2022
Eur. J. Nucl. Med. Mol. Imaging, DOI: 10.1007/s00259-022-05896-5 (2022)
Purpose: Modern neuroimaging lacks the tools necessary for whole-brain, anatomically dense neuronal damage screening. An ideal approach would include unbiased histopathologic identification of aging and neurodegenerative disease. Methods: We report the postmortem application of multiscale X-ray phase-contrast computed tomography (X-PCI-CT) for the label-free and dissection-free organ-level to intracellular-level 3D visualization of distinct single neurons and glia. In deep neuronal populations in the brain of aged wild-type and of 3xTgAD mice (a triply-transgenic model of Alzheimer’s disease), we quantified intracellular hyperdensity, a manifestation of aging or neurodegeneration. Results: In 3xTgAD mice, the observed hyperdensity was identified as amyloid-β and hyper-phosphorylated tau protein deposits with calcium and iron involvement, by correlating the X-PCI-CT data to immunohistochemistry, X-ray fluorescence microscopy, high-field MRI, and TEM. As a proof-of-concept, X-PCI-CT was used to analyze hippocampal and cortical brain regions of 3xTgAD mice treated with LY379268, selective agonist of group II metabotropic glutamate receptors (mGlu2/3 receptors). Chronic pharmacologic activation of mGlu2/3 receptors significantly reduced the hyperdensity particle load in the ventral cortical regions of 3xTgAD mice, suggesting a neuroprotective effect with locoregional efficacy. Conclusions: This multiscale micro-to-nano 3D imaging method based on X-PCI-CT enabled identification and quantification of cellular and sub-cellular aging and neurodegeneration in deep neuronal and glial cell populations in a transgenic model of Alzheimer’s disease. This approach quantified the localized and intracellular neuroprotective effects of pharmacological activation of mGlu2/3 receptors.
Wissenschaftlicher Artikel
Scientific Article
Neuropathol. Appl. Neurobiol. 48:e12750 (2022)
AIMS: We investigated N471D WASH complex subunit strumpellin (Washc5) knock-in and Washc5 knock-out mice as models for hereditary spastic paraplegia type 8 (SPG8). METHODS: We generated hetero- and homozygous N471D Washc5 knock-in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. RESULTS: Homozygous N471D Washc5 knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels, and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology, and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in hetero- and homozygous knock-in mice. WASHC5-related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock-out mice showing normal WASHC5 levels could not be bred to homozygosity. CONCLUSIONS: While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.
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Scientific Article
Am. J. Physiol. Endocrinol. Metab. 322, E85-E100 (2022)
Objective Activation of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) upon cold stimulation leads to substantial increase in energy expenditure to defend body temperature. Increases in energy expenditure after a high caloric food intake, termed diet-induced thermogenesis, are also attributed to BAT. These properties render BAT a potential target to combat diet-induced obesity. However, studies investigating the role of UCP1 to protect against diet-induced obesity are controversial and rely on the phenotyping of a single constitutive UCP1-knockout model. To address this issue, we generated a novel UCP1-knockout model by Cre-mediated deletion of Exon 2 in the UCP1 gene. We studied the effect of constitutive UCP1 knockout on metabolism and the development of diet-induced obesity. Methods UCP1 knockout and wildtype mice were housed at 30°C and fed a control diet for 4-weeks followed by 8-weeks of high-fat diet. Body weight and food intake were monitored continuously over the course of the study and indirect calorimetry was used to determine energy expenditure during both feeding periods. Results Based on Western blot analysis, thermal imaging and noradrenaline test, we confirmed the lack of functional UCP1 in knockout mice. However, body weight gain, food intake and energy expenditure were not affected by deletion of UCP1 gene function during both feeding periods. Conclusion We introduce a novel UCP1-KO mouse enabling the generation of conditional UCP1-knockout mice to scrutinize the contribution of UCP1 to energy metabolism in different cell types or life stages. Our results demonstrate that UCP1 does not protect against diet-induced obesity at thermoneutrality.
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Scientific Article
Biomedicines 10:616 (2022)
Glucotoxic metabolites and pathways play a crucial role in diabetic complications, and new treatment options which improve glucotoxicity are highly warranted. In this study, we analyzed bezafibrate (BEZ) treated, streptozotocin (STZ) injected mice, which showed an improved glucose metabolism compared to untreated STZ animals. In order to identify key molecules and pathways which participate in the beneficial effects of BEZ, we studied plasma, skeletal muscle, white adipose tissue (WAT) and liver samples using non-targeted metabolomics (NMR spectroscopy), targeted metabolomics (mass spectrometry), microarrays and mitochondrial enzyme activity measurements, with a particular focus on the liver. The analysis of muscle and WAT demonstrated that STZ treatment elevated inflammatory pathways and reduced insulin signaling and lipid pathways, whereas BEZ decreased inflammatory pathways and increased insulin signaling and lipid pathways, which can partly explain the beneficial effects of BEZ on glucose metabolism. Furthermore, lysophosphatidylcholine levels were lower in the liver and skeletal muscle of STZ mice, which were reverted in BEZ-treated animals. BEZ also improved circulating and hepatic glucose levels as well as lipid profiles. In the liver, BEZ treatment reduced elevated fumarate levels in STZ mice, which was probably due to a decreased expression of urea cycle genes. Since fumarate has been shown to participate in glucotoxic pathways, our data suggests that BEZ treatment attenuates the urea cycle in the liver, decreases fumarate levels and, in turn, ameliorates glucotoxicity and reduces insulin resistance in STZ mice.
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Scientific Article
BMJ Open 12:e058268 (2022)
INTRODUCTION: Even well-treated gestational diabetes mellitus (GDM) might still have impact on long-term health of the mother and her offspring, although this relationship has not yet been conclusively studied. Using in-depth phenotyping of the mother and her offspring, we aim to elucidate the relationship of maternal hyperglycaemia during pregnancy and adequate treatment, and its impact on the long-term health of both mother and child. METHODS: The multicentre PREG study, a prospective cohort study, is designed to metabolically and phenotypically characterise women with a 75-g five-point oral glucose tolerance test (OGTT) during, and repeatedly after pregnancy. Outcome measures are maternal glycaemia during OGTTs, birth outcome and the health and growth development of the offspring. The children of the study participants are followed up until adulthood with developmental tests and metabolic and epigenetic phenotyping in the PREG Offspring study. A total of 800 women (600 with GDM, 200 controls) will be recruited. ETHICS AND DISSEMINATION: The study protocol has been approved by all local ethics committees. Results will be disseminated via conference presentations and peer-reviewed publications. TRIAL REGISTRATION NUMBER: The PREG study and the PREG Offspring study are registered with Clinical Trials (ClinicalTrials.gov identifiers: NCT04270578, NCT04722900).
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Scientific Article
Dis. Model. Mech. 15:dmm049205 (2022)
Understanding the shared genetic aetiology of psychiatric and medical comorbidity in neurodevelopmental disorders (NDDs) could improve patient diagnosis, stratification and treatment options. Rare TANC2 (Tetratricopeptide Repeat, Ankyrin Repeat and Coiled-Coil Containing 2) disrupting variants were disease-causing in NDD patients. This post-synaptic scaffold protein, essential for dendrite formation in synaptic plasticity, plays an unclarified but critical role in development. We here report a novel homozygous-viable Tanc2 disrupted function model where mutant mice were hyperactive and had impaired sensorimotor gating consistent with NDD patient psychiatric endophenotypes. Yet, a multi-systemic analysis revealed the pleiotropic effects of Tanc2 outside the brain such as growth failure and hepatocellular damage. This was associated with aberrant liver function including altered hepatocellular metabolism. Integrative analysis indicates that these disrupted Tanc2 systemic effects relate to interaction with Hippo developmental signalling pathway proteins and will increase the risk for comorbid somatic disease. This highlights how NDD gene pleiotropy can augment medical comorbidity susceptibility underscoring the benefit of holistic NDD patient diagnosis and treatment for which large-scale preclinical functional genomics can provide complementary pleiotropic gene function information.
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Scientific Article
Nat. Methods 19, 803-811 (2022)
The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo. Proteogenomic comparison of mouse, human and Arabidopsis reveal common and distinct mechanisms of gene expression regulation and, despite many similarities, numerous differentially abundant orthologs that likely serve species-specific functions. We leverage the mouse proteome by integrating phenotypic drug (n > 400) and radiation response data with the proteomes of 66 pancreatic ductal adenocarcinoma (PDAC) cell lines to reveal molecular markers for sensitivity and resistance. This unique atlas complements other molecular resources for the mouse and can be explored online via ProteomicsDB and PACiFIC.
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Scientific Article
Nat. Genet. 54, 358-360 (2022)
In the version of this article initially published, members of the Eumorphia Consortium appeared in the Supplementary Information but were not included in the main article. The full list of members appears below.
PLoS Genet. 18:e1010190 (2022)
Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.
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Scientific Article
Comm. Biol. 5:408 (2022)
Suitable animal models are essential for translational research, especially in the case of complex, multifactorial conditions, such as obesity. The non-inbred mouse (Mus musculus) line Titan, also known as DU6, is one of the world’s longest selection experiments for high body mass and was previously described as a model for metabolic healthy (benign) obesity. The present study further characterizes the geno- and phenotypes of this non-inbred mouse line and tests its suitability as an interventional obesity model. In contrast to previous findings, our data suggest that Titan mice are metabolically unhealthy obese and short-lived. Line-specific patterns of genetic invariability are in accordance with observed phenotypic traits. Titan mice also show modifications in the liver transcriptome, proteome, and epigenome linked to metabolic (dys)regulations. Importantly, dietary intervention partially reversed the metabolic phenotype in Titan mice and significantly extended their life expectancy. Therefore, the Titan mouse line is a valuable resource for translational and interventional obesity research.
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Scientific Article
Transgenic Res. 31, 227-237 (2022)
The Hippo signal transduction network regulates transcription through Yap/Taz-Tead1-4 in many tissues including skeletal muscle. Whilst transgenic mice have been generated for many Hippo genes, the resultant skeletal muscle phenotypes were not always characterized. Here, we aimed to phenotype the hindlimb muscles of Hippo gene-mutated Lats1−/−, Mst2−/−, Vgll3−/−, and Vgll4+/− mice. This analysis revealed that Lats1−/− mice have 11% more slow type I fibers than age and sex-matched wild-type controls. Moreover, the mRNA expression of slow Myh7 increased by 50%, and the concentration of type I myosin heavy chain is 80% higher in Lats1−/− mice than in age and sex-matched wild-type controls. Second, to find out whether exercise-related stimuli affect Lats1, we stimulated C2C12 myotubes with the hypertrophy agent clenbuterol or the energy stress agent AICAR. We found that both stimulated Lats1 expression by 1.2 and 1.3 fold respectively. Third, we re-analyzed published datasets and found that Lats1 mRNA in muscle is 63% higher in muscular dystrophy, increases by 17–77% after cardiotoxin-induced muscle injury, by 41–71% in muscles during overload-induced hypertrophy, and by 19–21% after endurance exercise when compared to respective controls. To conclude, Lats1 contributes to the regulation of muscle fiber type proportions, and its expression is regulated by physiological and pathological situations in skeletal muscle.
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Scientific Article
Radiat. Res. 197, 7-21 (2022)
Ionizing radiation is widely known to induce various kinds of lens cataracts, of which posterior subcapsular cataracts (PSCs) have the highest prevalence. Despite some studies regarding the epidemiology and biology of radiation-induced PSCs, the mechanism underscoring the formation of this type of lesions and their dose dependency remain uncertain. Within the current study, our team investigated the in vivo characteristics of PSCs in B6C3F1 mice (F1-hybrids of BL6 × C3H) that received 0.5-2 Gy γ-ray irradiation after postnatal day 70. For purposes of assessing lenticular damages, spectral domain optical coherence tomography was utilized, and the visual acuity of the mice was measured to analyze their levels of visual impairment, and histological sections were then prepared in to characterize in vivo phenotypes. Three varying in vivo phenotype anterior and posterior lesions were thus revealed and correlated with the applied doses to understand their marginal influence on the visual acuity of the studied mice. Histological data indicated no significantly increased odds ratios for PSCs below a dose of 1 Gy at the end of the observation time. Furthermore, our team demonstrated that when the frequencies of the posterior and anterior lesions were calculated at early time points, their responses were in accordance with a deterministic model, whereas at later time points, their responses were better described via a stochastic model. The current study will aid in honing the current understanding of radiation-induced cataract formation and contributes greatly to addressing the fundamental questions of lens dose response within the field of radiation biology.
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Scientific Article
Nat. Cardio. Res. 1, 157-173 (2022)
Clinical presentation of congenital heart disease is heterogeneous,
making identification of the disease-causing genes and their genetic
pathways and mechanisms of action challenging. By using in vivo
electrocardiography, transthoracic echocardiography and microcomputed
tomography imaging to screen 3,894 single-gene-null mouse lines for
structural and functional cardiac abnormalities, here we identify 705
lines with cardiac arrhythmia, myocardial hypertrophy and/or ventricular
dilation. Among these 705 genes, 486 have not been previously
associated with cardiac dysfunction in humans, and some of them
represent variants of unknown relevance (VUR). Mice with mutations in Casz1, Dnajc18, Pde4dip, Rnf38 or Tmem161b
genes show developmental cardiac structural abnormalities, with their
human orthologs being categorized as VUR. Using UK Biobank data, we
validate the importance of the DNAJC18 gene for cardiac
homeostasis by showing that its loss of function is associated with
altered left ventricular systolic function. Our results identify
hundreds of previously unappreciated genes with potential function in
congenital heart disease and suggest causal function of five VUR in
congenital heart disease.
Wissenschaftlicher Artikel
Scientific Article
Front. Med. 9:875430 (2022)
Advanced age, followed by male sex, by far poses the greatest risk for severe COVID-19. An unresolved question is the extent to which modifiable comorbidities increase the risk of COVID-19-related mortality among younger patients, in whom COVID-19-related hospitalization strongly increased in 2021. A total of 3,163 patients with SARS-COV-2 diagnosis in the Lean European Open Survey on SARS-CoV-2-Infected Patients (LEOSS) cohort were studied. LEOSS is a European non-interventional multi-center cohort study established in March 2020 to investigate the epidemiology and clinical course of SARS-CoV-2 infection. Data from hospitalized patients and those who received ambulatory care, with a positive SARS-CoV-2 test, were included in the study. An additive effect of obesity, diabetes and hypertension on the risk of mortality was observed, which was particularly strong in young and middle-aged patients. Compared to young and middle-aged (18-55 years) patients without obesity, diabetes and hypertension (non-obese and metabolically healthy; n = 593), young and middle-aged adult patients with all three risk parameters (obese and metabolically unhealthy; n = 31) had a similar adjusted increased risk of mortality [OR 7.42 (95% CI 1.55-27.3)] as older (56-75 years) non-obese and metabolically healthy patients [n = 339; OR 8.21 (95% CI 4.10-18.3)]. Furthermore, increased CRP levels explained part of the elevated risk of COVID-19-related mortality with age, specifically in the absence of obesity and impaired metabolic health. In conclusion, the modifiable risk factors obesity, diabetes and hypertension increase the risk of COVID-19-related mortality in young and middle-aged patients to the level of risk observed in advanced age.
Wissenschaftlicher Artikel
Scientific Article
Sci. Rep. 12:6793 (2022)
This Article contains an error in Table 1 where the mean value and standard deviation of pregnancy week for the "screening group:NGT women" was incorrectly given as 23.0 +/- 9.5. The correct numbers are 26.5 +/- 2.1. Incorrect: (Table presented.) Correct: (Table presented.).
Sports Med. Open, DOI: 10.1007/s40279-022-01676-1 (2022)
In 1924, Otto Warburg asked "How does the metabolism of a growing tissue differ from that of a non-growing tissue?" Currently, we know that proliferating healthy and cancer cells reprogramme their metabolism. This typically includes increased glucose uptake, glycolytic flux and lactate synthesis. A key function of this reprogramming is to channel glycolytic intermediates and other metabolites into anabolic reactions such as nucleotide-RNA/DNA synthesis, amino acid-protein synthesis and the synthesis of, for example, acetyl and methyl groups for epigenetic modification. In this review, we discuss evidence that a hypertrophying muscle similarly takes up more glucose and reprogrammes its metabolism to channel energy metabolites into anabolic pathways. We specifically discuss the functions of the cancer-associated enzymes phosphoglycerate dehydrogenase and pyruvate kinase muscle 2 in skeletal muscle. In addition, we ask whether increased glucose uptake by a hypertrophying muscle explains why muscularity is often negatively associated with type 2 diabetes mellitus and obesity.
Review
Review
2021
Physiol. Res. 70, 227-236 (2021)
Mice are important models for biomedical research by providing the
possibility of standardizing genetic background and environmental
conditions, which both affect phenotypic variability. Use of both sexes
in experiments is strongly recommended because of possible differences
in the outcome. However, sex-specific phenotypic variability is
discussed with regard to putative consequences on the group size which
is necessary for achieving valid and reproducible results. Here, we
retrospectively analyzed the sex-specific variability of 25 blood
parameters of C3H inbred mice in two different mouse facilities within
the long-term, high-throughput Munich ENU mouse mutagenesis project.
Using the 95 % data range, data of 4,780-20,706 mice per parameter were
analyzed and resulted in ratios of the coefficient of variation (=
female CV / (female CV + male CV)) from 0.44 to 0.58 for the 25
parameters, with an overall mean of 0.51 in both facilities. Together
with data analyses of three additional, smaller studies with 72-247
animals per parameter examined and various genetic backgrounds (inbred
strains, F1 hybrids) included, hints for reproducible sex-specific
variability were observed for particular parameters. Thus, the overall
analysis comprising all 25 clinical chemical and hematological
parameters of the standardized, long-term analysis of a high number of
group housed, young adult, twelve-week-old C3H inbred mice showed no
evidence for substantial sex-specific variability. The results may
provide a basis for the examination of sex-specific variability in
particular blood parameters.
Wissenschaftlicher Artikel
Scientific Article
F1000 Res. 10:456 (2021)
In the last few decades, forward genetics approaches have been extensively used to identify gene function. Essentially, forward genetics is the elucidation of the genetic basis of a specific phenotype by screening a population containing random genomic modifications that alter gene function. These approaches have shed light on some essential gene functions in development and disease and have expanded the realm of understanding for genetic disorders. Due to the availability of efficient mutagenesis methods, phenotyping techniques, reliable validation, comprehensive sequence information and translational potential, mouse models are favored for forward genetics approaches. However, in this post-genomic CRISPR-Cas9 era, the relevance and future of forward genetics was brought into question. With more than 7300 mouse strains archived and close interactions with several leading mouse researchers around the world, INFRAFRONTIER - the European Research Infrastructure for mouse models organised a panel discussion on forward genetics at the International Mammalian Genome Conference 2018 to discuss the future of forward genetics as well as challenges faced by researchers using this approach in the current research environment. The commentary presents an overview of this discussion.
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Scientific Article
J. Clin. Invest. 131:e142202 (2021)
Cortical spreading depression (CSD), a wave of depolarization followed by depression of cortical activity, is a pathophysiological process implicated in migraine with aura and various other brain pathologies, such as ischemic stroke and traumatic brain injury. To gain insight into the pathophysiology of CSD, we generated a mouse model for a severe monogenic subtype of migraine with aura, familial hemiplegic migraine type 3 (FHM3). FHM3 is caused by mutations in SCN1A, encoding the voltage-gated Na+ channel NaV1.1 predominantly expressed in inhibitory interneurons. Homozygous Scn1aL1649Q knock-in mice died prematurely, whereas heterozygous mice had a normal lifespan. Heterozygous Scn1aL1649Q knock-in mice compared to wildtype mice displayed a significantly enhanced susceptibility to CSD. We found L1649Q to cause a gain-of-function effect with an impaired Na+-channel inactivation and increased ramp Na+-currents leading to hyperactivity of fast-spiking inhibitory interneurons. Brain slice recordings using K+-sensitive electrodes revealed an increase in extracellular K+ in the early phase of CSD in heterozygous mice, likely representing the mechanistic link between interneuron hyperactivity and CSD initiation. The neuronal phenotype and premature death of homozygous Scn1aL1649Q knock-in mice was partially rescued by GS967, a blocker of persistent Na+-currents. Collectively, our findings identify interneuron hyperactivity as a mechanism to trigger CSD.
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Scientific Article
Brain Behav. 11:e01928 (2021)
Background Dusp8 is the first GWAS-identified gene that is predominantly expressed in the brain and has previously been linked with the development of diabetes type 2 in humans. In this study, we unravel how Dusp8 is involved in the regulation of sucrose reward behavior.Methods Female, chow-fed global Dusp8 WT and KO mice were tested in an observer-independent IntelliCage setup for self-administrative sucrose consumption and preference followed by a progressive ratio task with restricted sucrose access to monitor seeking and motivation behavior. Sixty-three human carriers of the major C and minor T allele of DUSP8 SNP rs2334499 were tested for their perception of food cues by collecting a rating score for sweet versus savory high caloric food.Results Dusp8 KO mice showed a comparable preference for sucrose, but consumed more sucrose compared to WT mice. In a progressive ratio task, Dusp8 KO females switched to a "trial and error" strategy to find sucrose while control Dusp8 WT mice kept their previously established seeking pattern. Nonetheless, the overall motivation to consume sucrose, and the levels of dopaminergic neurons in the brain areas NAcc and VTA were comparable between genotypes. Diabetes-risk allele carriers of DUSP8 SNP rs2334499 preferred sweet high caloric food compared to the major allele carriers, rating scores for savory food remained comparable between groups.Conclusion Our data suggest a novel role for Dusp8 in the perception of sweet high caloric food as well as in the control of sucrose consumption and foraging in mice and humans.
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Scientific Article
J. Mol. Cell. Cardiol. 156, 45-56 (2021)
CRELD1 (Cysteine-Rich with EGF-Like Domains 1) is a risk gene for non-syndromic atrioventricular septal defects in human patients. In a mouse model, Creld1 has been shown to be essential for heart development, particularly in septum and valve formation. However, due to the embryonic lethality of global Creld1 knockout (KO) mice, its cell type-specific function during peri- and postnatal stages remains unknown. Here, we generated conditional Creld1 KO mice lacking Creld1 either in the endocardium (KOTie2) or the myocardium (KOMyHC). Using a combination of cardiac phenotyping, histology, immunohistochemistry, RNA-sequencing and flow cytometry, we demonstrate that Creld1 function in the endocardium is dispensable for heart development. Lack of myocardial Creld1 causes extracellular matrix remodeling and trabeculation defects by modulation of the Notch1 signaling pathway. Hence, KOMyHC mice die early postnatally due to myocardial hypoplasia. Our results reveal that Creld1 not only controls the formation of septa and valves at an early stage during heart development, but also cardiac maturation and function at a later stage. These findings underline the central role of Creld1 in mammalian heart development and function.
Wissenschaftlicher Artikel
Scientific Article
Mamm. Genome 32, 332-349 (2021)
Pathogenic variants in the WDR45 (OMIM: 300,526) gene on chromosome Xp11 are the genetic cause of a rare neurological disorder characterized by increased iron deposition in the basal ganglia. As WDR45 encodes a beta-propeller scaffold protein with a putative role in autophagy, the disease has been named Beta-Propeller Protein-Associated Neurodegeneration (BPAN). BPAN represents one of the four most common forms of Neurodegeneration with Brain Iron Accumulation (NBIA). In the current study, we generated and characterized a whole-body Wdr45 knock-out (KO) mouse model. The model, developed using TALENs, presents a 20-bp deletion in exon 2 of Wdr45. Homozygous females and hemizygous males are viable, proving that systemic depletion of Wdr45 does not impair viability and male fertility in mice. The in-depth phenotypic characterization of the mouse model revealed neuropathology signs at four months of age, neurodegeneration progressing with ageing, hearing and visual impairment, specific haematological alterations, but no brain iron accumulation. Biochemically, Wdr45 KO mice presented with decreased complex I (CI) activity in the brain, suggesting that mitochondrial dysfunction accompanies Wdr45 deficiency. Overall, the systemic Wdr45 KO described here complements the two mouse models previously reported in the literature (PMIDs: 26,000,824, 31,204,559) and represents an additional robust model to investigate the pathophysiology of BPAN and to test therapeutic strategies for the disease.
Wissenschaftlicher Artikel
Scientific Article
Nat. Genet. 53, 416-419 (2021)
The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for more than 5,000 genes, including 2,850 novel null, 2,987 novel conditional-ready and 4,433 novel reporter alleles.
Review
Review
Mol. Metab. 54:101334 (2021)
OBJECTIVE: Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to carry a vital role in β-cell dysfunction and diabetes. However, very little is known about the function of this protein in β-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on β-cell development and function. METHODS: We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the β-cell development at embryonic day (E)18.5 and β-cell identity as well as function at postnatal stages. RESULTS: Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although, no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing β-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP as well as PDI family member PDIA4, however without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of β-cell maturation and function, such as Ins2, Mafa and Slc2a2 along with increased expression of α-cell markers, Mafb and glucagon was observed in adult mice, suggesting loss of β-cell identity. CONCLUSIONS: The data demonstrates that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic β-cell function and identity, suggesting a critical role of PDIA6 specifically for β-cells.
Wissenschaftlicher Artikel
Scientific Article
Nature 589, E6 (2021)
In the HTML version of this Article, owing to a typesetting error, the affiliations for author Indrabahadur Singh were incorrect. The correct affiliation is ‘Emmy Noether Research Group Epigenetic Machineries and Cancer, Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany’. The PDF and print versions of the Article are correct. In addition, Ilias Angelidis should have been listed as an author, with the affiliation: ‘Comprehensive Pneumology Center (CPC), Institute of Lung Biology and Disease, Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Neuherberg, Germany’. They designed, undertook, and analysed scRNA-seq experiments, and analysed and interpreted data (see ‘Author contributions’). Finally, in the original Article, authors Mathias Heikenwalder and Ali Önder Yildirim were listed as ‘jointly supervising’ authors instead of ‘equally contributing’ authors, alongside authors Thomas M. Conlon and Gerrit John-Schuster. The original Article has been corrected online.
Diabetologe 17, 780–787 (2021)
Data in translational healthcare research is complex and highly connected. Information on wide-spread diseases like diabetes and cancer is extensive, heterogeneous and rapidly growing. Data are available at various locations and are neither interconnectable with other data sources nor searchable. Consequently, it is difficult for researchers to access data and to cope with the amount of literature. Collecting data and knowledge is still done manually by comparing data tables. However, a flexible and efficient approach to processing biomedical data is offered by graph databases. Based on the open source Neo4j graph database, the German Center for Diabetes Research (DZD) developed DZDconnect—a knowledge graph that links data from basic research and clinical studies across sites, disciplines and species with external knowledge. DZDconnect collects, structures, interconnects and makes available various data and information on wide-spread diseases and its long-term complications. Information from well-established databases is connected on the metadata level, raw data level as well as on the insight level. In addition, in-house data from translational research can be integrated. The enabling technology is a flexible and scalable graph database. DZDconnect thus bridges the gap between healthcare research and state-of-the-art information technology and helps to make disease research faster and more efficient. With DZDconnect scientists can quickly and efficiently generate hypotheses regarding the underlying mechanisms of these diseases and how to intervene medically. DZDconnect is developed as an open-source project.
Review
Review
Cells 10:3443 (2021)
Physical training improves insulin sensitivity and can prevent type 2 diabetes (T2D). However, approximately 20% of individuals lack a beneficial outcome in glycemic control. TGF-β, identified as a possible upstream regulator involved in this low response, is also a potent regulator of microRNAs (miRNAs). The aim of this study was to elucidate the potential impact of TGF-β-driven miRNAs on individual exercise response. Non-targeted long and sncRNA sequencing analyses of TGF-β1-treated human skeletal muscle cells corroborated the effects of TGF-β1 on muscle cell differentiation, the induction of extracellular matrix components, and identified several TGF-β1-regulated miRNAs. qPCR validated a potent upregulation of miR-143-3p/145-5p and miR-181a2-5p by TGF-β1 in both human myoblasts and differentiated myotubes. Healthy subjects who were overweight or obese participated in a supervised 8-week endurance training intervention (n = 40) and were categorized as responder or low responder in glycemic control based on fold change ISIMats (≥+1.1 or <+1.1, respectively). In skeletal muscle biopsies of low responders, TGF-β signaling and miR-143/145 cluster levels were induced by training at much higher rates than among responders. Target-mining revealed HDACs, MYHs, and insulin signaling components INSR and IRS1 as potential miR-143/145 cluster targets. All these targets were down-regulated in TGF-β1-treated myotubes. Transfection of miR-143-3p/145-5p mimics in differentiated myotubes validated MYH1, MYH4, and IRS1 as miR-143/145 cluster targets. Elevated TGF-β signaling and miR-143/145 cluster induction in skeletal muscle of low responders might obstruct improvements in insulin sensitivity by training in two ways: by a negative impact of miR-143-3p on muscle cell fusion and myofiber functionality and by directly impairing insulin signaling via a reduction in INSR by TGF-β and finetuned IRS1 suppression by miR-143-3p.
Wissenschaftlicher Artikel
Scientific Article
Front. Med. 8:751277 (2021)
Adipose tissue (AT) is no longer considered to be responsible for energy storage only but is now recognized as a major endocrine organ that is distributed across different parts of the body and is actively involved in regulatory processes controlling energy homeostasis. Moreover, AT plays a crucial role in the development of metabolic disease such as diabetes. Recent evidence has shown that adipokines have the ability to regulate blood glucose levels and improve metabolic homeostasis. While AT has been studied extensively in the context of type 2 diabetes, less is known about how different AT types are affected by absolute insulin deficiency in type 1 or permanent neonatal diabetes mellitus. Here, we analyzed visceral and subcutaneous AT in a diabetic, insulin-deficient pig model (MIDY) and wild-type (WT) littermate controls by RNA sequencing and quantitative proteomics. Multi-omics analysis indicates a depot-specific dysregulation of crucial metabolic pathways in MIDY AT samples. We identified key proteins involved in glucose uptake and downstream signaling, lipogenesis, lipolysis and β-oxidation to be differentially regulated between visceral and subcutaneous AT in response to insulin deficiency. Proteins related to glycogenolysis, pyruvate metabolism, TCA cycle and lipogenesis were increased in subcutaneous AT, whereas β-oxidation-related proteins were increased in visceral AT from MIDY pigs, pointing at a regionally different metabolic adaptation to master energy stress arising from diminished glucose utilization in MIDY AT. Chronic, absolute insulin deficiency and hyperglycemia revealed fat depot-specific signatures using multi-omics analysis. The generated datasets are a valuable resource for further comparative and translational studies in clinical diabetes research.
Wissenschaftlicher Artikel
Scientific Article
Diabetes 70, 2785-2795:2785-2795 (2021)
Lifestyle intervention (LI) can prevent type 2 diabetes, but response to LI varies depending on risk subphenotypes. We tested if prediabetic individuals with low risk benefit from conventional LI and individuals with high risk benefit from an intensification of LI in a multi-center randomized controlled intervention over 12 months with 2 years follow up. 1105 prediabetic individuals based on ADA glucose criteria were stratified into a high- and low-risk phenotype, based on previously described thresholds of insulin secretion, insulin sensitivity and liver fat content. Low-risk individuals were randomly assigned to conventional LI according to the DPP protocol or control (1:1), high-risk individuals to conventional or intensified LI with doubling of required exercise (1:1). A total of 908 (82%) participants completed the study. In high-risk individuals, the difference between conventional and intensified LI in post-challenge glucose change was -0.29 mmol/l [CI:-0.54;-0.04], p=0.025. Liver fat (-1.34 percentage points [CI:-2.17;-0.50], p=0.002) and cardiovascular risk (-1.82[CI:-3.13-0.50],p=0.007) underwent larger reductions with intensified than with conventional LI. During a follow up of 3 years, intensified compared to conventional LI had a higher probability to normalize glucose tolerance (p=0.008). In conclusion, it is possible in high-risk individuals with prediabetes to improve glycemic and cardiometabolic outcomes by intensification of LI. Individualized, risk-phenotype-based LI may be beneficial for the prevention of diabetes.
Wissenschaftlicher Artikel
Scientific Article
Diabetologe 17, 777-779 (2021)
Editorial
Editorial
Mol. Metab. 54:101359 (2021)
OBJECTIVE: Liver mitochondria adapt to high calorie intake. We investigated how exercise alters the early compensatory response of mitochondria and thus prevents fatty liver disease as a long-term consequence of overnutrition. METHODS: We compared the effects of a steatogenic high-energy diet (HED, for 6 weeks) on mitochondrial metabolism of sedentary and treadmill-trained C57BL/6N mice. We applied multi-OMICs analyses to study the alterations in the proteome, transcriptome and lipids in isolated mitochondria of liver and skeletal muscle as well as in whole tissue and examined the functional consequences by high resolution respirometry. RESULTS: HED increased the respiratory capacity of isolated liver mitochondria, both in sedentary and in trained mice. However, proteomics analysis of the mitochondria and transcriptomics indicated that training modified the adaptation of the hepatic metabolism to HED on the level of respiratory complex I, glucose oxidation, pyruvate and acetyl-CoA metabolism and lipogenesis. Training also counteracted the HED-induced increase in fasting insulin, glucose tolerance, and liver fat. This was accompanied by lower diacylglycerol species and JNK phosphorylation in the livers of trained HED-fed mice, two mechanisms that can reverse hepatic insulin resistance. In skeletal muscle, the combination of HED and training improved the oxidative capacity to a greater extent than training alone by increasing respiration of isolated mitochondria and total mitochondrial protein content. CONCLUSION: We provide a comprehensive insight into the early adaptations of mitochondria in liver and skeletal muscle to HED and endurance training. Our results suggest that exercise disconnects the HED-induced increase in mitochondrial substrate oxidation from pyruvate and acetyl-CoA-driven lipid synthesis. This could contribute to the prevention of deleterious long-term effects of high fat and sugar intake on hepatic mitochondrial function and insulin sensitivity.
Wissenschaftlicher Artikel
Scientific Article
Diabetol. Stoffwechs. 16, 473-475 (2021)
Genetische Prädisposition, d.h. das Vorhandensein bestimmter
Genvarianten, sowie unausgewogene Ernährung und zu wenig körperliche
Aktivität sind wesentliche Faktoren, die das Auftreten eines
Typ-2-Diabetes (T2D) beeinflussen. Der Lebensstil wirkt nicht nur direkt
auf den Stoffwechsel, sondern kann auch über epigenetische Mechanismen
zu langfristigen Änderungen in der Expression stoffwechselrelevanter
Gene führen. Diese Änderungen können sogar an die nächsten Generationen
weitervererbt werden. Aktuelle Studien helfen dabei, mehr und mehr zu
entschlüsseln, welchen Einfluss genetische und epigenetische Elemente
auf die Krankheitsentstehung haben. Die aktuellen Forschungsergebnisse
zeigen, wie komplex die Erkrankung ist; sie eröffnen aber auch neue
Ansätze für die Prävention und Therapie von Diabetes.
Review
Review
Metabolites 11:89 (2021)
Biological exploration of early biomarkers for chronic kidney disease (CKD) in (pre)diabetic individuals is crucial for personalized management of diabetes. Here, we evaluated two candidate biomarkers of incident CKD (sphingomyelin (SM) C18:1 and phosphatidylcholine diacyl (PC aa) C38:0) concerning kidney function in hyperglycemic participants of the Cooperative Health Research in the Region of Augsburg (KORA) cohort, and in two biofluids and six organs of leptin receptor-deficient (db/db) mice and wild type controls. Higher serum concentrations of SM C18:1 and PC aa C38:0 in hyperglycemic individuals were found to be associated with lower estimated glomerular filtration rate (eGFR) and higher odds of CKD. In db/db mice, both metabolites had a significantly lower concentration in urine and adipose tissue, but higher in the lungs. Additionally, db/db mice had significantly higher SM C18:1 levels in plasma and liver, and PC aa C38:0 in adrenal glands. This cross-sectional human study confirms that SM C18:1 and PC aa C38:0 associate with kidney dysfunction in pre(diabetic) individuals, and the animal study suggests a potential implication of liver, lungs, adrenal glands, and visceral fat in their systemic regulation. Our results support further validation of the two phospholipids as early biomarkers of renal disease in patients with (pre)diabetes.
Wissenschaftlicher Artikel
Scientific Article
Nat. Commun. 12:4957 (2021)
Influenza during pregnancy can affect the health of offspring in later life, among which neurocognitive disorders are among the best described. Here, we investigate whether maternal influenza infection has adverse effects on immune responses in offspring. We establish a two-hit mouse model to study the effect of maternal influenza A virus infection (first hit) on vulnerability of offspring to heterologous infections (second hit) in later life. Offspring born to influenza A virus infected mothers are stunted in growth and more vulnerable to heterologous infections (influenza B virus and MRSA) than those born to PBS- or poly(I:C)-treated mothers. Enhanced vulnerability to infection in neonates is associated with reduced haematopoetic development and immune responses. In particular, alveolar macrophages of offspring exposed to maternal influenza have reduced capacity to clear second hit pathogens. This impaired pathogen clearance is partially reversed by adoptive transfer of alveolar macrophages from healthy offspring born to uninfected dams. These findings suggest that maternal influenza infection may impair immune ontogeny and increase susceptibility to early life infections of offspring.
