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date: 14 October 2019

Molecular Biology and Physiology of Circadian Clocks

Summary and Keywords

Circadian rhythm is the approximately 24-hour rhythmicity that regulates physiology and behavior in a variety of organisms. The mammalian circadian system is organized in a hierarchical manner. Molecular circadian oscillations driven by genetic feedback loops are found in individual cells, whereas circadian rhythms in different systems of the body are orchestrated by the master clock in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. SCN receives photic input from retina and synchronizes endogenous rhythms with the external light/dark cycles. SCN regulates circadian rhythms in the peripheral oscillators via neural and humoral signals, which account for daily fluctuations of the physiological processes in these organs. Disruption of circadian rhythms can cause health problems and circadian dysfunction has been linked to many human diseases.

Keywords: circadian clock, SCN, light, clock genes, physiology, disease

Introduction of the Mammalian Circadian System

Living organisms track cyclic changes in the environment and coordinate their physiology and behavior based on these changes in a process termed “biological rhythmicity.” Many important biological cycles have been studied, including cycles longer than a day (24 hours), such as animals’ annual migration, and cycles shorter than a day, such as 90-minute rapid eye movement sleep cycles and roughly 12.4-hour marine tidal rhythms. The best-known biological rhythms, however, are the approximately 24-hour rhythms called “circadian rhythms.” The word “circadian” was first coined by Franz Halberg (1919–2013) in the 1950s (Halberg et al., 2003). “Circa” means “around” and “dian” means “day” in Latin’ Therefore, “circadian” means “around a day.” Circadian rhythmicity is an evolutionarily conserved property that has been observed in a wide spectrum of species from cyanobacteria to humans (Rosbash, 2009). It has evolved as an adaptation to the axial rotation of the earth and the ensuing cyclic environmental changes, such as light, temperature, humidity, and food availability. In this article describes the molecular basis of circadian rhythms and the physiology of the circadian system in mammals. The role of circadian rhythms in human health and disease is also briefly discussed.

Molecular Biology and Physiology of Circadian ClocksClick to view larger

Figure 1. Organization of the circadian system. In mammals, including the human beings, the mater circadian clock is located in the suprachiasmatic nucleus (SCN) in the hypothalamus. SCN receives photic input from a subpopulation of the retinal ganglion cells and is synchronized to environmental light-dark cycles. In turn, SCN generates daily rhythms to regulate various functions of the body, including stress response, hormone secretion, autonomic nerve activities, metabolism, and behavior.

The mammalian circadian system is organized in a hierarchical manner (Figure 1; Reppert & Weaver, 2002). The molecular circadian oscillations are found within individual cells (Takahashi, Hong, Ko, & McDearmon, 2008; see the section “Molecular Basis of the Cellular Circadian Clock), whereas cellular rhythms in different systems of the body must be orchestrated by the master pacemaker, which is located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus (Mohawk, Green, & Takahashi, 2012; see “SCN Is the Master Circadian Clock In Mammals”). SCN receives photic input from the melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) and is therefore entrained by the ambient light-dark cycles (see “Entrainment of the circadian clock”). In turn, SCN generates rhythms to regulate the rhythmic activities in other brain regions as well as peripheral organs. The neural and endocrine SCN outputs serve as the entrainment cues for the peripheral circadian oscillators (see “Output Signals of the Circadian Clock”). Thus, circadian rhythms in different parts of the body are synchronized under physiological conditions so that physiology and metabolism in different systems are coordinated temporally based on the time of day. Disruption of synchrony among the circadian oscillators in the body can be caused by a rapid shift of time zones, which leads to misalignments of the internal time with the local time and the groggy feelings of jet lag (Aton & Herzog, 2005). The circadian clock not only regulates the physiological functions of the body but also is involved in many human diseases (Takahashi et al., 2008). Circadian dysfunction has been linked to pathogenesis of human diseases (see “Clock Dysfunction and Human Diseases”).

