Depriving mice of sleep also deprives of food

Both sleep-wake behavior and circadian rhythms are tightly coupled to energy metabolism and food intake. Altered feeding times in mice are known to entrain clock-gene rhythms in brain and liver and sleep-deprived humans tend to eat more and gain weight. Previous observations in mice showing that sleep deprivation (SD) changes clock-gene expression might thus relate to altered food intake and not to the loss of sleep per se. Whether SD affects food intake in the mouse and how this might affect clock-gene expression is, however, unknown. We therefore quantified i) the cortical expression of the clock genes Per1, Per2, Dbp, and Cry1 in mice that had access to food or not during a 6h SD, and ii) food intake during baseline, SD, and recovery sleep. We found that food deprivation did not modify the SD-incurred clock-gene changes in the cortex. Moreover, we discovered that although food intake during SD did not differ from baseline, mice lost weight and increased food intake during subsequent recovery. We conclude that SD is associated with food deprivation and that the resulting energy deficit might contribute to the effects of SD that are commonly interpreted as a response to sleep loss.

Rhythmic Clock Gene Expression in Atlantic Salmon Parr Brain

To better understand the complexity of clock genes in salmonids, a taxon with an additional whole genome duplication, an analysis was performed to identify and classify gene family members (clock, arntl, period, cryptochrome, nr1d, ror, and csnk1). The majority of clock genes, in zebrafish and Northern pike, appeared to be duplicated. In comparison to the 29 clock genes described in zebrafish, 48 clock genes were discovered in salmonid species. There was also evidence of species-specific reciprocal gene losses conserved to the Oncorhynchus sister clade. From the six period genes identified three were highly significantly rhythmic, and circadian in their expression patterns (per1a.1, per1a.2, per1b) and two was significantly rhythmically expressed (per2a, per2b). The transcriptomic study of juvenile Atlantic salmon (parr) brain tissues confirmed gene identification and revealed that there were 2,864 rhythmically expressed genes (p < 0.001), including 1,215 genes with a circadian expression pattern, of which 11 were clock genes. The majority of circadian expressed genes peaked 2 h before and after daylight. These findings provide a foundation for further research into the function of clock genes circadian rhythmicity and the role of an enriched number of clock genes relating to seasonal driven life history in salmonids.

NF-κB modifies the mammalian circadian clock through interaction with the core clock protein BMAL1

In mammals, the circadian clock coordinates cell physiological processes including inflammation. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how inflammation affects the clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-κB in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-κB subunit RELA in the human U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude, its inhibition lengthened period length and caused amplitude phenotypes. NF-κB perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity behavior under different light/dark conditions. We show that RELA, like the clock repressor CRY1, repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA binds to the transactivation domain of BMAL1. These data support a model in which NF-kB competes with CRY1 and coactivator CBP/p300 for BMAL1 binding to affect circadian transcription. This is further supported by chromatin immunoprecipitation analysis showing that binding of RELA, BMAL1 and CLOCK converges on the E-boxes of clock genes. Taken together, these data support a significant role for NF-κB in directly regulating the circadian clock and highlight mutual regulation between the circadian and inflammatory pathways.

TAMI-44. ASSOCIATION OF CIRCADIAN CLOCK GENE EXPRESSION WITH GLIOMA TUMOR MICROENVIRONMENT AND PATIENT SURVIVAL

Circadian clock genes have been linked to differences in clinical outcomes in cancer, including gliomas. However, these studies have not accounted for established prognostic markers, including mutations in Isocitrate Dehydrogenase (IDH). To study the connection between circadian clock genes and glioma outcomes while accounting for IDH mutational status, we analyzed multiple publicly available gene expression datasets. Unsupervised clustering of 13 clock gene transcriptomic signatures from The Cancer Genome Atlas resulted in four distinct transcriptomic clusters, two clusters were enriched for IDH mutant (Circadian 1-2) and the others for IDH wild-type gliomas (Circadian 3-4). Within these clusters we observed differential prognosis of the patients by Kaplan–Meier analysis (Circadian 1-2, p=0.0001; Circadian 3-4, p=0.0002) suggesting that these transcriptomic circadian subtypes might reflect different disease states. Further analyses using Cox Proportional Hazards Regression showed that lower Period (PER) gene expression was associated with worse prognosis (increasing PER expression HR=0.655, p=0.007) independent of IDH wild-type status (HR=5.312, p< 0.001) and increasing age (HR = 1.04, p< 0.001). Lower PER expression was associated with enrichment of a number of immune signaling pathways. These findings prompted the exploration of the relationship between microenvironment and clock genes using the Ivy GAP dataset to explore tumor location-specific differences and single cell RNA sequencing data from Darmanis (accession: GSE84465) to explore cell-specific differences. Circadian clock genes were found to be differentially expressed across anatomical tumor locations and cell types, including microglia. In ongoing studies we are examining the role of the microenvironment and PER2 expression on tumor growth by disrupting PER2 expression in tumor cells and microglia using IDH mutant and wild-type in vitro models. Clock gene expression is a potential prognostic biomarker in glioma and further studies to elucidate the importance of circadian rhythms in other cell types beyond the tumor are warranted.

