V12 Circadian rhythms are coupled to metabolism

1. V12 Circadian rhythms are coupled to metabolism Review: The suprachiasmatic nuclei (SCN) of the hypothalamus are the principal circadian pacemaker in mammals, They drive the sleepwake cycle and coordinate subordinate clocks in other tissues. Current understanding: The molecular clockwork within the SCN is being modeled as a combination of transcriptional and posttranslational negative feedback loops. Protein products of Period and Cryptochrome genes periodically suppress their own expression. O‘Neill et al. Science, 320, 949 (2008) Biological Sequence Analysis

2. Circadian rhythms are coupled to metabolism Open question: It is unclear how long-term, high-amplitude oscillations with a daily period are maintained. In particular, transcriptional feedback loops are typically less precise than the oscillation of the circadian clock and oscillate at a higher frequency than one cycle per day. Possible explanations given in V11: - phosphorylation causes delay, - secondary loops give stabilization. O‘Neill et al. Science, 320, 949 (2008) Biological Sequence Analysis

3. Intro: Metabolites in E. coli Each distinct substrate occurs in an average of 2.1 reactions. Ouzonis, Karp, Genome Res. 10, 568 (2000)

4. Intro: Metabolism: Citrate Cycle (TCA cycle) in E.coli

5. Intro: Coupling of gene transcription and metabolites Solid arrows indicate direct associations between genes and proteins (via transcription and translation), between proteins and proteins (via direct physical interactions), between proteins and metabolites (via direct physical interactions or with proteins acting as enzymatic catalysts), and the effect of metabolite binding to genes (via direct interactions). Lines show direct effects, with arrows standing for activation, and bars for inhibition. The dashed lines represent indirect associations between genes that result from the projection onto 'gene space'. For example, gene 1 deactivates gene 2 via protein 1 resulting in an indirect interaction between gene 1 and gene 2 (drawn after [Brazhnik00]).

6. Review (V11): circadian rhythms in mammals Ko & Takahashi Hum Mol Genet 15, R271 (2006) SS 2009 lecture 11 Biological Sequence Analysis 6

7. Evidence for coupling of circadian clocks with metabolism • Recombinant cyanobacterial proteins can sustain circadian cycles of autophosphorylation in vitro, in the absence of transcription, • (2) intracellular signaling molecules cyclic adenosine diphosphate–ribose (cADPR) and Ca2+ are essential regulators of circadian oscillation in Arabidopsis and Drosophila. • This indicates that transcriptional mechanisms may not be the sole, or principal, mediator of circadian pacemaking. O‘Neill et al. Science, 320, 949 (2008)

8. Example of a gene regulatory network O’Neill and co-workers now show that the transcriptional feedback loops of theSCN are sustained by cytoplasmic cAMP signaling. cAMP signaling determines their canonical properties of amplitude, phase, and period. Roles of cAMP? In molluscs, birds, and the mammalian SCN, cAMP is implicated in entrainment or maintenance of clocks, or both, or mediation of clock output. It has not been considered as part of the core oscillator. This extends the concept of the mammalian pacemaker beyond transcriptional feedback to incorporate its integration with rhythmic cAMP-mediated cytoplasmic signaling. O‘Neill et al. Science, 320, 949 (2008)

9. What is cAMP Cyclic adenosine monophosphate (cAMP) is a second messenger that is important in many biological processes. cAMP is derived from ATP and used for intracellular signal transduction in many different organisms, conveying the cAMP dependent pathway. In humans, cyclic AMP works by activating cAMP-dependent protein kinase. Cyclic AMP binds to specific locations on the regulatory units of the protein kinase, and causes dissociation between the regulatory and catalytic subunits Thus it activates the catalytic units and enables them to phosphorylate substrate proteins. www.wikipedia.org

10. Side functions of cAMP There are some minor PKA-independent functions of cAMP, e.g. activation of calcium channels. This provides a minor pathway by which growth hormone releasing hormone causes release growth hormone Picture: Epinephrine (adrenaline) binds its receptor, that associates with an heterotrimeric G protein. The G protein associates with adenylyl cyclase that converts ATP to cAMP, spreading the signal www.wikipedia.org

11. Cyclic cAMP levels in mouse brain We tracked the molecular oscillations of the SCN as circadian emission of bioluminescence by organo-typical slices from transgenic mouse brain. Rhythmic luciferase activity controlled by the Per1 promoter (Per1::luciferase) revealed circadian transcription, and a fusion protein of mPER2 and LUCIFERASE (mPER2::LUC) reported circadian protein synthesis rhythms. Circadian oscillation of cAMP concentration (blue) and PER2::LUC bioluminescence (red), as well as cAMP concentration in SCN slices treated with MDL-12,330A (MDL) or with forskolin plus IBMX. O‘Neill et al. Science, 320, 949 (2008) Interpretation: Under these conditions, the cAMP content of the SCN was circadian.

