythmic expression of metabolic genes in peripheral tissues, notably the liver. For example, genetic or environmental disruption of the circadian clock is linked with metabolic disease, such as obesity and type 2 diabetes. Furthermore, cycling changes in cellular redox potential impact on the expression of circadian clock genes and influence energy metabolism. Therefore, it is vital to understand how animals integrate input from lighting conditions and food availability to ultimately coordinate their daily metabolic rhythms. In this regard, one key issue is whether there are genetically distinct light and food regulated circadian clock mechanisms. The Foulkes group has used zebrafish and blind cavefish models to demonstrate that certain metabolic pathways cycle according to the light dark cycle and are unaffected by the timing of feeding activity, while other pathways are predominantly feeding time regulated. Based on these preliminary data, this thesis project used fish models and fish-derived cell lines to explore the genetic mechanisms linking metabolism with light and food regulated circadian clocks. The first part of this project aimed to explore at which stage during early zebrafish development a feeding-regulated clock first appears. Due to reduced feeding activity in constant darkness it was not feasible to examine the impact of feeding on clock gene expression. However, it was revealed that regular handling and disturbance of fish larvae, under otherwise constant environmental conditions results in the emergence of circadian clock rhythmicity. Several lines of evidence indicate that stress serves as a Zeitgeber and results in the emergence of rhythmicity in clock gene expression as well as clock outputs such as the cell cycle. The second part of this thesis explored whether genetically distinct light and food regulated clocks coexist in fish cells and which transcriptional control mechanisms link food-regulated circadian clocks with metabolism. It was demonstrated that during restricted feeding in zebrafish, rhythmic expression of core clock genes in the liver is regulated according to the timing of light-dark cycles, whereas the expression of genes involved in the control of metabolism are influenced by feeding time. However, this study was unable to confirm previous data obtained using NMR, where it was shown that circadian rhythmicity in the levels of essential amino acids is regulated by the light-dark cycle while rhythmic non-essential amino acid levels are influenced by feeding time. Instead, by UPLC-MS/MS analysis, daily changes in the concentration of both essential and non-essential amino acids were shown to be set by the phase of regular timed feeding and not by the light dark cycle. Furthermore, the NAD+ biosynthesis pathway and autophagy were affected by a clock which is set by feeding time and not by light-dark cycles. In addition, regular nocturnal feeding resulted in an increase in obesity. These findings point to the presence of at least two distinct clock mechanism in the zebrafish liver. In order to explore in more detail, the nature of the multiple clock mechanisms in zebrafish cells, the next part of this project employed multi-omics approaches and revealed infradian rhythmicity in amino acid concentrations in cultured fish cell lines. However, neither the expression of amino acid transporters nor autophagy exhibited infradian rhythmicity, instead showing circadian rhythmicity. In order to explore the involvement of the classical circadian clock mechanism in generating infradian rhythmicity, a cell line expressing a dominant negative form of clock1 gene was examined and shown to lack infradian rhythmicity in amino acid levels. Interestingly, the mRNA expression of Asparagine synthetase (asns) shows infradian rhythmicity, which are disrupted in Δclock1 cells. These data lead to the hypothesis that asns may be involved in the regulation of infradian rhythms in amino acid levels and point to a complex interplay between circadian and infradian rhythmicity.

Protracted opiate withdrawal can extend for months of disrupted hormonal circadian rhythms. We examined rodent behaviors and these circadian disturbances in hormone and peptide levels as well as brain clock gene expression during 60 days of protracted withdrawal. Our behavioral tests included open field, elevated plus maze, and sucrose preference tests at 36 h, 10, 30, and 60 days after stopping chronic morphine. At these four assessment points, we collected samples every 4 h for 24 h to examine circadian rhythms in blood hormone and peptide levels and brain expression of rPER1, rPER2, and rPER3 clock genes. Decreased locomotor activity and elevated adrenocorticotropic hormone and melatonin levels persisted for 2 months after morphine withdrawal, but corticosterone was elevated only at 36 h and 10 days after withdrawal. Orexin levels were high at 36 h after withdrawal, but then reversed during protracted withdrawal to abnormally low levels. Beta-endorphin (β-EP) levels showed no differences from normal. However, circadian rhythms were blunted for all of these hormones. Corticosterone, adrenocorticotropic hormone, and orexin blunting persisted at least for 60 days. The blunted circadian rhythm of β-EP and melatonin recovered by day 60, but the peak phase of β-EP was delayed about 8 h. Blunted circadian rhythms and reduced expression of rPER1, rPER2, and rPER3 persisted at least for 60 days in the suprachiasmatic nucleus, prefrontal cortex, nucleus accumbens core, central nucleus of the amygdala, Hippocampus, and ventral tegmental area. Circadian rhythms of rPER1 in the nucleus accumbens shell and basolateral nucleus of the amygdala and of rPER2 in the central nucleus of the amygdala were reversed. Disrupted circadian rhythms of rPER1, rPER 2, and rPER3 expression in reward-related brain circuits and blunted circadian rhythms in peripheral hormones and peptides may play a role in protracted opiate withdrawal and contribute to relapse.

