Clock Protein PERIOD Research Articles

Abstract Tu132: Estrogen modulation of ethanol-evoked cardiac oxidative stress and dysfunction: Role of circadian clock period-2 and ferroptosis in estrogen deficient rats

Background: Alcohol triggers exacerbated cardiovascular effects in females, compared to males, in estrogen (E2) dependent manner. E2 provides cardiac protection in premenopausal women and models of menopause, yet the interplay between the circadian clock proteins and E2 in the female heart, particularly in the presence of ethanol (EtOH) remains unclear. Aim: This study aims to determine if the suppression of the circadian clock protein Period 2 (Per2) and associated redox enzymes contribute to E2-mediated exacerbation of the EtOH-induced cardiac ferroptosis, and dysfunction. Methods: Two groups of female Sprague-Dawley rats (n=6-8) were subjected to bilateral ovariectomy (OVX) and fed with EtOH (5% in liquid diet) in the presence of E2 or its vehicle for 8 weeks. Measurements of cardiac function using radiotelemetry and echocardiography along with ex vivo biochemical/molecular studies. Results: Compared to vehicle treated OVX+EtOH group, the E2-treated OVX+EtOH group exhibited: (1) reduced (p<0.05) body weight gain and fat mass; (2) higher (p<0.05) telemetry-measured blood pressure and heart rate; (3) reductions (p<0.05) in echocardiography derived contractility indices, ejection fraction and fractional shortening. In the same group, ex vivo molecular analysis demonstrated: (1) lower cardiac Per2 and suppressed (p<0.05) redox enzymes; (2) increased (p<0.05) ROS level and glutathione depletion (p<0.05), leading to the degradation of glutathione peroxidase 4 (GPX4); (3) heme oxygenase-1 upregulation leading to iron overload (p<0.05). These ferroptosis-related effects resulted in increased lipid peroxidation (p<0.05) compared to findings in OVX+EtOH rats in the absence of E2. Conclusion: This study is the first to suggest that abrogation of the E2-mediated Per2 upregulation unraveled cardiac ferroptosis and contributes to the detrimental cardiac effects of ethanol in E2-replete rats. These novel findings underscore the potential risk for myocardial ferroptosis and dysfunction in menopausal women who are consuming ethanol while on E2 replacement therapy.

Pigment-dispersing factor is present in circadian clock neurons of pea aphids and may mediate photoperiodic signalling to insulin-producing cells.

The neuropeptide pigment-dispersing factor (PDF) plays a pivotal role in the circadian clock of most Ecdysozoa and is additionally involved in the timing of seasonal responses of several photoperiodic species. The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species with an annual life cycle tightly coupled to the seasonal changes in day length. Nevertheless, PDF could not be identified in A. pisum so far. In the present study, we identified a PDF-coding gene that has undergone significant changes in the otherwise highly conserved insect C-terminal amino acid sequence. A newly generated aphid-specific PDF antibody stained four neurons in each hemisphere of the aphid brain that co-express the clock protein Period and have projections to the pars lateralis that are highly plastic and change their appearance in a daily and seasonal manner, resembling those of the fruit fly PDF neurons. Most intriguingly, the PDF terminals overlap with dendrites of the insulin-like peptide (ILP) positive neurosecretory cells in the pars intercerebralis and with putative terminals of Cryptochrome (CRY) positive clock neurons. Since ILP has been previously shown to be crucial for seasonal adaptations and CRY might serve as a circadian photoreceptor vital for measuring day length, our results suggest that PDF plays a critical role in aphid seasonal timing.

A New Clock is Running for Multiple Myeloma: Circadian Clock Protein-Period 3 (PER-3) Polymorphism.

