Circadian Rhythms and Astrocytes: The Good, the Bad, and the Ugly.

This review explores the interface between circadian timekeeping and the regulation of brain function by astrocytes. Although astrocytes regulate neuronal activity across many time domains, their cell-autonomous circadian clocks exert a particular role in controlling longer-term oscillations of brain function: the maintenance of sleep states and the circadian ordering of sleep and wakefulness. This is most evident in the central circadian pacemaker, the suprachiasmatic nucleus, where the molecular clock of astrocytes suffices to drive daily cycles of neuronal activity and behavior. In Alzheimer's disease, sleep impairments accompany cognitive decline. In mouse models of the disease, circadian disturbances accelerate astroglial activation and other brain pathologies, suggesting that daily functions in astrocytes protect neuronal homeostasis. In brain cancer, treatment in the morning has been associated with prolonged survival, and gliomas have daily rhythms in gene expression and drug sensitivity. Thus, circadian time is fast becoming critical to elucidating reciprocal astrocytic-neuronal interactions in health and disease.

The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

Bmal1 integrates circadian function and temperature sensing in the suprachiasmatic nucleus.

Circadian regulation and temperature dependency are important orchestrators of molecular pathways. How the integration between these two drivers is achieved, is not understood. We monitored circadian- and temperature-dependent effects on transcription dynamics of cold-response protein RNA Binding Motif 3 (Rbm3). Temperature changes in the mammalian master circadian pacemaker, the suprachiasmatic nucleus (SCN), induced Rbm3 transcription and regulated its circadian periodicity, whereas the core clock gene Per2 was unaffected. Rbm3 induction depended on a full Brain And Muscle ARNT-Like Protein 1 (Bmal1) complement: reduced Bmal1 erased Rbm3 responses and weakened SCN circuit resilience to temperature changes. By focusing on circadian and temperature dependency, we highlight weakened transmission between core clock and downstream pathways as a potential route for reduced circadian resilience.

Generation of circadian rhythms in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) of the hypothalamus is remarkable. Despite numbering only about 10,000 neurons on each side of the third ventricle, the SCN is our principal circadian clock, directing the daily cycles of behaviour and physiology that set the tempo of our lives. When this nucleus is isolated in organotypic culture, its autonomous timing mechanism can persist indefinitely, with precision and robustness. The discovery of the cell-autonomous transcriptional and post-translational feedback loops that drive circadian activity in the SCN provided a powerful exemplar of the genetic specification of complex mammalian behaviours. However, the analysis of circadian time-keeping is moving beyond single cells. Technical and conceptual advances, including intersectional genetics, multidimensional imaging and network theory, are beginning to uncover the circuit-level mechanisms and emergent properties that make the SCN a uniquely precise and robust clock. However, much remains unknown about the SCN, not least the intrinsic properties of SCN neurons, its circuit topology and the neuronal computations that these circuits support. Moreover, the convention that the SCN is a neuronal clock has been overturned by the discovery that astrocytes are an integral part of the timepiece. As a test bed for examining the relationships between genes, cells and circuits in sculpting complex behaviours, the SCN continues to offer powerful lessons and opportunities for contemporary neuroscience.

A spatio-temporal model to reveal oscillator phenotypes in molecular clocks: Parameter estimation elucidates circadian gene transcription dynamics in single-cells.

We propose a stochastic distributed delay model together with a Markov random field prior and a measurement model for bioluminescence-reporting to analyse spatio-temporal gene expression in intact networks of cells. The model describes the oscillating time evolution of molecular mRNA counts through a negative transcriptional-translational feedback loop encoded in a chemical Langevin equation with a probabilistic delay distribution. The model is extended spatially by means of a multiplicative random effects model with a first order Markov random field prior distribution. Our methodology effectively separates intrinsic molecular noise, measurement noise, and extrinsic noise and phenotypic variation driving cell heterogeneity, while being amenable to parameter identification and inference. Based on the single-cell model we propose a novel computational stability analysis that allows us to infer two key characteristics, namely the robustness of the oscillations, i.e. whether the reaction network exhibits sustained or damped oscillations, and the profile of the regulation, i.e. whether the inhibition occurs over time in a more distributed versus a more direct manner, which affects the cells' ability to phase-shift to new schedules. We show how insight into the spatio-temporal characteristics of the circadian feedback loop in the suprachiasmatic nucleus (SCN) can be gained by applying the methodology to bioluminescence-reported expression of the circadian core clock gene Cry1 across mouse SCN tissue. We find that while (almost) all SCN neurons exhibit robust cell-autonomous oscillations, the parameters that are associated with the regulatory transcription profile give rise to a spatial division of the tissue between the central region whose oscillations are resilient to perturbation in the sense that they maintain a high degree of synchronicity, and the dorsal region which appears to phase shift in a more diversified way as a response to large perturbations and thus could be more amenable to entrainment.