12. Effect of MDL Idea: can one show that cAMP is the reason for the oscillations? Realization: need to suppress cAMP-production in the cell. Experiment: treat SCN slices with MDL, a potent, irreversible inhibitor of adenylyl cyclase (that synthesizes cAMP) to reduce concentrations of cAMP to basal levels. Interpretation: MDL rapidly suppressed circadian CRE::luciferase activity, presumably through loss of cAMP-dependent activation of CRE sequences. This caused a dose-dependent decrease in the amplitude of cycles of circadian transcription and protein synthesis observed with mPer1::luciferase and mPER2::LUC. O‘Neill et al. Science, 320, 949 (2008)

13. MDL also affects the synchronization of the clock Prolonged exposure to mild levels of MDL (1.0 mM) suppressed and desynchro-nized the transcriptional cycles of SCN cells. O‘Neill et al. Science, 320, 949 (2008)

14. Can one block cAMP action? Time of application of ZD7288 Idea: If cAMP sustains the clock, interference with cAMP effectors should compromise pacemaking. PlanA: treat brain slices with inhibitors of cAMP-dependent protein kinase. This had no effect, however, on circadian gene expression in the SCN. PlanB: But cAMP also acts through hyperpolarizing cyclic nucleotide–gated ion (HCN) channels and through the guanine nucleotide–exchange factors Epac1 and Epac2 (Epac, exchange protein directly activated by cAMP). The irreversible HCN channel blocker ZD7288, which would be expected to hyperpolarize the neuronal membrane, dose-dependently damped circadian gene expression in the SCN. This is consistent with disruption of transcriptional feedback rhythms by other manipulations that hyperpolarize clock neurons. O‘Neill et al. Science, 320, 949 (2008)

15. Can cAMP stimulation be recoved? Idea: Direct activation of the effectors might compensate, therefore, for inactivation of Adenylate Cyclase by MDL. Observation: A hydrolysis-resistant Epac agonist transiently activated oscillations in transcriptional activity in SCN treated with MDL. O‘Neill et al. Science, 320, 949 (2008)

16. slowing cAMP synthesis Idea: if cAMP signaling is an integral component of the SCN pacemaker, altering the rate of cAMP synthesis should affect circadian period. Experiment: 9-(Tetrahydro-2-furyl)-adenine (THFA) is a noncompetitive inhibitor of adenylate cyclase that slows the rate of Gs-stimulated cAMP synthesis, which attenuates peak concentrations. Interpretation: THFA dose-dependently increased the period of circadian pacemaking in the SCN, from 24 to 31 hours, with rapid reversal upon washout O‘Neill et al. Science, 320, 949 (2008)

17. Conclusions on cAMP-coupling Circadian pacemaking in mammals is sustained. Its canonical properties of amplitude, phase, and period are determined by a reciprocal interplay in which transcriptional and posttranslational feedback loops drive rhythms of cAMP signaling. Dynamic changes in cAMP signaling, in turn, regulate transcriptional cycles. Thus, output from the current cycle constitutes an input into subsequent cycles. The interdependence between nuclear and cytoplasmic oscillator elements we describe for cAMP also occurs in the case of Ca2+ and cADPR. This highlights an important newly recognized common logic to circadian pacemaking in widely divergent taxa. O‘Neill et al. Science, 320, 949 (2008)

18. Implications? • These studies raise the question of which mechanisms couple oscillations of intracellular signaling molecules to the transcriptional feedback loops of circadian clocks. • Of the cAMP effectors studied by O’Neill et al., only inhibition of • the hyperpolarization-activated cyclic nucleotide–gated ion channel or • the guanine nucleotide–exchange factors Epac 1 and Epac 2 • suppressed circadian gene expression. Harrising & Nitabach Science, 320, 879 (2008)

19. Implications? Application of an Epac agonist resulted in the phosphorylation and increased activity of cAMP response element–binding (CREB) protein, a transcription factor. This suggests that changes in cAMP signaling could feed into the circadian transcriptional oscillator by regulating the expression of genes that contain binding sites for CREB. Such genes include the circadian clock genes Per1 and Per2. Harrising & Nitabach Science, 320, 879 (2008)

