Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster

Circadian clocks are highly conserved transcriptional regulators that control 24-hour oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila, which we term Locally Activatable BioLuminescence or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcription properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks; we found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. This result suggests that the presumed dominance of the brain clock in regulating peripheral clocks needs to be re-examined. This result further demonstrates that LABL allows rapid, affordable, and direct real-time monitoring of clocks in vivo.

Chronic Social Defeat Stress Shifts Peripheral Circadian Clocks in Male Mice in a Tissue-Specific and Time-of-Day Dependent Fashion

Uncontrollable stress is linked to the development of many diseases, some of which are associated with disrupted daily rhythms in physiology and behavior. While available data indicate that the master circadian pacemaker in the suprachiasmatic nucleus (SCN) is unaffected by stress, accumulating evidence suggest that circadian oscillators in peripheral tissues and organs can be shifted by a variety of stressors and stress hormones. In the present study, we examined effects of acute and chronic social defeat stress in mice and addressed the question of whether effects of uncontrollable stress on peripheral clocks are tissue specific and depend on time of day of stress exposure. We used mice that carry a luciferase reporter gene fused to the circadian clock gene Period2 (PER2::LUC) to examine daily rhythms of PER2 expression in various peripheral tissues. Mice were exposed to social defeat stress in the early (ZT13-14) or late (ZT21-22) dark phase, either once (acute stress) or repeatedly on 10 consecutive days (chronic stress). One hour after the last stressor, tissue samples from liver, lung, kidney, and white adipose tissue (WAT) were collected. Social defeat stress caused a phase delay of several hours in the rhythm of PER2 expression in lung and kidney, but this delay was stronger after chronic than after acute stress. Moreover, shifts only occurred after stress in the late dark phase, not in the early dark phase. PER2 rhythms in liver and WAT were not significantly shifted by social defeat, suggesting a different response of various peripheral clocks to stress. This study indicates that uncontrollable social defeat stress is capable of shifting peripheral clocks in a time of day dependent and tissue specific manner. These shifts in peripheral clocks were smaller or absent after a single stress exposure and may therefore be the consequence of a cumulative chronic stress effect.

Multi-Modal Regulation of Circadian Physiology by Interactive Features of Biological Clocks

The circadian clock is a fundamental biological timing mechanism that generates nearly 24 h rhythms of physiology and behaviors, including sleep/wake cycles, hormone secretion, and metabolism. Evolutionarily, the endogenous clock is thought to confer living organisms, including humans, with survival benefits by adapting internal rhythms to the day and night cycles of the local environment. Mirroring the evolutionary fitness bestowed by the circadian clock, daily mismatches between the internal body clock and environmental cycles, such as irregular work (e.g., night shift work) and life schedules (e.g., jet lag, mistimed eating), have been recognized to increase the risk of cardiac, metabolic, and neurological diseases. Moreover, increasing numbers of studies with cellular and animal models have detected the presence of functional circadian oscillators at multiple levels, ranging from individual neurons and fibroblasts to brain and peripheral organs. These oscillators are tightly coupled to timely modulate cellular and bodily responses to physiological and metabolic cues. In this review, we will discuss the roles of central and peripheral clocks in physiology and diseases, highlighting the dynamic regulatory interactions between circadian timing systems and multiple metabolic factors.

Methods for Detecting PER2:LUCIFERASE Bioluminescence Rhythms in Freely Moving Mice

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue circadian rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of circadian bioluminescence from a freely moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2::LUC, in freely behaving mice. The LumiCycle In Vivo applies a background subtraction that corrects for effects of room temperature on photomultiplier tube (PMT) output. We tested delivery of d-luciferin via a subcutaneous minipump and in the drinking water. We demonstrate spikes in bioluminescence associated with drinking bouts. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than d-luciferin, and can support longer-term studies. A small difference in phase of the PER2::LUC bioluminescence rhythms, with females phase leading males, can be detected with this technique. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.

