Detection of endogenous circadian rhythms of clock gene mRNA expression in mouse lung tissue using slice cultures.

Detection of endogenous circadian rhythms in clock gene mRNA expression requires that mice be sacrificed at regular time intervals over one or more days. This protocol uses culture tissue slices obtained from a single mouse to collect time-course samples. We describe the procedure from preparation of lung slices to rhythmicity analysis of mRNA expression, including details to create handmade culture inserts. This protocol is useful for many mammalian biological clock researchers because it allows a decrease in animal sacrifice. For complete details on the use and execution of this protocol, please refer to Matsumura et al. (2022).1.

Potential negative effect of total parenteral nutrition on the human circadian clock.

When patients cannot eat on their own, total parenteral nutrition (TPN) is a clinically beneficial method of maintaining nutrition. However, many animal studies have demonstrated that circadian rhythms are strongly affected by feeding time, raising the concern that continuous TPN around the clock may have an unexpected negative impact on the circadian clock of patients. To investigate this concern, we compared clock gene expression of aged subjects with or without TPN using hair follicle cells and found that while none of the non-TPN subjects showed any obvious defects in circadian rhythms of peripheral clock gene expression, a portion of aged subjects receiving continuous TPN showed abnormal circadian rhythms in peripheral clocks. Continuous TPN around the clock may therefore potentially perturb peripheral circadian rhythms, giving rise to the proposal that TPN needs to be administered with consideration to time factors.

The role of cell-autonomous circadian oscillation of Cry transcription in circadian rhythm generation.

The current model of the mammalian circadian clock describes cell-autonomous and negative feedback-driven circadian oscillation of Cry and Per transcription as the core circadian rhythm generator. However, the actual contribution of this oscillation to circadian rhythm generation remains undefined. Here we perform targeted disruption of cis elements indispensable for cell-autonomous Cry oscillation. Mice lacking overt cell-autonomous Cry oscillation show robust circadian rhythms in locomotor activity. In addition, tissue-autonomous circadian rhythms are robust in the absence of overt Cry oscillation. Unexpectedly, although the absence of overt Cry oscillation leads to severe attenuation of Per oscillation at the cell-autonomous level, circadian rhythms in Per2 accumulation remain robust. As a mechanism to explain this counterintuitive result, Per2 half-life shows cell-autonomous circadian rhythms independent of Cry and Per oscillation. The cell-autonomous circadian clock may therefore remain partially functional even in the absence of overt Cry and Per oscillation because of circadian oscillation in Per2 degradation.

A detection method for latent circadian rhythm sleep-wake disorder.

BACKGROUND: Individuals with typical circadian rhythm sleep-wake disorders (CRSWDs) have a habitual sleep timing that is desynchronized from social time schedules. However, it is possible to willfully force synchronisation against circadian-driven sleepiness, which causes other sleep problems. This pathology is distinguishable from typical CRSWDs and is referred to here as latent CRSWD (LCRSWD). Conventional diagnostic methods for typical CRSWDs are insufficient for detecting LCRSWD because sufferers have an apparently normal habitual sleep timing. METHODS: We first evaluated the reliability of circadian phase estimation based on clock gene expression using hair follicles collected at three time points without sleep interruption. Next, to identify detection criteria for LCRSWD, we compared circadian and sleep parameters according to estimated circadian phases, at the group and individual level, between subjects with low and high Pittsburgh Sleep Quality Index (PSQI) scores. To validate the reliability of identified detection criteria, we investigated whether the same subjects could be reproducibly identified at a later date and whether circadian amelioration resulted in sleep improvement. FINDINGS: We successfully validated the reliability of circadian phase estimation at three time points and identified potential detection criteria for individuals with LCRSWD attributed to delayed circadian-driven sleepiness. In particular, a criterion based on the interval between the times of the estimated circadian phase of clock gene expression and getting out of bed on work or school days was promising. We also successfully confirmed the reproducibility of candidate screening and sleep improvement by circadian amelioration, supporting the reliability of the detection criteria. INTERPRETATION: Although several limitations remain, our present study demonstrates a promising prototype of a detection method for LCRSWD attributed to delayed circadian-driven sleepiness. More extensive trials are needed to further validate this method. FUNDING: This study was supported mainly by JSPS, Japan.

