Expression Of Clock-controlled Genes Research Articles

Evidence for a role of human blood-borne factors in mediating age-associated changes in molecular circadian rhythms.

Aging is associated with a number of physiologic changes including perturbed circadian rhythms; however, mechanisms by which rhythms are altered remain unknown. To test the idea that circulating factors mediate age-dependent changes in peripheral rhythms, we compared the ability of human serum from young and old individuals to synchronize circadian rhythms in culture. We collected blood from apparently healthy young (age 25-30) and old (age 70-76) individuals at 14:00 and used the serum to synchronize cultured fibroblasts. We found that young and old sera are equally competent at initiating robust ~24 hr oscillations of a luciferase reporter driven by clock gene promoter. However, cyclic gene expression is affected, such that young and old sera promote cycling of different sets of genes. Genes that lose rhythmicity with old serum entrainment are associated with oxidative phosphorylation and Alzheimer's Disease as identified by STRING and IPA analyses. Conversely, the expression of cycling genes associated with cholesterol biosynthesis increased in the cells entrained with old serum. Genes involved in the cell cycle and transcription/translation remain rhythmic in both conditions. We did not observe a global difference in the distribution of phase between groups, but found that peak expression of several clock-controlled genes (PER3, NR1D1, NR1D2, CRY1, CRY2, and TEF) lagged in the cells synchronized ex vivo with old serum. Taken together, these findings demonstrate that age-dependent blood-borne factors affect circadian rhythms in peripheral cells and have the potential to impact health and disease via maintaining or disrupting rhythms respectively.

Plant Circadian Clocks: Unravelling the Molecular Rhythms of Nature

The circadian clock is a fundamental biological mechanism that allows organisms to synchronize their internal processes with the external environment, thereby optimizing growth, development, and physiology. In plants, circadian rhythms govern various aspects of their life cycle, including germination, leaf movement, flowering, and responses to environmental cues such as light and temperature. Understanding the molecular mechanisms underlying plant circadian clocks is essential not only for elucidating fundamental principles of plant biology but also for applications in agriculture and biotechnology. This review delves into the intricate molecular networks that comprise plant circadian clocks, focusing on key components and their interactions. At the core of these clocks are transcription-translation feedback loops (TTFLs) involving a set of clock genes. The central oscillator, composed of genes such as CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1), and PSEUDO-RESPONSE REGULATOR (PRR) genes, drives rhythmic expression of downstream clock-controlled genes (CCGs). The interplay between positive and negative regulators within the TTFLs generates robust oscillations with a period of approximately 24 hours. Additionally, post-translational modifications, protein-protein interactions, and chromatin remodeling contribute to fine-tuning the circadian system, allowing plants to adapt to changing environmental conditions. Light perception through photoreceptors such as phytochromes and cryptochromes plays a crucial role in entraining the circadian clock to external cues, ensuring synchronization with the day-night cycle. Furthermore, recent advancements in omics technologies, including genomics, transcriptomics, proteomics, and metabolomics, have provided unprecedented insights into the complexity and plasticity of plant circadian clocks. Integration of multi-omics data has facilitated the construction of comprehensive regulatory networks and computational models, enabling predictive understanding of clock function and behavior under diverse conditions. Overall, unraveling the molecular rhythms of plant circadian clocks not only enhances our knowledge of fundamental biological processes but also holds promise for improving crop productivity, stress resilience, and sustainability in agriculture through targeted manipulation of clock components and their associated pathways. Future research endeavors will undoubtedly continue to unveil the intricacies of these fascinating timekeeping mechanisms, further enriching our understanding of the molecular rhythms of nature.

Importance of Circadian Rhythms in the Ocular Surface.

Circadian rhythms are a ubiquitous feature throughout the organism. Accumulating evidence suggests that the dysfunction of circadian rhythms due to genetic mutations or environmental factors contributes to the genesis and progress of multiple diseases. The physiological homeostasis of the ocular surface, like any other tissue or organ, is also orchestrated by circadian rhythms. In this review, we summarize the molecular clocks and the expression of clock-controlled genes in the mammalian ocular surface. Based on the circadian expression of these genes, we conclude the diurnal oscillations of cellular biological activities in the mammalian ocular surface. Moreover, we evaluate the factors entraining circadian oscillators in the ocular surface. Finally, we further discuss the latest development of the close correlation between circadian rhythms and ocular health. Briefly, this review aimed to synthesize the previous studies to aid in understanding the importance of circadian rhythms in the ocular surface and the possible opportunities for circadian rhythm-based interventional strategies to restore the homeostasis of the ocular surface.

