Implications of biological clocks in pharmacology and pharmacokinetics of antitumor drugs.

Mammalians' circadian pacemaker resides in the paired suprachiasmatic nuclei (SCN). SCN control biological rhythms such as the sleep-wake rhythm and homeostatic functions of steroid hormones and their receptors. Alterations in these biological rhythms are implicated in the outcomes of pathogenic conditions such as depression, diabetes, and cancer. Chronotherapy is about optimizing treatment to combat risks and intensity of the disease symptoms that vary depending on the time of day. Thus, conditions/diseases such as allergic rhinitis, arthritis, asthma, myocardial infarction, congestive heart failure, stroke, and peptic ulcer disease, prone to manifest severe symptoms depending on the time of day, would be benefited from chronotherapy. Monitoring rhythm, overcoming rhythm disruption, and manipulating the rhythms from the viewpoints of underlying molecular clocks are essential to enhanced chronopharmacotherapy. New drugs focused on molecular clocks are being developed to improve therapeutics. In this review, we provide a critical summary of literature reports concerning (a) the rationale/mechanisms for time-dependent dosing differences in therapeutic outcomes and safety of antitumor drugs, (b) the molecular pathways underlying biological rhythms, and (c) the possibility of pharmacotherapy based on the intra- and inter-individual variabilities from the viewpoints of the clock genes.

Nuclear Receptors and Clock Components in Cardiovascular Diseases.

Cardiovascular diseases (CVD) are still the first cause of death worldwide. Their main origin is the development of atherosclerotic plaque, which consists in the accumulation of lipids and inflammatory leucocytes within the vascular wall of large vessels. Beyond dyslipidemia, diabetes, obesity, hypertension and smoking, the alteration of circadian rhythms, in shift workers for instance, has recently been recognized as an additional risk factor. Accordingly, targeting a pro-atherogenic pathway at the right time window, namely chronotherapy, has proven its efficiency in reducing plaque progression without affecting healthy tissues in mice, thus providing the rationale of such an approach to treat CVD and to reduce drug side effects. Nuclear receptors are transcriptional factors involved in the control of many physiological processes. Among them, Rev-erbs and RORs control metabolic homeostasis, inflammatory processes and the biological clock. In this review, we discuss the opportunity to dampen atherosclerosis progression by targeting such ligand-activated core clock components in a (chrono-)therapeutic approach in order to treat CVD.

Decreased expression of the clock gene Bmal1 is involved in the pathogenesis of temporal lobe epilepsy.

Clock genes not only regulate the circadian rhythm of physiological activities but also participate in the pathogenesis of many diseases. Previous studies have documented the abnormal expression of clock genes in epilepsy. However, the molecular mechanism of brain and muscle Arnt-like protein 1 (Bmal1), one of the core clock genes, in the epileptogenesis and seizures of temporal lobe epilepsy (TLE) remain unclear. We first investigated the levels of Bmal1 and other clock proteins in the hippocampus of subjects with epilepsy to define the function of Bmal1. The levels of Bmal1 were decreased during the latent and chronic phases in the experimental group compared with those in the control group. Knockout of Bmal1 in hippocampal dentate gyrus (DG) neurons of Bmal1flox/flox mice by Synapsin 1 (Syn1) promoter AAV (adeno-associated virus) lowered the threshold of seizures induced by pilocarpine administration. High-throughput sequencing analysis showed that PCDH19 (protocadherin 19), a gene associated with epilepsy, was regulated by Bmal1. PCDH19 expression was also decreased in the hippocampus of epileptic mice. Furthermore, the higher levels of Bmal1 and PCDH19 were detected in patients with no hippocampal sclerosis (no HS) than in patients with HS International League Against Epilepsy (ILAE) type I and III. Altogether, these data suggest that decreased expression of clock gene Bmal1 may participate in epileptogenesis and seizures via PCDH19 in TLE.

Long-term continuous positive airway pressure treatment ameliorates biological clock disruptions in obstructive sleep apnea.

