Clock Genes Per2 Research Articles

Maternal Obesity during Pregnancy Alters Daily Activity and Feeding Cycles, and Hypothalamic Clock Gene Expression in Adult Male Mouse Offspring.

An obesogenic diet adversely affects the endogenous mammalian circadian clock, altering daily activity and metabolism, and resulting in obesity. We investigated whether an obese pregnancy can alter the molecular clock in the offspring hypothalamus, resulting in changes to their activity and feeding rhythms. Female mice were fed a control (C, 7% kcal fat) or high fat diet (HF, 45% kcal fat) before mating and throughout pregnancy. Male offspring were fed the C or HF diet postweaning, resulting in four offspring groups: C/C, C/HF, HF/C, and HF/HF. Daily activity and food intake were monitored, and at 15 weeks of age were killed at six time-points over 24 h. The clock genes Clock, Bmal1, Per2, and Cry2 in the suprachiasmatic nucleus (SCN) and appetite genes Npy and Pomc in the arcuate nucleus (ARC) were measured. Daily activity and feeding cycles in the HF/C, C/HF, and HF/HF offspring were altered, with increased feeding bouts and activity during the day and increased food intake but reduced activity at night. Gene expression patterns and levels of Clock, Bmal1, Per2, and Cry2 in the SCN and Npy and Pomc in the ARC were altered in HF diet-exposed offspring. The altered expression of hypothalamic molecular clock components and appetite genes, together with changes in activity and feeding rhythms, could be contributing to offspring obesity.

Expression of miR-34a-5p is up-regulated in human colorectal cancer and correlates with survival and clock gene PER2 expression.

Colorectal cancer represents a leading cause of cancer death. MicroRNAs (miRNAs) are small non-coding RNA molecules that have been extensively studied in tumours, since changes in their levels can reveal patient prognosis. Cancer progression is also influenced by the circadian system whose functioning is based on the rhythmic expression of clock genes. Therefore, we performed macroarray screening of tumour and adjacent tissues in patients undergoing surgery for colorectal carcinoma. We identified 17 miRNAs showing expression that was more than 100 times higher in tumour tissue compared to adjacent tissue. From in silico analysis, miR-34a-5p was selected as showing a computer-predicted interaction with PER2. Real-time PCR revealed a negative correlation between expression of PER2 mRNA and miR-34a in patients with more advanced cancer stage. Expression of miR-34a was up-regulated in cancer tissue compared to adjacent tissue. High miR-34a expression was associated with better survival of patients. miR-34a showed lower expression levels in male patients with lymph node involvement, and a trend towards decreased expression in male patients with distant metastases. Male patients, but not female patients, with high expression of miR-34a and who were free of distant metastases and/or lymph node involvement showed better survival. Therefore, we proposed that expression of miR-34a was regulated in a sex-dependent manner and could be considered a marker of prognosis in earlier cancer stages in male patients.

Repetitive mild traumatic brain injury alters diurnal locomotor activity and response to the light change in mice

Mild traumatic brain injury (mTBI) is a common cause of brain damage with a high incidence of multiple mTBIs found among athletes and soldiers. The purpose of this study is to examine the diurnal behavioral changes after multiple mTBIs. Adult mice were anesthetized; mTBI was conducted by dropping a 30-g weight to the right temporal skull once (mTBI1) or three times (mTBI3) over 3-week. Open-field motor behavior was recorded for 3 days after the last mTBI. In the first 4-hour exploratory phase, mTBI1 or mTBI3 equally reduced locomotor activity. A significant reduction of locomotor activity was found in the dark cycle between 4–72 hour in mTBI1 or mTBI3 mice; higher motor activity was seen after mTBI3 compared to mTBI1. In the light cycle, mTBI3 mice demonstrated an earlier immobilization followed by hyperactivity. The response to light change significantly correlated with the number of impacts. The IBA1 and BAX protein levels were equally increased in the lesioned cortex after mTBI1 and mTBI3. mTBI3 selectively upregulated the expression of circadian clock gene Per1 in hypothalamus and hippocampus as well as iNOS expression in the lesioned side cortex. Our data suggest multiple mTBIs alter diurnal locomotor activity and response to the change of light, which may involve Per1 expression in the lesioned brain.