Circadian control of histone turnover during cardiac development and growth

During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting a role for the circadian clock in temporal control of histone turnover and coordinated cardiomyocyte gene expression. To elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Bmal1 knockdown in neonatal rat ventricular myocytes (NRVM) decreased myocyte size, total cellular protein synthesis, and transcription of the fetal hypertrophic gene Nppb following treatment with serum or the α-adrenergic agonist phenylephrine (PE). Depletion of Bmal1 decreased expression of clock-controlled genes Per2 and Tcap, as well as Sik1, a Bmal1 target upregulated in adult versus embryonic hearts. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by MNase-qPCR and impaired histone turnover as measured by metabolic labeling of acid-soluble chromatin fractions. Sik1 knockdown in turn decreased myocyte size, while simultaneously inhibiting Nppb transcription and activating Per2 transcription. Linking these changes to chromatin remodeling, depletion of the replication-independent histone variant H3.3a inhibited myocyte hypertrophy and prevented PE-induced changes in clock-controlled gene transcription. Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription. Replication-independent histone turnover is required for transcriptional remodeling of clock-controlled genes in cardiac myocytes in response to growth stimuli.

Sex and age-dependent characterization of the circadian clock as a potential biomarker for physical performance: A prospective study protocol.

Circadian rhythms (CR) regulate daily cycles in behavior, physiology and molecular processes. CRs are endogenous and vary across individuals. Seasonal changes can influence CR. Accordingly, rhythms with different characteristics (amplitude, phase) are depicted during the summer months, as compared to winter. Increasing evidence points to an influence of circadian regulation on physical performance. Here, we aim to obtain a comprehensive circadian gene expression profile for physically active individuals, which can potentially be used for the identification of optimal time intervals for physical exercise. To explore these different aspects, we propose a study where we will carry out a molecular analysis of CR by measuring the expression of specific clock and clock-controlled genes, based on a non-invasive approach using RNA extracted from saliva in physically active, healthy participants. We will collect data across two seasons and use computational algorithms to integrate the molecular data with hormonal data (cortisol and melatonin), and generate a profile of CR in healthy individuals of different sex and age groups. Finally, we will use computational tools to predict optimal time intervals for physical performance based on the above-described data, thereby retrieving valuable data on the circadian clock as a key factor for health maintenance and optimization.

Effect of miR-34a on the expression of clock and clock-controlled genes in DLD1 and Lovo human cancer cells with different backgrounds with respect to p53 functionality and 17β-estradiol-mediated regulation.

The small non-coding RNA miR-34a is a p53-regulated miRNA that acts as a tumour suppressor of colorectal cancer (CRC). Oncogenesis is also negatively influenced by deregulation of the circadian system in many types of tumours with various genetic backgrounds. As the clock gene per2 was recently recognized as one of the target genes of miR-34a, we focused on the miR-34a-mediated influence on the circadian oscillator in CRC cell lines DLD1 and LoVo, which differ in their p53 status. Previously, a sex-dependent association between the expression of per2 and that of miR-34a was demonstrated in CRC patients. Therefore, we also investigated the effect of 17β-estradiol (E2) on miR-34a oncostatic functions. miR-34a mimic caused a pronounced inhibition of per2 expression in both cell lines. Moreover, miR-34a mimic significantly inhibited bmal1 expression in LoVo and rev-erbα expression in DLD1 cells and induced clock gene expression in both cell lines. miR-34a mimic caused a pronounced decrease in sirt1 and cyclin D1 expression, which may be related to the inhibition of proliferation observed after mir-34a administration in DLD1 cells. E2 administration inhibited the migration and proliferation of DLD1 cells. E2 and miR-34a, when administered simultaneously, did not potentiate each other's effects. To conclude, miR-34a strongly influences the expression of components of the circadian oscillator without respect to p53 status and exerts its oncostatic effects via inhibition of sirt1 and cyclin D1 mRNA expression. E2 administration inhibits the growth of DLD1 cells; however, this effect seems to be independent of miR-34a-mediated action. With respect to the possible use of miR-34a in cancer treatment, clock genes can be considered as off-target genes, as changes in their expression induced by miR-34a treatment do not contribute to the oncostatic functions of miR-34a. Possible ambiguous oncogenic characteristics should be taken into consideration in future clinical studies focused on miR-34a.

