GSK3 activity regulates rhythms in hippocampal clock gene expression and synaptic plasticity.

Hippocampal rhythms in clock gene expression, enzymatic activity, and long-term potentiation (LTP) are thought to underlie day-night differences in memory acquisition and recall. Glycogen synthase kinase 3-beta (GSK3ß) is a known regulator of hippocampal function, and inhibitory phosphorylation of GSK3ß exhibits region-specific differences over the light-dark cycle. Here, we sought to determine whether phosphorylation of both GSK3α and GSK3ß isoforms has an endogenous circadian rhythm in specific areas of the hippocampus and whether chronic inhibition or activation alters the molecular clock and hippocampal plasticity (LTP). Results indicated a significant endogenous circadian rhythm in phosphorylation of GSK3ß, but not GSK3α, in hippocampal CA1 extracts from mice housed in constant darkness for at least 2 weeks. To examine the importance of this rhythm, genetic and pharmacological strategies were used to disrupt the GSK3 activity rhythm by chronically activating or inhibiting GSK3. Chronic activation of both GSK3 isoforms in transgenic mice (GSK3-KI mice) diminished rhythmic BMAL1 expression. On the other hand, chronic treatment with a GSK3 inhibitor significantly shortened the molecular clock period of organotypic hippocampal PER2::LUC cultures. While WT mice exhibited higher LTP magnitude at night compared to day, the day-night difference in LTP magnitude remained with greater magnitude at both times of day in mice with chronic GSK3 activity. On the other hand, pharmacological GSK3 inhibition impaired day-night differences in LTP by blocking LTP selectively at night. Taken together, these results support the model that circadian rhythmicity of hippocampal GSK3ß activation state regulates day/night differences in molecular clock periodicity and a major form of synaptic plasticity (LTP).

Motor restlessness, sleep disturbances, thermal sensory alterations and elevated serum iron levels in Btbd9 mutant mice.

Restless legs syndrome (RLS), also known as Willis-Ekbom disease, is a sensory-motor neurological disorder with a circadian component. RLS is characterized by uncomfortable sensations in the extremities, generally at night or during sleep, which often leads to an uncontrollable urge to move them for relief. Recently, genomic studies identified single-nucleotide polymorphisms in BTBD9, along with three other genes, as being associated with a higher risk of RLS. Little is known about the function of BTBD9 or its potential role in the pathophysiology of RLS. We therefore examined a line of Btbd9 mutant mice we recently generated for phenotypes similar to symptoms found in RLS patients. We observed that the Btbd9 mutant mice had motor restlessness, sensory alterations likely limited to the rest phase, and decreased sleep and increased wake times during the rest phase. Additionally, the Btbd9 mutant mice had altered serum iron levels and monoamine neurotransmitter systems. Furthermore, the sensory alterations in the Btbd9 mutant mice were relieved using ropinirole, a dopaminergic agonist widely used for RLS treatment. These results, taken together, suggest that the Btbd9 mutant mice model several characteristics similar to RLS and would therefore be the first genotypic mouse model of RLS. Furthermore, our data provide further evidence that BTBD9 is involved in RLS, and future studies of the Btbd9 mutant mice will help shine light on its role in the pathophysiology of RLS. Finally, our data argue for the utility of Btbd9 mutant mice to discover and screen novel therapeutics for RLS.

O-GlcNAcylation, novel post-translational modification linking myocardial metabolism and cardiomyocyte circadian clock.