Rhythmic Release of Corticosterone Induces Circadian Clock Gene Expression in the Cerebellum

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.

Effect of urban environment on pineal machinery and clock genes expression of tree sparrow (Passer montanus)

Increasing urbanisation is altering the physiology of wild animals and the mechanisms involved are largely unknown. We hypothesised that altering the physiology of urban organisms is due to the effect of extra light at night on the circadian clock by modulating the expression of pineal machinery and clock genes. Two experiments were performed. In Experiment 1, immediately after being procured from their respective sites (urban and rural sites), birds were released individually in LLdim light conditions. Circadian rhythm period, activity duration, and total activity count were calculated and did not differ between urban and rural birds. In Experiment 2, birds (from urban and rural habitats) were sampled at six time points at regular 4-h intervals, beginning 1 h after sunrise. We measured daily variations in plasma melatonin levels. We also analysed the expression levels of Aanat, Mel1A and Mel1B as an indicator of melatonin biosynthesis and action machinery. Clock and clock-controlled genes (Bmal1, Clock, Per2, Per3, Cry1 and Npas2) were studied in the hypothalamus, the pineal gland, and retina to investigate the effects of urban habitats on the circadian clock. Our results show that there is a lower expression of Aanat in the pineal gland and relatively low plasma melatonin levels in urban birds. Further, clock genes are also differentially expressed in all three central tissues of urban birds. We propose that alterations in the melatonin biosynthesis machinery and the expression of clock genes could result in miscalculations in the internal timing of the organism, with environmental timings leading to altered physiology in urban wild animals.

Molecular biology of periodontal ligament fibroblasts and orthodontic tooth movement : Evidence and possible role of the circadian rhythm.

The circadian clock plays an important role in many physiological states and pathologies. The significance of its core genes in bone formation and tooth development has already been demonstrated. However, regulation of these genes and their influence on periodontal and bone remodeling in periodontal ligament (PDL) fibroblasts remains to be elucidated. Our hypothesis was that the circadian clock influences markers for periodontal and bone remodeling and therefore orthodontic tooth movement itself. Human PDL fibroblasts were cultured and synchronized in circadian rhythms with the help of adexamethasone shock. Cells were harvested at 4 h intervals. Reverse transcription and quantitative RT PCR (real time polymerase chain reaction) were performed to assess the mRNA levels of the clock genes ARNTL, CLOCK1, PER1, and PER2. Subsequently, mRNA expression of important marker genes for periodontal and bone remodeling, OPG, RANKL, OCN, OPN, RUNX2, COL1A1, IL1β, KI67, and POSTN, were examined at time points of ARNTL amplitude expression. Gene expression of core clock genes varied over 48 h in accordance with the circadian rhythm. Functional markers, except KI67, showed significant differences at time points of maximum fluctuation especially of ARNTL. PDL fibroblasts express circadian clock genes. Our results suggest that genes associated with bone and periodontal remodeling are influenced by the circadian rhythm. Further research will have to refine the understanding of this influence for orthodontic treatment.

Persistent vs transient alteration of folliculogenesis and estrous cycle after neonatal vs adult exposure to Bisphenol A

Exposure to bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical (EDC), is known to produce variable effects on female puberty and ovulation. This variability of effects is possibly due to differences in dose and period of exposure. Little is known about the effects of adult exposure to environmentally relevant doses of this EDC and the differences in effect after neonatal exposure. This study sought to compare the effects of neonatal vs adult exposure to a very low dose or a high dose of BPA for 2 weeks on ovulation and folliculogenesis and to explore the hypothalamic mechanisms involved in such disruption by BPA. One-day-old and 90-day-old female rats received daily subcutaneous injections of corn oil (vehicle) or BPA (25 ng/kg/d or 5 mg/kg/d) for 15 days. Neonatal exposure to both BPA doses significantly disrupted the estrous cycle and induced a decrease in primordial follicles. Effects on estrous cyclicity and folliculogenesis persisted into adulthood, consistent with a disruption of organizational mechanisms. During adult exposure, both doses caused a reversible decrease in antral follicles and corpora lutea. A reversible disruption of the estrous cycle associated with a delay and a decrease in the amplitude of the LH surge was also observed. Alterations of the hypothalamic expression of the clock gene Per1 and the reproductive peptide phoenixin indicated a disruption of the hypothalamic control of the preovulatory LH surge by BPA.