Molecular Basis of the Cellular Circadian Clock

A major breakthrough in the field of chronobiology (the biology of biological rhythms) is the discovery of the clock genes as the genetic basis of circadian rhythms. Clock genes are about a dozen genes, the rhythmic expression of which generates daily rhythms in cells and is conserved in Drosophila and mammals. The first Drosophila clock gene Period (Per) was predicted by Benzer and Konopka. Using a classical chemical-based mutagenesis strategy, they isolated three different strains of mutant flies showing alterations in the normal 24-hour cycle of pupal eclosion and locomotor activity (Konopka & Benzer, 1971). The Per gene was molecularly cloned and sequenced in the mid-1980s by Jeffrey Hall and Michael Rosbash, who collaborated at Brandeis University, and Michael Young at Rockefeller University (Bargiello & Young, 1984; Bargiello, Jackson, & Young, 1984; Reddy et al., 1984; Zehring et al., 1984). Later, the concept of transcription-translation feedback loops (TTFL) was developed as a mechanism for autoregulatory clock gene cycling in Drosophila (Hardin, Hall, & Rosbash, 1990). In Drosophila, the transcription of Per and its partner Timeless (Tim) are driven by CLOCK(CLK): CYCLE(CYC) heterodimers. CLK and CYC both contain basic helix-loop-helix (bHLH) motifs and bind to specific elements in the Per and Tim genes (Rutila et al., 1998). TIM and PER bind to form protein complexes. Upon reaching a certain level, the PER:TIM complexes translocate into the cell nucleus and repress their own gene expression, forming an inhibitory feedback loop (Darlington et al., 1998; Hardin, 1998). In 2017, the Nobel prize in physiology or medicine was awarded jointly to Jeffrey C. Hall, Michael Rosbash, and Michael W. Young “for their discoveries of molecular mechanisms controlling the circadian rhythm” (Nobel Media, 2019).

In mammals, there are three Per homologs (Albrecht et al., 1997; Shigeyoshi et al., 1997; Sun et al., 1997; Zheng et al., 1999; Zylka et al., 1998). The first mammalian clock gene, the circadian locomotor output cycles kaput, or Clock, was discovered by Joseph Takahashi and colleagues using forward genetics and positional cloning techniques (King et al., 1997; Vitaterna et al., 1994). CLOCK form a heterodimeric complex with another bHLH-PAS domain-containing transcriptional activators, BMAL1 (Bunger et al., 2000; Hogenesch, Gu, Jain, & Bradfield, 1998), and activate E-box-mediated transcription of Per (Per1, 2, 3) and Cryptochrome (Cry1, 2) genes (Gekakis et al., 1998). In turn, PER and CRY proteins form protein complexes and accumulate in the cytosol. Upon reaching a certain level, the PER/CRY protein complexes translocate into the cell nucleus and interact with the CLOCK:BMAL1 heterodimers to repress their own gene transcription (Kume et al., 1999; Shearman et al., 2000). Later, it was found that the transcription factor NPAS2 (MOP4) functionally substitutes for CLOCK in the master brain clock to regulate circadian rhythmicity (DeBruyne, Weaver, & Reppert, 2007). In addition to the primary feedback loop, there is a second negative feedback loop. The transcription of the nuclear hormone receptors, Rev-erb (α‎ and β‎), is promoted by CLOCK:BMAL1. In turn REV-ERBs repress Bmal1 transcription (Preitner et al., 2002). Another nuclear hormone receptor, RORα‎, competes with REV-ERBs to activate Bmal1 transcription (Sato et al., 2004). CLOCK:BMAL1 is not only critical for sustaining the TTFL but also serves as an important clock output mechanism. Rhythmic expression of clock output genes (so-called clock-controlled genes) are transcriptionally regulated by CLOCK:BMAL1 acting on E-box elements in their regulatory regions (Jin et al., 1999).

In addition to transcriptional regulation, Per gene expression is controlled at post-transcriptional levels. In Drosophila, the RNA-binding proteins Ataxin-2 (Atx2) and Twenty-four (Tyf) interact to activate Per mRNA translation in pacemaker neurons to sustain robust behavioral rhythms in Drosophila (Lim et al., 2011; Lim & Allada, 2013; Zhang, Ling, Yuan,Dubruille, & Emery, 2013). A targeted RNAi screen revealed that the atypical translation factor NAT1 regulates PER protein levels in PDF neurons (Bradley, Narayanan, & Rosbash, 2012). PER phosphorylation is controlled by DOUBLETIME protein (DBT; Kloss et al., 1998), which is related to CK1ε‎ in mammals and the NEMO/NLK kinase (Chiu, Ko, & Edery, 2011). PER and TIM degradation is controlled by the F-box protein slimb (Grima et al., 2002)