BACKGROUND: Obstructive Sleep Apnea (OSA) is a highly prevalent and underdiagnosed sleep disorder. Recent studies suggest that OSA might disrupt the biological clock, potentially causing or worsening OSA-associated comorbidities. However, the effect of OSA treatment on clock disruption is not fully understood. METHODS: The impact of OSA and short- (four months) and long-term (two years) OSA treatment, with Continuous Positive Airway Pressure (CPAP), on the biological clock was investigated at four time points within 24 h, in OSA patients relative to controls subjects (no OSA) of the same sex and age group, in a case-control study. Plasma melatonin and cortisol, body temperature and the expression levels and rhythmicity of eleven clock genes in peripheral blood mononuclear cells (PBMCs) were assessed. Additional computational tools were used for a detailed data analysis. FINDINGS: OSA impacts on clock outputs and on the expression of several clock genes in PBMCs. Neither short- nor long-term treatment fully reverted OSA-induced alterations in the expression of clock genes. However, long-term treatment was able to re-establish levels of plasma melatonin and cortisol and body temperature. Machine learning methods could discriminate controls from untreated OSA patients. Following long-term treatment, the distinction between controls and patients disappeared, suggesting a closer similarity of the phenotypes. INTERPRETATION: OSA alters biological clock-related characteristics that differentially respond to short- and long-term CPAP treatment. Long-term CPAP was more efficient in counteracting OSA impact on the clock, but the obtained results suggest that it is not fully effective. A better understanding of the impact of OSA and OSA treatment on the clock may open new avenues to OSA diagnosis, monitoring and treatment.

Circadian control of hepatitis B virus replication.

Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide for which there are no curative therapies. The major challenge in curing infection is eradicating or silencing the covalent closed circular DNA (cccDNA) form of the viral genome. The circadian factors BMAL1/CLOCK and REV-ERB are master regulators of the liver transcriptome and yet their role in HBV replication is unknown. We establish a circadian cycling liver cell-model and demonstrate that REV-ERB directly regulates NTCP-dependent hepatitis B and delta virus particle entry. Importantly, we show that pharmacological activation of REV-ERB inhibits HBV infection in vitro and in human liver chimeric mice. We uncover a role for BMAL1 to bind HBV genomes and increase viral promoter activity. Pharmacological inhibition of BMAL1 through REV-ERB ligands reduces pre-genomic RNA and de novo particle secretion. The presence of conserved E-box motifs among members of the Hepadnaviridae family highlight an evolutionarily conserved role for BMAL1 in regulating this family of small DNA viruses.

Associations of Age, Sex, Race/Ethnicity, and Education With 13 Epigenetic Clocks in a Nationally Representative U.S. Sample: The Health and Retirement Study.

BACKGROUND: Many DNA methylation-based indicators have been developed as summary measures of epigenetic aging. We examine the associations between 13 epigenetic clocks, including 4 second generation clocks, as well as the links of the clocks to social, demographic, and behavioral factors known to be related to health outcomes: sex, race/ethnicity, socioeconomic status, obesity, and lifetime smoking pack-years. METHODS: The Health and Retirement Study is the data source which is a nationally representative sample of Americans over age 50. Assessment of DNA methylation was based on the EPIC chip and epigenetic clocks were developed based on existing literature. RESULTS: The clocks vary in the strength of their relationships with age, with each other and with independent variables. Second generation clocks trained on health-related characteristics tend to relate more strongly to the sociodemographic and health behaviors known to be associated with health outcomes in this age group. CONCLUSIONS: Users of this publicly available data set should be aware that epigenetic clocks vary in their relationships to age and to variables known to be related to the process of health change with age.

[Common cause and mechanism for all pathologies of aging?] / Cause commune et mécanisme commun aux maladies du vieillissement ?

Health is harmony, aging and its diseases (are) functional disharmony at the molecular, cellular and tissue levels. Our observations lead us to think that there seems to be a common cause and a common mechanism for aging and its many and diverse diseases. This common cause is the oxidative damage to particular proteins emerging from a combination of imperfect folding and oxidative stress. This common cause jointly goes with the biological clock common to various age-related diseases, whose the incidence increases exponentially over time and causes 90% of human mortality. Pharmacological interventions on the common cause could avoid and simultaneously attenuate all degenerative and malignant diseases, as it is the natural case of super-centenarians.