Comparison of expression patterns of six canonical clock genes of follicular phase and luteal phase in Small-tailed Han sheep

The circadian rhythm is a biological rhythm that is closely related to the rhythmic expression of a series of clock genes. Results from several studies have indicated that clock genes are associated with the estrous cycle in female animals. Until now, the relationship between estrus cycle transition and clock gene expression in reproductive-axis-related tissues has remained unknown in Small-tailed Han (STH) sheep. This study was conducted to analyze the expression patterns of six canonical clock genes (Clock, BMAL1, Per1, Per2, Cry1, and Cry2) in the follicle phase and luteal phase of STH sheep. We found that all six genes were expressed in the brain, cerebellum, hypothalamus, pituitary, ovary, uterus, and oviduct in follicle and luteal phases. The results indicated that Clock expression was significantly higher in the cerebellum, hypothalamus, and uterus of the luteal phase than that of the follicle phase, whereas BMAL1 expression was significantly higher in the hypothalamus of the luteal phase than that of the follicle phase. Per1 expression was significantly higher in the brain, cerebellum, hypothalamus, and pituitary of the luteal phase than that of the follicle phase, and Per2 expression was significantly higher in the hypothalamus, pituitary, and uterus of the luteal phase than that of the follicle phase. Cry1 expression was significantly higher in the brain, cerebellum, and hypothalamus of the luteal phase than that of the follicle phase, whereas Cry2 expression was significantly higher in the pituitary of the luteal phase than that of the follicle phase. The clock gene expression in all tissues was different between follicle and luteal phases, but all clock gene mRNA levels were found to exhibit higher expression among seven tissues in the luteal phase. Our results suggest that estrous cycles may be associated with clock gene expression in the STH sheep. This is the first study to systematically analyze the expression patterns of clock genes of different estrous cycle in ewes, which could form a basis for further studies to develop the relationship between clock genes and the estrous cycle.

The Circadian Oscillator of the Cerebellum: Triiodothyronine Regulates Clock Gene Expression in Granule Cells in vitro and in the Cerebellum of Neonatal Rats in vivo

The central circadian clock resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, but an SCN-dependent molecular circadian oscillator is present in the cerebellar cortex. Recent findings suggest that circadian release of corticosterone is capable of driving the circadian oscillator of the rat cerebellum. To determine if additional neuroendocrine signals act to shape cerebellar clock gene expression, we here tested the role of the thyroid hormone triiodothyronine (T3) in regulation of the cerebellar circadian oscillator. In cultured cerebellar granule cells from mixed-gender neonatal rats, T3 treatment affected transcript levels of the clock genes Per2, Arntl, Nr1d1, and Dbp, suggesting that T3 acts directly on granule cells to control the circadian oscillator. We then used two different in vivo protocols to test the role of T3 in adult female rats: Firstly, a single injection of T3 did not influence clock gene expression in the cerebellum. Secondly, we established a surgical rat model combining SCN lesion with a programmable micropump infusing circadian physiological levels of T3; however, rhythmic infusion of T3 did not reestablish differential clock gene expression between day and night in SCN lesioned rats. To test if the effects of T3 observed in vitro were related to the developmental stage, acute injections of T3 were performed in mixed-gender neonatal rats in vivo; this procedure significantly affected cerebellar expression of the clock genes Per1, Per2, Nr1d1, and Dbp. Developmental comparisons showed rhythmic expression of all clock genes analyzed in the cerebellum of adult rats only, whereas T3 responsiveness was limited to neonatal animals. Thus, T3 shapes cerebellar clock gene profiles in early postnatal stages, but it does not represent a systemic circadian regulatory mechanism linking the SCN to the cerebellum throughout life.

