Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes

Seizures, ataxia and parvalbumin-expressing interneurons respond to selenium supply in Selenop-deficient mice

Mice with constitutive disruption of the Selenop gene have been key to delineate the importance of selenoproteins in neurobiology. However, the phenotype of this mouse model is exquisitely dependent on selenium supply and timing of selenium supplementation. Combining biochemical, histological, and behavioral methods, we tested the hypothesis that parvalbumin-expressing interneurons in the primary somatosensory cortex and hippocampus depend on dietary selenium availability in Selenop−/− mice. Selenop-deficient mice kept on adequate selenium diet (0.15 mg/kg, i.e. the recommended dietary allowance, RDA) developed ataxia, tremor, and hyperexcitability between the age of 4–5 weeks. Video-electroencephalography demonstrated epileptic seizures in Selenop−/− mice fed the RDA diet, while Selenop ± heterozygous mice behaved normally. Both neurological phenotypes, hyperexcitability/seizures and ataxia/dystonia were successfully prevented by selenium supplementation from birth or transgenic expression of human SELENOP under a hepatocyte-specific promoter. Selenium supplementation with 10 μM selenite in the drinking water on top of the RDA diet increased the activity of glutathione peroxidase in the brains of Selenop−/− mice to control levels. The effects of selenium supplementation on the neurological phenotypes were dose- and time-dependent. Selenium supplementation after weaning was apparently too late to prevent ataxia/dystonia, while selenium withdrawal from rescued Selenop−/− mice eventually resulted in ataxia. We conclude that SELENOP expression is essential for preserving interneuron survival under limiting Se supply, while SELENOP appears dispensable under sufficiently high Se status

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy

Mechanisms controlling anaemia in Trypanosoma congolense infected mice.

Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia. The progress of anaemia after Trypanosoma congolense infection was followed in three mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore, functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive. However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the expression of the inflammatory cytokines Il6 and Ifng. The innate immune response appears to be the major contributor to the inflammation associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors regulating haematopoiesis, Tal1, Gata1, Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis induced anaemia after infection

Abnormal Brain Iron Metabolism in Irp2 Deficient Mice Is Associated with Mild Neurological and Behavioral Impairments

Iron Regulatory Protein 2 (Irp2, Ireb2) is a central regulator of cellular iron homeostasis in vertebrates. Two global knockout mouse models have been generated to explore the role of Irp2 in regulating iron metabolism. While both mouse models show that loss of Irp2 results in microcytic anemia and altered body iron distribution, discrepant results have drawn into question the role of Irp2 in regulating brain iron metabolism. One model shows that aged Irp2 deficient mice develop adult-onset progressive neurodegeneration that is associated with axonal degeneration and loss of Purkinje cells in the central nervous system. These mice show iron deposition in white matter tracts and oligodendrocyte soma throughout the brain. A contrasting model of global Irp2 deficiency shows no overt or pathological signs of neurodegeneration or brain iron accumulation, and display only mild motor coordination and balance deficits when challenged by specific tests. Explanations for conflicting findings in the severity of the clinical phenotype, brain iron accumulation and neuronal degeneration remain unclear. Here, we describe an additional mouse model of global Irp2 deficiency. Our aged Irp2−/− mice show marked iron deposition in white matter and in oligodendrocytes while iron content is significantly reduced in neurons. Ferritin and transferrin receptor 1 (TfR1, Tfrc), expression are increased and decreased, respectively, in the brain from Irp2−/− mice. These mice show impairments in locomotion, exploration, motor coordination/balance and nociception when assessed by neurological and behavioral tests, but lack overt signs of neurodegenerative disease. Ultrastructural studies of specific brain regions show no evidence of neurodegeneration. Our data suggest that Irp2 deficiency dysregulates brain iron metabolism causing cellular dysfunction that ultimately leads to mild neurological, behavioral and nociceptive impairments

Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology

Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOX(Rosa26) mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOX(Rosa26) mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.Peer reviewe