Circadian Clock Protein PERIOD 3 (PER-3) is situated on chromosome 1p36.23 and has a polymorphic domain that expresses 4 or 5 copies of the 54-bp tandem repeat sequence. PER-3 gene polymorphisms play a role in the dysregulation of the immune system. This study intended to investigate the distributions and clinical effectiveness of the PER-3 gene polymorphism in multiple myeloma (MM) patients. One hundred fifty patients diagnosed between January 2007-2009 and 100 healthy individuals were included in this study. All patients were suitable for autologous stem cell transplantation (ASCT) at first evaluation, and after 4 courses of VCD at least partial remission, ASCT was carried out. Later, LD was used as maintenance. Genotypes of PER-3 gene of patients and healthy controls were statistically compared before treatment. In addition, these genotypes' effects on overall and progression free survival (OS and PFS) were investigated. Median PFS in the 5R/5R genotype was found to be significantly longer, albeit low, at 86% (p = 0.046). In the statistical analysis performed between the 4R/4R genotype and others, the PFS of patients with 4R/4R was found to be significantly shorter at 40.4 months (p = 0.026). Patients with the 4R/4R genotype would have a risk of 2.049 times of a shorter PFS (p=0.009). With this first study investigating the effect of a circadian gene in MM, the net effect of PER-3 gene polymorphism on PFS was revealed, and it will be a guide for future studies.

Phosphorylation Promotes the Accumulation of PERIOD Protein Foci.

Circadian clock drives the 24-h rhythm in our behavior and physiology. The molecular clock consists of a series of transcriptional/translational feedback loops operated by a number of clock genes. A very recent study reported that the clock protein PERIOD (PER) is organized into discrete foci at the nuclear envelope in fly circadian neurons, which is believed to be important for controlling the subcellular localization of clock genes. Loss of inner nuclear membrane protein lamin B receptor (LBR) leads to disruption of these foci, but how they are regulated is yet unknown. Here, we found that PER foci are likely phase-separated condensates, the formation of which is mediated by intrinsically disordered region in PER. Phosphorylation promotes the accumulation of these foci. Protein phosphatase 2A, which is known to dephosphorylate PER, hampers the accumulation of the foci. On the other hand, the circadian kinase DOUBLETIME (DBT) which phosphorylates PER enhances the accumulation of the foci. LBR likely facilitates PER foci accumulation by destabilizing the catalytic subunit of protein phosphatase 2A, MICROTUBULE STAR (MTS). In conclusion, here, we demonstrate a key role for phosphorylation in promoting the accumulation of PER foci, while LBR modulates this process by impinging on the circadian phosphatase MTS.

Hyperammonaemia disrupts daily rhythms reversibly by elevating glutamate in the central circadian pacemaker.

Patients with cirrhosis exhibit features of circadian disruption. Hyperammonaemia has been suggested to impair both homeostatic and circadian sleep regulation. Here, we tested if hyperammonaemia directly disrupts circadian rhythm generation in the central pacemaker, the suprachiasmatic nuclei (SCN) of the hypothalamus. Wheel-running activity was recorded from mice fed with a hyperammonaemic or normal diet for ~35 days in a 12:12 light-dark (LD) cycle followed by ~15 days in constant darkness (DD). The expression of the clock protein PERIOD2 (PER2) was recorded from SCN explants before, during and after ammonia exposure, ±glutamate receptor antagonists. In LD, hyperammonaemic mice advanced their daily activity onset time by ~1h (16.8 ± 0.3 vs. 18.1 ± 0.04 h, p=.009) and decreased their total activity, concentrating it during the first half of the night. In DD, hyperammonaemia reduced the amplitude of daily activity (551.5 ± 27.7 vs. 724.9 ± 59 counts, p=.007), with no changes in circadian period. Ammonia (≥0.01 mM) rapidly and significantly reduced PER2 amplitude, and slightly increased circadian period. The decrease in PER2 amplitude correlated with decreased synchrony among circadian cells in the SCN and increased extracellular glutamate, which was rescued by AMPA glutamate receptor antagonists. These data suggest that hyperammonaemia affects circadian regulation of rest-activity behaviour by increasing extracellular glutamate in the SCN.

Hsp40 overexpression in pacemaker neurons delays circadian dysfunction in a Drosophila model of Huntington's disease.