The Circadian Nobiletin-ROR Axis Suppresses Adipogenic Differentiation and IκBα/NF-κB Signaling in Adipocytes.

Obesity is a known risk factor for metabolic diseases and is often associated with chronic inflammation in adipose tissue. We previously identified the polyethoxylated flavonoid Nobiletin (NOB) as a circadian clock modulator that directly binds to and activates the ROR receptors in the core oscillator, markedly improving metabolic fitness in obese mice. Here, we show that NOB enhanced the oscillation of core clock genes in differentiated 3T3-L1 adipocytes, including ROR target genes such as Bmal1, Cry1, Dec1, and Dec2. NOB inhibited lipid accumulation in 3T3-L1 and SVF cells, concomitant with the dysregulated circadian expression of adipogenic differentiation-related genes including Cebpb, Pparg, Lpl, Scd1, and Fas. Importantly, RORα/RORγ double knockdown in 3T3-L1 cells (Ror DKD) significantly attenuated the effects of NOB on circadian gene expression and lipid accumulation. Furthermore, whereas NOB upregulated the expression of IκBα, a target of RORs, to inhibit NF-κB activation and proinflammatory cytokine expression, Ror DKD cells exhibited a heightened activation of the NF-κB pathway, further indicating a requisite role of RORs for NOB efficacy in adipocytes. Together, these results highlight a significant regulatory function of the NOB-ROR axis in the circadian expression of clock and clock-controlled genes in adipocytes, thereby governing adipogenic differentiation, lipogenesis, and inflammation.

Abstract A027: Defining the role of PER1 and circadian rhythm dysregulation in KRAS/LKB1-mutant lung adenocarcinoma

Abstract KRAS and LKB1 are frequently co-mutated in lung adenocarcinoma and characterize a particularly aggressive, metastatic, and treatment-resistant subtype of this disease. We recently published that the outputs of biological rhythm pathways downstream of the AMPK kinase are altered in KRAS/LKB1-mutant tumors. In a live-cell phenotyping screen we used RNAseq to identify the circadian clock genes PER1 and PER2 as uniquely upregulated in partially transformed and invasive KRAS/LKB1-mutant human bronchial epithelial cells. We have analyzed expression of individual circadian clock genes in KRAS-mutant and KRAS/LKB1-mutant patient tumors and found that expression of the PER genes, particularly PER1, is increased in the KRAS/LKB1 subtype of lung adenocarcinoma. At the protein level, PER1 expression is increased in KRAS/LKB1-mutant HBECs as compared to KRAS-mutant cells. Addback of wild-type LKB1 in A549 cells, which are KRAS/LKB1-mutant, results in a decrease in PER1 protein levels, confirming that LKB1 has a role in controlling PER1 levels. Multiple phosphorylation events lead to PER1 nuclear translocation to control its role as a transcriptional repressor in the core clock. In circadian-synced HBECs, PER1 enters the nucleus in KRAS-mutant cells, but remains cytosolic in KRAS/LKB1-mutant cells. In circadian-synced A549 cells, we also observed a lack of nuclear PER1 localization. Addback of wild-type LKB1 restored nuclear localization of PER1, as did expression of kinase-dead LKB1, suggesting that LKB1’s regulation of PER1 localization is kinase-independent. Our current and future experiments aim to determine how clock-controlled gene expression may be altered in KRAS/LKB1-mutant cells, what the functional impact of altered PER1 expression and localization is on growth of KRAS/LKB1-mutant tumors, and whether pharmacological manipulation of LKB1-related signaling pathways can restore nuclear localization of PER1. Citation Format: Rebecca E. Parker, Junghui Koo, Bhakti Dwivedi, Adam I. Marcus, Melissa Gilbert-Ross. Defining the role of PER1 and circadian rhythm dysregulation in KRAS/LKB1-mutant lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A027.

