Magel2 knockout mice manifest altered social phenotypes and a deficit in preference for social novelty

MAGEL2 is one of five protein‐coding, maternally imprinted, paternally expressed genes in the Prader–Willi syndrome (PWS)‐critical domain on chromosome 15q11‐q13. Truncating pathogenic variants of MAGEL2 cause Schaaf‐Yang syndrome (SHFYNG) (OMIM #615547), a neurodevelopmental disorder related to PWS. Affected individuals manifest a spectrum of neurocognitive and behavioral phenotypes, including intellectual disability and autism spectrum disorder (ASD). Magel2 knockout mice carrying a maternally inherited, imprinted wild‐type (WT) allele and a paternally inherited Magel2‐lacZ knock‐in allele, which abolishes endogenous Magel2 gene function, exhibit several features reminiscent of the human Prader–Willi phenotypes, including neonatal growth retardation, excessive weight gain after weaning and increased adiposity in adulthood. They were shown to have altered circadian rhythm, reduced motor activity and reduced fertility. An extensive assessment for autism‐like behaviors in this mouse model was warranted, because of the high prevalence of ASD in human patients. The behavior of Magel2 knockout mice and their WT littermates were assayed via open field, elevated plus maze, tube, three‐chamber and partition tests. Our studies confirm decreased horizontal activity of male and female mice and increased vertical activity of females, in the open field. Both sexes spent more time in the open arm of the elevated plus maze, suggestive of reductions in anxiety. Both sexes displayed a lack of preference for social novelty, via a lack of discrimination between known and novel partners in the partition test. The in‐depth investigation of behavioral profiles caused by Magel2 loss‐of‐function helps to elucidate the etiology of behavioral phenotypes both for SHFYNG and PWS in general.     

Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice

Background

Cell proliferation in all rapidly renewing mammalian tissues follows a circadian rhythm that is often disrupted in advanced-stage tumors. Epidemiologic studies have revealed a clear link between disruption of circadian rhythms and cancer development in humans. Mice lacking the circadian genes Period1 and 2 (Per) or Cryptochrome1 and 2 (Cry) are deficient in cell cycle regulation and Per2 mutant mice are cancer-prone. However, it remains unclear how circadian rhythm in cell proliferation is generated in vivo and why disruption of circadian rhythm may lead to tumorigenesis.

Methodology/Principal Findings

Mice lacking Per1 and 2, Cry1 and 2, or one copy of Bmal1, all show increased spontaneous and radiation-induced tumor development. The neoplastic growth of Per-mutant somatic cells is not controlled cell-autonomously but is dependent upon extracellular mitogenic signals. Among the circadian output pathways, the rhythmic sympathetic signaling plays a key role in the central-peripheral timing mechanism that simultaneously activates the cell cycle clock via AP1-controlled Myc induction and p53 via peripheral clock-controlled ATM activation. Jet-lag promptly desynchronizes the central clock-SNS-peripheral clock axis, abolishes the peripheral clock-dependent ATM activation, and activates myc oncogenic potential, leading to tumor development in the same organ systems in wild-type and circadian gene-mutant mice.

Conclusions/Significance

Tumor suppression in vivo is a clock-controlled physiological function. The central circadian clock paces extracellular mitogenic signals that drive peripheral clock-controlled expression of key cell cycle and tumor suppressor genes to generate a circadian rhythm in cell proliferation. Frequent disruption of circadian rhythm is an important tumor promoting factor.     

AMPK regulates circadian rhythms in a tissue- and isoform-specific manner

Background

AMP protein kinase (AMPK) plays an important role in food intake and energy metabolism, which are synchronized to the light-dark cycle. In vitro, AMPK affects the circadian rhythm by regulating at least two clock components, CKIα and CRY1, via direct phosphorylation. However, it is not known whether the catalytic activity of AMPK actually regulates circadian rhythm in vivo.