The cardiomyocyte circadian clock directly regulates multiple myocardial functions in a time-of-day-dependent manner, including gene expression, metabolism, contractility, and ischemic tolerance. These same biological processes are also directly influenced by modification of proteins by monosaccharides of O-linked ß-N-acetylglucosamine (O-GlcNAc). Because the circadian clock and protein O-GlcNAcylation have common regulatory roles in the heart, we hypothesized that a relationship exists between the two. We report that total cardiac protein O-GlcNAc levels exhibit a diurnal variation in mouse hearts, peaking during the active/awake phase. Genetic ablation of the circadian clock specifically in cardiomyocytes in vivo abolishes diurnal variations in cardiac O-GlcNAc levels. These time-of-day-dependent variations appear to be mediated by clock-dependent regulation of O-GlcNAc transferase and O-GlcNAcase protein levels, glucose metabolism/uptake, and glutamine synthesis in an NAD-independent manner. We also identify the clock component Bmal1 as an O-GlcNAc-modified protein. Increasing protein O-GlcNAcylation (through pharmacological inhibition of O-GlcNAcase) results in diminished Per2 protein levels, time-of-day-dependent induction of bmal1 gene expression, and phase advances in the suprachiasmatic nucleus clock. Collectively, these data suggest that the cardiomyocyte circadian clock increases protein O-GlcNAcylation in the heart during the active/awake phase through coordinated regulation of the hexosamine biosynthetic pathway and that protein O-GlcNAcylation in turn influences the timing of the circadian clock.

Dysregulation of inflammatory responses by chronic circadian disruption.

Circadian rhythms modulate nearly every mammalian physiological process. Chronic disruption of circadian timing in shift work or during chronic jet lag in animal models leads to a higher risk of several pathologies. Many of these conditions in both shift workers and experimental models share the common risk factor of inflammation. In this study, we show that experimentally induced circadian disruption altered innate immune responses. Endotoxemic shock induced by LPS was magnified, leading to hypothermia and death after four consecutive weekly 6-h phase advances of the light/dark schedule, with 89% mortality compared with 21% in unshifted control mice. This may be due to a heightened release of proinflammatory cytokines in response to LPS treatment in shifted animals. Isolated peritoneal macrophages harvested from shifted mice exhibited a similarly heightened response to LPS in vitro, indicating that these cells are a target for jet lag. Sleep deprivation and stress are known to alter immune function and are potential mediators of the effects we describe. However, polysomnographic recording in mice exposed to the shifting schedule revealed no sleep loss, and stress measures were not altered in shifted mice. In contrast, we observed altered or abolished rhythms in the expression of clock genes in the central clock, liver, thymus, and peritoneal macrophages in mice after chronic jet lag. We conclude that circadian disruption, but not sleep loss or stress, are associated with jet lag-related dysregulation of the innate immune system. Such immune changes might be a common mechanism for the myriad negative health effects of shift work.

Nightshift Work and Nighttime Eating Are Associated With Higher Insulin and Leptin Levels in Hospital Nurses.

Background: Circadian misalignment between behaviors such as feeding and endogenous circadian rhythms, particularly in the context of shiftwork, is associated with poorer cardiometabolic health. We examined whether insulin and leptin levels differ between dayshift versus nightshift nurses, as well as explored whether the timing of food intake modulates these effects in nightshift workers. Methods: Female nurses (N=18; 8 dayshift and 10 nightshift) completed daily diet records for 8 consecutive days. The nurses then completed a 24-h inpatient stay, during which blood specimens were collected every 3 h (beginning at 09:00) and meals were consumed at regular 3-h intervals (09:00, 12:00, 15:00, and 18:00). Specimens were analyzed for insulin and leptin levels, and generalized additive models were used to examine differences in mean insulin and leptin levels. Results: Mean insulin and leptin levels were higher in nightshift nurses by 11.6 ± 3.8 mU/L (p=0.003) and 7.4 ± 3.4 ng/ml (p=0.03), respectively, compared to dayshift nurses. In an exploratory subgroup analysis of nightshift nurses, predominately eating at night (21:00 - 06:00) was associated with significantly higher insulin and leptin levels than consuming most calories during the daytime (06:00 - 21:00). Conclusions: In our study of hospital nurses, working the nightshift was associated with higher insulin and leptin levels, and these effects were driven by eating predominately at night. We conclude that although nightshift work may raise insulin and leptin levels, eating during the daytime may attenuate some of the negative effects of nightshift work on metabolic health.

Shift Work Disrupts Circadian Regulation of the Transcriptome in Hospital Nurses.