ABSTRACTCircadian disturbances are early features of neurodegenerative diseases, including Huntington's disease (HD). Emerging evidence suggests that circadian decline feeds into neurodegenerative symptoms, exacerbating them. Therefore, we asked whether known neurotoxic modifiers can suppress circadian dysfunction. We performed a screen of neurotoxicity-modifier genes to suppress circadian behavioural arrhythmicity in a Drosophila circadian HD model. The molecular chaperones Hsp40 and HSP70 emerged as significant suppressors in the circadian context, with Hsp40 being the more potent mitigator. Upon Hsp40 overexpression in the Drosophila circadian ventrolateral neurons (LNv), the behavioural rescue was associated with neuronal rescue of loss of circadian proteins from small LNv soma. Specifically, there was a restoration of the molecular clock protein Period and its oscillations in young flies and a long-lasting rescue of the output neuropeptide Pigment dispersing factor. Significantly, there was a reduction in the expanded Huntingtin inclusion load, concomitant with the appearance of a spot-like Huntingtin form. Thus, we provide evidence implicating the neuroprotective chaperone Hsp40 in circadian rehabilitation. The involvement of molecular chaperones in circadian maintenance has broader therapeutic implications for neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper.

Proteome analysis of the circadian clock protein PERIOD2.

Circadian rhythms are a series of endogenous autonomous 24-h oscillations generated by the circadian clock. At the molecular level, the circadian clock is based on a transcription-translation feedback loop, in which BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME (CRY) and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. There are three PER proteins, of which PER2 shows the strongest oscillation at both stability and cellular localization level. Protein-protein interactions (PPIs) or interactome of the circadian clock proteins have been investigated using classical methods such as two-dimensional gel electrophoresis, immunoprecipitation-coupled mass spectrometry, and yeast-two hybrid assay where the dynamic and weak interactions are difficult to catch. To identify the interactome of PER2 we have adopted proximity-dependent labeling with biotin and mass spectrometry-based identification of labeled proteins (BioID). In addition to known interactions with such as CRY1 and CRY2, we have identified several new PPIs for PER2 and confirmed some of them using co-immunoprecipitation technique. This study characterizes the PER2 protein interactions in depth, and it also implies that using a fast BioID method with miniTurbo or TurboID coupled to other major circadian clock proteins might uncover other interactors in the clock that have yet to be discovered.

The microtubule-associated protein Tau suppresses the axonal distribution of PDF neuropeptide and mitochondria in circadian clock neurons.

Disrupted circadian rhythms are a prominent feature of multiple neurodegenerative diseases. Yet mechanisms linking Tau to rhythmic behavior remain unclear. Here, we find that expression of a phosphomimetic human Tau mutant (TauE14) in Drosophila circadian pacemaker neurons disrupts free-running rhythmicity. While cell number and oscillations of the core clock protein PERIOD are unaffected in the small LNv (sLNv) neurons important for free running rhythms, we observe a near complete loss of the major LNv neuropeptide pigment dispersing factor (PDF) in the dorsal axonal projections of the sLNvs. This was accompanied by a ~50% reduction in the area of the dorsal terminals and a modest decrease in cell body PDF levels. Expression of wild-type Tau also reduced axonal PDF levels but to a lesser extent than TauE14. TauE14 also induces a complete loss of mitochondria from these sLNv projections. However, mitochondria were increased in sLNv cell bodies in TauE14 flies. These results suggest that TauE14 disrupts axonal transport of neuropeptides and mitochondria in circadian pacemaker neurons, providing a mechanism by which Tau can disrupt circadian behavior prior to cell loss.

Is There a Link between Circadian Clock Protein PERIOD 3 (PER3) (rs57875989) Variant and the Severity of COVID-19 Infection?

ObjectiveCorona Virus Disease-2019 (COVID-19) has been among the major infectious events of the century. In today’s literature where COVID-19 and host factor effects are frequently examined, we aimed to examine another factor: Circadian Clock Protein PERIOD 3 (PER3). There is a significant correlation between PER3 gene polymorphism and circadian rhythm disturbances and immune system dysregulation.MethodsIn our study, we recruited 200 patients diagnosed with COVID-19 in our hospital between April–June 2020, and 100 volunteers without known comorbidities to create a healthy control group. After comparing the initial gene polymorphisms of the patients with healthy controls, three separate clinical subgroups were formed. Gene polymorphism distribution and statistical significance were examined in the formed patient groups.ResultsNo significant difference was found between the patient group and the healthy controls (P>0.05, for all). When patients were divided into two separate clinical subgroups as exitus/alive according to their last condition during their 28-day follow-up, the 4R/5R genotype was significantly more common in patients with a mortal course (P=0.007). The PER3 4R/5R genotype was found at a significantly higher rate in the group of patients with the need for intensive care (P=0.034).ConclusionThe 4R/5R genotype may be associated with the need for intensive care and mortality in COVID-19 patients. These important results will be a guide for future studies.