Methodology/Principal Finding

The catalytic subunit of AMPK has two isoforms: α1 and α2. We investigate the circadian rhythm of behavior, physiology and gene expression in AMPKα1−/− and AMPKα2−/− mice. We found that both α1−/− and α2−/− mice are able to maintain a circadian rhythm of activity in dark-dark (DD) cycle, but α1−/− mice have a shorter circadian period whereas α2−/− mice showed a tendency toward a slightly longer circadian period. Furthermore, the circadian rhythm of body temperature was dampened in α1−/− mice, but not in α2−/− mice. The circadian pattern of core clock gene expression was severely disrupted in fat in α1−/− mice, but it was severely disrupted in the heart and skeletal muscle of α2−/− mice. Interestingly, other genes that showed circadian pattern of expression were dysreguated in both α1−/− and α2−/− mice. The circadian rhythm of nicotinamide phosphoryl-transferase (NAMPT) activity, which converts nicotinamide (NAM) to NAD⁺, is an important regulator of the circadian clock. We found that the NAMPT rhythm was absent in AMPK-deficient tissues and cells.

Conclusion/Significance

This study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners.     

Circadian Timekeeping Is Disturbed in Rheumatoid Arthritis at Molecular Level

Introduction

Patients with rheumatoid arthritis (RA) have disturbances in the hypothalamic-pituitary-adrenal (HPA) axis. These are reflected in altered circadian rhythm of circulating serum cortisol, melatonin and IL-6 levels and in chronic fatigue. We hypothesized that the molecular machinery responsible for the circadian timekeeping is perturbed in RA. The aim of this study was to investigate the expression of circadian clock in RA.

Methods

Gene expression of thirteen clock genes was analyzed in the synovial membrane of RA and control osteoarthritis (OA) patients. BMAL1 protein was detected using immunohistochemistry. Cell autonomous clock oscillation was started in RA and OA synovial fibroblasts using serum shock. The effect of pro-inflammatory stimulus on clock gene expression in synovial fibroblasts was studied using IL-6 and TNF-α.

Results

Gene expression analysis disclosed disconcerted circadian timekeeping and immunohistochemistry revealed strong cytoplasmic localization of BMAL1 in RA patients. Perturbed circadian timekeeping is at least in part inflammation independent and cell autonomous, because RA synovial fibroblasts display altered circadian expression of several clock components, and perturbed circadian production of IL-6 and IL-1β after clock resetting. However, inflammatory stimulus disturbs the rhythm in cultured fibroblasts. Throughout the experiments ARNTL2 and NPAS2 appeared to be the most affected clock genes in human immune-inflammatory conditions.

Conclusion

We conclude that the molecular machinery controlling the circadian rhythm is disturbed in RA patients.     

Characterization of Circadian Behavior in the Starlet Sea Anemone,Nematostella vectensis

Background

Although much is known about how circadian systems control daily cycles in the physiology and behavior of Drosophila and several vertebrate models, marine invertebrates have often been overlooked in circadian rhythms research. This study focuses on the starlet sea anemone, Nematostella vectensis, a species that has received increasing attention within the scientific community for its potential as a model research organism. The recently sequenced genome of N. vectensis makes it an especially attractive model for exploring the molecular evolution of circadian behavior. Critical behavioral data needed to correlate gene expression patterns to specific behaviors are currently lacking in N. vectensis.

Methodology/Principal Findings

To detect the presence of behavioral oscillations in N. vectensis, locomotor activity was evaluated using an automated system in an environmentally controlled chamber. Animals exposed to a 24 hr photoperiod (12 hr light: 12 hr dark) exhibited locomotor behavior that was both rhythmic and predominantly nocturnal. The activity peak occurred in the early half of the night with a 2-fold increase in locomotion. Upon transfer to constant lighting conditions (constant light or constant dark), an approximately 24 hr rhythm persisted in most animals, suggesting that the rhythm is controlled by an endogenous circadian mechanism. Fourier analysis revealed the presence of multiple peaks in some animals suggesting additional rhythmic components could be present. In particular, an approximately 12 hr oscillation was often observed. The nocturnal increase in generalized locomotion corresponded to a 24 hr oscillation in animal elongation.