In mammals, Per1 and Per2 mRNA processing is regulated by methylation (Fustin et al., 2013). PER protein abundance is controlled at the level of mRNA translation. The mitogen-activated protein kinases (MAPKs) interacting protein kinases (MNKs) phosphorylate the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and promotes Per1 and Per2 mRNA translation (Cao et al., 2015). PER protein abundance is also regulated by post-translational modifications and degradation. Phosphorylation of PER and CRY proteins targets them for polyubiquitination and degradation. The phosphorylation and degradation rate of PER proteins is a key determinant of circadian period. PER can be phosphorylated by Casein kinase 1δ‎ (CK1δ‎) and CK1ε‎ and dephosphorylated by PP2A (Lee, Etchegaray, Cagampang, Loudon, & Reppert, 2001; Lowrey et al., 2000; Meng et al., 2008). CRY proteins can be phosphorylated by AMPK (Lamia et al., 2009). The ubiquitin ligase the SCF (Skp1-Cul1-F-box protein) beta-TRCP1 and mouse double minute 2 homolog (MDM2) mediates ubiquitination of PER1 and PER2 proteins, respectively (Liu, Zou, et al., 2018; Shirogane, Jin, Ang, & Harper, 2005). The F-box proteins FBXL3 and FBXL21 regulate ubiquitination of cryptochromes (Busino et al., 2007; Hirano et al., 2013; Siepka et al., 2007; Yoo et al., 2013; see Figure 2).

Molecular Biology and Physiology of Circadian ClocksClick to view larger

Figure 2. Molecular mechanisms of the mammalian circadian clock. In a cell, CLOCK (CLK) forms a heterodimeric complex with BMAL1 (BM1) and activates E-box-mediated transcription of Period (Per) and Cryptochrome (Cry) genes. PER and CRY proteins form protein complexes and accumulate in the cytosol. Upon reaching a sufficient level, the PER/CRY protein complexes translocate into the cell nucleus and interact with the CLOCK:BMAL1 complexes to repress their own gene transcription. In addition, the transcription of the nuclear hormone receptor Rev-erbα‎ is promoted by CLOCK:BMAL1. REV-ERBα‎ in turn represses Bmal1 transcription. ROR is another the nuclear hormone receptor that activates Bmal1 transcription. As a clock output mechanism, CLOCK:BMAL1 acts on E-box elements within the clock-controlled genes (CCG) and regulates rhythmic gene expression and physiological processes, such as neuronal firing and hormone secretion. In addition to transcriptional regulation, Per gene expression is controlled at the mRNA translational level by MNK-mediated eIF4E phosphorylation. PER and CRY proteins are also regulated by post-translational modifications and ubiquitination. PER proteins are phosphorylated by Casein kinase (CSNK) 1δ‎ and ε‎ and dephosphorylated by PP2A. CRY proteins are phosphorylated by AMPK. Phosphorylated PER and CRY proteins are degraded by ubiquitination. In the SCN, heterogenous SCN neurons are coupled to form a neuronal network via complex mechanisms involving VIP signaling, GABAergic neurotransmission, gap junctions, and so on. Photic input from the retina evokes neurotransmitter release in the SCN, activating a number of intracellular signaling transduction pathways (MAPK, mTOR, etc.). MAPK/MSK activation leads to CREB phosphorylation, which promotes Per and Cry transcription and resetting of the clock. Under constant conditions, intracellular signals (Ca2+, cAMP, redox state, etc.) couple cellular physiology and metabolism to the clock machinery.

SCN: Master Circadian Clock in Mammals

Although clock genes are expressed in a variety of cells and tissues, the master circadian pacemaker is thought to be located in the SCN of hypothalamus for the following reasons: (1) ablation of SCN abolishes circadian behavioral and endocrine rhythms in animals (Moore & Eichler, 1972; Stephan & Zucker, 1972); (2) SCN receives direct synaptic input from RHT, the pathway essential for photic entrainment (Moore, 1973); (3) isolated SCN tissue continues to show robust circadian rhythmicity in vitro (Green & Gillette, 1982; Inouye & Kawamura, 1979; Yamazaki et al., 2000); and (4) SCN transplantation restores circadian behavioral rhythms in arrhythmic hosts with the period determined by the donor SCN (Ralph, Foster, Davis, & Menaker, 1990).