Antibodies Against the Clock Proteins Period and Cryptochrome Reveal the Neuronal Organization of the Circadian Clock in the Pea Aphid.

Circadian clocks prepare the organism to cyclic environmental changes in light, temperature, or food availability. Here, we characterized the master clock in the brain of a strongly photoperiodic insect, the aphid Acyrthosiphon pisum, immunohistochemically with antibodies against A. pisum Period (PER), Drosophila melanogaster Cryptochrome (CRY1), and crab Pigment-Dispersing Hormone (PDH). The latter antibody detects all so far known PDHs and PDFs (Pigment-Dispersing Factors), which play a dominant role in the circadian system of many arthropods. We found that, under long days, PER and CRY are expressed in a rhythmic manner in three regions of the brain: the dorsal and lateral protocerebrum and the lamina. No staining was detected with anti-PDH, suggesting that aphids lack PDF. All the CRY1-positive cells co-expressed PER and showed daily PER/CRY1 oscillations of high amplitude, while the PER oscillations of the CRY1-negative PER neurons were of considerable lower amplitude. The CRY1 oscillations were highly synchronous in all neurons, suggesting that aphid CRY1, similarly to Drosophila CRY1, is light sensitive and its oscillations are synchronized by light-dark cycles. Nevertheless, in contrast to Drosophila CRY1, aphid CRY1 was not degraded by light, but steadily increased during the day and decreased during the night. PER was always located in the nuclei of the clock neurons, while CRY was predominantly cytoplasmic and revealed the projections of the PER/CRY1-positive neurons. We traced the PER/CRY1-positive neurons through the aphid protocerebrum discovering striking similarities with the circadian clock of D. melanogaster: The CRY1 fibers innervate the dorsal and lateral protocerebrum and putatively connect the different PER-positive neurons with each other. They also run toward the pars intercerebralis, which controls hormone release via the neurohemal organ, the corpora cardiaca. In contrast to Drosophila, the CRY1-positive fibers additionally travel directly toward the corpora cardiaca and the close-by endocrine gland, corpora allata. This suggests a direct link between the circadian clock and the photoperiodic control of hormone release that can be studied in the future.

An Intact Krüppel-like factor 9 Gene Is Required for Acute Liver Period 1 mRNA Response to Restraint Stress.

The clock protein period 1 (PER1) is a central component of the core transcription-translation feedback loop governing cell-autonomous circadian rhythms in animals. Transcription of Per1 is directly regulated by the glucocorticoid (GC) receptor (GR), and Per1 mRNA is induced by stressors or injection of GC. Circulating GCs may synchronize peripheral clocks with the central pacemaker located in the suprachiasmatic nucleus of the brain. Krüppel-like factor 9 (KLF9) is a zinc finger transcription factor that, like Per1, is directly regulated by liganded GR, and it associates in chromatin at clock and clock-output genes, including at Per1. We hypothesized that KLF9 modulates stressor-dependent Per1 transcription. We exposed wild-type (WT) and Klf9 null mice (Klf9-/-) of both sexes to 1 hour restraint stress, which caused similar 2- to 2.5-fold increases in plasma corticosterone (B) in each genotype and sex. Although WT mice of both sexes showed a 2-fold increase in liver Per1 mRNA level after restraint stress, this response was absent in Klf9-/- mice. However, injection of B in WT and Klf9-/- mice induced similar increases in Per1 mRNA. Our findings support that an intact Klf9 gene is required for liver Per1 mRNA responses to an acute stressor, but a possible role for GCs in this response requires further investigation.

Induction of diapause by clock proteins period and timeless via changes in PTTH and ecdysteroid titer in the sugar beet moth, Scrobipalpa ocellatella (Lepidoptera: Gelechiidae).

