Period determination in the mammalian circadian clock involves the turnover rate of the repressors CRY and PER. We show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss of function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.

Fluphenazine is a potent antipsychotic drug that can increase action potential duration and induce QT prolongation in several animal models and in humans. As the block of cardiac human ether-a-go-go-related gene (hERG) channels is one of the leading causes of acquired long QT syndrome, we investigated the acute effects of fluphenazine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. Fluphenazine at concentrations of 0.1-1.0 μM increased the action potential duration at 90% of repolarization (APD90) and action potential duration at 50% of repolarization (APD50) in 5 min when action potentials were elicited under current-clamp conditions in guinea pig ventricular myocytes. We examined the effects of fluphenazine on hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. The IC50 for the fluphenazine-induced block of hERG currents in HEK293 cells at 36 °C was 0.102 μM at +20 mV. Fluphenazine-induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The fluphenazine-dependent hERG block in Xenopus oocytes increased progressively relative to the degree of depolarization. Fluphenazine affected the channels in the activated and inactivated states but not in the closed states, and the S6 domain mutation from tyrosine to alanine at amino acid 652 (Y652A) attenuated the hERG current block. These results suggest that the antipsychotic drug fluphenazine is a potent blocker of hERG channels, providing a molecular mechanism for the drug-induced arrhythmogenic side effects.

The circadian regulatory network is organized in a hierarchical fashion, with a central oscillator in the suprachiasmatic nuclei (SCN) orchestrating circadian oscillations in peripheral tissues. The nature of the relationship between central and peripheral oscillators, however, is poorly understood. We used the tetOFF expression system to specifically restore Clock function in the brains of Clock(Δ19) mice, which have compromised circadian clocks. Rescued mice showed normal locomotor rhythms in constant darkness, with activity period lengths approximating wildtype controls. We used microarray analysis to assess whether brain-specific rescue of circadian rhythmicity was sufficient to restore circadian transcriptional output in the liver. Compared to Clock mutants, Clock-rescue mice showed significantly larger numbers of cycling transcripts with appropriate phase and period lengths, including many components of the core circadian oscillator. This indicates that the SCN oscillator overcomes local circadian defects and signals directly to the molecular clock. Interestingly, the vast majority of core clock genes in liver were responsive to Clock expression in the SCN, suggesting that core clock genes in peripheral tissues are intrinsically sensitive to SCN cues. Nevertheless, most circadian output in the liver was absent or severely low-amplitude in Clock-rescue animals, demonstrating that the majority of peripheral transcriptional rhythms depend on a fully functional local circadian oscillator. We identified several new system-driven rhythmic genes in the liver, including Alas1 and Mfsd2. Finally, we show that 12-hour transcriptional rhythms (i.e., circadian "harmonics") are disrupted by Clock loss-of-function. Brain-specific rescue of Clock converted 12-hour rhythms into 24-hour rhythms, suggesting that signaling via the central circadian oscillator is required to generate one of the two daily peaks of expression. Based on these data, we conclude that 12-hour rhythms are driven by interactions between central and peripheral circadian oscillators.

Protriptyline, a tricyclic antidepressant for psychiatric disorders, can induce prolonged QT, torsades de pointes, and sudden death. We studied the effects of protriptyline on human ether-à-go-go-related gene (HERG) channels expressed in Xenopus oocytes and HEK293 cells. Protriptyline induced a concentration-dependent decrease in current amplitudes at the end of the voltage steps and HERG tail currents. The IC(50) for protriptyline block of HERG current in Xenopus oocytes progressively decreased relative to the degree of depolarization, from 142.0 microM at -40 mV to 91.7 microM at 0 mV to 52.9 microM at +40 mV. The voltage dependence of the block could be fit with a monoexponential function, and the fractional electrical distance was estimated to be delta=0.93. The IC(50) for the protriptyline-induced blockade of HERG currents in HEK293 cells at 36 degrees C was 1.18 microM at +20 mV. Protriptyline affected channels in the activated and inactivated states, but not in the closed states. HERG blockade by protriptyline was use-dependent, exhibiting a more rapid onset and a greater steady-state block at higher frequencies of activation. Our findings suggest that inhibition of HERG currents may contribute to the arrhythmogenic side effects of protriptyline.

