Long-Term Imaging Reveals a Circadian Rhythm of Intracellular Chloride in Neurons of the Suprachiasmatic Nucleus

Both inhibitory and excitatory GABA transmission exist in the mature suprachiasmatic nucleus (SCN), the master pacemaker of circadian physiology. Whether GABA is inhibitory or excitatory depends on the intracellular chloride concentration ([Cl−]i). Here, using the genetically encoded ratiometric probe Cl-Sensor, we investigated [Cl−]i in AVP and VIP-expressing SCN neurons for several days in culture. The chloride ratio (RCl) demonstrated circadian rhythmicity in AVP + neurons and VIP + neurons, but was not detected in GFAP + astrocytes. RCl peaked between ZT 7 and ZT 8 in both AVP + and VIP + neurons. RCl rhythmicity was not dependent on the activity of several transmembrane chloride carriers, action potential generation, or the L-type voltage-gated calcium channels, but was sensitive to GABA antagonists. We conclude that [Cl−]i is under circadian regulation in both AVP + and VIP + neurons.

A multidisciplinary perspective on the complex interactions between sleep, circadian, and metabolic disruption in cancer patients

Sleep is a basic need that is frequently set aside in modern societies. This leads to profound but complex physiological maladaptations in the body commonly referred to as circadian disruption, which recently has been characterized as a carcinogenic factor and reason for poor treatment outcomes, shortened survival, and reduced quality of life in cancer patients. As sleep and circadian physiology in cancer patients spans several disciplines including nursing science, neurology, oncology, molecular biology and medical technology, there is a lack of comprehensive and integrated approaches to deal with this serious and growing issue and at best a fractionated understanding of only part of the problem among researchers within each of these segments. Here, we take a multidisciplinary approach to comprehensively review the diagnosis and impact of sleep and circadian disruption in cancer patients. We discuss recent discoveries on molecular regulation of the circadian clock in healthy and malignant cells, the neurological and endocrine pathways controlling sleep and circadian rhythmicity, and their inputs to and outputs from the organism. The benefits and drawbacks of the various technologies, devices, and instruments used to assess sleep and circadian function, as well as the known consequences of sleep disruption and how sleep can be corrected in cancer patients, will be analyzed. We will throughout the review highlight the extensive crosstalk between sleep, circadian rhythms, and metabolic pathways involved in malignancy and identify current knowledge gaps and barriers for addressing the issue of sleep and circadian disruption in cancer patients. By addressing these issues, we hope to provide a foundation for further research as well as better and more effective care for the patients in the future.

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.

Melatonin Ameliorates Valproic Acid-Induced Neurogenesis Impairment: The Role of Oxidative Stress in Adult Rats

Background. Valproic acid (anticonvulsant medication) has been found to inhibit histone deacetylase activity and suppress hippocampal neurogenesis, which causes memory impairment in both humans and rodents. The neurohormone melatonin, which regulates mammalian seasonal and circadian physiology, has recently been shown to have neuroprotective properties, counteracting memory impairment associated with VPA-caused hippocampal neurogenesis reduction. This study is aimed at investigating the molecular mechanisms of melatonin associated with VPA-induced hippocampal neurogenesis and memory impairment. Methods. Male Spraque-Dawley rats received VPA (300 mg/kg) twice daily or melatonin (8 mg/kg/day) or some rats were given melatonin for 14 days during VPA administration. Results. The VPA-treated rats showed a significant increase in malondialdehyde (MDA) levels in the hippocampus and p21-positive cells in the subgranular zone (SGZ) of the dentate gyrus (DG) but decreased superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities. Moreover, VPA significantly decreased levels of nestin, Notchl, nuclear factor erythroid 2-related factor 2 (Nrf2), doublecortin (DCX), sex determining region Y-box 2 (SOX2), and brain-derived neurotrophic factor (BDNF). Conclusions. We found that melatonin was able to counteract these neurotoxic effects, acting as a neuroprotectant in VPA-induced memory hippocampal neurogenesis impairment by preventing intracellular oxidative stress and increasing antioxidant activity.

Susceptibility rhythm to bacterial endotoxin in myeloid clock-knockout mice

Local circadian clocks are active in most cells of our body. However, their impact on circadian physiology is still under debate. Mortality by endotoxic (LPS) shock is highly time-of-day dependent and local circadian immune function such as the cytokine burst after LPS challenge has been assumed to be causal for the large differences in survival. Here, we investigate the roles of light and myeloid clocks on mortality by endotoxic shock. Strikingly, mice in constant darkness (DD) show a three-fold increased susceptibility to LPS as compared to mice in light-dark conditions. Mortality by endotoxic shock as a function of circadian time is independent of light-dark cycles as well as myeloid CLOCK or BMAL1 as demonstrated in conditional knockout mice. Unexpectedly, despite the lack of a myeloid clock these mice still show rhythmic patterns of pro- and anti-inflammatory cytokines such as TNF,α MCP-1, IL-18 and IL-10 in peripheral blood as well as time-of-day and site dependent traffc of myeloid cells. We speculate that systemic time-cues are sufficient to orchestrate innate immune response to LPS by driving immune functions such as cell traffcking and cytokine expression.