SCN consists of about 20,000 neurons with great heterogeneity (Herzog, Hermanstyne, Smyllie, & Hastings, 2017). The SCN neurons express different neuropeptides and have distinct neuroanatomical features. In general, the neurons in the ventral SCN express vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) and receive direct innervation from the RHT. In contrast, the neurons in the dorsal SCN express arginine vasopressin (AVP) and do not receive direct RHT input. Instead, they express VIP receptor 2 (Vipr2) and receive input from the ventral SCN neurons (Antle & Silver, 2005). To generate coherent rhythms and function as a circadian pacemaker, SCN neurons must be coupled and synchronized (Aton & Herzog, 2005).

VIPergic signaling is essential for synchronization among SCN neurons as well as robustness of rhythmicity in individual SCN cells. Microinjection of VIP induces phase shifts in the SCN circadian pacemaker, and VIP antagonists disrupt circadian function (Cutler et al., 2003; Gozes et al., 1995; Piggins, Antle, & Rusak, 1995; Reed, Meyer-Spasche, Cutler, Coen, & Piggins, 2001). Transgenic mice overexpressing VPAC2 (a VIP receptor) show accelerated re-entrainment to a shifted light-dark cycle (Shen et al., 2000). VIP- (Colwell et al., 2003) and VPAC2-deficient mice (Harmer et al., 2002) show weakened circadian rhythmicity under constant conditions and disrupted circadian behavior under the skeleton photoperiod. Electrophysiological recordings show that SCN neurons in slices from Vip-/- and Vipr2-/- (encoding VPAC2) mice do not exhibit circadian rhythms of SCN neuronal firing and lack interneuronal synchrony (Aton, Colwell, Harmar, Waschek, & Herzog, 2005). Daily application of a VIP agonist to the Vip-/- SCN restores synchrony (Aton et al., 2005).

Similarly, bioluminescent recordings of PER2 rhythms from Vipr2-/- SCN slices suggest that VIP signaling is necessary to synchronize individual SCN neurons as well as to maintain intracellular rhythms within these cells (Maywood et al., 2006). Levels of VIP and its precursor protein prepro-VIP is controlled by the mammalian target of rapamycin complex (mTORC) 1 signaling through the translational repressor eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Mice lacking 4E-BP1 show VIP overexpression in the SCN and enhanced SCN synchrony, whereas mTOR heterozygotes exhibit decreased VIP level, weakened PER2 rhythms in the SCN, and vulnerability to constant light (Cao, Butcher, Karelina, Arthur, & Obrietan, Impey, Smith, Athos, & Storm, 2013; Ramanathan et al., 2018). Thus, mTORC1 signaling regulates SCN cell synchrony through VIP (Liu, Stowie, et al., 2018).

AVP signaling, on the other hand, confers on the SCN an intrinsic resistance to external perturbations and accounts for the jet-lag responses following abrupt shifts in light-dark cycles (Yamaguchi et al., 2013). In mice lacking vasopressin receptors V1a and V1b, circadian rhythms of locomotor activities, clock gene expression, and body temperature are normal under a stable light-dark cycle. However, these rhythms can immediately re-entrain to an abrupt shift of light-dark cycles, compared to the gradual re-entrainment in WT mice. Thus, internal synchronization of SCN cells are not only important for autonomous circadian oscillations but also for proper response to environmental cues that reset the clock.

SCN synchrony can be disrupted by strong environmental signals such as constant light. Animals exposed to constant light can exhibit behavioral arrhythmicity or “splitting” of behavioral rhythms. By imaging molecular rhythms of individual clock neurons in explanted mouse clock nuclei, it is found that constant light desynchronizes clock neurons but does not compromise cellular circadian rhythms (Ohta, Yamazaki, & McMahon, 2005). SCN synchrony can also be disrupted by pharmacological agents that impact on the molecular mechanisms of synchronization. MDL-12,330A (MDL) is a potent, irreversible inhibitor of adenylyl cyclase to reduce concentrations of cAMP to basal levels. As cAMP signaling is involved in intrinsic cellular clock oscillations, decreasing cAMP will attenuate circadian clock oscillations. Thus, prolonged exposure to MDL suppresses and desynchronizes the transcriptional cycles of SCN cells (O’Neill, Maywood, Chesham, Takahashi, & Hastings, 2008). Disruption of VIP signaling by VPAC2 antagonist PG99-465 also desynchronizes SCN neurons (Aton et al., 2005; Cutler et al., 2003). PG99-465 can reverse the enhanced synchrony phenotypes in the 4E-BP1 KO mice by antagonizing the VPAC2 receptor (Cao et al., 2013). Surprisingly, VIP, the essential peptide for SCN synchrony, can also desynchronize SCN neurons and damp SCN rhythms when applied exogenously. The degree and duration of desynchronization among SCN neurons depended on both the phase and the dose of VIP (An et al., 2013; Hamnett, Crosby, Chesham, & Hastings, 2019).