The mechanism of circadian oscillations in mammals is cell autonomous and is generated by a set of genes that form a transcriptional autoregulatory feedback loop. While these "clock genes" are well conserved among animals, their specific functions remain to be fully understood and their roles in central versus peripheral circadian oscillators remain to be defined. We utilized the in vivo inducible tetracycline-controlled transactivator (tTA) system to regulate Clock gene expression conditionally in a tissue-specific and temporally controlled manner. Through the use of Secretogranin II to drive tTA expression, suprachiasmatic nucleus- and brain-directed expression of a tetO::Clock(Delta19) dominant-negative transgene lengthened the period of circadian locomotor rhythms in mice, whereas overexpression of a tetO::Clock(wt) wild-type transgene shortened the period. Low doses (10 mug/ml) of doxycycline (Dox) in the drinking water efficiently inactivated the tTA protein to silence the tetO transgenes and caused the circadian periodicity to return to a wild-type state. Importantly, low, but not high, doses of Dox were completely reversible and led to a rapid reactivation of the tetO transgenes. The rapid time course of tTA-regulated transgene expression demonstrates that the CLOCK protein is an excellent indicator for the kinetics of Dox-dependent induction/repression in the brain. Interestingly, the daily readout of circadian period in this system provides a real-time readout of the tTA transactivation state in vivo. In summary, the tTA system can manipulate circadian clock gene expression in a tissue-specific, conditional, and reversible manner in the central nervous system. The specific methods developed here should have general applicability for the study of brain and behavior in the mouse.

The mammalian circadian clock is encoded by an autoregulatory transcription feedback loop that drives rhythmic behavior and gene expression in the brain and peripheral tissues. Transcriptomic analyses indicate cell type-specific effects of circadian cycles on rhythmic physiology, although how clock cycles respond to environmental stimuli remains incompletely understood. Here, we show that activation of the inducible transcription factor NF-κB in response to inflammatory stimuli leads to marked inhibition of clock repressors, including the Period, Cryptochrome, and Rev-erb genes, within the negative limb. Furthermore, activation of NF-κB relocalizes the clock components CLOCK/BMAL1 genome-wide to sites convergent with those bound by NF-κB, marked by acetylated H3K27, and enriched in RNA polymerase II. Abrogation of NF-κB during adulthood alters the expression of clock repressors, disrupts clock-controlled gene cycles, and impairs rhythmic activity behavior, revealing a role for NF-κB in both unstimulated and activated conditions. Together, these data highlight NF-κB-mediated transcriptional repression of the clock feedback limb as a cause of circadian disruption in response to inflammation.

Lindera erythrocarpa Makino (Lauraceae) is used as a traditional medicine for analgesic, antidote, and antibacterial purposes and shows anti-tumor activity. We studied the effects of Lindera erythrocarpa on the human ether-a-go-go-related gene (HERG) channel, which appears of importance in favoring cancer progression in vivo and determining cardiac action potential duration. Application of MeOH extract of Lindera erythrocarpa showed a dose-dependent decrease in the amplitudes of the outward currents measured at the end of the pulse (I(HERG)) and the tail currents of HERG (I(tail)). When the BuOH fraction and H(2)O fraction of Lindera erythrocarpa were added to the perfusate, both I(HERG) and I(tail) were suppressed, while the hexane fraction, CHCl(3) fraction, and EtOAc fraction did not inhibit either I(HERG) or I(tail). The potential required for half-maximal activation caused by EtOAc fraction, BuOH fraction, and H(2)O fraction shifted significantly. The BuOH fraction and H(2)O fraction (100 microg/mL) decreased g(max) by 59.6% and 52.9%, respectively. The H(2)O fraction- and BuOH fraction-induced blockades of I(tail) progressively decreased with increasing depolarization, showing the voltage-dependent block. Our findings suggest that Lindera erythrocarpa, a traditional medicine, blocks HERG channel, which could contribute to its anticancer and cardiac arrhythmogenic effect.

