Relaxin-3 null mutation mice display a circadian hypoactivity phenotype

Characterizing the neurocircuits and neurotransmitters that underlie arousal and circadian sleep/wake patterns is an important goal of neuroscience research, with potential implications for understanding human mental illnesses, such as major depression. Recent anatomical and functional studies suggest that relaxin-3 neurons and their ascending projections contribute to these functions via actions on key cortical, limbic and hypothalamic circuits. This study reports the behavioral phenotype of C57BL/6J backcrossed relaxin-3 knockout (KO) mice. Cohorts of adult, male and female relaxin-3 KO and wild-type (WT) littermate mice were subjected to a battery of behavioral tests to assess sensorimotor function and complex behavior. No overt deficits were detected in motor-coordination, spatial memory, sensorimotor gating, anxiety-like behavior or locomotor behavior in novel environments; and no marked genotype differences were observed in response to a chronic stress protocol. Notably however, compared to WT mice, relaxin-3 KO mice displayed robust hypoactivity during the dark/active phase when provided with free home-cage access to voluntary running wheels. This circadian hypoactivity was reflected by reduced time spent and distance traveled on running wheels, coupled with an increase in the time spent immobile, possibly reflecting increased sleeping. Overall, these studies support a role for relaxin-3 signaling in the control of arousal and sleep/wakefulness, and identify the relaxin-3 KO mouse as a useful model to study this role further.     

Diversity in cardiac sodium channel disease phenotype in transgenic mice carrying a single SCN5A mutation

Lethal ventricular arrhythmias are increasingly considered an important cause of sudden death in relatively young individuals. A genetic predisposition has been recognised in many cases, and research in the last decade has focused on underlying inherited mutations in cardiac ion channels.     

Liver steatosis and increased ChREBP expression in mice carrying a liver specific SIRT1 null mutation under a normal feeding condition

SIRT1, a homolog of yeast Sir2, is a type III NAD(+) dependent histone and protein deacetylase. Previous studies of mice carrying liver specific deletion of exon 4 of the Sirt1 gene revealed opposite responses of mutant mice to a high-fat diet in terms of fatty liver formation, which obscures the function of SRIT1 in liver development and lipid metabolism. To investigate this, we deleted exons 5 and 6 of Sirt1 in the liver by using a Cre-loxP approach. Western blot using an antibody to N-terminal SIRT1 does not detect a truncated protein in the liver of the mutant mice (Sirt1(flox5-6/flox5-6);Alb-Cre), suggesting a null mutation for SIRT1 is generated in the liver. Unlike the previously reported phenotypes, the Sirt1(flox5-6/flox5-6);Alb-Cre mice develop fatty liver under a normal feeding condition. The disease starts at two months of age and incidence increases as the animals become older, affecting 78% of them when they are over one year of age. We showed that the steatosis is accompanied by altered expression of a number of genes, including increased expression of ChREBP, which acts as one of the central determinants of lipid synthesis in the liver. This data uncovers an important role of SIRT1 in regulating lipid metabolism in the liver, and the SIRT1 mutant mice may serve as an animal model for studying human fatty liver disease and facilitate the development of effective therapeutic approach for the disease.     

A null mutation of cytoplasmic malic enzyme in mice

In the course of conducting a biochemical screening program for mutant enzymes in mice, individuals with an apparent nonfunctional allele at the locus (Mod-1) responsible for cytoplasmic malic enzyme were observed. The variant, later attributed to a germinal mutation, was identified by starch gel electrophoresis and by enzyme activity measurements. A series of matings were made, and mice homozygous for the nonfunctional, null, allele (Mod-1) were produced. In liver, kidney, and testis homogenates, the homozygous mutant exhibited less than 10% of the enzyme activity of the control mice. By an enzyme immuno-inactivation study, the residual enzyme activity was shown to be mitochondrial malic enzyme in all of the tissues examined. By double immuno-diffusion experiments, the kidney homogenate of the mutant formed no precipitin lines with the antiserum to cytoplasmic malic enzyme. Thus, the null mutants express no proteins that crossreact with the antiserum to cytoplasmic malic enzyme (CRM negative). Tissue enzyme assays revealed no significant differences between the normal and the mutant mice in activities of other enzymes in the related metabolic pathways. Because malic acid and malic enzyme together are reported to serve as a pump for NADPH generation in cytoplasm, total cellular NADP⁺ and NADPH concentrations in liver were determined for the control and the mutant mice. In liver from two individual mutant mice, lower NADPH/NADP⁺ ratio was detected in comparison to the level in liver from control mice. In spite of the lower levels of NADPH in the mutant mice, their body weight and lipid content were not significantly altered. Mice without cytoplasmic malic enzyme exhibited no striking deficiencies in metabolism or viability.     