Standardized, systemic phenotypic analysis reveals kidney dysfunction as main alteration of Kctd1 I27N mutant mice

Background: Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically dominant mutant line HST014 was established and further analyzed. Methods: Analysis of the causative mutation as well as the standardized, systemic phenotypic analysis of the mutant line was carried out. Results: The causative mutation was detected in the potassium channel tetramerization domain containing 1 (Kctd1) gene which leads to the amino acid exchange Kctd1 I27N thereby affecting the functional BTB domain of the protein. This line is the first mouse model harboring a Kctd1 mutation. Kctd1 I27N homozygous mutant mice die perinatally. Standardized, systemic phenotypic analysis of Kctd1 I27N heterozygous mutants was carried out in the German Mouse Clinic (GMC). Systematic morphological investigation of the external physical appearance did not detect the specific alterations that are described in KCTD1 mutant human patients affected by the scalp-ear-nipple (SEN) syndrome. The main pathological phenotype of the Kctd1 I27N heterozygous mutant mice consists of kidney dysfunction and secondary effects thereof, without gross additional primary alterations in the other phenotypic parameters analyzed. Genome-wide transcriptome profiling analysis at the age of 4 months revealed about 100 differentially expressed genes (DEGs) in kidneys of Kctd1 I27N heterozygous mutants as compared to wild-type controls. Conclusions: In summary, the main alteration of the Kctd1 I27N heterozygous mutants consists in kidney dysfunction. Additional analyses in 9–21 week-old heterozygous mutants revealed only few minor effects

Dll1 Haploinsufficiency in Adult Mice Leads to a Complex Phenotype Affecting Metabolic and Immunological Processes

BACKGROUND: The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligands is less well understood. Loss-of-function of Dll1 leads to embryonic lethality, but reduction of Delta-like 1 protein levels has not been studied in adult stage. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the haploinsufficient phenotype of Dll1 and a missense mutant Dll1 allele (Dll1(C413Y)). Haploinsufficiency leads to a complex phenotype with several biological processes altered. These alterations reveal the importance of Dll1 mainly in metabolism, energy balance and in immunology. The animals are smaller, lighter, with altered fat to lean ratio and have increased blood pressure and a slight bradycardia. The animals have reduced cholesterol and triglyceride levels in blood. At the immunological level a subtle phenotype is observed due to the effect and fine-tuning of the signaling network at the different levels of differentiation, proliferation and function of lymphocytes. Moreover, the importance of the proteolytic regulation of the Notch signaling network emphasized. CONCLUSIONS/SIGNIFICANCE: In conclusion, slight alterations in one player of Notch signaling alter the entire organism, emphasizing the fine-tuning character of this pathway in a high number of processes

Neurobeachin, a Regulator of Synaptic Protein Targeting, Is Associated with Body Fat Mass and Feeding Behavior in Mice and Body-Mass Index in Humans

Neurobeachin (Nbea) regulates neuronal membrane protein trafficking and is required for the development and functioning of central and neuromuscular synapses. In homozygous knockout (KO) mice, Nbea deficiency causes perinatal death. Here, we report that heterozygous KO mice haploinsufficient for Nbea have higher body weight due to increased adipose tissue mass. In several feeding paradigms, heterozygous KO mice consumed more food than wild-type (WT) controls, and this consumption was primarily driven by calories rather than palatability. Expression analysis of feeding-related genes in the hypothalamus and brainstem with real-time PCR showed differential expression of a subset of neuropeptide or neuropeptide receptor mRNAs between WT and Nbea+/− mice in the sated state and in response to food deprivation, but not to feeding reward. In humans, we identified two intronic NBEA single-nucleotide polymorphisms (SNPs) that are significantly associated with body-mass index (BMI) in adult and juvenile cohorts. Overall, data obtained in mice and humans suggest that variation of Nbea abundance or activity critically affects body weight, presumably by influencing the activity of feeding-related neural circuits. Our study emphasizes the importance of neural mechanisms in body weight control and points out NBEA as a potential risk gene in human obesity