Defective Reelin/Dab1 signaling pathways associated with disturbed hippocampus development of homozygous yotari mice.

Homozygous Dab1 yotari mutant mice, Dab1yot (yot/yot) mice, have an autosomal recessive mutation of Dab1 and show reeler-like phenotype including histological abnormality of the cerebellum, hippocampus, and cerebral cortex. We here show abnormal hippocampal development of yot/yot mice where granule cells and pyramidal cells fail to form orderly rows but are dispersed diffusely in vague multiplicative layers. Possibly due to the positioning failure of granule cells and pyramidal cells and insufficient synaptogenesis, axons of the granule cells did not extend purposefully to connect with neighboring regions in yot/yot mice. We found that both hippocampal granule cells and pyramidal cells of yot/yot mice expressed proteins reactive with the anti-Dab1 antibody. We found that Y198- phosphorylated Dab1 of yot/yot mice was greatly decreased. Accordingly the downstream molecule, Akt was hardly phosphorylated. Especially, synapse formation was defective and the distribution of neurons was scattered in hippocampus of yot/yot mice. Some of neural cell adhesion molecules and hippocampus associated transcription factors of the neurons were expressed aberrantly, suggesting that the Reelin-Dab1 signaling pathway seemed to be importantly involved in not only neural migration as having been shown previously but also neural maturation and/or synaptogenesis of the mice. It is interesting to clarify whether the defective neural maturation is a direct consequence of the dysfunctional Dab1, or alternatively secondarily due to the Reelin-Dab1 intracellular signaling pathways.

Metabolic Profiling of Multiorgan Samples: Evaluation of MODY5/RCAD Mutant Mice.

In the present study, we performed a metabolomics analysis to evaluate a MODY5/RCAD mouse mutant line as a potential model for HNF1B-associated diseases. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) of gut, kidney, liver, muscle, pancreas, and plasma samples uncovered the tissue specific metabolite distribution. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) was used to identify the differences between MODY5/RCAD and wild-type mice in each of the tissues. The differences included, for example, increased levels of amino acids in the kidneys and reduced levels of fatty acids in the muscles of the MODY5/RCAD mice. Interestingly, campesterol was found in higher concentrations in the MODY5/RCAD mice, with a four-fold and three-fold increase in kidneys and pancreas, respectively. As expected, the MODY5/RCAD mice displayed signs of impaired renal function in addition to disturbed liver lipid metabolism, with increased lipid and fatty acid accumulation in the liver. From a metabolomics perspective, the MODY5/RCAD model was proven to display a metabolic pattern similar to what would be suspected in HNF1B-associated diseases. These findings were in line with the presumed outcome of the mutation based on the different anatomy and function of the tissues as well as the effect of the mutation on development.

Fluoxetine induces paradoxical effects in C57BL6/J mice: comparison with BALB/c mice.

The C57BL6/J mouse is the most commonly used strain in genetic investigations and behavioural tests. However, only a few studies have used C57BL6/J mice to assess the effects of antidepressant compounds. We carried out a study to compare the behavioural effects of fluoxetine (FLX) in a model of depression in two mice strains: C57BL6/J and BALB/c. We used an 8-week unpredictable chronic mild stress (UCMS) protocol during which FLX was administered (15 mg/kg, oral) from the third week to the end of the protocol. We found that UCMS induced degradation of the coat state in the two strains. Moreover, as expected, we observed that FLX elicited antidepressant-like effects in the BALB/c mice by reducing the coat state deterioration and the latency of grooming in splash test. However, in the C57BL6/J mice, it did not induce this action, but instead triggered an opposite effect: an increased sniffing latency in the novelty suppression of feeding test. We conclude that FLX exerts a paradoxical effect in the C57Bl6/J strain. This observation is consistent with some clinical features of hyper-reactivity to FLX observed in humans. Therefore, the UCMS protocol used in C57Bl6/J mice could be a good model to study the mechanisms of the paradoxical effects caused by selective serotonin reuptake inhibitors.

Myosins.

The number and variety of myosins that have been identified has increased greatly over the past few years, and is still growing. Myosins have been classified into at least six distinct classes. Research during the last year has concentrated on identifying the roles of various myosins.

