John S. O'Neill

Synchronization and maintenance of timekeeping in suprachiasmatic circadian clock cells by neuropeptidergic signaling.

Circadian timekeeping in mammals is driven by transcriptional/posttranslational feedback loops that are active within both peripheral tissues and the circadian pacemaker of the suprachiasmatic nuclei (SCN). Spontaneous synchronization of these molecular loops between SCN neurons is a primary requirement of its pacemaker role and distinguishes it from peripheral tissues, which require extrinsic, SCN-dependent cues to impose cellular synchrony. Vasoactive intestinal polypeptide (VIP) is an intrinsic SCN factor implicated in acute activation and electrical synchronization of SCN neurons and coordination of behavioral rhythms. Using real-time imaging of cellular circadian gene expression across entire SCN slice cultures, we show for the first time that the Vipr2 gene encoding the VPAC2 receptor for VIP is necessary both to maintain molecular timekeeping within individual SCN neurons and to synchronize molecular timekeeping between SCN neurons embedded within intact, organotypical circuits. Moreover, we demonstrate that both depolarization and a second SCN neuropeptide, gastrin-releasing peptide (GRP), can acutely enhance and synchronize molecular timekeeping in Vipr2-/- SCN neurons. Nevertheless, transiently activated and synchronized Vipr2-/- cells cannot sustain synchrony in the absence of VIP-ergic signaling. Hence, neuropeptidergic interneuronal signaling confers a canonical property upon the SCN: spontaneous synchronization of the intracellular molecular clockworks of individual neurons.

cAMP-Dependent Signaling as a Core Component of the Mammalian Circadian Pacemaker

The mammalian circadian clockwork is modeled as transcriptional and posttranslational feedback loops, whereby circadian genes are periodically suppressed by their protein products. We show that adenosine 3′,5′-monophosphate (cAMP) signaling constitutes an additional, bona fide component of the oscillatory network. cAMP signaling is rhythmic and sustains the transcriptional loop of the suprachiasmatic nucleus, determining canonical pacemaker properties of amplitude, phase, and period. This role is general and is evident in peripheral mammalian tissues and cell lines, which reveals an unanticipated point of circadian regulation in mammals qualitatively different from the existing transcriptional feedback model. We propose that daily activation of cAMP signaling, driven by the transcriptional oscillator, in turn sustains progression of transcriptional rhythms. In this way, clock output constitutes an input to subsequent cycles.

Circadian clocks: regulators of endocrine and metabolic rhythms

Daily and seasonal rhythms in the endocrine system are co-ordinated by a hypothalamic pacemaker, the suprachiasmatic nuclei (SCN) that is synchronised to solar time by direct retinal afferents. Individual SCN neurons are circadian clocks, their intrinsic oscillator consisting of a series of interlinked autoregulatory transcriptional/post-translational feedback loops incorporating Period (Per) and Cryptochrome (Cry) genes. Mutations that alter the rate of transcription of Per and Cry genes or the stability of Per and Cry proteins affect clock speed. Molecular timekeeping in SCN neurons is synchronised and sustained by interneuronal neuropeptidergic signals. A molecular clock mechanism comparable to that of the SCN is present in most major organ systems. These tissue clocks are synchronised by endocrine, autonomic and behavioural cues that are dependent on the SCN, and in turn they drive the circadian expression of local transcriptomes, thereby co-ordinating circadian metabolism and physiology. Rhythmic glucocorticoid signalling is a prominent mediator of SCN output and internal synchroniser. The role of local SCN-synchronised clocks in controlling vital processes, including xenobiotic detoxification, cell division and nutrient metabolism, is essential to health, and disturbances to circadian timing arising from modern working schedules are becoming recognised as an increasingly relevant factor in major systemic illness. Moreover, the newly identified molecular components of circadian control systems provide novel avenues for therapeutic intervention.

Sperm Deliver a New Second Messenger: NAADP

NAADP is a highly potent mobilizer of Ca(2+), which in turn triggers Ca(2+)-induced Ca(2+) release pathways in a wide range of species. Nevertheless, NAADP is not presently classified as a second messenger because it has not been shown to increase in response to a physiological stimulus. We now report a dramatic increase in NAADP during sea urchin egg fertilization that was largely due to production in sperm upon contacting egg jelly. The NAADP bolus plays a physiological role upon delivery to the egg based on its ability to induce a cortical flash, a depolarization-induced activation of L-type Ca(2+) channels. Moreover, the sperm-induced cortical flash was eliminated in eggs desensitized to NAADP. We conclude that an NAADP increase plays a physiologically relevant role during fertilization and provides the first conclusive demonstration that NAADP is a genuine second messenger.

Multiple light inputs to a simple clock circuit allow complex biological rhythms

Circadian clocks are biological timekeepers that allow living cells to time their activity in anticipation of predictable environmental changes. Detailed understanding of the circadian network of higher plants, such as Arabidopsis thaliana, is hampered by the high number of partially redundant genes. However, the picoeukaryotic alga Ostreococcus tauri, which was recently shown to possess a small number of non-redundant clock genes, presents an attractive alternative target for detailed modelling of circadian clocks in the green lineage. Based on extensive time-series data from in vivo reporter gene assays, we developed a model of the Ostreococcus clock as a feedback loop between the genes TOC1 and CCA1. The model reproduces the dynamics of the transcriptional and translational reporters over a range of photoperiods. Surprisingly, the model is also able to predict the transient behaviour of the clock when the light conditions are altered. Despite the apparent simplicity of the clock circuit, it displays considerable complexity in its response to changing light conditions. Systematic screening of the effects of altered day length revealed a complex relationship between phase and photoperiod, which is also captured by the model. The complex light response is shown to stem from circadian gating of light-dependent mechanisms. This study provides insights into the contributions of light inputs to the Ostreococcus clock. The model suggests that a high number of light-dependent reactions are important for flexible timing in a circadian clock with only one feedback loop.

