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.

We investigated the effect of methamphetamine (MA) injections on the circadian organization of behavior and individual tissues in the mouse. Scheduled, daily injections of MA resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. Daily MA also shifted the peak time of PER2 expression in the liver, pituitary, and salivary glands. It has been suggested that reward pathways, and dopamine signaling in particular, may underlie the effects of MA on the circadian system. To test this hypothesis, we examined the effect of the D1 receptor antagonist SCH23390 (SCH) on circadian rhythms. The MA-induced shift in the phase of pituitary and salivary glands was attenuated by pretreatment with the D1 antagonist SCH23390 (SCH). Interestingly, daily SCH, administered alone, also affected some circadian oscillators. The livers and lungs (but not pituitaries or salivary glands) of mice treated with daily injections of SCH displayed disrupted rhythms of PER2 expression, suggesting that D1 receptor signaling is important for entrainment of these organs. From these results, we conclude that MA has widespread effects within the circadian system, and that these effects are mediated, at least in part, by the dopaminergic system. This study also identifies a role for dopamine signaling in normal entrainment of circadian oscillators.

The transcription factor, myocyte enhancer factor-2 (MEF2), is required for normal circadian behavior in Drosophila; however, its role in the mammalian circadian system has not been established. Of the four mammalian Mef2 genes, Mef2d is highly expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus, a region critical for coordinating peripheral circadian clocks. Using both conventional and brain-specific Mef2d KO (Mef2d-/-) mouse lines, we demonstrate that MEF2D is essential for maintaining the length of the circadian free-running period of locomotor activity and normal sleep patterns in male mice. Crossing Mef2d-/- with Per2::luc reporter mice, we show that these behavioral changes are achieved without altering the endogenous period of the master circadian oscillator in the SCN. Together, our data suggest that alterations in behavior in Mef2d-/- mice may be the result of an effect on SCN output, rather than an effect on timekeeping within the SCN itself. These findings add to the growing body of evidence that MEF2 proteins play important roles in the brain.SIGNIFICANCE STATEMENT These studies are the first to show a role for MEF2 proteins in the brain outside of the hippocampus, and our findings suggest that these proteins may play diverse roles in the CNS. It is important to continue to build on our understanding of the roles of proteins acting in the SCN because SCN dysfunction underlies jet lag in humans and influences the response to shift work schedules, which are now known as risk factors for the development of cancer. Our work on MEF2D could be the basis for opening new lines of research in the development and regulation of circadian rhythms.

Transcriptional regulation of numerous interferon-regulated genes, including Toll-like receptor 3 (Tlr3), which encodes an innate immune sensor of viral double-stranded RNA, depends on the interferon regulatory factor 1 (IRF1) and IRF2 transcription factors. We detected specific abrogation of macrophage responses to polyinosinic-polycytidylic acid (poly(I:C)) resulting from three independent N-ethyl-N-nitrosourea-induced mutations in host cell factor C2 (Hcfc2). Hcfc2 mutations compromised survival during influenza virus and herpes simplex virus 1 infections. HCFC2 promoted the binding of IRF1 and IRF2 to the Tlr3 promoter, without which inflammatory cytokine and type I IFN responses to the double-stranded RNA analogue poly(I:C) are reduced in mouse macrophages. HCFC2 was also necessary for the transcription of a large subset of other IRF2-dependent interferon-regulated genes. Deleterious mutations of Hcfc2 may therefore increase susceptibility to diverse infectious diseases.

Previous studies have demonstrated a positive correlation between glucocorticoid levels and circadian reentrainment time following a shift in the light:dark (LD) cycle. We conducted a series of experiments to examine the circadian dependence of the corticosterone (CORT) response to light. Exp. 1 measured CORT release in rats exposed to light at six timepoints. Light presented during the subjective night increased CORT (p<0.05), while light presented during the subjective day did not. In Exp. 2, we documented the time course of the CORT response to light in entrained animals. Rats exposed to light at zeitgeber time (ZT) 18 had a maximal increase in CORT levels following 60 min of stimulus presentation (p<0.05). There was also an increase in adrenocorticotropic hormone following 15 min of light at ZT18 (p<0.05). In an effort to elucidate the effect of changes in the LD cycle on the circadian profile of CORT, Exp. 3 followed the CORT rhythm (in cerebrospinal fluid) of rats prior to and following a shift in the LD cycle. The CORT nadir was elevated following a 6 h photic advance (p<0.05), as was the mean CORT concentration during the peak phase (p<0.05). Most components of the circadian CORT rhythm, however, failed to show an immediate shift towards the change in the light cycle. Together, these data support the hypothesis that a photic phase-shift results in elevated CORT levels, while the rhythm of CORT secretion is robust against changes in the photic environment.

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.

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.