Wissenschaftlicher Artikel
Scientific Article
Nat. Commun. 12:1804 (2021)
The original version of the Peer Review File associated with this Article was updated after publication to redact two figures in the interest of confidentiality.
Sci. Adv. 7:eabg6424 (2021)
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.
Wissenschaftlicher Artikel
Scientific Article
Exp. Eye Res. 204:108432 (2021)
Ionising radiation interacts with lenses and retinae differently. In human lenses, posterior subcapsular cataracts are the predominant observation, whereas retinae of adults are comparably resistant to even relatively high doses. In this study, we demonstrate the effects of 2 Gy of low linear energy transfer ionising radiation on eyes of B6C3F1 mice aged postnatal day 2. Optical coherence tomography and Scheimpflug imaging were utilised for the first time to monitor murine lenses and retinae in vivo. The visual acuity of the mice was determined and histological analysis was conducted. Our results demonstrated that visual acuity was reduced by as much as 50 % approximately 9 months after irradiation in irradiated mice. Vision impairment was caused by retinal atrophy and inner cortical cataracts. These results help to further our understanding of the risk of ionising radiation for human foeti (∼ 8 mo), which follow the same eye development stages as neonatal mice.
Wissenschaftlicher Artikel
Scientific Article
Genes 12:1732 (2021)
Trpc7 (transient receptor potential cation channel, subfamily C, member 7; 862 amino acids) knockout mice are described showing no clear phenotypic alterations, therefore, the functional relevance of the gene remains unclear. A complementary approach for the functional analysis of a given gene is the examination of individuals harbouring a mutant allele of the gene. In the phenotype-driven Munich ENU mouse mutagenesis project, a high number of phenotypic parameters was used for establishing novel mouse models on the genetic background of C3H inbred mice. The phenotypically dominant mutant line SMA002 was established and further examined. Analysis of the causative mutation as well as the phenotypic characterization of the mutant line were carried out. The causative mutation was detected in the gene Trpc7 which leads to the production of a truncated protein due to the novel stop codon at amino acid position 810 thereby affecting the highly conserved cytoplasmic C terminus of the protein. Trpc7 heterozygous mutant mice of both sexes were viable and fertile, but showed distinct morphological and behavioural alterations which is in contrast to the published phenotype of Trpc7 knockout mice. Thus, the Trpc7K810Stop mutation leads to a dominant negative effect of the mutant protein.
Wissenschaftlicher Artikel
Scientific Article
Antioxidants 10:1103 (2021)
The NADPH oxidase Nox4 is a hydrogen peroxide (H2 O2)-producing enzyme, with the highest expression in the kidney. As the kidney is involved in volume and blood pressure control through sodium handling, we set out to determine the impact of a low sodium diet on these parameters in WT and Nox4-/-mice. Nox4 expression in the murine kidney was restricted to the proximal tubule. Nevertheless, low-sodium-induced weight loss and sodium sparing function was similar in WT and Nox4-/-mice, disputing an important function of renal Nox4 in sodium handling. In contrast, a low sodium diet resulted in a reduction in systolic blood pressure in Nox4-/-as compared to WT mice. This was associated with a selectively lower pressure to heart-rate ratio, as well as heart to body weight ratio. In general, a low sodium diet leads to activation of sympathetic tone and the renin angiotensin system, which subsequently increases peripheral resistance. Our observations suggest that the control by this system is attenuated in Nox4-/-mice, resulting in lower blood pressure in response to low sodium.
Wissenschaftlicher Artikel
Scientific Article
Lancet Diabet. Endocrinol. 9, 786-798 (2021)
Up to 50% of the people who have died from COVID-19 had metabolic and vascular disorders. Notably, there are many direct links between COVID-19 and the metabolic and endocrine systems. Thus, not only are patients with metabolic dysfunction (eg, obesity, hypertension, non-alcoholic fatty liver disease, and diabetes) at an increased risk of developing severe COVID-19 but also infection with SARS-CoV-2 might lead to new-onset diabetes or aggravation of pre-existing metabolic disorders. In this Review, we provide an update on the mechanisms of how metabolic and endocrine disorders might predispose patients to develop severe COVID-19. Additionally, we update the practical recommendations and management of patients with COVID-19 and post-pandemic. Furthermore, we summarise new treatment options for patients with both COVID-19 and diabetes, and highlight current challenges in clinical management.
Review
Review
Dis. Model. Mech. 14:dmm049285 (2021)
Large animal models for Duchenne muscular dystrophy (DMD) are crucial for evaluation of diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMDΔ52) resemble molecular, clinical and pathological hallmarks of DMD, but die before sexual maturity and cannot be propagated by breeding. Therefore, we generated female DMD+/- carriers. A single founder animal had 11 litters with 29 DMDY/-, 34 DMD+/- as well as 36 male and 29 female wild-type offspring. Breeding with F1 and F2 DMD+/- carriers resulted in additional 114 DMDY/- piglets. With intensive neonatal management, the majority survived for 3-4 months, providing statistically relevant cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance of human disease mechanisms. Importantly, DMDY/- pigs reveal progressive myocardial fibrosis and increased expression of connexin-43, associated with significantly reduced left ventricular ejection fraction already at age 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability. Our breeding cohort of DMDΔ52 pigs and standardized tissue repositories provide important resources for studying DMD disease mechanisms and for testing novel treatment strategies.
Wissenschaftlicher Artikel
Scientific Article
PLoS Genet. 16:e1009190 (2021)
The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.
Wissenschaftlicher Artikel
Scientific Article
PLoS ONE 16:e0248594 (2021)
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.
Wissenschaftlicher Artikel
Scientific Article
Int. J. Radiat. Biol. 97, 156-169 (2021)
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.
Wissenschaftlicher Artikel
Scientific Article
EMBO Mol. Med.:e14397 (2021)
Mitochondrial disorders are clinically and genetically diverse, with isolated complex III (CIII) deficiency being relatively rare. Here, we describe two affected cousins, presenting with recurrent episodes of severe lactic acidosis, hyperammonaemia, hypoglycaemia and encephalopathy. Genetic investigations in both cases identified a homozygous deletion of exons 2 and 3 of UQCRH, which encodes a structural complex III (CIII) subunit. We generated a mouse model with the equivalent homozygous Uqcrh deletion (Uqcrh−/−), which also presented with lactic acidosis and hyperammonaemia, but had a more severe, non-episodic phenotype, resulting in failure to thrive and early death. The biochemical phenotypes observed in patient and Uqcrh−/− mouse tissues were remarkably similar, displaying impaired CIII activity, decreased molecular weight of fully assembled holoenzyme and an increase of an unexpected large supercomplex (SXL), comprising mostly of one complex I (CI) dimer and one CIII dimer. This phenotypic similarity along with lentiviral rescue experiments in patient fibroblasts verifies the pathogenicity of the shared genetic defect, demonstrating that the Uqcrh−/− mouse is a valuable model for future studies of human CIII deficiency.
Wissenschaftlicher Artikel
Scientific Article
Nat. Med. 27, 49-57 (2021)
The state of intermediate hyperglycemia is indicative of elevated risk of developing type 2 diabetes1. However, the current definition of prediabetes neither reflects subphenotypes of pathophysiology of type 2 diabetes nor is predictive of future metabolic trajectories. We used partitioning on variables derived from oral glucose tolerance tests, MRI-measured body fat distribution, liver fat content and genetic risk in a cohort of extensively phenotyped individuals who are at increased risk for type 2 diabetes2,3 to identify six distinct clusters of subphenotypes. Three of the identified subphenotypes have increased glycemia (clusters 3, 5 and 6), but only individuals in clusters 5 and 3 have imminent diabetes risks. By contrast, those in cluster 6 have moderate risk of type 2 diabetes, but an increased risk of kidney disease and all-cause mortality. Findings were replicated in an independent cohort using simple anthropomorphic and glycemic constructs4. This proof-of-concept study demonstrates that pathophysiological heterogeneity exists before diagnosis of type 2 diabetes and highlights a group of individuals who have an increased risk of complications without rapid progression to overt type 2 diabetes.
Wissenschaftlicher Artikel
Scientific Article
2020
Sci. Rep. 10:18334 (2020)
Sex steroids, such as estrogens and androgens, are important regulators of the humoral immune response. Studies in female mice have demonstrated that alteration of circulating estrogen concentration regulates antibody-mediated immunity. As males have normally little endogenous estrogen, we hypothesized that in males high estrogens and low androgens affect the immune system and enhance the allergic inflammatory response. Here, we studied transgenic male mice expressing human aromatase (AROM+). These animals have a high circulating estrogen to androgen ratio (E/A), causing female traits such as gynecomastia. We found that AROM+ male mice had significantly higher plasma immunoglobulin levels, particularly IgE. Flow cytometry analyses of splenocytes revealed changes in mature/immature B cell ratio together with a transcriptional upregulation of the Igh locus. Furthermore, higher proliferation rate and increased IgE synthesis after IgE class-switching was found. Subsequently, we utilized an ovalbumin airway challenge model to test the allergic response in AROM+ male mice. In line with above observations, an increase in IgE levels was measured, albeit no impact on immune cell infiltration into the lungs was detected. Together, our findings suggest that high circulating E/A in males significantly alters B cell function without any significant enhancement in allergic inflammation.
Wissenschaftlicher Artikel
Scientific Article
Haematologica 105, 937-950 (2020)
Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing . oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the ation of mitochondria but is now known to occur through mitophary Yet, ggenetic ablation of the Alox15 gene in mice failed to provice evidence or this hyppothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullaty erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation.
Wissenschaftlicher Artikel
Scientific Article
J. Neurosci. Methods 339:108744 (2020)
Background: Dense and unbiased cellular-resolution representations of extended volumetric central nervous system soft-tissue anatomy are difficult to obtain, even in experimental post-mortem settings. Interestingly, X-ray phase-contrast computed tomography (X-PCI-CT), an emerging soft-tissue-sensitive volumetric imaging technique, can provide multiscale organ- to cellular-level morphological visualizations of neuroanatomical structure.New Method: Here, we tested different nervous-tissue fixation procedures, conventionally used for transmission electron microscopy, to better establish X-PCI-CT-specific sample-preparation protocols. Extracted rat spinal medullas were alternatively fixed with a standard paraformaldehyde-only aldehyde-based protocol, or in combination with glutaraldehyde. Some specimens were additionally post-fixed with osmium tetroxide. Multiscale X-PCI-CT datasets were collected at several synchrotron radiation facilities, using state-of-the-art setups with effective image voxel sizes of 3.0(3) to 0.3(3) mu m(3), and compared to high-field magnetic resonance imaging, histology and vascular fluorescence microscopy data.Results: Multiscale X-PCI-CT of aldehyde-fixed spinal cord specimens resulted in dense histology-like volumetric representations and quantifications of extended deep spinal micro-vascular networks and of intra-medullary cell populations. Osmium post-fixation increased intra-medullary contrast between white and gray-matter tissues, and enhanced delineation of intra-medullary cellular structure, e.g. axon fibers and motor neuron perikarya.Comparison with Existing Methods: Volumetric X-PCI-CT provides complementary contrast and higher spatial resolution compared to 9.4T MRI. X-PCI-CT's advantage over planar histology is the volumetric nature of the cellular-level data obtained, using samples much larger than those fit for volumetric vascular fluorescence microscopy.Conclusions: Deliberately choosing (post-)fixation protocols tailored for optimal nervous-tissue structural preservation is of paramount importance in achieving effective and targeted neuroimaging via the X-PCI-CT technique.
Wissenschaftlicher Artikel
Scientific Article
Mamm. Genome, DOI: 10.1007/s00335-020-09843-3 (2020)
Editorial
Editorial
Neurosci. Lett. 735:135206 (2020)
Inositol-1,4,5-trisphosphate 3-kinase-A (ITPKA) is the neuronal isoform of ITPKs and exhibits both actin bundling and InsP3kinase activity. In addition to neurons, ITPKA is ectopically expressed in tumor cells, where its oncogenic activity increases tumor cell malignancy. In order to analyze the physiological relevance of ITPKA, here we performed a broad phenotypic screening of itpka deficient mice. Our data show that among the neurobehavioral tests analyzed, itpka deficient mice reacted faster to a hotplate, prepulse inhibition was impaired and the accelerating rotarod test showed decreased latency of itpka deficient mice to fall. These data indicate that ITPKA is involved in the regulation of nociceptive pathways, sensorimotor gating and motor learning. Analysis of extracerebral functions in control and itpka deficient mice revealed significantly reduced glucose, lactate, and triglyceride plasma concentrations in itpka deficient mice. Based on this finding, expression of ITPKA was analyzed in extracerebral tissues and the highest level was found in the small intestine. However, functional studies on CaCo-2 control and ITPKA depleted cells showed that glucose, as well as triglyceride uptake, were not significantly different between the cell lines. Altogether, these data show that ITPKA exhibits distinct functions in the central nervous system and reveal an involvement of ITPKA in energy metabolism.
Wissenschaftlicher Artikel
Scientific Article
Sci. Rep. 10:12407 (2020)
Among obese subjects, metabolically healthy (MHO) and unhealthy obese (MUHO) subjects exist, the latter being characterized by whole-body insulin resistance, hepatic steatosis, and subclinical inflammation. Insulin resistance and obesity are known to associate with alterations in mitochondrial density, morphology, and function. Therefore, we assessed mitochondrial function in human subcutaneous preadipocytes as well as in differentiated adipocytes derived from well-matched donors. Primary subcutaneous preadipocytes from 4 insulin-resistant (MUHO) versus 4 insulin-sensitive (MHO), non-diabetic, morbidly obese Caucasians (BMI > 40 kg/m(2)), matched for sex, age, BMI, and percentage of body fat, were differentiated in vitro to adipocytes. Real-time cellular respiration was measured using an XF24 Extracellular Flux Analyzer (Seahorse). Lipolysis was stimulated by forskolin (FSK) treatment. Mitochondrial respiration was fourfold higher in adipocytes versus preadipocytes (p = 1.6*10(-9)). In adipocytes, a negative correlation of mitochondrial respiration with donors' insulin sensitivity was shown (p = 0.0008). Correspondingly, in adipocytes of MUHO subjects, an increased basal respiration (p = 0.002), higher proton leak (p = 0.04), elevated ATP production (p = 0.01), increased maximal respiration (p = 0.02), and higher spare respiratory capacity (p = 0.03) were found, compared to MHO. After stimulation with FSK, the differences in ATP production, maximal respiration and spare respiratory capacity were blunted. The differences in mitochondrial respiration between MUHO/MHO were not due to altered mitochondrial content, fuel switch, or lipid metabolism. Thus, despite the insulin resistance of MUHO, we could clearly show an elevated mitochondrial respiration of MUHO adipocytes. We suggest that the higher mitochondrial respiration reflects a compensatory mechanism to cope with insulin resistance and its consequences. Preserving this state of compensation might be an attractive goal for preventing or delaying the transition from insulin resistance to overt diabetes.
Wissenschaftlicher Artikel
Scientific Article
Nat. Commun. 11:655 (2020)
The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery.
Wissenschaftlicher Artikel
Scientific Article
Comm. Biol. 3:628 (2020)
The transcription factor PAX6 is involved in the development of the eye and pancreatic islets, besides being associated with sleep-wake cycles. Here, we investigated a point mutation in the RED subdomain of PAX6, previously described in a human patient, to present a comprehensive study of a homozygous Pax6 mutation in the context of adult mammalian metabolism and circadian rhythm. Pax6(Leca2) mice lack appropriate retinal structures for light perception and do not display normal daily rhythmic changes in energy metabolism. Despite beta cell dysfunction and decreased insulin secretion, mutant mice have normal glucose tolerance. This is associated with reduced hepatic glucose production possibly due to altered circadian variation in expression of clock and metabolic genes, thereby evading hyperglycemia. Hence, our findings show that while the RED subdomain is important for beta cell functional maturity, the Leca2 mutation impacts peripheral metabolism via loss of circadian rhythm, thus revealing pleiotropic effects of PAX6. Nirav Chhabra et al. characterize adult mice carrying a homozygous mutation in Pax6 that was identified in a patient with foveal hypoplasia. They find that the Pax6 point mutation has pleiotropic effects, including defects in the mouse retinal structures, loss of the optic nerve, changes in energy metabolism and circadian rhythms, and dysregulation of genes expressed in the pancreas.
Wissenschaftlicher Artikel
Scientific Article
Nature 588, 151–156 (2020)
Blockade of lymphotoxin beta-receptor (LT beta R) signalling restores WNT signalling and epithelial repair in a model of chronic obstructive pulmonary disease.Lymphotoxin beta-receptor (LT beta R) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures(1,2), which are associated with severe chronic inflammatory diseases that span several organ systems(3-6). How LT beta R signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LT beta R blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LT beta R ligands in adaptive and innate immune cells, enhanced non-canonical NF-kappa B signalling, and enriched LT beta R target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LT beta R signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LT beta R signalling dampened epithelial non-canonical activation of NF-kappa B, reduced TGF beta signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/beta-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LT beta R signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures(1) and inhibition of apoptosis with tissue-regenerative strategies.
Wissenschaftlicher Artikel
Scientific Article
Cell Rep. 30, 3183-3194.e4 (2020)
Biofluids contain various circulating cell-free RNAs (ccfRNAs). The composition of these ccfRNAs varies among biofluids. They constitute tantalizing biomarker candidates for several pathologies and have been demonstrated to be mediators of cellular communication. Little is known about their function in physiological and developmental settings, and most works are limited to in vitro studies. Here, we develop iTAG-RNA, a method for the unbiased tagging of RNA transcripts in mice in vivo. We use iTAG-RNA to isolate hepatocytes and kidney proximal epithelial cell-specific transcriptional responses to a dietary challenge without interfering with the tissue architecture and to identify multiple hepatocyte-secreted ccfRNAs in plasma. We also identify specific transfer of liver-derived ccfRNAs to adipose tissue and skeletal muscle, where they likely constitute a buffering system to maintain lipid homeostasis under acute high-fat-diet feeding. Our findings directly demonstrate in vivo transfer of RNAs between tissues and highlight its implications for endocrine signaling and homeostasis.
Wissenschaftlicher Artikel
Scientific Article
Nat. Commun. 11:296 (2020)
Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking iron-regulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in beta cells. This impairs Fe-S cluster biosynthesis, reducing the function of Cdkal1, an Fe-S cluster enzyme that catalyzes methylthiolation of t(6)A37 in tRNA(UUU)(Lys) to ms(2)t(6)A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms(2)t(6)A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2(-/-) beta cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans.
Wissenschaftlicher Artikel
Scientific Article
J. Pers. Med. 10:E124 (2020)
Aldo-keto reductase family 1 (AKR1) enzymes play a crucial role in diabetic complications. Since type 2 diabetes (T2D) is associated with cancer progression, we investigated the impact of diabetes onAKR1gene expression in the context of prostate cancer (PCa) development. In this study, we analyzed benign (BEN) prostate and PCa tissue of patients with and without T2D. Furthermore, to replicate hyperglycemia in vitro, we treated the prostate adenocarcinoma cell line PC3 with increasing glucose concentrations. Gene expression was quantified using real-time qPCR. In the prostate tissue of patients with T2D,AKR1C1andAKR1C2transcripts were higher compared to samples of patients without diabetes. In PC3 cells, high glucose treatment induced the gene expression levels ofAKR1C1,C2,andC3. Furthermore, both in human tissue and in PC3 cells, the transcript levels ofAKR1C1, C2,andC3showed positive associations with oncogenes, which are involved in proliferation processes and HIF1 alpha and NF kappa B pathways. These results indicate that in the prostate glands of patients with T2D, hyperglycemia could play a pivotal role by inducing the expression ofAKR1C1, C2,andC3. The higher transcript level ofAKR1Cwas furthermore associated with upregulated HIF1 alpha and NF kappa B pathways, which are major drivers of PCa carcinogenesis.
Wissenschaftlicher Artikel
Scientific Article
Genes 11:1174 (2020)
Prostate cancer (PCa), the most incident cancer in men, is tightly regulated by endocrine signals. A number of different PCa cell lines are commonly used for in vitro experiments, but these are of diverse origin, and have very different cell-proliferation rates and hormone-response capacities. By analyzing the gene-expression pattern of main hormone pathways, we systematically compared six PCa cell lines and parental primary cells. We compared these cell lines (i) with each other and (ii) with PCa tissue samples from 11 patients. We found major differences in the gene-expression levels of androgen, insulin, estrogen, and oxysterol signaling between PCa tissue and cell lines, and between different cell lines. Our systematic characterization gives researchers a solid basis to choose the appropriate PCa cell model for the hormone pathway of interest.
Wissenschaftlicher Artikel
Scientific Article
Biomedicines 8:507 (2020)
Type 2 diabetes (T2D) is associated with worse prognosis of prostate cancer (PCa). The molecular mechanisms behind this association are still not fully understood. The aim of this study was to identify key factors, which contribute to the more aggressive PCa phenotype in patients with concurrent T2D. Therefore, we investigated benign and PCa tissue of PCa patients with and without diabetes using real time qPCR. Compared to patients without diabetes, patients with T2D showed a decreased E‐cadherin/N‐cadherin (CDH1/CDH2) ratio in prostate tissue, indicating a switch of epithelial‐mesenchymal transition (EMT), which is a pivotal process in carcinogenesis. In addition, the gene expression levels of matrix metalloproteinases (MMPs) and CC chemokine ligands (CCLs) were higher in prostate samples of T2D patients. Next, prostate adenocarcinoma PC3 cells were treated with increasing glucose concentrations to replicate hyperglycemia in vitro. In these cells, high glucose induced expressions of MMPs and CCLs, which showed significant positive associations with the proliferation marker proliferating cell nuclear antigen (PCNA). These results indicate that in prostate tissue of men with T2D, hyperglycemia may induce EMT, increase MMP and CCL gene expressions, which in turn activate invasion and inflammatory processes accelerating the progression of PCa.
Wissenschaftlicher Artikel
Scientific Article
Transl. Psychiatry 10:66 (2020)
Neurodevelopmental disorders are heterogeneous and identifying shared genetic aetiologies and converging signalling pathways affected could improve disease diagnosis and treatment. Truncating mutations of the abnormal spindle-like microcephaly associated (ASPM) gene cause autosomal recessive primary microcephaly (MCPH) in humans. ASPM is a positive regulator of Wnt/beta-Catenin signalling and controls symmetric to asymmetric cell division. This process balances neural progenitor proliferation with differentiation during embryogenesis, the malfunction of which could interfere with normal brain development. ASPM mutations may play a role also in other neurodevelopmental disorders, nevertheless, we lack the details of how or to what extent. We therefore assessed neurodevelopmental disease and circuit endophenotypes in mice with a truncating Aspm(1-7) mutation. Aspm(1-7) mice exhibited impaired short- and long-term object recognition memory and markedly enhanced place learning in the IntelliCage (R). This behaviour pattern is reminiscent of a cognitive phenotype seen in mouse models and patients with a rare form of autism spectrum disorder (ASD) as well as in mouse models of altered Wnt signalling. These alterations were accompanied by ventriculomegaly, corpus callosum dysgenesis and decreased parvalbumin (PV)+ interneuron numbers in the hippocampal Cornu Ammonis (CA) region and thalamic reticular nucleus (TRN). PV+ cell number correlated to object recognition (CA and TRN) and place learning (TRN). This opens the possibility that, as well as causing MCPH, mutant ASPM potentially contributes to other neurodevelopmental disorders such as ASD through altered parvalbuminergic interneuron development affecting cognitive behaviour. These findings provide important information for understanding the genetic overlap and improved treatment of neurodevelopmental disorders associated with ASPM.
Wissenschaftlicher Artikel
Scientific Article
Bioinformatics 36, 1492-1500 (2020)
Motivation: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors.Results: Here we introduce 'soft windowing', a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources.
Wissenschaftlicher Artikel
Scientific Article
R. Soc. Open Sci. 7:200701 (2020)
Astrocytes provide neurons with structural support and energy in form of lactate, modulate synaptic transmission, are insulin sensitive and act as gatekeeper for water, ions, glutamate and second messengers. Furthermore, astrocytes are important for glucose sensing, possess neuroendocrine functions and also play an important role in cerebral lipid metabolism. To answer the question, if there is a connection between lipid metabolism and insulin action in human astrocytes, we investigated if storage of ectopic lipids in human astrocytes has an impact on insulin signalling in those cells. Human astrocytes were cultured in the presence of a lipid emulsion, consisting of fatty acids and triglycerides, to induce ectopic lipid storage. After several days, cells were stimulated with insulin and gene expression profiling was performed. In addition, phosphorylation of Akt as well as glycogen synthesis and cell proliferation was assessed. Ectopic lipid storage was detected in human astrocytes after lipid exposure and lipid storage was persistent even when the fat emulsion was removed from the cell culture medium. Chronic exposure to lipids induced profound changes in the gene expression profile, whereby some genes showed a reversible gene expression profile upon removal of fat, and some did not. This included FOXO-dependent expression patterns. Furthermore, insulin-induced phosphorylation of Akt was diminished and also insulin-induced glycogen synthesis and proliferation was impaired in lipid-laden astrocytes. Chronic lipid exposure induces lipid storage in human astrocytes accompanied by insulin resistance. Analyses of the gene expression pattern indicated the potential of a partially reversible gene expression profile. Targeting astrocytic insulin resistance by reducing ectopic lipid load might represent a promising treatment target for insulin resistance of the brain in obesity, diabetes and neurodegeneration.
Wissenschaftlicher Artikel
Scientific Article
PLoS ONE 15:e0230162 (2020)
Dislocation in hindlimb tarsals are being observed at a low, but persistent frequency in group-housed adult male mice from C57BL/6N substrains. Clinical signs included a sudden onset of mild to severe unilateral or bilateral tarsal abduction, swelling, abnormal hindlimb morphology and lameness. Contraction of digits and gait abnormalities were noted in multiple cases. Radiographical and histological examination revealed caudal dislocation of the calcaneus and partial dislocation of the calcaneoquartal (calcaneus-tarsal bone IV) joint. The detection, frequency, and cause of this pathology in five large mouse production and phenotyping centres (MRC Harwell, UK; The Jackson Laboratory, USA; The Centre for Phenogenomics, Canada; German Mouse Clinic, Germany; Baylor College of Medicine, USA) are discussed.
Wissenschaftlicher Artikel
Scientific Article
Xenotransplantation, DOI: 10.1111/xen.12664 (2020)
Background: Many genetically multi-modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno-organs may impair their long-term function after orthotopic transplantation in non-human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR-KO) to reduce the size of donor pigs in one step. Methods: Heart weight and proteome profile of myocardium were investigated in GHR-KO and control pigs. GHR-KO mutations were introduced using CRISPR/Cas9 in an α1,3-galactosyltransferase (GGTA1)-deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple-modified (4GM) pigs. Results: At age 6 months, GHR-KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin-like growth factor 1 (IGF1) levels (24 ± 1 ng/mL) and reached body weights of 70.3 and 73.4 kg at 9 months. Control pigs with IGF1 levels of 228 ± 24 ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi-modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes. Conclusion: GHR-KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno-organ overgrowth in preclinical studies.
Wissenschaftlicher Artikel
Scientific Article
Diabetes 69, 2756-2765 (2020)
Early and precise identification of individuals with pre-diabetes and type 2 diabetes (T2D) at risk for progressing to chronic kidney disease (CKD) is essential to prevent complications of diabetes. Here, we identify and evaluate prospective metabolite biomarkers and the best set of predictors of CKD in the longitudinal, population-based Cooperative Health Research in the Region of Augsburg (KORA) cohort by targeted metabolomics and machine learning approaches. Out of 125 targeted metabolites, sphingomyelin C18:1 and phosphatidylcholine diacyl C38:0 were identified as candidate metabolite biomarkers of incident CKD specifically in hyperglycemic individuals followed during 6.5 years. Sets of predictors for incident CKD developed from 125 metabolites and 14 clinical variables showed highly stable performances in all three machine learning approaches and outperformed the currently established clinical algorithm for CKD. The two metabolites in combination with five clinical variables were identified as the best set of predictors, and their predictive performance yielded a mean area value under the receiver operating characteristic curve of 0.857. The inclusion of metabolite variables in the clinical prediction of future CKD may thus improve the risk prediction in people with prediabetes and T2D. The metabolite link with hyperglycemia-related early kidney dysfunction warrants further investigation.
Wissenschaftlicher Artikel
Scientific Article
Genes Dev. 34, 715-729 (2020)
Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.
Wissenschaftlicher Artikel
Scientific Article
Sci. Adv. 6:eaaz4551 (2020)
Recently, covalent modifications of RNA, such as methylation, have emerged as key regulators of all aspects of RNA biology and have been implicated in numerous diseases, for instance, cancer. Here, we undertook a combination of in vitro and in vivo screens to test 78 potential methyltransferases for their roles in hepatocellular carcinoma (HCC) cell proliferation. We identified methyltransferase-like protein 6 (METTL6) as a crucial regulator of tumor cell growth. We show that METTL6 is a bona fide transfer RNA (tRNA) methyltransferase, catalyzing the formation of 3-methylcytidine at C32 of specific serine tRNA isoacceptors. Deletion of Mettl6 in mouse stem cells results in changes in ribosome occupancy and RNA levels, as well as impaired pluripotency. In mice, Mettl6 knockout results in reduced energy expenditure. We reveal a previously unknown pathway in the maintenance of translation efficiency with a role in maintaining stem cell self-renewal, as well as impacting tumor cell growth profoundly.
Wissenschaftlicher Artikel
Scientific Article
In: Beyond Our Genes. 2020. 175-208
It has been demonstrated originally in plants that phenotypic traits,
such as floral symmetry, can be caused by changes of methylation
patterns of specific genes. Such traits can be transgenerationally
inherited for multiple generations and remain associated with cytosine
methylation patterns. Whether genomic methylation may also contribute to
epigenetic inheritance across generations in vertebrates and notably in
mammals is still more controversial. One reason for this tentativeness
is the dual occurrence of global genomic de-methylation first in
pre-implantation embryos and subsequently in primordial germ cells
(PGCs) of mammals. Although gene focused cases of epigenetic inheritance
associated with genomic DNA methylation have been well studied mostly
in rodents (such as imprinted genes and the Agouti viable yellow, Avy,
allele), it is still a matter of debate whether genomic DNA methylation
may provide a more general mechanism for the epigenetic inheritance of
acquired traits across generations. We review the current literature on
this topic with a focus on the potential role of DNA methylation for
epigenetic inheritance across generations in mammals.
Mamm. Genome 31, 119–133 (2020)
Nutritional constraints including not only caloric restriction or protein deficiency, but also energy-dense diets affect metabolic health and frequently lead to obesity and insulin resistance, as well as glucose intolerance and type 2 diabetes. The effects of these environmental factors are often mediated via epigenetic modifiers that target the expression of metabolic genes. More recently, it was discovered that such parentally acquired metabolic changes can alter the metabolic health of the filial and grand-filial generations. In mammals, this epigenetic inheritance can either follow an intergenerational or transgenerational mode of inheritance. In the case of intergenerational inheritance, epimutations established in gametes persist through the first round of epigenetic reprogramming occurring during preimplantation development. For transgenerational inheritance, epimutations persist additionally throughout the reprogramming that occurs during germ cell development later in embryogenesis. Differentially expressed transcripts, genomic cytosine methylations, and several chemical modifications of histones are prime candidates for tangible marks which may serve as epimutations in inter- and transgenerational inheritance and which are currently being investigated experimentally. We review, here, the current literature in support of epigenetic inheritance of metabolic traits caused by nutritional constraints and potential mechanisms in man and in rodent model systems.
Review
Review
Nat. Commun. 11:624 (2020)
Brown adipose thermogenesis increases energy expenditure and relies on uncoupling protein 1 (UCP1), however, UCP1 knock-out mice show resistance to diet-induced obesity at room temperature. Here, the authors show that this resistance relies on FGF21-signaling, inducing the browning of white adipose tissue.Uncoupling protein 1 (UCP1) executes thermogenesis in brown adipose tissue, which is a major focus of human obesity research. Although the UCP1-knockout (UCP1 KO) mouse represents the most frequently applied animal model to judge the anti-obesity effects of UCP1, the assessment is confounded by unknown anti-obesity factors causing paradoxical obesity resistance below thermoneutral temperatures. Here we identify the enigmatic factor as endogenous FGF21, which is primarily mediating obesity resistance. The generation of UCP1/FGF21 double-knockout mice (dKO) fully reverses obesity resistance. Within mild differences in energy metabolism, urine metabolomics uncover increased secretion of acyl-carnitines in UCP1 KOs, suggesting metabolic reprogramming. Strikingly, transcriptomics of metabolically important organs reveal enhanced lipid and oxidative metabolism in specifically white adipose tissue that is fully reversed in dKO mice. Collectively, this study characterizes the effects of endogenous FGF21 that acts as master regulator to protect from diet-induced obesity in the absence of UCP1.
Wissenschaftlicher Artikel
Scientific Article
Mamm. Genome 31, 30-48 (2020)
The collaborative cross (CC) is a large panel of mouse-inbred lines derived from eight founder strains (NOD/ShiLtJ, NZO/HILtJ, A/J, C57BL/6J, 129S1/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). Here, we performed a comprehensive and comparative phenotyping screening to identify phenotypic differences and similarities between the eight founder strains. In total, more than 300 parameters including allergy, behavior, cardiovascular, clinical blood chemistry, dysmorphology, bone and cartilage, energy metabolism, eye and vision, immunology, lung function, neurology, nociception, and pathology were analyzed; in most traits from sixteen females and sixteen males. We identified over 270 parameters that were significantly different between strains. This study highlights the value of the founder and CC strains for phenotype-genotype associations of many genetic traits that are highly relevant to human diseases. All data described here are publicly available from the mouse phenome database for analyses and downloads.
Wissenschaftlicher Artikel
Scientific Article
Acta Neuropathol. 140, 121–142 (2020)
Expansion of a (G(4)C(2))(n)repeat inC9orf72causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the link of the five repeat-encoded dipeptide repeat (DPR) proteins to neuroinflammation, TDP-43 pathology, and neurodegeneration is unclear. Poly-PR is most toxic in vitro, but poly-GA is far more abundant in patients. To directly compare these in vivo, we created congenic poly-GA and poly-PR mice. 40% of poly-PR mice were affected with ataxia and seizures, requiring euthanasia by 6 weeks of age. The remaining poly-PR mice were asymptomatic at 14 months of age, likely due to an 80% reduction of the transgene mRNA in this subgroup. In contrast, all poly-GA mice showed selective neuron loss, inflammation, as well as muscle denervation and wasting requiring euthanasia before 7 weeks of age. In-depth analysis of peripheral organs and blood samples suggests that peripheral organ failure does not drive these phenotypes. Although transgene mRNA levels were similar between poly-GA and affected poly-PR mice, poly-GA aggregated far more abundantly than poly-PR in the CNS and was also found in skeletal muscle. In addition, TDP-43 and other disease-linked RNA-binding proteins co-aggregated in rare nuclear inclusions in the hippocampus and frontal cortex only in poly-GA mice. Transcriptome analysis revealed activation of an interferon-responsive pro-inflammatory microglial signature in end-stage poly-GA but not poly-PR mice. This signature was also found in all ALS patients and enriched inC9orf72cases. In summary, our rigorous comparison of poly-GA and poly-PR toxicity in vivo indicates that poly-GA, but not poly-PR at the same mRNA expression level, promotes interferon responses inC9orf72disease and contributes to TDP-43 abnormalities and neuron loss selectively in disease-relevant regions.
Wissenschaftlicher Artikel
Scientific Article
Biochim. Biophys. Acta-Mol. Basis Dis. 1866:165622 (2020)
Isolated methylmalonic aciduria (MMAuria) is primarily caused by deficiency of methylmalonyl-CoA mutase (MMUT or MUT). Biochemically, MUT deficiency results in the accumulation of methylmalonic acid (MMA), propionyl-camitine (C3) and other metabolites. Patients often exhibit lethargy, failure to thrive and metabolic decompensation leading to coma or even death, with kidney and neurological impairment frequently identified in the long-term. Here, we report a hemizygous mouse model which combines a knock-in (ki) missense allele of Mut with a knock-out (ko) allele (Mut-ko/ki mice) that was fed a 51%-protein diet from day 12 of life, constituting a bespoke model of MMAuria. Under this diet, mutant mice developed a pronounced metabolic phenotype characterized by drastically increased blood levels of MMA and C3 compared to their littermate controls (Mut-ki/wt). With this bespoke mouse model, we performed a standardized phenotypic screen to assess the whole-body impairments associated with this strong metabolic condition. We found that Mut-ko/ki mice show common clinical manifestations of MMAuria, including pronounced failure to thrive, indications of mild neurological and kidney dysfunction, and degenerative morphological changes in the liver, along with less well described symptoms such as cardiovascular and hematological abnormalities. The analyses also reveal so far unknown disease characteristics, including low bone mineral density, anxiety-related behaviour and ovarian atrophy. This first phenotypic screening of a MMAuria mouse model confirms its relevance to human disease, reveals new alterations associated with MUT deficiency, and suggests a series of quantifiable readouts that can be used to evaluate potential treatment strategies.
