Ubiquitin-Specific Peptidase 46 (Usp46) Regulates Mouse Immobile Behavior in the Tail Suspension Test through the GABAergic System

The tail suspension test (TST) is widely recognized as a useful experimental paradigm for assessing antidepressant activity and depression-like behavior. We have previously identified ubiquitin-specific peptidase 46 (Usp46) as a quantitative trait gene responsible for decreasing immobility time in the TST in mice. This Usp46 mutation has a 3-bp deletion coding for lysine in the open reading frame, and we indicated that Usp46 is implicated in the regulation of the GABAergic system. However, it is not known precisely how the immobile behavior is regulated by the GABAergic system. Therefore, in the present study, we examined whether the immobility time is influenced by drugs affecting the action mediated by GABAA receptor using both 3-bp deleted (the Usp46 mutant) and null Usp46 (Usp46 KO) mice. Nitrazepam, an agonist at the benzodiazepine-binding site of the GABAA receptor, which potentiates the action of GABA, produced a dose-dependent increase in TST immobility time in the Usp46 mutant mice without affecting general behaviors. The Usp46 KO mice exhibited short immobility times comparable to the Usp46 mutant mice, which was also increased by nitrazepam administration. The effects of nitrazepam in the Usp46 mutant and KO mice were antagonized by flumazenil. These results indicate that the 3-bp deleted Usp46 mutation causes a loss-of-function phenotype, and that the GABAA receptor might participate in the regulation of TST immobility time

PRMT1 Deficiency in Mouse Juvenile Heart Induces Dilated Cardiomyopathy and Reveals Cryptic Alternative Splicing Products

Protein arginine methyltransferase 1 (PRMT1) catalyzes the asymmetric dimethylation of arginine residues in proteins and methylation of various RNA-binding proteins and is associated with alternative splicing in vitro. Although PRMT1 has essential in vivo roles in embryonic development, CNS development, and skeletal muscle regeneration, the functional importance of PRMT1 in the heart remains to be elucidated. Here, we report that juvenile cardiomyocyte-specific PRMT1-deficient mice develop severe dilated cardiomyopathy and exhibit aberrant cardiac alternative splicing. Furthermore, we identified previously undefined cardiac alternative splicing isoforms of four genes (Asb2, Fbxo40, Nrap, and Eif4a2) in PRMT1-cKO mice and revealed that eIF4A2 protein isoforms translated from alternatively spliced mRNA were differentially ubiquitinated and degraded by the ubiquitin-proteasome system. These findings highlight the essential roles of PRMT1 in cardiac homeostasis and alternative splicing regulation

Tissue-Specific Function of Period3 in Circadian Rhythmicity

The mammalian circadian system is composed of multiple central and peripheral clocks that are temporally coordinated to synchronize physiology and behavior with environmental cycles. Mammals have three homologs of the circadian Period gene (Per1, 2, 3). While numerous studies have demonstrated that Per1 and Per2 are necessary for molecular timekeeping and light responsiveness in the master circadian clock in the suprachiasmatic nuclei (SCN), the function of Per3 has been elusive. In the current study, we investigated the role of Per3 in circadian timekeeping in central and peripheral oscillators by analyzing PER2::LUCIFERASE expression in tissues explanted from C57BL/6J wild-type and Per3−/− mice. We observed shortening of the periods in some tissues from Per3−/− mice compared to wild-types. Importantly, the periods were not altered in other tissues, including the SCN, in Per3−/− mice. We also found that Per3-dependent shortening of endogenous periods resulted in advanced phases of those tissues, demonstrating that the in vitro phenotype is also present in vivo. Our data demonstrate that Per3 is important for endogenous timekeeping in specific tissues and those tissue-specific changes in endogenous periods result in internal misalignment of circadian clocks in Per3−/− mice. Taken together, our studies demonstrate that Per3 is a key player in the mammalian circadian system

Photoperiod Regulates Corticosterone Rhythms by Altered Adrenal Sensitivity via Melatonin-Independent Mechanisms in Fischer 344 Rats and C57BL/6J Mice