Shotgun proteomic analysis of the unicellular alga Ostreococcus tauri

Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living eukaryotes. The O. tauri genome sequence was determined in 2006. Molecular, physiological and taxonomic data that has been generated since then highlight its potential as a simple model species for algae and plants. However, its proteome remains largely unexplored. This paper describes the global proteomic study of O. tauri, using mass spectrometry-based approaches: phosphopeptide enrichment, cellular fractionation, label-free quantification and (15)N metabolic labeling. The O. tauri proteome was analyzed under the following conditions: sampling at different times during the circadian cycle, after 24h of illumination, after 24h of darkness and under various nitrogen source supply levels. Cell cycle related proteins such as dynamin and kinesin were significantly up-regulated during the daylight-to-darkness transition. This is reflected by their higher intensity at ZT13 and this transition phase coincides with the end of mitosis. Proteins involved in several metabolic mechanisms were found to be up-regulated under low nitrogen conditions, including carbon storage pathways, glycolysis, phosphate transport, and the synthesis of inorganic polyphosphates. Ostreococcus tauri responds to low nitrogen conditions by reducing its nitrogen assimilation machinery which suggests an atypical adaptation mechanism for coping with a nutrient-limited environment.

Circadian regulation of olfaction and an evolutionarily conserved, nontranscriptional marker in Caenorhabditis elegans

Circadian clocks provide a temporal structure to processes from gene expression to behavior in organisms from all phyla. Most clocks are synchronized to the environment by alternations of light and dark. However, many organisms experience only muted daily environmental cycles due to their lightless spatial niches (e.g., caves or soil). This has led to speculation that they may dispense with the daily clock. However, recent reports contradict this notion, showing various behavioral and molecular rhythms in Caenorhabditis elegans and in blind cave fish. Based on the ecology of nematodes, we applied low-amplitude temperature cycles to synchronize populations of animals through development. This entrainment regime reveals rhythms on multiple levels: in olfactory cued behavior, in RNA and protein abundance, and in the oxidation state of a broadly conserved peroxiredoxin protein. Our work links the nematode clock with that of other clock model systems; it also emphasizes the importance of daily rhythms in sensory functions that are likely to impact on organism fitness and population structure.

The essential role of cAMP/Ca2+ signalling in mammalian circadian timekeeping

Approximately daily, or circadian, rhythms are ubiquitous across eukaryotes. They are manifest in the temporal co-ordination of metabolism, physiology and behaviour, thereby allowing organisms to anticipate and synchronize with daily environmental cycles. Although cellular rhythms are self-sustained and cell-intrinsic, in mammals, the master regulator of timekeeping is localized within the hypothalamic SCN (suprachiasmatic nucleus). Molecular models for mammalian circadian rhythms have focused largely on transcriptional-translational feedback loops, but recent data have revealed essential contributions by intracellular signalling mechanisms. cAMP and Ca2+ signalling are not only regulated by the cellular clock, but also contribute directly to the timekeeping mechanism, in that appropriate manipulations determine the canonical pacemaker properties of amplitude, phase and period. It is proposed that daily auto-amplification of second messenger activity, through paracrine neuropeptidergic coupling, is necessary and sufficient to account for the increased amplitude, accuracy and robustness of SCN timekeeping.

Increased Coherence of Circadian Rhythms in Mature Fibroblast Cultures

Circadian transcriptional rhythms in confluent NIH 3T3 and PER2::LUCIFERASE fibroblast cultures become more coherent as they mature. This enhanced coherence correlated with increased culture density, while inhibition of the cell cycle increased coherence in immature cultures. Mature cultures may therefore express more coherent rhythms for longer because of their reduced rate of cell division. The authors conclude that mature fibroblast cultures will be of great utility for high-throughput pharmacological and mutational circadian screening.

Cry1 Circadian Phase in vitro: Wrapped Up with an E-Box

The circadian timing of gene expression is determined by transcriptional regulation through upstream response elements present throughout the genome. Central to this regulation are the actions of a core group of transcriptional activators and repressors, which act through, and are themselves regulated by, a small set of canonical circadian response elements. Among these, the E-box (CACGTG) is crucial for daytime transcriptional activity. The mammalian Period (Per1-3) and Cryptochrome (Cry1-2) genes are E-box—regulated genes, but in peripheral tissues peak Cry1 mRNA expression is delayed by several hours relative to that of Per. It has been proposed that this delay originates from interactions between the proximal E-box and retinoic acid—related orphan receptor response elements (RORE) present in the Cry1 promoter. By using real-time luciferase reporter assays in NIH3T3 cells the authors show here that a proximal 47-bp E-box containing region of the Cry1 promoter is both necessary and sufficient to drive circadian Cry1 transcription with an appropriate phase delay (around 4 h) relative to Per2. The results therefore suggest that, at least in this in vitro model of the clock, RORE are not necessary for the appropriate circadian regulation of Cry1 expression and rather suggest that sequences surrounding the proximal E-boxes confer gene-specific circadian phasing.