Circadian misalignment between sleep and behavioral/feeding rhythms is thought to lead to various health impairments in shift workers. Therefore, we investigated how shift work leads to genome-wide circadian dysregulation in hospital nurses. Female nurses from the University of Alabama at Birmingham (UAB) Hospital working night shift ( n = 9; 29.6 ± 11.4 y) and day shift ( n = 8; 34.9 ± 9.4 y) participated in a 9-day study measuring locomotor activity and core body temperature (CBT) continuously. Additionally, cortisol and melatonin were assayed and peripheral blood mononuclear cells (PBMCs) were harvested for RNA extraction every 3 h on a day off from work. We saw phase desynchrony of core body temperature, peak cortisol, and dim light melatonin onset in individual night-shift subjects compared with day-shift subjects. This variability was evident even though day- and night-shift nurses had similar sleep timing and scheduled meal times on days off. Surprisingly, the phase and rhythmicity of the expression of the clock gene, PER1, in PBMCs were similar for day-shift and night-shift subjects. Genome-wide microarray analysis of PBMCs from a subset of nurses revealed distinct gene expression patterns between night-shift and day-shift subjects. Enrichment analysis showed that day-shift subjects expressed pathways involved in generic transcription and regulation of signal transduction, whereas night-shift subjects expressed pathways such as RNA polymerase I promoter opening, the matrisome, and endocytosis. In addition, there was large variability in the number of rhythmic transcripts among subjects, regardless of shift type. Interestingly, the amplitude of the CBT rhythm appeared to be more consistent with the number of cycling transcripts for each of the 6 subjects than was melatonin rhythm. In summary, we show that shift-work patterns affect circadian alignment and gene expression in PBMCs.

The impact of meal timing on cardiometabolic syndrome indicators in shift workers.

The aims of this study were to 1) compare the inflammatory potential of night- and day-shift nurses' diets with regard to time of day and work status and 2) explore how the timing of food intake during work and off-work is associated with cardiometabolic syndrome (CMS) risk factors between these two groups. Female nurses (N = 17; 8 day-shift and 9 night-shift) reported food intake over 9 days. On a middle day off of work, metabolic parameters were measured after an overnight fast. Energy/macronutrient intake and inflammatory potential of dietary intake (as assessed via the Dietary Inflammatory IndexTM) were calculated for nurses' workdays, work nights, off-work days, and off-work nights. Work-night total food intake (grams) accounted for a significant amount of variance in CMS risk factors for night-shift nurses only. Increased total gram consumption during night-shift nurses' work nights was associated with increased lipid levels - independent of the macronutrient composition of the food consumed. Alternatively, for night-shift nurses, work-day intake of several food parameters accounted for a significant proportion of variance in HDL cholesterol levels, with higher intake associated with higher HDL levels. For both day- and night-shift nurses, food intake during the day was more pro-inflammatory regardless of shift type or work status. Our novel approach of combining time-of-day-specific and work-day-specific analyses of dietary inflammatory factors and macronutrient composition with measurement of CMS risk factors suggests a link between meal timing and cardiometabolic health for shift-working nurses.

Circadian rhythmicity of active GSK3 isoforms modulates molecular clock gene rhythms in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) drives and synchronizes daily rhythms at the cellular level via transcriptional-translational feedback loops comprising clock genes such as Bmal1 and Period (Per). Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, phosphorylates at least 5 core clock proteins and shows diurnal variation in phosphorylation state (inactivation) of the GSK3ß isoform. Whether phosphorylation of the other primary isoform (GSK3α) varies across the subjective day-night cycle is unknown. The purpose of this study was to determine if the endogenous rhythm of GSK3 (α and ß) phosphorylation is critical for rhythmic BMAL1 expression and normal amplitude and periodicity of the molecular clock in the SCN. Significant circadian rhythmicity of phosphorylated GSK3 (α and ß) was observed in the SCN from wild-type mice housed in constant darkness for 2 weeks. Importantly, chronic activation of both GSK3 isoforms impaired rhythmicity of the GSK3 target BMAL1. Furthermore, chronic pharmacological inhibition of GSK3 with 20 µM CHIR-99021 enhanced the amplitude and shortened the period of PER2::luciferase rhythms in organotypic SCN slice cultures. These results support the model that GSK3 activity status is regulated by the circadian clock and that GSK3 feeds back to regulate the molecular clock amplitude in the SCN.