Senescence-induced alteration of circadian phagocytic activity of retinal pigment epithelium cell line ARPE-19

Renewal of retinal photoreceptor outer segments is conducted through daily shedding of distal photoreceptor outer segment tips and subsequent their phagocytosis by the adjacent retinal pigment epithelium (RPE) monolayer. Dysregulation of the diurnal clearance of photoreceptor outer segment tips has been implicated in age-related retinal degeneration, but it remains to be clarified how the circadian phagocytic activity of RPE cells is modulated by senescence. In this study, we used the human RPE cell line ARPE-19 to investigate whether hydrogen peroxide (H2O2)-induced senescence in ARPE-19 cells alters the circadian rhythm of their phagocytic activity. After synchronization of the cellular circadian clock by dexamethasone treatment, the phagocytic activity of normal ARPE-19 cells exhibited significant 24-h oscillation, but this oscillation was modulated by senescence. The phagocytic activity of senescent ARPE-19 cells increased constantly throughout the 24-h period, which still exhibited blunted circadian oscillation, accompanied by an alteration in the rhythmic expression of circadian clock genes and clock-controlled phagocytosis-related genes. The expression levels of REV-ERBα, a molecular component of the circadian clock, were constitutively increased in senescent ARPE-19 cells. Furthermore, pharmacological activation of REV-ERBα by its agonist SR9009 enhanced the phagocytic activity of normal ARPE-19 cells and increased the expression of clock-controlled phagocytosis-related genes. Our present findings extend to understand the role of circadian clock in the alteration of phagocytic activity in RPE during aging. Constitutive enhancement of phagocytic activity of senescent RPE may contribute to age-related retinal degeneration.

Cell-specific clock-controlled gene expression program regulates rhythmic fiber cell growth in cotton

BackgroundThe epidermis of cotton ovule produces fibers, the most important natural cellulose source for the global textile industry. However, the molecular mechanism of fiber cell growth is still poorly understood.ResultsHere, we develop an optimized protoplasting method, and integrate single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) to systematically characterize the cells of the outer integument of ovules from wild type and fuzzless/lintless (fl) cotton (Gossypium hirsutum). By jointly analyzing the scRNA-seq data from wildtype and fl, we identify five cell populations including the fiber cell type and construct the development trajectory for fiber lineage cells. Interestingly, by time-course diurnal transcriptomic analysis, we demonstrate that the primary growth of fiber cells is a highly regulated circadian rhythmic process. Moreover, we identify a small peptide GhRALF1 that circadian rhythmically controls fiber growth possibly through oscillating auxin signaling and proton pump activity in the plasma membrane. Combining with scATAC-seq, we further identify two cardinal cis-regulatory elements (CREs, TCP motif, and TCP-like motif) which are bound by the trans factors GhTCP14s to modulate the circadian rhythmic metabolism of mitochondria and protein translation through regulating approximately one third of genes that are highly expressed in fiber cells.ConclusionsWe uncover a fiber-specific circadian clock-controlled gene expression program in regulating fiber growth. This study unprecedentedly reveals a new route to improve fiber traits by engineering the circadian clock of fiber cells.