D-Ser2-oxyntomodulin ameliorated Aβ31-35-induced circadian rhythm disorder in mice.

IntroductionThe occurrence of circadian rhythm disorder in patients with Alzheimer's disease (AD) is closely related to the abnormal deposition of amyloid‐β (Aβ), and d‐Ser2‐oxyntomodulin (Oxy) is a protease‐resistant oxyntomodulin analogue that has been shown to exert neuroprotective effects.AimsThis study aimed to explore whether Oxy, a new GLP‐1R/GCGR dual receptor agonist, can improve the Aβ‐induced disrupted circadian rhythm and the role of GLP‐1R.MethodsA mouse wheel‐running experiment was performed to explore the circadian rhythm, and western blotting and real‐time PCR were performed to assess the expression of the circadian clock genes Bmal1 and Per2. Furthermore, a lentivirus encoding an shGLP‐1R‐GFP‐PURO was used to interfere with GLP‐1R gene expression and so explore the role of GLP‐1R.ResultsThe present study has confirmed that Oxy could restore Aβ31‐35‐induced circadian rhythm disorders and improve the abnormal expression of Bmal1 and Per2. After interfering the GLP‐1R gene, we found that Oxy could not improve the Aβ31‐35‐induced circadian rhythm disorder and abnormal expression of clock genes.ConclusionThis study demonstrated that Oxy could improve Aβ31‐35‐induced circadian rhythm disorders, and GLP‐1R plays a critical role. This study thus describes a novel target that may be potentially used in the treatment of AD.

Angptl8 mediates food-driven resetting of hepatic circadian clock in mice

Diurnal light-dark cycle resets the master clock, while timed food intake is another potent synchronizer of peripheral clocks in mammals. As the largest metabolic organ, the liver sensitively responds to the food signals and secretes hepatokines, leading to the robust regulation of metabolic and clock processes. However, it remains unknown which hepatokine mediates the food-driven resetting of the liver clock independent of the master clock. Here, we identify Angptl8 as a hepatokine that resets diurnal rhythms of hepatic clock and metabolic genes in mice. Mechanistically, the resetting function of Angptl8 is dependent on the signal relay of the membrane receptor PirB, phosphorylation of kinases and transcriptional factors, and consequently transient activation of the central clock gene Per1. Importantly, inhibition of Angptl8 signaling partially blocks food-entrained resetting of liver clock in mice. We have thus identified Angptl8 as a key regulator of the liver clock in response to food.

Illuminated night alters behaviour and negatively affects physiology and metabolism in diurnal zebra finches

Light at night (LAN) negatively impacts the behaviour and physiology; however, very little is known about molecular correlates of LAN-induced effects in diurnal animals. Here, we assessed LAN-induced effects on behaviour and physiology, and examined molecular changes in the liver of diurnal zebra finches (Taeniopygia guttata). Birds were exposed to dim LAN (dLAN: 12L = 150 lux: 12D = 5 lux), with controls on 12L (150 lux): 12D (0 lux). dLAN altered daily activity-rest and eating patterns, induced nocturnal eating and caused body fattening and weight gain, and reduced nocturnal melatonin levels. Concomitant increased nighttime glucose levels, decreased daytime thyroxine and triglycerides levels, and hepatic lipid accumulation suggested the impairment of metabolism under dLAN. Transcriptional assays evidenced dLAN-induced negative effects on metabolism in the liver, the site of metabolic homeostasis. Particularly, increased g6pc and foxo1 mRNA expressions suggested an enhanced gluconeogenesis, while increased egr1 and star expressions suggested enhanced cholesterol biosynthesis and lipid metabolism, respectively. Similarly, overexpressed sirt1 indicated protection from the metabolic damage due to elevated gluconeogenesis and cholesterol biosynthesis under dLAN. However, no effect on genes involved in lipogenesis (fasn) and insulin signalling pathway (socs3 and insig1) might indicate for the post transcriptional/post translational modification effects or the involvement of other genetic pathways in LAN-induced effects. We also found daily rhythm in the hepatic expression of selected clock and clock-controlled genes (per2, bmal1 and reverb-beta), with an elevated mesor and amplitude of per2 oscillation, suggesting a role of per2 in the liver metabolism. These results demonstrate dLAN-induced negative effects on the behaviour and physiology, and provide molecular insights into metabolic risks of the exposure to illuminated nights to diurnal animals including humans in an urban setting.