Promethazine is a phenothiazine derivative with antihistaminic (H(1)), sedative, antiemetic, anticholinergic, and antimotion sickness properties that can induce QT prolongation, which may lead to torsades de pointes. Since block of cardiac human ether-a-go-go-related gene (hERG) channels is one of the leading causes of acquired long QT syndrome, we investigated the acute effects of promethazine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. Promethazine increased the action potential duration at 90% of repolarization (APD(90)) in a concentration-dependent manner, with an IC(50) of 0.73microM when action potentials were elicited under current clamp in guinea pig ventricular myocytes. We examined the effects of promethazine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Promethazine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC(50) of promethazine dependent hERG block in Xenopus oocytes decreased progressively relative to the degree of depolarization. The IC(50) for the promethazine-induced block of the hERG currents in HEK293 cells at 36 degrees C was 1.46microM at +20mV. Promethazine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG current block. These results suggest that promethazine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of promethazine.

The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways, but a mechanistic understanding of how these pathways collaborate in animals is lacking. Here we demonstrate in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light- and clock-regulated gene timeless (tim). Moreover, we show that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light, and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, our studies reveal that PRL-1-dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to change day-night cycles.

While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases.

Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockΔ19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockΔ19 mutation to a ∼900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKΔ19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals. DOI:http://dx.doi.org/10.7554/eLife.00426.001.

Forward genetic approaches (phenotype to gene) are powerful methods to identify mouse circadian clock components. The success of these approaches, however, is highly dependent on the quality of the phenotype--specifically, the ability to measure circadian rhythms in individual mice. This article outlines the factors necessary to measure mouse circadian rhythms, including choice of mouse strain, facilities and equipment design and construction, experimental design, high-throughput methods, and finally methods for data analysis.

Using a forward genetics ENU mutagenesis screen for recessive mutations that affect circadian rhythmicity in the mouse, we isolated a long period (approximately 26 hr) circadian mutant named Overtime (Ovtm). Positional cloning and genetic complementation reveal that Ovtm is encoded by the F-box protein FBXL3, a component of the SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligase complex. The Ovtm mutation causes an isoleucine to threonine (I364T) substitution leading to a loss of function in FBXL3, which interacts specifically with the CRYPTOCHROME (CRY) proteins. In Ovtm mice, expression of the PERIOD proteins PER1 and PER2 is reduced; however, the CRY proteins CRY1 and CRY2 are unchanged. The loss of FBXL3 function leads to a stabilization of the CRY proteins, which in turn leads to a global transcriptional repression of the Per and Cry genes. Thus, Fbxl3(Ovtm) defines a molecular link between CRY turnover and CLOCK/BMAL1-dependent circadian transcription to modulate circadian period.

In mammals, circadian rhythms are entrained to the light cycle and drive daily oscillations in levels of NAD+, a cosubstrate of the class III histone deacetylase sirtuin 1 (SIRT1) that associates with clock transcription factors. Although NAD+ also participates in redox reactions, the extent to which NAD(H) couples nutrient state with circadian transcriptional cycles remains unknown. Here we show that nocturnal animals subjected to time-restricted feeding of a calorie-restricted diet (TRF-CR) only during night-time display reduced body temperature and elevated hepatic NADH during daytime. Genetic uncoupling of nutrient state from NADH redox state through transduction of the water-forming NADH oxidase from Lactobacillus brevis (LbNOX) increases daytime body temperature and blood and liver acyl-carnitines. LbNOX expression in TRF-CR mice induces oxidative gene networks controlled by brain and muscle Arnt-like protein 1 (BMAL1) and peroxisome proliferator-activated receptor alpha (PPARα) and suppresses amino acid catabolic pathways. Enzymatic analyses reveal that NADH inhibits SIRT1 in vitro, corresponding with reduced deacetylation of SIRT1 substrates during TRF-CR in vivo. Remarkably, Sirt1 liver nullizygous animals subjected to TRF-CR display persistent hypothermia even when NADH is oxidized by LbNOX. Our findings reveal that the hepatic NADH cycle links nutrient state to whole-body energetics through the rhythmic regulation of SIRT1.