Marc Ruben

A Mechanism for Circadian Control of Pacemaker Neuron Excitability

Although the intracellular molecular clocks that regulate circadian (~24 h) behavioral rhythms are well understood, it remains unclear how molecular clock information is transduced into rhythmic neuronal activity that in turn drives behavioral rhythms. To identify potential clock outputs, the authors generated expression profiles from a homogeneous population of purified pacemaker neurons (LNvs) from wild-type and clock mutant Drosophila. They identified a group of genes with enriched expression in LNvs and a second group of genes rhythmically expressed in LNvs in a clock-dependent manner. Only 10 genes fell into both groups: 4 core clock genes, including period (per) and timeless (tim), and 6 genes previously unstudied in circadian rhythms. The authors focused on one of these 6 genes, Ir, which encodes an inward rectifier K+ channel likely to regulate resting membrane potential, whose expression peaks around dusk. Reducing Ir expression in LNvs increased larval light avoidance and lengthened the period of adult locomotor rhythms, consistent with increased LNv excitability. In contrast, increased Ir expression made many adult flies arrhythmic and dampened PER protein oscillations. The authors propose that rhythmic Ir expression contributes to daily rhythms in LNv neuronal activity, which in turn feed back to regulate molecular clock oscillations.

Guidelines for Genome-Scale Analysis of Biological Rhythms

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding “big data” that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

What Is There Left to Learn about the Drosophila Clock

Circadian rhythms offer probably the best understanding of how genes control behavior, and much of this understanding has come from studies in Drosophila. More recently, genetic manipulation of clock neurons in Drosophila has helped identify how daily patterns of activity are programmed by different clock neuron groups. Here, we review some of the more recent findings on the fly molecular clock and ask what more the fly model can offer to circadian biologists.

Population level rhythms in human skin: implications for circadian medicine

Skin is the largest organ in the body and serves important barrier, regulatory, and sensory functions. Like other tissues, skin is subject to temporal fluctuations in physiological responses under both homeostatic and stressed states. To gain insight into these fluctuations, we investigated the role of the circadian clock in the transcriptional regulation of epidermis using a hybrid experimental design, where a limited set of human subjects (n=20) were sampled throughout the 24 h cycle and a larger population (n=219) were sampled once. By looking at pairwise correlations of core clock genes in 298 skin samples, we found a robust circadian oscillator in skin at the population level. Encouraged by this, we used CYCLOPS to reconstruct the temporal order of all samples and identified hundreds of rhythmically-expressed genes at the population level in human skin. We compared these results with published time-series skin data from mouse and show strong concordance in circadian phase across species for both transcripts and pathways. Further, like blood, skin is readily accessible and a potential source of biomarkers. Using ZeitZeiger, we identified a biomarker set for human skin that is capable of reporting circadian phase to within 3 h from a single sample. In summary, we show rhythms in human skin that persist at the population scale and a path to develop robust single-sample circadian biomarkers. One Sentence Summary Human epidermis shows strong circadian rhythms at the population scale and provides a better source for developing robust, single-sample circadian phase biomarkers than human blood.

Circadian Rhythms: Move Over Neurons — Astrocytes Mediate SCN Clock Function

Neurons in the suprachiasmatic nuclei (SCN) of the hypothalamus are described as master pacemaker cells for biological rhythms. However, a series of recent studies demonstrate the importance of another cell type, astrocytes, for biological timekeeping.

