CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping.

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ε activity to be maintained. In the absence of CRY-mediated feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational.

The VIP-VPAC2 neuropeptidergic axis is a cellular pacemaking hub of the suprachiasmatic nucleus circadian circuit.

The hypothalamic suprachiasmatic nuclei (SCN) are the principal mammalian circadian timekeeper, co-ordinating organism-wide daily and seasonal rhythms. To achieve this, cell-autonomous circadian timing by the ~20,000 SCN cells is welded into a tight circuit-wide ensemble oscillation. This creates essential, network-level emergent properties of precise, high-amplitude oscillation with tightly defined ensemble period and phase. Although synchronised, regional cell groups exhibit differentially phased activity, creating stereotypical spatiotemporal circadian waves of cellular activation across the circuit. The cellular circuit pacemaking components that generate these critical emergent properties are unknown. Using intersectional genetics and real-time imaging, we show that SCN cells expressing vasoactive intestinal polypeptide (VIP) or its cognate receptor, VPAC2, are neurochemically and electrophysiologically distinct, but together they control de novo rhythmicity, setting ensemble period and phase with circuit-level spatiotemporal complexity. The VIP/VPAC2 cellular axis is therefore a neurochemically and topologically specific pacemaker hub that determines the emergent properties of the SCN timekeeper.

Cell-autonomous clock of astrocytes drives circadian behavior in mammals.

Circadian (~24-hour) rhythms depend on intracellular transcription-translation negative feedback loops (TTFLs). How these self-sustained cellular clocks achieve multicellular integration and thereby direct daily rhythms of behavior in animals is largely obscure. The suprachiasmatic nucleus (SCN) is the fulcrum of this pathway from gene to cell to circuit to behavior in mammals. We describe cell type-specific, functionally distinct TTFLs in neurons and astrocytes of the SCN and show that, in the absence of other cellular clocks, the cell-autonomous astrocytic TTFL alone can drive molecular oscillations in the SCN and circadian behavior in mice. Astrocytic clocks achieve this by reinstating clock gene expression and circadian function of SCN neurons via glutamatergic signals. Our results demonstrate that astrocytes can autonomously initiate and sustain complex mammalian behavior.

Circadian rhythms and metabolism: from the brain to the gut and back again.

This paper focuses on the relationship between the circadian system and glucose metabolism. Research across the translational spectrum confirms the importance of the circadian system for glucose metabolism and offers promising clues as to when and why these systems go awry. In particular, basic research has started to clarify the molecular and genetic mechanisms through which the circadian system regulates metabolism. The study of human behavior, especially in the context of psychiatric disorders, such as bipolar disorder and major depression, forces us to see how inextricably linked mental health and metabolic health are. We also emphasize the remarkable opportunities for advancing circadian science through big data and advanced analytics. Advances in circadian research have translated into environmental and pharmacological interventions with tremendous therapeutic potential.

Rhythmic expression of cryptochrome induces the circadian clock of arrhythmic suprachiasmatic nuclei through arginine vasopressin signaling.

Circadian rhythms in mammals are coordinated by the suprachiasmatic nucleus (SCN). SCN neurons define circadian time using transcriptional/posttranslational feedback loops (TTFL) in which expression of Cryptochrome (Cry) and Period (Per) genes is inhibited by their protein products. Loss of Cry1 and Cry2 stops the SCN clock, whereas individual deletions accelerate and decelerate it, respectively. At the circuit level, neuronal interactions synchronize cellular TTFLs, creating a spatiotemporal wave of gene expression across the SCN that is lost in Cry1/2-deficient SCN. To interrogate the properties of CRY proteins required for circadian function, we expressed CRY in SCN of Cry-deficient mice using adeno-associated virus (AAV). Expression of CRY1::EGFP or CRY2::EGFP under a minimal Cry1 promoter was circadian and rapidly induced PER2-dependent bioluminescence rhythms in previously arrhythmic Cry1/2-deficient SCN, with periods appropriate to each isoform. CRY1::EGFP appropriately lengthened the behavioral period in Cry1-deficient mice. Thus, determination of specific circadian periods reflects properties of the respective proteins, independently of their phase of expression. Phase of CRY1::EGFP expression was critical, however, because constitutive or phase-delayed promoters failed to sustain coherent rhythms. At the circuit level, CRY1::EGFP induced the spatiotemporal wave of PER2 expression in Cry1/2-deficient SCN. This was dependent on the neuropeptide arginine vasopressin (AVP) because it was prevented by pharmacological blockade of AVP receptors. Thus, our genetic complementation assay reveals acute, protein-specific induction of cell-autonomous and network-level circadian rhythmicity in SCN never previously exposed to CRY. Specifically, Cry expression must be circadian and appropriately phased to support rhythms, and AVP receptor signaling is required to impose circuit-level circadian function.

Genetic analysis of circadian responses to low frequency electromagnetic fields in Drosophila melanogaster.