Circadian Rhythm Disruption and Sepsis in Severe Trauma Patients.

Circadian rhythms are important regulators of immune functions. Admission to an intensive care unit may impact molecular clock activity and host response. Our objective was to assess and compare the immune circadian rhythms in trauma patients who develop and in those who do not develop sepsis. Blood samples were collected from severe trauma patients within 4 days after admission, with collections taking place every 4 h over a 24-h period. Cortisol and cytokines were measured with immunoassays. Whole-blood expression of 3 clock genes (Bmal1, Per2, and Per3) was studied by reverse transcription quantitative polymerase chain reaction. Neutrophils, monocytes, and lymphocytes were analyzed by flow cytometry. Patients with and without sepsis were compared with the cosinor mixed model to estimate mesors, amplitudes, and acrophases. Thirty-eight patients were enrolled in the study, and 13 developed at least 1 septic episode. The septic patients had higher levels of cortisol than the nonseptic patients (mesor at 489 nmol/L vs. 405 nmol/L, P < 0.05) and delayed acrophases (22 h vs. 15 h, P < 0.05). They also had lower lymphocyte counts (mesor at 785 vs. 1,012 cells/μL, P < 0.05), higher neutrophil counts (mesor at 7,648 vs. 7,001 cells/μL, P < 0.05), and monocyte counts (mesor at 579 vs. 473 cells/μL, P < 0.05) than the nonseptic patients. Although no amplitude difference was identified, the acrophases were significantly different between the 2 groups for lymphocytes, interleukin 10 and tumor necrosis factor. We demonstrated that all trauma patients had impaired circadian rhythms of cortisol, cytokines, leukocytes, and clock genes. Early circadian disruption was associated with the occurrence of sepsis and might be a marker of sepsis severity.

Abstract 1729: Investigating deregulated circadian clock machinery in cancer cells

Abstract The circadian clock plays an integral role in cellular functioning by temporally controlling gene expression, and there is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study we aimed to investigate circadian clock gene expression and oscillation patterns in cervical and oesophageal cancers, in order to determine whether disruptions in circadian clock functioning occur in these cancer types. Microarray gene expression analysis revealed the circadian clock gene, Per2, to be one of the most significantly downregulated genes in cervical cancer patient tissue compared to normal epithelium. In addition, Oncomine data-mining revealed significant downregulation of not only Per2, but multiple members of the circadian clock gene family (Clock, Bmal1, Per1, Per2, Cry1, Rev-erbα and RORα) in cervical cancer tissue compared to normal. Real-time RT-PCR analysis of circadian clock genes in oesophageal cancer patient tissue compared to matched normal epithelium revealed significant downregulation of Clock, Per2, Cry1 and RORα in oesophageal tumour tissue. Significant positive correlations were observed where circadian clock genes were simultaneously dysregulated in tumour tissue specimens. In cell line models, Clock, Bmal1, Per2, Cry1 and RORα circadian clock genes were significantly downregulated in transformed cells, compared to their untransformed counterparts, as well as in most cervical and oesophageal cancer cell lines compared to non-cancer epithelial cells. Patterns of protein expression did not accurately match mRNA expression, likely due to extensive post-translational processing, but Clock and Cry1 protein levels were considerably reduced in the cancer cell lines compared to normal. Overexpression of circadian clock genes in cancer cell lines negatively affected cell proliferation, highlighting the tumour suppressor properties of these genes in cervical and oesophageal cancer cells. Despite downreglated expression of circadian clock genes, cervical and oesophageal cancer cells maintain functional circadian oscillations, after synchronisation with Dexamethasone, as shown using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are still intact. Together, this study is a first to describe deregulated circadian clock machinery in cervical and oesophageal cancer cells, although the cells maintain a functional circadian rhythm. The study has relevance to the field of chronotherapy, where elucidating differences in circadian clock functioning in normal and cancer cells could yield better insights into the timing of administration of chemotherapy, ultimately ensuring a better patient response. Citation Format: Pauline J. van der Watt, Kate Davis, Virna Leaner. Investigating deregulated circadian clock machinery in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1729.

