RNA-Interference Knockdown of Drosophila Pigment Dispersing Factor in Neuronal Subsets: The Anatomical Basis of a Neuropeptide's Circadian Functions

Background

In animals, neuropeptide signaling is an important component of circadian timekeeping. The neuropeptide pigment dispersing factor (PDF) is required for several aspects of circadian activity rhythms in Drosophila.

Methodology/Principal Findings

Here we investigate the anatomical basis for PDF''s various circadian functions by targeted PDF RNA-interference in specific classes of Drosophila neuron. We demonstrate that PDF is required in the ventro-lateral neurons (vLNs) of the central brain and not in the abdominal ganglion for normal activity rhythms. Differential knockdown of PDF in the large or small vLNs indicates that PDF from the small vLNs is likely responsible for the maintenance of free-running activity rhythms and that PDF is not required in the large vLNs for normal behavior. PDF''s role in setting the period of free-running activity rhythms and the proper timing of evening activity under light:dark cycles emanates from both subtypes of vLN, since PDF in either class of vLN was sufficient for these aspects of behavior.

Conclusions/Significance

These results reveal the neuroanatomical basis PDF''s various circadian functions and refine our understanding of the clock neuron circuitry of Drosophila.     

Metabolic cycles are linked to the cardiovascular diurnal rhythm in rats with essential hypertension

Background

The loss of diurnal rhythm in blood pressure (BP) is an important predictor of end-organ damage in hypertensive and diabetic patients. Recent evidence has suggested that two major physiological circadian rhythms, the metabolic and cardiovascular rhythms, are subject to regulation by overlapping molecular pathways, indicating that dysregulation of metabolic cycles could desynchronize the normal diurnal rhythm of BP with the daily light/dark cycle. However, little is known about the impact of changes in metabolic cycles on BP diurnal rhythm.

Methodology/Principal Findings

To test the hypothesis that feeding-fasting cycles could affect the diurnal pattern of BP, we used spontaneously hypertensive rats (SHR) which develop essential hypertension with disrupted diurnal BP rhythms and examined whether abnormal BP rhythms in SHR were caused by alteration in the daily feeding rhythm. We found that SHR exhibit attenuated feeding rhythm which accompanies disrupted rhythms in metabolic gene expression not only in metabolic tissues but also in cardiovascular tissues. More importantly, the correction of abnormal feeding rhythms in SHR restored the daily BP rhythm and was accompanied by changes in the timing of expression of key circadian and metabolic genes in cardiovascular tissues.

Conclusions/Significance

These results indicate that the metabolic cycle is an important determinant of the cardiovascular diurnal rhythm and that disrupted BP rhythms in hypertensive patients can be normalized by manipulating feeding cycles.     

Shift work in nurses: contribution of phenotypes and genotypes to adaptation

Background

Daily cycles of sleep/wake, hormones, and physiological processes are often misaligned with behavioral patterns during shift work, leading to an increased risk of developing cardiovascular/metabolic/gastrointestinal disorders, some types of cancer, and mental disorders including depression and anxiety. It is unclear how sleep timing, chronotype, and circadian clock gene variation contribute to adaptation to shift work.

Methods

Newly defined sleep strategies, chronotype, and genotype for polymorphisms in circadian clock genes were assessed in 388 hospital day- and night-shift nurses.

Results

Night-shift nurses who used sleep deprivation as a means to switch to and from diurnal sleep on work days (∼25%) were the most poorly adapted to their work schedule. Chronotype also influenced efficacy of adaptation. In addition, polymorphisms in CLOCK, NPAS2, PER2, and PER3 were significantly associated with outcomes such as alcohol/caffeine consumption and sleepiness, as well as sleep phase, inertia and duration in both single- and multi-locus models. Many of these results were specific to shift type suggesting an interaction between genotype and environment (in this case, shift work).

Conclusions

Sleep strategy, chronotype, and genotype contribute to the adaptation of the circadian system to an environment that switches frequently and/or irregularly between different schedules of the light-dark cycle and social/workplace time. This study of shift work nurses illustrates how an environmental “stress” to the temporal organization of physiology and metabolism can have behavioral and health-related consequences. Because nurses are a key component of health care, these findings could have important implications for health-care policy.     