Nitecap: An Exploratory Circadian Analysis Web Application

Circadian omics analyses present investigators with large amounts of data to consider and many choices for methods of analysis. Visualization is crucial as rhythmicity can take many forms and p-values offer an incomplete picture. Yet statically viewing the entirety of high-throughput datasets is impractical, and there is often limited ability to assess the impact of choices, such as significance threshold cutoffs. Nitecap provides an intuitive and unified web-based solution to these problems. Through highly responsive visualizations, Nitecap enables investigators to see dataset-wide behavior. It supports deep analyses, including comparisons of two conditions. Moreover, it focuses upon ease-of-use and enables collaboration through dataset sharing. As an application, we investigated cross talk between peripheral clocks in adipose and liver tissues and determined that adipocyte clock disruption does not substantially modulate the transcriptional rhythmicity of liver but does advance the phase of core clock gene Bmal1 (Arntl) expression in the liver. Nitecap is available at nitecap.org and is free-to-use.

Circadian Clock, Sleep, and Diet

Circadian rhythm is a fundamental process of sustaining metabolic homeostasis by predicting changes in the environment. This is driven by biological clocks, which operate within a 24-h period to orchestrate daily variation of metabolism and sleep. The central clock in the hypothalamus is the master keeper of the circadian rhythm and is primarily reset by light, while the feeding-fasting rhythm, that is, nutritional stimulus, entrains peripheral clocks in peripheral organs such as the intestine and liver. Nutritional stimuli are important modulators of peripheral circadian rhythms and may affect the central clock and sleep homeostasis through metabolic alterations. In this chapter, I will summarize the significance of circadian rhythm and sleep in metabolic regulation as well as discuss the impact that diet has on circadian rhythm and sleep.

Circadian Rhythm, Clock Genes, and Hypertension: Recent Advances in Hypertension

Accumulating evidence suggests that the molecular circadian clock is crucial in blood pressure (BP) control. Circadian rhythms are controlled by the central clock, which resides in the suprachiasmatic nucleus of the hypothalamus and peripheral clocks throughout the body. Both light and food cues entrain these clocks but whether these cues are important for the circadian rhythm of BP is a growing area of interest. The peripheral clocks in the smooth muscle, perivascular adipose tissue, liver, adrenal gland, and kidney have been recently implicated in the regulation of BP rhythm. Dysregulation of the circadian rhythm of BP is associated with adverse cardiorenal outcomes and increased risk of cardiovascular mortality. In this review, we summarize the most recent advances in peripheral clocks as BP regulators, highlight the adverse outcomes of disrupted circadian BP rhythm in hypertension, and provide insight into potential future work in areas exploring the circadian clock in BP control and chronotherapy. A better understanding of peripheral clock function in regulating the circadian rhythm of BP will help pave the way for targeted therapeutics in the treatment of circadian BP dysregulation and hypertension.

CDK5 influences the organization of the Circadian Machinery in peripheral clocks

Circadian rhythms are self-sustained physiological changes that drive rhythmicity within the 24-hours cycles. Posttranslational modifications (PMTs), such as protein phosphorylation, acetylation, sumoylation, and ubiquitination, are biochemical processes that modify protein structure and functions, ensuring circadian rhythm precision. For example, phosphorylation is considered the most important hallmark of rhythmicity from cyanobacteria to mammals. Cyclin-dependent kinase 5 (CDK5) has been shown to regulate the mammalian SCN’s circadian clock via phosphorylation of PER2. Here, we show that CDK5 influences the clock machinery assembling, using immortalized mouse embryonic fibroblast as an in vitro model for studying the peripheral clock. In fact, the circadian period at the cellular level is lengthened. Furthermore, the clock-controlled gene’s expression amplitude is dampened in Cdk5 ko cell lines, while the phase is delayed about 4 hours.Taken together, we show in vitro that CDK5 is critically involved in regulating the peripheral clocks, influencing their temporal and spatial dynamics.