Roman V. Kondratov

The circadian clock and pathology of the ageing brain

Ageing leads to a functional deterioration of many brain systems, including the circadian clock — an internal time-keeping system that generates ∼24-hour rhythms in physiology and behaviour. Numerous clinical studies have established a direct correlation between abnormal circadian clock functions and the severity of neurodegenerative and sleep disorders. Latest data from experiments in model organisms, gene expression studies and clinical trials imply that dysfunctions of the circadian clock contribute to ageing and age-associated pathologies, thereby suggesting a functional link between the circadian clock and age-associated decline of brain functions. Potential molecular mechanisms underlying this link include the circadian control of physiological processes such as brain metabolism, reactive oxygen species homeostasis, hormone secretion, autophagy and stem cell proliferation.

Circadian rhythms govern cardiac repolarization and arrhythmogenesis.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease, but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) or pattern (for example, Brugada’s syndrome) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current. Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis.

Dual effect of p53 on radiation sensitivity in vivo: p53 promotes hematopoietic injury, but protects from gastro-intestinal syndrome in mice

Ionizing radiation (IR) induces p53-dependent apoptosis in radiosensitive tissues, suggesting that p53 is a determinant of radiation syndromes. In fact, p53-deficient mice survive doses of IR that cause lethal hematopoietic syndrome in wild-type animals. Surprisingly, p53 deficiency results in sensitization of mice to higher doses of IR, causing lethal gastro-intestinal (GI) syndrome. While cells in the crypts of p53-wild-type epithelium undergo prolonged growth arrest after irradiation, continuous cell proliferation ongoing in p53-deficient epithelium correlates with accelerated death of damaged cells followed by rapid destruction of villi and accelerated lethality. p21-deficient mice are also characterized by increased sensitivity to GI syndrome-inducing doses of IR. We conclude that p53/p21-mediated growth arrest plays a protective role in the epithelium of small intestine after severe doses of IR. Pharmacological inhibition of p53 by a small molecule that can rescue from lethal hematopoietic syndrome has no effect on the lethality from gastro-intestinal syndrome, presumably because of a temporary and reversible nature of its action.

Disruption of the circadian clock due to the Clock mutation has discrete effects on aging and carcinogenesis.

The mammalian circadian system has been implicated in the regulation of various biological processes including those involved in genotoxic stress responses and tumor suppression. Here we report that mice with the functional deficiency in circadian transcription factor CLOCK (Clock/Clock mutant mice) do not display predisposition to tumor formation both during their normal lifespan or when challenged by γ-radiation. This phenotype is consistent with high apoptotic and low proliferation rate in lymphoid tissues of Clock mutant mice and is supported by the gene expression profiling of a number of apoptosis and cell cycle-related genes, as well as by growth inhibition of cells with CLOCK downregulation. At the same time, Clock mutant mice respond to low-dose irradiation by accelerating their aging program, and develop phenotypes that are reminiscent of those in Bmal1-deficient mice. Taken together, our results demonstrate the dichotomy in biological consequences of the disruption of the circadian clock with respect to ageing and cancer. They also highlight the existence of a complex interconnection between ageing, carcinogenesis and individual components of the circadian clock machinery.

Small molecules that dramatically alter multidrug resistance phenotype by modulating the substrate specificity of P-glycoprotein

By screening a chemical library for the compounds protecting cells from adriamycin (Adr), a series of small molecules was isolated that interfered with the accumulation of Adr in mouse fibroblasts by enhancing efflux of the drug. Isolated compounds also stimulated efflux of Rhodamine 123 (Rho-123), another substrate of multidrug transporters. Stimulation of drug efflux was detectable in the cells expressing P-glycoprotein (P-gp), but not in their P-gp-negative variants, and was completely reversible by the P-gp inhibitors. A dramatic stimulation of P-gp activity against Adr and Rho-123 by the identified compounds was accompanied by suppression of P-gp-mediated efflux of other substrates, such as Taxol (paclitaxel) or Hoechst 33342, indicating that they act as modulators of substrate specificity of P-gp. Consistently, P-gp modulators dramatically altered the pattern of cross-resistance of P-gp-expressing cells to different P-gp substrates: an increase in resistance to Adr, daunorubicin, and etoposide was accompanied by cell sensitization to Vinca alkaloids, gramicidin D, and Taxol with no effect on cell sensitivity to colchicine, actinomycin D, puromycin, and colcemid, as well as to several non-P-gp substrates. The relative effect of P-gp modulators against different substrates varied among the isolated compounds that can be used as fine tools for analyzing mechanisms of drug selectivity of P-gp. These results raise the possibility of a rational control over cell sensitivity to drugs and toxins through modulation of P-gp activity by small molecules.

A role of the circadian system and circadian proteins in aging

Circadian rhythms are genetically determined biological rhythms that are considered an important adaptive mechanism to the cyclical light/dark alterations in the Earth environment. Age-related changes in the circadian time-keeping mechanism are well known, and seemingly contribute to various pathologies of aging. Recent findings demonstrate that the circadian system and circadian proteins play direct roles in many physiological processes, including those associated with aging. The core circadian proteins BMAL1 and PERIODs, in addition to their known functions in the circadian oscillator, play essential non-redundant roles in the control of tissue homeostasis and aging. Although the exact mechanisms are unknown, the involvement of circadian proteins in the regulation of metabolism, genotoxic stress response and reactive oxygen species (ROS) homeostasis can be responsible for the premature aging, observed in some circadian mutants. The understanding of the molecular mechanisms of these non-circadian activities of the circadian proteins will ultimately lead to the improvement in prevention and treatment of age-related pathologies.