Bright light improves sleep in patients with Parkinson's disease: possible role of circadian restoration.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders. Among the most common manifestations of PD are sleep problems, which are coupled with the adverse effects of dopaminergic therapies (DT). A non-pharmacological solution for these sleep problems has been sought to avoid additional pharmacological intervention. Here, we show that bright light therapy (BLT) is effective for improving sleep in Japanese PD patients receiving DT. Furthermore, experimental evaluation of peripheral clock gene expression rhythms revealed that most PD patients receiving DT who experienced improved sleep following BLT showed a circadian phase shift, indicating the existence of a correlation between circadian modulation and sleep improvement. Conversely, this result indicates that sleep problems in PD patients receiving DT may arise at least in part as a result of circadian dysfunction. Indeed, we found that chronic dopaminergic stimulation induced a rapid attenuation of autonomous oscillations of clock gene expression in ex vivo cultured mouse suprachiasmatic nucleus (SCN) at the single neuron level. In conclusion, BLT is a promising medical treatment for improving sleep in PD patients receiving DT. This BLT-induced improvement may be due to the restoration of circadian function.

In vivo evaluation of the effect of lithium on peripheral circadian clocks by real-time monitoring of clock gene expression in near-freely moving mice.

Lithium has been used as a mood stabilizer to treat human bipolar disorders for over half a century. Several studies have suggested the possibility that the efficacy of lithium treatment results in part from the amelioration of circadian dysfunction. However, the effect of lithium on clock gene expression has not yet been investigated in vivo because continuous measurement of gene expression in organs with high time resolution over a period of several days is difficult. To resolve this issue, we attached a small photo multiplier tube (PMT) tightly to the body surface of transgenic mice carrying a reporter gene such that the photon input window faced target organs such as the liver and kidney and succeeded in long-term continuous measurement of circadian gene expression in semi-freely moving mice over periods of several weeks. Using this simple method, we clearly showed that lithium causes circadian period elongation in peripheral clock gene expression rhythms in vivo. Further development of our detection system to maturity will aid a wide range of research fields in medicine and biology.

Role of the clock gene Period3 in the human cell-autonomous circadian clock.

Previous studies have shown that mouse Period3 (mPer3) is dispensable for the generation of autonomous oscillations in the circadian clock. However, human studies have suggested that human Period3 (hPer3) may have more important roles in the core clock machinery than mPer3. To investigate the role of hPer3 protein in the cell-autonomous circadian oscillator, we conducted gene knockout of the hPer3 gene in human bone osteosarcoma epithelial cells using genome-editing technology. We examined the circadian transcription of endogenous clock genes in hPer3-deficient cell clones and found that hPer3-deficient cells showed a phase advance in circadian transcription compared to wild-type cells. We subsequently transfected wild-type and mutant cells with an adenovirus carrying a luciferase gene whose expression was driven by a clock gene promotor, and monitored bioluminescence in real time. Cosinor analysis showed that the circadian period length in all hPer3-deficient cells was significantly shorter than that in wild-type cells, demonstrating that the phase advance in endogenous clock gene expression in hPer3-deficient cell clones was attributable to a shortened circadian period length rather than a phase shift. Together these findings are consistent with previous studies in mice lacking functional mPer3, indicating that the Per3 protein functions similarly in both mice and humans.

Normal peripheral circadian phase in the old-old with abnormal circadian behavior.

Almost all organisms maintain a circadian clock from birth to death to synchronize their own physiology and behavior with the earth's rotation. However, extensive studies based on animal experiments have showed that aging results in circadian dysfunction. Human studies have also indicated age-associated abnormal phase, reduced amplitude and enhanced fragmentation in circadian physiology and behavior, thereby strongly implying age-related dysfunction of the clock machinery. Here, we carried out functional assessment of the circadian clock machinery in elderly patients aged 83-94 with severe dementia who showed abnormal circadian behavior. To investigate whether or not the systemic pathway from the circadian input to peripheral clocks functioned normally, the circadian phase in peripheral clock gene expression rhythms was evaluated using plucked hair tissues. Unexpectedly, the phase in all volunteer patients was within a range similar to that of healthy subjects. The circadian pathway from external inputs to peripheral clocks may therefore function normally, even in the old-old with severe dementia.

Involvement of the luteinizing hormone surge in the regulation of ovary and oviduct clock gene expression in mice.