The blue-light sensitive photoreceptor cryptochrome (CRY) may act as a magneto-receptor through formation of radical pairs involving a triad of tryptophans. Previous genetic analyses of behavioral responses of Drosophila to electromagnetic fields using conditioning, circadian and geotaxis assays have lent some support to the radical pair model (RPM). Here, we describe a new method that generates consistent and reliable circadian responses to electromagnetic fields that differ substantially from those already reported. We used the Schuderer apparatus to isolate Drosophila from local environmental variables, and observe extremely low frequency (3 to 50 Hz) field-induced changes in two locomotor phenotypes, circadian period and activity levels. These field-induced phenotypes are CRY- and blue-light dependent, and are correlated with enhanced CRY stability. Mutational analysis of the terminal tryptophan of the triad hypothesised to be indispensable to the electron transfer required by the RPM reveals that this residue is not necessary for field responses. We observe that deletion of the CRY C-terminus dramatically attenuates the EMF-induced period changes, whereas the N-terminus underlies the hyperactivity. Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change. Finally, we observe that hCRY2, but not hCRY1, transformants can detect EMFs, suggesting that hCRY2 is blue light-responsive. In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect. Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.

Analysis of core circadian feedback loop in suprachiasmatic nucleus of mCry1-luc transgenic reporter mouse.

The suprachiasmatic nucleus (SCN) coordinates circadian rhythms that adapt the individual to solar time. SCN pacemaking revolves around feedback loops in which expression of Period (Per) and Cryptochrome (Cry) genes is periodically suppressed by their protein products. Specifically, PER/CRY complexes act at E-box sequences in Per and Cry to inhibit their transactivation by CLOCK/BMAL1 heterodimers. To function effectively, these closed intracellular loops need to be synchronized between SCN cells and to the light/dark cycle. For Per expression, this is mediated by neuropeptidergic and glutamatergic extracellular cues acting via cAMP/calcium-responsive elements (CREs) in Per genes. Cry genes, however, carry no CREs, and how CRY-dependent SCN pacemaking is synchronized remains unclear. Furthermore, whereas reporter lines are available to explore Per circadian expression in real time, no Cry equivalent exists. We therefore created a mouse, B6.Cg-Tg(Cry1-luc)01Ld, carrying a transgene (mCry1-luc) consisting of mCry1 elements containing an E-box and E'-box driving firefly luciferase. mCry1-luc organotypic SCN slices exhibited stable circadian bioluminescence rhythms with appropriate phase, period, profile, and spatial organization. In SCN lacking vasoactive intestinal peptide or its receptor, mCry1 expression was damped and desynchronized between cells. Despite the absence of CREs, mCry1-luc expression was nevertheless (indirectly) sensitive to manipulation of cAMP-dependent signaling. In mPer1/2-null SCN, mCry1-luc bioluminescence was arrhythmic and no longer suppressed by elevation of cAMP. Finally, an SCN graft procedure showed that PER-independent as well as PER-dependent mechanisms could sustain circadian expression of mCry1. The mCry1-luc mouse therefore reports circadian mCry1 expression and its interactions with vasoactive intestinal peptide, cAMP, and PER at the heart of the SCN pacemaker.

Evaluating the frequency of bacterial co-infections in children recruited into a malaria pathogenesis study in The Gambia, West Africa using molecular methods.

Systemic bacteraemia has been reported in children with severe Plasmodium falciparum malaria in Sub Saharan Africa, making the identification or exclusion of concurrent infections a prerequisite for adequate treatment and studies of the immune responses to particular infections. Given the overlap in clinical signs in humans between malaria and, for example, pneumonia, the true cause of severe illness is sometimes difficult to establish. Traditional microbiological culture methods employed to detect systemic bacteraemia are often time consuming and have modest sensitivity. Therefore, molecular methods have become increasingly used in the diagnosis of septicaemia. Here, we evaluated the usefulness of both broad-range 16S rRNA PCR, in conjunction with DNA sequencing and species-specific PCR targeting of Streptococcus pneumoniae and non-typhoidal Salmonella, to screen for bacterial co-infections in blood samples from children enrolled in a malaria pathogenesis study. PCR revealed no test-positive results for these pathogens and DNA sequencing of 16S rRNA amplicons identified the presence of bacterial genomic DNA (most probably from environmental bacterial sources) in a large proportion of samples. We demonstrate that the issue of potential mixed bacteraemic infection and/or background bacterial genomic DNA, which may relate to co-migration of PCR amplicons on agarose gels, can be overcome by using denaturing gradient gel electrophoresis (DGGE). PCR for Plasmodium spp. was also performed on genomic DNA from bloods from Gambian children with pneumonia, in order to estimate the prevalence of Plasmodium/pneumonia co-infections in the study population. While 12.2% of samples were test-positive, parasite density was very low and did not vary significantly between cases and controls.