ENU mutagenesis screen to establish motor phenotypes in wild-type mice and modifiers of a pre-existing motor phenotype in tau mutant mice.

Modifier screening is a powerful genetic tool. While not widely used in the vertebrate system, we applied these tools to transgenic mouse strains that recapitulate key aspects of Alzheimer's disease (AD), such as tau-expressing mice. These are characterized by a robust pathology including both motor and memory impairment. The phenotype can be modulated by ENU mutagenesis, which results in novel mutant mouse strains and allows identifying the underlying gene/mutation. Here we discuss this strategy in detail. We firstly obtained pedigrees that modify the tau-related motor phenotype, with mapping ongoing. We further obtained transgene-independent motor pedigrees: (i) hyperactive, circling ENU 37 mice with a causal mutation in the Tbx1 gene-the complete knock-out of Tbx1 models DiGeorge Syndrome; (ii) ENU12/301 mice that show sudden jerky movements and tremor constantly; they have a causal mutation in the Kcnq1 gene, modelling aspects of the Romano-Ward and Jervell and Lange-Nielsen syndromes; and (iii) ENU16/069 mice with tremor and hypermetric gait that have a causal mutation in the Mpz (Myelin Protein Zero) gene, modelling Charcot-Marie-Tooth disease type 1 (CMT1B). Together, we provide evidence for a real potential of an ENU mutagenesis to dissect motor functions in wild-type and tau mutant mice.

BiP, an endoplasmic reticulum chaperone, modulates the development of morphine antinociceptive tolerance.

Morphine is a potent analgesic, but the molecular mechanism for tolerance formation after repeated use is not fully understood. Binding immunoglobulin protein (BiP) is an endoplasmic reticulum (ER) chaperone that is central to ER function. We examined knock-in mice expressing a mutant BiP with the retrieval sequence deleted in order to elucidate physiological processes that are sensitive to BiP functions. We tested the thermal antinociceptive effect of morphine in heterozygous mutant BiP mice in a hot plate test. Paw withdrawal latencies before and after a single administration of morphine were not significantly different between the wild-type and mutant BiP mice. Repeated morphine administration caused the development of morphine tolerance in the wild-type mice. The activation of glycogen synthase kinase 3b (GSK-3b) was associated with morphine tolerance, because an inhibitor of GSK-3ß prevented it. On the other hand, the mutant BiP mice showed less morphine tolerance, and the activation of GSK-3b was suppressed in their brain. These results suggest that BiP may play an important role in the development of morphine tolerance. Furthermore, we found that a chemical chaperone which improves ER protein folding capacity also attenuated the development of morphine tolerance in wild-type mice, suggesting a possible clinical application of chemical chaperones in preventing morphine tolerance.

Nephron-deficient Fvb mice develop rapidly progressive renal failure and heavy albuminuria involving excess glomerular GLUT1 and VEGF.

Reduced nephron numbers may predispose to renal failure. We hypothesized that glucose transporters (GLUTs) may contribute to progression of the renal disease, as GLUTs have been implicated in diabetic glomerulosclerosis and hypertensive renal disease with mesangial cell (MC) stretch. The Os (oligosyndactyly) allele that typically reduces nephron number by approximately 50%, was repeatedly backcrossed from ROP (Ra/+ (ragged), Os/+ (oligosyndactyly), and Pt/+ (pintail)) Os/+ mice more than six times into the Fvb mouse background to obtain Os/+ and +/+ mice with the Fvb background for study. Glomerular function, GLUT1, signaling, albumin excretion, and structural and ultrastructural changes were assessed. The FvbROP Os/+ mice (Fvb background) exhibited increased glomerular GLUT1, glucose uptake, VEGF, glomerular hypertrophy, hyperfiltration, extensive podocyte foot process effacement, marked albuminuria, severe extracellular matrix (ECM) protein deposition, and rapidly progressive renal failure leading to their early demise. Glomerular GLUT1 was increased 2.7-fold in the FvbROP Os/+ mice vs controls at 4 weeks of age, and glucose uptake was increased 2.7-fold. These changes were associated with the activation of glomerular PKCbeta1 and NF-kappaB p50 which contribute to ECM accumulation. The cyclic mechanical stretch of MCs in vitro, used as a model for increased MC stretch in vivo, reproduced increased GLUT1 at 48 h, a stimulus for increased VEGF expression which followed at 72 h. VEGF was also shown to act in a positive feedback manner on MC GLUT1, increasing GLUT1 expression, glucose uptake and fibronectin (FN) accumulation in vitro, whereas antisense suppression of GLUT1 largely blocked FN upregulation by VEGF. The FvbROP Os/+ mice exhibited an early increase in glomerular GLUT1 leading to increased glomerular glucose uptake PKCbeta1, and NF-kappaB activation, with excess ECM accumulation. A GLUT1-VEGF-GLUT1 positive feedback loop may play a key role in contributing to renal disease in this model of nondiabetic glomerulosclerosis.