Abstract 1888: Circadian rhythm modulation of breast cancer gene expression depends on stem cell properties induced in culture

Abstract It is important to know whether breast cancer cells within tumors have functional circadian clocks. If so, they may benefit from the ability provided by the clock to predict when important daily events occur such as nutrient availability in blood following meals or elevated immune cell surveillance at night. If instead the circadian clock is nonfunctional, then clinical treatments could be directed towards activating the clock because evidence indicates circadian rhythm loss leads to more aggressive tumor growth. Here, we examined the role of circadian clocks in metastasis-related events, focusing on epithelial-mesenchymal transition (EMT), including cancer stem cell (CSC) generation, and mesenchymal-epithelial transition (MET). Because of their differing circadian properties two tumor-forming cell lines were compared, MCF-7 human breast cancer and C6 rat glioma cells. C6 monolayer and tumorsphere cultures express distinct circadian rhythms in gene expression. In contrast, monolayer MCF-7 cell cultures are reported to be arrhythmic and so appear to lack a functional circadian clock. Nevertheless, MCF-7 cells may contain inducible circadian oscillators that fail to generate circadian rhythms under standard culture conditions but may function in tumor microenvironments. Tumorspheres simulate the three-dimensional tumor structure and were used here to test for favorable conditions enabling circadian rhythms in cancer cells. Circadian rhythms in EMT, MET, mitosis, migration, and cell death were evaluated through time-lapse imaging over several days. Bioluminescence imaging and quantitative RT-PCR were used to test for circadian rhythms in expression of the core circadian clock gene Per2 in MCF-7 cells. Following treatment with serum-free culture medium designed for stem cells, EMT and CSC-related protein markers of MCF-7 and C6 cells were confirmed by immunofluorescence: MSI1 and CD133 in MCF-7; OCT4, TWIST1, ZEB1, vimentin in C6. Morphometry showed distinct changes in cell shape following EMT as shown by nearly all MCF-7 cells having an average roundness of 0.815 ±0.09 SD and a C6 roundness of 0.855 ±0.029. This medium increased the number of MCF-7 tumorspheres expressing circadian rhythms by 31.5%, and average amplitude of rhythms increased 11.6-fold. MET was then induced by returning cells to serum-containing medium, which produced cells with a flattened cell morphology indicating epithelial or pseudoepithelial differentiation. A circadian clock in C6 cells appeared to gate MET events after the return to serum (95% confidence limit of period: 20.8-26.9 hrs, p&amp;lt;0.05). The circadian timing properties of MCF-7 tumorspheres suggest that the circadian clock may also be present in tumors and that cell-cell interactions or paracrine factors could sustain otherwise poorly performing circadian clocks in breast cancer. Citation Format: Arpan De, Vishal Premdev Sharma, Dilshan Harshajith Beligala, Angelia Marie Lee, Michael Eric Geusz. Circadian rhythm modulation of breast cancer gene expression depends on stem cell properties induced in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1888.