20. Effect of cADPR in plants Dodd et al. determined that cADPR concentration peaks during the early hours of the day. This fluctuation was abolished in plants with defective clock function, indicating that the circadian clock regulates cADPR concentration. cADPR is synthesized from nicotinamide adenine dinucleotide by the enzyme ADP ribosyl cyclase. Nicotinamide, at 10 to 50 mM concentrations, inhibited ADP ribosyl cyclase and weakened circadian [Ca2+]i oscillation in plant cells. Dodd et al. also found a correlation between the expression of circadian- and cADPR-regulated genes. Moreover, decreasing the cellular concentration of cADPR lengthened the period of circadian gene expression. The authors suggest that circadian- regulated cADPR-derived Ca2+ signaling may configure part of the feedback loop that controls the clock (see the figure). Imaizumi et al. Science, 318, 1730 (2007)

21. Example of a gene regulatory network The results of Dodd et al. raise interesting questions. The phytohormone abscisic acid, thought to lengthen the clock period, induces cADPR production, and cADPR gene expression overlaps with that of genes controlled by abscisic acid. Does abscisic acid affect the clock partly through cADPR derived signals? Also, assuming that both IP3-and cADPR-dependent pathways are involved in generating circadian [Ca2+]i oscillation, do they interact with each other? Imaizumi et al. Science, 318, 1730 (2007)

22. Example of a gene regulatory network Dodd et al. found that a pharmacological inhibitor (U73122 at 1 μM) of IP3 production did not affect daily [Ca2+]i oscillation. Because IP3 concentrations were not analyzed, more research is needed to understand the relative roles of both cADPR and IP3. In particular, identification of the plant genes that encode the enzymes that produce cADPR and the proteins that control Ca2+ release by cADPR and IP3 are required to analyze the functions of these signaling molecules in plants. Imaizumi et al. Science, 318, 1730 (2007)

23. Current evidence Recent finding: activity of sirtuin-1 (Sirt1), a longevity-associated protein belonging to a family of NAD+-activated histone deacetylases oscillates in a circadian fashion. Eckel-Mahan & Sassone-Corsi, Nat Struct Mol Biol. 16, 462 (2009)

24. Outlook1 Whether there are mammalian metabolite oscillations analogous to those of the yeast metabolic cycle is still unclear, but it remains a tantalizing possibility. The fact that cellular demands are met temporally as a function of the cell’s metabolic cycle is likely to be true for all cells, regardless of the organism. In the context of mammalian Sirt1 circadian activity, it seems likely that metabolite oscillations in the coenzyme NAD+ must also occur in a cyclical, circadian manner. If metabolite fluctuations are organized temporally in a circadian manner, what might this mean physiologically? Eckel-Mahan & Sassone-Corsi, Nat Struct Mol Biol. 16, 462 (2009)

25. Outlook 2 The central functions of NAD+ in DNA repair, gene silencing, the cell cycle and circadian control indicate that the consequences of its aberrant regulation could be numerous and physiologically severe. It is conceivable that food restriction impinges on circadian rhythms because it disrupts NAD+-NADH cycling, essentially allowing the redox state of individual cells and tissues to alter rhythmicity. Eckel-Mahan & Sassone-Corsi, Nat Struct Mol Biol. 16, 462 (2009)

26. Outlook 3 The absence of Clock–Bmal1 dimerization in the presence of increased levels of oxidized NAD is one piece of evidence supporting this idea. As such, it is easy to imagine sophisticated schemes coordinating SCN-driven rhythms with those of a phase-shifted periphery for drug administration and efficacy. Already there are numerous drugs, perhaps most commonly known within cancer chemotherapeutic strategies, administered following a circadian protocol so that the maximal benefit might be achieved from their use. Eckel-Mahan & Sassone-Corsi, Nat Struct Mol Biol. 16, 462 (2009)

27. Additional slides

28. Cross-talk By activating Sirt1, NAD+ conjoins two feedback loops necessary for cross-talk between the circadian clock and metabolite production. The NAD+-salvage pathway is important for regulating intracellular NAD+ levels. After the conversion of nicotinamide (NAM) into nicotinamide mononucleotide (NMN) by NAM phosphoribosyl transferase (NAMPT), NMN is further modified into NAD+ by the nicotinamide mononucleotide adenylyl transferases (Nmnat1, –2 and –3). Whereas NAM inhibits Sirt1 activity, NAD+-activated Sirt1 feeds back into the NAD+-salvage pathway by directly regulating Nampt gene expression in a Clock–Bmal1-dependent manner. By this mechanism, NAD+ conjoins the two feedback loops, contributing to the fine tuning necessary for achieving energy balance. Eckel-Mahan & Sassone-Corsi, Nat Struct Mol Biol. 16, 462 (2009)