Dual role of the CLOCK/BMAL1 circadian complex in transcriptional regulation

The basic helix–loop–helix (bHLH) –PAS domain containing transcription factors CLOCK and BMAL1 are two major components of the circadian molecular oscillator. It is known that the CLOCK/BMAL1 complex positively regulates the activity of E‐box containing promoters. Here we demonstrate that the CLOCK/BMAL1 complex can also suppress the activity of some promoters upon its interaction with CRYPTOCHROME (CRY). Such a dual function of the circadian transcriptional complex provides a mechanistic explanation for the unpredicted pattern of circadian gene expression in the tissues of Bmal1 null mice. We speculate that the switch from transcriptional activation to transcriptional repression may provide a highly efficient mechanism for circadian control of gene expression. We also show that CLOCK/BMAL1 can interfere with promoter regulation by other, non‐circadian, transcription factors including N‐MYC and ETS, leading to attenuation or abrogation of transcription of CLOCK/BMAL1‐controlled stress‐induced genes. We propose that, based upon these results, both circadian repression and activation of the transcription of different target genes are required for circadian responses to various external stimuli, including genotoxic stress induced by anticancer treatment.

Post-translational regulation of circadian transcriptional CLOCK(NPAS2)/BMAL1 complex by CRYPTOCHROMES.

Mammalian CLOCK(NPAS2), BMAL1 and CRYPTOCHROMEs are core components of the circadian oscillatory mechanism. The active CLOCK/BMAL1 or NPAS2/BMAL1 complexes regulate expression of numerous genes including two Cryptochromes. The products of these genes, CRY1 and CRY2, in turn repress CLOCK/BMAL1 transcriptional activity by an unknown mechanism. We have examined the effect of CRYPTOCHROMEs on posttranslational modifications and intracellular distribution of endogenous and ectopically expressed CLOCK(NPAS2) and BMAL1 proteins. We found that ectopic coexpression with CRY led to stabilization and nuclear accumulation of unphosphorylated forms of the proteins, which directly correlated with the inhibition of their transcriptional activity. This effect was CRY-specific, as other known repressors of CLOCK/BMAL1 and NPAS2/BMAL1 transcriptional activity were not able to induce similar effects. CRYs had no effect on CLOCK(NPAS2)/BMAL1 complex formation or its ability to bind DNA. Altogether, these results demonstrate that CRYs regulate the functional activity of circadian transcriptional complex at the posttranslational level. Importantly, the posttranslational modifications and intracellular distribution of CLOCK and BMAL1 proteins were critically impaired in the tissues of mice with targeted disruption of both Cry genes, thus confirming the suggested role of CRY in clock function in vivo. Based on these findings we propose a modified model of the circadian transcriptional control, which implies CRY-mediated periodic rotation of transcriptionally active and inactive forms of CLOCK/BMAL1 on the promoter. This model provides mechanistic explanation for previously reported dual functional activity of CLOCK/BMAL1 and highlights the involvement of the circadian system in modulating the organism’s response to various types of genotoxic stress, including chemotherapy and radiation.

Circadian regulation of cell cycle: Molecular connections between aging and the circadian clock

Abstract The circadian clock generates oscillations in physiology and behavior, known as circadian rhythms. Links between the circadian clock genes Periods, Bmal1, and Cryptochromes and aging and cancer are emerging. Circadian clock gene expression is changed in human pathologies, and transgenic mice with mutations in clock genes develop cancer and premature aging. Control of genome integrity and cell proliferation play key roles in the development of age-associated pathologies and carcinogenesis. Here, we review recent data on the connection between the circadian clock and control of the cell cycle. The circadian clock regulates the activity and expression of several critical cell cycle and cell cycle check-point-related proteins, and in turn cell cycle-associated proteins regulate circadian clock proteins. DNA damage can reset the circadian clock, which provides a molecular mechanism for reciprocal regulation between the circadian clock and the cell cycle. This circadian clock-dependent control of cell proliferation, together with other known physiological functions of the circadian clock such as the control of metabolism, oxidative and genotoxic stress response, and DNA repair, opens new horizons for understanding the mechanisms behind aging and carcinogenesis.

The Role of Mammalian Circadian Proteins in Normal Physiology and Genotoxic Stress Responses

The last two decades have significantly advanced our understanding of the organization of the circadian system at all levels of regulation-molecular, cellular, tissue, and systemic. It has been recognized that the circadian system represents a complex temporal regulatory network, which plays an important role in synchronizing various biological processes within an organism and coordinating them with the environment. It is believed that deregulation of this synchronization may result in the development of various pathologies. However, recent studies using various circadian mutant mouse models have demonstrated that at least some of the components of the molecular oscillator are actively involved in physiological processes not directly related to their role in the circadian clock. The growing amount of evidence suggests that, in addition to their circadian function, circadian proteins are important in maintaining tissue homeostasis under normal and stress conditions. In this chapter, we will summarize recent data about the regulation of the mammalian molecular circadian oscillator and will focus on a new role of the circadian system and individual circadian proteins in the organisms physiology and response to genotoxic stress in connection with diseases treatment and prevention.