Circadian Rhythms, Disease and Chronotherapy

Circadian clocks are biological timing mechanisms that generate 24-h rhythms of physiology and behavior, exemplified by cycles of sleep/wake, hormone release, and metabolism. The adaptive value of clocks is evident when internal body clocks and daily environmental cycles are mismatched, such as in the case of shift work and jet lag or even mistimed eating, all of which are associated with physiological disruption and disease. Studies with animal and human models have also unraveled an important role of functional circadian clocks in modulating cellular and organismal responses to physiological cues (ex., food intake, exercise), pathological insults (e.g. virus and parasite infections), and medical interventions (e.g. medication). With growing knowledge of the molecular and cellular mechanisms underlying circadian physiology and pathophysiology, it is becoming possible to target circadian rhythms for disease prevention and treatment. In this review, we discuss recent advances in circadian research and the potential for therapeutic applications that take patient circadian rhythms into account in treating disease.

Hexosamine biosynthetic pathway and O-GlcNAc-processing enzymes regulate daily rhythms in protein O-GlcNAcylation

The integration of circadian and metabolic signals is essential for maintaining robust circadian rhythms and ensuring efficient metabolism and energy use. Using Drosophila as an animal model, we show that cellular protein O-GlcNAcylation exhibits robust 24-hour rhythm and represents a key post-translational mechanism that regulates circadian physiology. We observe strong correlation between protein O-GlcNAcylation rhythms and clock-controlled feeding-fasting cycles, suggesting that O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, daily O-GlcNAcylation rhythms are severely dampened when we subject flies to time-restricted feeding at unnatural feeding time. This suggests the presence of clock-regulated buffering mechanisms that prevent excessive O-GlcNAcylation at non-optimal times of the day-night cycle. We show that this buffering mechanism is mediated by the expression and activity of GFAT, OGT, and OGA, which are regulated through integration of circadian and metabolic signals. Finally, we generate a mathematical model to describe the key factors that regulate daily O-GlcNAcylation rhythm.

Chronic Jetlag-Induced Alterations in Pancreatic Diurnal Gene Expression

Cell-autonomous circadian clocks exist in nearly every organ and function to maintain homeostasis through a complex series of transcriptional-translational feedback loops. The response of these peripheral clocks to external perturbations, such as chronic jetlag and shiftwork, has been extensively investigated. However, an evaluation of the effects of chronic jetlag on the mouse pancreatic transcriptome is still lacking. Herein we report an evaluation of the diurnal variations encountered in the pancreatic transcriptome following exposure to an established chronic jetlag protocol. We found approximately 5.4% of the pancreatic transcriptome was rhythmic. Following chronic jetlag, we found the number of rhythmic transcripts decreased to approximately 3.6% of the transcriptome. Analysis of the core clock genes, which orchestrate circadian physiology, revealed that nearly all exhibited a shift in the timing of peak gene expression - known as a phase shift. Similarly, over 95% of the rhythmically expressed genes in the pancreatic transcriptome exhibited a phase shift, many of which were found to be important for metabolism. Evaluation of the genes involved in pancreatic exocrine secretion and insulin signaling revealed many pancreas-specific genes were also rhythmically expressed and several displayed a concomitant phase shift with chronic jetlag. Phase differences were found 9 days after normalization, indicating a persistent failure to reentrain to the new light-dark cycle. This study is the first to evaluate the endogenous pancreatic clock and rhythmic gene expression in whole pancreas over 48 hours, and how the external perturbation of chronic jetlag affects the rhythmic expression of genes in the pancreatic

Attenuated SIRT1 Activity Leads to PER2 Cytoplasmic Localization and Dampens the Amplitude of Bmal1 Promoter-Driven Circadian Oscillation

The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD+), which is associated with posttranslational modifications of acetylation and poly-ADP-ribosylation status of circadian clock proteins and decreases with aging. However, how low NAD+ condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. Here, we show that low NAD+ in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD+/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD+ amount, and a higher cytoplasmic PER2 localization was observed under low NAD+ condition. We further found that NAD+-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. Thus, we anticipate that the altered PER2 subcellular localization by low NAD+ is one of the complex changes that occurs in the aged circadian clock.