PER3 variable number tandem repeat (VNTR) polymorphism modulates the circadian variation of the descending pain modulatory system in healthy subjects

We evaluated the circadian pattern of variation of the descending pain modulatory system (DPMS) using a conditioned pain modulation (CPM) paradigm according to the variable-number tandem-repeat (VNTR) of the clock gene PER3 polymorphism. We assessed the relationship between the genotypes PER34/4 and PER35/5 and the temporal pattern of variation across the day using the following measures: the heat pain threshold (HPT), the cold pressure test (CPT), and the serum levels of BDNF and S100-B protein. The ∆-values (from afternoon to morning) of these measures were used for the analysis. The circadian phenotype was according to the mid-point sleep time established by the Munich ChronoType Questionnaire (MCTQ). We included 18 healthy volunteers (15 women) ages 18 to 30. A Generalized Linear Model (GLM) revealed a significant difference in the ∆-CPM-task between Per34/4 and Per35/5 genotypes, with means (SDs) of −0.41 (0.78) vs. 0.67 (0.90) (χ2 = 7.256; df = 1′ P = 0.007), respectively. Both sleep deprivation of at least 2 h/day (B = −0.96, 95% confidence interval (CI) = −1.86 to −0.11)) and the ∆-S100-B protein (−0.03, 95% CI = −0.06 to −0.02) were negatively correlated with the ∆-CPM-task, while the ∆-BDNF was positively correlated with the ∆-CPM-task (0.015, 95% CI = 0.01 to 0.03). We observed a difference in the ∆-CPT between PER34/4 and PER35/5 (0.11 (4.51) vs. 4.00 (2.60), respectively) (χ2 = 22.251; df = 1 P = 0.001). These findings suggest that the polymorphism of PER35/5 is associated with a decrease in the inhibitory function of the DPMS over the course of the day. However, sleep deprivation is an independent factor that also reduces the inhibitory function of the DPMS, regardless of the PER3 VNTR polymorphism.

Non-coding cis-element of Period2 is essential for maintaining organismal circadian behaviour and body temperature rhythmicity

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive. Cis-element-based transcriptional regulation is believed to be crucial for generating circadian rhythms in behavior and physiology. However, genetic evidence supporting this model is based on mutations in the protein-coding sequences of clock genes. Here, we report generation of mutant mice carrying a mutation only at the E′-box cis-element in the promoter region of the core clock gene Per2. The Per2 E′-box mutation abolishes sustainable molecular clock oscillations and renders circadian locomotor activity and body temperature rhythms unstable. Without the E′-box, Per2 messenger RNA and protein expression remain at mid-to-high levels. Our work delineates the Per2 E′-box as a critical nodal element for keeping sustainable cell-autonomous circadian oscillation and reveals the extent of the impact of the non-coding cis-element in daily maintenance of animal locomotor activity and body temperature rhythmicity.

The Absence of Pineal Melatonin Abolishes the Daily Rhythm of Tph1 (Tryptophan Hydroxylase 1), Asmt (Acetylserotonin O-Methyltransferase), and Aanat (Aralkylamine N-Acetyltransferase) mRNA Expressions in Rat Testes.

This study examined the effects of pinealectomy in Wistar rats and melatonin replacement therapy on the daily mRNA expression of melatonin (Tph1, Aanat, Asmt, Mt1, Mt2, and Rorα), and steroidogenic (Star, 17βhsd3, and Lhr) related genes as well as clock genes (Rev-erbα, Bmal1, Per1, Per2, Cry1, and Cry2) in testes. The testes of control animals express the Tph1, Aanat, and Asmt and Per2 genes with 24-h rhythms in mRNA, reaching the maximal values during the dark phase. Pinealectomy abolished and melatonin treatment restored the 24-h rhythmicity. Daytime differences in mRNA expression were significant for Star, Lhr, Mt1, Mt2, Rorα, Rev-erbα, Bmal1, Cry1, and Cry2 genes in testes of control rats. Conversely, 17βhsd3 and Per1 mRNA expression did not show a daytime difference in testes of control animals. Pinealectomy abolished the peak time of Mt1 and Mt2 mRNA expression, phase shifted the peak time of Star, Rorα, Rev-erbα, Bmal1, and Cry2 mRNA expression, downregulated the 24-h Lhr mRNA expression, and inverted the peak time of Per1, Per2, and Cry1 mRNA expression to the light phase. The melatonin replacement therapy completely restored the control levels of Lhr, Rev-erbα, and Per1 mRNA expression patterns, partially restored the daily control of Star, Mt2, Rorα, Bmal1, Cry1, and Cry2 mRNA expression but did not re-establish the daily control of Mt1 mRNA expression. This suggests that the daily mRNA expression of these genes is probably driven by pineal melatonin and melatonin treatment restores (partially or completely) the daily control of gene expression patterns.