Astrocyte Circadian Timekeeping in Brain Health and Neurodegeneration.

Almost three decades ago, astrocytes neighbouring clock neurons of the suprachiasmatic nucleus, the hypothalamic tissue responsible for synchronising circadian timekeeping in mammals, were found to undergo morphological and protein expression changes in a cyclic 24-h pattern, suggesting that glia could harbour circadian timekeeping mechanisms and that neuron-glia interactions could play a part in the daily organisation of rhythms of physiology and behaviour. Recently, it has become clear that astrocytes are circadian timekeepers, capable of initiating daily patterns of behaviour and imposing their intrinsic circadian tempo in mammals. In this chapter, we will describe properties of intracellular timekeeping of astrocytes and the mechanisms by which astrocytes functionally integrate in brain circuits underlying circadian, sleep, and cognitive behaviours in mammals. We will then discuss how altered astrocyte timekeeping may be involved in early brain vulnerability underpinning neurodegeneration. We will focus on Alzheimer's disease as a template of how altered astrocyte timekeeping may be involved in neurodegeneration, both directly via unbalancing of inflammatory and oxidative stress cellular pathways, and indirectly, by altering sleep and cognitive functions.

Translational switching of Cry1 protein expression confers reversible control of circadian behavior in arrhythmic Cry-deficient mice.

The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional-translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.

Filtering and inference for stochastic oscillators with distributed delays.

MOTIVATION: The time evolution of molecular species involved in biochemical reaction networks often arises from complex stochastic processes involving many species and reaction events. Inference for such systems is profoundly challenged by the relative sparseness of experimental data, as measurements are often limited to a small subset of the participating species measured at discrete time points. The need for model reduction can be realistically achieved for oscillatory dynamics resulting from negative translational and transcriptional feedback loops by the introduction of probabilistic time-delays. Although this approach yields a simplified model, inference is challenging and subject to ongoing research. The linear noise approximation (LNA) has recently been proposed to address such systems in stochastic form and will be exploited here. RESULTS: We develop a novel filtering approach for the LNA in stochastic systems with distributed delays, which allows the parameter values and unobserved states of a stochastic negative feedback model to be inferred from univariate time-series data. The performance of the methods is tested for simulated data. Results are obtained for real data when the model is fitted to imaging data on Cry1, a key gene involved in the mammalian central circadian clock, observed via a luciferase reporter construct in a mouse suprachiasmatic nucleus. AVAILABILITY AND IMPLEMENTATION: Programmes are written in MATLAB and Statistics Toolbox Release 2016 b, The MathWorks, Inc., Natick, Massachusetts, USA. Sample code and Cry1 data are available on GitHub https://github.com/scalderazzo/FLNADD. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

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.

Network-mediated encoding of circadian time: the suprachiasmatic nucleus (SCN) from genes to neurons to circuits, and back.

The transcriptional architecture of intracellular circadian clocks is similar across phyla, but in mammals interneuronal mechanisms confer a higher level of circadian integration. The suprachiasmatic nucleus (SCN) is a unique model to study these mechanisms, as it operates as a ∼24 h clock not only in the living animal, but also when isolated in culture. This "clock in a dish" can be used to address fundamental questions, such as how intraneuronal mechanisms are translated by SCN neurons into circuit-level emergent properties and how the circuit decodes, and responds to, light input. This review addresses recent developments in understanding the relationship between electrical activity, [Ca(2+)]i, and intracellular clocks. Furthermore, optogenetic and chemogenetic approaches to investigate the distinct roles of neurons and glial cells in circuit encoding of circadian time will be discussed, as well as the epigenetic and circuit-level mechanisms that enable the SCN to translate light input into coherent daily rhythms.

The circadian clock time tunes axonal regeneration.