Conclusions/Significance

These data confirm the presence of a light-entrainable circadian clock in Nematostella vectensis. Additional components observed in some individuals indicate that an endogenous clock of approximately 12 hr frequency may also be present. By describing rhythmic locomotor behavior in N. vectensis, we have made important progress in developing the sea anemone as a model organism for circadian rhythm research.     

Reduced Viability,Fertility and Fecundity in Mice Lacking the Cajal Body Marker Protein,Coilin

Background

Coilin is the signature protein of the Cajal body, a conserved nuclear organelle involved in multiple aspects of small ribonucleoprotein (RNP) biogenesis. Coilin is required for Cajal body homeostasis in both plants and animals. Mice lacking coilin are viable when the mutation is crossed to an outbred strain but only partially viable when crossed to inbred lines.

Methodology/Principal Findings

In order to clarify this issue, we backcrossed the coilin deletion onto the C57BL6/J background for ten generations and then investigated the consequences of coilin removal on overall viability and reproductive success. We conclude that semi-lethal phenotype observed in mixed-background crosses is due to loss of the Coilin gene (or a very tightly-linked locus). Interestingly, coilin knockout embryos die relatively late in gestation, between E13.5 and birth. We show that the maternal contribution of coilin is not important for organismal viability. Importantly, coilin knockout mice display significant fertility and fecundity defects. Mutant males that escape the embryonic lethality display reduced testis size, however, both males and females contribute to the observed reduction in reproductive fitness.

Conclusions/Significance

The evolutionary conservation of coilin from plants to animals suggests that the protein plays an important role, perhaps coordinating the activities of various RNA-processing machineries. Our observations are consistent with the idea that coilin functions to ensure robust organismal development, especially during periods of rapid growth.     

Shifting the Circadian Rhythm of Feeding in Mice Induces Gastrointestinal,Metabolic and Immune Alterations Which Are Influenced by Ghrelin and the Core Clock Gene Bmal1

Background

In our 24-hour society, an increasing number of people are required to be awake and active at night. As a result, the circadian rhythm of feeding is seriously compromised. To mimic this, we subjected mice to restricted feeding (RF), a paradigm in which food availability is limited to short and unusual times of day. RF induces a food-anticipatory increase in the levels of the hunger hormone ghrelin. We aimed to investigate whether ghrelin triggers the changes in body weight and gastric emptying that occur during RF. Moreover, the effect of genetic deletion of the core clock gene Bmal1 on these physiological adaptations was studied.

Methods

Wild-type, ghrelin receptor knockout and Bmal1 knockout mice were fed ad libitum or put on RF with a normal or high-fat diet (HFD). Plasma ghrelin levels were measured by radioimmunoassay. Gastric contractility was studied in vitro in muscle strips and in vivo (¹³C breath test). Cytokine mRNA expression was quantified and infiltration of immune cells was assessed histologically.

Results

The food-anticipatory increase in plasma ghrelin levels induced by RF with normal chow was abolished in HFD-fed mice. During RF, body weight restoration was facilitated by ghrelin and Bmal1. RF altered cytokine mRNA expression levels and triggered contractility changes resulting in an accelerated gastric emptying, independent from ghrelin signaling. During RF with a HFD, Bmal1 enhanced neutrophil recruitment to the stomach, increased gastric IL-1α expression and promoted gastric contractility changes.

Conclusions

This is the first study demonstrating that ghrelin and Bmal1 regulate the extent of body weight restoration during RF, whereas Bmal1 controls the type of inflammatory infiltrate and contractility changes in the stomach. Disrupting the circadian rhythm of feeding induces a variety of diet-dependent metabolic, immune and gastrointestinal alterations, which may explain the higher prevalence of obesity and immune-related gastrointestinal disorders among shift workers.     