TITLE: Cause commune et mécanisme commun aux maladies du vieillissement ? ABSTRACT: La santé est l'harmonie, le vieillissement et ses maladies la dysharmonie fonctionnelle aux niveaux moléculaire, cellulaire et tissulaire. Nos observations semblent suggérer une cause commune et un mécanisme commun du vieillissement et de ses nombreuses et diverses maladies. Cette cause commune est le dommage oxydatif de protéines particulières, résultant à la fois de leur mauvais repliement et du stress oxydatif. La cause commune va de pair avec l'horloge biologique des diverses maladies du vieillissement, dont l'incidence augmente exponentiellement avec l'âge, responsables de 90 % de la mortalité humaine. Des interventions pharmacologiques sur la cause commune pourraient éviter et atténuer simultanément toutes les maladies dégénératives et malignes, comme c'est le cas naturellement chez les super-centenaires.

Cell wall remodeling and vesicle trafficking mediate the root clock in Arabidopsis.

In Arabidopsis thaliana, lateral roots initiate in a process preceded by periodic gene expression known as the root clock. We identified the vesicle-trafficking regulator GNOM and its suppressor, ADENOSINE PHOSPHATE RIBOSYLATION FACTOR GTPase ACTIVATION PROTEIN DOMAIN3, as root clock regulators. GNOM is required for the proper distribution of pectin, a mediator of intercellular adhesion, whereas the pectin esterification state is essential for a functional root clock. In sites of lateral root primordia emergence, both esterified and de-esterified pectin variants are differentially distributed. Using a reverse-genetics approach, we show that genes controlling pectin esterification regulate the root clock and lateral root initiation. These results indicate that the balance between esterified and de-esterified pectin states is essential for proper root clock function and the subsequent initiation of lateral root primordia.

Analysis of epigenetic aging in vivo and in vitro: Factors controlling the speed and direction.

IMPACT STATEMENT: Aging is associated with DNA methylation (DNAm) changes. Recent advancement of the whole-genome DNAm analysis technology allowed scientists to develop DNAm-based age estimators. A majority of these estimators use DNAm data from a single tissue type such as blood. In 2013, a multi-tissue age estimator using DNAm pattern of 353 CpGs was developed by Steve Horvath. This estimator was named "epigenetic clock", and the improved version using DNAm pattern of 391 CpGs was developed in 2018. The estimated age by epigenetic clock is named DNAmAge. DNAmAge can be used as a biomarker of aging predicting the risk of age-associated diseases and mortality. Although the DNAm-based age estimators were developed, the mechanism of epigenetic aging is still enigmatic. The biological significance of epigenetic aging is not well understood, either. This minireview discusses the current understanding of the mechanism of epigenetic aging and the future direction of aging research.

A minor population of macrophage-tropic HIV-1 variants is identified in recrudescing viremia following analytic treatment interruption.

HIV-1 persists in cellular reservoirs that can reignite viremia if antiretroviral therapy (ART) is interrupted. Therefore, insight into the nature of those reservoirs may be revealed from the composition of recrudescing viremia following treatment cessation. A minor population of macrophage-tropic (M-tropic) viruses was identified in a library of recombinant viruses constructed with individual envelope genes that were obtained from plasma of six individuals undergoing analytic treatment interruption (ATI). M-tropic viruses could also be enriched from post-ATI plasma using macrophage-specific (CD14) but not CD4+ T cell-specific (CD3) antibodies, suggesting that M-tropic viruses had a macrophage origin. Molecular clock analysis indicated that the establishment of M-tropic HIV-1 variants predated ATI. Collectively, these data suggest that macrophages are a viral reservoir in HIV-1-infected individuals on effective ART and that M-tropic variants can appear in rebounding viremia when treatment is interrupted. These findings have implications for the design of curative strategies for HIV-1.