Light-emitting diodes that emit high-intensity blue light are associated with blue-light hazard. Here, we report that blue light disturbs circadian rhythms by interfering with the clock gene in the suprachiasmatic nucleus (SCN) and that suppression of blue light at night ameliorates metabolic abnormalities by controlling circadian rhythms. C57BL/6J mice were exposed to 10-lux light for 30 minutes at Zeitgeber time 14 for light pulse with blue light or blue-light cut light to induce phase shift of circadian rhythms. Phase shift, clock gene expression in SCN, and metabolic parameters were analyzed. In the clinical study, healthy participants wore blue-light shield eyewear for 2 to 3 hours before bed. Anthropometric data analyses, laboratory tests, and sleep quality questionnaires were performed before and after the study. In mice, phase shift induced with a blue-light cut light pulse was significantly shorter than that induced with a white light pulse. The phase of Per2 expression in the SCN was also delayed after a white light pulse. Moreover, blood glucose levels 48 hours after the white light pulse were higher than those after the blue-cut light pulse. Irs2 expression in the liver was decreased with white light but significantly recovered with the blue-cut light pulse. In a clinical study, after 1 month of wearing blue-light shield eyeglasses, there were improvements in fasting plasma glucose levels, insulin resistance, and sleep quality. Our results suggest that suppression of blue light at night effectively maintains circadian rhythms and metabolism.

Circadian rhythms are endogenous and self-sustained oscillations of multiple biological processes with approximately 24-h rhythmicity. Circadian genes and their protein products constitute the molecular components of the circadian oscillator that form positive/negative feedback loops and generate circadian rhythms. The circadian regulation extends from core clock genes to various clock-controlled genes that include various cell cycle genes. Aberrant expression of circadian clock genes, therefore, may lead to genomic instability and accelerated cellular proliferation potentially promoting carcinogenesis. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of serum melatonin levels in peripheral blood samples of 37 CLL (chronic lymphocytic leukemia) patients and equal number of age- and sex-matched healthy controls in order to indicate association between deregulated circadian clock and manifestation of CLL. Results showed significantly down-regulated expression of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc and Cyclin D1 (P < 0.0001) in CLL patients as compared to healthy controls. When expression of these genes was compared between shift-workers and non-shift-workers within the CLL group, the expression was found more aberrant in shift-workers as compared to non-shift-workers. However, this difference was found statistically significant for Myc and Cyclin D1 only (P < 0.05). Serum melatonin levels were found significantly low (P < 0.0001) in CLL subjects as compared to healthy controls whereas melatonin levels were found still lower in shift-workers as compared to non-shift-workers within CLL group (P < 0.01). Our results suggest that aberrant expression of circadian clock genes can lead to aberrant expression of their downstream targets that are involved in cell proliferation and apoptosis and hence may result in manifestation of CLL. Moreover, shift-work and low melatonin levels may also contribute in etiology of CLL by further perturbing of circadian clock.

Soriano award lecture: Light, circadian rhythms and sleep: Mechanisms to new therapeutics

Alzheimer’s Disease (AD) is the most common type of dementia. The circadian system, which is controlled by the master clock in the Suprachiasmatic Nucleus (SCN) of the hypothalamus, is crucial for various physiological processes. Studies have shown that changes in the circadian rhythms can deteriorate neurodegenerative diseases. Changes in the SCN are associated with cognitive decline in AD. The cognitive impairments in AD, especially memory dysfunctions, may be related to Circadian Rhythm Disturbances (CRDs). Moreover, rhythmic expression of clock genes is disrupted in AD patients. There is a circadian pattern of inflammatory processes in AD, and dysregulation of core clock genes promotes neuroinflammation. The present narrative review addresses the intricate link between CRDs and AD, revisiting the relevant cellular and molecular mechanisms. The association between CRDs and AD highlights the need for further investigation of the underlying mechanisms.