Sleep Strategies of Night-Shift Nurses on Days Off: Which Ones are Most Adaptive?

OBJECTIVES: To determine the off-shift sleep strategies of bi-ethnic night-shift nurses, the relationship between these sleep strategies and adaptation to shift work, and identify the participant-level characteristics associated with a given sleep strategy. METHODS: African-American and non-Hispanic White female, night-shift nurses from an academic hospital were recruited to complete a survey on sleep-wake patterns (n = 213). Participants completed the standard shiftwork index and the biological clocks questionnaire to determine sleep strategies and adaptation to night-shift work. In addition, chronotype was determined quantitatively with a modified version of the Munich ChronoType Questionnaire. Most participants worked ~3 consecutive 12-h night-shifts followed by several days off. RESULTS: Five sleep strategies used on days off were identified: (a) night stay, (b) nap proxy, (c) switch sleeper, (d) no sleep, and (e) incomplete switcher. Nap proxy and no sleep types were associated with poorer adaptation to night-shift work. The switch sleeper and incomplete switcher types were identified as more adaptive strategies that were associated with less sleep disturbance, a later chronotype, and less cardiovascular problems. CONCLUSION: Behavioral sleep strategies are related to adaptation to a typical night-shift schedule among hospital nurses. Nurses are crucial to the safety and well-being of their patients. Therefore, adoption of more adaptive sleep strategies may reduce sleep/wake dysregulation in this population, and improve cardiovascular outcomes.

Neuropeptide Y-induced phase shifts of PER2::LUC rhythms are mediated by long-term suppression of neuronal excitability in a phase-specific manner.

Endogenous circadian rhythms are entrained to the 24-h light/dark cycle by both light and nonphotic stimuli. During the day, nonphotic stimuli, such as novel wheel-induced exercise, produce large phase advances. Neuropeptide Y (NPY) release from the thalamus onto suprachiasmatic nucleus (SCN) neurons at least partially mediates this nonphotic signal. The authors examined the hypothesis that NPY-induced phase advances are accompanied by suppression of PER2 and are mediated by long-term depression of neuronal excitability in a phase-specific manner. First, it was found that NPY-induced phase advances in PER2::LUC SCN cultures are largest when NPY (2.35 µM) is given in the early part of the day (circadian time [CT] 0-6). In addition, PER2::LUC levels in NPY-treated (compared to vehicle-treated) samples were suppressed beginning 6-7 h after treatment. Similar NPY application to organotypic Per1::GFP SCN cultures resulted in long-term suppression of spike rate of green fluorescent protein-positive (GFP+) cells when slices were treated with NPY during the early or middle of the day (zeitgeber time [ZT] 2 or 6), but not during the late day (ZT 10). Furthermore, 1-h bath application of NPY to acute SCN brain slices decreased general neuronal activity measured through extracellular recordings. Finally, NPY-induced phase advances of PER2::LUC rhythms were blocked by latent depolarization with 34.5 mM K(+) 3 h after NPY application. These results suggest that NPY-induced phase advances may be mediated by long-term depression of neuronal excitability. This model is consistent with findings in other brain regions that NPY-induced persistent hyperpolarization underlies mechanisms of energy homeostasis, anxiety-related behavior, and thalamocortical synchronous firing.

'Disease is unrhythmical': jazz, health, and disability in 1920s America.

The 1920s in the United States are commonly remembered as the Jazz Age. Although historians have focused on the African American origins of the music, another theme was also prominent in the public discourse surrounding jazz: disability. Critics saw jazz and its associated dances as defective, causing both mental and physical impairments in their devotees. In other words, jazz music and dance were disabled and disabling. Proponents of jazz responded in kind, asserting that jazz did not cause impairments, it cured them; similarly, jazz was not defective music or dance, but a revitalisation of the art forms. On the one hand, these reactions might have been expected, given the long history of belief in a relationship between music and health. However, the importance of health issues such as eugenics and rehabilitation in the 1920s also clearly influenced the responses of opinion leaders, politicians, academics, music professionals, and others to jazz music and dance.