Relative Metabolic Stability,but Disrupted Circadian Cortisol Secretion during the Fasting Month of Ramadan

Background

Chronic feeding and sleep schedule disturbances are stressors that exert damaging effects on the organism. Practicing Muslims in Saudi Arabia go through strict Ramadan fasting from dawn till sunset for one month yearly. Modern era Ramadan practices in Saudi Arabia are associated with disturbed feeding and sleep patterns, namely abstaining from food and water and increasing daytime sleep, and staying awake and receiving food and water till dawn.

Hypothesis

Strict Ramadan practices in Saudi Arabia may influence metabolism, sleep and circadian cortisol secretion.

Protocol

Young, male Ramadan practitioners were evaluated before and two weeks into the Ramadan. Blood samples were collected at 9.00 am and 9.00 pm for measurements of metabolic parameters and cortisol. Saliva was collected serially during the day for cortisol determinations.

Results

Ramadan practitioners had relative metabolic stability or changes expected by the pattern of feeding. However, the cortisol circadian rhythm was abolished and circulating insulin levels and HOMA index were increased during this period.

Discussion

The flattening of the cortisol rhythm is typical of conditions associated with chronic stress or endogenous hypercortisolism and associated with insulin resistance.

Conclusions

Modern Ramadan practices in Saudi Arabia are associated with evening hypercortisolism and increased insulin resistance. These changes might contribute to the high prevalence of chronic stress-related conditions, such as central obesity, hypertension, metabolic syndrome and diabetes mellitus type 2, and their cardiovascular sequelae observed in the Kingdom.     

Effects of thioglycolic acid on parthenogenetic activation of Xenopus oocytes

Background

Existing in Permanent-wave solutions (PWS), thioglycolic acid (TGA) is widely used in hairdressing industry for its contribution to hair styling. However, the toxicity of TGA, especially its reproductive toxicity, gradually calls the attention of more and more researchers.

Method

In this work, xenopus oocytes were pretreated with different concentration of TGA, and then activated by calcium ionophore A23187. During culture, the oocytes activation rates were taken note at different time after adding calcium ionophore A23187. At the end of the culture period, the nuclear status was detected under confocal microscope. In addition, some other samples were collected for Western-Blotting analysis.

Result

TGA significantly inhibited the oocytes activation rate and pronuclear formation. It may be resulted from the inhibition of the degradation of p-ERK1, Mos and CyclinB2.

Conclusion

TGA inhibits in vitro parthenogenetic activation of xenopus oocytes with inhibited the degradation of proteins involved in mitogenic-activated protein kinase (MAPK) and maturation-promoting factor (MPF) pathways.     

Strain- and sex-dependent circadian changes in abcc2 transporter expression: implications for irinotecan chronotolerance in mouse ileum

Background

ATP-binding cassette transporter abcc2 is involved in the cellular efflux of irinotecan. The drug is toxic for mouse ileum, where abcc2 is highly expressed. Here, we investigate whether circadian changes in local abcc2 expression participate in the circadian rhythm of irinotecan toxicity for ileum mucosa, and further assess whether genetic background or sex modify this relation.

Methodology/Principal Findings

Ileum mucosa was obtained every 3–4 h for 24 h in male and female B6D2F₁ and B6CBAF₁ mice synchronized with light from Zeitgeber Time (ZT)0 to ZT12 alternating with 12 h of darkness. Irinotecan (50 mg/kg i.v. daily for 4 days) was administered at the sex- and strain-specific times corresponding to least (ZT11-15) or largest drug-induced body weight loss (ZT23-03-07). Abcc2 expression was determined with qRT-PCR for mRNA and with immunohistochemistry and confocal microscopy for protein. Histopathologic lesions were graded in ileum tissues obtained 2, 4 or 6 days after treatment. Two- to six-fold circadian changes were demonstrated for mRNA and protein mean expressions of abcc2 in mouse ileum (p<0.05). ZT12 corresponded to high mRNA and protein expressions, with circadian waveforms differing according to genetic background and sex. The proportion of mice spared from ileum lesions varied three-fold according to irinotecan timing, with best tolerability at ZT11-15 (p = 0.00003). Irinotecan was also best tolerated in males (p = 0.05) and in B6CBAF₁ (p = 0.0006).