A population-based human enCYCLOPedia for circadian medicine

The discovery that half of the mammalian protein-coding genome is clock-regulated has clear implications for medicine. Indeed, recent studies demonstrate time-of-day impact on therapeutic outcomes in human heart disease and cancer. Yet biological time is rarely given clinical consideration. A key barrier is the absence of information on the what and where of molecular rhythms in the human body. Here, we have applied CYCLOPS, an algorithm designed to reconstruct sample order in the absence of time-of-day information, to the GTEx collection of 632 human donors contributing 4,292 RNA-seq samples from 13 distinct human tissue types. We identify rhythms in expression across the body that persist at the population-level. This includes a set of ‘ubiquitous cyclers’ comprised of well-established circadian clock factors but also many genes without prior circadian context. Among thousands of tissue-divergent rhythms, we discover a set of genes robustly oscillating in cardiovascular tissue, including key drug targets relevant to heart disease. These results also have implications for genetic studies where circadian variability may have masked genetic influence. It is our hope that the human enCYCLOPedia helps drive the translation of circadian biology into prospective clinical trials in cardiology and many other therapeutic areas. One Sentence Summary Bioinformatic analyses on thousands of human tissue samples reveals an enCYCLOPedia of rhythmic gene expression across the body and identifies key translational opportunities for circadian medicine in cardiovascular disease.

A database of tissue-specific rhythmically expressed human genes has potential applications in circadian medicine

Bioinformatic analyses of human tissue samples were used to build a database of rhythmically expressed genes across the body. Body timing Although the existence of circadian clock–dependent modulation of gene expression in humans has been known for more than a decade, the relevance of the circadian clock in drug response and therapeutic outcome has been only recently appreciated. Now, Ruben et al. used an algorithm called cyclic ordering by periodic structure (CYCLOPS) to create a database of cycling genes in 13 human tissues. The authors show that several rhythmically expressed genes code for known drug targets or for proteins involved in drug transport and metabolism. The data represent a useful resource for circadian medicine and strengthen the notion that circadian rhythms should be considered when determining therapeutic interventions. The discovery that half of the mammalian protein-coding genome is regulated by the circadian clock has clear implications for medicine. Recent studies demonstrated that the circadian clock influences therapeutic outcomes in human heart disease and cancer. However, biological time is rarely given clinical consideration. A key barrier is the absence of information on tissue-specific molecular rhythms in the human body. We have applied the cyclic ordering by periodic structure (CYCLOPS) algorithm, designed to reconstruct sample temporal order in the absence of time-of-day information, to the gene expression collection of 13 tissues from 632 human donors. We identified rhythms in gene expression across the body; nearly half of protein-coding genes were shown to be cycling in at least 1 of the 13 tissues analyzed. One thousand of these cycling genes encode proteins that either transport or metabolize drugs or are themselves drug targets. These results provide a useful resource for studying the role of circadian rhythms in medicine and support the idea that biological time might play a role in determining drug response.

Circadian Dysregulation: The Next Frontier in Obstructive Sleep Apnea Research

Objective To review the effects of the circadian clock on homeostasis, the functional interaction between the circadian clock and hypoxia-inducible factors, and the role of circadian dysregulation in the progression of cardiopulmonary disease in obstructive sleep apnea (OSA). Data Sources The MEDLINE database was accessed through PubMed. Review Methods A general review is presented on molecular pathways disrupted in OSA, circadian rhythms and the role of the circadian clock, hypoxia signaling, crosstalk between the circadian and hypoxia systems, the role of the circadian clock in cardiovascular disease, and implications for practice. Studies included in this State of the Art Review demonstrate the potential contribution of the circadian clock and hypoxia in animal models or human disease. Conclusions Molecular crosstalk between the circadian clock and hypoxia-inducible factors has not been evaluated in disease models of OSA. Implications for Practice Pediatric OSA is highly prevalent and, if left untreated, may lead to cardiopulmonary sequelae. Changes in inflammatory markers that normally demonstrate circadian rhythmicity are also seen among patients with OSA. Hypoxia-inducible transcription factors interact with core circadian clock transcription factors; however, the interplay between these pathways has not been elucidated in the cardiopulmonary system. This gap in knowledge hinders our ability to identify potential biomarkers of OSA and develop alternative therapeutic strategies. A deeper understanding of the mechanisms by which OSA impinges on clock function and the impact of clock dysregulation on the cardiopulmonary system may lead to future advancements for the care of patients with OSA. The aim of this review is to shed light on this important clinical topic.