Mice carrying a R142C Notch 3 knock-in mutation do not develop a CADASIL-like phenotype

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy, MIM 125310) is a genetic vascular dementia disease that is linked to missense mutations, small in-frame deletions, and splice site mutations in the human Notch 3 gene. Here we describe the generation of a mouse knockin model for one of the most prevalent CADASIL mutations, an arginine to cysteine transition at position 141, R141C, which corresponds to mutation R142C in mouse NOTCH 3. CADASIL(R142C) mice show no apparent CADASIL-like phenotype after histological and MRI analysis. The NOTCH 3 (R142C) receptor is processed normally and does not appear to accumulate the ectodomain, which has been observed in CADASIL patients. We discuss possible reasons for the different outcomes of the same germline CADASIL mutation in mice and humans.     

Liraglutide dictates macrophage phenotype in apolipoprotein E null mice during early atherosclerosis

Background

Macrophages play a pivotal role in atherosclerotic plaque development. Recent evidence has suggested the glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, can attenuate pro-inflammatory responses in macrophages. We hypothesized that liraglutide could limit atherosclerosis progression in vivo via modulation of the inflammatory response.

Methods

Human THP-1 macrophages and bone marrow-derived macrophages, from both wild-type C57BL/6 (WT) and apolipoprotein E null mice (ApoE^?/?) were used to investigate the effect of liraglutide on the inflammatory response in vitro. In parallel, ApoE^?/? mice were fed a high-fat (60% calories from fat) high-cholesterol (1%) diet for 8 weeks to induce atherosclerotic disease progression with/without daily 300 μg/kg liraglutide administration for the final 6 weeks. Macrophages were analysed for MΦ1 and MΦ2 macrophage markers by Western blotting, RT-qPCR, ELISA and flow cytometry. Atherosclerotic lesions in aortae from ApoE^?/? mice were analysed by en face staining and monocyte and macrophage populations from bone marrow derived cells analysed by flow cytometry.

Results

Liraglutide decreased atherosclerotic lesion formation in ApoE^?/? mice coincident with a reduction in pro-inflammatory and increased anti-inflammatory monocyte/macrophage populations in vivo. Liraglutide decreased IL-1beta in MΦ0 THP-1 macrophages and bone marrow-derived macrophages from WT mice and induced a significant increase in the MΦ2 surface marker mannose receptor in both MΦ0 and MΦ2 macrophages. Significant reduction in total lesion development was found with once daily 300 μg/kg liraglutide treatment in ApoE^?/? mice. Interestingly, liraglutide inhibited disease progression at the iliac bifurcation suggesting that it retards the initiation and development of disease. These results corresponded to attenuated MΦ1 markers (CCR7, IL-6 and TNF-alpha), augmented MΦ2 cell markers (Arg-1, IL-10 and CD163) and finally decreased MΦ1-like monocytes and macrophages from bone marrow-derived cells.

Conclusions

This data supports a therapeutic role for liraglutide as an atheroprotective agent via modulating macrophage cell fate towards MΦ2 pro-resolving macrophages.

     

Eye-open at birth phenotype with reduced keratinocyte motility in LGR4 null mice

We observed a consistent eye-open at birth (EOB) phenotype in mouse pups homozygous for a leucine-rich repeat containing G-protein coupled receptor 4 (Lgr4) allele deleting the whole transmembrane domain coding region. An in vitro wound-healing scratch assay showed notably reduced keratinocyte motility in the null mice. Phalloidin staining of F-actin in the eyelid epidermis was also reduced. We also generated keratinocyte-specific Lgr4 deficient mice, circumventing the embryonic/neonatal lethality and kidney abnormalities. Most of the conditional Lgr4 knockout mice showed the EOB phenotype. Thus, Lgr4 might be a novel gene class regulating cell motility.     

The myostatin null mutation and clenbuterol administration elicit additive effects in mice

In mice, the myostatin (Mstn) null mutation and treatment with clenbuterol both increase muscle growth and decrease fat mass. Our objective was to determine whether mechanistic overlap exists by administering clenbuterol to Mstn null mice. Male Mstn null and wild-type mice of similar genetic backgrounds received either 0 (control) or 20 p.p.m. clenbuterol in tap water free choice for 14 days. Several traits were measured to estimate muscle and fat growth. The Mstn null mutation resulted in increased body and empty carcass weight, increased muscle weights and decreased fat pad weights. Fat content was reduced and protein content was increased in the empty carcasses of Mstn null mice. Similarly, treatment with clenbuterol resulted in increased body and empty carcass weight, increased muscle weights and reduced fat pad weights. Fat content of empty carcasses and viscera was reduced and protein content of empty carcasses was increased with clenbuterol treatment. A significant interaction of genotype and clenbuterol treatment would indicate an altered responsiveness of Mstn null mice to clenbuterol. However, only the weight of gastrocnemius muscles exhibited a significant (P = 0.01) interaction of genotype and clenbuterol treatment, indicating that Mstn null mice were less responsive to clenbuterol compared with wild-type mice. Thus, for all other traits, the impact of Mstn null mutation and clenbuterol treatment was completely additive. These data suggest that disruption of Mstn function does not alter the response of mice to β-adrenergic agonists.     