Genetic adaptation of the human circadian clock to day-length latitudinal variations and relevance for affective disorders

Background

The temporal coordination of biological processes into daily cycles is a common feature of most living organisms. In humans, disruption of circadian rhythms is commonly observed in psychiatric diseases, including schizophrenia, bipolar disorder, depression and autism. Light therapy is the most effective treatment for seasonal affective disorder and circadian-related treatments sustain antidepressant response in bipolar disorder patients. Day/night cycles represent a major circadian synchronizing signal and vary widely with latitude.

Results

We apply a geographically explicit model to show that out-of-Africa migration, which led humans to occupy a wide latitudinal area, affected the evolutionary history of circadian regulatory genes. The SNPs we identify using this model display consistent signals of natural selection using tests based on population genetic differentiation and haplotype homozygosity. Signals of natural selection driven by annual photoperiod variation are detected for schizophrenia, bipolar disorder, and restless leg syndrome risk variants, in line with the circadian component of these conditions.

Conclusions

Our results suggest that human populations adapted to life at different latitudes by tuning their circadian clock systems. This process also involves risk variants for neuropsychiatric conditions, suggesting possible genetic modulators for chronotherapies and candidates for interaction analysis with photoperiod-related environmental variables, such as season of birth, country of residence, shift-work or lifestyle habits.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0499-7) contains supplementary material, which is available to authorized users.     

Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation

Background

Circadian rhythms govern a large array of physiological and metabolic functions. To achieve plasticity in circadian regulation, proteins constituting the molecular clock machinery undergo various post-translational modifications (PTMs), which influence their activity and intracellular localization. The core clock protein BMAL1 undergoes several PTMs. Here we report that the Akt-GSK3β signaling pathway regulates BMAL1 protein stability and activity.

Principal Findings

GSK3β phosphorylates BMAL1 specifically on Ser 17 and Thr 21 and primes it for ubiquitylation. In the absence of GSK3β-mediated phosphorylation, BMAL1 becomes stabilized and BMAL1 dependent circadian gene expression is dampened. Dopamine D2 receptor mediated signaling, known to control the Akt-GSK3β pathway, influences BMAL1 stability and in vivo circadian gene expression in striatal neurons.

Conclusions

These findings uncover a previously unknown mechanism of circadian clock control. The GSK3β kinase phosphorylates BMAL1, an event that controls the stability of the protein and the amplitude of circadian oscillation. BMAL1 phosphorylation appears to be an important regulatory step in maintaining the robustness of the circadian clock.     

The physiological period length of the human circadian clock in vivo is directly proportional to period in human fibroblasts

Background

Diurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype (“larks” and “owls”), clock properties measured in human fibroblasts correlated with extreme diurnal behavior.

Methodology/Principal Findings

In this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer.

Conclusions/Significance

We conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness.     

Characterization of Circadian Behavior in the Starlet Sea Anemone,Nematostella vectensis

Background

Although much is known about how circadian systems control daily cycles in the physiology and behavior of Drosophila and several vertebrate models, marine invertebrates have often been overlooked in circadian rhythms research. This study focuses on the starlet sea anemone, Nematostella vectensis, a species that has received increasing attention within the scientific community for its potential as a model research organism. The recently sequenced genome of N. vectensis makes it an especially attractive model for exploring the molecular evolution of circadian behavior. Critical behavioral data needed to correlate gene expression patterns to specific behaviors are currently lacking in N. vectensis.

Methodology/Principal Findings

To detect the presence of behavioral oscillations in N. vectensis, locomotor activity was evaluated using an automated system in an environmentally controlled chamber. Animals exposed to a 24 hr photoperiod (12 hr light: 12 hr dark) exhibited locomotor behavior that was both rhythmic and predominantly nocturnal. The activity peak occurred in the early half of the night with a 2-fold increase in locomotion. Upon transfer to constant lighting conditions (constant light or constant dark), an approximately 24 hr rhythm persisted in most animals, suggesting that the rhythm is controlled by an endogenous circadian mechanism. Fourier analysis revealed the presence of multiple peaks in some animals suggesting additional rhythmic components could be present. In particular, an approximately 12 hr oscillation was often observed. The nocturnal increase in generalized locomotion corresponded to a 24 hr oscillation in animal elongation.

Conclusions/Significance

These data confirm the presence of a light-entrainable circadian clock in Nematostella vectensis. Additional components observed in some individuals indicate that an endogenous clock of approximately 12 hr frequency may also be present. By describing rhythmic locomotor behavior in N. vectensis, we have made important progress in developing the sea anemone as a model organism for circadian rhythm research.