Long-term administration of massive doses of Sn-protoporphyrin in anemic mutant mice (sphha/sphha).

The effects of long-term administration of very large doses of Sn-protoporphyrin on hematological indices, histological changes, plasma bilirubin levels, tissue heme oxygenase activity, and activities of heme biosynthetic enzymes, were examined in genetically anemic mutant mice with hemolytic anemia (sphha/sphha). Long-term weekly treatment with Sn-protoporphyrin (100 mumol/kg body weight for 32 wk) did not alter hematological indices, histological findings, or enzyme activities related to heme biosynthesis, even though it resulted in sustained decreases in microsomal heme oxygenase activity in the liver, kidney, and spleen, and a prolonged decrease in plasma bilirubin concentration. Inhibition of heme oxygenase did not alter the level of cytochrome P-450 in the liver and the kidney. The results indicate that long-term treatment with massive doses of Sn-protoporphyrin suppresses bilirubin formation but does not produce significant histopathological changes or appreciably interfere with heme synthesis, in this strain of genetically anemic mice. These findings provide further support for the idea that suppression of heme degradation to bile pigment by the inhibition of heme oxygenase may prove useful to the prevention of severe hyperbilirubinemia in humans.

Raf-1 sets the threshold of Fas sensitivity by modulating Rok-alpha signaling.

Ablation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hypersensitivity to Fas-induced cell death. Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha. In this study, we show that the kinase-independent modulation of Rok-alpha signaling is also the basis of the antiapoptotic function of Raf-1. Fas activation stimulates the formation of Raf-1-Rok-alpha complexes, and Rok-alpha signaling is up-regulated in Raf-1-deficient cells. This leads to increased clustering and membrane expression of Fas, which is rescued both by kinase-dead Raf-1 and by interfering with Rok-alpha or its substrate ezrin. Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts. Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.

An Overview of Rodent Models of Obesity and Type 2 Diabetes.

Many animal models that are currently used in appetite and obesity research share at least some main features of human obesity and its comorbidities. Hence, even though no animal model replicates all aspects of "common" human obesity, animal models are imperative in studying the control of energy balance and reasons for its imbalance that may eventually lead to overt obesity. The most frequently used animal models are small rodents that may be based on mutations or manipulations of individual or several genes and on the exposure to obesogenic diets or other manipulations that predispose the animals to gaining or maintaining excessive weight. Characteristics include hyperphagia or changes in energy metabolism and at least in some models the frequent comorbidities of obesity, like hyperglycemia, insulin resistance, or diabetes-like syndromes. Some of the most frequently used animal models of obesity research involve animals with monogenic mutations of the leptin pathway which in fact are useful to study specific mechanistic aspects of eating controls, but typically do not recapitulate "common" obesity in the human population. Hence, this review will mention advantages and disadvantages of respective animal models in order to build a basis for the most appropriate use in biomedical research.