Role of Per3, a circadian clock gene, in embryonic development of mouse cerebral cortex

Per3 is one of the primary components of circadian clock system. While circadian dysregulation is known to be involved in the pathogenesis of several neuropsychiatric diseases. It remains largely unknown whether they participate in embryonic brain development. Here, we examined the role of clock gene Per3 in the development of mouse cerebral cortex. In situ hybridization analysis revealed that Per3 is expressed in the developing mouse cortex. Acute knockdown of Per3 with in utero electroporation caused abnormal positioning of cortical neurons, which was rescued by RNAi-resistant Per3. Per3-deficient cells showed abnormal migration phenotypes, impaired axon extension and dendritic arbor formation. Taken together, Per3 was found to play a pivotal role in corticogenesis via regulation of excitatory neuron migration and synaptic network formation.

Pathophysiological significance of clock genes BMAL1 and PER2 as erythropoietin-controlling factors in acute blood hemorrhage.

This study aimed to characterize the pathophysiology, including possible correlations, of clock gene expression and erythropoietin (EPO) production in the acute stage of blood hemorrhage. Specimens of human cortical tissues (right and left kidneys) and cardiac blood were collected at autopsy from 52 cases following mortality due to acute-stage blood hemorrhage following sharp instrument injury. BMAL1 and PER2 mRNA levels were determined by reverse transcription-polymerase chain reaction; BMAL1 and PER2 protein levels were assessed using immunohistochemistry; BMAL1 protein levels were quantitatively measured by western blotting; and serum EPO levels were measured by chemiluminescent enzyme immunoassay. Separately, a rat model of hemorrhagic conditions was generated and used to confirm the results obtained with autopsy-derived specimens. A positive correlation was observed between BMAL1 protein and serum EPO levels, but not between BMAL1 mRNA levels and serum EPO levels. We also noted that Per2 mRNA expression became elevated in humans who survived for > 3h after acute hemorrhagic events, with subsequent decreases in serum EPO levels. The rat model showed that even short (30-min) intervals of blood loss yielded increases in both Bmal1 mRNA and serum EPO levels; longer (60-min) intervals resulted in increases in Per2 mRNA expression along with decreases in serum EPO. Thus, the acute-stage human hemorrhage cases and the rat hemorrhage model yielded similar tendencies for clock gene expression and EPO secretion. In conclusion, our results indicated that clock genes are involved in the regulation of EPO production during the early stages of hypoxia/ischemia resulting from the acute hemorrhagic events.