Most species living in temperate zones adapt their physiology and behavior to seasonal changes in the environment by using the photoperiod as a primary cue. The mechanisms underlying photoperiodic regulation of stress-related functions are not well understood. In this study, we analyzed the effects of photoperiod on the hypothalamic-pituitary-adrenal axis in photoperiod-sensitive Fischer 344 rats. We first examined how photoperiod affects diurnal variations in plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone. ACTH levels did not exhibit diurnal variations under long- and short-day conditions. On the other hand, corticosterone levels exhibited a clear rhythm under short-day condition with a peak during dark phase. This peak was not observed under long-day condition in which a significant rhythm was not detected. To analyze the mechanisms responsible for the photoperiodic regulation of corticosterone rhythms, ACTH was intraperitoneally injected at the onset of the light or dark phase in dexamethasone-treated rats maintained under long- and short-day conditions. ACTH induced higher corticosterone levels in rats examined at dark onset under short-day condition than those maintained under long-day condition. Next, we asked whether melatonin signals are involved in photoperiodic regulation of corticosterone rhythms, and rats were intraperitoneally injected with melatonin at late afternoon under long-day condition for 3 weeks. However, melatonin injections did not affect the corticosterone rhythms. In addition, photoperiodic changes in the amplitude of corticosterone rhythms were also observed in melatonin-deficient C57BL/6J mice, in which expression profiles of several clock genes and steroidgenesis genes in adrenal gland were modified by the photoperiod. Our data suggest that photoperiod regulates corticosterone rhythms by altered adrenal sensitivity through melatonin-independent mechanisms that may involve the adrenal clock

Evidence for Time-of-Day Dependent Effect of Neurotoxic Dorsomedial Hypothalamic Lesions on Food Anticipatory Circadian Rhythms in Rats

The dorsomedial hypothalamus (DMH) is a site of circadian clock gene and immediate early gene expression inducible by daytime restricted feeding schedules that entrain food anticipatory circadian rhythms in rats and mice. The role of the DMH in the expression of anticipatory rhythms has been evaluated using different lesion methods. Partial lesions created with the neurotoxin ibotenic acid (IBO) have been reported to attenuate food anticipatory rhythms, while complete lesions made with radiofrequency current leave anticipatory rhythms largely intact. We tested a hypothesis that the DMH and fibers of passage spared by IBO lesions play a time-of-day dependent role in the expression of food anticipatory rhythms. Rats received intra-DMH microinjections of IBO and activity and body temperature (Tb) rhythms were recorded by telemetry during ad-lib food access, total food deprivation and scheduled feeding, with food provided for 4-h/day for 20 days in the middle of the light period and then for 20 days late in the dark period. During ad-lib food access, rats with DMH lesions exhibited a lower amplitude and mean level of light-dark entrained activity and Tb rhythms. During the daytime feeding schedule, all rats exhibited food anticipatory activity and Tb rhythms that persisted during 2 days without food in constant dark. In some rats with partial or total DMH ablation, the magnitude of the anticipatory rhythm was weak relative to most intact rats. When mealtime was shifted to the late night, the magnitude of the food anticipatory activity rhythms in these cases was restored to levels characteristic of intact rats. These results confirm that rats can anticipate scheduled daytime or nighttime meals without the DMH. Improved anticipation at night suggests a modulatory role for the DMH in the expression of food anticipatory activity rhythms during the daily light period, when nocturnal rodents normally sleep

Whole-genome resequencing shows numerous genes with nonsynonymous SNPs in the Japanese native cattle Kuchinoshima-Ushi

<p>Abstract</p> <p>Background</p> <p>Because the Japanese native cattle <it>Kuchinoshima-Ushi </it>have been isolated in a small island and their lineage has been intensely protected, it has been assumed to date that numerous and valuable genomic variations are conserved in this cattle breed.</p> <p>Results</p> <p>In this study, we evaluated genetic features of this breed, including single nucleotide polymorphism (SNP) information, by whole-genome sequencing using a Genome Analyzer II. A total of 64.2 Gb of sequence was generated, of which 86% of the obtained reads were successfully mapped to the reference sequence (Btau 4.0) with BWA. On an average, 93% of the genome was covered by the reads and the number of mapped reads corresponded to 15.8-fold coverage across the covered region. From these data, we identified 6.3 million SNPs, of which more than 5.5 million (87%) were found to be new. Out of the SNPs annotated in the bovine sequence assembly, 20,432 were found in protein-coding regions containing 11,713 nonsynonymous SNPs in 4,643 genes. Furthermore, phylogenetic analysis using sequence data from 10 genes (more than 10 kbp) showed that <it>Kuchinoshima-Ushi </it>is clearly distinct from European domestic breeds of cattle.</p> <p>Conclusions</p> <p>These results provide a framework for further genetic studies in the <it>Kuchinoshima-Ushi </it>population and research on functions of SNP-containing genes, which would aid in understanding the molecular basis underlying phenotypic variation of economically important traits in cattle and in improving intrinsic defects in domestic cattle breeds.</p

Treatment- and Population-Dependent Activity Patterns of Behavioral and Expression QTLs