The sugar beet moth, Scrobipalpa ocellatella (Boyd), one of the most severe sugar beet pests, causes quantitative and qualitative yield losses late in the autumn. Previously, it was shown that low temperature and short-day photoperiod together cause diapause induction in pupae. Here, the interaction of the critical elements of the diapause induction, including the period (PER), timeless (TIM), prothoracicotropic hormone (PTTH), and ecdysteroid titer, were investigated. Immunohistochemistry results showed that the number of period immunoreactivity (PER-ir) and TIM-ir cells in nondiapause pupae (NDP) was lower than in the brain of the diapause pupae (DP). Moreover, the number of PER-ir and TIM-ir cells in the protocerebrum and optic lobe (OL) of NDP was lower than DP. Moreover, lower PTTH content in the brain and hemolymph of DP was confirmed by competitive enzyme-linked immunosorbent assay. Enzyme immunoassay showed a lower 20-hydroxyecdysone (20E) titer in the hemolymph of the DP compared with the NDP. Within a short-day condition, PER and TIM titers increased in the brain leading to decreasing PTTH titers in the brain and hemolymph that caused decreasing 20E titer in the hemolymph, leading to the induction of diapause. This study suggests that PER and TIM could be one of the brain factors that play an essential role in regulating diapause in S. ocellatella.

Timing of Food Intake Drives the Circadian Rhythm of Blood Pressure.

Timing of food intake has become a critical factor in determining overall cardiometabolic health. We hypothesized that timing of food intake entrains circadian rhythms of blood pressure (BP) and renal excretion in mice. Male C57BL/6J mice were fed ad libitum or reverse feeding (RF) where food was available at all times of day or only available during the 12-h lights-on period, respectively. Mice eating ad libitum had a significantly higher mean arterial pressure (MAP) during lights-off compared to lights-on (113 ± 2 mmHg vs 100 ± 2 mmHg, respectively; P < 0.0001); however, RF for 6 days inverted the diurnal rhythm of MAP (99 ± 3 vs 110 ± 3 mmHg, respectively; P < 0.0001). In contrast to MAP, diurnal rhythms of urine volume and sodium excretion remained intact after RF. Male Bmal1 knockout mice (Bmal1KO) underwent the same feeding protocol. As previously reported, Bmal1KO mice did not exhibit a diurnal MAP rhythm during ad libitum feeding (95 ± 1 mmHg vs 92 ± 3 mmHg, lights-off vs lights-on; P > 0.05); however, RF induced a diurnal rhythm of MAP (79 ± 3 mmHg vs 95 ± 2 mmHg, lights-off vs lights-on phase; P < 0.01). Transgenic PERIOD2::LUCIFERASE knock-in mice were used to assess the rhythm of the clock protein PERIOD2 in ex vivo tissue cultures. The timing of the PER2::LUC rhythm in the renal cortex and suprachiasmatic nucleus was not affected by RF; however, RF induced significant phase shifts in the liver, renal inner medulla, and adrenal gland. In conclusion, the timing of food intake controls BP rhythms in mice independent of Bmal1, urine volume, or sodium excretion.

&lt;p&gt;Circadian Clock Protein PERIOD2 Suppresses the PI3K/Akt Pathway and Promotes Cisplatin Sensitivity in Ovarian Cancer&lt;/p&gt;

BackgroundThe mortality rate of ovarian cancer is the highest among gynecological tumors. The two factors leading to high mortality of ovarian cancer are late clinical stage and chemotherapy resistance. It is very important to reverse or intervene chemotherapy resistance. Abnormal circadian rhythm is related to the occurrence of tumor, and circadian clock protein PERIOD2 (PER2) acts as a tumor suppressor in cancer; however, little is known about its involvement in chemosensitivity.MethodsThis study aimed to investigate the role and underlying mechanisms of PER2 in ovarian cancer sensitivity to cisplatin. Overexpression and knockdown of PER2 were performed to explore its role in ovarian cancer cell sensitivity to cisplatin both in vitro and in vivo. The protein levels of PI3K, AKT, caspase 3, E-cadherin, and other drug resistance-related molecules were determined in parental SKOV3 and SKOV3/DDP cells as well as in xenograft tumor tissues.ResultsCompared with parental cells, SKOV3/DDP cells had dramatically decreased PER2 expression, possibly due to hypermethylation in the PER2 promoter. PER2 overexpression significantly inhibited proliferation while promoting cisplatin-induced apoptosis in SKOV3 and SKOV3/DDP cells. In agreement, PER2-overexpressing SKOV3/DPP cells yielded significantly reduced tumor mass in cisplatin-treated mice compared with control cells. Mechanistically, PER2 overexpression remarkably reduced the protein amounts of PI3K, AKT, and MDR1 while increasing those of caspase 3 and E-cadherin in tumor tissues. Knockdown of PER2 exhibited opposite effects. PER2 overexpression also reduced the serum levels of TNF-α and IL-6 in tumor-bearing mice before the initiation of cisplatin treatment.ConclusionThis study suggests that loss of PER2 contributes to cisplatin resistance in SKOV3 cells, possibly by activating the PI3K/AKT pathway and EMT, inhibiting apoptosis, and promoting drug efflux and inflammatory responses. Overexpression of PER2 could reverse these alterations and sensitize both parental SKOV3 and SKOV3/DDP cells to cisplatin.