CORRELATION BETWEEN PERIODONTAL HEALTH INDICES AND PECULIARITIES OF CIRCADIAN RHYTHM IN BUCCAL EPITHELIAL CELLS

There is a complex system of biological rhythms arranging the coordinationion of physiological functions and the environmental rhythms. The leading place it taken by the circadian rhythm, a 24-hour cycle that is a part of the body's internal clock. Circadian regulation of normal physiological and metabolic processes is carried out by the fluctuations in the expression of genes (clock-controlled genes (CCGs)) and the proteins they encode, which differ in each individual organ or tissue. The circadian rhythm of gene expression was detected in the basal cells of the oral epithelium, in the cells of ameloblasts and odontoblasts, cells of the dental pulp, cells of the periodontal ligament, osteoblasts and osteoclasts of the alveolar bone. However, so far little is known about the dependence of clock controlled gene expression on chronotype in buccal epithelial cells.
 The purpose of the study is to determine the expression level of the clock controlled genes Per1, Cry1, Bmal1 and Clock in the buccal epithelial cells of healthy people with different chronotype.
 Materials and methods. In order to achieve the goal we surveyed 14 people (7 men (50%) and 7 women (50%) aged from 36 to 45 years with a healthy periodontium without any somatic pathology or with compensated one. All participants signed an informed consent forms. The chronotype of the participants was determined by the Horn-Ostberg test modified by S.I. Stepanova and determined the mRNA expression of Clock, Bmal1, Per1 and Cry1 in buccal epithelium samples at 7:00 am and 7:00 pm in one day in the autumn-winter period.
 Results. The Per1 is better expressed early in the morning than in the evening, regardless of the chronotype (р1<0.05). The level of Clock expression is higher in the evening in both groups (р1<0.05). The difference between the indicators of the evening expression of the Clock in the two groups is statistically significant (р2<0.05). Bmal1 expression is higher in the evening, regardless of chronotype (р1<0.05). The Cry1 shows the same evening predominance among participants of both chronotypes, but it is not statistically significant.
 Conclusion. The study has revealed the correlation between chronotype and the evening expression of the Clock that is higher in people with the evening chronotype. Belonging to the morning or evening chronotype did not reveal any effect on the expression level of Per1, Bmal1 and Cry1.

Abstract 13596: Effects of Plasminogen Activator Inhibitor-1 on the Circadian Clock in Liver

Introduction: Plasminogen activator inhibitor-1 (PAI-1, encoded by Serpine1 ) plays a major role in the mammalian fibrinolytic system, but elevated PAI-1 activity contributes to other biological processes including the metabolic syndrome. Multiple studies suggest that disruption of circadian rhythms also promotes features of the metabolic syndrome including obesity, hypertension, and type 2 diabetes. While it has been long known that PAI-1 plasma levels exhibit a marked circadian variation, and that the expression of PAI-1 is strongly influenced by the clock, a reciprocal role for PAI-1 in regulating components of the clock itself has never been described. Hypothesis: We hypothesized that changes in PAI-1 activity influence the expression of core circadian clock genes and clock-controlled genes in the liver. Methods: We used two mouse models of reduced PAI-1 activity: oral PAI-1 inhibitor TM5614 (20 mg/kg/day) versus vehicle in C57BL/6J wild-type mice (n = 5 per group), and Serpine +/- heterozygous mice versus wild-type littermate control mice (n = 3 per group). Mice were maintained on a 14:10 light:dark cycle. Livers were collected between 3 and 4 hours after lights-on, which corresponds to Zeitgeber Time (ZT) 3-4. RNA was isolated for RNA-seq and downstream analyses. Results: Between ZT 3-4, we found that reduced PAI-1 activity led to marked changes in circadian gene expression in the liver. Both PAI-1 inhibition with TM5614 and PAI-1 haploinsufficiency significantly increased expression of the core circadian clock components Arntl (aka Bmal1) and Clock while significantly reducing expression of core clock components Per1, Per2, Per3, and clock output genes. Furthermore, we found that PAI-1 inhibition in aged transgenic mice overexpressing the stable variant of human SERPINE1 rescued two markers of impaired circadian function: 1) the premature onset of activity prior to lights-off, and 2) reduced feeding during the lights-off period. Conclusions: Taken together, these data support a reciprocal role for PAI-1 in regulating components of the circadian clock. Elevated PAI-1 activity may contribute to the metabolic syndrome through effects on the circadian clock and may provide a mechanistic link between PAI-1, the circadian clock, and aging.

