Unbiased analysis of obesity related, fat depot specific changes of adipocyte volumes and numbers using light sheet fluorescence microscopy

In translational obesity research, objective assessment of adipocyte sizes and numbers is essential to characterize histomorphological alterations linked to obesity, and to evaluate the efficacies of experimental medicinal or dietetic interventions. Design-based quantitative stereological techniques based on the analysis of 2D-histological sections provide unbiased estimates of relevant 3D-parameters of adipocyte morphology, but often involve complex and time-consuming tissue processing and analysis steps. Here we report the application of direct 3D light sheet fluorescence microscopy (LSFM) for effective and accurate analysis of adipocyte volumes and numbers in optically cleared adipose tissue samples from a porcine model of diet-induced obesity (DIO). Subcutaneous and visceral adipose tissue samples from DIO-minipigs and lean controls were systematically randomly sampled, optically cleared with 3DISCO (3-dimensional imaging of solvent cleared organs), stained with eosin, and subjected to LSFM for detection of adipocyte cell membrane autofluorescence. Individual adipocytes were unbiasedly sampled in digital 3D reconstructions of the adipose tissue samples, and their individual cell volumes were directly measured by automated digital image analysis. Adipocyte numbers and mean volumes obtained by LSFM analysis did not significantly differ from the corresponding values obtained by unbiased quantitative stereological analysis techniques performed on the same samples, thus proving the applicability of LSFM for efficient analysis of relevant morphological adipocyte parameters. The results of the present study demonstrate an adipose tissue depot specific plasticity of adipocyte growth responses to nutrient oversupply. This was characterized by an exclusively hypertrophic growth of visceral adipocytes, whereas adipocytes in subcutaneous fat tissue depots also displayed a marked (hyperplastic) increase in cell number. LSFM allows for accurate and efficient determination of relevant quantitative morphological adipocyte parameters. The applied stereological methods and LSFM protocols are described in detail and can serve as a guideline for unbiased quantitative morphological analyses of adipocytes in other studies and species

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

Dusp8 affects hippocampal size and behavior in mice and humans

Dual-specificity phosphatase 8 (Dusp8) acts as physiological inhibitor for the MAPKs Jnk, Erk and p38 which are involved in regulating multiple CNS processes. While Dusp8 expression levels are high in limbic areas such as the hippocampus, the functional role of Dusp8 in hippocampus morphology, MAPK-signaling, neurogenesis and apoptosis as well as in behavior are still unclear. It is of particular interest whether human carriers of a DUSP8 allelic variant show similar hippocampal alterations to mice. Addressing these questions using Dusp8WT and KO mouse littermates, we found that KOs suffered from mildly impaired spatial learning, increased locomotor activity and elevated anxiety. Cell proliferation, apoptosis and p38 and Jnk phosphorylation were unaffected, but phospho-Erk levels were higher in hippocampi of the KOs. Consistent with a decreased hippocampus size in Dusp8 KO mice, we found reduced volumes of the hippocampal subregions subiculum and CA4 in humans carrying the DUSP8 allelic variant SNP rs2334499:C > T. Overall, aberrations in morphology and behavior in Dusp8 KO mice and a decrease in hippocampal volume of SNP rs2334499:C > T carriers point to a novel, translationally relevant role of Dusp8 in hippocampus function that warrants further studies on the role of Dusp8 within the limbic network

KIT is required for hepatic function during mouse post-natal development

<p>Abstract</p> <p>Background</p> <p>The <it>Kit </it>gene encodes a receptor tyrosine kinase involved in various biological processes including melanogenesis, hematopoiesis and gametogenesis in mice and human. A large number of <it>Kit </it>mutants has been described so far showing the pleiotropic phenotypes associated with partial loss-of-function of the gene. Hypomorphic mutations can induce a light coat color phenotype while complete lack of KIT function interferes with embryogenesis. Interestingly several intermediate hypomorphic mutations induced in addition growth retardation and post-natal mortality.</p> <p>Results</p> <p>In this report we investigated the post-natal role of <it>Kit </it>by using a panel of chemically-induced hypomorphic mutations recently isolated in the mouse. We found that, in addition to the classical phenotypes, mutations of <it>Kit </it>induced juvenile steatosis, associated with the downregulation of the three genes, <it>VldlR</it>, <it>Lpin1 </it>and <it>Lpl</it>, controlling lipid metabolism in the post-natal liver. Hence, <it>Kit </it>loss-of-functions mimicked the inactivation of genes controlling the hepatic metabolism of triglycerides, the major source of energy from maternal milk, leading to growth and viability defects during neonatal development.</p> <p>Conclusion</p> <p>This is a first report involving KIT in the control of lipid metabolism in neonates and opening new perspectives for understanding juvenile steatosis. Moreover, it reinforces the role of Kit during development of the liver and underscores the caution that should be exerted in using KIT inhibitors during anti-cancer treatment.</p

Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy

Alternative oxidase (AOX) is a non-mammalian enzyme that can bypass blockade of the complex III-IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)-deficient Bcs1l(p.S78G) knock-in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue-specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.Peer reviewe

MTO1 mediates tissue specificity of OXPHOS defects via tRNA modification and translation optimization, which can be bypassed by dietary intervention

Mitochondrial diseases often exhibit tissue-specific pathologies, but this phenomenon is poorly understood. Here we present regulation of mitochondrial translation by the Mitochondrial Translation Optimization Factor 1, MTO1, as a novel player in this scenario. We demonstrate that MTO1 mediates tRNA modification and controls mitochondrial translation rate in a highly tissue-specific manner associated with tissue-specific OXPHOS defects. Activation of mitochondrial proteases, aberrant translation products, as well as defects in OXPHOS complex assembly observed in MTO1 deficient mice further imply that MTO1 impacts translation fidelity. In our mouse model, MTO1-related OXPHOS deficiency can be bypassed by feeding a ketogenic diet. This therapeutic intervention is independent of the MTO1-mediated tRNA modification and involves balancing of mitochondrial and cellular secondary stress responses. Our results thereby establish mammalian MTO1 as a novel factor in the tissue-specific regulation of OXPHOS and fine tuning of mitochondrial translation accurac

Dose-dependent long-term effects of a single radiation event on behaviour and glial cells

Purpose The increasing use of low-dose ionizing radiation in medicine requires a systematic study of its long-term effects on the brain, behaviour and its possible association with neurodegenerative disease vulnerability. Therefore, we analysed the long-term effects of a single low-dose irradiation exposure at 10 weeks of age compared to medium and higher doses on locomotor, emotion-related and sensorimotor behaviour in mice as well as on hippocampal glial cell populations. Materials and methods We determined the influence of radiation dose (0, 0.063, 0.125 or 0.5 Gy), time post-irradiation (4, 12 and 18 months p.i.), sex and genotype (wild type versus mice with Ercc2 DNA repair gene point mutation) on behaviour. Results The high dose (0.5 Gy) had early-onset adverse effects at 4 months p.i. on sensorimotor recruitment and late-onset negative locomotor effects at 12 and 18 months p.i. Notably, the low dose (0.063 Gy) produced no early effects but subtle late-onset (18 months) protective effects on sensorimotor recruitment and exploratory behaviour. Quantification and morphological characterization of the microglial and the astrocytic cells of the dentate gyrus 24 months p.i. indicated heightened immune activity after high dose irradiation (0.125 and 0.5 Gy) while conversely, low dose (0.063 Gy) induced more neuroprotective features. Conclusion This is one of the first studies demonstrating such long-term and late-onset effects on brain and behaviour after a single radiation event in adulthood

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

Disruption of paternal circadian rhythm affects metabolic health in male offspring via nongerm cell factors

Circadian rhythm synchronizes each body function with the environment and regulates physiology. Disruption of normal circadian rhythm alters organismal physiology and increases disease risk. Recent epidemiological data and studies in model organisms have shown that maternal circadian disruption is important for offspring health and adult phenotypes. Less is known about the role of paternal circadian rhythm for offspring health. Here, we disrupted circadian rhythm in male mice by night-restricted feeding and showed that paternal circadian disruption at conception is important for offspring feeding behavior, metabolic health, and oscillatory transcription. Mechanistically, our data suggest that the effect of paternal circadian disruption is not transferred to the offspring via the germ cells but initiated by corticosterone-based parental communication at conception and programmed during in utero development through a state of fetal growth restriction. These findings indicate paternal circadian health at conception as a newly identified determinant of offspring phenotypes

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