Conclusions/Significance

Strain- and sex-dependent circadian patterns in abcc2 expressions displayed robust relations with the chronotolerance of ileum mucosa for irinotecan. This finding has strong potential implications for improving the intestinal tolerability of anticancer drugs through circadian delivery.     

Circadian Control of Dendrite Morphology in the Visual System of Drosophila melanogaster

Background

In the first optic neuropil (lamina) of the fly''s visual system, monopolar cells L1 and L2 and glia show circadian rhythms in morphological plasticity. They change their size and shape during the day and night. The most pronounced changes have been detected in circadian size of the L2 axons. Looking for a functional significance of the circadian plasticity observed in axons, we examined the morphological plasticity of the L2 dendrites. They extend from axons and harbor postsynaptic sites of tetrad synaptic contacts from the photoreceptor terminals.

Methodology/Principal Findings

The plasticity of L2 dendrites was evaluated by measuring an outline of the L2 dendritic trees. These were from confocal images of cross sections of L2 cells labeled with GFP. They were in wild-type and clock mutant flies held under different light conditions and sacrified at different time points. We found that the L2 dendrites are longest at the beginning of the day in both males and females. This rhythm observed under a day/night regime (LD) was maintained in constant darkness (DD) but not in continuous light (LL). This rhythm was not present in the arrhythmic per⁰¹ mutant in LD or in DD. In the clock photoreceptor cry^b mutant the rhythm was maintained but its pattern was different than that observed in wild-type flies.

Conclusions/Significance

The results obtained showed that the L2 dendrites exhibit circadian structural plasticity. Their morphology is controlled by the per gene-dependent circadian clock. The L2 dendrites are longest at the beginning of the day when the daytime tetrad presynaptic sites are most numerous and L2 axons are swollen. The presence of the rhythm, but with a different pattern in cry^b mutants in LD and DD indicates a new role of cry in the visual system. The new role is in maintaining the circadian pattern of changes of the L2 dendrite length and shape.     

Period2 Deficiency Blunts Hypoxia-Induced Mobilization and Function of Endothelial Progenitor Cells

Background

In the clinic, variations in circadian rhythm are evident in patients with cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. In this study, we focused on the role of the circadian gene period2 (per2) in mobilization and function of endothelial progenitor cells (EPCs) in vitro and in vivo after myocardial infarction (MI) in mice.

Methods and Results

MI was produced by surgical ligation of the left anterior descending coronary artery in mice with and without per2 deficiency. Trans-thoracic echocardiography was used to evaluate cardiac function in mice. Per2^−/− mice with MI showed decreased cardiac function and increased infarct size. The number of CD34+ cells and capillary density were decreased in the myocardium of per2^−/− mice on immunohistochemistry. Flow cytometry revealed decreased number of circulating EPCs in per2^−/− mice after MI. In vitro, per2^−/− EPCs showed decreased migration and tube formation capacity under hypoxia. Western blot analysis revealed inhibited activation of extracellular signal-regulated kinase and Akt signaling in the bone marrow of per2^−/− mice and inhibited PI3K/Akt expression in per2^−/− EPCs under hypoxia.

Conclusions

Per2 modulates EPC mobilization and function after MI, which is important to recovery after MI in mice.     

Health Impact of Fasting in Saudi Arabia during Ramadan: Association with Disturbed Circadian Rhythm and Metabolic and Sleeping Patterns

Background

Muslims go through strict Ramadan fasting from dawn till sunset for one month yearly. These practices are associated with disturbed feeding and sleep patterns. We recently demonstrated that, during Ramadan, circadian cortisol rhythm of Saudis is abolished, exposing these subjects to continuously increased cortisol levels.

Hypothesis

Secretory patterns of other hormones and metabolic parameters associated with cortisol, and insulin resistance, might be affected during Ramadan.

Protocol

Ramadan practitioners (18 males, 5 females; mean age ±SEM = 23.16±1.2 years) were evaluated before and two weeks into Ramadan. Blood was collected for measurements of endocrine and metabolic parameters at 9 am (±1 hour) and again twelve hours later.