Circadian dysfunction perturbs the female reproductive cycle. In particular, mice lacking the clock gene Bmal1 show severe infertility, implying that BMAL1 plays roles in ovulation and luteinization. Here, we examined temporal changes in clock gene expression in the ovary and oviduct before and during gonadotropin-induced follicular growth, ovulation, and luteinization in sexually immature mice. While the oviduct did not show a drastic change in clock gene expression, Bmal1 expression in the ovary was higher than that in control mice during the period from 4 to 16 hr after human chorionic gonadotropin (hCG) administration. Bmal1 expression reached a maximum at 16 hr after hCG administration, when follicle luteinization occurred. In an interesting manner, administration of hCG to ex vivo-cultured oviduct triggered a shorter circadian period and inevitably resulted in phase advance. Together, our present data suggest that LH surge induces continuous expression of BMAL1 in the mouse ovary and modulates circadian phase in the mouse oviduct.

Potential role of the pancreatic hormone insulin in resetting human peripheral clocks.

Mammalian circadian rhythms are phase-adjusted and amplified by external cues such as light and food. While the light input pathway via the central clock, the suprachiasmatic nucleus, has been well defined, the mechanism of feeding-induced circadian resetting remains undefined, particularly in humans. Animal studies have indicated that insulin, a pancreatic hormone that is secreted rapidly in response to feeding, is an input factor for a few peripheral clocks, such as liver and adipose tissue. In this study, using plucked and cultured hair follicles as a representative human peripheral clock, we examined the effect of insulin on circadian characteristics of clock gene expression. Our results demonstrate that insulin phase-shifts or amplifies the clock gene expression rhythms of ex vivo cultured hair follicles in a phase-responsive manner. To reduce the possibility that differences in species, genetic or environmental background, and experimental methods affected experimental outcomes, we also treated surgically extracted whisker follicles of Period2::Luciferase (Per2Luc ) mice with insulin and found that the effect of insulin on clock gene expression was reproducible. These results suggest the possibility that feeding-induced insulin resets peripheral circadian clocks in humans.

Effect of different light-dark schedules on estrous cycle in mice, and implications for mitigating the adverse impact of night work.

Approximately 20% of workers in developed countries are involved in night work. Nevertheless, many studies have strongly suggested that night-work-induced chronic circadian misalignment increases the risk of a diverse range of health problems. Although a relation between night work and irregular menstrual cycles has been indicated epidemiologically, a direct causal link remains elusive. Here, we report that repetitive reversal of light-dark (LD) cycles triggers irregular estrous cycles in mice. The findings showed that the estrous cycle remained irregular for more than four weeks after the mice were returned to regular LD cycles. Importantly, the magnitude of the negative impact of reversed LD cycles on the estrous cycle, or more specifically the decreased number of normal estrous cycles during the observation period, was dependent on the difference in the frequency of LD reversal. Presently, no clear solution to prevent night-work-mediated menstrual abnormalities is available, and reducing night work in modern society is difficult. Our findings indicate that optimizing work schedules could significantly prevent menstrual problems without reducing total night-work time.

Multiple circadian transcriptional elements cooperatively regulate cell-autonomous transcriptional oscillation of Period3, a mammalian clock gene.

Cell-autonomous oscillation in clock gene expression drives circadian rhythms. The development of comprehensive analytical techniques, such as bioinformatics and ChIP-sequencing, has enabled the genome-wide identification of potential circadian transcriptional elements that regulate the transcriptional oscillation of clock genes. However, detailed analyses using traditional biochemical and molecular-biological approaches, such as binding and reporter assays, are still necessary to determine whether these potential circadian transcriptional elements are actually functional and how significantly they contribute to driving transcriptional oscillation. Here, we focused on the molecular mechanism of transcriptional oscillations in the mammalian clock gene Period3 (Per3). The PER3 protein is essential for robust peripheral clocks and is a key component in circadian output processes. We found three E box-like elements located upstream of human Per3 transcription start sites that additively contributed to cell-autonomous transcriptional oscillation. However, we also found that Per3 is still expressed in a circadian manner when all three E box-like elements are functionally impaired. We noted that Per3 transcription was activated by the synergistic actions of two D box-like elements and the three E box-like elements, leading to a drastic increase in circadian amplitude. Interestingly, circadian expression of Per3 was completely disrupted only when all five transcriptional elements were functionally impaired. These results indicate that three E box-like and two D box-like elements cooperatively and redundantly regulate cell-autonomous transcriptional oscillation of Per3.