Entrainment of the Circadian Clock

As an adaptation to the ever-changing environment, the circadian clock is continuously adjusted by external cues such as light. For example, with seasonal changes the photoperiod continuously changes throughout the year. To be synchronized with the ambient light-dark cycles, the circadian clock needs to be reset by light, which is important for the survival of animals. For example, for the nocturnal rodents, failure to reset their clock and exposure during the day can increase the risk of being killed by their predators.

Neural Pathway

Photic input is relayed to the SCN via the retinohypothalamic tract (RHT) from the retina. Light-induced phase shifts of the SCN clock, inhibition of nocturnal melatonin production, inhibition of activity by light (negative masking), and pupillary constrictor reflexes are all mediated by a nonrod, noncone system. The pathway is distinct from the image-forming visual pathway. Reception of light is mediated by intrinsically photosensitive retina ganglion cells (ipRGCs). This special population of retinal ganglion cells have large dendritic fields. They express melanopsin and use the excitatory neurotransmitter glutamate and the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) as their neurotransmitters. They project directly to the SCN and intergeniculate leaflet (Panda, 2007; Peirson & Foster, 2006). Most RHT terminals end in the SCN and form synaptic connections with the ventral SCN neurons. In response to a light stimulus at night, the RHT terminals release glutamate and PACAP, which in turn bind to their respective postsynaptic receptors on the SCN neurons and evoke activation of intracellular signal transduction events that regulate clock gene expression and trigger clock resetting (Golombek & Rosenstein, 2010). Photic and nonphotic input also reaches the SCN indirectly through the intergeniculate leaflet and midbrain, with γ‎-aminobutyric acid (GABA), neuropeptide Y, and serotonin as neurotransmitters (Mrosovsky, 1996).

Signaling Mechanisms in the SCN Neurons

The intracellular signaling events that couple light to clock gene transcription are being increasingly uncovered. A number of cell signaling pathways have been found to regulate resetting of the circadian clock, including the MAPK pathway (Butcher et al., 2002; Coogan & Piggins, 2003; Obrietan, Impey, & Storm, 1998), the protein kinase A (PKA) pathway (Lee, Schak, & Harrington, 1999; Meyer-Spasche & Piggins, 2004; Prosser, Heller, & Miller, 1994; Schurov, Hepworth, & Hastings, 2002; Sterniczuk, Yamakawa, Pomeroy, & Antle, 2014; Tischkau, Gallman, Buchanan, & Gillette, 2000), the nitric oxide/protein kinase G (NO/PKG) pathway (Ding et al., 1994; Mathur, Golombek, & Ralph, 1996; Tischkau et al., 2004; Weber, Gannon, & Rea, 1995), the protein kinase C (PKC) pathway (Jakubcakova et al., 2007; Lee, Almad, Butcher, & Obrietan, 2007; Schak & Harrington, 1999), and the mTOR pathway (Cao, Li, Cho, Lee, & Obrietan, 2010). These pathways are regulated by light-evoked synaptic response at the RHT terminal, and couple light information to clock gene expression and entrainment of the circadian clock.

The MAPK pathway plays a central role in photic entrainment of the circadian clock (Butcher et al., 2002; Coogan & Piggins, 2003; Obrietan et al., 1998). Transient light entrainment cues trigger a concordant increase in ERK activation at Thr202/Tyr204 (Coogan & Piggins, 2003; Obrietan et al., 1998). Light-induced ERK activation only occurs during the subjective nighttime, corresponding to the time period when light can reset the behavioral rhythms. Disruption of light-induced MAPK activation by MEK inhibitor U0126 or SL-327 significantly attenuates light-induced phase delay at night (Butcher et al., 2002). MAPK functions through activation of multiple downstream effectors. Along these lines, photic stimulation has been shown to trigger activation of the ERK/MAPK effectors RSK and mitogen- and stress-activated protein kinase (MSK; Butcher, Lee, Hsieh, & Obrietan, 2004; Butcher, Lee, Cheng, & Obrietan, 2005). The light-activated MSK1 lead to the phosphorylation of cAMP response element-binding proteins (CREB) at Ser-133, histone phosphorylation, and Per1 transcription (Cao et al., 2013).