Wissenschaftlicher Artikel
Scientific Article
Metabolites 10:472 (2020)
A large part of metabolomics research relies on experiments involving mouse models, which are usually 6 to 20 weeks of age. However, in this age range mice undergo dramatic developmental changes. Even small age differences may lead to different metabolomes, which in turn could increase inter-sample variability and impair the reproducibility and comparability of metabolomics results. In order to learn more about the variability of the murine plasma metabolome, we analyzed male and female C57BL/6J, C57BL/6NTac, 129S1/SvImJ, and C3HeB/FeJ mice at 6, 10, 14, and 20 weeks of age, using targeted metabolomics (BIOCRATES AbsoluteIDQ™ p150 Kit). Our analysis revealed high variability of the murine plasma metabolome during adolescence and early adulthood. A general age range with minimal variability, and thus a stable metabolome, could not be identified. Age-related metabolomic changes as well as the metabolite profiles at specific ages differed markedly between mouse strains. This observation illustrates the fact that the developmental timing in mice is strain specific. We therefore stress the importance of deliberate strain choice, as well as consistency and precise documentation of animal age, in metabolomics studies.
Wissenschaftlicher Artikel
Scientific Article
Exp. Eye Res. 190:107871 (2020)
The eye lens displays a variety of phenotypes in the wake of genetic modifications or environmental influences. Therefore, a high-resolution in vivo imaging method for the lens is desirable. Optical coherence tomography (OCT) has become a powerful imaging tool in ophthalmology, especially for retinal imaging in small animal models such as mice. Here, we demonstrate an optimized approach specifically for anterior eye segment imaging with spectral domain OCT (SD-OCT) on several known murine lens cataract mutants. Scheimpflug and histological section images on the same eye were used in parallel to assess the observed pathologies. With SD-OCT images, we obtained detailed information about the different alterations from the anterior to the posterior pole of the lens. This capability makes OCT a valuable high-resolution imaging modality for the anterior eye segment in mouse.
Wissenschaftlicher Artikel
Scientific Article
J. Clin. Endocrinol. Metab. 105, 14-25:dgz020 (2020)
Context: Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a heterotrimeric enzyme and central regulator of cellular energy metabolism. The impact of single nucleotide polymorphisms (SNPs) in all 7 AMPK subunit genes on adiposity, glucose metabolism, and lipid metabolism has not yet been systematically studied.Objective: To analyze the associations of common SNPs in all AMPK genes, and of different scores thereof, with adiposity, insulin sensitivity, insulin secretion, blood glucose, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, total cholesterol, and triglycerides.Study Design and Methods: A cohort of 2789 nondiabetic participants from the Tubingen Family study of type 2 diabetes, metabolically characterized by oral glucose tolerance test and genotyped by genome-wide SNP array, was analyzed.Results: We identified 6 largely nonoverlapping SNP sets across 4 AMPK genes (PRKAA1, PRKAA2, PRKAG2, PRKAG3) associated with adiposity, insulin sensitivity, insulin secretion, blood glucose, total/LDL cholesterol, or HDL cholesterol, respectively. A genetic score of body-fat-increasing alleles revealed per-allele effect sizes on body mass index (BMI) of +0.22 kg/m(2) (P = 2.3 x 10(-7)), insulin sensitivity of -0.12 x 10(19) L-2/mol(2) (P = 9.9 x 10(-6)) and 2-hour blood glucose of +0.02 mmol/L (P = 0.0048). Similar effects on blood glucose were observed with scores of insulin-sensitivity-reducing, insulin-secretion-reducing and glucose-raising alleles, respectively. A genetic cholesterol score increased total and LDL cholesterol by 1.17 mg/dL per allele (P = 0.0002 and P = 3.2 x 10(-5), respectively), and a genetic HDL score decreased HDL cholesterol by 0.32 mg/dL per allele (P = 9.1 x 10(-6)).Conclusions: We describe largely nonoverlapping genetic determinants in AMPK genes for diabetes-/atherosclerosis-related traits, which reflect the metabolic pathways controlled by the enzyme. Formation of trait-specific genetic scores revealed additivity of allele effects, with body-fat-raising alleles reaching a marked effect size.
Wissenschaftlicher Artikel
Scientific Article
Mol. Metab. 36:100978 (2020)
Objective: The liver is a central target organ of growth hormone (GH), which stimulates the synthesis of insulin-like growth factor 1 (IGF1) and affects multiple biochemical pathways. A systematic multi-omics analysis of GH effects in the liver has not been performed. GH receptor (GHR) deficiency is a unique model for studying the consequences of lacking GH action. In this study, we used molecular profiling techniques to capture a broad spectrum of these effects in the liver of a clinically relevant large animal model for Laron syndrome.Methods: We performed holistic proteome and targeted metabolome analyses of liver samples from 6-month-old GHR-deficient (GHR-KO) pigs and GHR-expressing controls (four males, four females per group).Results: GHR deficiency resulted in an increased abundance of enzymes involved in amino acid degradation, in the urea cycle, and in the tricarboxylic acid cycle. A decreased ratio of long-chain acylcarnitines to free carnitine suggested reduced activity of carnitine palmitoyltransferase 1A and thus reduced mitochondrial import of fatty acids for beta-oxidation. Increased levels of short-chain acylcarnitines in the liver and in the circulation of GHR-KO pigs may result from impaired beta-oxidation of short-chain fatty acids or from increased degradation of specific amino acids. The concentration of mono-unsaturated glycerophosphocholines was significantly increased in the liver of GHR-KO pigs without morphological signs of steatosis, although the abundances of several proteins functionally linked to non-alcoholic fatty liver disease (fetuin B, retinol binding protein 4, several mitochondrial proteins) were increased. Moreover, GHR-deficient liver samples revealed distinct changes in the methionine and glutathione metabolic pathways, in particular, a significantly increased level of glycine N-methyltransferase and increased levels of total and free glutathione. Several proteins revealed a sex-related abundance difference in the control group but not in the GHR-KO group.Conclusions: Our integrated proteomics/targeted metabolomics study of GHR-deficient and control liver samples from a clinically relevant large animal model identified a spectrum of biological pathways that are significantly altered in the absence of GH action. Moreover, new insights into the role of GH in the sex-related specification of liver functions were provided.
Wissenschaftlicher Artikel
Scientific Article
Mamm. Genome 31, 49-53 (2020)
Design and production of genetically engineered mouse strains by individual research laboratories, research teams, large-scale consortia, and the biopharmaceutical industry have magnified the need for qualified personnel to identify, annotate, and validate (phenotype) these potentially new mouse models of human disease. The PATHBIO project has been recently established and funded by the European Union's ERASMUS+ Knowledge Alliance program to address the current shortfall in formally trained personnel. A series of teaching workshops will be given by experts on anatomy, histology, embryology, imaging, and comparative pathology to increase the availability of individuals with formal training to contribute to this important niche of Europe's biomedical research enterprise. These didactic and hands-on workshops are organized into three modules: (1) embryology, anatomy, histology, and the anatomical basis of imaging, (2) image-based phenotyping, and (3) pathology. The workshops are open to all levels of participants from recent graduates to Ph.D., M.D., and veterinary scientists. Participation is available on a competitive basis at no cost for attending. The first series of Workshop Modules was held in 2019 and these will continue for the next 2 years.
Sonstiges: Meinungsartikel
Other: Opinion
Diabetes 69, 915-926 (2020)
Genes of the Notch signaling pathway are expressed in different cell types and organs at different time points during embryonic development and adulthood. The Notch ligand Delta-like 1 (DLL1) controls the decision between endocrine and exocrine fates of multipotent progenitors in the developing pancreas, and loss of Dll1 leads to premature endocrine differentiation. However, the role of Delta-Notch signaling in adult tissue homeostasis is not well understood. Here, we describe the spatial expression pattern of Notch pathway components in adult murine pancreatic islets and show that DLL1 and DLL4 are specifically expressed in beta-cells, whereas JAGGED1 is expressed in alpha-cells. We show that mice lacking both DLL1 and DLL4 in adult beta-cells display improved glucose tolerance, increased glucose-stimulated insulin secretion, and hyperglucagonemia. In contrast, overexpression of the intracellular domain of DLL1 in adult murine pancreatic beta-cells results in impaired glucose tolerance and reduced insulin secretion, both in vitro and in vivo. These results suggest that Notch ligands play specific roles in the adult pancreas and highlight a novel function of the Delta/Notch pathway in beta-cell insulin secretion.
Wissenschaftlicher Artikel
Scientific Article
J. Clin. Invest. 130, 6093-6108 (2020)
Recent genome-wide association studies (GWAS) identified DUSP8, encoding a dual-specificity phosphatase targeting mitogen-activated protein kinases, as a type 2 diabetes (T2D) risk gene. Here, we reveal that Dusp8 is a gatekeeper in the hypothalamic control of glucose homeostasis in mice and humans. Male, but not female, Dusp8 loss-of-function mice, either with global or corticotropin-releasing hormone neuron-specific deletion, had impaired systemic glucose tolerance and insulin sensitivity when exposed to high-fat diet (HFD). Mechanistically, we found impaired hypothalamic-pituitary-adrenal axis feedback, blunted sympathetic responsiveness, and chronically elevated corticosterone levels driven by hypothalamic hyperactivation of Jnk signaling. Accordingly, global/Jnk1 ablation, AAV-mediated Dusp8 overexpression in the mediobasal hypothalamus, or metyrapone-induced chemical adrenalectomy rescued the impaired glucose homeostasis of obese male Dusp8-KO mice, respectively. The sex-specific role of murine Dusp8 in governing hypothalamic Jnk signaling, insulin sensitivity, and systemic glucose tolerance was consistent with functional MRI data in human volunteers that revealed an association of the DUSP8 rs2334499 risk variant with hypothalamic insulin resistance in men. Further, expression of DUSP8 was increased in the infundibular nucleus of T2D humans. In summary, our findings suggest the GWAS-identified gene Dusp8 as a novel hypothalamic factor that plays a functional role in the etiology of T2D.
Wissenschaftlicher Artikel
Scientific Article
Cancer Res. 80, 5231-5244 (2020)
Post-translational modifications are essential for regulating the
transcription factor p53 which binds DNA in a highly cooperative manner
to control expression of a plethora of tumor suppressive programs. Here
we show at the biochemical, cellular, and organismal level that the
cooperative nature of DNA binding is reduced by phosphorylation of
highly conserved serine residues (human S183/S185, mouse S180) in the
DNA binding domain. To explore the role of this inhibitory
phosphorylation in vivo, new phosphorylation-deficient p53-S180A
knock-in mice were generated. ChIP-seq and RNA-seq studies of S180A
knock-in cells demonstrated enhanced DNA binding and increased target
gene expression. In vivo, this translated into a tissue-specific
vulnerability of the bone marrow that caused depletion of hematopoietic
stem cells and impaired proper regeneration of hematopoiesis after DNA
damage. Median lifespan was significantly reduced by 20% from 709 days
in wild-type to only 568 days in S180A littermates. Importantly,
lifespan was reduced by a loss of general fitness and increased
susceptibility to age-related diseases, not by increased cancer
incidence as often seen in other p53 mutant mouse models. For example,
S180A knock-in mice showed markedly reduced spontaneous tumorigenesis
and increased resistance to Myc-driven lymphoma and Eml4-Alk-driven lung
cancer. Preventing phosphorylation of S183/S185 in human cells boosted
p53 activity and allowed tumor cells to be killed more efficiently.
Together out data identify p53 DNA binding domain phosphorylation as a
druggable mechanism that balances tumorigenesis and aging.
Wissenschaftlicher Artikel
Scientific Article
Invest. Ophthalmol. Vis. Sci. 61:44 (2020)
PURPOSE. The clinical phenotype of retinal gliosis occurs in different forms; here, we characterize one novel genetic feature, (i.e., signaling via BMP-receptor 1b).METHODS. Mouse mutants were generated within a recessive ENU mutagenesis screen; the underlying mutation was identified by linkage analysis and Sanger sequencing. The eye phenotype was characterized by fundoscopy, optical coherence tomography, optokinetic drum, electroretinography, and visual evoked potentials, by histology, immunohistology, and electron-microscopy.RESULTS. The mutation affects intron 10 of the Bmpr1b gene, which is causative for skipping of exon 10. The expression levels of pSMAD1/5/8 were reduced in the mutant retina. The loss of BMPR1B-mediated signaling leads to optic nerve coloboma, gliosis in the optic nerve head and ventral retina, defective optic nerve axons, and irregular retinal vessels. The ventral retinal gliosis is proliferative and hypertrophic, which is concomitant with neuronal delamination and the reduction of retinal ganglion cells (RGCs); it is dominated by activated astrocytes overexpressing PAX2 and SOX2 but not PAX6, indicating that they may retain properties of gliogenic precursor cells. The expression pattern of PAX2 in the optic nerve head and ventral retina is altered during embryonic development. These events finally result in reduced electrical transmission of the retina and optic nerve and significantly reduced visual acuity.CONCLUSIONS. Our study demonstrates that BMPR1B is necessary for the development of the optic nerve and ventral retina. This study could also indicate a new mechanism in the formation of retinal gliosis; it opens new routes for its treatment eventually preventing scar formation in the retina.
Wissenschaftlicher Artikel
Scientific Article
Cell 181, 1189-1193 (2020)
Researchers around the globe have been mounting, accelerating, and redeploying efforts across disciplines and organizations to tackle the SARS-CoV-2 outbreak. However, humankind continues to be afflicted by numerous other devastating diseases in increasing numbers. Here, we outline considerations and opportunities toward striking a good balance between maintaining and redefining research priorities.
Review
Review
PLoS Genet. 16:e1008577 (2020)
Circadian systems provide a fitness advantage to organisms by allowing them to adapt to daily changes of environmental cues, such as light/dark cycles. The molecular mechanism underlying the circadian clock has been well characterized. However, how internal circadian clocks are entrained with regular daily light/dark cycles remains unclear. By collecting and analyzing indirect calorimetry (IC) data from more than 2000 wild-type mice available from the International Mouse Phenotyping Consortium (IMPC), we show that the onset time and peak phase of activity and food intake rhythms are reliable parameters for screening defects of circadian misalignment. We developed a machine learning algorithm to quantify these two parameters in our misalignment screen (SyncScreener) with existing datasets and used it to screen 750 mutant mouse lines from five IMPC phenotyping centres. Mutants of five genes (Slc7a11, Rhbdl1, Spop, Ctc1 and Oxtr) were found to be associated with altered patterns of activity or food intake. By further studying the Slc7a11tm1a/tm1a mice, we confirmed its advanced activity phase phenotype in response to a simulated jetlag and skeleton photoperiod stimuli. Disruption of Slc7a11 affected the intercellular communication in the suprachiasmatic nucleus, suggesting a defect in synchronization of clock neurons. Our study has established a systematic phenotype analysis approach that can be used to uncover the mechanism of circadian entrainment in mice.
Wissenschaftlicher Artikel
Scientific Article
2019
Proc. Natl. Acad. Sci. U.S.A. 116, 11872-11877 (2019)
Autoinflammatory syndromes are characterized by dysregulation of the innate immune response with subsequent episodes of acute spontaneous inflammation. Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder that presents with bone pain and localized swelling. Ali18 mice, isolated from a mutagenesis screen, exhibit a spontaneous inflammatory paw phenotype that includes sterile osteomyelitis and systemic reduced bone mineral density. To elucidate the molecular basis of the disease, positional cloning of the causative gene for Ali18 was attempted. Using a candidate gene approach, a missense mutation in the C-terminal region of Fgr, a member of Src family tyrosine kinases (SFKs), was identified. For functional confirmation, additional mutations at the N terminus of Fgr were introduced in Ali18 mice by CRISPR/Cas9-mediated genome editing. N-terminal deleterious mutations of Fgr abolished the inflammatory phenotype in Ali18 mice, but in-frame and missense mutations in the same region continue to exhibit the phenotype. The fact that Fgr null mutant mice are morphologically normal suggests that the inflammation in this model depends on Fgr products. Furthermore, the levels of C-terminal negative regulatory phosphorylation of Fgr(Ali18) are distinctly reduced compared with that of wild-type Fgr. In addition, whole-exome sequencing of 99 CRMO patients including 88 trios (proband and parents) identified 13 patients with heterozygous coding sequence variants in FGR, including two missense mutant proteins that affect kinase activity. Our results strongly indicate that gain-of-function mutations in Fgr are involved in sterile osteomyelitis, and thus targeting SFKs using specific inhibitors may allow for efficient treatment of the disease.
Wissenschaftlicher Artikel
Scientific Article
Mol. Metab. 26, 30-44 (2019)
Objective: The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically.Methods: We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics.Results: Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples.Conclusions: The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets. (C) 2019 The Authors. Published by Elsevier GmbH.
Wissenschaftlicher Artikel
Scientific Article
Sci. Rep. 9:19483 (2019)
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.
Wissenschaftlicher Artikel
Scientific Article
J. Clin. Invest. 129, 1772-1778 (2019)
lschemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic option. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier. Its mechanism, however, is unknown. Here, we identified type 5 NADPH oxidase (NOX5), a calciumactivated, ROS-forming enzyme, as the missing link. Using a humanized knockin (KI) mouse model and in vitro organotypic cultures, we found that reoxygenation or calcium overload increased brain ROS levels in a NOX5-dependent manner. In vivo, postischemic ROS formation, infarct volume, and functional outcomes were worsened in NOXS-KI mice. Of clinical and therapeutic relevance, in a human blood-barrier model, pharmacological NOX inhibition also prevented acute reoxygenationinduced leakage. Our data support further evaluation of poststroke recanalization in the presence of NOX inhibition for limiting stroke-induced damage.
Wissenschaftlicher Artikel
Scientific Article
Fluids barriers CNS 16:30 (2019)
BACKGROUND: The blood-brain barrier (BBB) ensures central nervous system (CNS) homeostasis by strictly controlling the passage of molecules and solutes from the bloodstream into the CNS. Complex and continuous tight junctions (TJs) between brain endothelial cells block uncontrolled paracellular diffusion of molecules across the BBB, with claudin-5 being its dominant TJs protein. However, claudin-5 deficient mice still display ultrastructurally normal TJs, suggesting the contribution of other claudins or tight-junction associated proteins in establishing BBB junctional complexes. Expression of claudin-12 at the BBB has been reported, however the exact function and subcellular localization of this atypical claudin remains unknown. METHODS: We created claudin-12-lacZ-knock-in C57BL/6J mice to explore expression of claudin-12 and its role in establishing BBB TJs function during health and neuroinflammation. We furthermore performed a broad standardized phenotypic check-up of the mouse mutant. RESULTS: Making use of the lacZ reporter allele, we found claudin-12 to be broadly expressed in numerous organs. In the CNS, expression of claudin-12 was detected in many cell types with very low expression in brain endothelium. Claudin-12lacZ/lacZ C57BL/6J mice lacking claudin-12 expression displayed an intact BBB and did not show any signs of BBB dysfunction or aggravated neuroinflammation in an animal model for multiple sclerosis. Determining the precise localization of claudin-12 at the BBB was prohibited by the fact that available anti-claudin-12 antibodies showed comparable detection and staining patterns in tissues from wild-type and claudin-12lacZ/lacZ C57BL/6J mice. CONCLUSIONS: Our present study thus shows that claudin-12 is not essential in establishing or maintaining BBB TJs integrity. Claudin-12 is rather expressed in cells that typically lack TJs suggesting that claudin-12 plays a role other than forming classical TJs. At the same time, in depth phenotypic screening of clinically relevant organ functions of claudin-12lacZ/lacZ C57BL/6J mice suggested the involvement of claudin-12 in some neurological but, more prominently, in cardiovascular functions.
Wissenschaftlicher Artikel
Scientific Article
Diabetes akt. 17, 96 (2019)
Editorial
Editorial
Mol. Neurobiol. 56, 4215-4230 (2019)
As part of the -superfamily, B2-crystallin (CRYBB2) is an ocular structural protein in the lens, and mutation of the corresponding gene can cause cataracts. CRYBB2 also is expressed in non-lens tissue such as the adult mouse brain and is associated with neuropsychiatric disorders such as schizophrenia. Nevertheless, the robustness of this association as well as how CRYBB2 may contribute to disease-relevant phenotypes is unknown. To add further clarity to this issue, we performed a comprehensive analysis of behavioral and neurohistological alterations in mice with an allelic series of mutations in the C-terminal end of the Crybb2 gene. Behavioral phenotyping of these three B2-mutant lines Crybb2(O377), Crybb2(Philly), and Crybb2(Aey2) included assessment of exploratory activity and anxiety-related behavior in the open field, sensorimotor gating measured by prepulse inhibition (PPI) of the acoustic startle reflex, cognitive performance measured by social discrimination, and spontaneous alternation in the Y-maze. In each mutant line, we also quantified the number of parvalbumin-positive (PV+) GABAergic interneurons in selected brain regions that express CRYBB2. While there were allele-specific differences in individual behaviors and affected brain areas, all three mutant lines exhibited consistent alterations in PPI that paralleled alterations in the PV+ cell number in the thalamic reticular nucleus (TRN). The direction of the PPI change mirrored that of the TRN PV+ cell number thereby suggesting a role for TRN PV+ cell number in modulating PPI. Moreover, as both altered PPI and PV+ cell number are schizophrenia-associated endophenotypes, our result implicates mutated Crybb2 in the development of this neuropsychiatric disorder.
Wissenschaftlicher Artikel
Scientific Article
Allergy 74, 197-198 (2019)
Meeting abstract
Meeting abstract
J. Clin. Endocrinol. Metab. 104, 1090-1098 (2019)
Context: Primary dysregulation of adipose tissue lipolysis caused by genetic variation and independent of insulin resistance could explain unhealthy body fat distribution and its metabolic consequences.Objective: To analyze common single nucleotide polymorphisms (SNPs) in 48 lipolysis-, but not insulin-signaling-related genes, to form polygenic risk scores of lipolysis-associated SNPs, and to investigate their effects on body fat distribution, glycemia, insulin sensitivity, insulin secretion, and proinsulin conversion.Study Design, Participants, and Methods: SNP array, anthropometric, and metabolic data were available from up to 2789 participants without diabetes of the Tubingen Family study of type 2 diabetes characterized by oral glucose tolerance tests. In a subgroup (n = 942), magnetic resonance measurements of body fat stores were available.Results: We identified insulin-sensitivity-independent nominal associations (P < 0.05) of SNPs in 10 genes with plasma free fatty acids (FFAs), in 7 genes with plasma glycerol and in 6 genes with both, plasma FFAs and glycerol. A score formed of the latter SNPs (in ADCY4, CIDEA, GNAS, PDE8B, PRKAA1, PRKAG2) was associated with plasma FFA and glycerol measurements (1.4*10(-9) <= P <= 1.2*10(-5)), visceral adipose tissue mass (P = 0.0326), and proinsulin conversion (P <= 0.0272). The more lipolysis-increasing alleles a subject had, the lower was the visceral fat mass and the lower the proinsulin conversion.Conclusions: We found evidence for a genetic basis of adipose tissue lipolysis resulting from common SNPs in CIDEA, AMP-activated protein kinase subunits, and cAMP signaling components. A genetic score of lipolysis-increasing alleles determined lower visceral fat mass and lower proinsulin conversion.
Wissenschaftlicher Artikel
Scientific Article
Preadipocytes of obese humans display gender-specific bioenergetic responses to glucose and insulin.
Mol. Metab. 20, 28-37 (2019)
Background/Objectives: Although the prevalence of obesity and its associated metabolic disorders is increasing in both sexes, the clinical phenotype differs between men and women, highlighting the need for individual treatment options. Mitochondrial dysfunction in various tissues, including white adipose tissue (WAT), has been accepted as a key factor for obesity-associated comorbidities such as diabetes. Given higher expression of mitochondria-related genes in the WAT of women, we hypothesized that gender differences in the bioenergetic profile of white (pre-) adipocytes from obese (age-and BMI-matched) donors must exist.Subjects/Methods: Using Seahorse technology, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of (pre-)adipocytes from male (n = 10) and female (n = 10) deeply-phenotyped obese donors under hypo-, normo- and hyperglycemic (0, 5 and 25 mM glucose) and insulin-stimulated conditions. Additionally, expression levels (mRNA/protein) of mitochondria-related genes (e.g. UQCRC2) and glycolytic enzymes (e.g. PKM2) were determined.Results: Dissecting cellular OCR and ECAR into different functional modules revealed that preadipocytes from female donors show significantly higher mitochondrial to glycolytic activity (higher OCR/ECAR ratio, p = 0.036), which is supported by a higher ratio of UQCRC2 to PKM2 mRNA levels (p = 0.021). However, no major gender differences are detectable in in vitro differentiated adipocytes (e.g. OCR/ECAR, p = 0.248). Importantly, glucose and insulin suppress mitochondrial activity (i.e. ATP-linked respiration) significantly only in preadipocytes of female donors, reflecting their trends towards higher insulin sensitivity.Conclusions: Collectively, we show that preadipocytes, but not in vitro differentiated adipocytes, represent a model system to reveal gender differences with clinical importance for metabolic disease status. In particular preadipocytes of females maintain enhanced mitochondrial flexibility, as demonstrated by pronounced responses of ATP-linked respiration to glucose. (C) 2018 The Authors. Published by Elsevier GmbH.
Wissenschaftlicher Artikel
Scientific Article
Comm. Biol. 2:97 (2019)
This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply. In the original published version of the article, Valerie Vancollie was mistakenly omitted from the list of members of the International Mouse Phenotyping Consortium. In addition, recognition of funding from Wellcome Trust grant WT098051 was mistakenly omitted from the Acknowledgements.The errors have been corrected in both the PDF and HTML versions of the paper.
Sci. Rep. 9:11211 (2019)
Oculocutaneous syndromes are often due to mutations in single genes. In some cases, mouse models for these diseases exist in spontaneously occurring mutations, or in mice resulting from forward mutatagenesis screens. Here we present novel genes that may be causative for oculocutaneous disease in humans, discovered as part of a genome-wide screen of knockout-mice in a targeted single-gene deletion project. The International Mouse Phenotyping Consortium (IMPC) database (data release 10.0) was interrogated for all mouse strains with integument abnormalities, which were then cross-referenced individually to identify knockouts with concomitant ocular abnormalities attributed to the same targeted gene deletion. The search yielded 307 knockout strains from unique genes with integument abnormalities, 226 of which have not been previously associated with oculocutaneous conditions. Of the 307 knockout strains with integument abnormalities, 52 were determined to have ocular changes attributed to the targeted deletion, 35 of which represent novel oculocutaneous genes. Some examples of various integument abnormalities are shown, as well as two examples of knockout strains with oculocutaneous phenotypes. Each of the novel genes provided here are potentially relevant to the pathophysiology of human integumentary, or oculocutaneous conditions, such as albinism, phakomatoses, or other multi-system syndromes. The novel genes reported here may implicate molecular pathways relevant to these human diseases and may contribute to the discovery of novel therapeutic targets.
Wissenschaftlicher Artikel
Scientific Article
Conservation genetics 20, 135-136 (2019)
The original publication of this article unfortunately contained the following mistakes.
Front. Physiol. 10:262 (2019)
Endurance is not only a key factor in many sports but endurance-related variables are also associated with good health and low mortality. Twin and family studies suggest that several endurance-associated traits are ≈50% inherited. However, we still poorly understand what DNA sequence variants contribute to endurance heritability. To address this issue, we conducted a systematic review to identify genes whose experimental loss or gain-of-function increases endurance capacity in mice. We found 31 genes including two isoforms of Ppargc1a whose manipulation increases running or swimming endurance performance by up to 1800%. Genes whose gain-of-function increases endurance are Adcy5, Adcy8, Hk2, Il15, Mef2c, Nr4a3, Pck1 (Pepck), Ppard, Ppargc1a (both the a and b isoforms of the protein Pgc-1α), Ppargc1b, Ppp3ca (calcineurin), Scd1, Slc5a7, Tfe3, Tfeb, Trib3 & Trpv1. Genes whose loss-of-function increases endurance in mice are Actn3, Adrb2, Bdkrb2, Cd47, Crym, Hif1a, Myoz1, Pappa, Pknox1, Pten, Sirt4, Thbs1, Thra, and Tnfsf12. Of these genes, human DNA sequence variants of ACTN3, ADCY5, ADRB2, BDKRB2, HIF1A, PPARD, PPARGC1A, PPARGC1B, and PPP3CA are also associated with endurance capacity and/or VO2max trainability suggesting evolutionary conservation between mice and humans. Bioinformatical analyses show that there are numerous amino acid or copy number-changing DNA variants of endurance genes in humans, suggesting that genetic variation of endurance genes contributes to the variation of human endurance capacity, too. Moreover, several of these genes/proteins change their expression or phosphorylation in skeletal muscle or the heart after endurance exercise, suggesting a role in the adaptation to endurance exercise.
Wissenschaftlicher Artikel
Scientific Article
Dis. Model. Mech. 12:dmm039156 (2019)
Alongside the obesity epidemic, the prevalence of maternal diabetes is rising worldwide, and adverse effects on fetal development and metabolic disturbances in the offspring's later life have been described. To clarify whether metabolic programming effects are due to mild maternal hyperglycemia without confounding obesity, we investigated wild-type offspring of INSC93S transgenic pigs, which are a novel genetically modified large-animal model expressing mutant insulin (INS) C93S in pancreatic beta-cells. This mutation results in impaired glucose tolerance, mild fasting hyperglycemia and insulin resistance during late pregnancy. Compared with offspring from wildtype sows, piglets from hyperglycemic mothers showed impaired glucose tolerance and insulin resistance (homeostatic model assessment of insulin resistance: +3-fold in males; +4.4-fold in females) prior to colostrum uptake. Targeted metabolomics in the fasting and insulin-stimulated state revealed distinct alterations in the plasma metabolic profile of piglets from hyperglycemic mothers. They showed increased levels of acylcarnitines, gluconeogenic precursors such as alanine, phospholipids (in particular lysophosphatidylcholines) and a-aminoadipic acid, a potential biomarker for type 2 diabetes. These observations indicate that mild gestational hyperglycemia can cause impaired glucose tolerance, insulin resistance and associated metabolic alterations in neonatal offspring of a large-animal model born at a developmental maturation status comparable to human babies.
Wissenschaftlicher Artikel
Scientific Article
Allergy 74, 1549-1560 (2019)
Background: Allergen-specific immunotherapy (AIT) is the only causal treatment for allergy. However, success rates vary depending on the type of allergy and disease background of the patient. Hence, strategies targeting an increased therapeutic efficacy are urgently needed. Here, the effects of blockade of IL-4 and IL-13 signaling on different phases of AIT were addressed.Methods: The impact of the recombinantly produced IL-4 and IL-13 antagonist IL-4 mutein (IL-4M) on allergic sensitization and AIT outcome in experimental allergic asthma were analyzed in a murine model. The effects of IL-4M administration were assessed prior/during sensitization, immediately after AIT under allergen challenge, and two weeks post-treatment.Results: Intervention with IL-4M prior/during sensitization led to strong induction of IgG1, IgG2a, IgG2b, and IgG3, decrease of specific and total IgE, as well as of IL-5 in serum. Similar effects on the serum immunoglobulin levels were observed immediately after IL4M-supplemented AIT during allergen challenge. Additionally, IL4M markedly suppressed type-2 cytokine secretion of splenocytes beyond the effect of AIT alone. These effects were equaled to those of AIT alone two weeks post-treatment. Intriguingly, here, IL-4M induced a sustained decrease of Th2-biased Tregs (ST2(+) FOXP3(+)GATA3(intermediate)).Conclusions: IL-4 and IL-13 blockade during experimental AIT demonstrates beneficial effects on immunological key parameters such as immunoglobulin and cytokine secretion immediately after AIT. Although two weeks later these effects were dropped to those of AIT alone, the number of potentially disease-triggering Th2-biased Tregs was further significantly decreased by IL-4M treatment. Hence, IL-4/IL13-targeting therapies prime the immune memory in therapy success-favoring manner.
Wissenschaftlicher Artikel
Scientific Article
Transl. Psychiatry 9:7 (2019)
In humans, genetic variants of DLGAP1-4 have been linked with neuropsychiatric conditions, including autism spectrum disorder (ASD). While these findings implicate the encoded postsynaptic proteins, SAPAP1-4, in the etiology of neuropsychiatric conditions, underlying neurobiological mechanisms are unknown. To assess the contribution of SAPAP4 to these disorders, we characterized SAPAP4-deficient mice. Our study reveals that the loss of SAPAP4 triggers profound behavioural abnormalities, including cognitive deficits combined with impaired vocal communication and social interaction, phenotypes reminiscent of ASD in humans. These behavioural alterations of SAPAP4-deficient mice are associated with dramatic changes in synapse morphology, function and plasticity, indicating that SAPAP4 is critical for the development of functional neuronal networks and that mutations in the corresponding human gene, DLGAP4, may cause deficits in social and cognitive functioning relevant to ASD-like neurodevelopmental disorders.
Wissenschaftlicher Artikel
Scientific Article
J. Inherit. Metab. Dis. 42, 839-849 (2019)
Triosephosphate isomerase (TPI) deficiency is a fatal genetic disorder characterized by hemolytic anemia and neurological dysfunction. Although the enzyme defect in TPI was discovered in the 1960s, the exact etiology of the disease is still debated. Some aspects indicate the disease could be caused by insufficient enzyme activity, whereas other observations indicate it could be a protein misfolding disease with tissue-specific differences in TPI activity. We generated a mouse model in which exchange of a conserved catalytic amino acid residue (isoleucine to valine, Ile170Val) reduces TPI specific activity without affecting the stability of the protein dimer. TPIIle170Val/Ile170Val mice exhibit an approximately 85% reduction in TPI activity consistently across all examined tissues, which is a stronger average, but more consistent, activity decline than observed in patients or symptomatic mouse models that carry structural defect mutant alleles. While monitoring protein expression levels revealed no evidence for protein instability, metabolite quantification indicated that glycolysis is affected by the active site mutation. TPIIle170Val/Ile170Val mice develop normally and show none of the disease symptoms associated with TPI deficiency. Therefore, without the stability defect that affects TPI activity in a tissue-specific manner, a strong decline in TPI catalytic activity is not sufficient to explain the pathological onset of TPI deficiency.
Wissenschaftlicher Artikel
Scientific Article
Nat. Metab. 1, 1009-1026 (2019)
Non-alcoholic fatty liver disease (NAFLD) represents a key feature of obesity-related type 2 diabetes with increasing prevalence worldwide. To our knowledge, no treatment options are available to date, paving the way for more severe liver damage, including cirrhosis and hepatocellular carcinoma. Here, we show an unexpected function for an intracellular trafficking regulator, the small Rab GTPase Rab24, in mitochondrial fission and activation, which has an immediate impact on hepatic and systemic energy homeostasis. RAB24 is highly upregulated in the livers of obese patients with NAFLD and positively correlates with increased body fat in humans. Liver-selective inhibition of Rab24 increases autophagic flux and mitochondrial connectivity, leading to a strong improvement in hepatic steatosis and a reduction in serum glucose and cholesterol levels in obese mice. Our study highlights a potential therapeutic application of trafficking regulators, such as RAB24, for NAFLD and establishes a conceptual functional connection between intracellular transport and systemic metabolic dysfunction.
Wissenschaftlicher Artikel
Scientific Article
Haematologica 105, 2484-2495 (2019)
T issue factor (TF) is highly expressed in sub-endothelial tissue. The extracellular allosteric disulfide bond Cys186-Cys209 of human TF shows high evolutionary conservation and in vitro evidence suggests that it significantly contributes to TF procoagulant activity. To investigate the role of this allosteric disulfide bond in vivo, we generated a C213G mutant Tf mouse by replacing Cys213 of the corresponding disulfide Cys190-Cys213 in murine Tf A bleeding phenotype was prominent in homozygous C213G Tf mice. Pre-natal lethality of one third of homozygous offspring was observed between embryonic (E) day E9.5 and E14.5 associated with placental hemorrhages. After birth, homozygous mice suffered from bleedings in different organs and reduced survival. Homozygous C213G Tf male mice showed higher incidence of lung bleedings and lower survival rates than females. In both sexes, C213G mutation evoked a reduced protein expression (about 10-fold) and severely reduced pro-coagulant activity (at least 100-fold). Protein glycosylation was impaired and cell membrane exposure decreased in macrophages in vivo. Single housing of homozygous C213G Tf males reduced the occurrence of severe bleeding and significantly improved survival, suggesting that inter-male aggressiveness might significantly account for the sex differences. These experiments show that the TF allosteric disulfide bond is of crucial importance for normal in vivo expression, post-translational processing and activity of murine TF. Although C213G Tf mice do not display the severe embryonic lethality of Tf knock-out mice, their postnatal bleeding phenotype emphasizes the importance of fully functional TF for hemostasis.