A simple method using ex vivo culture of hair follicle tissue to investigate intrinsic circadian characteristics in humans.

Almost all organisms maintain a circadian clock from birth to death to synchronize their own physiology and behavior with the earth's rotation. Because the in vivo evaluation of human circadian characteristics is labor-intensive, in vitro or ex vivo approaches could provide advantages. In this study, to enable the simple and non-invasive evaluation of autonomous circadian oscillation, we established a method for monitoring clock gene expression by performing ex vivo culture of whole hair root tissue. This method is extremely simple and imposes little burden on subjects. Results obtained using Cryptochrome-deficient mice support that circadian period length in hair tissue correlates with intrinsic period length observed in physiology and behavior. We then applied this method to old-old subjects with severe dementia, who showed abnormal circadian behavior, and found that their peripheral clocks autonomously oscillated in a manner similar to those of healthy or younger subjects, indicating that the effect of cellular senescence on the autonomous clock oscillator is limited at least in some cell types. Although further validation may be required, the hair tissue-based culture assay would be a tool to investigate intrinsic circadian characteristics in humans.

Potential contribution of tandem circadian enhancers to nonlinear oscillations in clock gene expression.

Limit-cycle oscillations require the presence of nonlinear processes. Although mathematical studies have long suggested that multiple nonlinear processes are required for autonomous circadian oscillation in clock gene expression, the underlying mechanism remains controversial. Here we show experimentally that cell-autonomous circadian transcription of a mammalian clock gene requires a functionally interdependent tandem E-box motif; the lack of either of the two E-boxes results in arrhythmic transcription. Although previous studies indicated the role of the tandem motifs in increasing circadian amplitude, enhancing amplitude does not explain the mechanism for limit-cycle oscillations in transcription. In this study, mathematical analysis suggests that the interdependent behavior of enhancer elements including not only E-boxes but also ROR response elements might contribute to limit-cycle oscillations by increasing transcriptional nonlinearity. As expected, introduction of the interdependence of circadian enhancer elements into mathematical models resulted in autonomous transcriptional oscillation with low Hill coefficients. Together these findings suggest that interdependent tandem enhancer motifs on multiple clock genes might cooperatively enhance nonlinearity in the whole circadian feedback system, which would lead to limit-cycle oscillations in clock gene expression.

Hypercholesterolemia Causes Circadian Dysfunction: A Potential Risk Factor for Cardiovascular Disease.

Hypercholesterolemia is a well-known risk factor for a wide range of diseases in developed countries. Here, we report that mice lacking functional LDLR (low density lipoprotein receptor), an animal model of human familial hypercholesterolemia, show circadian abnormalities. In free running behavioral experiments in constant darkness, these mice showed a prolonged active phase and distinctly bimodal rhythms. Even when the circadian rhythms were entrained by light and dark cycles, these mice showed a significant attenuation of behavioral onset intensity at the start of the dark period. Further, we hypothesized that the combination of hypercholesterolemia and circadian abnormalities may affect cardiovascular disease progression. To examine this possibility, we generated LDLR-deficient mice with impaired circadian rhythms by simultaneously introducing a mutation into Period2, a core clock gene, and found that these mice showed a significant enlargement of artery plaque area with an increase in inflammatory cytokine IL-6 levels. These results suggest that circadian dysfunction may be associated with the development or progression of cardiovascular diseases.

Estimation methods for human circadian phase by use of peripheral tissues.

Almost all living organisms, including humans, exhibit diurnal rhythms of physiology and behavior, which are driven by the circadian clock. Many studies have found that chronic misalignment between circadian and environmental/social rhythms carries a significant risk of various disorders, including sleep disorders, metabolic syndrome, cardiovascular diseases and cancer. However, irregular sleep-wake cycles and circadian maladjustment often cause 'social jet lag', which is minor but chronic jet-lag in our daily lives. Establishment of objective and convenient circadian-phase estimation methods in the clinical setting would therefore greatly contribute not only to resolving this global health problem but also to developing chronomedicine, a clinical approach for optimizing the time of day of treatments. Traditional melatonin-based methods have limitations with respect to circadian-phase evaluation; however, estimation methods based on clock gene expression may be able to compensate for these limitations. As a representative application of circadian-phase estimation based on clock gene expression, our method of using hair follicle cells may aid in the rapid clinical detection of circadian-related sleep problems, especially circadian rhythm sleep disorders that are masked because of forced adaptation to social time schedules.