The Clock‐NAD+‐Sirtuin connection in nonalcoholic fatty liver disease

Nonalcoholic or metabolic associated fatty liver disease (NAFLD/MAFLD) is a hepatic reflection of metabolic derangements characterized by excess fat deposition in the hepatocytes. Identifying metabolic regulatory nodes in fatty liver pathology is essential for effective drug targeting. Fatty liver is often associated with circadian rhythm disturbances accompanied with alterations in physical and feeding activities. In this regard, both sirtuins and clock machinery genes have emerged as critical metabolic regulators in maintaining liver homeostasis. Knockouts of either sirtuins or clock genes result in obesity associated with the fatty liver phenotype. Sirtuins (SIRT1-SIRT7) are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, protecting cells from metabolic stress by deacetylating vital proteins associated with lipid metabolism. Circadian rhythm is orchestrated by oscillations in expression of master regulators (BMAL1 and CLOCK), which in turn regulate rhythmic expression of clock-controlled genes involved in lipid metabolism. The circadian metabolite, NAD+ , serves as a crucial link connecting clock genes to sirtuin activity. This is because, NAMPT which is a rate limiting enzyme in NAD+ biosynthesis is transcriptionally regulated by the clock genes and NAD+ in turn is a cofactor regulating the deacetylation activity of sirtuins. Intriguingly, on one hand the core circadian clock regulates the sirtuin activity and on the other hand the activated sirtuins regulate the acetylation status of clock proteins thereby affecting their transcriptional functions. Thus, the Clock-NAD+-Sirtuin connection represents a novel "feedback loop" circuit that regulates the metabolic machinery. The current review underpins the importance of NAD+ on the sirtuin and clock connection in preventing fatty liver disorder.

Organoids as Model Systems to Investigate Circadian Clock-Related Diseases and Treatments.

Circadian rhythms exist in most cell types in mammals regulating temporal organization of numerous cellular and physiological processes ranging from cell cycle to metabolism. The master clock, suprachiasmatic nucleus (SCN) in the hypothalamus, processes light input and coordinates peripheral clocks optimizing organisms’ survival and functions aligning with external conditions. Intriguingly, it was demonstrated that circadian rhythms in the mouse liver can be decoupled from the master clock under time-restricted feeding regimen when food was provided during their inactive phase. Furthermore, mouse liver showed clock-controlled gene expression even in the absence of the master clock demonstrating independent functions of peripheral clocks apart from the SCN. These findings suggest a dynamic relationship between the master and peripheral clocks and highlight potential functions of peripheral clocks independent of the master clock. Importantly, disruption of circadian rhythms correlates with numerous human ailments including cancer and metabolic diseases, suggesting that diseases may be exacerbated by disruption of circadian rhythms in the SCN and/or peripheral clocks. However, molecular mechanisms providing causative links between circadian rhythms and human diseases remain largely unknown. Recent technical advances highlighted PCS- and tissue-derived 3-dimensional organoids as in vitro organs that possess numerous applications ranging from disease modeling to drug screening. In this mini-review, we highlight recent findings on the importance and contributions of peripheral clocks and potential uses of 3D organoids investigating complex circadian clock-related diseases.

Transcriptional Feedback Loops in the Caprine Circadian Clock System.

The circadian clock system is based on interlocked positive and negative transcriptional and translational feedback loops of core clock genes and their encoded proteins. The mammalian circadian clock system has been extensively investigated using mouse models, but has been poorly investigated in diurnal ruminants. In this study, goat embryonic fibroblasts (GEFs) were isolated and used as a cell model to elucidate the caprine circadian clock system. Real-time quantitative PCR analysis showed that several clock genes and clock-controlled genes were rhythmically expressed in GEFs over a 24 h period after dexamethasone stimulation. Immunofluorescence revealed that gBMAL1 and gNR1D1 proteins were expressed in GEFs, and western blotting analysis further verified that the proteins were expressed with circadian rhythmic changes. Diurnal changes in clock and clock-controlled gene expression at the mRNA and protein levels were also observed in goat liver and kidney tissues at two representative time points in vivo. Amino acid sequences and tertiary structures of goat BMAL1 and CLOCK proteins were found to be highly homologous to those in mice and humans. In addition, a set of goat representative clock gene orthologs and the promoter regions of two clock genes of goats and mice were cloned. Dual-luciferase reporter assays showed that gRORα could activate the promoter activity of the goat BMAL1, while gNR1D1 repressed it. The elevated pGL4.10-gNR1D1-Promoter-driven luciferase activity induced by mBMAL1/mCLOCK was much higher than that induced by gBMAL1/gCLOCK, and the addition of gCRY2 or mPER2 repressed it. Real-time bioluminescence assays revealed that the transcriptional activity of BMAL1 and NR1D1 in goats and mice exhibited rhythmic changes over a period of approximately 24 h in NIH3T3 cells or GEFs. Notably, the amplitudes of gBMAL1 and gNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells were higher than those in GEFs, while mBMAL1 and mNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells had the highest amplitude. In sum, transcriptional and translational loops of the mammalian circadian clock system were found to be broadly conserved in goats and not as robust as those found in mice, at least in the current experimental models. Further studies are warranted to elucidate the specific molecular mechanisms involved.