Mutants suppressing novobiocin hypersensitivity of a mukB null mutation

The mukB gene is essential for the partitioning of sister chromosomes in Escherichia coli. A mukB null mutant is hypersensitive to the DNA gyrase inhibitor novobiocin. In this work, we isolated mutants suppressing the novobiocin hypersensitivity of the mukB null mutation. All suppressor mutations are localized in or near the gyrB gene, and the four tested clones have an amino acid substitution in the DNA gyrase beta subunit. We found that in the mukB mutant, the process of sister chromosome segregation is strikingly hypersensitive to novobiocin; however, the effect of novobiocin on growth, which was measured by culture turbidity, is the same as that of the wild-type strain.     

Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene

Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apc(Min/+)) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca(2+) activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin-luciferase in isolated erythrocytes from apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent clearance of apc(Min/+) erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.     

Partial pneumonectomy of telomerase null mice carrying shortened telomeres initiates cell growth arrest resulting in a limited compensatory growth response

Telomerase mutations and significantly shortened chromosomal telomeres have recently been implicated in human lung pathologies. Natural telomere shortening is an inevitable consequence of aging, which is also a risk factor for development of lung disease. However, the impact of shortened telomeres and telomerase dysfunction on the ability of lung cells to respond to significant challenge is still largely unknown. We have previously shown that lungs of late generation, telomerase null B6.Cg-Terc(tm1Rdp) mice feature alveolar simplification and chronic stress signaling at baseline, a phenocopy of aged lung. To determine the role telomerase plays when the lung is challenged, B6.Cg-Terc(tm1Rdp) mice carrying shortened telomeres and wild-type controls were subjected to partial pneumonectomy. We found that telomerase activity was strongly induced in alveolar epithelial type 2 cells (AEC2) of the remaining lung immediately following surgery. Eighty-six percent of wild-type animals survived the procedure and exhibited a burst of early compensatory growth marked by upregulation of proliferation, stress response, and DNA repair pathways in AEC2. In B6.Cg-Terc(tm1Rdp) mice carrying shortened telomeres, response to pneumonectomy was characterized by decreased survival, diminished compensatory lung growth, attenuated distal lung progenitor cell response, persistent DNA damage, and cell growth arrest. Overall, survival correlated strongly with telomere length. We conclude that functional telomerase and properly maintained telomeres play key roles in both long-term survival and the early phase of compensatory lung growth following partial pneumonectomy.     

The skeletal responsiveness to mechanical loading is enhanced in mice with a null mutation in estrogen receptor-beta

Mechanical loading caused by physical activity can stimulate bone formation and strengthen the skeleton. Estrogen receptors (ERs) play some role in the signaling cascade that is initiated in bone cells after a mechanical load is applied. We hypothesized that one of the ERs, ER-beta, influences the responsiveness of bone to mechanical loads. To test our hypothesis, 16-wk-old male and female mice with null mutations in ER-beta (ER-beta(-/-)) had their right forelimbs subjected to short daily loading bouts. The loading technique used has been shown to increase bone formation in the ulna. Each loading bout consisted of 60 compressive loads within 30 s applied daily for 3 consecutive days. Bone formation was measured by first giving standard fluorochrome bone labels 1 and 6 days after loading and using quantitative histomorphometry to assess bone sections from the midshaft of the ulna. The left nonloaded ulna served as an internal control for the effects of loading. Mechanical loading increased bone formation rate at the periosteal bone surface of the mid-ulna in both ER-beta(-/-) and wild-type (WT) mice. The ulnar responsiveness to loading was similar in male ER-beta(-/-) vs. WT mice, but for female mice bone formation was stimulated more effectively in ER-beta(-/-) mice (P < 0.001). We conclude that estrogen signaling through ER-beta suppresses the mechanical loading response on the periosteal surface of long bones.     