Spatiotemporal imaging documented the maturation of the cardiomyocytes from human induced pluripotent stem cells.

OBJECTIVES: Cardiomyocytes derived from human induced pluripotent stem cells are a promising source of cells for regenerative medicine. However, contractions in such derived cardiomyocytes are often irregular and asynchronous, especially at early stages of differentiation. This study aimed to determine the differentiation stage of initiation of synchronized and regular contractions, using spatiotemporal imaging and physiological and genetic analyses. METHODS: Knock-in human induced pluripotent stem cell lines were established with clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 to analyze cardiac and pacemaker cell maturation. Time-frequency analysis and Ca2+ imaging were performed, and the expression of related proteins and specific cardiac/pacemaker mRNAs in contracting embryoid bodies was analyzed at various differentiation stages. RESULTS: Time-frequency analysis and Ca2+ imaging revealed irregular, asynchronous contractions at the early stage of differentiation with altered electrophysiological properties upon differentiation. Genes associated with electrophysiological properties were upregulated after 70 days of culturing in differentiation media, whereas pacemaker genes were initially upregulated during the early stage and downregulated at the later stage. CONCLUSIONS: A differentiation period >70 days is required for adequate development of cardiac elements including ion channels and gap junctions and for sarcomere maturation.

The histone demethylase KDM5 controls developmental timing in Drosophila by promoting prothoracic gland endocycles.

In Drosophila, the larval prothoracic gland integrates nutritional status with developmental signals to regulate growth and maturation through the secretion of the steroid hormone ecdysone. While the nutritional signals and cellular pathways that regulate prothoracic gland function are relatively well studied, the transcriptional regulators that orchestrate the activity of this tissue remain less characterized. Here, we show that lysine demethylase 5 (KDM5) is essential for prothoracic gland function. Indeed, restoring kdm5 expression only in the prothoracic gland in an otherwise kdm5 null mutant animal is sufficient to rescue both the larval developmental delay and the pupal lethality caused by loss of KDM5. Our studies show that KDM5 functions by promoting the endoreplication of prothoracic gland cells, a process that increases ploidy and is rate limiting for the expression of ecdysone biosynthetic genes. Molecularly, we show that KDM5 activates the expression of the receptor tyrosine kinase torso, which then promotes polyploidization and growth through activation of the MAPK signaling pathway. Taken together, our studies provide key insights into the biological processes regulated by KDM5 and expand our understanding of the transcriptional regulators that coordinate animal development.

Adipose­derived stem cells overexpressing SK4 calcium­activated potassium channel generate biological pacemakers.

Recent studies have suggested that calcium­activated potassium channel (KCa) agonists increase the proportion of mouse embryonic stem cell­derived cardiomyocytes and promote the differentiation of pacemaker cells. In the present study, it was hypothesized that adipose­derived stem cells (ADSCs) can differentiate into pacemaker­like cells via overexpression of the SK4 gene. ADSCs were transduced with a recombinant adenovirus vector carrying the mouse SK4 gene, whereas the control group was transduced with GFP vector. ADSCs transduced with SK4 vector were implanted into the rat left ventricular free wall. Complete atrioventricular block (AVB) was established in isolated perfused rat hearts after 2 weeks. SK4 was successfully and stably expressed in ADSCs following transduction. The mRNA levels of the pluripotent markers Oct­4 and Sox­2 declined and that of the transcription factor Shox2 was upregulated following SK4 transduction. The expression of α­actinin and hyperpolarization­activated cyclic nucleotide­gated potassium channel 4 (HCN4) increased in the SK4 group. The hyperpolarizing activated pacemaker current If (8/20 cells) was detected in ADSCs transduced with SK4, but not in the GFP group. Furthermore, SK4 transduction induced the expression of p­ERK1/2 and p­p38 MAPK. In the ex vivo experiments, the heart rate of the SK4 group following AVB establishment was significantly higher compared with that in the GFP group. Immunofluorescence revealed that the transduced ADSCs were successfully implanted and expressed HCN4 in the SK4 group. In conclusion, SK4 induced ADSCs to differentiate into cardiomyocyte­like and pacemaker­like cells via activation of the extracellular signal­regulated kinase 1/2 and p38 mitogen­activated protein kinase pathways. Therefore, ADSCs transduced with SK4 may be used to generate biological pacemakers in ex vivo rat hearts.