NR1D1 downregulation in astrocytes induces a phenotype that is detrimental to cocultured motor neurons.

Nuclear receptor subfamily 1 group D member 1 (NR1D1, also known as Rev‐erbα) is a nuclear transcription factor that is part of the molecular clock encoding circadian rhythms and may link daily rhythms with metabolism and inflammation. NR1D1, unlike most nuclear receptors, lacks a ligand‐dependent activation function domain 2 and is a constitutive transcriptional repressor. Amyotrophic lateral sclerosis (ALS) is the most common adult‐onset motor neuron disease, caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Approximately 10%–20% of familial ALS is caused by a toxic gain‐of‐function induced by mutations of the Cu/Zn superoxide dismutase (SOD1). Dysregulated clock and clock‐controlled gene expression occur in multiple tissues from mutant hSOD1‐linked ALS mouse models. Here we explore NR1D1 dysregulation in the spinal cord of ALS mouse models and its consequences on astrocyte–motor neuron interaction. NR1D1 protein and mRNA expression are significantly downregulated in the spinal cord of symptomatic mice expressing mutant hSOD1, while no changes were observed in age‐matched animals overexpressing wild‐type hSOD1. In addition, NR1D1 downregulation in primary astrocyte cultures induces a pro‐inflammatory phenotype and decreases the survival of cocultured motor neurons. NR1D1 orchestrates the cross talk between physiological pathways identified to be disrupted in ALS (e.g., metabolism, inflammation, redox homeostasis, and circadian rhythms) and we observed that downregulation of NR1D1 alters astrocyte–motor neuron interaction. Our results suggest that NR1D1 could be a potential therapeutic target to prevent astrocyte‐mediated motor neuron toxicity in ALS.

Loss of circadian rhythmicity in bdnf knockout zebrafish larvae

SummaryBrain-derived neurotrophic factor (BDNF) plays a pivotal role in neuronal growth and differentiation, neuronal plasticity, learning, and memory. Using CRISPR/Cas9 technology, we generated a vital Bdnf null mutant line in zebrafish and carried out its molecular and behavioral characterization. Although no defects are evident on a morphological inspection, 66% of coding genes and 37% of microRNAs turned out to be differentially expressed in bdnf−/− compared with wild type sibling embryos. We deeply investigated the circadian clock pathway and confirmed changes in the rhythmic expression of clock (arntl1a, clock1a and clock2) and clock-controlled (aanat2) genes. The modulatory role of Bdnf on the zebrafish circadian clock was then validated by behavioral tests highlighting the absence of circadian activity rhythms in bdnf−/− larvae. The circadian behavior was partially rescued by pharmacological treatment. The bdnf−/− zebrafish line presented here is the first valuable and stable vertebrate model for the study of BDNF-related neurodevelopmental diseases

The clock modulator Nobiletin mitigates astrogliosis-associated neuroinflammation and disease hallmarks in an Alzheimer's disease model.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, and there is a pressing need to identify disease‐modifying factors and devise interventional strategies. The circadian clock, our intrinsic biological timer, orchestrates various cellular and physiological processes including gene expression, sleep, and neuroinflammation; conversely, circadian dysfunctions are closely associated with and/or contribute to AD hallmarks. We previously reported that the natural compound Nobiletin (NOB) is a clock‐enhancing modulator that promotes physiological health and healthy aging. In the current study, we treated the double transgenic AD model mice, APP/PS1, with NOB‐containing diets. NOB significantly alleviated β‐amyloid burden in both the hippocampus and the cortex, and exhibited a trend to improve cognitive function in these mice. While several systemic parameters for circadian wheel‐running activity, sleep, and metabolism were unchanged, NOB treatment showed a marked effect on the expression of clock and clock‐controlled AD gene expression in the cortex. In accordance, cortical proteomic profiling demonstrated circadian time‐dependent restoration of the protein landscape in APP/PS1 mice treated with NOB. More importantly, we found a potent efficacy of NOB to inhibit proinflammatory cytokine gene expression and inflammasome formation in the cortex, and immunostaining further revealed a specific effect to diminish astrogliosis, but not microgliosis, by NOB in APP/PS1 mice. Together, these results underscore beneficial effects of a clock modulator to mitigate pathological and cognitive hallmarks of AD, and suggest a possible mechanism via suppressing astrogliosis‐associated neuroinflammation.