Wissenschaftlicher Artikel
Scientific Article
Eur. J. Cell Biol. 98:151046 (2019)
CRN2 is an actin filament binding protein involved in the regulation of various cellular processes including cell migration and invasion. CRN2 has been implicated in the malignant progression of different types of human cancer. We used CRN2 knock-out mice for analyses as well as for crossbreeding with a Tp53/Pten knock-out glioblastoma mouse model. CRN2 knock-out mice were subjected to a phenotyping screen at the German Mouse Clinic. Murine glioblastoma tissue specimens as well as cultured murine brain slices and glioblastoma cell lines were investigated by immunohistochemistry, immunofluorescence, and cell biological experiments. Protein interactions were studied by immunoprecipitation, pull-down, and enzyme activity assays. CRN2 knock-out mice displayed neurological and behavioural alterations, e.g. reduced hearing sensitivity, reduced acoustic startle response, hypoactivity, and less frequent urination. While glioblastoma mice with or without the additional CRN2 knock-out allele exhibited no significant difference in their survival rates, the increased levels of CRN2 in transplanted glioblastoma cells caused a higher tumour cell encasement of murine brain slice capillaries. We identified two important factors of the tumour microenvironment, the tissue inhibitor of matrix metalloproteinase 4 (TIMP4) and the matrix metalloproteinase 14 (MMP14, synonym: MT1-MMP), as novel binding partners of CRN2. All three proteins mutually interacted and co-localised at the front of lamellipodia, and CRN2 was newly detected in exosomes. On the functional level, we demonstrate that CRN2 increased the secretion of TIMP4 as well as the catalytic activity of MMP14. Our results imply that CRN2 represents a pro-invasive effector within the tumour cell microenvironment of glioblastoma multiforme.
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Scientific Article
Exp. Eye Res. 188:107632 (2019)
During an ENU (N-ethyl-N-nitrosourea) mutagenesis screen, we observed a dominant small-eye mutant mouse with viable homozygotes. A corresponding mutant line was established and referred to as Aey69 (abnormality of the eye #69). Comprehensive phenotyping of the homozygous Aey69 mutants in the German Mouse Clinic revealed only a subset of statistically significant alterations between wild types and homozygous mutants. The mutation causes microphthalmia without a lens but with retinal hyperproliferation. Linkage was demonstrated to mouse chromosome 3 between the markers D3Mit188 and D3Mit11. Sequencing revealed a 358 A- > C mutation (I1e120Leu) in the Hist2h3c1 gene and a 71 T- > C (Val24Ala) mutation in the Gja8 gene. Detailed analysis of eye development in the homozygous mutant mice documented a perturbed lens development starting -from the lens vesicle stage including decreasing expression of crystallins as well as of lens-specific transcription - factors like PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by several markers including BRN3 (retinal ganglion cells) and OTX2 (cone photoreceptors). The changes in the retina at the early embryonic stages of E11.5-E15.5 happen in parallel with apoptotic processes in the lens at the respective stages. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. The hyperproliferation, however, does not disrupt the differentiation and appearance of the principal retinal cell types at postnatal stages, even if the overgrowing retina covers finally the entire bulbus of the eye. Morpholino-mediated knock-down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations, ranging from cyclopia to severe microphthalmia. The wild-type Hist2h3c1 mRNA can rescue the morpholino-induced defects corroborating its specific function in lens development. Based upon these data, it is concluded that the ocular function of the Hist2h3c1 gene (encoding a canonical H3.2 variant) is conserved throughout evolution. Moreover, the data highlight also the importance of Hist2h3c1 in the coordinated formation of lens and retina during eye development.
Wissenschaftlicher Artikel
Scientific Article
Eur. J. Hum. Genet. 27, 818-819 (2019)
Meeting abstract
Meeting abstract
2018
PLoS Biol. 16:e2005019 (2018)
Animal welfare requires the adequate housing of animals to ensure health and well-being. The application of environmental enrichment is a way to improve the well-being of laboratory animals. However, it is important to know whether these enrichment items can be incorporated in experimental mouse husbandry without creating a divide between past and future experimental results. Previous small-scale studies have been inconsistent throughout the literature, and it is not yet completely understood whether and how enrichment might endanger comparability of results of scientific experiments. Here, we measured the effect on means and variability of 164 physiological parameters in 3 conditions: with nesting material with or without a shelter, comparing these 2 conditions to a “barren” regime without any enrichments. We studied a total of 360 mice from each of 2 mouse strains (C57BL/6NTac and DBA/2NCrl) and both sexes for each of the 3 conditions. Our study indicates that enrichment affects the mean values of some of the 164 parameters with no consistent effects on variability. However, the influence of enrichment appears negligible compared to the effects of other influencing factors. Therefore, nesting material and shelters may be used to improve animal welfare without impairment of experimental outcome or loss of comparability to previous data collected under barren housing conditions.
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Scientific Article
Front. Endocrin. 9:82 (2018)
Introduction: Impaired glucose tolerance (IGT) is diagnosed by a standardized oral glucose tolerance test (OGTT). However, the OGTT is laborious, and when not performed, glucose tolerance cannot be determined from fasting samples retrospectively. We tested if glucose tolerance status is reasonably predictable from a combination of demographic, anthropometric, and laboratory data assessed at one time point in a fasting state. Methods: Given a set of 22 variables selected upon clinical feasibility such as sex, age, height, weight, waist circumference, blood pressure, fasting glucose, HbA1c, hemoglobin, mean corpuscular volume, serum potassium, fasting levels of insulin, C-peptide, triglyceride, non-esterified fatty acids (NEFA), proinsulin, prolactin, cholesterol, low-density lipoprotein, HDL, uric acid, liver transaminases, and ferritin, we used supervised machine learning to estimate glucose tolerance status in 2,337 participants of the TUEF study who were recruited before 2012. We tested the performance of 10 different machine learning classifiers on data from 929 participants in the test set who were recruited after 2012. In addition, reproducibility of IGT was analyzed in 78 participants who had 2 repeated OGTTs within 1 year. Results: The most accurate prediction of IGT was reached with the recursive partitioning method (accuracy = 0.78). For all classifiers, mean accuracy was 0.73 +/- 0.04. The most important model variable was fasting glucose in all models. Using mean variable importance across all models, fasting glucose was followed by NEFA, triglycerides, HbA1c, and C-peptide. The accuracy of predicting IGT from a previous OGTT was 0.77. Conclusion: Machine learning methods yield moderate accuracy in predicting glucose tolerance from a wide set of clinical and laboratory variables. A substitution of OGTT does not currently seem to be feasible. An important constraint could be the limited reproducibility of glucose tolerance status during a subsequent OGTT.
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Scientific Article
Int. J. Radiat. Oncol. Biol. Phys. 101, 965-984 (2018)
Purpose Experimental neuroimaging provides a wide range of methods for the visualization of brain anatomic morphology down to subcellular detail. Still, each technique-specific detection mechanism presents compromises among the achievable field-of-view size, spatial resolution, and nervous tissue sensitivity, leading to partial sample coverage, unresolved morphologic structures, or sparse labeling of neuronal populations and often also to obligatory sample dissection or other sample invasive manipulations. X-ray phase-contrast imaging computed tomography (PCI-CT) is an experimental imaging method that simultaneously provides micrometric spatial resolution, high soft-tissue sensitivity, and ex vivo full organ rodent brain coverage without any need for sample dissection, staining or labeling, or contrast agent injection. In the present study, we explored the benefits and limitations of PCI-CT use for in vitro imaging of normal and cancerous brain neuromorphology after in vivo treatment with synchrotron-generated x-ray microbeam radiation therapy (MRT), a spatially fractionated experimental high-dose radiosurgery. The goals were visualization of the MRT effects on nervous tissue and a qualitative comparison of the results to the histologic and high-field magnetic resonance imaging findings. Methods and Materials MRT was administered in vivo to the brain of both healthy and cancer-bearing rats. At 45 days after treatment, the brain was dissected out and imaged ex vivo using propagation-based PCI-CT. Results PCI-CT visualizes the brain anatomy and microvasculature in 3 dimensions and distinguishes cancerous tissue morphology, necrosis, and intratumor accumulation of iron and calcium deposits. Moreover, PCI-CT detects the effects of MRT throughout the treatment target areas (eg, the formation of micrometer-thick radiation-induced tissue ablation). The observed neurostructures were confirmed by histologic and immunohistochemistry examination and related to the micro-magnetic resonance imaging data. Conclusions PCI-CT enabled a unique 3D neuroimaging approach for ex vivo studies on small animal models in that it concurrently delivers high-resolution insight of local brain tissue morphology in both normal and cancerous micro-milieu, localizes radiosurgical damage, and highlights the deep microvasculature. This method could assist experimental small animal neurology studies in the postmortem evaluation of neuropathology or treatment effects.
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Scientific Article
Front. Immunol. 9:587 (2018)
Aicardi-Goutières syndrome (AGS) is a rare early onset childhood encephalopathy caused by persistent neuroinflammation of autoimmune origin. AGS is a genetic disorder and >50% of affected individuals bear hypomorphic mutations in ribonuclease H2 (RNase H2). All available RNase H2 mouse models so far fail to mimic the prominent CNS involvement seen in AGS. To establish a mouse model recapitulating the human disease, we deleted RNase H2 specifically in the brain, the most severely affected organ in AGS. Although RNase H2δGFAPmice lacked the nuclease in astrocytes and a majority of neurons, no disease signs were apparent in these animals. We additionally confirmed these results in a second, neuron-specific RNase H2 knockout mouse line. However, when astrocytes were isolated from brains of RNase H2δGFAPmice and cultured under mitogenic conditions, they showed signs of DNA damage and premature senescence. Enhanced expression of interferon-stimulated genes (ISGs) represents the most reliable AGS biomarker. Importantly, primary RNase H2δGFAPastrocytes displayed significantly increased ISG transcript levels, which we failed to detect in in vivo in brains of RNase H2δGFAPmice. Isolated astrocytes primed by DNA damage, including RNase H2-deficiency, exhibited a heightened innate immune response when exposed to bacterial or viral antigens. Taken together, we established a valid cellular AGS model that utilizes the very cell type responsible for disease pathology, the astrocyte, and phenocopies major molecular defects observed in AGS patient cells.
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Scientific Article
EMBO Mol. Med. 10:e8613 (2018)
Most antidiabetic drugs treat disease symptoms rather than adipose tissue dysfunction as a key pathogenic cause in the metabolic syndrome and type 2 diabetes. Pharmacological targeting of adipose tissue through the nuclear receptor PPARg, as exemplified by glitazone treatments, mediates efficacious insulin sensitization. However, a better understanding of the context-specific PPARg responses is required for the development of novel approaches with reduced side effects. Here, we identified the transcriptional cofactor Cited4 as a target and mediator of rosiglitazone in human and murine adipocyte progenitor cells, where it promoted specific sets of the rosiglitazone-dependent transcriptional program. In mice, Cited4 was required for the proper induction of thermogenic expression by Rosi specifically in subcutaneous fat. This phenotype had high penetrance in females only and was not evident in beta-adrenergically stimulated browning. Intriguingly, this specific defect was associated with reduced capacity for systemic thermogenesis and compromised insulin sensitization upon therapeutic rosiglitazone treatment in female but not male mice. Our findings on Cited4 function reveal novel unexpected aspects of the pharmacological targeting of PPARg.
Wissenschaftlicher Artikel
Scientific Article
Sci. Rep. 8:4199 (2018)
Epigenetic deregulation, such as the reduction of histone acetylation levels, is thought to be causally linked to various maladies associated with aging. Consequently, histone deacetylase inhibitors are suggested to serve as epigenetic therapy by increasing histone acetylation. However, previous work suggests that many non-histone proteins, including metabolic enzymes, are also acetylated and that post transitional modifications may impact their activity. Furthermore, deacetylase inhibitors were recently shown to impact the acetylation of a variety of proteins. By utilizing a novel technique to measure oxygen consumption rate from whole living tissue, we demonstrate that treatment of whole living fly heads by the HDAC/KDAC inhibitors sodium butyrate and Trichostatin A, induces a rapid and transient increase of oxygen consumption rate. In addition, our study indicates that the rate increase is markedly attenuated in midlife fly head tissue. Overall, our data suggest that HDAC/KDAC inhibitors may induce enhanced mitochondrial activity in a rapid manner. This observed metabolic boost provides further, but novel evidence, that treating various maladies with deacetylase inhibitors may be beneficial.
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Scientific Article
Ageing Res. Rev. 47, 214-277 (2018)
Objective: Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel.Methods: Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several "hallmark of aging" pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six "hallmark of aging" pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers.Results: A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified.Conclusion: Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) alpha-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin alpha, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGF beta (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (REIN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.
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Scientific Article
Biochem. Biophys. Res. Commun. 503, 2770-2777 (2018)
Heterozygous missense mutations in the human VCP gene cause inclusion body myopathy associated with Paget disease of bone and fronto-temporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). The exact molecular mechanisms by which VCP mutations cause disease manifestation in different tissues are incompletely understood. In the present study, we report the comprehensive analysis of a newly generated R155C VCP knock-in mouse model, which expresses the ortholog of the second most frequently occurring human pathogenic VCP mutation. Heterozygous R155C VCP knock-in mice showed decreased plasma lactate, serum albumin and total protein concentrations, platelet numbers, and liver to body weight ratios, and increased oxygen consumption and CD8+/Ly6C + T-cell fractions, but none of the typical human IBMPFD or ALS pathologies. Breeding of heterozygous mice did not yield in the generation of homozygous R155C VCP knock-in animals. Immunoblotting showed identical total VCP protein levels in human IBMPFD and murine R155C VCP knock-in tissues as compared to wild-type controls. However, while in human IBMPFD skeletal muscle tissue 70% of the total VCP mRNA was derived from the mutant allele, in R155C VCP knock-in mice only 5% and 7% mutant mRNA were detected in skeletal muscle and brain tissue, respectively. The lack of any obvious IBMPFD or ALS pathology could thus be a consequence of the very low expression of mutant VCP. We conclude that the increased and decreased fractions of the R155C mutant VCP mRNA in man and mice, respectively, are due to missense mutation-induced, divergent alterations in the biological half-life of the human and murine mutant mRNAs. Furthermore, our work suggests that therapy approaches lowering the expression of the mutant VCP mRNA below a critical threshold may ameliorate the intrinsic disease pathology.
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Scientific Article
J. Bone Min. Res. 33, 875-887 (2018)
The X-linked WTX/AMER1 protein constitutes an important component of the β-catenin destruction complex that can both enhance and suppress canonical β-catenin signaling. Somatic mutations in WTX/AMER1 have been found in a proportion of the pediatric kidney cancer Wilms' tumor. By contrast, germline mutations cause the severe sclerosing bone dysplasia osteopathia striata congenita with cranial sclerosis (OSCS), a condition usually associated with fetal or perinatal lethality in male patients. Here we address the developmental and molecular function of WTX by generating two novel mouse alleles. We show that in addition to the previously reported skeletal abnormalities, loss of Wtx causes severe midline fusion defects including cleft palate and ectopic synostosis at the base of the skull. By contrast, deletion of the C-terminal part of the protein results in only mild developmental abnormalities permitting survival beyond birth. Adult analysis, however, revealed skeletal defects including changed skull morphology and an increased whole-body bone density, resembling a subgroup of male patients carrying a milder, survivable phenotype. Molecular analysis in vitro showed that while β-catenin fails to co-immunoprecipitate with the truncated protein, partial recruitment appears to be achieved in an indirect manner using AXIN/AXIN2 as a molecular bridge. Taken together our analysis provides a novel model for WTX-caused bone diseases and explains on the molecular level how truncation mutations in this gene may retain some of WTX-protein functions.
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Scientific Article
J. Vis. Exp. 143, DOI: 10.3791/58601 (2018)
Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell's metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.
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Scientific Article
Int. J. Obes. 42, 507-517 (2018)
Background/Objectives:Dieting is a popular yet often ineffective way to lower body weight, as the majority of people regain most of their pre-dieting weights in a relatively short time. The underlying molecular mechanisms driving weight regain and the increased risk for metabolic disease are still incompletely understood. Here we investigate the molecular alterations inherited from a history of obesity.Methods:In our model, male high-fat diet (HFD)-fed obese C57BL/6J mice were switched to a low caloric chow diet, resulting in a decline of body weight to that of lean mice. We measured body composition, as well as metrics of glucose, insulin and lipid homeostasis. This was accompanied by histological and gene expression analysis of adipose tissue and liver to assess adipose tissue inflammation and hepatosteatosis. Moreover, acute hypothalamic response to (re-) exposure to HFD was assessed by qPCR.Results & Conclusions:Within 7 weeks after diet switch, most obesity-associated phenotypes, such as body mass, glucose intolerance and blood metabolite levels were reversed. However, hepatic inflammation, hepatic steatosis as well as hypertrophy and inflammation of perigonadal, but not subcutaneous, adipocytes persisted in formerly obese mice. Transcriptional profiling of liver and perigonadal fat revealed an upregulation of pathways associated with immune function and cellularity. Thus, we show that weight reduction leaves signs of inflammation in liver and perigonadal fat, indicating that persisting proinflammatory signals in liver and adipose tissue could contribute to an increased risk of formerly obese subjects to develop the metabolic syndrome upon recurring weight gain.
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Scientific Article
Sci. Data 5:180002 (2018)
Sci. Rep. 8:1116 (2018)
The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application.
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Scientific Article
Mol. Metab. 18, 42-50 (2018)
Objective: Although debated, metabolic health characterizes 10-25% of obese individuals and reduces risk of developing life-threatening comorbidities. Adipose tissue is a recognized endocrine organ important for the maintenance of whole-body metabolic health. Adipocyte transcriptional signatures of healthy and unhealthy obesity are largely unknown.Methods: Here, we used a small cohort of highly characterized obese individuals discordant for metabolic health, characterized their adipocytes transcriptional signatures, and cross-referenced them to mouse phenotypic and human GWAs databases.Results and conclusions: Our study showed that glucose intolerance and insulin resistance co-operate to remodel adipocyte transcriptome. We also identified the Nuclear Export Mediator Factor (NEMF) and the Ectoderm-Neural Cortex 1 (ENC1) as novel potential targets in the management of metabolic health in human obesity. (C) 2018 The Authors. Published by Elsevier GmbH.
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Scientific Article
Anal. Chim. Acta 1032, 18-31 (2018)
Urinary analyte data has to be corrected for the sample specific dilution as the dilution varies intra-and interpersonally dramatically, leading to non-comparable concentration measures. Most methods of dilution correction utilized nowadays like probabilistic quotient normalization or total spectra normalization result in a division of the raw data by a dilution correction factor. Here, however, we show that the implicit assumption behind the application of division, log-linearity between the urinary flow rate and the raw urinary concentration, does not hold for analytes which are not in steady state in blood. We explicate the physiological reason for this short-coming in mathematical terms and demonstrate the empirical consequences via simulations and on multiple time-point metabolomic data, showing the insufficiency of division-based normalization procedures to account for the complex non-linear analyte specific dependencies on the urinary flow rate. By reformulating normalization as a regression problem, we propose an analyte specific way to remove the dilution variance via a flexible non-linear regression methodology which then was shown to be more effective in comparison to division-based normalization procedures. In the progress, we developed several, easily applicable methods of normalization diagnostics to decide on the method of dilution correction in a given sample. On the way, we identified furthermore the time-span since last urination as an important variance factor in urinary metabolome data which is until now completely neglected. In conclusion, we present strong theoretical and empirical evidence that normalization has to be analyte specific in dynamically influenced data. Accordingly, we developed a normalization methodology for removing the dilution variance in urinary data respecting the single analyte kinetics.
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Scientific Article
Mol. Metab. 11, 113-128 (2018)
Objective: Laron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a large animal model for LS, pigs with GHR knockout (KO) mutations were generated and characterized.Methods: CRISPR/Cas9 technology was applied to mutate exon 3 of the GHR gene in porcine zygotes. Two heterozygous founder sows with a 1bp or 7-bp insertion in GHR exon 3 were obtained, and their heterozygous F1 offspring were intercrossed to produce GHR-KO, heterozygous GHR mutant, and wild-type pigs. Since the latter two groups were not significantly different in any parameter investigated, they were pooled as the GHR expressing control group. The characterization program included body and organ growth, body composition, endocrine and clinical-chemical parameters, as well as signaling studies in liver tissue.Results: GHR-KO pigs lacked GHR and had markedly reduced serum insulin-like growth factor 1 (IGF1) levels and reduced IGF-binding protein 3 (IGFBP3) activity but increased IGFBP2 levels. Serum GH concentrations were significantly elevated compared with control pigs. GHR-KO pigs had a normal birth weight. Growth retardation became significant at the age of five weeks. At the age of six months, the body weight of GHR-KO pigs was reduced by 60% compared with controls. Most organ weights of GHR-KO pigs were reduced proportionally to body weight. However, the weights of liver, kidneys, and heart were disproportionately reduced, while the relative brain weight was almost doubled. GHR-KO pigs had a markedly increased percentage of total body fat relative to body weight and displayed transient juvenile hypoglycemia along with decreased serum triglyceride and cholesterol levels. Analysis of insulin receptor related signaling in the liver of adult fasted pigs revealed increased phosphorylation of IRS1 and PI3K. In agreement with the loss of GHR, phosphorylation of STAT5 was significantly reduced. In contrast, phosphorylation of JAK2 was significantly increased, possibly due to the increased serum leptin levels and increased hepatic leptin receptor expression and activation in GHR-KO pigs. In addition, increased mTOR phosphorylation was observed in GHR-KO liver samples, and phosphorylation studies of downstream substrates suggested the activation of mainly mTOR complex 2.Conclusion: GHR-KO pigs resemble the pathophysiology of LS and are an interesting model for mechanistic studies and treatment trials.
Wissenschaftlicher Artikel
Scientific Article
Nutrients 10:547 (2018)
Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOy lated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4-and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.
Wissenschaftlicher Artikel
Scientific Article
Sci. Rep. 8:737 (2018)
Measuring mitochondrial respiration in cultured cells is a valuable tool to investigate the influence of physiological and disease-related factors on cellular metabolism; however, the details of the experimental workflow greatly influence the informative value of the results. Working with primary cells and cell types capable of differentiation can be particularly challenging. We present a streamlined workflow optimised for investigation of primary human skeletal muscle cells. We applied the workflow to differentiated and undifferentiated cells and we investigated the effect of TGF beta 1 treatment. Differentiation of myoblasts to myotubes increased mitochondrial respiration and abundance of mitochondrial enzymes and mitochondrial marker proteins. Differentiation also induced qualitative changes in mitochondrial protein composition and respiration. TGF beta 1 reduced complex IV protein MTCO1 abundance in both myoblasts and myotubes. In myoblasts, spare electron transport system (ETS) capacity was reduced due to a reduction in maximal oxygen consumption. In TGF beta 1-treated myotubes, the reduction in spare ETS capacity is mainly a consequence of increased oxidative phosphorylation capacity and complex III protein UQCRC2. Taken together, our data shows that it is important to monitor muscle cell differentiation when mitochondrial function is studied. Our workflow is not only sensitive enough to detect physiological-sized differences, but also adequate to form mechanistic hypotheses.
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Scientific Article
EMBO J.:e94813 (2018)
Epitranscriptomic events such as adenosine-to-inosine (A-to-I) RNA editing by ADAR can recode mRNAs to translate novel proteins. Editing of the mRNA that encodes actin crosslinking protein Filamin A (FLNA) mediates a Q-to-R transition in the interactive C-terminal region. While FLNA editing is conserved among vertebrates, its physiological function remains unclear. Here, we show that cardiovascular tissues in humans and mice show massive editing and that FLNA RNA is the most prominent substrate. Patient-derived RNA-Seq data demonstrate a significant drop in FLNA editing associated with cardiovascular diseases. Using mice with only impaired FLNA editing, we observed increased vascular contraction and diastolic hypertension accompanied by increased myosin light chain phosphorylation, arterial remodeling, and left ventricular wall thickening, which eventually causes cardiac remodeling and reduced systolic output. These results demonstrate a causal relationship between RNA editing and the development of cardiovascular disease indicating that a single epitranscriptomic RNA modification can maintain cardiovascular health.
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Scientific Article
Sci. Comp. World August/September, 24-25 (2018)
Diabetologe 14, 486-492 (2018)
Seit 1980 vervierfachte sich die Zahl der Menschen mit Diabetes weltweit. Allein in Deutschland leiden knapp 7 Mio. Menschen an dieser Stoffwechselerkrankung, und jedes Jahr erkranken bis zu 500.000 neu daran. Diese Zahlen machen deutlich, wie dringend neue wirksame Präventionsmaßnahmen und innovative Behandlungsformen benötigt werden. Die Digitalisierung ermöglicht es, die Volkskrankheit Diabetes in einer neuen Dimension zu erforschen, um sehr früh Subtypen dieser Stoffwechselerkrankung zu erkennen und geeignete personalisierte Präventionsmaßnahmen anzubieten. Mit dem Aufbau eines digitalen Diabetespräventionszentrums könnten Gesundheits- und Forschungsdaten aus unterschiedlichsten Quellen zusammengeführt und mit innovativen Informationstechnologien (IT: Informationstechnik) analysiert und ausgewertet werden, um unterschiedliche Diabetessubtypen identifizieren und spezifische Präventions- und Therapiemaßnahmen anbieten zu können, die durch die enge Zusammenarbeit mit der Bevölkerung direkt einsetzbar wären.
Review
Review
Biochim. Biophys. Acta-Mol. Basis Dis. 1865, 2083-2093 (2018)
Mutations in the X chromosomal tRNA 2'-O-methyltransferase FTSJ1 cause intellectual disability (ID). Although the gene is ubiquitously expressed affected individuals present no consistent clinical features beyond ID. In order to study the pathological mechanism involved in the aetiology of FTSJ1 deficiency-related cognitive impairment, we generated and characterized an Ftsj1 deficient mouse line based on the gene trapped stem cell line RRD143. Apart from an impaired learning capacity these mice presented with several statistically significantly altered features related to behaviour, pain sensing, bone and energy metabolism, the immune and the hormone system as well as gene expression. These findings show that Ftsj1 deficiency in mammals is not phenotypically restricted to the brain but affects various organ systems. Re-examination of ID patients with FTSJ1 mutations from two previously reported families showed that several features observed in the mouse model were recapitulated in some of the patients. Though the clinical spectrum related to Ftsj1 deficiency in mouse and man is variable, we suggest that an increased pain threshold may be more common in patients with FTSJ1 deficiency. Our findings demonstrate novel roles for Ftsj1 in maintaining proper cellular and tissue functions in a mammalian organism.
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Scientific Article
EMBO Mol. Med. 10:e8349 (2018)
The development of chronic obstructive pulmonary disease (COPD) pathogenesis remains unclear, but emerging evidence supports a crucial role for inducible bronchus-associated lymphoid tissue (iBALT) in disease progression. Mechanisms underlying iBALT generation, particularly during chronic CS exposure, remain to be defined. Oxysterol metabolism of cholesterol is crucial to immune cell localization in secondary lymphoid tissue. Here, we demonstrate that oxysterols also critically regulate iBALT generation and the immune pathogenesis of COPD. In both COPD patients and cigarette smoke (CS)-exposed mice, we identified significantly upregulated CH25H and CYP7B1 expression in airway epithelial cells, regulating CS-induced B-cell migration and iBALT formation. Mice deficient in CH25H or the oxysterol receptor EBI2 exhibited decreased iBALT and subsequent CS-induced emphysema. Further, inhibition of the oxysterol pathway using clotrimazole resolved iBALT formation and attenuated CS-induced emphysema in vivo therapeutically. Collectively, our studies are the first to mechanistically interrogate oxysterol-dependent iBALT formation in the pathogenesis of COPD, and identify a novel therapeutic target for the treatment of COPD and potentially other diseases driven by the generation of tertiary lymphoid organs.
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Scientific Article
J. Breath Res. 12:017102 (2018)
Volatile organic compounds (VOCs) from breath can successfully be used to diagnose disease-specific pathological alterations in metabolism. However, the exact origin and underlying biochemical pathways that could be mapped to VOC signatures are mainly unknown. There is a knowledge gap regarding the contribution of tissues, organs, the gut microbiome, and exogenous factors to the "sum signal" from breath samples. Animal models for human disease such as mutant mice provide the possibility to reproduce genetic predisposition to disease, thereby allowing the in-depth analysis of metabolic and biochemical functions. We hypothesized that breath VOCs can be traced back to origins and organ-specific metabolic functions by combining breath concentrations with systemic levels detected in different organs and biological media (breath, blood, faeces and urine). For this we fed C57Bl/6N mice a grain-based chow or a purified low-fat diet, thereby modifying the emission of methanol in breath whereas acetone levels were unaffected. We then measured headspace concentrations of both VOCs in ex-vivo samples of several biological media. Especially cecum content was identified as a likely source of systemic methanol, whereas liver showed highest acetone concentrations. Our findings are a first step to the systemic mapping of VOC patterns to metabolic functions in mice because differences between VOCs could be traced to different sources in the body. As a future aim, different levels of so-called omics technologies (genomics, proteomics, metabolomics, and breathomics) could be mapped to metabolic pathways in multiple tissues deepening our understanding of VOC metabolism and possibly leading to early non-invasive biomarkers for human pathologies.
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Scientific Article
Nat. Rev. Endocrinol. 14, 140-162 (2018)
More than one-third of the worldwide population is overweight or obese and therefore at risk of developing type 2 diabetes mellitus. In order to mitigate this pandemic, safer and more potent therapeutics are urgently required. This necessitates the continued use of animal models to discover, validate and optimize novel therapeutics for their safe use in humans. In order to improve the transition from bench to bedside, researchers must not only carefully select the appropriate model but also draw the right conclusions. In this Review, we consolidate the key information on the currently available animal models of obesity and diabetes and highlight the advantages, limitations and important caveats of each of these models.
Review
Review
J. Neurochem. 145, 258-270 (2018)
Delayed cell death in the penumbra region of acute ischemic stroke occurs through apoptotic mechanisms, making it amenable to therapeutic interventions. Fas/CD95 mediates apoptotic cell death in response to external stimuli. In mature neurons, Fas/CD95 signaling is modulated by Fas-apoptotic inhibitory molecule 2 (Faim2), which reduces cell death in animal models of stroke, meningitis, and Parkinson disease. Erythropoietin (EPO) has been studied as a therapeutic strategy in ischemic stroke. Erythropoietin stimulates the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway, which regulates Faim2 expression. Therefore, up-regulation of Faim2 may contribute to neuroprotection by EPO. Male Faim2-deficient mice (Faim2 -/- ) and wild-type littermates (WT) were subjected to 30 min of middle cerebral artery occlusion (MCAo) followed by 72 h of reperfusion. EPO was applied before (30 min) and after (24 and 48 h) MCAo. In WT mice application of EPO at a low dose (5000 U/kg) significantly reduced stroke volume, whereas treatment with high dose (90 000 U/kg) did not. In Faim2 -/- animals administration of low-dose EPO did not result in a significant reduction in stroke volume. Faim2 expression as measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) increased after low-dose EPO but not with high dose. An extensive phenotyping including analysis of cerebral vessel architecture did not reveal confounding differences between the genotypes. In human post-mortem brain Faim2 displayed a differential expression in areas of penumbral ischemia. Faim2 up-regulation may contribute to the neuroprotective effects of low-dose erythropoietin in transient brain ischemia. The dose-dependency may explain mixed effects of erythropoietin observed in clinical stroke trials.
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Scientific Article
Adv. Func. Mat. 28:1706793 (2018)
Nanomaterials are of enormous value for biomedical applications because of their customizable features. However, the material properties of nanomaterials can be altered substantially by interactions with tissue thus making it important to assess them in the specific biological context to understand and tailor their effects. Here, a genetically controlled system is optimized for cellular uptake of superparamagnetic ferritin and subsequent trafficking to lysosomes. High local concentrations of photoabsorbing magnetoferritin give robust contrast in optoacoustic imaging and allow for selective photoablation of cells overexpressing ferritin receptors. Genetically controlled uptake of the biomagnetic nanoparticles also strongly enhances third-harmonic generation due to the change of refractive index caused by the magnetite-protein interface of ferritins entrapped in lysosomes. Selective uptake of magnetoferritin furthermore enables sensitive detection of receptor-expressing cells by magnetic resonance imaging, as well as efficient magnetic cell sorting and manipulation. Surprisingly, a substantial increase in the blocking temperature of lysosomally entrapped magnetoferritin is observed, which allows for specific ablation of genetically defined cell populations by local magnetic hyperthermia. The subcellular confinement of superparamagnetic ferritins thus enhances their physical properties to empower genetically controlled interrogation of cellular processes with deep tissue penetration.
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Scientific Article
Comm. Biol. 1:236 (2018)
Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.
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Scientific Article
Conservation genetics 19, 995-1005 (2018)
The International Mouse Phenotyping Consortium (IMPC) is building a catalogue of mammalian gene function by producing and phenotyping a knockout mouse line for every protein-coding gene. To date, the IMPC has generated and characterised 5186 mutant lines. One-third of the lines have been found to be non-viable and over 300 new mouse models of human disease have been identified thus far. While current bioinformatics efforts are focused on translating results to better understand human disease processes, IMPC data also aids understanding genetic function and processes in other species. Here we show, using gorilla genomic data, how genes essential to development in mice can be used to help assess the potentially deleterious impact of gene variants in other species. This type of analyses could be used to select optimal breeders in endangered species to maintain or increase fitness and avoid variants associated to impaired-health phenotypes or loss-of-function mutations in genes of critical importance. We also show, using selected examples from various mammal species, how IMPC data can aid in the identification of candidate genes for studying a condition of interest, deliver information about the mechanisms involved, or support predictions for the function of genes that may play a role in adaptation. With genotyping costs decreasing and the continued improvements of bioinformatics tools, the analyses we demonstrate can be routinely applied.
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Scientific Article
Exp. Clin. Endocrinol. Diabet. 126, 20-29 (2018)
Aims and Methods Glucose homeostasis and energy balance are under control by peripheral and brain processes. Especially insulin signaling in the brain seems to impact whole body glucose homeostasis and interacts with fatty acid signaling. In humans circulating saturated fatty acids are negatively associated with brain insulin action while animal studies suggest both positive and negative interactions of fatty acids and insulin brain action. This apparent discrepancy might reflect a difference between acute and chronicfatty acid signaling. To address this question we investigated the acute effect of an intracere-broventricular palmitic acid administration on peripheral glucose homeostasis. We developed and implemented a method for simultaneous monitoring of brain activity and peripheral insulin action in freely moving mice by combining radiotelemetry electrocorticography (ECoG) and euglycemic-hyperin-sulinemic clamps. This method allowed gaining insight in the early kinetics of brain fatty acid signaling and its contemporaneous effect on liverfunction in vivo, which, to our knowledge, has not been assessed so far in mice.Results Insulin-induced brain activity in the theta and beta band was decreased by acute intracerebroventricular application of palmitic acid. Peripherally it amplified insulin action as demonstrated by a significant inhibition of endogenous glucose production and increased glucose infusion rate. Moreover, our results further revealed that the brain effect of peripheral insulin is modulated by palm itic acid load in the brain.Conclusion These findings suggest that insulin action is amplified in the periphery and attenuated in the brain by acute palmitic acid application. Thus, our results indicate that acute palmitic acid signaling in the brain may be different from chronic effects.
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Scientific Article
Sci. Rep. 8:11335 (2018)
Diabetes mellitus is a group of disorders characterized by prolonged high levels of circulating blood glucose. Type 1 diabetes is caused by decreased insulin production in the pancreas whereas type 2 diabetes may develop due to obesity and lack of exercise; it begins with insulin resistance whereby cells fail to respond properly to insulin and it may also progress to decreased insulin levels. The brain is an important target for insulin, and there is great interest in understanding how diabetes affects the brain. In addition to the direct effects of insulin on the brain, diabetes may also impact the brain through modulation of the inflammatory system. Here we investigate how perturbation of circulating insulin levels affects the expression of Hes3, a transcription factor expressed in neural stem and progenitor cells that is involved in tissue regeneration. Our data show that streptozotocin-induced beta-cell damage, high fat diet, as well as metformin, a common type 2 diabetes medication, regulate Hes3 levels in the brain. This work suggests that Hes3 is a valuable biomarker helping to monitor the state of endogenous neural stem and progenitor cells in the context of diabetes mellitus.