Results

In Ramadan, glucose concentration was kept within normal range, with a significant increase in the morning. Mean morning concentration of leptin was significantly higher than pre-Ramadan values (p = 0.001), in contrast to that of adiponectin, which was significantly lower (p<0.001). These changes were associated with increased insulin resistance in morning and evening. Concentrations of hsCRP were lower during Ramadan than those during regular living conditions, however, normal circadian fluctuation was abolished (p = 0.49). Even though means of liver enzymes, total bilirubin, total protein and albumin were all decreased during Ramadan, statistically lower means were only noted for GGT, total protein, and albumin (p = 0.018, 0.002 and 0.001 respectively).

Discussion

Saudi Ramadan practitioners have altered adipokine patterns, typical of insulin resistance. The noted decreases of hsCRP, liver enzymes, total protein, and albumin, are most likely a result of fasting, while loss of circadian rhythmicity of hsCRP is probably due to loss of circadian cortisol rhythm.

Conclusions

Modern Ramadan practices in Saudi Arabia, which are associated with evening hypercortisolism, are also characterized by altered adipokines patterns, and an abolished hsCRP circadian rhythm, all likely to increase cardiometabolic risk.     

Female reproductive success decreases with display size in monkshood, Aconitum kusnezoffii (Ranunculaceae)

Background and Aims

Reduction in female fitness in large clones can occur as a result of increased geitonogamous self-fertilization and its influence through inbreeding depression. This possibility was investigated in the self-compatible, bee-pollinated perennial herb Aconitum kusnezoffii which varies in clone size.

Methods

Field investigations were conducted on pollinator behaviour, flowering phenology and variation in seed set. The effects of self-pollination following controlled self- and cross-pollination were also examined. Selfing rates of differently sized clones were assessed using allozyme markers.

Key Results

High rates of geitonogamous pollination were associated with large display size. Female fitness at the ramet level decreased with clone size. Fruit and seed set under cross-pollination were significantly higher than those under self-pollination. The pre-dispersal inbreeding depression was estimated as 0·502 based on the difference in seed set per flower between self- and cross-pollinated flowers. Selfing rates of differently sized clones did not differ.

Conclusions

It is concluded that in A. kusnezoffii the negative effects of self-pollination causing reduced female fertility with clone size arise primarily from a strong early-acting inbreeding depression leading to the abortion of selfed embryos prior to seed maturation.Key words: Early-acting inbreeding depression, Aconitum kusnezoffii, clone size, female reproductive success, geitonogamy     

Metabolic cycles are linked to the cardiovascular diurnal rhythm in rats with essential hypertension

Background

The loss of diurnal rhythm in blood pressure (BP) is an important predictor of end-organ damage in hypertensive and diabetic patients. Recent evidence has suggested that two major physiological circadian rhythms, the metabolic and cardiovascular rhythms, are subject to regulation by overlapping molecular pathways, indicating that dysregulation of metabolic cycles could desynchronize the normal diurnal rhythm of BP with the daily light/dark cycle. However, little is known about the impact of changes in metabolic cycles on BP diurnal rhythm.

Methodology/Principal Findings

To test the hypothesis that feeding-fasting cycles could affect the diurnal pattern of BP, we used spontaneously hypertensive rats (SHR) which develop essential hypertension with disrupted diurnal BP rhythms and examined whether abnormal BP rhythms in SHR were caused by alteration in the daily feeding rhythm. We found that SHR exhibit attenuated feeding rhythm which accompanies disrupted rhythms in metabolic gene expression not only in metabolic tissues but also in cardiovascular tissues. More importantly, the correction of abnormal feeding rhythms in SHR restored the daily BP rhythm and was accompanied by changes in the timing of expression of key circadian and metabolic genes in cardiovascular tissues.

Conclusions/Significance

These results indicate that the metabolic cycle is an important determinant of the cardiovascular diurnal rhythm and that disrupted BP rhythms in hypertensive patients can be normalized by manipulating feeding cycles.