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  • Journal article
    Patton AP, Edwards MD, Smyllie NJ, Hamnett R, Chesham JE, Brancaccio M, Maywood ES, Hastings MHet al., 2020,

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

    , NATURE COMMUNICATIONS, Vol: 11, ISSN: 2041-1723
  • Journal article
    Calderazzo S, Brancaccio M, Finkenstadt B, 2019,

    Filtering and inference for stochastic oscillators with distributed delays

    , Bioinformatics, Vol: 35, Pages: 1380-1387, ISSN: 1367-4803

    MotivationThe 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.ResultsWe 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 implementationProgrammes 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

  • Journal article
    Hastings MH, Maywood ES, Brancaccio M, 2019,

    The mammalian circadian timing system and the suprachiasmatic nucleus as its pacemaker

    , Biology, Vol: 8, ISSN: 2079-7737

    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 exmachina 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.

  • Journal article
    Brancaccio M, Edwards MD, Patton AP, Smyllie NJ, Chesham JE, Maywood ES, Hastings MHet al., 2019,

    Cell-autonomous clock of astrocytes drives circadian behavior in mammals

    , Science, Vol: 363, Pages: 187-192, ISSN: 0036-8075

    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.

  • Journal article
    Maywood ES, Elliott TS, Patton AP, Krogager TP, Chesham JE, Ernst RJ, Beranek V, Brancaccio M, Chin JW, Hastings MHet al., 2018,

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

    , Proceedings of the National Academy of Sciences of the United States of America, Vol: 115, Pages: E12388-E12397, ISSN: 0027-8424

    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.

  • Journal article
    Hastings MH, Maywood ES, Brancaccio M, 2018,

    Generation of circadian rhythms in the suprachiasmatic nucleus

    , NATURE REVIEWS NEUROSCIENCE, Vol: 19, Pages: 453-469, ISSN: 1471-003X
  • Journal article
    Brancaccio M, Patton AP, Chesham JE, Maywood ES, Hastings MHet al., 2017,

    Astrocytes control circadian timekeeping in the suprachiasmatic nucleus via glutamatergic signaling

    , Neuron, Vol: 93, Pages: 1420-1435.e5, ISSN: 0896-6273

    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.

  • Journal article
    Feeney KA, Putker M, Brancaccio M, O'Neill JSet al., 2016,

    In-depth characterization of firefly luciferase as a reporter of circadian gene expression in mammalian cells

    , Journal of Biological Rhythms, Vol: 31, Pages: 540-550, ISSN: 0748-7304

    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.

  • Journal article
    Edwards MD, Brancaccio M, Chesham JE, Maywood ES, Hastings MHet al., 2016,

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

    , Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: 2732-2737, ISSN: 0027-8424

    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.

  • Journal article
    Parsons MJ, Brancaccio M, Sethi S, Maywood ES, Satija R, Edwards JK, Jagannath A, Couch Y, Finelli MJ, Smyllie NJ, Esapa C, Butler R, Barnard AR, Chesham JE, Saito S, Joynson G, Wells S, Foster RG, Oliver PL, Simon MM, Mallon A-M, Hastings MH, Nolan PMet al., 2015,

    The regulatory factor ZFHX3 modifies circadian function in SCN via an AT motif-driven axis

    , Cell, Vol: 162, Pages: 607-621, ISSN: 0092-8674

    We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3Sci), 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 Zfhx3Sci/+ 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 Zfhx3Sci/+ SCN slices. In conclusion, by cloning Zfhx3Sci, we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

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