cAMP signaling constitutes a critical component of cellular circadian oscillations, and daily activation of cAMP signaling sustains transcriptional rhythms (O’Neill et al., 2008). cAMP-responsive elements (CREs) have been identified in the promoter regions of Per1 and Per2 (Albrecht, Sun, Eichele, & Lee, 1997; Travnickova-Bendova, Cermakian, Reppert, & Sassone-Corsi, 2002). It has been shown that light exposure at night leads to CRE-mediated gene transcription in the SCN (Obrietan, 1999). CRE activation appears to be necessary for the light entrainment process (Tischkau Mitchell, Tyan, Buchanan, & Gillette, 2003). CREB binds to CREs and promotes CRE-mediated gene transcription (Sakamoto, Karelina, & Obrietan, 2011). CREB activities are regulated by phosphorylation. Activation of the CREB is under circadian control (Belvin, Zhou, and Yin (1999); Gau et al., 2002; Obrietan et al., 1999) and is stimulated by light via the MAPK/MSK1 pathway (Cao et al., 2013; Ginty et al., 1993; Obrietan et al., 1999). CREB and its regulation by phosphorylation is important not only for SCN clock entrainment (Ding et al., 1997; Gau et al., 2002; Gamble et al., 2007; Wheaton et al., 2018) but also for its autonomous rhythmicity (Lee at al., 2010; Wheaton et al., 2018). CREB-dependent transcription is also regulated by the CREB coactivator CREB-regulated transcription coactivator (CRTC; Jagannath et al., 2013; Sakamoto et al., 2013). microRNA132 can also be induced by photic entrainment cues via a MAPK/CREB-dependent mechanism and facilitates Per1 transcription (Cheng et al., 2007).

mTORC1 signaling can also be activated by light. Light at night activates S6K1 by inducing its phosphorylation at Thr389 and increases phosphorylation of 4E-BP1 at Thr37/46 in the SCN (Cao, Lee, Cho, Saklayen, & Obrietan, 2008). In turn, activated S6K1 phosphorylates its downstream translation effectors, including the ribosomal protein S6 (S6), a component of the 40S ribosomal subunit, and potentially stimulates translation of a subset of mRNAs (Cao et al., 2010). Light-activated mTOR signaling may regulate light-responsive mRNA translation in the SCN (Cao R, 2018).

Light-Induced Clock Gene Expression

Light exposure at night leads to an increase in Per gene expression, which is thought to be the molecular mechanism underlying photic entrainment of the circadian clock. The mammalian Per1 and Per2 genes are photo-inducible in the rodent SCN with phase-dependence similar to that in behavioral rhythmicity (Albrecht et al., 1997; Zylka, Shearman, Weaver, & Reppert, 1998). Using Per1 antisense, Akiyama et al. (1999) inhibited light- and glutamate-induced phase delays of the locomotor rhythm in mice and Wakamatsu et al. (2001) eliminated the delays in vivo by injecting antisense oligos to both Per1 and Per2. However, three lines of mice genetically deficient in Per1 (all lack PER1 protein expression) have been reported and not all mice exhibited impaired phase shift by light (Bae et al., 2001; Cermakian, Monaco, Pando, Dierich, & Sassone-Corsi, 2001; Zheng et al., 2001).

The Per mRNA levels begin to rise 10 to 15 minutes after light onset during subjective night, peak at about 1 to 2 hours and return to baseline after about 3 hours. The protein immunoreactivities also rise following the mRNA level increase, but are technically difficult to detect until the basal levels decrease to a very low level (e.g., 4 hours after light at CT15; Yan & Silver, 2004). The increased levels of PER1 and PER2 proteins early in the night could extend the period of transcriptional inhibition by the PER/CRY complex, whereas the increase late in the night could lead to an earlier initiation of the PER/CRY mediated inhibition. This may be the mechanism by which light in the early night causes a phase delay and light in the late night causes a phase advance (Reppert & Weaver, 2002).