Circadian Clock Gene Expression in Regions of Interest to Respiratory Control

Circadian rhythms are endogenous, ~24‐hour cycles that synchronize biological functions to external cues known as zeitgebers. Circadian rhythms emerge from self‐sustaining oscillations in a transcription‐translation feedback loop of core clock genes (Bmal1, Clock, Per1 and Per2). The central clock (suprachiasmatic nucleus) and peripheral tissues throughout the body utilize this same circadian machinery. Core clock components also exert tissue‐specific effects, including tissue‐appropriate gene regulation. Although clock gene networks have been established throughout the body, little is known concerning clock genes in the neuromuscular system regulating breathing. We tested the hypothesis that clock genes oscillate in key regions of the respiratory circuit by characterizing mRNA expression patterns of core clock genes in the medulla, ventral spinal cord and diaphragm throughout the day via qPCR. Samples were collected from Sprague Dawley rats entrained to a 12‐hour light/dark cycle at 4 zeitgeber times (ZT), including (n = 8 per group): ZT5 (11am), ZT11 (5pm), ZT17 (11pm) and ZT23 (5am); ZT0 corresponds to beginning of light cycle. Cosinor analysis revealed that Bmal1, Clock, Per1 and Per2 clock genes were rhythmic in all 3 regions (all p&lt;0.01). The expression pattern of Per1 and Per2 was antiphase to Bmal1 and Clock in all regions. We hypothesize that clock genes may influence the potential to elicit serotonin‐dependent respiratory motor plasticity, such as intermittent‐hypoxia‐induced phrenic long‐term facilitation. Thus, we tested the specific hypotheses that BMAL1 protein is expressed within phrenic motor neurons and molecules necessary for phrenic long‐term facilitation (5‐HT2A receptors, BDNF and TrkB) exhibit rhythmic gene expression in brainstem and spinal regions involved in this form of plasticity. Using immunofluorescence, we demonstrate that the clock protein BMAL1 is expressed within cholera toxin B labelled phrenic motor neurons. The plasticity‐related gene Bdnf was expressed rhythmically in medulla and in ventral spinal (C3–C5) homogenates (p&lt;0.001), peaking in the active phase. TrkB mRNA (Ntrk2) was expressed rhythmically within the medulla (p&lt;0.01), but not the ventral spinal cord. 5‐HT2A receptor mRNA (Htr2a) did not exhibit rhythmic expression in either region. In conclusion, daily rhythms are observed in transcripts from clock genes and molecules necessary and sufficient for phrenic long‐term facilitation (e.g. BDNF). Although we did not uncouple cellular clocks from zeitgebers (light/dark) in this initial study, we confirm the potential for local circadian regulation in regions of interest to respiratory motor control.Support or Funding InformationUniversity of Florida Graduate School Preeminence Award (MNK), NIH T32 HD043730 (MDS) and the McKnight Brain Institute (GSM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of gene expression in adrenal glands of mice lacking the circadian clock protein PER1

The circadian clock regulates a variety of physiological responses throughout a 24‐hour period. At a molecular level, the clock is governed by four core transcription factors, including PER1 and CRY, which are found in every cell in the human body. All reported circadian clock gene KO mice have a blood pressure (BP) phenotype. Aldosterone, a mineralocorticoid hormone and product of the renin‐angiotensin‐aldosterone system (RAAS), is secreted by the adrenal cortex and leads to increased sodium reabsorption by the kidneys in response to low BP or low blood volume. Previously we reported that 129/sv mice lacking the circadian clock gene Per1 had reduced BP and reduced plasma aldosterone levels associated with decreased expression of HSD3B1, a key enzyme in the steroid hormone synthesis pathway. Conversely, we found that male C57BL/6 Per1 KO mice have higher BP than control mice and develop non‐dipping hypertension (a lack of dip in BP during the rest period) on a high salt diet plus mineralocorticoid treatment. Non‐dipping hypertension has been associated with an increased risk for cardiovascular disease in humans. Interestingly, female Per1 KO mice do not develop non‐dipping hypertension on a high salt diet plus mineralocorticoid treatment. The goal of this study was to determine if expression of genes involved in the aldosterone synthesis pathway is altered in C57BL/6 Per1 KO mice. These deviances could explain the BP phenotype observed at the physiological level in male mice. We obtained adrenal glands from male and female WT and Per1 KO mice at noon (midpoint of the mouse rest phase), the same time at which serum was collected for aldosterone measurements. RNA was isolated from these tissues, converted to cDNA, and mRNA levels of genes involved in circadian rhythm and aldosterone synthesis were measured by quantitative real time PCR (qPCR). Preliminary data show that Cry1 is significantly down‐regulated in female Per1 KO mice by about 50% versus WT (n=5/6 per group, p&lt;0.05). Expression of aldosterone synthesis genes CYP11B and HSD3B1 did not appear to be significantly altered in the adrenal tissue of male or female Per1 KO mice compared to WT. In contrast to what was observed with 129/sv Per1 KO, plasma aldosterone levels did not differ between C57BL/6 Per1 KO vs. WT. The observed difference in Cry1 expression suggests that RAAS and the circadian clock may work together in the maintenance of homeostatic BP. In the future we expect to perform similar experiments using male and female WT and Per1 KO mice adrenal tissue collected at a midnight time point in order to further investigate the communication occurring at the molecular level between RAAS and the circadian clock.Support or Funding InformationNIH/NIDDK 1R01DK109570; The Gatorade Trust through the UF Department of MedicineThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.