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Scientific Article
Pharmacogenomics 19, 577-587 (2018)
Type 2 diabetes prevalence is still on the rise worldwide. Antidiabetic drugs are widely prescribed to patients with Type 2 diabetes. Most patients start with metformin which is mostly well tolerated. However, a high percentage of patients fail to achieve glycemic control. The effectiveness of metformin as well as most other antidiabetic drugs depends among other factors on interindividual genetic differences that are up to now ignored in the treatment of Type 2 diabetes. Interestingly, many genes influencing the effectiveness of antidiabetic drugs are Type 2 diabetes risk genes making matters worse. Here, we shed light on these interindividual genetic differences.
Review
Review
Kidney Int. 94, 49-59 (2018)
High circulating fibroblast growth factor 23 (FGF23) levels are probably a major risk factor for cardiovascular disease in chronic kidney disease. FGF23 interacts with the receptor FGFR4 in cardiomyocytes inducing left ventricular hypertrophy. Moreover, in the liver FGF23 via FGFR4 increases the risk of inflammation which is also found in chronic kidney disease. In contrast, X-linked hypophosphatemia is characterized by high FGF23 circulating levels due to loss of function mutations of the phosphate-regulating gene with homologies to an endopeptidase on the X chromosome (PHEX), but is not characterized by high cardiovascular morbidity. Here we used a novel murine X-linked hypophosphatemia model, the PhexC733RMhda mouse line, bearing an amino acid substitution (p.Cys733Arg) to test whether high circulating FGF23 in the absence of renal injury would trigger cardiovascular disease. As X-linked hypophosphatemia patient mimics, these mice show high FGF23 levels, hypophosphatemia, normocalcemia, and low/normal vitamin D levels. Moreover, these mice show hyperparathyroidism and low circulating soluble alpha Klotho levels. At the age of 27 weeks we found no left ventricular hypertrophy and no alteration of cardiac function as assessed by echocardiography. These mice also showed no activation of the calcineurin/NFAT pathway in heart and liver and no tissue and systemic signs of inflammation. Importantly, blood pressure, glomerular filtration rate and urea clearance were similar between genotypes. Thus, the presence of high circulating FGF23 levels alone in the absence of renal impairment and normal/high phosphate levels is not sufficient to cause cardiovascular disease.
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Scientific Article
EMBO Mol. Med. 10:e9456 (2018)
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.
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Scientific Article
Mol. Metab., DOI: 10.1016/j.molmet.2018.06.015 (2018)
© 2018 The Authors Objective: The worldwide prevalence of obesity has increased to 10% in men and 15% in women and is associated with severe comorbidities such as diabetes, cancer, and cardiovascular disease. Animal models of obesity are central to experimental studies of disease mechanisms and therapeutic strategies. Diet-induced obesity (DIO) models in rodents have provided important insights into the pathophysiology of obesity and, in most instances, are the first in line for exploratory pharmacology studies. To deepen the relevance towards translation to human patients, we established a corresponding DIO model in Göttingen minipigs (GM). Methods: Young adult female ovariectomized GM were fed a high-fat/high-energy diet for a period of 70 weeks. The ration was calculated to meet the requirements and maintain body weight (BW) of lean adult minipigs (L-GM group) or increased stepwise to achieve an obese state (DIO-GM group). Body composition, blood parameters and intravenous glucose tolerance were determined at regular intervals. A pilot chronic treatment trial with a GLP1 receptor agonist was conducted in DIO-GM. At the end of the study, the animals were necropsied and a biobank of selected tissues was established. Results: DIO-GM developed severe subcutaneous and visceral adiposity (body fat >50% of body mass vs. 22% in L-GM), increased plasma cholesterol, triglyceride, and free fatty acid levels, insulin resistance (HOMA-IR >5 vs. 2 in L-GM), impaired glucose tolerance and increased heart rate when resting and active. However, fasting glucose concentrations stayed within normal range throughout the study. Treatment with a long-acting GLP1 receptor agonist revealed substantial reduction of food intake and body weight within four weeks, with increased drug sensitivity relative to observations in other DIO animal models. Extensive adipose tissue inflammation and adipocyte necrosis was observed in visceral, but not subcutaneous, adipose tissue of DIO-GM. Conclusions: The Munich DIO-GM model resembles hallmarks of the human metabolic syndrome with extensive adipose tissue inflammation and adipocyte necrosis reported for the first time. DIO-GM may be used for evaluating novel treatments of obesity and associated comorbidities. They may help to identify triggers and mechanisms of fat tissue inflammation and mechanisms preventing complete metabolic decompensation despite morbid obesity.
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Scientific Article
PLoS Genet. 14:e1007329 (2018)
As part of a broader collaborative network of exome sequencing studies, we developed a jointly called data set of 5,685 Ashkenazi Jewish exomes. We make publicly available a resource of site and allele frequencies, which should serve as a reference for medical genetics in the Ashkenazim (hosted in part at https://ibd.broadinstitute.org, also available in gnomAD at http://gnomad.broadinstitute.org). We estimate that 34% of protein-coding alleles present in the Ashkenazi Jewish population at frequencies greater than 0.2% are significantly more frequent (mean 15-fold) than their maximum frequency observed in other reference populations. Arising via a well-described founder effect approximately 30 generations ago, this catalog of enriched alleles can contribute to differences in genetic risk and overall prevalence of diseases between populations. As validation we document 148 AJ enriched protein-altering alleles that overlap with "pathogenic" ClinVar alleles (table available at https://github.com/macarthur-lab/clinvar/blob/master/output/clinvar.tsv), including those that account for 10-100 fold differences in prevalence between AJ and non-AJ populations of some rare diseases, especially recessive conditions, including Gaucher disease (GBA, p.Asn409Ser, 8-fold enrichment); Canavan disease (ASPA, p.Glu285Ala, 12-fold enrichment); and Tay-Sachs disease (HEXA, c.1421+1G>C, 27-fold enrichment; p.Tyr427IlefsTer5, 12-fold enrichment). We next sought to use this catalog, of well-established relevance to Mendelian disease, to explore Crohn's disease, a common disease with an estimated two to four-fold excess prevalence in AJ. We specifically attempt to evaluate whether strong acting rare alleles, particularly protein-truncating or otherwise large effect-size alleles, enriched by the same founder-effect, contribute excess genetic risk to Crohn's disease in AJ, and find that ten rare genetic risk factors in NOD2 and LRRK2 are enriched in AJ (p < 0.005), including several novel contributing alleles, show evidence of association to CD. Independently, we find that genomewide common variant risk defined by GWAS shows a strong difference between AJ and non-AJ European control population samples (0.97 s.d. higher, p<10-16). Taken together, the results suggest coordinated selection in AJ population for higher CD risk alleles in general. The results and approach illustrate the value of exome sequencing data in case-control studies along with reference data sets like ExAC (sites VCF available via FTP at ftp.broadinstitute.org/pub/ExAC_release/release0.3/) to pinpoint genetic variation that contributes to variable disease predisposition across populations.
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Scientific Article
Chem. Commun. 54, 5426-5429 (2018)
In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in β-sheet rich oligomers with increased β-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.
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Scientific Article
Metabolites 8:45 (2018)
Night shift work can have a serious impact on health. Here, we assess whether and how night shift work influences the metabolite profiles, specifically with respect to different chronotype classes. We have recruited 100 women including 68 nurses working both, day shift and night shifts for up to 5 consecutive days and collected 3640 spontaneous urine samples. About 424 waking-up urine samples were measured using a targeted metabolomics approach. To account for urine dilution, we applied three methods to normalize the metabolite values: creatinine-, osmolality- and regression-based normalization. Based on linear mixed effect models, we found 31 metabolites significantly (false discovery rate <0.05) affected in nurses working in night shifts. One metabolite, acylcarnitine C10:2, was consistently identified with all three normalization methods. We further observed 11 and 4 metabolites significantly associated with night shift in early and late chronotype classes, respectively. Increased levels of medium- and long chain acylcarnitines indicate a strong impairment of the fatty acid oxidation. Our results show that night shift work influences acylcarnitines and BCAAs, particularly in nurses in the early chronotype class. Women with intermediate and late chronotypes appear to be less affected by night shift work.
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Scientific Article
Nat. Commun. 9:288 (2018)
Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome.
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Scientific Article
Allergy 73, 233-233 (2018)
Meeting abstract
Meeting abstract
Allergy 73, 232-232 (2018)
Meeting abstract
Meeting abstract
JCI insight 3:98864 (2018)
Mesenchymal TNF signaling is etiopathogenic for inflammatory diseases such as rheumatoid arthritis and spondyloarthritis (SpA). The role of Tnfr1 in arthritis has been documented; however, Tnfr2 functions are unknown. Here, we investigate the mesenchymal-specific role of Tnfr2 in the Tnf(1ARE) mouse model of SpA in arthritis and heart valve stenosis comorbidity by cell-specific, Col6a1-cre-driven gene targeting. We find that TNF/Tnfr2 signaling in resident synovial fibroblasts (SFs) and valvular interstitial cells (VICs) is detrimental for both pathologies, pointing to common cellular mechanisms. In contrast, systemic Tnfr2 provides protective signaling, since its complete deletion leads to severe deterioration of both pathologies. SFs and VICs lacking Tnfr2 fail to acquire pathogenic activated phenotypes and display increased expression of antiinflammatory cytokines associated with decreased Akt signaling. Comparative RNA sequencing experiments showed that the majority of the deregulated pathways in Tnf(1ARE) mesenchymal-origin SFs and VICs, including proliferation, inflammation, migration, and disease-specific genes, are regulated by Tnfr2; thus, in its absence, they are maintained in a quiescent nonpathogenic state. Our data indicate a pleiotropy of Tnfr2 functions, with mesenchymal Tnfr2 driving cell activation and arthritis/valve stenosis pathogenesis only in the presence of systemic Tnfr2, whereas nonmesenchymal Tnfr2 overcomes this function, providing protective signals and, thus, containing both pathologies.
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Scientific Article
J. Invest. Dermatol. 138, 179-188 (2018)
Fibroblast growth factor-binding protein 1 (FGFBP1, FGF-BP) is a secreted chaperone that mobilizes paracrine-acting FGFs, stored in the extracellular matrix, and presents them to their cognate receptors. FGFBP1 enhances FGF signaling including angiogenesis during cancer progression, and is upregulated in various cancers. Here we evaluated the contribution of endogenous FGFBP1 to development and homeostasis as well as to skin pathologies utilizing Fgfbp1-knockout (KO) mice. Relative to wild-type (WT) littermates KO mice showed no gross pathologies. Still, in KO mice a significant thickening of the epidermis associated with a decreased transepidermal water loss and increased pro-inflammatory gene expression in the skin was detected. Also, skin carcinogen challenge by DMBA/TPA resulted in delayed and reduced papillomatosis in KO mice. This was paralleled by delayed healing of skin wounds and reduced angiogenic sprouting in subcutaneous matrigel plugs. Heterozygous GFP-knock-in mice revealed rapid induction of gene expression during papilloma induction and during wound healing. Examination of WT skin grafted onto Fgfbp1 GFP knockin reporter hosts and bone marrow transplants from the GFP reporter model into WT hosts revealed that circulating Fgfbp1-expressing cells migrate into healing wounds. We conclude that tissue-resident and circulating Fgfbp1-expressing cells modulate skin carcinogenesis and inflammation.
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Scientific Article
Life Sci. All. 1:e201800106 (2018)
Locomotion is coordinated by neuronal circuits of the spinal cord. Recently, dI6 neurons were shown to participate in the control of locomotion. A subpopulation of dI6 neurons expresses the Wilms tumor suppressor gene Wt1. However, the function of Wt1 in these cells is not understood. Here, we aimed to identify behavioral changes and cellular alterations in the spinal cord associated with Wt1 deletion. Locomotion analyses of mice with neuron-specific Wt1 deletion revealed a slower walk with a decreased stride frequency and an increased stride length. These mice showed changes in their fore-/hindlimb coordination, which were accompanied by a loss of contralateral projections in the spinal cord. Neonates with Wt1 deletion displayed an increase in uncoordinated hindlimb movements and their motor neuron output was arrhythmic with a decreased frequency. The population size of dI6, V0, and V2a neurons in the developing spinal cord of conditional Wt1 mutants was significantly altered. These results show that the development of particular dI6 neurons depends on Wt1 expression and that loss of Wt1 is associated with alterations in locomotion.
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Scientific Article
Nat. Commun. 9:1990 (2018)
We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus xanthus auto-assembles into nanocompartments inside mammalian cells to which sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase leads to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT). Co-expression of ferritin-like native cargo (EncB,C) results in efficient iron sequestration producing substantial contrast by magnetic resonance imaging (MRI) and allowing for magnetic cell sorting. The monodisperse, spherical, and iron-loading nanoshells are also excellent genetically encoded reporters for electron microscopy (EM). In general, eukaryotically expressed encapsulins enable cellular engineering of spatially confined multicomponent processes with versatile applications in multiscale molecular imaging, as well as intriguing implications for metabolic engineering and cellular therapy.
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Scientific Article
Nat. Microbiol. 3, 1161–1174 (2018)
Congenital Zika virus (ZIKV) syndrome may cause fetal microcephaly in -1% of affected newborns. Here, we investigate whether the majority of clinically inapparent newborns might suffer from long-term health impairments not readily visible at birth. Infection of immunocompetent pregnant mice with high-dose ZIKV caused severe offspring phenotypes, such as fetal death, as expected. By contrast, low-dose (LD) maternal ZIKV infection resulted in reduced fetal birth weight but no other obvious phenotypes. Male offspring born to LD ZIKV-infected mothers had increased testosterone (TST) levels and were less likely to survive in utero infection compared to their female littermates. Males also presented an increased number of immature neurons in apical and basal hippocampal dendrites, while female offspring had immature neurons in basal dendrites only. Moreover, male offspring with high but not very high (storm) TST levels were more likely to suffer from learning and memory impairments compared to females. Future studies are required to understand the impact of TST on neuropathological and neurocognitive impairments in later life. In summary, increased sex-specific vigilance is required in countries with high ZIKV prevalence, where impaired neurodevelopment may be camouflaged by a healthy appearance at birth.
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Scientific Article
Sci. Rep. 8:1366 (2018)
The number of pregnancies complicated by gestational diabetes (GDM) is increasing worldwide. To identify novel characteristics of GDM, we studied miRNA profiles of maternal and fetal whole blood cells (WBCs) from GDM and normal glucose tolerant (NGT) pregnant women matched for body mass index and maternal age. After adjustment for maternal weight gain and pregnancy week, we identified 29 mature micro-RNAs (miRNAs) up-regulated in GDM, one of which, i.e., miRNA-340, was validated by qPCR. mRNA and protein expression of PAIP1, a miRNA-340 target gene, was found down-regulated in GDM women, accordingly. In lymphocytes derived from the mothers' blood and treated in vitro, insulin increased and glucose reduced miRNA-340 expression. In fetal cord blood samples, no associations of miRNA-340 with maternal GDM were observed. Our results provide evidence for insulin-induced epigenetic, i.e., miRNA-dependent, programming of maternal WBCs in GDM.
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Scientific Article
Pigment Cell Melanoma Res. 31, 693-707 (2018)
The mouse tail has an important role in the study of melanogenesis, because mouse tail skin can be used to model human skin pigmentation. To better understand the development of melanocytes in the mouse tail, we cloned two dominant ENU-generated mutations of the Adamts9 gene, Und3 and Und4, which cause an unpigmented ring of epidermis in the middle of the tail, but do not alter pigmentation in the rest of the mouse. Adamts9 encodes a widely expressed zinc metalloprotease with thrombospondin type 1 repeats with few known substrates. Melanocytes are lost in the Adamts9 mutant tail epidermis at a relatively late stage of development, around E18.5. Studies of our Adamts9 conditional allele suggest that there is a melanocyte cell-autonomous requirement for Adamts9. In addition, we used a proteomics approach, TAILS N-terminomics, to identify new Adamts9 candidate substrates in the extracellular matrix of the skin. The tail phenotype of Adamts9 mutants is strikingly similar to the unpigmented trunk belt in Adamts20 mutants, which suggests a particular requirement for Adamts family activity at certain positions along the anteriorposterior axis.
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Scientific Article
Sci. Rep. 8:5975 (2018)
By N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated the mutant mouse line TUB6 that is characterised by severe combined immunodeficiency (SCID) and systemic sterile autoinflammation in homozygotes, and a selective T cell defect in heterozygotes. The causative missense point mutation results in the single amino acid exchange G170W in multicatalytic endopeptidase complex subunit-1 (MECL-1), the β2i-subunit of the immuno- and thymoproteasome. Yeast mutagenesis and crystallographic data suggest that the severe TUB6-phenotype compared to the MECL-1 knockout mouse is caused by structural changes in the C-terminal appendage of β2i that prevent the biogenesis of immuno- and thymoproteasomes. Proteasomes are essential for cell survival, and defective proteasome assembly causes selective death of cells expressing the mutant MECL-1, leading to the severe immunological phenotype. In contrast to the immunosubunits β1i (LMP2) and β5i (LMP7), mutations in the gene encoding MECL-1 have not yet been assigned to human disorders. The TUB6 mutant mouse line exemplifies the involvement of MECL-1 in immunopathogenesis and provides the first mouse model for primary immuno- and thymoproteasome-associated immunodeficiency that may also be relevant in humans.
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Scientific Article
Front. Physiol. 9:553 (2018)
Skeletal muscle mass differs greatly in mice and humans and this is partially inherited. To identify muscle hypertrophy candidate genes we conducted a systematic review to identify genes whose experimental loss or gain-of-function results in significant skeletal muscle hypertrophy in mice. We found 47 genes that meet our search criteria and cause muscle hypertrophy after gene manipulation. They are from high to small effect size: Ski, Fst, Acvr2b, Akt1, Mstn, Klf10, Rheb, Igf1, Pappa, Ppard, Ikbkb, Fstl3, Atgr1a, Ucn3, Mcu, Junb, Ncor1, Gprasp1, Grb10, Mmp9, Dgkz, Ppargc1a (specifically the Ppargc1a4 isoform), Smad4, Ltbp4, Bmpr1a, Crtc2, Xiap, Dgat1, Thra, Adrb2, Asb15, Cast, Eif2b5, Bdkrb2, Tpt1, Nr3c1, Nr4a1, Gnas, Pld1, Crym, Camkk1, Yap1, Inhba, Tp53inp2, Inhbb, Nol3, Esr1. Knock out, knock down, overexpression or a higher activity of these genes causes overall muscle hypertrophy as measured by an increased muscle weight or cross sectional area. The mean effect sizes range from 5 to 345% depending on the manipulated gene as well as the muscle size variable and muscle investigated. Bioinformatical analyses reveal that Asb15, Klf10, Tpt1 are most highly expressed hypertrophy genes in human skeletal muscle when compared to other tissues. Many of the muscle hypertrophy-regulating genes are involved in transcription and ubiquitination. Especially genes belonging to three signaling pathways are able to induce hypertrophy: (a) Igf1-Akt-mTOR pathway, (b) myostatin-Smad signaling, and (c) the angiotensin-bradykinin signaling pathway. The expression of several muscle hypertrophy-inducing genes and the phosphorylation of their protein products changes after human resistance and high intensity exercise, in maximally stimulated mouse muscle or in overloaded mouse plantaris.
Review
Review
Mol. Metab. 9, 57-68 (2018)
Objective: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing beta-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult beta-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. Results: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and ME1S1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in the literature in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand of the Notch receptor DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult beta-cell function, respectively. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes. Conclusions: Our data provide stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a beta-cell developmental program.
Wissenschaftlicher Artikel
Scientific Article
Proc. Natl. Acad. Sci. U.S.A. 115, E2348-E2357 (2018)
Advanced age is not only a major risk factor for a range of disorders within an aging individual but may also enhance susceptibility for disease in the next generation. In humans, advanced paternal age has been associated with increased risk for a number of diseases. Experiments in rodent models have provided initial evidence that paternal age can influence behavioral traits in offspring animals, but the overall scope and extent of paternal age effects on health and disease across the life span remain underexplored. Here, we report that old father offspring mice showed a reduced life span and an exacerbated development of aging traits compared with young father offspring mice. Genome-wide epigenetic analyses of sperm from aging males and old father offspring tissue identified differentially methylated promoters, enriched for genes involved in the regulation of evolutionarily conserved longevity pathways. Gene expression analyses, biochemical experiments, and functional studies revealed evidence for an overactive mTORC1 signaling pathway in old father offspring mice. Pharmacological mTOR inhibition during the course of normal aging ameliorated many of the aging traits that were exacerbated in old father offspring mice. These findings raise the possibility that inherited alterations in longevity pathways contribute to intergenerational effects of aging in old father offspring mice.
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Scientific Article
Poster: Federation of European Neuroscience Societies Forum, 7-11 July 2018, Berlin. (2018)
Metabolomics 14:18 (2018)
Introduction Global metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases. However, the mechanism of a disease is often tissue-based and it is advantageous to analyze metabolomic changes directly in the tissue. Metabolomics from tissue samples faces many challenges like tissue collection, homogenization, and metabolite extraction. Objectives We aimed to establish a metabolite extraction protocol optimized for tissue metabolite quantification by the targeted metabolomics AbsoluteIDQ (TM) p180 Kit (Biocrates). The extraction method should be non-selective, applicable to different kinds and amounts of tissues, monophasic, reproducible, and amenable to high throughput. Methods We quantified metabolites in samples of eleven murine tissues after extraction with three solvents (methanol, phosphate buffer, ethanol/phosphate buffer mixture) in two tissue to solvent ratios and analyzed the extraction yield, ionization efficiency, and reproducibility. Results We found methanol and ethanol/phosphate buffer to be superior to phosphate buffer in regard to extraction yield, reproducibility, and ionization efficiency for all metabolites measured. Phosphate buffer, however, outperformed both organic solvents for amino acids and biogenic amines but yielded unsatisfactory results for lipids. The observed matrix effects of tissue extracts were smaller or in a similar range compared to those of human plasma. Conclusion We provide for each murine tissue type an optimized high-throughput metabolite extraction protocol, which yields the best results for extraction, reproducibility, and quantification of metabolites in the p180 kit. Although the performance of the extraction protocol was monitored by the p180 kit, the protocol can be applicable to other targeted metabolomics assays.
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Scientific Article
2017
PLoS ONE 12:e0178563 (2017)
BACKGROUND: Allergen-specific immunotherapy (AIT) is the only curative treatment for type-1 allergies, but sometimes shows limited therapeutic response as well as local and systemic side effects. Limited control of local inflammation and patient symptoms hampers its widespread use in severe allergic asthma. OBJECTIVE: Our aim was to evaluate whether AIT is more effective in suppression of local inflammation if performed under the umbrella of short-term non-specific immunomodulation using a small molecule inhibitor of JAK pathways. METHODS: In C57BL/6J mice, a model of ovalbumin (OVA)-induced allergic airway inflammation and allergen-specific immunotherapy was combined with the administration of Tofacitinib (TOFA, a FDA-approved JAK inhibitor) from 48 hours prior to 48 hours after therapeutic OVA-injection. The effect of TOFA on human FOXP3+CD4+ T cells was studied in vitro. RESULTS: AIT combined with short-term TOFA administration was significantly more effective in suppressing total cell and eosinophil infiltration into the lung, local cytokine production including IL-1β and CXCL1 and showed a trend for the reduction of IL-4, IL-13, TNF-α and IL-6 compared to AIT alone. Furthermore, TOFA co-administration significantly reduced systemic IL-6, IL-1β and OVA-specific IgE levels and induced IgG1 to the same extent as AIT alone. Additionally, TOFA enhanced the induction of human FOXP3+CD4+ T cells. CONCLUSIONS: This proof of concept study shows that JAK inhibition did not inhibit tolerance induction, but improved experimental AIT at the level of local inflammation. The improved control of local inflammation might extend the use of AIT in more severe conditions such as polyallergy, asthma and high-risk patients suffering from mastocytosis or anaphylaxis.
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Scientific Article
Cell Metab. 25, 1334-1347.e4 (2017)
The processes contributing to β cell dysfunction in type 2 diabetes (T2D) are uncertain, largely because it is difficult to access β cells in their intact immediate environment. We examined the pathophysiology of β cells under T2D progression directly in pancreatic tissues. We used MALDI imaging of Langerhans islets (LHIs) within mouse tissues or from human tissues to generate in situ-omics data, which we supported with in vitro experiments. Molecular interaction networks provided information on functional pathways and molecules. We found that stearoylcarnitine accumulated in β cells, leading to arrest of insulin synthesis and energy deficiency via excessive β-oxidation and depletion of TCA cycle and oxidative phosphorylation metabolites. Acetylcarnitine and an accumulation of N-acyl taurines, a group not previously detected in β cells, provoked insulin secretion. Thus, β cell dysfunction results from enhanced insulin secretion combined with an arrest of insulin synthesis.
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Scientific Article
J. Clin. Invest. 127, 695-708 (2017)
BACKGROUND: Dietary intake of saturated fat is a likely contributor to nonalcoholic fatty liver disease (NAFLD) and insulin resistance, but the mechanisms that initiate these abnormalities in humans remain unclear. We examined the effects of a single oral saturated fat load on insulin sensitivity, hepatic glucose metabolism, and lipid metabolism in humans. Similarly, initiating mechanisms were examined after an equivalent challenge in mice. METHODS: Fourteen lean, healthy individuals randomly received either palm oil (PO) or vehicle (VCL). Hepatic metabolism was analyzed using in vivo 13C/31P/1H and ex vivo 2H magnetic resonance spectroscopy before and during hyperinsulinemic-euglycemic clamps with isotope dilution. Mice underwent identical clamp procedures and hepatic transcriptome analyses. RESULTS: PO administration decreased whole-body, hepatic, and adipose tissue insulin sensitivity by 25%, 15%, and 34%, respectively. Hepatic triglyceride and ATP content rose by 35% and 16%, respectively. Hepatic gluconeogenesis increased by 70%, and net glycogenolysis declined by 20%. Mouse transcriptomics revealed that PO differentially regulates predicted upstream regulators and pathways, including LPS, members of the TLR and PPAR families, NF-κB, and TNF-related weak inducer of apoptosis (TWEAK). CONCLUSION: Saturated fat ingestion rapidly increases hepatic lipid storage, energy metabolism, and insulin resistance. This is accompanied by regulation of hepatic gene expression and signaling that may contribute to development of NAFLD.REGISTRATION. ClinicalTrials.gov NCT01736202. FUNDING: Germany: Ministry of Innovation, Science, and Research North Rhine-Westfalia, German Federal Ministry of Health, Federal Ministry of Education and Research, German Center for Diabetes Research, German Research Foundation, and German Diabetes Association. Portugal: Portuguese Foundation for Science and Technology, FEDER - European Regional Development Fund, Portuguese Foundation for Science and Technology, and Rede Nacional de Ressonância Magnética Nuclear.
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Scientific Article
Diabetes akt. 15, 104-107 (2017)
Wer sich längere Zeit ungesund ernährt, verändert womöglich wichtige Schaltstellen in seinem Erbgut. Eltern steigern dadurch nicht nur das eigene Risiko auf Stoffwechselerkrankungen, sondern auch das ihrer Nachkommen. Verschiedene Studien des Deutschen Zentrums für Diabetesforschung (DZD) weisen darauf hin, dass die Ernährung als Umweltfaktor den Aktivitätszustand von Genen nachhaltig beeinflussen kann – zum Beispiel durch chemische Veränderungen der DNA-Bausteine. Wissenschaftler sprechen hier von Epigenetik (griechisch epi: auf, an, bei). So führt ein erhöhter Body-Mass-Index zu epigenetischen Veränderungen an fast 200 Stellen des Erbguts. Unter anderem können epigenetische Modifikationen eine Leberverfettung bei Maus und Mensch fördern.
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Scientific Article
Mol. Metab. 6, 931-940 (2017)
Objective: The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INS C94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. Methods: Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. Results: MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. Conclusions: The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension.
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Scientific Article
Nat. Commun. 8:886 (2017)
The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.
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Scientific Article
Cell Rep. 21, 1507-1520 (2017)
Regular endurance training improves muscle oxidative capacity and reduces the risk of age-related disorders. Understanding the molecular networks underlying this phenomenon is crucial. Here, by exploiting the power of computational modeling, we show that endurance training induces profound changes in gene regulatory networks linking signaling and selective control of translation to energy metabolism and tissue remodeling. We discovered that knockdown of the mTOR-independent factor Eif6, which we predicted to be a key regulator of this process, affects mitochondrial respiration efficiency, ROS production, and exercise performance. Our work demonstrates the validity of a data-driven approach to understanding muscle homeostasis.
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Scientific Article
Water Res. 122, 290-298 (2017)
Iopromide is frequently detected in water bodies due to its widespread use as an X-ray contrast agent in medicine. Due to its rapid clearance from the human body and its incomplete removal by wastewater treatment, an elevation of its concentration in the environment is observed that might lead to a serious impact on human and environmental health. Alternative or additional removal technologies may be more effective to remove iopromide from the effluents of wastewater treatment facilities, like phytoremediation with aquatic macrophytes. To test this, a hydroponic experiment was carried out to assess the fate of iopromide in Typha latifolia. The transformation products (TPs) in the plant were investigated to predict possible transformation mechanisms. The removal process followed first order kinetics with a linear regression R(2) value of 0.983. The iopromide concentration in roots and rhizomes reached a maximum value of 20.70 ± 0.81 and 16.82 ± 1.78 nmol g(-1) on the 7th day, respectively, thereafter decreased until the end of experiment. A different result was found in leaves, where iopromide concentration decreased over the whole experimental period. A total of eight transformation products were detected in T. latifolia, including 23 isomers. The relative content of aldehyde and ketone TPs decreased in roots and rhizomes while the relative content of carboxylic TPs increased. However, the relative content of aldehyde and ketone TPs only showed a slight decrease in leaves while the relative content of carboxylic TPs remained stable during the experimental period. In addition, a significant increase of decarboxylated TPs was found in leaves, but not in roots and rhizomes. These results indicate that a difference in transformation mechanisms exists among plant tissues. The findings of this study are important to better understand the transformation mechanisms of iopromide in plants and to improve phytoremediation technologies for such kind of compounds.
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Scientific Article
Curr. Protoc. Mouse Biol. 7, 88-99 (2017)
The cell composition of bronchoalveolar lavage fluid (BAL) is an important indicator of airway inflammation. It is commonly determined by cytocentrifuging leukocytes on slides, then staining, identifying, and counting them as eosinophils, neutrophils, macrophages, or lymphocytes according to morphological criteria under light microscopy, where it is not always easy to distinguish macrophages from lymphocytes. We describe here a one-step, easy-to-use, and easy-to-customize 8-color flow cytometric method for performing differential cell count and comparing it to morphological counts on stained cytospins. This method identifies BAL cells by a simultaneous one-step immunolabeling procedure using antibodies to identify T cells, B cells, neutrophils, eosinophils, and macrophages. Morphological analysis of flow-sorted cell subsets is used to validate this protocol. An important advantage of this basic flow cytometry protocol is the ability to customize it by the addition of antibodies to study receptor expression at leukocyte cell surfaces and identify subclasses of inflammatory cells as needed. © 2017 by John Wiley & Sons, Inc.
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Scientific Article
Nucleic Acids Res. 45, 3031-3045 (2017)
An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior.
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Scientific Article
Ann. Rheum. Dis. 76, A49-A49 (2017)
Meeting abstract
Meeting abstract
Ann. Rheum. Dis. 76, 500-500 (2017)
Meeting abstract
Meeting abstract
Nature 551, 398 (2017)
This corrects the article DOI: 10.1038/nature19356.
Sci. Rep. 7:15453 (2017)
Paladin (Pald1, mKIAA1274 or x99384) was identified in screens for vascular-specific genes and is a putative phosphatase. Paladin has also been proposed to be involved in various biological processes such as insulin signaling, innate immunity and neural crest migration. To determine the role of paladin we have now characterized the Pald1 knock-out mouse in a broad array of behavioral, physiological and biochemical tests. Here, we show that female, but not male, Pald1 heterozygous and homozygous knock-out mice display an emphysema-like histology with increased alveolar air spaces and impaired lung function with an obstructive phenotype. In contrast to many other tissues where Pald1 is restricted to the vascular compartment, Pald1 is expressed in both the epithelial and mesenchymal compartments of the postnatal lung. However, in Pald1 knock-out females, there is a specific increase in apoptosis and proliferation of endothelial cells, but not in non-endothelial cells. This results in a transient reduction of endothelial cells in the maturing lung. Our data suggests that Pald1 is required during lung vascular development and for normal function of the developing and adult lung in a sex-specific manner. To our knowledge, this is the first report of a sex-specific effect on endothelial cell apoptosis.
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Scientific Article
Immunity 47, 1067-1082.e12 (2017)
Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17 92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhan ced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells. Essig et al. show that spontaneous activation and aberrant differentiation of Roquin-deficient T cells involves cell-intrinsic causes in not only conventional T cells but also impaired Treg cell function. In both cell types, Roquin inhibits the PI3K-mTOR signaling pathway at several levels, thereby controlling protein biosynthesis and limiting differentiation toward Th17 and Tfh cells as well as preventing the conversion and functional specialization of Treg into Tfr cells.
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Scientific Article
Sci. Data 4:170179 (2017)
To investigate the genetic basis of type 2 diabetes (T2D) to high resolution, the GoT2D and T2D-GENES consortia catalogued variation from whole-genome sequencing of 2,657 European individuals and exome sequencing of 12,940 individuals of multiple ancestries. Over 27M SNPs, indels, and structural variants were identified, including 99% of low-frequency (minor allele frequency [MAF] 0.1-5%) non-coding variants in the whole-genome sequenced individuals and 99.7% of low-frequency coding variants in the whole-exome sequenced individuals. Each variant was tested for association with T2D in the sequenced individuals, and, to increase power, most were tested in larger numbers of individuals (>80% of low-frequency coding variants in ~82 K Europeans via the exome chip, and ~90% of low-frequency non-coding variants in ~44 K Europeans via genotype imputation). The variants, genotypes, and association statistics from these analyses provide the largest reference to date of human genetic information relevant to T2D, for use in activities such as T2D-focused genotype imputation, functional characterization of variants or genes, and other novel analyses to detect associations between sequence variation and T2D.
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Scientific Article
Behav. Brain Res. 352, 187-196 (2017)
Since decades, model organisms have provided an important approach for understanding the mechanistic basis of human diseases. The German Mouse Clinic (GMC) was the first phenotyping facility that established a collaboration-based platform for phenotype characterization of mouse lines. In order to address individual projects by a tailor-made phenotyping strategy, the GMC advanced in developing a series of pipelines with tests for the analysis of specific disease areas. For a general broad analysis, there is a screening pipeline that covers the key parameters for the most relevant disease areas. For hypothesis-driven phenotypic analyses, there are thirteen additional pipelines with focus on neurological and behavioral disorders, metabolic dysfunction, respiratory system malfunctions, immune-system disorders and imaging techniques. In this article, we give an overview of the pipelines and describe the scientific rationale behind the different test combinations.
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Scientific Article
Mol. Neurobiol. 55, 4580–4595 (2017)
In neuropsychiatric diseases, such as major depression and anxiety, pathogenic vulnerability is partially dictated by a genetic predisposition. The search continues to define this genetic susceptibility and establish new genetic elements as potential therapeutic targets. The fibroblast growth factors (FGFs) could be interesting in this regard. This family of signaling molecules plays important roles in development while also functioning within the adult. This includes effects on aspects of brain function such as neurogenesis and synapse formation. Of this family, Fgf9 is expressed in the adult brain, but its functional role is less well defined. In this study, we examined the role of Fgf9 in different brain functions by analyzing the behavior of Fgf9 (Y162C) mutant mice, an Fgf9 allele without the confounding systemic effects of other Fgf9 genetic models. Here, we show that this mutation caused altered locomotor and exploratory reactivity to novel, mildly stressful environments. In addition, mutants showed heightened acoustic startle reactivity as well as impaired social discrimination memory. Notably, there was a substantial decrease in the level of adult olfactory bulb neurogenesis with no difference in hippocampal neurogenesis. Collectively, our findings indicate a role for the Fgf9 (Y162C) mutation in information processing and perception of aversive situations as well as in social memory. Thus, genetic alterations in Fgf9 could increase vulnerability to developing neuropsychiatric disease, and we propose the Fgf9 (Y162C) mutant mice as a valuable tool to study the predictive etiological aspects.