Circadian neurons in the paraventricular nucleus entrain and sustain daily rhythms in glucocorticoids

Signals from the central circadian pacemaker, the suprachiasmatic nucleus (SCN), must be decoded to generate daily rhythms in hormone release. Here, we hypothesized that the SCN entrains rhythms in the paraventricular nucleus (PVN) to time the daily release of corticosterone. In vivo recording revealed a critical circuit from SCN vasoactive intestinal peptide (SCNVIP)-producing neurons to PVN corticotropin-releasing hormone (PVNCRH)-producing neurons. PVNCRH neurons peak in clock gene expression around midday and in calcium activity about three hours later. Loss of the clock gene Bmal1 in CRH neurons results in arrhythmic PVNCRH calcium activity and dramatically reduces the amplitude and precision of daily corticosterone release. SCNVIP activation reduces (and inactivation increases) corticosterone release and PVNCRH calcium activity, and daily SCNVIP activation entrains PVN clock gene rhythms by inhibiting PVNCRH neurons. We conclude that daily corticosterone release depends on coordinated clock gene and neuronal activity rhythms in both SCNVIP and PVNCRH neurons.

In utero Exposure to Valproic-Acid Alters Circadian Organisation and Clock-Gene Expression: Implications for Autism Spectrum Disorders

Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder characterised by restrictive patterns of behaviour and alterations in social interaction and communication. Up to 80% of children with ASD exhibit sleep-wake cycle disturbances, emphasising the pressing need for novel approaches in the treatment of ASD-associated comorbidities. While sleep disturbances have been identified in ASD individuals, little has been done to assess the contribution of the circadian system to these findings. The objective of this study is to characterise circadian behaviour and clock-gene expression in a valproic acid (VPA)-induced animal model of autism to highlight perturbations potentially contributing to these disturbances. Male and female VPA-exposed offspring underwent circadian challenges, including baseline light-dark cycles, constant dark/light and light pulse protocols. Baseline analysis showed that VPA-exposed males, but not females, had a greater distribution of wheel-running behaviour across light-dark phases and a later activity offset (p < 0.0001), while controls showed greater activity confinement to the dark phase (p = 0.0256). Constant light analysis indicated an attenuated masking response and an increase in the number of days to reach arrhythmicity (p < 0.0001). A 1-h light pulse (150 lux) at CT 15 after 6 days of constant dark showed that both sexes exposed to VPA exhibited a lesser phase-shift when compared to controls (p = 0.0043). Immunohistochemical and western-blot assays reveal no alterations in retinal organisation or function. However, immunohistochemical assay of the SCN revealed altered expression of BMAL1 expression in VPA-exposed males (p = 0.0016), and in females (p = 0.0053). These findings suggest alterations within the core clockwork of the SCN and reduced photic-entrainment capacity, independent of retinal dysfunction. The results of this study shed light on the nature of circadian dysregulation in VPA-exposed animals and highlights the urgent need for novel perspectives in the treatment of ASD-associated comorbidities.

Disrupted Sleep Homeostasis and Altered Expressions of Clock Genes in Rats with Chronic Lead Exposure

Sleep disturbance is one of the neurobehavioral complications of lead neurotoxicity. The present study evaluated the impacts of chronic lead exposure on alteration of the sleep–wake cycle in association with changes of clock gene expression in the hypothalamus. Sprague–Dawley rats with chronic lead exposure consumed drinking water that contained 250 ppm of lead acetate for five weeks. Electroencephalography and electromyography were recorded for scoring the architecture of the sleep–wake cycle in animals. At six Zeitgeber time (ZT) points (ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22), three clock genes, including rPer1, rPer2, and rBmal1b, were analyzed. The rats with chronic lead exposure showed decreased slow wave sleep and increased wakefulness in the whole light period (ZT1 to ZT12) and the early dark period (ZT13 to ZT15) that was followed with a rebound of rapid-eye-movement sleep at the end of the dark period (ZT22 to ZT24). The disturbance of the sleep–wake cycle was associated with changes in clock gene expression that was characterized by the upregulation of rPer1 and rPer2 and the feedback repression of rBmal1b. We concluded that chronic lead exposure has a negative impact on the sleep–wake cycle in rats that predominantly disrupts sleep homeostasis. The disruption of sleep homeostasis was associated with a toxic effect of lead on the clock gene expression in the hypothalamus.