The mammalian circadian clock protein period counteracts cryptochrome in phosphorylation dynamics of circadian locomotor output cycles kaput (CLOCK).

The circadian transcription factor CLOCK exhibits a circadian oscillation in its phosphorylation levels. Although it remains unclear whether this phosphorylation contributes to circadian rhythm generation, it has been suggested to be involved in transcriptional activity, intracellular localization, and degradative turnover of CLOCK. Here, we obtained direct evidence that CLOCK phosphorylation may be essential for autonomous circadian oscillation in clock gene expression. Importantly, we found that the circadian transcriptional repressors Cryptochrome (CRY) and Period (PER) showed an opposite effect on CLOCK phosphorylation; CRY impaired BMAL1-dependent CLOCK phosphorylation, whereas PER protected the phosphorylation against CRY. Interestingly, unlike PER1 and PER2, PER3 did not exert a protective action, which correlates with the phenotypic differences among mice lacking the Per genes. Further studies on the regulatory mechanism of CLOCK phosphorylation would thus lead to elucidation of the mechanism of CRY-mediated transcriptional repression and an understanding of the true role of PER in the negative feedback system.

The role of the endocrine system in feeding-induced tissue-specific circadian entrainment.

The circadian clock is entrained to environmental cycles by external cue-mediated phase adjustment. Although the light input pathway has been well defined, the mechanism of feeding-induced phase resetting remains unclear. The tissue-specific sensitivity of peripheral entrainment to feeding suggests the involvement of multiple pathways, including humoral and neuronal signals. Previous in vitro studies with cultured cells indicate that endocrine factors may function as entrainment cues for peripheral clocks. However, blood-borne factors that are well characterized in actual feeding-induced resetting have yet to be identified. Here, we report that insulin may be involved in feeding-induced tissue-type-dependent entrainment in vivo. In ex vivo culture experiments, insulin-induced phase shift in peripheral clocks was dependent on tissue type, which was consistent with tissue-specific insulin sensitivity, and peripheral entrainment in insulin-sensitive tissues involved PI3K- and MAPK-mediated signaling pathways. These results suggest that insulin may be an immediate early factor in feeding-mediated tissue-specific entrainment.

Compensation for intracellular environment in expression levels of mammalian circadian clock genes.

The circadian clock is driven by transcriptional oscillation of clock genes in almost all body cells. To investigate the effect of cell type-specific intracellular environment on the circadian machinery, we examined gene expression profiles in five peripheral tissues. As expected, the phase relationship between expression rhythms of nine clock genes was similar in all tissues examined. We also compared relative expression levels of clock genes among tissues, and unexpectedly found that quantitative variation remained within an approximately three-fold range, which was substantially smaller than that of metabolic housekeeping genes. Interestingly, circadian gene expression was little affected even when fibroblasts were cultured with different concentrations of serum. Together, these findings support a hypothesis that expression levels of clock genes are quantitatively compensated for the intracellular environment, such as redox potential and metabolite composition. However, more comprehensive studies are required to reach definitive conclusions.

Nuclear receptor-mediated cell-autonomous oscillatory expression of the circadian transcription factor, neuronal PAS domain protein 2 (NPAS2).

NPAS2 (MOP4) is a heme-containing sensor transcription factor responsive to a wide range of intra- and extracellular stimuli, which also functions as a circadian transcription factor. This molecule forms a heterodimer with another circadian transcription factor, BMAL1, and activates transcription via E-box elements, indicating that circadian phase synchronization between NPAS2 and BMAL1 expression is important for the efficient transcriptional activation of target genes. However, details of the mechanism of cell-autonomous circadian transcription of Npas2 remain unclear. Here, we show that one of the ROREs (retinoid-related orphan receptor response elements) in the upstream region of the transcription start site is essential for circadian transcription of the Npas2 gene. Furthermore, we also show that endogenous RORα indeed plays an essential role in cell-autonomous circadian transcription of Npas2, because a damped transcriptional oscillation was observed not only by introduction of a dominant negative form or small interfering RNA but also in embryonic fibroblasts obtained from RORα-mutant (sg/sg) mice. These results indicate that circadian transcription of Npas2 is synchronized with that of Bmal1 in a cell-autonomous nuclear receptor-mediated fashion.