Differential Expression of Hippocampal Circular RNAs in the BTBR Mouse Model for Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition with unknown etiology. Recent experimental evidences suggest the contribution of non-coding RNAs (ncRNAs) in the pathophysiology of ASD. In this work, we aimed to investigate the expression profile of the ncRNA class of circular RNAs (circRNAs) in the hippocampus of the BTBR T + tf/J (BTBR) mouse model and age-matched C57BL/6J (B6) mice. Alongside, we analyzed BTBR hippocampal gene expression profile to evaluate possible correlations between the differential abundance of circular and linear gene products. From RNA sequencing data, we identified circRNAs highly modulated in BTBR mice. Thirteen circRNAs and their corresponding linear isoforms were validated by RT-qPCR analysis. The BTBR-regulated circCdh9 was better characterized in terms of molecular structure and expression, highlighting altered levels not only in the hippocampus, but also in the cerebellum, prefrontal cortex, and amygdala. Finally, gene expression analysis of the BTBR hippocampus pinpointed altered biological and molecular pathways relevant for the ASD phenotype. By comparison of circRNA and gene expression profiles, we identified 6 genes significantly regulated at either circRNA or mRNA gene products, suggesting low overall correlation between circRNA and host gene expression. In conclusion, our results indicate a consistent deregulation of circRNA expression in the hippocampus of BTBR mice. ASD-related circRNAs should be considered in functional studies to identify their contribution to the etiology of the disorder. In addition, as abundant and highly stable molecules, circRNAs represent interesting potential biomarkers for autism.

Correction of abnormal matrix formed by cmd/cmd chondrocytes in culture by exogenously added cartilage proteoglycan.

The cartilage matrix deficiency (cmd/cmd) mouse fails to synthesize the core protein of cartilage-characteristic proteoglycan (cartilage PG). Chondrocytes from the cmd/cmd cartilage cultured in vitro produced nodules with greatly reduced extracellular matrix. Immunofluorescence staining revealed that the nodules of mutant cells differed from the normal in lacking cartilage PG and in uneven and reduced deposition of type II collagen. Exogenously added cartilage PG prepared from either normal mouse cartilage or Swarm rat chondrosarcoma to the culture medium was incorporated exclusively into the extracellular matrices of the nodules, with a concurrent correction of the abnormal distribution pattern of type II collagen. The incorporation of cartilage PG into the matrix was disturbed by hyaluronic acid or decasaccharide derived therefrom, suggesting that the incorporation process involves the interaction of added proteoglycan with hyaluronic acid. Both the hyaluronic acid-binding region and the protein-enriched core molecule prepared from rat chondrosarcoma cartilage PG could also be incorporated but, unlike the intact cartilage PG, they were distributed equally in the surrounding zones where fibroblast-like cells predominate. The results indicate that the intact form of cartilage PG is required for specific incorporation into the chondrocyte nodules, and further suggest that cartilage PG plays a regulatory role in the assembly of the matrix macromolecules.

Persistence of crystallin messenger RNA's with reduced translation in hereditary cataracts in mice.

In vitro translation experiments showed that the lens fiber cells of two hereditary cataracts in mice (Nakano and Philly) possessed a full complement of crystallin messenger RNA's, despite severely reduced synthesis of crystallin in these cells. The reduction in synthesis in the lens fiber cells correlated with the increase in Na+ and the decrease in K+, which occurs during cataractogenesis. In contrast to the fiber cells, the epithelial cells continued to synthesize crystallins in the cataractous lenses. Crystallin synthesis was stimulated in the fiber cells by raising the K+ concentration and lowering the Na+ concentration in the cultured lenses. The reduction in crystallin synthesis in the initial stages of cataractogenesis in the Nakano and Philly lenses thus appears to be due to poor utilization of crystallin messenger RNA's in the fiber cells.

Notch signaling in the mammalian central nervous system: insights from mouse mutants.

The Notch pathway, although originally identified in fruit flies, is now among the most heavily studied in mammalian biology. In mice, loss-of-function and gain-of-function work has demonstrated that Notch signaling is essential both during development and in the adult in a multitude of tissues. Prominent among these is the CNS, where Notch has been implicated in processes ranging from neural stem cell regulation to learning and memory. Here we review the role of Notch in the mammalian CNS by focusing specifically on mutations generated in mice. These mutations have provided critical insight into Notch function in the CNS and have led to the identification of promising new directions that are likely to generate important discoveries in the future.

The laminin alpha2 expressed by dystrophic dy(2J) mice is defective in its ability to form polymers.