As mentioned previously, Per1 and Per2 harbor CREs in their promoter regions. Photic induction of Per1 and Per2 gene transcription is mediated by CREs. Less is known about the role of translational control in light-induced gene expression. Light-induced mTORC1 activation appears to be important for photic entrainment of the SCN clock, as rapamycin inhibits light-induced behavioral phase delay in mice (Cao et al., 2010). Effects of rapamycin on behavioral phase shift are consistent with its inhibitory effect on light-induced increase of PER1 and PER2 protein levels in the SCN, which is partially independent of transcription. Interestingly, rapamycin also inhibits light-induced increase of proteins of which mRNAs are with a 5′terminal oligopyrimidine (TOP) tract such as eukaryotic elongation factor 1A and Jun B. Together, these results demonstrate that the mTORC1/S6K1 pathway is an integral part of the photic entrainment pathway that regulates mRNA translation in the SCN. In addition, light regulates the activity of MNKs, which phosphorylate the cap-binding protein eIF4E in the SCN. Phosphorylation of eIF4E promotes light-activated Per1 and Per2 mRNA translation. In the mice with a nonphosphorylatable form of eIF4E, light-induced phase delay is diminished (Cao et al., 2015). Thus, the MAPK pathway regulates coordinated transcription (through CREB phosphorylation) and translation (through eIE4E phosphorylation) in the SCN in photic entrainment of the clock.

Output Signals From the Circadian Clock

Most physiological (temperature, hormones, blood pressure) and behavioral (mood, alertness, sleep, performance) functions of the body exhibit daily rhythms. These rhythms ensure best performance of the system at a specific time of day and rhythms in different systems are coordinated for the well-being of the body as a whole. The peripheral rhythms are orchestrated by the SCN via neural and endocrine signals.

Neural Innervation of Other Brain Areas by SCN

SCN neurons show autonomous circadian rhythms in spontaneous firing frequency as output signals to other brain regions (Kuhlman & McMahon, 2006). In vivo recordings reveal circadian rhythms in the electrical activity in the SCN, with spike activity being high during the day and low at night (Inouye & Kawamura, 1979). SCN neurons project mostly to adjacent brain regions (Abrahamson & Moore, 2001), including the subparaventricular zone, the preoptic area, bed nucleus of the stria terminalis, the lateral septum, retrochiasmatic area, the arcuate nucleus, and the dorsomedial hypothalamus (Morin, 2013). The shell SCN receives inputs from the core region and send outputs to these brain regions. There are also limited projections to the forebrain, midline thalamus, intergeniculate leaflet, and periaqueductal gray. The direct SCN recipient areas in turn signal more brain regions so that all brain regions get direct or indirect rhythmic inputs and show daily rhythms of activity. In a mapping study, robust PER2 rhythms are found in numerous forebrain regions including the hippocampus, prefrontal cortex, amygdala, and striatum (Harbour, Weigl, Robinson, & Amir, 2013). These regions modulate various neural processes. Indeed, a variety of neurophysiological functions exhibit daily fluctuations, which accounts for the time-of-day variations in sensation, motion, learning, memory, and mood (Krishnan & Lyons, 2015; McClung, 2013). Timing signals from the SCN travel to peripheral organs by the sympathetic and parasympathetic nervous systems and by circadian fluctuations in levels of various hormones (Ota, Fustin, Yamada, Doi, & Okamura, 2012).

Endocrine Output by SCN

Besides rhythmic firing, SCN neurons synthesize hormones and growth factors and release them to influence circadian rhythms of peripheral oscillators. The first demonstrated SCN output hormone was AVP (Goodwin, Jenner, & Slater, 1968). AVP is produced in the dorsal SCN and rhythmically released into the CSF (Reppert, Artman, Swaminathan, & Fisher, 1981). The function of such rhythmic activity is not clear. It has also been demonstrated that the transplanted SCN tissue surrounded by semipermeable membrane can still restore rhythmic locomotor activity in animals although synaptic connections between the SCN and surrounding brain regions cannot be formed (Silver, LeSauter, Tresco, & Lehman, 1996). This indicates that some diffusible factors can be released across the membrane from the transplanted SCN tissue. Subsequently, factors such as TGF-α‎, prokineticin-2, and cardiotrophin-like cytokine have been proposed as humoral outputs of the circadian pacemaker that control rhythmic locomotor activity in animals (Cheng et al., 2002; Kramer et al., 2001; Kraves & Weitz, 2006).