Genetic control of gene expression and higher-order phenotypes is almost invariably dependent on environment and experimental conditions. We use two families of recombinant inbred strains of mice (LXS and BXD) to study treatment- and genotype-dependent control of hippocampal gene expression and behavioral phenotypes. We analyzed responses to all combinations of two experimental perturbations, ethanol and restraint stress, in both families, allowing for comparisons across 8 combinations of treatment and population. We introduce the concept of QTL activity patterns to characterize how associations between genomic loci and traits vary across treatments. We identified several significant behavioral QTLs and many expression QTLs (eQTLs). The behavioral QTLs are highly dependent on treatment and population. We classified eQTLs into three groups: cis-eQTLs (expression variation that maps to within 5 Mb of the cognate gene), syntenic trans-eQTLs (the gene and the QTL are on the same chromosome but not within 5 Mb), and non-syntenic trans-eQTLs (the gene and the QTL are on different chromosomes). We found that most non-syntenic trans-eQTLs were treatment-specific whereas both classes of syntenic eQTLs were more conserved across treatments. We also found there was a correlation between regions along the genome enriched for eQTLs and SNPs that were conserved across the LXS and BXD families. Genes with eQTLs that co-localized with the behavioral QTLs and displayed similar QTL activity patterns were identified as potential candidate genes associated with the phenotypes, yielding identification of novel genes as well as genes that have been previously associated with responses to ethanol

Genetic and Molecular Analysis of Wild-Derived Arrhythmic Mice

A new circadian variant was isolated by screening the intercross offspring of wild-caught mice (Mus musculus castaneus). This variant was characterized by an initial maintenance of damped oscillations and subsequent loss of rhythmicity after being transferred from light-dark (LD) cycles to constant darkness (DD). To map the genes responsible for the persistence of rhythmicity (circadian ratio) and the length of free-running period (τ), quantitative trait locus (QTL) analysis was performed using F2 mice obtained from an F1 cross between the circadian variant and C57BL/6J mice. As a result, a significant QTL with a main effect for circadian ratio (Arrhythmicity; Arrh-1) was mapped on Chromosome (Chr) 8. For τ, four significant QTLs, Short free-running period (Sfp-1) (Chr 1), Sfp-2 (Chr 6), Sfp-3 (Chr 8), Sfp-4 (Chr 11) were determined. An epistatic interaction was detected between Chr 3 (Arrh-2) and Chr 5 (Arrh-3). An in situ hybridization study of clock genes and mouse Period1::luciferase (mPer1::luc) real-time monitoring analysis in the suprachiasmatic nucleus (SCN) suggested that arrhythmicity in this variant might not be attributed to core circadian mechanisms in the SCN neurons. Our strategy using wild-derived variant mice may provide a novel opportunity to evaluate circadian and its related disorders in human that arise from the interaction between multiple variant genes

Characterisation of the maternal response to chronic phase shifts during gestation in the rat: implications for fetal metabolic programming

Disrupting maternal circadian rhythms through exposure to chronic phase shifts of the photoperiod has lifelong consequences for the metabolic homeostasis of the fetus, such that offspring develop increased adiposity, hyperinsulinaemia and poor glucose and insulin tolerance. In an attempt to determine the mechanisms by which these poor metabolic outcomes arise, we investigated the impact of chronic phase shifts (CPS) on maternal and fetal hormonal, metabolic and circadian rhythms. We assessed weight gain and food consumption of dams exposed to either CPS or control lighting conditions throughout gestation. At day 20, dams were assessed for plasma hormone and metabolite concentrations and glucose and insulin tolerance. Additionally, the expression of a range of circadian and metabolic genes was assessed in maternal, placental and fetal tissue. Control and CPS dams consumed the same amount of food, yet CPS dams gained 70% less weight during the first week of gestation. At day 20, CPS dams had reduced retroperitoneal fat pad weight (−15%), and time-of-day dependent decreases in liver weight, whereas fetal and placental weight was not affected. Melatonin secretion was not altered, yet the timing of corticosterone, leptin, glucose, insulin, free fatty acids, triglycerides and cholesterol concentrations were profoundly disrupted. The expression of gluconeogenic and circadian clock genes in maternal and fetal liver became either arrhythmic or were in antiphase to the controls. These results demonstrate that disruptions of the photoperiod can severely disrupt normal circadian profiles of plasma hormones and metabolites, as well as gene expression in maternal and fetal tissues. Disruptions in the timing of food consumption and the downstream metabolic processes required to utilise that food, may lead to reduced efficiency of growth such that maternal weight gain is reduced during early embryonic development. It is these perturbations that may contribute to the programming of poor metabolic homeostasis in the offspring.Tamara J. Varcoe, Michael J. Boden, Athena Voultsios, Mark D. Salkeld, Leewen Rattanatray, David J. Kennawa