Evaluation of stable reference genes for qPCR normalization in circadian studies related to lung inflammation and injury in mouse model

Circadian rhythms have a profound effect on lung function and immune-inflammatory response in chronic airway diseases. Thus, understanding the molecular mechanisms of circadian gene expression of core clock-controlled genes (CCGs) may help better understand how it contributes to the physiology and pathology of lung diseases. Ongoing studies have been analyzing gene expression levels of CCGs in mouse lungs using quantitative real-time PCR (qRT-PCR). However, to date, there are no reports on the most stable reference gene in the mouse lung for circadian studies. Herein, we utilized an acute house dust mite (HDM)-sensitization mouse model to evaluate the stability of 10 reference genes commonly used for qRT-PCR normalization using 5 unique algorithms: GeNorm, NormFinder, BestKeeper, RefFinder and Qbase+. Rn18s was determined as the most stable reference gene across all samples evaluated, and Actb, the least stable reference gene. Furthermore, CircWave analysis showed no diurnal variation in the expression pattern for Rn18s but Actb showed strong diurnal changes in the lungs of both PBS (control) and HDM groups. We demonstrate systematically how using Actb as a housekeeping gene offsets the diurnal expression patterns of the CCGs and leads to statistically significant results which may not be the true reflection of the qRT-PCR analysis.

A time-restricted feeding intervention reduces alterations in circadian behaviors and pathology in a mouse model of Alzheimer's disease.

Circadian rhythm disruptions impact nearly all Alzheimer's disease (AD) patients, with daytime sleepiness, sundowning, sleep fragmentation, and night-time insomnia being common disturbances. These early and progressive declines support the role of circadian dysfunction as a driver of AD pathology, hence the therapeutic potential of circadian-modulating interventions needs to be investigated. One of the most powerful regulators of the circadian system is the daily feed/fast cycle. We have recently shown that early, presymptomatic time-restricted feeding (TRF) can ameliorate a range of symptoms related to neurodegenerative conditions, including improving sleep/wake timing and circadian rhythmicity. At 12 months of age, the APP23 mouse model of AD (expressing the human APP gene containing the Swedish mutation) exhibits disrupted behavior resembling sundowning. We hypothesized that imposing early-age TRF in APP23 mice would improve circadian rhythmicity and deter the disruptions observed to emerge and progress with advancing neuropathology. Adult male and female APP23 transgenic (AD) and littermate non-transgenic (control) mice were housed individually and subjected to ab libitum (ALF) feeding or a TRF protocol consisting of 6-hours feeding in the middle of the active phase followed by 18-hours fasting for 3 months. Behavioral sleep (48 hours) and ongoing cage locomotor activity were recorded from an early age. Our analysis shows that AD mice exhibit disruptions in total sleep (P < 0.001) and the emergence of excessive wakefulness (P < 0.001). AD mice also show significant active-phase agitation (P < 0.05) accompanied by excessive sleep-phase fragmentation (P < 0.05). TRF induced nutritional ketosis (β-Hydroxybutyrate > 0.5 mM) accompanied by reduced blood glucose when compared to ALF mice, although mice consumed equivalent volumes of food in both experimental groups. Importantly, TRF improved total and sleep-phase sleep in AD mice (P < 0.05) and significantly reduced agitation and fragmentation (P < 0.05). Early application of TRF can forestall circadian disruptions in AD mice. TRF has been reported to modulate expression of clock and clock-controlled genes, transcriptional regulators, metabolism related genes, BDNF, and other neurotrophic factors. Our results suggest that these changes may also impact sleep, circadian behaviors, and agitation associated with AD.