A Functional Clock Within the Main Morning and Evening Neurons of D. melanogaster Is Not Sufficient for Wild-Type Locomotor Activity Under Changing Day Length.

A major challenge for all organisms that live in temperate and subpolar regions is to adapt physiology and activity to different photoperiods. A long-standing model assumes that there are morning (M) and evening (E) oscillators with different photoreceptive properties that couple to dawn and dusk, respectively, and by this way adjust activity to the different photoperiods. In the fruit fly Drosophila melanogaster, M and E oscillators have been localized to specific circadian clock neurons in the brain. Here, we investigate under different photoperiods the activity pattern of flies expressing the clock protein PERIOD (PER) only in subsets of M and E oscillators. We found that all fly lines that expressed PER only in subsets of the clock neurons had difficulties to track the morning and evening in a wild-type manner. The lack of the E oscillators advanced M activity under short days, whereas the lack of the M oscillators delayed E activity under the same conditions. In addition, we found that flies expressing PER only in subsets of clock neurons showed higher activity levels at certain times of day or night, suggesting that M and E clock neurons might inhibit activity at specific moments throughout the 24 h. Altogether, we show that the proper interaction between all clock cells is important for adapting the flies’ activity to different photoperiods and discuss our findings in the light of the current literature.

Social stress and glucocorticoids alter PERIOD2 rhythmicity in the liver, but not in the suprachiasmatic nucleus

Circadian (~24 h) rhythms in behavior and physiological functions are under control of an endogenous circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN directly drives some of these rhythms or serves as a coordinator of peripheral oscillators residing in other tissues and organs. Disruption of the circadian organization may contribute to disease, including stress-related disorders. Previous research indicates that the master clock in the SCN is resistant to stress, although it is unclear whether stress affects rhythmicity in other tissues, possibly mediated by glucocorticoids, released in stressful situations. In the present study, we examined the effect of uncontrollable social defeat stress and glucocorticoid hormones on the central and peripheral clocks, respectively in the SCN and liver. Transgenic PERIOD2::LUCIFERASE knock-in mice were used to assess the rhythm of the clock protein PERIOD2 (PER2) in SCN slices and liver tissue collected after 10 consecutive days of social defeat stress. The rhythmicity of PER2 expression in the SCN was not affected by stress exposure, whereas in the liver the expression showed a delayed phase in defeated compared to non-defeated control mice. In a second experiment, brain slices and liver samples were collected from transgenic mice and exposed to different doses of corticosterone. Corticosterone did not affect PER2 rhythm of the SCN samples, but caused a phase shift in PER2 expression in liver samples. This study confirms earlier findings that the SCN is resistant to stress and shows that clocks in the liver are affected by social stress, which might be due to the direct influence of glucocorticoids released from the adrenal gland.

Temperature synchronization of the Drosophila circadian clock protein PERIOD is controlled by the TRPA channel PYREXIA

Circadian clocks are endogenous molecular oscillators that temporally organize behavioral activity thereby contributing to the fitness of organisms. To synchronize the fly circadian clock with the daily fluctuations of light and temperature, these environmental cues are sensed both via brain clock neurons, and by light and temperature sensors located in the peripheral nervous system. Here we demonstrate that the TRPA channel PYREXIA (PYX) is required for temperature synchronization of the key circadian clock protein PERIOD. We observe a molecular synchronization defect explaining the previously reported defects of pyx mutants in behavioral temperature synchronization. Surprisingly, surgical ablation of pyx-mutant antennae partially rescues behavioral synchronization, indicating that antennal temperature signals are modulated by PYX function to synchronize clock neurons in the brain. Our results suggest that PYX protects antennal neurons from faulty signaling that would otherwise interfere with temperature synchronization of the circadian clock neurons in the brain.