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Scientific Article
Biochim. Biophys. Acta-Mol. Basis Dis. 1863, 1605-1614 (2017)
Peri-conceptional exposure to maternal obesogenic nutrition is associated with in utero programming of later-life overweight and metabolic disease in the offspring. We aimed to investigate whether dietary intervention with a modified fatty acid quality in an obesogenic high-calorie (HC) diet during the preconception and gestational phases can improve unfavourable effects of an adipogenic maternal environment. In NMRI mice, peri-conceptional and gestational obesity was induced by feeding a HC diet (controls), and they were compared with dams on a fat-modified (Fat-mod) HC diet of the same energy content but enriched with medium-chain fatty acids (MCFAs) and adjusted to a decreased ratio of n-6 to n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). Effects on maternal and placental outcomes at delivery (day 17.5 post coitum) were investigated. Despite comparable energy assimilation between the two groups of dams, the modified fatty acid composition of the high-caloric diet induced lower maternal body weight, weights of fat depots, adipocyte size, and hepatic fat accumulation compared to the unmodified HC diet group. Further, there was a trend towards lower fasting glucose, insulin and leptin concentrations in dams fed the Fat-mod HC diet. Phenotypic changes were accompanied by inhibition of transcript and protein expression of genes involved in hepatic de novo lipogenesis comprising PPARG2 and its target genes Fasn, Acaca, Fabp4, whereas regulation of other lipogenic factors (Srebf1, Nr1h3, Abca1) appeared to be more complex. The modified diet led to a sex-specific placental response by upregulating PPARG-dependent fatty acid transport gene expression in female versus male placentae. Qualitative modification of the fatty acid spectrum of a high-energy maternal diet, using a combination of both MCFAs and n-3 LC-PUFAs, seems to be a promising interventional approach to ameliorate the adipogenic milieu of mice before and during gestation.
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Scientific Article
Allergy 72, 40-40 (2017)
Meeting abstract
Meeting abstract
Proc. Natl. Acad. Sci. U.S.A. 114, E11323-E11332 (2017)
Thyroid hormone (TH) and TH receptors (TRs) α and β act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type 1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger signaling pathways independently of gene expression (noncanonical or type 3 TR signaling). To test the physiological relevance of noncanonical TR signaling, we generated knockin mice with a mutation in the TR DNA-binding domain that abrogates binding to DNA and leads to complete loss of canonical TH action. We show that several important physiological TH effects are preserved despite the disruption of DNA binding of TRα and TRβ, most notably heart rate, body temperature, blood glucose, and triglyceride concentration, all of which were regulated by noncanonical TR signaling. Additionally, we confirm that TRE-binding–defective TRβ leads to disruption of the hypothalamic–pituitary–thyroid axis with resistance to TH, while mutation of TRα causes a severe delay in skeletal development, thus demonstrating tissue- and TR isoform-specific canonical signaling. These findings provide in vivo evidence that noncanonical TR signaling exerts physiologically important cardiometabolic effects that are distinct from canonical actions. These data challenge the current paradigm that in vivo physiological TH action is mediated exclusively via regulation of gene transcription at the nuclear level.
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Scientific Article
Mol. Metab. 6, 1286-1295 (2017)
Objective Angiopoietin-like protein-4 (ANGPTL4) is a circulating protein that is highly expressed in liver and implicated in regulation of plasma triglyceride levels. Systemic ANGPTL4 increases during prolonged fasting and is suggested to be secreted from skeletal muscle following exercise. Methods We investigated the origin of exercise-induced ANGPTL4 in humans by measuring the arterial-to-venous difference over the leg and the hepato-splanchnic bed during an acute bout of exercise. Furthermore, the impact of the glucagon-to-insulin ratio on plasma ANGPTL4 was studied in healthy individuals. The regulation of ANGPTL4 was investigated in both hepatic and muscle cells. Results The hepato-splanchnic bed, but not the leg, contributed to exercise-induced plasma ANGPTL4. Further studies using hormone infusions revealed that the glucagon-to-insulin ratio is an important regulator of plasma ANGPTL4 as elevated glucagon in the absence of elevated insulin increased plasma ANGPTL4 in resting subjects, whereas infusion of somatostatin during exercise blunted the increase of both glucagon and ANGPTL4. Moreover, activation of the cAMP/PKA signaling cascade let to an increase in ANGPTL4 mRNA levels in hepatic cells, which was prevented by inhibition of PKA. In humans, muscle ANGPTL4 mRNA increased during fasting, with only a marginal further induction by exercise. In human muscle cells, no inhibitory effect of AMPK activation could be demonstrated on ANGPTL4 expression. Conclusions The data suggest that exercise-induced ANGPTL4 is secreted from the liver and driven by a glucagon-cAMP-PKA pathway in humans. These findings link the liver, insulin/glucagon, and lipid metabolism together, which could implicate a role of ANGPTL4 in metabolic diseases.
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Scientific Article
Sci. Rep. 7:2397 (2017)
The human growth hormone (hGH) minigene used for transgene stabilization in mice has been recently identified to be locally expressed in the tissues where transgenes are active and associated with phenotypic alterations. Here we extend these findings by analyzing the effect of the hGH minigene in TgC6hp55 transgenic mice which express the human TNFR1 under the control of the mesenchymal cell-specific CollagenVI promoter. These mice displayed a fully penetrant phenotype characterized by growth enhancement accompanied by perturbations in metabolic, skeletal, histological and other physiological parameters. Notably, this phenotype was independent of TNF-TNFR1 signaling since the genetic ablation of either Tnf or Tradd did not rescue the phenotype. Further analyses showed that the hGH minigene was expressed in several tissues, also leading to increased hGH protein levels in the serum. Pharmacological blockade of GH signaling prevented the development of the phenotype. Our results indicate that the unplanned expression of the hGH minigene in CollagenVI expressing mesenchymal cells can lead through local and/or systemic mechanisms to enhanced somatic growth followed by a plethora of primary and/or secondary effects such as hyperphagia, hypermetabolism, disturbed glucose homeostasis, altered hematological parameters, increased bone formation and lipid accumulation in metabolically critical tissues.
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Scientific Article
Drug Discov. Today, DOI: 10.1016/j.ddmod.2017.08.002 (2017)
© 2017 Elsevier Ltd. The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6000 mutant mouse strains have been generated by the IKMC in collaboration with the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 C57BL/6 cre-inducible mouse strains. Complementing the cre-driver resource, a collection comprising 27 rAAVs expressing cre in a tissue-specific manner has also been produced. All resources are easily accessible from the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds enabling the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research.
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Scientific Article
Nat. Commun. 8:15475 (2017)
The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans.
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Scientific Article
Mol. Metab. 6, 1226-1239 (2017)
Objective Obesity-associated WAT inflammation is characterized by the accumulation and local activation of macrophages (MΦs), and recent data from mouse studies suggest that macrophages are modifiers of adipocyte energy metabolism and mitochondrial function. As mitochondrial dysfunction has been associated with obesity and the metabolic syndrome in humans, herein we aimed to delineate how human macrophages may affect energy metabolism of white adipocytes. Methods Human adipose tissue gene expression analysis for markers of macrophage activation and tissue inflammation (CD11c, CD40, CD163, CD206, CD80, MCP1, TNFα) in relationship to mitochondrial complex I (NDUFB8) and complex III (UQCRC2) was performed on subcutaneous WAT of 24 women (BMI 20–61 kg/m2). Guided by these results, the impact of secreted factors of LPS/IFNγ- and IL10/TGFβ-activated human macrophages (THP1, primary blood-derived) on mitochondrial function in human subcutaneous white adipocytes (SGBS, primary) was determined by extracellular flux analysis (Seahorse technology) and gene/protein expression. Results Stepwise regression analysis of human WAT gene expression data revealed that a linear combination of CD40 and CD163 was the strongest predictor for mitochondrial complex I (NDUFB8) and complex III (UQCRC2) levels, independent of BMI. IL10/TGFβ-activated MΦs displayed high CD163 and low CD40 expression and secreted factors that decreased UQCRC2 gene/protein expression and ATP-linked respiration in human white adipocytes. In contrast, LPS/IFNγ-activated MΦs showed high CD40 and low CD163 expression and secreted factors that enhanced adipocyte mitochondrial activity resulting in a total difference of 37% in ATP-linked respiration of white adipocytes (p = 0.0024) when comparing the effect of LPS/IFNγ- vs IL10/TGFβ-activated MΦs. Conclusion Our data demonstrate that macrophages modulate human adipocyte energy metabolism via an activation-dependent paracrine mechanism.
Wissenschaftlicher Artikel
Scientific Article
J. Biomed. Sci. 24:57 (2017)
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.
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Scientific Article
Curr. Opin. Syst. Biol. 4, 97-104 (2017)
Systemic phenotyping of mutant mice has been established at large scale in the last decade as a new tool to uncover the relations between genotype, phenotype and environment. Recent advances in that field led to the generation of a valuable open access data resource that can be used to better understanding the underlying causes for human diseases. From an ethical perspective, systemic phenotyping significantly contributes to the reduction of experimental animals and the refinement of animal experiments by enforcing standardisation efforts. There are particular logistical, experimental and analytical challenges of systemic large-scale mouse phenotyping. On all levels, IT solutions are critical to implement and efficiently support breeding, phenotyping and data analysis processes that lead to the generation of high-quality systemic phenotyping data accessible for the scientific community.
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Scientific Article
Dev. Comp. Immunol. 77, 69-76 (2017)
Eukaryotic Initiation Factor 6 (eIF6) is required for 60S ribosomal subunit biogenesis and efficient initiation of translation. Intriguingly, in both mice and humans, endogenous levels of eIF6 are detrimental, as they act as tumor and obesity facilitators, raising the question on the evolutionary pressure that maintains high eIF6 levels. Here we show that in mice and humans, high levels of eIF6 are required for proper immune functions. First, eIF6 heterozygous (het) mice show an increased mortality during viral infection and a reduction of peripheral blood CD4(+) Effector Memory T cells. In human CD4(+) T cells, eIF6 levels rapidly increase upon T-cell receptor activation and drive the glycolytic switch and the acquisition of effector functions. Importantly, in CD4(+) T cells, eIF6 levels control interferon-γ (IFN-γ) secretion without affecting proliferation. In conclusion, the immune system has a high evolutionary pressure for the maintenance of a dynamic and powerful regulation of the translational machinery.
Wissenschaftlicher Artikel
Scientific Article
Data Brief 14, 653-658 (2017)
The data described in this article are related to “High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4 + T cells in both mice and humans” (Manfrini et al., in press) [1]. eIF6 is a translation initiation factor required for ribosomal biogenesis (Sanvito et al., 1999) [2] and for proper translational initiation (Gallo and Manfrini, 2015; Miluzio et al., 2016) [3,4] whose protein abundance requires tight regulation. Here we analyze by flow cytometry the effects of eIF6 depletion on proportions of specific innate and adaptive immune system subpopulations and on thymocyte maturation in mice.
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Scientific Article
Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.
Nat. Genet. 49, 1231-1238 (2017)
Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.
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Scientific Article
Cell Metab. 26, 620-632.e6 (2017)
Chronic inflammation has been proposed to contribute to the pathogenesis of diet-induced obesity. However, scarce therapeutic options are available to treat obesity and the associated immunometabolic complications. Glucocorticoids are routinely employed for the management of inflammatory diseases, but their pleiotropic nature leads to detrimental metabolic side effects. We developed a glucagon-like peptide-1 (GLP-1)-dexamethasone co-agonist in which GLP-1 selectively delivers dexamethasone to GLP-1 receptor-expressing cells. GLP-1-dexamethasone lowers body weight up to 25% in obese mice by targeting the hypothalamic control of feeding and by increasing energy expenditure. This strategy reverses hypothalamic and systemic inflammation while improving glucose tolerance and insulin sensitivity. The selective preference for GLP-1 receptor bypasses deleterious effects of dexamethasone on glucose handling, bone integrity, and hypothalamus-pituitary-adrenal axis activity. Thus, GLP-1-directed glucocorticoid pharmacology represents a safe and efficacious therapy option for diet-induced immunometabolic derangements and the resulting obesity.
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Scientific Article
Mol. Psychiatry 23, 1345-1355 (2017)
Dietary intake of methyl donors, such as folic acid and methionine, shows considerable intra-individual variation in human populations. While it is recognized that maternal departures from the optimum of dietary methyl donor intake can increase the risk for mental health issues and neurological disorders in offspring, it has not been explored whether paternal dietary methyl donor intake influences behavioral and cognitive functions in the next generation. Here, we report that elevated paternal dietary methyl donor intake in a mouse model, transiently applied prior to mating, resulted in offspring animals (methyl donor-rich diet (MD) F1 mice) with deficits in hippocampus-dependent learning and memory, impaired hippocampal synaptic plasticity and reduced hippocampal theta oscillations. Gene expression analyses revealed altered expression of the methionine adenosyltransferase Mat2a and BK channel subunit Kcnmb2, which was associated with changes in Kcnmb2 promoter methylation in MD F1 mice. Hippocampal overexpression of Kcnmb2 in MD F1 mice ameliorated altered spatial learning and memory, supporting a role of this BK channel subunit in the MD F1 behavioral phenotype. Behavioral and gene expression changes did not extend into the F2 offspring generation. Together, our data indicate that paternal dietary factors influence cognitive and neural functions in the offspring generation.Molecular Psychiatry advance online publication, 4 April 2017; doi:10.1038/mp.2017.53.
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Scientific Article
Mol. Metab. 6, 1304-1312 (2017)
Objective The fatty acid receptor 1 (FFAR1/GPR40) mediates fatty acid-dependent augmentation of glucose-induced insulin secretion (GIIS) in pancreatic β-cells. Genetically engineered Ffar1-knockout/congenic mice univocally displayed impaired fatty acid-mediated insulin secretion, but in vivo experiments delivered controversial results regarding the function of FFAR1 in glucose homeostasis and liver steatosis. This study presents a new coisogenic mouse model carrying a point mutation in Ffar1 with functional consequence. These mice reflect the situations in humans in which point mutations can lead to protein malfunction and disease development. Methods The Munich N-ethyl-N-nitrosourea (ENU) mutagenesis-derived F1 archive containing over 16,800 sperms and corresponding DNA samples was screened for mutations in the coding region of Ffar1. Two missense mutations (R258W and T146S) in the extracellular domain of the protein were chosen and homozygote mice were generated. The functional consequence of these mutations was examined in vitro in isolated islets and in vivo in chow diet and high fat diet fed mice. Results Palmitate, 50 μM, and the FFAR1 agonist TUG-469, 3 μM, stimulated insulin secretion in islets of Ffar1T146S/T146S mutant mice and of wild-type littermates, while in islets of Ffar1R258W/R258W mutant mice, these stimulatory effects were abolished. Insulin content and mRNA levels of Ffar1, Glp1r, Ins2, Slc2a2, Ppara, and Ppard were not significantly different between wild type and Ffar1R258W/R258W mouse islets. Palmitate exposure, 600 μM, significantly increased Ppara mRNA levels in wild type but not in Ffar1R258W/R258W mouse islets. On the contrary, Slc2a2 mRNA levels were significantly reduced in both wild type and Ffar1R258W/R258W mouse islets after palmitate treatment. HFD feeding induced glucose intolerance in wild-type mice. Ffar1R258W/R258W mutant mice remained glucose tolerant although their body weight gain, liver steatosis, insulin resistance, and plasma insulin levels were not different from those of wild-type littermates. Worth mentioning, fasting plasma insulin levels were lower in Ffar1R258W/R258W mice. Conclusion A point mutation in Ffar1 abrogates the stimulatory effect of palmitate on GIIS, an effect that does not necessarily translate to HFD-induced glucose intolerance.
Wissenschaftlicher Artikel
Scientific Article
Dis. Model. Mech. 10, 981-991 (2017)
MEIS1 is a developmental transcription factor linked to restless legs syndrome (RLS) in genome-wide association studies. RLS is a movement disorder leading to severe sleep reduction and with significant impact on the quality-of-life of patients. In genome-wide association studies, MEIS1 has consistently been the gene with the highest effect size and functional studies suggest a disease-relevant downregulation. Therefore, haploinsufficiency of Meis1 could be the most potential system for modeling RLS in animals. We used heterozygous Meis1 knock-out mice to study the effects of Meis1 haploinsufficiency on mouse behavioral and neurological phenotypes, and to relate the findings to human RLS. We exposed the Meis1-deficient mice to assays of motor, sensorimotor and cognitive ability and assessed the effect of a dopaminergic receptor 2/3 agonist commonly used in the treatment of RLS. The mutant mice showed a pattern of circadian hyperactivity, compatible with human RLS. Moreover, we discovered a replicable prepulse inhibition (PPI) deficit in the Meis1-deficient animals. In addition, these mice were hyposensitive to the PPI-reducing effect of the dopaminergic receptor agonist, highlighting a role of Meis1 in the dopaminergic system. Other reported phenotypes include enhanced social recognition at an older age that was not related to alterations in adult olfactory bulb neurogenesis previously shown to be implicated in this behavior. In conclusion, the Meis1-deficient mice fulfill some of the hallmarks of an RLS animal model, and revealed the role of Meis1 in sensorimotor gating and in the dopaminergic systems modulating it.
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Scientific Article
Sleep Med. 40, E290-E291 (2017)
Meeting abstract
Meeting abstract
Am. J. Respir. Crit. Care Med. 195 (2017)
Meeting abstract
Meeting abstract
Acta Neuropathol. 134, 241–254 (2017)
Translation of the expanded (ggggcc)n repeat in C9orf72 patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) causes abundant poly-GA inclusions. To elucidate their role in pathogenesis, we generated transgenic mice expressing codon-modified (GA)149 conjugated with cyan fluorescent protein (CFP). Transgenic mice progressively developed poly-GA inclusions predominantly in motoneurons and interneurons of the spinal cord and brain stem and in deep cerebellar nuclei. Poly-GA co-aggregated with p62, Rad23b and the newly identified Mlf2, in both mouse and patient samples. Consistent with the expression pattern, 4-month-old transgenic mice showed abnormal gait and progressive balance impairment, but showed normal hippocampus-dependent learning and memory. Apart from microglia activation we detected phosphorylated TDP-43 but no neuronal loss. Thus, poly-GA triggers behavioral deficits through inflammation and protein sequestration that likely contribute to the prodromal symptoms and disease progression of C9orf72 patients.
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Scientific Article
Mol. Metab. 6, 737-747 (2017)
Objective Metformin, the first line drug for treatment of type 2 diabetes, suppresses hepatic gluconeogenesis and reduces body weight in patients, the latter by an unknown mechanism. Methods Mice on a high fat diet were continuously fed metformin in a therapeutically relevant dose, mimicking a retarded formulation. Results Feeding metformin in pharmacologically relevant doses to mice on a high fat diet normalized HbA1c levels and ameliorated glucose tolerance, as expected, but also considerably slowed down weight gain. This was due to increased energy expenditure, since food intake was unchanged and locomotor activity was even decreased. Metformin caused lactate accumulation in the intestinal wall and in portal venous blood but not in peripheral blood or the liver. Increased conversion of glucose-1-13C to glucose-1,6-13C under metformin strongly supports a futile cycle of lactic acid production in the intestinal wall, and usage of the produced lactate for gluconeogenesis in liver. Conclusions The reported glucose–lactate–glucose cycle is a highly energy consuming process, explaining the beneficial effects of metformin given continuously on the development of a type 2 diabetic-like state in our mice.
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Scientific Article
Endocr. Rev. 38, 468-488 (2017)
ince its identification in 2000, the interest of scientists in the hepatokine fibroblast growth factor (FGF) 21 has tremendously grown, and still remains high, due to a wealth of very robust data documenting this factor’s favorable effects on glucose and lipid metabolism in mice. For more than ten years now, intense in vivo and ex vivo experimentation addressed the physiological functions of FGF21 in humans as well as its pathophysiological role and pharmacological effects in human metabolic disease. This work produced a comprehensive collection of data revealing overlaps in FGF21 expression and function but also significant differences between mice and men that have to be considered before translation from bench to bedside can be successful. This review summarizes what is known about FGF21 in mice and humans with a special focus on this factor’s role in glucose and lipid metabolism and in metabolic diseases, such as obesity and type-2 diabetes mellitus. We highlight the discrepancies between mice and men and try to decipher their underlying reasons.
Review
Review
Dis. Model. Mech. 10, 163-171 (2017)
Plants and many lower organisms, but not mammals, express alternative oxidases (AOX) 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 AOXRosa26 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, whilst animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.
Wissenschaftlicher Artikel
Scientific Article
Mol. Cell. Pediatr. 4, 1:5 (2017)
Meeting abstract
Meeting abstract
EMBO Rep. 18, 2015-2029 (2017)
Lipopolysaccharide-responsive beige-like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain-containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein-truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.
Wissenschaftlicher Artikel
Scientific Article
Nat. Commun. 8:155 (2017)
Dietary restriction regimes extend lifespan in various animal models. Here we show that longevity in male C57BL/6J mice subjected to every-other-day feeding is associated with a delayed onset of neoplastic disease that naturally limits lifespan in these animals. We compare more than 200 phenotypes in over 20 tissues in aged animals fed with a lifelong every-other-day feeding or ad libitum access to food diet to determine whether molecular, cellular, physiological and histopathological aging features develop more slowly in every-other-day feeding mice than in controls. We also analyze the effects of every-other-day feeding on young mice on shorter-term every-other-day feeding or ad libitum to account for possible aging-independent restriction effects. Our large-scale analysis reveals overall only limited evidence for a retardation of the aging rate in every-other-day feeding mice. The data indicate that every-other-day feeding-induced longevity is sufficiently explained by delays in life-limiting neoplastic disorders and is not associated with a more general slowing of the aging process in mice.Dietary restriction can extend the life of various model organisms. Here, Xie et al. show that intermittent periods of fasting achieved through every-other-day feeding protect mice against neoplastic disease but do not broadly delay organismal aging in animals.
Wissenschaftlicher Artikel
Scientific Article
J. Neurosci. Methods, DOI: 10.1016/j.jneumeth.2017.05.005 (2017)
BACKGROUND: Generation and phenotyping of mutant mouse models continues to increase along with the search for the most efficient phenotyping tests. Here we asked if a combination of different locomotor tests is necessary for comprehensive locomotor phenotyping, or if a large data set from an automated gait analysis with the CatWalk system would suffice. NEW METHOD: First we endeavored to meaningfully reduce the large CatWalk data set by Principal Component Analysis (PCA) to decide on the most relevant parameters. We analyzed the influence of sex, body weight, genetic background and age. Then a combination of different locomotor tests was analyzed to investigate the possibility of redundancy between tests. RESULT: The extracted 10 components describe 80% of the total variance in the CatWalk, characterizing different aspects of gait. With these, effects of CatWalk version, sex, body weight, age and genetic background were detected. In addition, the PCA on a combination of locomotor tests suggests that these are independent without significant redundancy in their locomotor measures. COMPARISON WITH EXISTING METHODS: The PCA has permitted the refinement of the highly dimensional CatWalk (and other tests) data set for the extraction of individual component scores and subsequent analysis. CONCLUSION: The outcome of the PCA suggests the possibility to focus on measures of the front and hind paws, and one measure of coordination in future experiments to detect phenotypic differences. Furthermore, although the CatWalk is sensitive for detecting locomotor phenotypes pertaining to gait, it is necessary to include other tests for comprehensive locomotor phenotyping.
Wissenschaftlicher Artikel
Scientific Article
2016
Allergy 71, 507-507 (2016)
Meeting abstract
Meeting abstract
Basic Res. Cardiol. 111:6 (2016)
Cardiomyopathy is one of the most common causes of chronic heart failure worldwide. Mutations in the gene encoding nexilin (NEXN) occur in patients with both hypertrophic and dilated cardiomyopathy (DCM); however, little is known about the pathophysiological mechanisms and relevance of NEXN to these disorders. Here, we evaluated the functional role of NEXN using a constitutive Nexn knock-out (KO) mouse model. Heterozygous (Het) mice were inter-crossed to produce wild-type (WT), Het, and homozygous KO mice. At birth, 32, 46, and 22 % of the mice were WT, Het, and KO, respectively, which is close to the expected Mendelian ratio. After postnatal day 6, the survival of the Nexn KO mice decreased dramatically and all of the animals died by day 8. Phenotypic characterizations of the WT and KO mice were performed at postnatal days 1, 2, 4, and 6. At birth, the relative heart weights of the WT and KO mice were similar; however, at day 4, the relative heart weight of the KO group was 2.3-fold higher than of the WT group. In addition, the KO mice developed rapidly progressive cardiomyopathy with left ventricular dilation and wall thinning and decreased cardiac function. At day 6, the KO mice developed a fulminant DCM phenotype characterized by dilated ventricular chambers and systolic dysfunction. At this stage, collagen deposits and some elastin deposits were observed within the left ventricle cavity, which resembles the features of endomyocardial fibroelastosis (EFE). Overall, these results further emphasize the role of NEXN in DCM and suggest a novel role in EFE.
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Scientific Article
Haematologica 101, e48-e51 (2016)
Wissenschaftlicher Artikel
Scientific Article
Diabetologia 59, S259-S259 (2016)
Meeting abstract
Meeting abstract
PLoS ONE 11:e0152996 (2016)
The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2AHOZ) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2AHOZ mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4+ T-cells and CD8+ effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2AHOZ as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects.
Wissenschaftlicher Artikel
Scientific Article
Am. J. Respir. Crit. Care Med. 193 (2016)
Meeting abstract
Meeting abstract
Nature 537, 508-514 (2016)
Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
Wissenschaftlicher Artikel
Scientific Article
Mamm. Genome 27, 111-121 (2016)
We performed exome sequencing for mutation discovery of an ENU (N-ethyl-N-nitrosourea)-derived mouse model characterized by significant elevated plasma alkaline phosphatase (ALP) activities in female and male mutant mice, originally named BAP014 (bone screen alkaline phosphatase #14). We identified a novel loss-of-function mutation within the Fam46a (family with sequence similarity 46, member A) gene (NM_001160378.1:c.469G>T, NP_001153850.1:p.Glu157*). Heterozygous mice of this mouse line (renamed Fam46a (E157*Mhda)) had significantly high ALP activities and apparently no other differences in morphology compared to wild-type mice. In contrast, homozygous Fam46a (E157*Mhda) mice showed severe morphological and skeletal abnormalities including short stature along with limb, rib, pelvis, and skull deformities with minimal trabecular bone and reduced cortical bone thickness in long bones. ALP activities of homozygous mutants were almost two-fold higher than in heterozygous mice. Fam46a is weakly expressed in most adult and embryonic tissues with a strong expression in mineralized tissues as calvaria and femur. The FAM46A protein is computationally predicted as a new member of the superfamily of nucleotidyltransferase fold proteins, but little is known about its function. Fam46a (E157*Mhda) mice are the first mouse model for a mutation within the Fam46a gene.
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Scientific Article
Nat. Commun. 7:11317 (2016)
Calcium signalling plays a critical role in the pathogenesis of heart failure. Here we describe a cardiac protein named Myoscape/FAM40B/STRIP2, which directly interacts with the L-type calcium channel. Knockdown of Myoscape in cardiomyocytes decreases calcium transients associated with smaller Ca(2+) amplitudes and a lower diastolic Ca(2+) content. Likewise, L-type calcium channel currents are significantly diminished on Myoscape ablation, and downregulation of Myoscape significantly reduces contractility of cardiomyocytes. Conversely, overexpression of Myoscape increases global Ca(2+) transients and enhances L-type Ca(2+) channel currents, and is sufficient to restore decreased currents in failing cardiomyocytes. In vivo, both Myoscape-depleted morphant zebrafish and Myoscape knockout (KO) mice display impairment of cardiac function progressing to advanced heart failure. Mechanistically, Myoscape-deficient mice show reduced L-type Ca(2+)currents, cell capacity and calcium current densities as a result of diminished LTCC surface expression. Finally, Myoscape expression is reduced in hearts from patients suffering of terminal heart failure, implying a role in human disease.
Wissenschaftlicher Artikel
Scientific Article
Nat. Med. 22, 1428-1438 (2016)
Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.
Wissenschaftlicher Artikel
Scientific Article
Cell 167, 843-857.e14 (2016)
Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.
Wissenschaftlicher Artikel
Scientific Article
Diabetes 65, 2540-2552 (2016)
Bezafibrate (BEZ), a pan activator of peroxisome proliferator-activated receptors (PPARs), has been generally used to treat hyperlipidemia for decades. Clinical trials with type 2 diabetes patients indicated that BEZ also has beneficial effects on glucose metabolism, although the underlying mechanisms of these effects remain elusive. Even less is known about a potential role for BEZ in treating type 1 diabetes. Here we show that BEZ markedly improves hyperglycemia and glucose and insulin tolerance in streptozotocin (STZ)-treated mice, an insulin-deficient mouse model of type 1 diabetes. BEZ treatment of STZ mice significantly suppressed the hepatic expression of genes that are annotated in inflammatory processes, whereas the expression of PPAR and insulin target gene transcripts was increased. Furthermore, BEZ-treated mice also exhibited improved metabolic flexibility as well as an enhanced mitochondrial mass and function in the liver. Finally, we show that the number of pancreatic islets and the area of insulin positive cells tended to be higher in BEZ-treated mice. Our data suggest that BEZ may improve impaired glucose metabolism by augmenting hepatic mitochondrial performance, suppressing hepatic inflammatory pathways, and improving insulin sensitivity and metabolic flexibility. Thus, BEZ treatment might also be useful for patients with impaired glucose tolerance or diabetes.
Wissenschaftlicher Artikel
Scientific Article
Glycoconj. J. 34, 393–404 (2016)
Secondary polycythemia, a disease characterized by a selective increase in circulating mature erythrocytes, is caused by enhanced erythropoietin (Epo) concentrations triggered by hypoxia-inducible factor-2α (HIF-2α). While mechanisms of hypoxia-dependent stabilization of HIF-2α protein are well established, data regarding oxygen-independent regulation of HIF-2α are sparse. In this study, we generated a novel transgenic mouse model, in which biglycan was constitutively overexpressed and secreted by hepatocytes (BGN (Tg)), thereby providing a constant source of biglycan released into the blood stream. We discovered that although the mice were apparently normal, they harbored an increase in mature circulating erythrocytes. In addition to erythrocytosis, the BGN (Tg) mice showed elevated hemoglobin concentrations, hematocrit values and enhanced total iron binding capacity, revealing a clinical picture of polycythemia. In BGN (Tg) mice markedly enhanced Epo mRNA expression was observed in the liver and kidney, while elevated Epo protein levels were found in liver, kidney and blood. Mechanistically, we showed that the transgenic animals had an abundance of HIF-2α protein in the liver and kidney. Finally, by transiently overexpressing circulating biglycan in mice deficient in various Toll-like receptors (TLRs), we determined that this novel function of biglycan to promote Epo synthesis was specifically mediated by a selective interaction with TLR2. Thus, we discovered a novel biological pathway of soluble biglycan inducing HIF-2α protein stabilization and Epo production presumably in an oxygen-independent manner, ultimately giving rise to secondary polycythemia.
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Scientific Article
Genes Genomes Genetics G3 6, 4035-4046 (2016)
The vertebrate Scube (Signal peptide, CUB and EGF-like domain-containing protein) family consists of three independent members Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K) that clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to understand SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3. In addition, alterations in energy metabolism, behavior and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.
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Scientific Article
Nat. Genet. 48, 497-499 (2016)
There is considerable controversy regarding epigenetic inheritance in mammalian gametes. Using in vitro fertilization to ensure exclusive inheritance via the gametes, we show that a parental high-fat diet renders offspring more susceptible to developing obesity and diabetes in a sex- and parent of origin-specific mode. The epigenetic inheritance of acquired metabolic disorders may contribute to the current obesity and diabetes pandemic.
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Scientific Article
J. Breath Res. 10:016009 (2016)
The prevalence of obesity is still rising in many countries, resulting in an increased risk of associated metabolic diseases. In this study we aimed to describe the volatile organic compound (VOC) patterns symptomatic for obesity. We analyzed high fat diet (HFD) induced obese and mono-genetic obese mice (global knock-in mutation in melanocortin-4 receptor MC4R-ki). The source strengths of 208 VOCs were analyzed in ad libitum fed mice and after overnight food restriction. Volatiles relevant for a random forest-based separation of obese mice were detected (26 in MC4R-ki, 22 in HFD mice). Eight volatiles were found to be important in both obesity models. Interestingly, by creating a partial correlation network of the volatile metabolites, the chemical and metabolic origins of several volatiles were identified. HFD-induced obese mice showed an elevation in the ketone body acetone and acrolein, a marker of lipid peroxidation, and several unidentified volatiles. In MC4R-ki mice, several yet-unidentified VOCs were found to be altered. Remarkably, the pheromone (methylthio)methanethiol was found to be reduced, linking metabolic dysfunction and reproduction. The signature of volatile metabolites can be instrumental in identifying and monitoring metabolic disease states, as shown in the screening of the two obese mouse models in this study. Our findings show the potential of breath gas analysis to non-invasively assess metabolic alterations for personalized diagnosis.
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Scientific Article
J. Inherit. Metab. Dis. 39, 309-319 (2016)
Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15 % of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90 % reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.
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Scientific Article
PLoS ONE 11:e0150472 (2016)
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 recessive mutant line HST011 was established and further analyzed. The causative mutation was detected in the POU domain, class 3 transcription factor 3 (Pou3f3) gene, which leads to the amino acid exchange Pou3f3L423P thereby affecting the conserved homeobox domain of the protein. Pou3f3 homozygous knockout mice are published and show perinatal death. Line Pou3f3L423P is a viable mouse model harboring a homozygous Pou3f3 mutation. Standardized, systemic phenotypic analysis of homozygous mutants was carried out in the German Mouse Clinic. Main phenotypic changes were low body weight and a state of low energy stores, kidney dysfunction and secondary effects thereof including low bone mineralization, multiple behavioral and neurological defects including locomotor, vestibular, auditory and nociceptive impairments, as well as multiple subtle changes in immunological parameters. Genome-wide transcriptome profiling analysis of kidney and brain of Pou3f3L423P homozygous mutants identified significantly regulated genes as compared to wild-type controls.
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Scientific Article
Am. J. Physiol.-Renal Physiol. 310, F785-F795 (2016)
Chronic kidney disease (CKD) research is limited by the lack of convenient inducible models mimicking human CKD and its complications in experimental animals. We demonstrate that a soluble oxalate-rich diet induces stable stages of CKD in male and female C57BL/6 mice. Renal histology is characterized by tubular damage, remnant atubular glomeruli, interstitial inflammation, and fibrosis with the extent of tissue involvement depending on the duration of oxalate feeding. Expression profiling of markers and magnetic resonance imaging findings established to reflect inflammation and fibrosis parallel the histological changes. Within 3 weeks the mice reproducibly develop normochromic anemia, metabolic acidosis, hyperkalemia, FGF23 activation, hyperphosphatemia and hyperparathyroidism. In addition, the model is characterized by profound arterial hypertension as well as cardiac fibrosis that persist following the switch to a control diet. Together, this new model of inducible CKD overcomes a number of previous experimental limitations and should serve useful in research related to CKD and its complications.
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Scientific Article
J. Am. Soc. Nephrol. 28, 761-768 (2016)
Intrarenal crystals trigger inflammation and renal cell necroptosis, processes that involve TNF receptor (TNFR) signaling. Here, we tested the hypothesis that TNFRs also have a direct role in tubular crystal deposition and progression of hyperoxaluria-related CKD. Immunohistochemical analysis revealed upregulated tubular expression of TNFR1 and TNFR2 in human and murine kidneys with calcium oxalate (CaOx) nephrocalcinosis-related CKD compared with controls. Western blot and mRNA expression analyses in mice yielded consistent data. When fed an oxalate-rich diet, wild-type mice developed progressive CKD, whereas Tnfr1-, Tnfr2-, and Tnfr1/2-deficient mice did not. Despite identical levels of hyperoxaluria, Tnfr1-, Tnfr2-, and Tnfr1/2-deficient mice also lacked the intrarenal CaOx deposition and tubular damage observed in wild-type mice. Inhibition of TNFR signaling prevented the induced expression of the crystal adhesion molecules, CD44 and annexin II, in tubular epithelial cells in vitro and in vivo, and treatment with the small molecule TNFR inhibitor R-7050 partially protected hyperoxaluric mice from nephrocalcinosis and CKD. We conclude that TNFR signaling is essential for CaOx crystal adhesion to the luminal membrane of renal tubules as a fundamental initiating mechanism of oxalate nephropathy. Furthermore, therapeutic blockade of TNFR might delay progressive forms of nephrocalcinosis in oxalate nephropathy, such as primary hyperoxaluria.
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Scientific Article
J. Amer. Assoc. Lab. Anim. Sci. 55, 375-386 (2016)
Although various mouse inbred strains are widely used to investigate disease mechanisms and to establish new therapeutic strategies, sex-specific reference intervals for laboratory diagnostic analytes that are generated from large numbers of animals have been unavailable. In this retrospective study, we screened data from more than 12,000 mice phenotyped in the German Mouse Clinic from January 2006 through June 2014 and selected animals with the genetic background of C57BL/6J, C57BL/6N, or C3HeB/FeJ. In addition, we distinguished between the C57BL/6NTac substrain and C57BL/6N mice received from other vendors. The corresponding data sets of electrolytes (sodium, potassium, calcium, chloride, inorganic phosphate), lipids (cholesterol, triglyceride), and enzyme activities (ALT, AST, ALP, α-amylase) and urea, albumin, and total protein levels were analyzed. Significant effects of age and sex on these analytes were identified, and strain- or substrain- and sex-specific reference intervals for 90- to 135-d-old mice were calculated. In addition, we include an overview of the literature that reports clinical chemistry values for wild-type mice of different strains. Our results support researchers interpreting clinical chemistry values from various mouse mutants and corresponding wild-type controls based on the examined strains and substrains.