Clockophagy is a novel selective autophagy process favoring ferroptosis.

Ferroptosis is a form of nonapoptotic regulated cell death driven by iron-dependent lipid peroxidation. Autophagy involves a lysosomal degradation pathway that can either promote or impede cell death. A high level of autophagy has been associated with ferroptosis, but the mechanisms underpinning this relationship are largely elusive. We characterize the contribution of autophagy to ferroptosis in human cancer cell lines and mouse tumor models. We show that "clockophagy," the selective degradation of the core circadian clock protein ARNTL by autophagy, is critical for ferroptosis. We identify SQSTM1 as a cargo receptor responsible for autophagic ARNTL degradation. ARNTL inhibits ferroptosis by repressing the transcription of Egln2, thus activating the prosurvival transcription factor HIF1A. Genetic or pharmacological interventions blocking ARNTL degradation or inhibiting EGLN2 activation diminished, whereas destabilizing HIF1A facilitated, ferroptotic tumor cell death. Thus, our findings reveal a new pathway, initiated by the autophagic removal of ARNTL, that facilitates ferroptosis induction.

Genome-wide association study of breakfast skipping links clock regulation with food timing.

BACKGROUND: Little is known about the contribution of genetic variation to food timing, and breakfast has been determined to exhibit the most heritable meal timing. As breakfast timing and skipping are not routinely measured in large cohort studies, alternative approaches include analyses of correlated traits. OBJECTIVES: The aim of this study was to elucidate breakfast skipping genetic variants through a proxy-phenotype genome-wide association study (GWAS) for breakfast cereal skipping, a commonly assessed correlated trait. METHODS: We leveraged the statistical power of the UK Biobank (n = 193,860) to identify genetic variants related to breakfast cereal skipping as a proxy-phenotype for breakfast skipping and applied several in silico approaches to investigate mechanistic functions and links to traits/diseases. Next, we attempted validation of our approach in smaller breakfast skipping GWAS from the TwinUK (n = 2,006) and the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium (n = 11,963). RESULTS: In the UK Biobank, we identified 6 independent GWAS variants, including those implicated for caffeine (ARID3B/CYP1A1), carbohydrate metabolism (FGF21), schizophrenia (ZNF804A), and encoding enzymes important for N6-methyladenosine RNA transmethylation (METTL4, YWHAB, and YTHDF3), which regulates the pace of the circadian clock. Expression of identified genes was enriched in the cerebellum. Genome-wide correlation analyses indicated positive correlations with anthropometric traits. Through Mendelian randomization (MR), we observed causal links between genetically determined breakfast skipping and higher body mass index, more depressive symptoms, and smoking. In bidirectional MR, we demonstrated a causal link between being an evening person and skipping breakfast, but not vice versa. We observed association of our signals in an independent breakfast skipping GWAS in another British cohort (P = 0.032), TwinUK, but not in a meta-analysis of non-British cohorts from the CHARGE consortium (P = 0.095). CONCLUSIONS: Our proxy-phenotype GWAS identified 6 genetic variants for breakfast skipping, linking clock regulation with food timing and suggesting a possible beneficial role of regular breakfast intake as part of a healthy lifestyle.

Circadian (De)regulation in Head and Neck Squamous Cell Carcinoma.

Head and neck cancer encompass different malignancies that develop in and around the throat, larynx, nose, sinuses and mouth. Most head and neck cancers are squamous cell carcinomas (HNSCC) that arise in the flat squamous cells that makeup the thin layer of tissue on the surface of anatomical structures in the head and neck. Each year, HNSCC is diagnosed in more than 600,000 people worldwide, with about 50,000 new cases. HNSCC is considered extremely curable if detected early. But the problem remains in treatment of inoperable cases, residues or late stages. Circadian rhythm regulation has a big role in developing various carcinomas, and head and neck tumors are no exception. A number of studies have reported that alteration in clock gene expression is associated with several cancers, including HNSCC. Analyses on circadian clock genes and their association with HNSCC have shown that expression of PER1, PER2, PER3, CRY1, CRY2, CKIε, TIM, and BMAL1 are deregulated in HNSCC tissues. This review paper comprehensively presents data on deregulation of circadian genes in HNSCC and critically evaluates their potential diagnostics and prognostics role in this type of pathology.