Assessing the Role of the Transcriptional Repressor BLMP-1 in the Molting Timer of C. elegans [University of California, Los Angeles

Biological rhythms are key features of animal development; however, the underlying timers are not well understood. A novel biological oscillator, perhaps ancestrally related to the human circadian clock, times the molting cycle in C. elegans. The molting timer consists of interconnected feedback loops among the nuclear hormone receptor NHR-23, the microRNA let-7 and LIN-42, the worm homolog of the human circadian clock protein PERIOD. However, other components of the timer are not known. I hypothesized that the BLMP-1 protein is a core component of the molting timer in C. elegans. The extent to which BLMP-1 times the molting cycle was determined by comparing the duration of larval stages in blmp-1 mutants to that of wild-type animals. I characterized the temporal expression profile of the blmp-1 gene using the blmp-1p::gfp reporter gene which I introduced to the animals through transfection. In order to determine if NHR-23 activates blmp-1, I used the reporter gene to observe blmp-1 expression in stage-specific knockdowns of nhr-23. I found that blmp-1 mutants reach adulthood earlier relative to wild type animals, when both are released from starvation at the same time. Additionally, lower fluorescence intensity of blmp-1p::gfp is observed in nhr-23 mutants than in control animals, signifying that blmp-1 levels are lower in the absence of NHR-23. A deeper understanding of the molting timer can lead to insights in the biological clocks pertinent to humans which can be applied both to the circadian rhythm and to aging.

Direct and indirect inhibition of the circadian clock protein Per1: effects on ENaC and blood pressure.

Circadian rhythms govern physiological functions and are important for overall health. The molecular circadian clock comprises several transcription factors that mediate circadian control of physiological function, in part, by regulating gene expression in a tissue-specific manner. These connections are well established, but the underlying mechanisms are incompletely understood. The overall goal of this study was to examine the connection among the circadian clock protein Period 1 (Per1), epithelial Na+ channel (ENaC), and blood pressure (BP) using a multipronged approach. Using global Per1 knockout mice on a 129/sv background in combination with a high-salt diet plus mineralocorticoid treatment, we demonstrated that loss of Per1 in this setting is associated with protection from hypertension. Next, we used the ENaC inhibitor benzamil to demonstrate a role for ENaC in BP regulation and urinary Na+ excretion in 129/sv mice. We targeted Per1 indirectly using pharmacological inhibition of Per1 nuclear entry in vivo to demonstrate altered expression of known Per1 target genes as well as a BP-lowering effect in 129/sv mice. Finally, we directly inhibited Per1 via genetic knockdown in amphibian distal nephron cells to demonstrate, for the first time, that reduced Per1 expression is associated with decreased ENaC activity at the single channel level.

Female C57BL/6J mice lacking the circadian clock protein PER1 are protected from nondipping hypertension.

The circadian clock is integral to the maintenance of daily rhythms of many physiological outputs, including blood pressure. Our laboratory has previously demonstrated the importance of the clock protein period 1 (PER1) in blood pressure regulation in male mice. Briefly, a high-salt diet (HS; 4% NaCl) plus injection with the long-acting mineralocorticoid deoxycorticosterone pivalate (DOCP) resulted in nondipping hypertension [<10% difference between night and day blood pressure (BP) in Per1-knockout (KO) mice but not in wild-type (WT) mice]. To date, there have been no studies that have examined the effect of a core circadian gene KO on BP rhythms in female mice. The goal of the present study was to determine whether female Per1-KO mice develop nondipping hypertension in response to HS/DOCP treatment. For the first time, we demonstrate that loss of the circadian clock protein PER1 in female mice does not significantly change mean arterial pressure (MAP) or the BP rhythm relative to female C57BL/6 WT control mice. Both WT and Per1-KO female mice experienced a significant increase in MAP in response to HS/DOCP. Importantly, however, both genotypes maintained a >10% dip in BP on HS/DOCP. This effect is distinct from the nondipping hypertension seen in male Per1-KO mice, demonstrating that the female sex appears to be protective against PER1-mediated nondipping hypertension in response to HS/DOCP. Together, these data suggest that PER1 acts in a sex-dependent manner in the regulation of cardiovascular rhythms.