Nerve injuries cause permanent neurological disability due to limited axonal regeneration. Injury-dependent and -independent mechanisms have provided important insight into neuronal regeneration, however, common denominators underpinning regeneration remain elusive. A comparative analysis of transcriptomic datasets associated with neuronal regenerative ability revealed circadian rhythms as the most significantly enriched pathway. Subsequently, we demonstrated that sensory neurons possess an endogenous clock and that their regenerative ability displays diurnal oscillations in a murine model of sciatic nerve injury. Consistently, transcriptomic analysis showed a time-of-day-dependent enrichment for processes associated with axonal regeneration and the circadian clock. Conditional deletion experiments demonstrated that Bmal1 is required for neuronal intrinsic circadian regeneration and target re-innervation. Lastly, lithium enhanced nerve regeneration in wild-type but not in clock-deficient mice. Together, these findings demonstrate that the molecular clock fine-tunes the regenerative ability of sensory neurons and propose compounds affecting clock pathways as a novel approach to nerve repair.

Glia-neuron interplay drives circadian glycosphingolipid homeostasis and structural brain plasticity.

Glia-neuronal interplay is critical for circadian regulation of physiology and behavior. In this issue of Neuron, Vaughen et al. identify daily variations of glycosphingolipids that depend on glia and whose disruption alters proteostasis and daily structural plasticity of Drosophila circadian circuits.

Emx2 and Foxg1 inhibit gliogenesis and promote neuronogenesis.

Neural stem cells (NSCs) give rise to all cell types forming the cortex: neurons, astrocytes, and oligodendrocytes. The transition from the former to the latter ones takes place via lineage-restricted progenitors in a highly regulated way. This process is mastered by large sets of genes, among which some implicated in central nervous system pattern formation. The aim of this study was to disentangle the kinetic and histogenetic roles exerted by two of these genes, Emx2 and Foxg1, in cortico-cerebral precursors. For this purpose, we set up a new integrated in vitro assay design. Embryonic cortical progenitors were transduced with lentiviral vectors driving overexpression of Emx2 and Foxg1 in NSCs and neuronal progenitors. Cells belonging to different neuronogenic and gliogenic compartments were labeled by spectrally distinguishable fluoroproteins driven by cell type-specific promoters and by cell type-specific antibodies and were scored via multiplex cytofluorometry and immunocytofluorescence. A detailed picture of Emx2 and Foxg1 activities in cortico-cerebral histogenesis resulted from this study. Unexpectedly, we found that both genes inhibit gliogenesis and promote neuronogenesis, through distinct mechanisms, and Foxg1 also dramatically stimulates neurite outgrowth. Remarkably, such activities, alone or combined, may be exploited to ameliorate the neuronal output obtainable from neural cultures, for purposes of cell-based brain repair.

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.

The Mammalian Circadian Timing System and the Suprachiasmatic Nucleus as Its Pacemaker.

The past twenty years have witnessed the most remarkable breakthroughs in our understanding of the molecular and cellular mechanisms that underpin circadian (approximately one day) time-keeping. Across model organisms in diverse taxa: cyanobacteria (Synechococcus), fungi (Neurospora), higher plants (Arabidopsis), insects (Drosophila) and mammals (mouse and humans), a common mechanistic motif of delayed negative feedback has emerged as the Deus ex machina for the cellular definition of ca. 24 h cycles. This review will consider, briefly, comparative circadian clock biology and will then focus on the mammalian circadian system, considering its molecular genetic basis, the properties of the suprachiasmatic nucleus (SCN) as the principal circadian clock in mammals and its role in synchronising a distributed peripheral circadian clock network. Finally, it will consider new directions in analysing the cell-autonomous and circuit-level SCN clockwork and will highlight the surprising discovery of a central role for SCN astrocytes as well as SCN neurons in controlling circadian behaviour.

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.

Astrocytes Control Circadian Timekeeping in the Suprachiasmatic Nucleus via Glutamatergic Signaling.

The suprachiasmatic nucleus (SCN) of the hypothalamus orchestrates daily rhythms of physiology and behavior in mammals. Its circadian (∼24 hr) oscillations of gene expression and electrical activity are generated intrinsically and can persist indefinitely in temporal isolation. This robust and resilient timekeeping is generally regarded as a product of the intrinsic connectivity of its neurons. Here we show that neurons constitute only one "half" of the SCN clock, the one metabolically active during circadian daytime. In contrast, SCN astrocytes are active during circadian nighttime, when they suppress the activity of SCN neurons by regulating extracellular glutamate levels. This glutamatergic gliotransmission is sensed by neurons of the dorsal SCN via specific pre-synaptic NMDA receptor assemblies containing NR2C subunits. Remarkably, somatic genetic re-programming of intracellular clocks in SCN astrocytes was capable of remodeling circadian behavioral rhythms in adult mice. Thus, SCN circuit-level timekeeping arises from interdependent and mutually supportive astrocytic-neuronal signaling.

In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells.

Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.