Mutations in LAMA2 cause severe congenital muscular dystrophy accompanied by nervous system defects [1]. Mice homozygous for the dy(2J) allele of LAMA2 express a laminin alpha2 subunit that has a deletion in the amino-terminal domain VI, providing an animal model for study of the molecular basis of congenital muscular dystrophy [2] [3]. Domain VI is predicted to be involved in laminin polymerization, along with amino-terminal domains from laminin beta and gamma chains [4]. In a solution-polymerization assay, we found that purified dy(2J) laminin assembled poorly and formed little polymer, in contrast to wild-type muscle laminin. Furthermore, dissolution of the collagen IV network caused dy(2J) laminin to be released into solution, indicating that laminin polymers within the skeletal muscle basement membrane were defective. In addition to loss of polymerization, dy(2J) laminin had a reduced affinity for heparin. Finally, recombinant laminin engineered with the dy(2J) deletion was more sensitive to proteolysis and was readily cleaved near the junction of domains V and VI. Thus, the dy(2J) deletion selectively disrupts polymer formation, reduces affinity for heparin, and destabilizes domain VI. These are the first specific functional defects to be identified in a muscular dystrophy laminin, and it is likely that these defects contribute to the abnormalities seen in dy(2J)/dy(2J) muscle and nerve.

The eumelanin and pheomelanin contents in dorsal hairs of female recessive yellow mice are greater than in male.

BACKGROUND: The murine recessive yellow (Mc1r(e)) is a loss-of-function mutation in the receptor for alpha-melanocyte-stimulating hormone (MSH), melanocortin receptor 1 (MC1R), and produces yellow coats by inducing pheomelanin synthesis in hair follicular melanocytes. OBJECTIVE: It is not known whether the Mc1r(e) mutation affects pheomelanin synthesis in other skin sites. In this study, the eumelanin and pheomelanin contents in the epidermis and dermis as well as hairs of wild-type and mutant mice were measured. MATERIALS AND METHODS: The content of melanin was measured by high performance liquid chromatography. RESULTS: The eumelanin contents in the epidermis and dermis of newborn wild-type (Mc1r(+)/Mc1r(+)) mice (0.5, 3.5, 5.5, and 7.5 days) were much greater than those of mutant (Mc1r(e)/Mc1r(e)) mice, whereas the pheomelanin contents in the epidermis and dermis of mutant mice were much greater than those of wild-type mice. No sex differences in the contents of eumelanin and pheomalanin in the epidermis and dermis both in mutant and wild-type mice were observed. The eumelanin contents in mutant hairs (5-week-old) was much smaller than in wild-type hairs, whereas the pheomelanin contents in mutant hairs was much greater than in wild-type hairs. However, the eumelanin and pheomelanin contents in mutant female hairs were greater than in male. These sex differences were not observed in wild-type mice. CONCLUSION: The Mc1r(e) gene stimulates pheomelanin synthesis in the epidermis, dermis and hair follicles. In addition, eumelanin and pheomelanin contents in Mc1r(e)/Mc1r(e) hairs may be influenced by the sex difference.

Mice null for sox18 are viable and display a mild coat defect.

We have previously shown that Sox18 is expressed in developing vascular endothelium and hair follicles during mouse embryogenesis and that point mutations in Sox18 are the underlying cause of cardiovascular and hair follicle defects in ragged (Ra) mice. Here we describe the analysis of Sox18(-/-) mice produced by gene targeting. Despite the profound defects seen in Ra mice, Sox18(-/-) mice have no obvious cardiovascular defects and only a mild coat defect with a reduced proportion of zigzag hairs. A reduction in the amount of pheomelanin pigmentation in hair shafts was also observed; later-forming hair follicles showed a reduced subapical pheomelanin band, giving Sox18(-/-) mice a slightly darker appearance than Sox18(+/+) and Sox18(+/-) siblings. Sox18(-/-) mice are viable and fertile and show no difference in the ability to thrive relative to littermates. Because of the mild effect of the mutation on the phenotype of Sox18(-/-) mice, we conclude that the semidominant nature of the Ra mutations is due to a trans-dominant negative effect mediated by the mutant SOX18 proteins rather than haploinsufficiency as has been observed for other SOX genes. Due to the similarity of SOX18 to other subgroup F SOX proteins, SOX7 and -17, and the overlap in expression of these genes, functional redundancy amongst these SOX proteins could also account for the mild phenotype of Sox18(-/-) mice.