Characteristics of the brain dopamine system in mice carrying the quaking neurological mutation

Dopamine (DA) metabolism and the response to dopaminergic drugs were studied in quaking (QK) mice with neurological mutation expressed in demyelinization of the brain neurons and constant shaking. It has been shown that apomorphine in a low dose (0.25 mg/kg) produced a more significant decrease in locomotor activity in Qk than in control mice. Qk mice appeared to be less sensitive to the blockade by haloperidol of apomorphine (2.5 mg/kg)-induced climbing. DA1 receptor agonist, SKF-38393 caused less pronounced climbing in Qk mice than in the control. There were no changes in DA level in striatum and n. accumbens, whereas 3,4-dihydroxyphenylacetic acid in n. accumbens and homovanillic acid level in striatum were elevated. It was suggested that the increased DA metabolism and the altered sensitivity of pre- and postsynaptic DA receptors are involved in the shaking behaviour of Qk mice.     

Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene

CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock‐out mice. This represents creation of the first gene knock‐out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock‐in (Clic1^FN) allele, followed by Clic1 knock‐out (Clic1^−/−) mice by crossing Clic1^FN allele with TNAP‐cre mice, resulting in germline gene deletion through Cre‐mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1^−/− mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y₁₂ receptor signaling. genesis 48:127–136, 2010. © 2010 Wiley‐Liss, Inc.     

Increased production of VLDL apoB-100 in subjects with familial hypercholesterolemia carrying the same null LDL receptor gene mutation

Early radiokinetic studies revealed that the classical metabolic defect in patients with familial hypercholesterolemia (FH) is hypocatabolism of LDL due to decreased LDL receptor activity. However, recent studies have suggested that hepatic oversecretion of apolipoprotein B-100 (apoB-100)-containing lipoproteins could also contribute to the markedly elevated plasma concentrations of LDL-cholesterol found in FH. The aim of this study was to examine the kinetics of apoB-100 labeled with a stable isotope (l-[5,5,5-D(3)] leucine) in five normolipidemic controls and in seven well-characterized FH subjects that included six FH heterozygotes and one FH homozygote carrying the same null LDL receptor gene mutation. As compared with controls, the VLDL apoB-100 production rate was increased by 50% in the FH heterozygotes and by 109% in the FH homozygote. Furthermore, FH subjects had significantly higher LDL apoB-100 pool size and lower LDL apoB-100 fractional catabolic rate than controls. These results indicate that the elevation of plasma LDL-cholesterol found in FH is attributable to both decreased clearance of LDL and increased hepatic production of apoB-100-containing lipoproteins.     

Effect of interleukin-10 null mutation on maternal immune response and reproductive outcome in mice

Interleukin-10 (IL-10) is an anti-inflammatory and immune-deviating cytokine expressed in the endometrium and placenta. IL-10 null mutant (IL-10-/-) mice have been employed to examine the role of IL-10 in regulating immune events in early pregnancy and its significance in implantation and pregnancy success. The inflammatory response elicited in endometrial tissue by insemination was amplified in IL-10-/- mice, with a 66% increase in leukocytes in the endometrial stroma on Day 3 of pregnancy. Despite this, no evidence of abnormal type 1/type 2 skewing was seen in T-lymphocytes from lymph nodes draining the uterus. On Day 18 of gestation, IL-10-/- females mated with IL-10-/- males had 15% more implantation sites and 27% more viable fetuses than pregnant wild-type (IL-10+/+) mice. Placental weight was unaffected, but fetal weight and the fetal:placental weight ratio were higher in IL-10-/- pregnancies. Similar data were obtained in allogeneic pregnancies when IL-10-/- females were mated with major-histocompatibility complex (MHC) disparate IL-10-/- males. Pups delivered by IL-10-/- mothers had increased birth weight and followed an altered growth trajectory, with growth impairment evident from early postnatal life into adulthood, which was reflected in alterations in body composition at 14 wk of age. This study shows that although IL-10 is not essential for maternal immune tolerance or successful pregnancy irrespective of MHC disparity in the fetus, maternal IL-10 is a determinant of growth trajectory in progeny in utero and after birth.     

Discovery of a null mutation in a human trace amine receptor gene

G-protein-coupled receptors (GPCRs) are important mediators of signal transduction, and mutations in GPCR-encoding genes can lead to disease states. Here we describe a null mutation in an orphan GPCR-encoding gene that is predicted to inactivate completely the encoded receptor. The TA(3) receptor is a putative member of the recently described mammalian trace amine receptor family, and it is expressed in the pituitary gland and skeletal muscle. We tested for the presence of the mutant form of TA(3) (named TA(3)-TR) in a normal population, as well as in two disease groups (ADHD and bipolar affective disorder). We found TA(3)-TR to be commonly expressed in all groups, with approximately 20% allele frequency. We did not find any statistically significant correlation between either disease and the presence of TA(3)-TR.