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Scientific Article
Mamm. Genome 27, 445-450 (2016)
Ageing research and more generally the study of the functional basis of human diseases profit enormously from the large-scale approaches and resources in mouse functional genomics: systematic targeted mutation of the mouse genome, systemic phenotyping in mouse clinics, and the archiving and distribution of the mouse resources in public repositories. INFRAFRONTIER, the European research infrastructure for the development, systemic phenotyping, archiving and distribution of mammalian models, offers access to sustainable mouse resources for biomedical research. INFRAFRONTIER promotes the global sharing of high-quality resources and data and thus contributes to data reproducibility and animal welfare. INFRAFRONTIER puts great effort into international standardisation and quality control and into technology development to improve and expand experimental protocols, reduce the use of animals in research and increase the reproducibility of results. In concert with the research community and the International Mouse Phenotyping Consortium (IMPC), INFRAFRONTIER is currently developing new pilot platforms and services for the research on ageing and age-related diseases.
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Scientific Article
J. Invest. Dermatol. 136, 2, S231-S231 (2016)
Meeting abstract
Meeting abstract
Allergy 71, 47-47 (2016)
Meeting abstract
Meeting abstract
Mamm. Genome 27, 587-598 (2016)
Animal models resembling human mutations are valuable tools to research the features of complex human craniofacial syndromes. This is the first report on a viable dominant mouse model carrying a non-synonymous sequence variation within the endothelin receptor type A gene (Ednra c.386A>T, p.Tyr129Phe) derived by an ENU mutagenesis program. The identical amino acid substitution was reported recently as disease causing in three individuals with the mandibulofacial dysostosis with alopecia (MFDA, OMIM 616367) syndrome. We performed standardized phenotyping of wild-type, heterozygous, and homozygous Ednra (Y129F) mice within the German Mouse Clinic. Mutant mice mimic the craniofacial phenotypes of jaw dysplasia, micrognathia, dysplastic temporomandibular joints, auricular dysmorphism, and missing of the squamosal zygomatic process as described for MFDA-affected individuals. As observed in MFDA-affected individuals, mutant Ednra (Y129F) mice exhibit hearing impairment in line with strong abnormalities of the ossicles and further, reduction of some lung volumetric parameters. In general, heterozygous and homozygous mice demonstrated inter-individual diversity of expression of the craniofacial phenotypes as observed in MFDA patients but without showing any cleft palates, eyelid defects, or alopecia. Mutant Ednra (Y129F) mice represent a valuable viable model for complex human syndromes of the first and second pharyngeal arches and for further studies and analysis of impaired endothelin 1 (EDN1)-endothelin receptor type A (EDNRA) signaling. Above all, Ednra (Y129F) mice model the recently published human MFDA syndrome and may be helpful for further disease understanding and development of therapeutic interventions.
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J. Diabetes Res. 2016:4208156 (2016)
© 2016 Tanja Telieps et al. Immune phenotyping provides insight into disease pathogenesis and prognostic markers. Trajectories from age of 4 to 36 weeks were modeled for insulin autoantibodies and for leukocyte subpopulations in peripheral blood from female NOD (n = 58) and NOR (n = 22) mice. NOD mice had higher trajectories of insulin autoantibodies, CD4+ and CD8+ T lymphocytes, B lymphocytes, IgD+IgM- B lymphocytes, and NK cells and lower trajectories of CD4+CD25+ T lymphocytes, IgM+ B lymphocytes, granulocytes, and monocytes than NOR mice (all p < 0.001). Of these, only the increased IAA trajectory was observed in NOD mice that developed diabetes as compared to NOD mice that remained diabetes-free. Therefore, the profound differences in peripheral blood leukocyte proportions observed between the diabetes-prone NOD mice and the diabetes-resistant mice do not explain the variation in diabetes development within NOD mice and do not provide markers for diabetes prediction in this model.
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Liver lipid metabolism is altered by increased circulating estrogen to androgen ratio in male mouse.
J. Proteomics 133, 66-75 (2016)
Estrogens are suggested to lower the risk of developing metabolic syndrome in both sexes. In this study, we investigated how the increased circulating estrogen-to-androgen ratio (E/A) alters liver lipid metabolism in males. The cytochrome P450 aromatase (P450arom) is an enzyme converting androgens to estrogens. Male mice overexpressing human aromatase enzyme (AROM+ mice), and thus have high circulating E/A, were used as a model in this study. Proteomics and gene expression analyses indicated an increase in the peroxisomal β-oxidation in the liver of AROM+ mice as compared with their wild type littermates. Correspondingly, metabolomic analysis revealed a decrease in the amount of phosphatidylcholines with long-chain fatty acids in the plasma. With interest we noted that the expression of Cyp4a12a enzyme, which specifically metabolizes arachidonic acid (AA) to 20-hydroxy AA, was dramatically decreased in the AROM+ liver. As a consequence, increased amounts of phospholipids having AA as a fatty acid tail were detected in the plasma of the AROM+ mice. Overall, these observations demonstrate that high circulating E/A in males is linked to indicators of higher peroxisomal β-oxidation and lower AA metabolism in the liver. Furthermore, the plasma phospholipid profile reflects the changes in the liver lipid metabolism. BIOLOGICAL SIGNIFICANCE: The role of sex steroid hormones in the development of metabolic diseases is a topical issue. Lipid metabolism in both sexes supposedly benefits from estrogens, and low circulating estrogen to androgen ratio has been shown to lead to liver steatosis in males. However, there are no comprehensive studies showing the effects of sex steroid hormones on the expression of genes regulating liver lipid metabolism on both mRNA and protein levels. In this study a combination of quantitative MS-based proteome measurements and mRNA microarray both consistently indicated a set of genes that are deregulated in the liver of male mice having high circulating E/A. Interestingly, the results of targeted profiling of phospholipids in the plasma by LC-MS/MS were in line with the mRNA and protein measurements carried out in the liver, suggesting that plasma phospholipid profile could be used as an indicator of altered liver lipid metabolism.
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Scientific Article
PLoS ONE 11:e0164298 (2016)
Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility.
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Scientific Article
Mol. Neurobiol. 54, 8242–8262 (2016)
Stress experience modulates behavior, metabolism, and energy expenditure of organisms. One molecular hallmark of an acute stress response is a rapid induction of immediate early genes (IEGs) such as c-Fos and Egr family members. IEG transcription in neurons is mediated by the neuronal activity-driven gene regulator serum response factor (SRF). We show a first role of SRF in immediate and long-lasting acute restraint stress (AS) responses. For this, we employed a standardized mouse phenotyping protocol at the German Mouse Clinic (GMC) including behavioral, metabolic, and cardiologic tests as well as gene expression profiling to analyze the consequences of forebrain-specific SRF deletion in mice exposed to AS. Adult mice with an SRF deletion in glutamatergic neurons (Srf;CaMKIIa-CreERT2) showed hyperactivity, decreased anxiety, and impaired working memory. In response to restraint AS, instant stress reactivity including locomotor behavior and corticosterone induction was impaired in Srf mutant mice. Interestingly, even several weeks after previous AS exposure, SRF-deficient mice showed long-lasting AS-associated changes including altered locomotion, metabolism, energy expenditure, and cardiovascular changes. This suggests a requirement of SRF for mediating long-term stress coping mechanisms in wild-type mice. SRF ablation decreased AS-mediated IEG induction and activity of the actin severing protein cofilin. In summary, our data suggest an SRF function in immediate AS reactions and long-term post-stress-associated coping mechanisms.
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2015
Mamm. Genome 27, 17-28 (2015)
Epigenetic inheritance (EI) of metabolic phenotypes via the paternal lineage has been shown in rodent models of diet-induced obesity (DIO). However, the factors involved in soma-to-germline information transfer remain elusive. Here, we address the role of alterations in insulin, leptin, and adiponectin levels for EI of metabolic phenotypes by treating C57BL/6NTac male mice (F0) with the synthetic glucocorticoid dexamethasone and generating offspring (F1) either by in vitro fertilization or by natural fecundation. Dexamethasone treatment slightly alters F0 body composition by increasing fat mass and decreasing lean mass, and significantly improves glucose tolerance. Moreover, it increases insulin and leptin levels and reduces adiponectin levels in F0 fathers as observed in mouse models of DIO. However, these paternal changes of metabolic hormones do not alter metabolic parameters, such as body weight, body composition and glucose homeostasis in male and female F1 mice even when these are challenged with a high-fat diet. Accordingly, sperm transcriptomes are not altered by dexamethasone treatment. Our results suggest that neither increased glucocorticoid, insulin, and leptin levels, nor decreased adiponectin levels in fathers are sufficient to confer soma-to-germline information transfer in EI of obesity via the paternal lineage.
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Scientific Article
Nat. Commun. 6:8261 (2015)
Insulin regulates glycaemia, lipogenesis and increases mRNA translation. Cells with reduced eukaryotic initiation factor 6 (eIF6) do not increase translation in response to insulin. The role of insulin-regulated translation is unknown. Here we show that reduction of insulin-regulated translation in mice heterozygous for eIF6 results in normal glycaemia, but less blood cholesterol and triglycerides. eIF6 controls fatty acid synthesis and glycolysis in a cell autonomous fashion. eIF6 acts by exerting translational control of adipogenic transcription factors like C/EBPβ, C/EBPδ and ATF4 that have G/C rich or uORF sequences in their 5' UTR. The outcome of the translational activation by eIF6 is a reshaping of gene expression with increased levels of lipogenic and glycolytic enzymes. Finally, eIF6 levels modulate histone acetylation and amounts of rate-limiting fatty acid synthase (Fasn) mRNA. Since obesity, type 2 diabetes, and cancer require a Fasn-driven lipogenic state, we propose that eIF6 could be a therapeutic target for these diseases.
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Functional compensation among HMGN variants modulates the DNase I hypersensitive sites at enhancers.
Genome Res. 25, 1295-1308 (2015)
DNase I hypersensitive sites (DHSs) are a hallmark of chromatin regions containing regulatory DNA such as enhancers and promoters; however, the factors affecting the establishment and maintenance of these sites are not fully understood. We now show that HMGN1 and HMGN2, nucleosome-binding proteins that are ubiquitously expressed in vertebrate cells, maintain the DHSs landscape of mouse embryonic fibroblasts (MEFs) synergistically. Loss of one of these HMGN variants led to a compensatory increase of binding of the remaining variant. Genome wide mapping of the DHSs in Hmgn1-/-, Hmgn2-/- and Hmgn1-/-n2-/- MEFs reveals that loss of both, but not a single HMGN variant, leads to significant remodeling of the DHSs landscape, especially at enhancer regions marked by H3K4me1 and H3K27ac. Loss of HMGN variants affects the induced expression of stress responsive genes in MEFs, the transcription profiles of several mouse tissues, and leads to altered phenotypes that are not seen in mice lacking only one variant. We conclude that the compensatory binding of HMGN variants to chromatin maintains the DHSs landscape and the transcription fidelity and is necessary to retain wild type phenotypes. Our study provides insights into mechanisms that maintain regulatory sites in chromatin and into functional compensation among nucleosome binding architectural proteins.
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Scientific Article
Front. Behav. Neurosci. 9:302 (2015)
Adult neurogenesis occurs in the adult mammalian subventricular zone (SVZ) along the walls of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus. While a burgeoning body of research implicates adult neurogenesis in olfactory bulb (OB)- and hippocampal-related behaviors, the precise function continues to elude. To further assess the behavioral importance of adult neurogenesis, we herein generated a novel inducible transgenic mouse model of adult neurogenesis reduction where mice with CreERT2 under doublecortin (DCX) promoter control were crossed with mice where diphtheria toxin A (DTA) was driven by the Rosa26 promoter. Activation of DTA, through the administration of tamoxifen (TAM), results in a specific reduction of DCX+ immature neurons in both the hippocampal dentate gyrus and OB. We show that the decrease of DCX+ cells causes impaired social discrimination ability in both young adult (from 3 months) and middle aged (from 10 months) mice. Furthermore, these animals showed an age-independent altered coping behavior in the Forced Swim Test without clear changes in anxiety-related behavior. Notably, these behavior changes were reversible on repopulating the neurogenic zones with DCX+ cells on cessation of the TAM treatment, demonstrating the specificity of this effect. Overall, these results support the notion that adult neurogenesis plays a role in social memory and in stress coping but not necessarily in anxiety-related behavior.
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Scientific Article
Front. Behav. Neurosci. 9:302 (2015)
Adult neurogenesis occurs in the adult mammalian subventricular zone (SVZ) along the walls of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus. While a burgeoning body of research implicates adult neurogenesis in olfactory bulb (OB)- and hippocampal-related behaviors, the precise function continues to elude. To further assess the behavioral importance of adult neurogenesis, we herein generated a novel inducible transgenic mouse model of adult neurogenesis reduction where mice with CreERT2 under doublecortin (DCX) promoter control were crossed with mice where diphtheria toxin A (DTA) was driven by the Rosa26 promoter. Activation of DTA, through the administration of tamoxifen (TAM), results in a specific reduction of DCX+ immature neurons in both the hippocampal dentate gyrus and OB. We show that the decrease of DCX+ cells causes impaired social discrimination ability in both young adult (from 3 months) and middle aged (from 10 months) mice. Furthermore, these animals showed an age-independent altered coping behavior in the Forced Swim Test without clear changes in anxiety-related behavior. Notably, these behavior changes were reversible on repopulating the neurogenic zones with DCX+ cells on cessation of the TAM treatment, demonstrating the specificity of this effect. Overall, these results support the notion that adult neurogenesis plays a role in social memory and in stress coping but not necessarily in anxiety-related behavior.
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Dev. Biol. 399, 2-14 (2015)
The correct wiring of neuronal circuits is of crucial importance for the function of the vertebrate nervous system. Guidance cues like the neuropilin receptors (Npn) and their ligands, the semaphorins (Sema) provide a tight spatiotemporal control of sensory and motor axon growth and guidance. Among this family of guidance partners the Sema3A-Npn1 interaction has been shown to be of great importance, since defective signaling leads to wiring deficits and defasciculation. For the embryonic stage these defects have been well described, however, also after birth the organism can adapt to new challenges by compensational mechanisms. Therefore, we used the mouse lines Olig2-Cre;Npn1(cond) and Npn1(Sema-) to investigate how postnatal organisms cope with the loss of Npn1 selectively from motor neurons or a systemic dysfunctional Sema3A-Npn1 signaling in the entire organism, respectively. While in Olig2-Cre(+);Npn1(cond-/-) mice clear anatomical deficits in paw posturing, bone structure, as well as muscle and nerve composition became evident, Npn1(Sema-) mutants appeared anatomically normal. Furthermore, Olig2-Cre(+);Npn1(cond) mutants revealed a dysfunctional extensor muscle innervation after single-train stimulation of the N.radial. Interestingly, these mice did not show obvious deficits in voluntary locomotion, however, skilled motor function was affected. In contrast, Npn1(Sema-) mutants were less affected in all behavioral tests and able to improve their performance over time. Our data suggest that loss of Sema3A-Npn1 signaling is not the only cause for the observed deficits in Olig2-Cre(+);Npn1(cond-/-) mice and that additional, yet unknown binding partners for Npn1 may be involved that allow Npn1(Sema-) mutants to compensate for their developmental deficits.
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Am. J. Physiol. Lung Cell Mol. Physiol. 308, L1114-L1124 (2015)
Microfibrillar-associated protein 4 (MFAP4) is localized to elastic fibers in blood vessels and the interalveolar septa of the lungs and is further present in bronchoalveolar lavage. Mfap4 has been previously suggested to be involved in elastogenesis in the lung. We tested this prediction and aimed to characterize the pulmonary function changes and emphysematous changes that occur in Mfap4 deficient (Mfap4-/-) mice. Significant changes included increases in total lung capacity and compliance, which were evident in Mfap4-/- mice at 6 months and 8 months, but not at 3 months of age. Using in vivo breath-hold gated micro-computed tomography (micro-CT) in 8-month-old Mfap4-/- mice, we found that the mean density of the lung parenchyma was decreased, and the low-attenuation area (LAA) was significantly increased by 14 % compared to Mfap4+/+ mice. Transmission electron microscopy (TEM) did not reveal differences in the organization of elastic fibers, and there was no difference in elastin content, but borderline significant increase in elastin mRNA expression in 3-month-old mice. Stereological analysis showed that alveolar surface density in relation to the lung parenchyma and total alveolar surface area inside of the lung were both significantly decreased in Mfap4-/- mice by 25 % and 15 %, respectively. The data did not support an essential role of MFAP4 in pulmonary elastic fiber organization or content, but indicated increased turnover in young Mfap4-/- mice. However, Mfap4-/- mice developed a spontaneous loss of lung function, which was evident at 6 months of age, and moderate airspace enlargement, with emphysema-like changes.
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Curr. Protoc. Mouse Biol. 5, 291-301:doi: 10.1002/9780470942390.mo160 (2015)
Phenotyping of inbred mouse strains and genetically modified mouse models for characteristics related to neuropsychiatric diseases includes assessing their anxiety-related behavior. A variety of tests have been developed to measure anxiety in laboratory rodents and these tests have been placed under scrutiny over the years concerning their validity. Here we describe the most widely used tests for anxiety in mice. The protocols we present are established methods used in the German Mouse Clinic (GMC), with which alterations in anxiety could successfully be discovered in mouse mutants. Moreover, since baseline anxiety levels in mice are easily influenced by a great variety of disturbances, we carefully outline the critical parameters that need to be considered.
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Scientific Article
Curr. Protoc. Mouse Biol. 5, 331-358:doi: 10.1002/9780470942390.mo160 (2015)
Genetically modified mouse models have proven useful to study learning and memory processes and the neurocircuitry and molecular mechanisms involved, as well as to develop therapies for diseases involving cognitive impairment. A variety of tests have been developed to measure cognition in mice, and here we present those established and regularly used in the German Mouse Clinic. The test paradigms have been carefully chosen according to reliability of results and disease relevance of the cognitive functions assessed. Further criteria were time efficiency and ease of application. All tests assess slightly different but also overlapping or interacting aspects of learning and memory so that they can be used to complement each other in a comprehensive assessment of cognitive function. The five protocols described are for spontaneous alternation in the Y-maze, social discrimination, object recognition, automated assessment of learning and memory using the IntelliCage, and olfactory discrimination learning.
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Scientific Article
PLoS ONE 10:e0134503 (2015)
We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype - envirotype interactions for other diseases.
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Scientific Article
Nat. Genet. 47, 969-978 (2015)
The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.
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Scientific Article
Mol. Metab. 4, 39-50 (2015)
Objective Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. Methods We induced hepatosteatosis by feeding C3HeB/FeJ male mice a HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. Results Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. Conclusions We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties – in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.
Wissenschaftlicher Artikel
Scientific Article
PLoS Biol. 13:e1002151 (2015)
The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were developed to address the lack of reproducibility in biomedical animal studies and improve the communication of research findings. While intended to guide the preparation of peer-reviewed manuscripts, the principles of transparent reporting are also fundamental for in vivo databases. Here, we describe the benefits and challenges of applying the guidelines for the International Mouse Phenotyping Consortium (IMPC), whose goal is to produce and phenotype 20,000 knockout mouse strains in a reproducible manner across ten research centres. In addition to ensuring the transparency and reproducibility of the IMPC, the solutions to the challenges of applying the ARRIVE guidelines in the context of IMPC will provide a resource to help guide similar initiatives in the future.
Wissenschaftlicher Artikel
Scientific Article
Mamm. Genome 26, 33-42 (2015)
Sequences encoding DUF1220 protein domains show the most extreme human lineage-specific copy number increase of any coding region in the genome and have been linked to human brain evolution. In addition, DUF1220 copy number (dosage) has been implicated in influencing brain size within the human species, both in normal populations and in individuals associated with brain size pathologies (1q21-associated microcephaly and macrocephaly). More recently, increasing dosage of a subtype of DUF1220 has been linked with increasing severity of the primary symptoms of autism. Despite these intriguing associations, a function for these domains has not been described. As a first step in addressing this question, we have developed the first transgenic model of DUF1220 function by removing the single DUF1220 domain (the ancestral form) encoded in the mouse genome. In a hypothesis generating exercise, these mice were evaluated by 197 different phenotype measurements. While resulting DUF1220-minus (KO) mice show no obvious anatomical peculiarities, they exhibit a significantly reduced fecundity (χ (2) = 19.1, df = 2, p = 7.0 × 10(-5)). Further extensive phenotypic analyses suggest hyperactivity (p < 0.05) of DUF1220 mice and changes in gene expression levels of brain associated with distinct neurological functions and disease. Other changes that met statistical significance include an increase in plasma glucose concentration (as measured by area under the curve, AUC 0-30 and AUC 30-120) in male mutants, fasting glucose levels, reduce sodium levels in male mutants, increased levels of the liver functional indicator ALAT/GPT in males, levels of alkaline phosphatase (also an indicator of liver function), mean R and SR amplitude by electrocardiography, elevated IgG3 levels, a reduced ratio of CD4:CD8 cells, and a reduced frequency of T cells; though it should be noted that many of these differences are quite small and require further examination. The linking of DUF1220 loss to a hyperactive phenotype is consistent with separate findings in which DUF1220 over expression results in a down-regulation of mitochondrial function, and potentially suggests a role in developmental metabolism. Finally, the substantially reduced fecundity we observe associated with KO mice argues that the ancestral DUF1220 domain provides an important biological functionthat is critical to survivability and reproductive success.
Wissenschaftlicher Artikel
Scientific Article
Circulation 131, 1191-1201 (2015)
BACKGROUND: -ADAMTS-7, a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, is recently identified to be genome-wide significantly associated with coronary artery disease (CAD). However, the mechanisms that link ADAMTS-7 and CAD risk remain elusive. We have previously demonstrated that ADAMTS-7 promotes vascular smooth muscle cell migration and post-injury neointima formation via degradation of a matrix protein cartilage oligomeric matrix protein (COMP). Because delayed endothelium repair renders neointima and atherosclerosis plaque formation after vessel injury, we examined whether ADAMTS-7 also inhibits re-endothelialization. METHODS AND RESULTS: -Wire-injury of the carotid artery and Evans blue staining were performed in Adamts7(-/-) and wildtype mice. Adamts-7 deficiency greatly promoted re-endothelialization at 3, 5, and 7 days after injury. Consequently, Adamts-7 deficiency substantially ameliorated neointima formation in mice at days 14 and 28 after injury compared with the wildtype. In vitro studies further indicated that ADAMTS-7 inhibited both endothelial cell proliferation and migration. Surprisingly, COMP deficiency did not affect endothelial cell proliferation/migration and re-endothelialization in mice. In a further examination of other potential vascular substrates of ADAMTS-7, a label-free LC MS/MS secretome analysis revealed thrombospondin-1 (TSP-1) as a potential ADAMTS-7 target. The subsequent studies showed that ADAMTS-7 was directly associated with TSP-1 by its C-terminus and degraded TSP-1 in vivo and in vitro. The inhibitory effect of ADAMTS-7 on post-injury endothelium recovery was circumvented in Tsp1(-/-) mice. CONCLUSIONS: -Our study revealed a novel mechanism by which ADAMTS-7 affects neointima formation. Thus, ADAMTS-7 is a promising treatment target for post-injury vascular intima hyperplasia.
Wissenschaftlicher Artikel
Scientific Article
J. Am. Heart Assoc. 4:e002153 (2015)
BACKGROUND: Ubiquitous deletion of thioredoxin reductase 2 (Txnrd2) in mice is embryonically lethal and associated with abnormal heart development, while constitutive, heart-specific Txnrd2 inactivation leads to dilated cardiomyopathy and perinatal death. The significance of Txnrd2 in aging cardiomyocytes, however, has not yet been examined. METHODS AND RESULTS: The tamoxifen-inducible heart-specific αMHC-MerCreMer transgene was used to inactivate loxP-flanked Txnrd2 alleles in adult mice. Hearts and isolated mitochondria from aged knockout mice were morphologically and functionally analyzed. Echocardiography revealed a significant increase in left ventricular end-systolic diameters in knockouts. Fractional shortening and ejection fraction were decreased compared with controls. Ultrastructural analysis of cardiomyocytes of aged mice showed mitochondrial degeneration and accumulation of autophagic bodies. A dysregulated autophagic activity was supported by higher levels of lysosome-associated membrane protein 1 (LAMP1), microtubule-associated protein 1A/1B-light chain 3-I (LC3-I), and p62 in knockout hearts. Isolated Txnrd2-deficient mitochondria used less oxygen and tended to produce more reactive oxygen species. Chronic hypoxia inducible factor 1, α subunit stabilization and altered transcriptional and metabolic signatures indicated that energy metabolism is deregulated. CONCLUSIONS: These results imply a novel role of Txnrd2 in sustaining heart function during aging and suggest that Txnrd2 may be a modifier of heart failure.
Wissenschaftlicher Artikel
Scientific Article
PLoS ONE 10:e0125304 (2015)
Cataracts are the major eye disorder and have been associated mainly with mutations in lens-specific genes, but cataracts are also frequently associated with complex syndromes. In a large-scale high-throughput ENU mutagenesis screen we analyzed the offspring of paternally treated C3HeB/FeJ mice for obvious dysmorphologies. We identified a mutant suffering from rough coat and small eyes only in homozygotes; homozygous females turned out to be sterile. The mutation was mapped to chromosome 7 between the markers 116J6.1 and D7Mit294;4 other markers within this interval did not show any recombination among 160 F2-mutants. The critical interval (8.6 Mb) contains 3 candidate genes (Apoe, Six5, Opa3); none of them showed a mutation. Using exome sequencing, we identified a c.2209T>C mutation in the Xpd/Ercc2 gene leading to a Ser737Pro exchange. During embryonic development, the mutant eyes did not show major changes. Postnatal histological analyses demonstrated small cortical vacuoles; later, cortical cataracts developed. Since XPD/ERCC2 is involved in DNA repair, we checked also for the presence of the repair-associated histone γH2AX in the lens. During the time, when primary lens fiber cell nuclei are degraded, γH2AX was strongly expressed in the cell nuclei; later, it demarcates clearly the border of the lens cortex to the organelle-free zone. Moreover, we analyzed also whether seemingly healthy heterozygotes might be less efficient in repair of DNA damage induced by ionizing radiation than wild types. Peripheral lymphocytes irradiated by 1Gy Cs137 showed 6 hrs after irradiation significantly more γH2AX foci in heterozygotes than in wild types. These findings demonstrate the importance of XPD/ERCC2 not only for lens fiber cell differentiation, but also for the sensitivity to ionizing radiation. Based upon these data, we hypothesize that variations in the human XPD/ERCC2 gene might increase the susceptibility for several disorders besides Xeroderma pigmentosum in heterozygotes under particular environmental conditions.
Wissenschaftlicher Artikel
Scientific Article
PLoS Genet. 11:e1005423 (2015)
We have studied the in vivo role of SLIRP in regulation of mitochondrial DNA (mtDNA) gene expression and show here that it stabilizes its interacting partner protein LRPPRC by protecting it from degradation. Although SLIRP is completely dependent on LRPPRC for its stability, reduced levels of LRPPRC persist in the absence of SLIRP in vivo. Surprisingly, Slirp knockout mice are apparently healthy and only display a minor weight loss, despite a 50-70% reduction in the steady-state levels of mtDNA-encoded mRNAs. In contrast to LRPPRC, SLIRP is dispensable for polyadenylation of mtDNA-encoded mRNAs. Instead, deep RNA sequencing (RNAseq) of mitochondrial ribosomal fractions and additional molecular analyses show that SLIRP is required for proper association of mRNAs to the mitochondrial ribosome and efficient translation. Our findings thus establish distinct functions for SLIRP and LRPPRC within the LRPPRC-SLIRP complex, with a novel role for SLIRP in mitochondrial translation. Very surprisingly, our results also demonstrate that mammalian mitochondria have a great excess of transcripts under basal physiological conditions in vivo.
Wissenschaftlicher Artikel
Scientific Article
Hum. Mol. Genet. 24, 7286-7294 (2015)
Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus maternally inherited traits. The pathophysiology induced by mtDNA mutations has traditionally been attributed to deficient oxidative phosphorylation, which causes energy crisis with functional impairment of multiple cellular processes. In contrast, it was recently reported that signaling induced by 'hypermethylation' of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness. In support for this concept, it was reported that overexpression of the essential mitochondrial methyltransferase TFB1M in the mouse was sufficient to induce mitoribosomal hypermethylation and deafness. At variance with this model, we show here that 12S rRNA is near fully methylated in vivo in the mouse and thus cannot be further methylated to any significant extent. Furthermore, bacterial artificial chromosome transgenic mice overexpressing TFB1M have no increase of 12S rRNA methylation levels and hear normally. We thus conclude that therapies directed against mitoribosomal methylation are unlikely to be beneficial to patients with sensorineural hearing loss or other types of mitochondrial disease.
Wissenschaftlicher Artikel
Scientific Article
Mamm. Genome 26, 467-481 (2015)
Large-scale systemic mouse phenotyping, as performed by mouse clinics for more than a decade, requires thousands of mice from a multitude of different mutant lines to be bred, individually tracked and subjected to phenotyping procedures according to a standardised schedule. All these efforts are typically organised in overlapping projects, running in parallel. In terms of logistics, data capture, data analysis, result visualisation and reporting, new challenges have emerged from such projects. These challenges could hardly be met with traditional methods such as pen & paper colony management, spreadsheet-based data management and manual data analysis. Hence, different Laboratory Information Management Systems (LIMS) have been developed in mouse clinics to facilitate or even enable mouse and data management in the described order of magnitude. This review shows that general principles of LIMS can be empirically deduced from LIMS used by different mouse clinics, although these have evolved differently. Supported by LIMS descriptions and lessons learned from seven mouse clinics, this review also shows that the unique LIMS environment in a particular facility strongly influences strategic LIMS decisions and LIMS development. As a major conclusion, this review states that there is no universal LIMS for the mouse research domain that fits all requirements. Still, empirically deduced general LIMS principles can serve as a master decision support template, which is provided as a hands-on tool for mouse research facilities looking for a LIMS.
Wissenschaftlicher Artikel
Scientific Article
Int. J. Cardiovasc. Imaging 31, 669-679 (2015)
Consistent protocols for the assessment of diastolic and systolic cardiac function to assure the comparability of existing data on preclinical models are missing. Calcineurin transgene (CN) mice are a preclinical model for hypertrophic and failing hearts. We aimed at evaluating left and right ventricular structural and functional remodeling in CN hearts with an optimized phenotyping protocol. We developed a protocol using techniques and indices comparable to those from human diagnostics for comprehensive in vivo cardiac screening using high-frequency echocardiography, Doppler, electrocardiography and cardiac magnetic resonance (CMR) techniques. We measured left and right ventricular dimensions and function, pulmonary and mitral flow pattern and the hearts electrophysiology non-invasively in <1 h per mouse. We found severe biventricular dilation and a drastic decline in performance in accordance with a condition of heart failure (HF), diastolic dysfunction and defects in electrical conduction in 8-week-old calcineurin transgenic mice. Echocardiography of the left ventricle was performed with and without anesthesia. In all cases absolute values on echocardiography compared with CMR were smaller for LV dimension and wall thickness, resulting in higher fractional shorting and ejection fraction. The study protocol described here opens opportunities to assess the added value of combined echocardiography, Doppler, CMR and ECG recording techniques for the diagnosis of biventricular cardiac pathologies i.e. of HF and to study symptom occurrence and disease progression non-invasively in high-throughput. Phenotyping CN hearts revealed new symptom occurrence and allowed insights into the diverse phenotype of hypertrophic failing hearts.
Wissenschaftlicher Artikel
Scientific Article
Int. J. Cardiovasc. Imaging 31, 1137 (2015)
Letter to the Editor
Letter to the Editor
Diabetes 64, 284-290 (2015)
Combined use of metformin and a sodium glucose cotransporter 2 inhibitor (SGLT2I) is a promising treatment strategy for type 2 diabetes. The mechanism by which combination treatment provides better glycemic control than metformin or SGLT2I monotherapy remains elusive. Therefore, we investigated the physiological mechanism, by which both compounds lower blood glucose concentrations in diabetic mice. We compared the potential of metformin and the SGLT2I AVE2268 alone or in combination to mitigate hyperglycemia and modulate glucose fluxes in diabetic db/db and Tallyho/JngJ mice. SGLT2I treatment alone elicited a rapid decline in circulating blood glucose levels, which appeared to induce endogenous glucose production. Supplementation of metformin dampened this counter-response and, therefore, combination therapy more efficiently maintained glycemic control. Finally, combination treatment blunted postprandial glucose excursions and improved HbA1c levels within two weeks. Taken together, we conclude that co-application of metformin enhances the glucose-lowering actions of SGLT2I by restraining endogenous glucose production what may provide long-term improvement of glycemic control in type 2 diabetic patients.
Wissenschaftlicher Artikel
Scientific Article
Cell Metab. 22, 838-850 (2015)
Canonical protein phosphatase 3/calcineurin signaling is central to numerous physiological processes. Here we provide evidence that calcineurin plays a pivotal role in controlling systemic energy and body weight homeostasis. Knockdown of calcineurin in Drosophila melanogaster led to a decrease in body weight and energy stores, and increased energy expenditure. In mice, global deficiency of catalytic subunit Ppp3cb, and tissue-specific ablation of regulatory subunit Ppp3r1 from skeletal muscle, but not adipose tissue or liver, led to protection from high-fat-diet-induced obesity and comorbid sequelæ. Ser637 hyperphosphorylation of dynamin-related protein 1 (Drp1) in skeletal muscle of calcineurin-deficient mice was associated with mitochondrial elongation into power-cable-shaped filaments and increased mitochondrial respiration, but also with attenuated exercise performance. Our data suggest that calcineurin acts as highly conserved pivot for the adaptive metabolic responses to environmental changes such as high-fat, high-sugar diets or exercise.
Wissenschaftlicher Artikel
Scientific Article
Thorax 70, 862-872 (2015)
BACKGROUND: Recently, several proteins of the extracellular matrix have been characterised as active contributors to allergic airway disease. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein abundant in the lung, whose biological functions remain poorly understood. In the current study we investigated the role of MFAP4 in experimental allergic asthma. METHODS: MFAP4-deficient mice were subjected to alum/ovalbumin and house dust mite induced models of allergic airway disease. In addition, human healthy and asthmatic primary bronchial smooth muscle cell cultures were used to evaluate MFAP4-dependent airway smooth muscle responses. RESULTS: MFAP4 deficiency attenuated classical hallmarks of asthma, such as eosinophilic inflammation, eotaxin production, airway remodelling and hyperresponsiveness. In wild-type mice, serum MFAP4 was increased after disease development and correlated with local eotaxin levels. MFAP4 was expressed in human bronchial smooth muscle cells and its expression was upregulated in asthmatic cells. Regarding the underlying mechanism, we showed that MFAP4 interacted with integrin αvβ5 and promoted asthmatic bronchial smooth muscle cell proliferation and CCL11 release dependent on phosphatidyloinositol-3-kinase but not extracellular signal-regulated kinase pathway. CONCLUSIONS: MFAP4 promoted the development of asthmatic airway disease in vivo and pro-asthmatic functions of bronchial smooth muscle cells in vitro. Collectively, our results identify MFAP4 as a novel contributor to experimental asthma, acting through modulation of airway smooth muscle cells.
Wissenschaftlicher Artikel
Scientific Article
Biometals 28, 293-306 (2015)
Iron is essential for numerous cellular processes. For diagnostic purposes iron-related parameters in patients are assessed by clinical chemical blood analysis including the analysis of ferritin, transferrin and iron levels. Here, we retrospectively evaluated the use of these parameters in the phenotype-driven Munich N-ethyl-N-nitrosourea mouse mutagenesis project for the generation of novel animal models for human diseases. The clinical chemical blood analysis was carried out on more than 10,700 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma levels of iron-related plasma parameters. We identified animals consistently exhibiting altered plasma ferritin or transferrin values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of three mutant lines with increased plasma ferritin levels. For two of these lines the causative mutations were identified in the Fth1gene and the Ireb2 gene, respectively. Thus, novel mouse models for the functional analysis of iron homeostasis were established by a phenotype-driven screen for mutant mice.
Wissenschaftlicher Artikel
Scientific Article
Curr. Protoc. Mouse Biol. 5, 65-84 (2015)
This article presents a detailed description of intraperitoneal and oral glucose tolerance tests in mice. The former is widely used in initial high-throughput phenotyping of mutant mice to assess a diabetic phenotype and alterations in glucose homeostasis. Each protocol provides a comprehensive description of each step in the workflow, including variation of the standard protocol under particular circumstances (e.g., sensitivity to food deprivation, excessive deviations in body composition, or need for extra blood samples for additional analyses). We also describe how reduction of body mass and body temperature can be used as additional readouts to monitor metabolic function in response to food deprivation.