Circadian Dysfunction and Human Diseases

In the modern society, transmeridian travel, night-shift work, artificial light at night, and night-owl lifestyles all become health hazards that disrupt the circadian rhythm and impact on the body clock. Whereas it may not be a matter of life and death for humans, disruption of circadian rhythms does cause health problems. Some disorders, such as jet lag and shift work sleep disorder, are caused by direct disruption of circadian rhythms and can be cured with a regular day/night schedule. Some inherited diseases are caused by mutations on clock genes, such as familial advanced sleep phase syndrome (FASPS; Patke et al., 2017; Xu et al., 2005, 2007). Perhaps more important, circadian clock malfunction has been linked to a number of common human diseases including cancer, metabolic syndromes, and neurological diseases.

The best-known example is jet lag, characterized by groggy, disoriented feelings and sleep deprivation. As experienced by millions of travelers, jet lag is caused by misalignment of the internal body time with the local time. As mentioned previously, clock genes are expressed in various tissues and organs outside of the SCN. Circadian oscillations of clock genes in these peripheral clocks are usually orchestrated by the SCN, so that the rhythms of these circadian oscillators keep a stable phase relationship (so-called circadian synchrony). Transmeridian travel can desynchronize the body’s circadian rhythms due to the different speeds of re-entrainment in different organs. Symptoms are caused by misalignment of the body’s internal time with the local time and transient desynchronization of rhythms among different organs.

Similar to jet lag, desynchronization of the body’s clock with the local time happens in patients with non-24-hour sleep wake disorder (Non-24). People with Non-24 have circadian rhythms that are not synchronized with the 24-hour day-night cycle, either through a failure of light to reach the SCN, as in total blindness, or due to various other reasons in sighted people (Quera Salva, Hartley, Leger, & Dauvilliers, 2017). The patients cannot synchronize their body clocks with the social schedule and are therefore unable to keep up with their daily life. They may fall asleep during the day and wake up in the middle of the night.

FASPS is a rare inherited circadian rhythm sleep disorder characterized by very early sleep onset and offset that segregates in a highly penetrant autosomal dominant manner. Polysomnographic measurements of sleep and body rhythms of temperature and melatonin level indicate that all measures are phase advanced by about 4 hours. Xu et al. (2005) found that hPer2, a human homolog of the Drosophila period gene, was mutated at position 2106 (A to G) in a family with FASPS. The point mutation leads to a substitution of a serine for a glycine (S662G) at amino acid 662 in the CKIε‎ binding domain of the hPER2 protein and results in hypophosphorylation of PER2 in vitro. Later, Xu et al. (2007) identified a missense mutation (T44A) in the human CKIδ‎ gene leading to FASPS. These findings directly connect fundamental molecular clock mechanisms to human diseases.

Disruption of circadian clock functions has been linked to a number of common human diseases. For example, epidemiological studies have established shift work as a risk factor for breast cancer (Wise, 2009). Women who routinely work overnight shifts (such as nurses and flight attendants) may have a slightly increased risk of breast cancer. One possible reason is the exposure to light related to these types of jobs. Being exposed to light throughout the night affects some hormone functions in the body that may be related to breast cancer.

The interactions among the circadian clock and various pathological processes are being actively studied using animal models with clock gene mutations. Mice lacking the core clock genes Cry1 and Cry2 show salt-sensitive hypertension due to abnormally high synthesis of the mineralocorticoid aldosterone by the adrenal gland (Doi et al., 2010). Pancreatic islets possess self-sustained circadian gene oscillations. The circadian clock in beta cells coordinate insulin secretion. Ablation of the pancreatic clock can trigger the onset of diabetes mellitus. Both Clock and Bmal1 mutants show impaired glucose tolerance, reduced insulin secretion, and defects in size and proliferation of pancreatic islets that worsen with age (Marcheva et al., 2010). Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins. Deletion of Bmal1 or Clock in combination with neuronal PAS domain protein 2 (Npas2) can induce severe age-dependent astrogliosis in the cortex and hippocampus (Kondratov, Kondratova, Gorbacheva, Vykhovanets, & Antoch, 2006; Musiek et al., 2013). These results indicate that impaired clock gene function is linked to neurodegeneration. Given the ubiquitous nature and hierarchical structure of the circadian system, much work needs to be done to fully understand circadian regulation of health and disease.

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