Fat aussie--a new Alström syndrome mouse showing a critical role for ALMS1 in obesity, diabetes, and spermatogenesis.

Mutations in the human ALMS1 gene are responsible for Alström syndrome, a disorder in which key metabolic and endocrinological features include childhood-onset obesity, metabolic syndrome, and diabetes, as well as infertility. ALMS1 localizes to the basal bodies of cilia and plays a role in intracellular trafficking, but the biological functions of ALMS1 and how these relate to the pathogenesis of obesity, diabetes, and infertility remain unclear. Here we describe a new mouse model of Alström syndrome, fat aussie, caused by a spontaneous mutation in the Alms1 gene. Fat aussie (Alms1 foz/foz) mice are of normal weight when young but, by 120 d of age, they become obese and hyperinsulinemic. Diabetes develops in Alms1 foz/foz mice accompanied by pancreatic islet hyperplasia and islet cysts. Female mice are fertile before the onset of obesity and metabolic syndrome; however, male fat aussie mice are sterile due to a progressive germ cell loss followed by an almost complete block of development at the round-to-elongating spermatid stage of spermatogenesis. In conclusion, Alms1 foz/foz mouse is a new animal model in which to study the pathogenesis of the metabolic and fertility defects of Alström syndrome, including the role of ALMS1 in appetite regulation, pathogenesis of the metabolic syndrome, pancreatic islet physiology, and spermatogenesis.

Defective carbohydrate metabolism in mice homozygous for the tubby mutation.

Tub is a member of a small gene family, the tubby-like proteins (TULPs), with predominant expression in neurons. Mice carrying a mutation in Tub develop retinal and cochlear degeneration as well as late-onset obesity with insulin resistance. During behavioral and metabolic testing, we found that homozygous C57BL/6J-Tub(tub) mice have a lower respiratory quotient than C57BL/6J controls before the onset of obesity, indicating that tubby homozygotes fail to activate carbohydrate metabolism and instead rely on fat metabolism for energy needs. In concordance with this, tubby mice show higher excretion of ketone bodies and accumulation of glycogen in the liver. Quantitation of liver mRNA levels shows that, during the transition from light to dark period, tubby mice fail to induce glucose-6-phosphate dehydrogenase (G6pdh), the rate-limiting enzyme in the pentose phosphate pathway that normally supplies NADPH for de novo fatty acid synthesis and glutathione reduction. Reduced G6PDH protein levels and enzymatic activity in tubby mice lead accordingly to lower levels of NADPH and reduced glutathione (GSH), respectively. mRNA levels for the lipolytic enzymes acetyl-CoA synthetase and carnitine palmitoyltransferase are increased during the dark cycle and decreased during the light period, and several citric acid cycle genes are dysregulated in tubby mice. Examination of hypothalamic gene expression showed high levels of preproorexin mRNA leading to accumulation of orexin peptide in the lateral hypothalamus. We hypothesize that abnormal hypothalamic orexin expression leads to changes in liver carbohydrate metabolism and may contribute to the moderate obesity observed in tubby mice.

Neural recognition molecules in disease and regeneration.

The genetic analysis of inherited human diseases of the nervous system and the characterization of transgenic mice deficient in neural recognition molecules is opening up a new dimension in understanding the cellular and molecular mechanisms underlying neuro-developmental and -degenerative diseases, as well as in delineating the functions of recognition molecules in cell-cell interactions. Progress in identifying recognition molecules that inhibit neurite outgrowth and further characterization of the mechanisms that promote neurite outgrowth are shedding more light on the processes of regeneration in the mature nervous system. In the adult, recognition functions are fine-tuned by glycan moeities associated with neural recognition molecules, and successful neurite outgrowth is likely to depend on the delicate balance between growth-promoting and inhibitory recognition cues.