Desynchronization of the molecular clock contributes to the heterogeneity of the inflammatory response.

Heterogeneity in the behavior of genetically and developmentally equivalent cells is becoming increasingly appreciated. There are several sources of cellular heterogeneity, including both intrinsic and extrinsic noise. We found that some aspects of heterogeneity in the response of macrophages to bacterial lipopolysaccharide (LPS) were due to intercellular desynchronization of the molecular clock, a cell-intrinsic oscillator. We found that the ratio of the relative expression of two clock genes, Nfil3 and Dbp, expressed in opposite phases of the clock, determined the fraction of cells that produced the cytokine IL-12p40 in response to LPS. The clock can be entrained by various environmental stimuli, making it a mechanism by which population-level heterogeneity and the inflammatory response can be regulated.

DNA Methylation Clocks in Aging: Categories, Causes, and Consequences.

Age-associated changes to the mammalian DNA methylome are well documented and thought to promote diseases of aging, such as cancer. Recent studies have identified collections of individual methylation sites whose aggregate methylation status measures chronological age, referred to as the DNA methylation clock. DNA methylation may also have value as a biomarker of healthy versus unhealthy aging and disease risk; in other words, a biological clock. Here we consider the relationship between the chronological and biological clocks, their underlying mechanisms, potential consequences, and their utility as biomarkers and as targets for intervention to promote healthy aging and longevity.

Myc activity is required for maintenance of the neuromesodermal progenitor signalling network and for segmentation clock gene oscillations in mouse.

The Myc transcriptional regulators are implicated in a range of cellular functions, including proliferation, cell cycle progression, metabolism and pluripotency maintenance. Here, we investigated the expression, regulation and function of the Myc family during mouse embryonic axis elongation and segmentation. Expression of both cMyc (Myc - Mouse Genome Informatics) and MycN in the domains in which neuromesodermal progenitors (NMPs) and underlying caudal pre-somitic mesoderm (cPSM) cells reside is coincident with WNT and FGF signals, factors known to maintain progenitors in an undifferentiated state. Pharmacological inhibition of Myc activity downregulates expression of WNT/FGF components. In turn, we find that cMyc expression is WNT, FGF and Notch protein regulated, placing it centrally in the signalling circuit that operates in the tail end that both sustains progenitors and drives maturation of the PSM into somites. Interfering with Myc function in the PSM, where it displays oscillatory expression, delays the timing of segmentation clock oscillations and thus of somite formation. In summary, we identify Myc as a component that links NMP maintenance and PSM maturation during the body axis elongation stages of mouse embryogenesis.

Transcriptional regulation of the cardiac conduction system.

The rate and rhythm of heart muscle contractions are coordinated by the cardiac conduction system (CCS), a generic term for a collection of different specialized muscular tissues within the heart. The CCS components initiate the electrical impulse at the sinoatrial node, propagate it from atria to ventricles via the atrioventricular node and bundle branches, and distribute it to the ventricular muscle mass via the Purkinje fibre network. The CCS thereby controls the rate and rhythm of alternating contractions of the atria and ventricles. CCS function is well conserved across vertebrates from fish to mammals, although particular specialized aspects of CCS function are found only in endotherms (mammals and birds). The development and homeostasis of the CCS involves transcriptional and regulatory networks that act in an embryonic-stage-dependent, tissue-dependent, and dose-dependent manner. This Review describes emerging data from animal studies, stem cell models, and genome-wide association studies that have provided novel insights into the transcriptional networks underlying CCS formation and function. How these insights can be applied to develop disease models and therapies is also discussed.