Wissenschaftlicher Artikel
Scientific Article
Allergy 70, 459-460 (2015)
Meeting abstract
Meeting abstract
Dev. Cell 33, 644-659 (2015)
Proper morphogenesis of neuronal dendritic spines is essential for the formation of functional synaptic networks. However, it is not known how spines are initiated. Here, we identify the inverse-BAR (I-BAR) protein MIM/MTSS1 as a nucleator of dendritic spines. MIM accumulated to future spine initiation sites in a PIP2-dependent manner and deformed the plasma membrane outward into a proto-protrusion via its I-BAR domain. Unexpectedly, the initial protrusion formation did not involve actin polymerization. However, PIP2-dependent activation of Arp2/3-mediated actin assembly was required for protrusion elongation. Overexpression of MIM increased the density of dendritic protrusions and suppressed spine maturation. In contrast, MIM deficiency led to decreased density of dendritic protrusions and larger spine heads. Moreover, MIM-deficient mice displayed altered glutamatergic synaptic transmission and compatible behavioral defects. Collectively, our data identify an important morphogenetic pathway, which initiates spine protrusions by coupling phosphoinositide signaling, direct membrane bending, and actin assembly to ensure proper synaptogenesis.
Wissenschaftlicher Artikel
Scientific Article
Arterioscler. Thromb. Vasc. Biol. 36, 122-133 (2015)
OBJECTIVE: Arterial injury stimulates remodeling responses that, when excessive, lead to stenosis. These responses are influenced by integrin signaling in vascular smooth muscle cells (VSMCs). Microfibrillar-associated protein 4 (MFAP4) is an integrin ligand localized to extracellular matrix fibers in the vascular wall. The role of MFAP4 in vascular biology is unknown. We aimed to test the hypothesis that MFAP4 would enhance integrin-dependent VSMC activation. APPROACH AND RESULTS: We produced Mfap4-deficient (Mfap4(-/-)) mice and performed carotid artery ligation to explore the role of MFAP4 in vascular biology in vivo. Furthermore, we investigated the effects of MFAP4 in neointimal formation ex vivo and in primary VSMC and monocyte cultures in vitro. When challenged with carotid artery ligation, Mfap4(-/-) mice exhibited delayed neointimal formation, accompanied by early reduction in the number of proliferating medial and neointimal cells, as well as infiltrating leukocytes. Delayed neointimal formation was associated with decreased cross-sectional area of ligated Mfap4(-/-) carotid arteries resulting in lumen narrowing 28 days after ligation. MFAP4 blockade prohibited the formation of neointimal hyperplasia ex vivo. Moreover, we demonstrated that MFAP4 is a ligand for integrin αVβ3 and mediates VSMC phosphorylation of focal adhesion kinase, migration, and proliferation in vitro. MFAP4-dependent VSMC activation was reversible by treatment with MFAP4-blocking antibodies and inhibitors of focal adhesion kinase and downstream kinases. In addition, we showed that MFAP4 promotes monocyte chemotaxis in integrin αVβ3-dependent manner. CONCLUSIONS: MFAP4 regulates integrin αVβ3-induced VSMC proliferation and migration, as well as monocyte chemotaxis, and accelerates neointimal hyperplasia after vascular injury.
Wissenschaftlicher Artikel
Scientific Article
Hum. Mol. Genet. 24, 2247-2266 (2015)
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 KO 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 accuracy.
Wissenschaftlicher Artikel
Scientific Article
2014
PLoS ONE 9:e114918 (2014)
Recently, mutations in the mitochondrial translation optimization factor 1 gene (MTO1) were identified as causative in children with hypertrophic cardiomyopathy, lactic acidosis and respiratory chain defect. Here, we describe an MTO1-deficient mouse model generated by gene trap mutagenesis that mirrors the human phenotype remarkably well. As in patients, the most prominent signs and symptoms were cardiovascular and included bradycardia and cardiomyopathy. In addition, the mutant mice showed a marked worsening of arrhythmias during induction and reversal of anaesthesia. The detailed morphological and biochemical workup of murine hearts indicated that the myocardial damage was due to complex I deficiency and mitochondrial dysfunction. In contrast, neurological examination was largely normal in Mto1-deficient mice. A translational consequence of this mouse model may be to caution against anaesthesia-related cardiac arrhythmias which may be fatal in patients.
Wissenschaftlicher Artikel
Scientific Article
EMBO J. 33, 2020-2039 (2014)
Mutations in the cytosine-5 RNA methyltransferase NSun2 cause microcephaly and other neurological abnormalities in mice and human. How post-transcriptional methylation contributes to the human disease is currently unknown. By comparing gene expression data with global cytosine-5 RNA methylomes in patient fibroblasts and NSun2-deficient mice, we find that loss of cytosine-5 RNA methylation increases the angiogenin-mediated endonucleolytic cleavage of transfer RNAs (tRNA) leading to an accumulation of 5' tRNA-derived small RNA fragments. Accumulation of 5' tRNA fragments in the absence of NSun2 reduces protein translation rates and activates stress pathways leading to reduced cell size and increased apoptosis of cortical, hippocampal and striatal neurons. Mechanistically, we demonstrate that angiogenin binds with higher affinity to tRNAs lacking site-specific NSun2-mediated methylation and that the presence of 5' tRNA fragments is sufficient and required to trigger cellular stress responses. Furthermore, the enhanced sensitivity of NSun2-deficient brains to oxidative stress can be rescued through inhibition of angiogenin during embryogenesis. In conclusion, failure in NSun2-mediated tRNA methylation contributes to human diseases via stress-induced RNA cleavage.
Wissenschaftlicher Artikel
Scientific Article
Biochim. Biophys. Acta-Mol. Basis Dis. 1842, 304–317 (2014)
Vulnerability of the fetus upon maternal obesity can potentially occur during all developmental phases. We aimed at elaborating longer-term health outcomes of fetal overnutrition during the earliest stages of development. We utilized NMRI mice to induce pre-conceptional and gestational obesity and followed offspring outcomes in the absence of any postnatal obesogenic influences. Male adult offspring developed overweight, insulin resistance, hyperleptinemia, hyperuricemia and hepatic steatosis; all these features not being observed in females. Instead, those showed impaired fasting glucose and a reduced fat mass and adipocyte size. Influences of the interaction of maternal diet*sex concerned offspring genes involved in fatty liver disease, lipid droplet size regulation and fat mass expansion. These data suggest that a peri-conceptional obesogenic exposure is sufficient to shape offspring gene expression patterns and health outcomes in a sex- and organ-specific manner, indicating varying developmental vulnerabilities between sexes towards metabolic disease in response to maternal overnutrition.
Wissenschaftlicher Artikel
Scientific Article
Eur. J. Immunol. 44, 3232-3239 (2014)
AOC3 (Amine oxidase, copper containing 3, also known as vascular adhesion protein-1 (VAP-1)) is an endothelial adhesion molecule that contributes to the extravasation of neutrophils, macrophages and lymphocytes to sites of inflammation. However, the role of AOC3/VAP-1 in allergic responses remains unknown. Here we studied eosinophil and CD4+ T-cell recruitment to the airways using AOC3/VAP-1-deficient mice. In an OVA-triggered asthma model, AOC3/VAP-1 slightly contributed to the accumulation of leukocytes in lungs in an age-dependent manner. We then established a new model to kinetically measure recruitment of OVA-specific CD4+ T cells to different airway immune compartments during the priming and effector phases of an adaptive immune response. The results showed that in the absence of AOC3/VAP-1, recruitment of antigen-specific CD4+ T cells to draining bronchial lymph nodes is reduced by 89% on day 3 after tracheal allergen exposure, but this difference was not observed on day 6. The dispersal of effector cells to lung and tracheal mucosa is AOC3/VAP-1-independent. Thus, in allergic airway reactions, AOC3/VAP-1 transiently contributes to the antigen-specific, CD4+ T-cell traffic to secondary lymphatic tissues, but not to airway mucosa or lung parenchyma. Our results suggest a largely redundant function for AOC3/VAP-1 in allergic inflammatory responses of the airways.
Wissenschaftlicher Artikel
Scientific Article
PLoS ONE 9:e113125 (2014)
Uromodulin-associated kidney disease (UAKD) is a hereditary progressive renal disease which can lead to renal failure and requires renal replacement therapy. UAKD belongs to the endoplasmic reticulum storage diseases due to maturation defect of mutant uromodulin and its retention in the enlarged endoplasmic reticulum in the cells of the thick ascending limb of Henle's loop (TALH). Dysfunction of TALH represents the key pathogenic mechanism of UAKD causing the clinical symptoms of this disease. However, the molecular alterations underlying UAKD are not well understood. In this study, transcriptome profiling of whole kidneys of two mouse models of UAKD, UmodA227T and UmodC93F, was performed. Genes differentially abundant in UAKD affected kidneys of both Umod mutant lines at different disease stages were identified and verified by RT-qPCR. Additionally, differential protein abundances of SCD1 and ANGPTL7 were validated by immunohistochemistry and Western blot analysis. ANGPTL7 expression was down-regulated in TALH cells of Umod mutant mice which is the site of the mutant uromodulin maturation defect. SCD1 was expressed selectively in the S3 segment of proximal tubule cells, and SCD1 abundance was increased in UAKD affected kidneys. This finding demonstrates that a cross talk between two functionally distinct tubular segments of the kidney, the TALH segment and the S3 segment of proximal tubule, exists.
Wissenschaftlicher Artikel
Scientific Article
Diabetologia 57, S172 (2014)
Meeting abstract
Meeting abstract
PLoS ONE 9:e111239 (2014)
A significant challenge facing high-throughput phenotyping of in-vivo knockout mice is ensuring phenotype calls are robust and reliable. Central to this problem is selecting an appropriate statistical analysis that models both the experimental design (the workflow and the way control mice are selected for comparison with knockout animals) and the sources of variation. Recently we proposed a mixed model suitable for small batch-oriented studies, where controls are not phenotyped concurrently with mutants. Here we evaluate this method both for its sensitivity to detect phenotypic effects and to control false positives, across a range of workflows used at mouse phenotyping centers. We found the sensitivity and control of false positives depend on the workflow. We show that the phenotypes in control mice fluctuate unexpectedly between batches and this can cause the false positive rate of phenotype calls to be inflated when only a small number of batches are tested, when the effect of knockout becomes confounded with temporal fluctuations in control mice. This effect was observed in both behavioural and physiological assays. Based on this analysis, we recommend two approaches (workflow and accompanying control strategy) and associated analyses, which would be robust, for use in high-throughput phenotyping pipelines. Our results show the importance in modelling all sources of variability in high-throughput phenotyping studies.
Wissenschaftlicher Artikel
Scientific Article
Nat. Commun. 5:5215 (2014)
The hormone calcitonin (CT) is primarily known for its pharmacologic action as an inhibitor of bone resorption, yet CT-deficient mice display increased bone formation. These findings raised the question about the underlying cellular and molecular mechanism of CT action. Here we show that either ubiquitous or osteoclast-specific inactivation of the murine CT receptor (CTR) causes increased bone formation. CT negatively regulates the osteoclast expression of Spns2 gene, which encodes a transporter for the signalling lipid sphingosine 1-phosphate (S1P). CTR-deficient mice show increased S1P levels, and their skeletal phenotype is normalized by deletion of the S1P receptor S1P3. Finally, pharmacologic treatment with the nonselective S1P receptor agonist FTY720 causes increased bone formation in wild-type, but not in S1P3-deficient mice. This study redefines the role of CT in skeletal biology, confirms that S1P acts as an osteoanabolic molecule in vivo and provides evidence for a pharmacologically exploitable crosstalk between osteoclasts and osteoblasts.
Wissenschaftlicher Artikel
Scientific Article
J. Biol. Chem. 289, 10715-10726 (2014)
Uromodulin (UMOD)-associated kidney disease (UAKD) belongs to the hereditary progressive ER storage diseases caused by maturation defects of mutant UMOD protein. Current treatments of UAKD patients are symptomatic and cannot prevent disease progression. Two in vitro studies reported a positive effect of the chemical chaperone sodium-4-phenylbutyrate (4-PBA) on mutant UMOD maturation. Thus, 4-PBA was suggested as potential treatment for UAKD. This study evaluated the effects of 4-PBA in two mouse models of UAKD. In contrast to previous in vitro studies, treatment with 4-PBA did not increase HSP70 expression or improve maturation and trafficking of mutant UMOD in vivo. Kidney function of UAKD mice was actually deteriorated by 4-PBA-treatment. In transfected tubular epithelial cells, 4-PBA did not improve maturation, but increased the expression level of both mutant and wild-type UMOD protein. Activation of NF-κB pathway in thick ascending limb of Henle's loop cells of UAKD mice was detected by increased abundance of RelB and phospho-IKKα/β, an indirect activator of NF-κB. Further, the abundance of NF-κB1 p105/p50, NF-κB2 p100/p52 and TRAF2 was increased in UAKD. NF-κB activation was identified as a novel disease mechanism of UAKD and might be a target for therapeutic intervention.
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Scientific Article
J. Biomed. Sci. 21:68 (2014)
BackgroundType I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na+-K+-2Cl¿ cotransporter NKCC2. We recently established the mutant mouse line Slc12a1 I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye.ResultsTo examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1 I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1 I299F in the German Mouse Clinic. Slc12a1 I299F homozygous mutant mice and Slc12a1 I299F heterozygous mutant littermates as controls were tested at the age of 4¿6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1 I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed.ConclusionsIn this systemic analysis no clear primary effects of the Slc12a1 I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.
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J. Breath Res. 8:016004 (2014)
Breath gas analysis in humans proved successful in identifying disease states and assessing metabolic functions in a non-invasive way. While many studies report diagnostic capability using volatile organic compounds (VOC) in breath, the inter-individual variability even in healthy human cohorts is rather large and not completely understood in its biochemical origin. Laboratory mice are the predominant animal model system for human disorders and are analysed under highly standardized and controlled conditions. We established a novel setup to monitor VOCs as biomarkers for disease in the breath gas of non-anesthetized, non-restrained mice using a proton transfer reaction mass spectrometer with time of flight detection. In this study, we implemented breath gas analysis in a dietary intervention study in C57BL/6J mice with the aim to assess the variability in VOC signatures due to a change in the diet matrix. Mice were fed a standard laboratory chow and then exposed to four semi-purified low- or high-fat diets for four weeks. Random forest (RF++) was used to identify VOCs that specifically respond to the diet matrix change. Interestingly, we found that the change from a chow diet to semi-purified diets resulted in a considerable drop of several VOC levels. Our results suggest that the diet matrix impacts VOC signatures and the underlying metabolic functions and may be one source of variability in exhaled volatiles.
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PLoS ONE 9:e104568 (2014)
The transcription factor Zscan10 had been attributed a role as a pluripotency factor in embryonic stem cells based on its interaction with Oct4 and Sox2 in in vitro assays. Here we suggest a potential role of Zscan10 in controlling progenitor cell populations in vivo. Mice homozygous for a Zscan10 mutation exhibit reduced weight, mild hypoplasia in the spleen, heart and long bones and phenocopy an eye malformation previously described for Sox2 hypomorphs. Phenotypic abnormalities are supported by the nature of Zscan10 expression in midgestation embryos and adults suggesting a role for Zscan10 in either maintaining progenitor cell subpopulation or impacting on fate choice decisions thereof.
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J. Comp. Physiol. B 184, 763-775 (2014)
Mice with genetic alterations are used in heart research as model systems of human diseases. In the last decade there was a marked increase in the recognition of genetic diversity within inbred mouse strains. Increasing numbers of inbred mouse strains and substrains and analytical variation of cardiac phenotyping methods require reproducible, high-throughput methods to standardize murine cardiovascular physiology. We describe methods for non-invasive, reliable, easy and fast to perform echocardiography and electrocardiography on awake mice. This method can be used for primary screening of the murine cardiovascular system in large-scale analysis. We provide insights into the physiological divergence of C57BL/6N, C57BL/6J, C3HeB/FeJ and 129P2/OlaHsd mouse hearts and define the expected normal values. Our report highlights that compared to the other three strains tested C57BL/6N hearts reveal features of heart failure such as hypertrophy and reduced contractile function. We found several features of the mouse ECG to be under genetic control and obtained several strain-specific differences in cardiac structure and function.
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Eur. J. Pain 18, 249-257 (2014)
BACKGROUND: Smad-interacting protein 1 (also named Zeb2 and Zfhx1b) is a transcription factor that plays an important role in neuronal development and, when mutated, causes Mowat-Wilson syndrome (MWS). A corresponding mouse model carrying a heterozygous Zeb2 deletion was comprehensively analysed in the German Mouse Clinic. The most prominent phenotype was the reduced pain sensitivity. In this study, we investigated the role of Zeb2 in inflammatory and neuropathic pain. METHODS: For this, we tested mutant Zeb2 animals in different models of inflammatory pain like abdominal constriction, formalin and carrageenan test. Furthermore, we studied the pain reactivity of the mice after peripheral nerve ligation. To examine the nociceptive transmission of primary sensory dorsal root ganglia (DRG) neurons, we determined the neuronal activity in the spinal dorsal horn after the formalin test using staining of c-Fos. Next, we characterized the neuronal cell population in the DRGs and in the sciatic nerve to study the effect of the Zeb2 mutation on peripheral nerve morphology. RESULTS: The present data show that Zeb2 is involved in the development of primary sensory DRG neurons, especially of C- and Aδ fibres. These alterations contribute to a hypoalgesic phenotype in inflammatory but not in neuropathic pain in these Zeb2+/- mice. CONCLUSION: Our data suggest that the under-reaction to pain observed in MWS patients results from a reduced responsivity to nociceptive stimulation rather than an inability to communicate discomfort.
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Mamm. Genome 25, 497-507 (2014)
Metabolic phenotyping of genetically modified animals aims to detect new candidate genes and related metabolic pathways that result in dysfunctional energy balance regulation and predispose for diseases such as obesity or type 2 diabetes mellitus. In this review, we provide a comprehensive overview on the technologies available to monitor energy flux (food uptake, bomb calorimetry of feces and food, and indirect calorimetry) and body composition (qNMR, DXA, and MRI) in animal models for human diseases with a special focus on phenotyping methods established in genetically engineered mice. We use an energy flux model to illustrate the principles of energy allocation, describe methodological aspects how to monitor energy balance, and introduce strategies for data analysis and presentation.
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Genome Res. 24, 592-603 (2014)
Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS.
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J. Biol. Chem. 289, 10769-10784 (2014)
The majority of Amyotrophic Lateral Sklerosis (ALS) cases as well as many patients suffering from Frontotemporal Lobar Dementia (FTLD) with ubiquitinated inclusion bodies show TDP-43 pathology, the protein encoded by the TAR-DNA binding protein (Tardbp) gene. We used recombinase-mediated cassette exchange (RMCE) to introduce an ALS patient cDNA into the mouse Tdp-43 locus. Expression levels of human A315T TDP-43 protein were 300% elevated in heterozygotes while the endogenous mouse Tdp-43 was decreased to 20% of wildtype levels as a result of disturbed feedback regulation. Heterozygous TDP-43A315TKi mutants lost 10% of their body weight and developed insoluble TDP-43 protein starting as early as 3 months after birth, a pathology that was exacerbated with age. We analyzed the splicing patterns of known Tdp-43 target genes, as well as genome-wide gene expression levels in different tissues that indicated mitochondrial dysfunction. In heterozygous mutant animals we observed a relative decrease in expression of Parkin (Park2) and the fatty acid transporter CD36 along with an increase in fatty acids, HDL cholesterol and glucose in the blood. As seen in Transmission Electron Microscopy, neuronal cells in motor cortices of TDP-43A315TKi animals had abnormal neuronal mitochondrial cristae formation. Motor neurons were reduced to 90% but only slight motoric impairment was detected. The observed phenotype was interpreted as a pre-disease model which might be valuable for the identification of further environmental or genetic triggers of neurodegeneration.
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Mamm. Genome 25, 129-140 (2014)
The phenotyping of genetic mouse models for human disorders may greatly benefit from breath gas analysis as a noninvasive tool to identify metabolic alterations in mice. Phenotyping screens such as the German Mouse Clinic demand investigations in unrestrained mice. Therefore, we adapted a breath screen in which exhaled volatile organic compounds (VOCs) were online monitored by proton transfer reaction mass spectrometry (hs-PTR-MS). The source strength of VOCs was derived from the dynamics in the accumulation profile of exhaled VOCs of a single mouse in a respirometry chamber. A careful survey of the accumulation revealed alterations in the source strength due to confounders, e.g., urine and feces. Moreover changes in the source strength of humidity were triggered by changes in locomotor behavior as mice showed a typical behavioral pattern from activity to settling down in the course of subsequent accumulation profiles. We demonstrated that metabolic changes caused by a dietary intervention, e.g., after feeding a high-fat diet (HFD) a sample of 14 male mice, still resulted in a statistically significant shift in the source strength of exhaled VOCs. Applying a normalization which was derived from the distribution of the source strength of humidity and accounted for varying locomotor behaviors improved the shift. Hence, breath gas analysis may provide a noninvasive, fast access to monitor the metabolic adaptation of a mouse to alterations in energy balance due to overfeeding or fasting and dietary macronutrient composition as well as a high potential for systemic phenotyping of mouse mutants, intervention studies, and drug testing in mice.
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Int. J. Cancer 136, 2293-2303 (2014)
Previous studies have evaluated the role of miRNAs in cancer initiation and progression. MiR-34a was found to be downregulated in several tumors, including medulloblastomas. Here we employed targeted transgenesis to analyze the function of miR-34a in vivo. We generated mice with a constitutive deletion of the miR-34a gene. These mice were devoid of mir-34a expression in all analyzed tissues, but were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters revealed no apparent phenotype. Analysis of miR-34a expression in human medulloblastomas and medulloblastoma cell lines revealed significantly lower levels than in normal human cerebellum. Re-expression of miR-34a in human medulloblastoma cells reduced cell viability and proliferation, induced apoptosis and downregulated the miR-34a target genes, MYCN and SIRT1. Activation of the Shh pathway by targeting SmoA1 transgene overexpression causes medulloblastoma in mice, which is dependent on the presence and upregulation of Mycn. Analysis of miR-34a in medulloblastomas derived from ND2:SmoA1(tg) mice revealed significant suppression of miR-34a compared to normal cerebellum. Tumor incidence was significantly increased and tumor formation was significantly accelerated in mice transgenic for SmoA1 and lacking miR-34a. Interestingly, Mycn and Sirt1 were strongly expressed in medulloblastomas derived from these mice. We here demonstrate that miR-34a is dispensable for normal development, but that its loss accelerates medulloblastomagenesis. Strategies aiming to re-express miR-34a in tumors could, therefore, represent an efficient therapeutic option.
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J. Proteome Res. 13, 4220-4231 (2014)
A metabolic disorder such as Type 2 Diabetes mellitus (T2DM) is a complex disease induced by genetic, environmental and nutritional factors. The db/db mouse model, bearing a non-functional leptin receptor is widely used to investigate the pathophysiology of T2DM. Fecal extracts of db/db and wild type littermates were studied to unravel a broad spectrum of new and relevant metabolites related to T2DM as proxies of the interplay of gut microbiome and murine metabolomes. The non-targeted metabolomics approach consists of an integrated analytical concept of high-resolution mass spectrometry FT-ICR-MS followed by UPLC-TOF-MS/MS experiments. We demonstrate that a metabolic disorder such as T2DM affects the gastrointestinal tract environment thereby influencing different metabolic pathways and their respective metabolites in diabetic mice. Fatty acids, bile acids concerning cholic and deoxycholic acid and steroid metabolism were highly discriminative comparing fecal metabolomes of wt and db/db mice. Furthermore, sulfur-(S)-containing metabolites including N-acyl taurines were altered in diabetic mice, enabling to focus on S-containing metabolites, especially the sulfate and taurine conjugates of bile and fatty acids. Different sulfate containing bile acids including sulfocholic acid, oxocholic acid sulfate, taurocholic acid sulfate and cyprinol sulfate were significantly altered in diabetic mice. Moreover, we identified twelve new sulfate and taurine conjugates of hydroxylated fatty acids with significant importance in T2DM metabolism in db/db mice.
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Hum. Mol. Genet. 23, 5597-5614 (2014)
Mutations in Peroxidasin (PXDN) cause severe inherited eye disorders in humans, such as congenital cataract, corneal opacity, and developmental glaucoma. The role of peroxidasin during eye development is poorly understood. Here we describe the first Pxdn mouse mutant which was induced by ENU (N-ethyl-N-nitrosourea) and led to a recessive phenotype. Sequence analysis of cDNA revealed a T3816A mutation resulting in a premature stop codon (Cys1272X) in the peroxidase domain. This mutation causes severe anterior segment dysgenesis and microphthalmia resembling the manifestations in patients with PXDN mutations. The proliferation and differentiation of the lens is disrupted in association with aberrant expression of transcription factor genes (Pax6 and Foxe3) in mutant eyes. Additionally, Pxdn is involved in the consolidation of the basement membrane and lens epithelium adhesion in the ocular lens. Lens material including γ-crystallin is extruded into the anterior and posterior chamber due to local loss of structural integrity of the lens capsule as a secondary damage to the anterior segment development leading to congenital ocular inflammation. Moreover, Pxdn mutants exhibited an early-onset glaucoma and progressive retinal dysgenesis. Transcriptome profiling revealed that peroxidasin affects the transcription of developmental and eye diseases-related genes at early eye development. These findings suggest that peroxidasin is necessary for cell proliferation and differentiation and for basement membrane consolidation during eye development. Our studies provide pathogenic mechanisms of PXDN mutation-induced congenital eye diseases.
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Front. Behav. Neurosci. 8:125 (2014)
Stress and an altered stress response have been associated with many multifactorial diseases, such as psychiatric disorders or neurodegenerative diseases. As currently mouse mutants for each single gene are generated and phenotyped in a large-scale manner, it seems advisable also to test these mutants for alterations in their stress responses. Here we present the determinants of a robust and reliable non-invasive test for stress-responsivity in mice. Stress is applied through restraining the mice in tubes and recording behavior in the Open Field 20 min after cessation of the stress. Two hours, but not 15 or 50 min of restraint lead to a robust and reproducible increase in distance traveled and number of rearings during the first 5 min in the Open Field in C57BL/6 mice. This behavioral response is blocked by the corticosterone synthesis inhibitor metyrapone, but not by RU486 treatment, indicating that it depends on corticosteroid secretion, but is not mediated via the glucocorticoid receptor type II. We assumed that with a stress duration of 15 min one could detect hyper-responsivity, and with a stress duration of 2 h hypo-responsivity in mutant mouse lines. This was validated with two mutant lines known to show opposing effects on corticosterone secretion after stress exposure, corticotropin-releasing hormone (CRH) over-expressing mice and CRH receptor 1 knockout (KO) mice. Both lines showed the expected phenotype, i.e., increased stress responsivity in the CRH over-expressing mouse line (after 15 min restraint stress) and decreased stress responsivity in the CRHR1-KO mouse line (after 2 h of restraint stress). It is possible to repeat the acute stress test several times without the stressed animal adapting to it, and the behavioral response can be robustly evoked at different ages, in both sexes and in different mouse strains. Thus, locomotor and rearing behavior in the Open Field after an acute stress challenge can be used as reliable, non-invasive indicators of stress responsivity and corticosterone secretion in mice.
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PLoS ONE 9:e98072 (2014)
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.
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2013
Oncogene 32, 2586–2591 (2013)
AVEN has been identified as an inhibitor of apoptosis, which binds to the adaptor protein, APAF-1, and thereby prevents apoptosome formation and mitochondrial apoptosis. Recent data have demonstrated high expression levels of AVEN messenger RNA in acute leukemias as well as a positive correlation between AVEN mRNA overexpression and poor prognosis in childhood acute lymphoblastic leukemia. On the basis of these data, we investigated the potential involvement of AVEN in tumorigenesis. First, we confirmed the overexpression of AVEN in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) patient samples. We then established a transgenic mouse model with T-cell-specific overexpression of AVEN, with which we demonstrated the oncogenic cooperation of AVEN with heterozygous loss of p53. Finally, we used a subcutaneous xenograft mouse model to show that AVEN knockdown in the T-ALL cell lines, MOLT-4 and CCRF-CEM, and in the acute myeloblastic leukemia cell line, Kasumi-1, leads to a halt in tumor growth owing to the increased apoptosis and decreased proliferation of tumor cells. Collectively, our data demonstrate that the anti-apoptotic molecule, AVEN, functions as an oncoprotein in hematopoietic neoplasms.
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Mamm. Genome 24, 376-388 (2013)
Dual-energy X-ray absorption (DEXA) is commonly used to measure bone mineral density (BMD), bone mineral content (BMC), and body composition data (fat mass and lean mass) for phenotype assessment in mice. We were interested in the long-term development of BMD, BMC, lean mass, and fat mass of mice, also taking into account sex and genetic background. The dataset was used to analyze correlations among the different parameters. We analyzed males and females from inbred strains C3HeB/FeJ and C57BL/6J, starting from 42 until 528 days of age. To evaluate the effect of husbandry systems, we repeated a part of the study in a second facility with a different caging system. We also assessed different DEXA settings and repeatability of the scans. The results of this study were used to draw conclusions for the use of DEXA analysis in mouse phenotyping approaches.
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Hum. Mol. Genet. 22, 4871-4887 (2013)
The peptidase CLPP is conserved from bacteria to humans. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder together with the chaperone CLPX. In spite of a known relevance for the mitochondrial unfolded protein response, its substrates and tissue-specific roles are unclear in mammals. Recessive CLPP mutations were recently observed in the human Perrault variant of ovarian failure and sensorineural hearing loss. Here, a first characterization of Clpp null mice demonstrated complete female and male infertility and auditory deficits. Disrupted spermatogenesis already at the spermatid stage and ovarian follicular differentiation failure were evident. Reduced pre-/post-natal survival and marked ubiquitous growth retardation contrasted with only light impairment of movement and respiratory activities. Interestingly, the mice showed resistance to ulcerative dermatitis. Systematic expression studies detected up-regulation of other mitochondrial chaperones, accumulation of CLPX and mtDNA as well as inflammatory factors throughout tissues. T-lymphocytes in the spleen were activated. Thus, murine Clpp deletion represents a faithful Perrault model. The disease mechanism probably involves deficient clearance of mitochondrial components and inflammatory tissue destruction.
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Bone 55, 501-511 (2013)
Hepatic osteodystrophy (HOD) denotes the alterations in bone morphology and metabolism frequently observed in patients with chronic liver diseases, in particular in case of cholestatic conditions. The molecular mechanisms underlying HOD are only partially understood. In the present study, we characterized the bone phenotypes of the ATP-binding cassette transporter B4 knockout mouse (Abcb4-/-), a well-established mouse model of chronic cholestatic liver disease, with the aim of identifying and characterizing a mouse model for HOD. Furthermore, we investigated the influence of vitamin D on bone quality in this model. The bone morphology analyses revealed reduced bone mineral contents as well as changes in trabecular bone architecture and decreased cortical bone densities in Abcb4-/- mice with severe liver fibrosis. We observed dysregulation of genes involved in bone remodeling (osteoprotegerin, osteocalcin, osteopontin) and vitamin D metabolism (7-dehydrocholesterol reductase, Gc-globulin, Cyp2r1, Cyp27a1) as well as alterations in calcium and vitamin D homeostasis. In addition, serum RANKL and TGF-β levels were increased in Abcb4-/- mice. Vitamin D dietary intervention did not restore the bone phenotypes of Abcb4-/- animals. We conclude that the Abcb4-/- mouse provides an experimental framework and a preclinical model to gain further insights into the molecular pathobiology of HOD and to study the systemic effects of therapeutic interventions.
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In:. 2013. K283 (Z. Gastroenterol. ; 51)
Aims: Depression is commonly associated with chronic liver disease; however, its causality remains largely unknown (Lee et al. Psychosomatics 2013;54:52 – 9). Quantitative phenotypes are linked to psychiatric disorders, thus genetic studies in mice might help to identify modifier genes that influence behavior. Our aim now was to characterize the behavioral phenotype in the ABCB4-deficient (Abcb4–/– ) mouse, a well-established model for chronic cholestatic liver disease. Methods: We used the Open Field (OF) test, which evaluates spontaneous exploratory drive, reactivity to novelty and emotionality. Specifically, time courses of distance travelled, including speed of movement and time spent in the center, and rearing frequencies were noted in 5-min-intervals. Additionally, we assessed sensorimotor gating by Prepulse Inhibition (PPI) one week after OF at different intensities. The tests were carried out with 18 Abcb4–/– mice and 20 BALB/cJ wild-type controls, aged 10 to 13 weeks. Results: OF revealed significant genotype effects (p < 0.001) with consistently decreased locomotor activity characterized by reduced speed of movement and increased avoidance of the peripheral and central zones in Abcb4–/– mice. The time course of unconditioned behavior of Abcb4–/– mice in the OF revealed a significant genotype effect on the curve shape of the measured parameters. Furthermore, we detected a significant decrease in rearing activity for Abcb4–/– mice as compared to controls (total frequency 47.8 ± 8.4 vs. 99.0 ± 8.6 [events], p < 0.001). Assessment of PPI revealed a deficit at 69 dB intensity in female Abcb4–/– mice only. Conclusions: Abcb4–/– mice show greater anxiety behavior and reduced exploratory activity compared to wild-type controls. However, given that ABCB4 is almost exclusively expressed in liver but not in brain, the induced locomotor behavioral alterations are most likely secondary, e.g. caused by neurochemical changes associated with the significantly reduced serum vitamin D levels recently described by us in Abcb4–/– mice. Of note, this model resembles the link between chronic liver diseases and increased stress susceptibility observed in humans.
PLoS ONE 8:e80923 (2013)
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing.
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DNA Repair 12, 356-366 (2013)
Smc5-6 is a highly conserved protein complex related to cohesin and condensin involved in the structural maintenance of chromosomes. In yeasts the Smc5-6 complex is essential for proliferation and is involved in DNA repair and homologous recombination. siRNA depletion of genes involved in the Smc5-6 complex in cultured mammalian cells results in sensitivity to some DNA damaging agents. In order to gain further insight into its role in mammals we have generated mice mutated in the Smc6 gene. A complete knockout resulted in early embryonic lethality, demonstrating that this gene is essential in mammals. However, mutation of the highly conserved serine-994 to alanine in the ATP hydrolysis motif in the SMC6 C-terminal domain, resulted in mice with a surprisingly mild phenotype. With the neo gene selection marker in the intron following the mutation, resulting in reduced expression of the SMC6 gene, the mice were reduced in size, but fertile and had normal lifespans. When the neo gene was removed, the mice had normal size, but detailed phenotypic analysis revealed minor abnormalities in glucose tolerance, haematopoiesis, nociception and global gene expression patterns. Embryonic fibroblasts derived from the ser994 mutant mice were not sensitive to killing by a range of DNA damaging agents, but they were sensitive to the induction of sister chromatid exchanges induced by ultraviolet light or mitomycin C. They also accumulated more oxidative damage than wild-type cells.
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Mol. Metab. 2, 435-446 (2013)
Genetic predisposition and environmental factors contribute to an individual's susceptibility to develop hepatosteatosis. In a systematic, comparative survey we focused on genotype-dependent and -independent adaptations early in the pathogenesis of hepatosteatosis by characterizing C3HeB/FeJ, C57BL/6NTac, C57BL/6J, and 129P2/OlaHsd mice after 7, 14, or 21 days high-fat-diet exposure. Strain-specific metabolic responses during diet challenge and liver transcript signatures in mild hepatosteatosis outline the suitability of particular strains for investigating the relationship between hepatocellular lipid content and inflammation, glucose homeostasis, insulin action, or organelle physiology. Genetic background-independent transcriptional adaptations in liver paralleling hepatosteatosis suggest an early increase in the organ's vulnerability to oxidative stress damage what could advance hepatosteatosis to steatohepatitis. “Universal“ adaptations in transcript signatures and transcription factor regulation in liver link insulin resistance, type 2 diabetes mellitus, cancer, and thyroid hormone metabolism with hepatosteatosis, hence, facilitating the search for novel molecular mechanisms potentially implicated in the pathogenesis of human non-alcoholic-fatty-liver-disease.
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Hum. Mol. Genet. 22, 4148-4163 (2013)
Uromodulin-associated kidney disease (UAKD) is a dominant heritable renal disease in humans which is caused by mutations in the uromodulin (UMOD) gene and characterized by heterogeneous clinical appearance. To get insights into possible causes of this heterogeneity of UAKD, we describe the new mutant mouse line UmodC93F, leading to disruption of a putative disulfide bond which is also absent in a known human UMOD mutation, and compare the phenotype of this new mouse line with the recently published mouse line UmodA227T. In both mutant mouse lines, which were both bred on the C3H background, the Umod mutations cause a gain-of-toxic function due to a maturation defect of the mutant uromodulin leading to a dysfunction of thick ascending limb of Henle's loop (TALH) cells of the kidney. Umod mutant mice exhibit increased plasma urea and cystatin levels, impaired urinary concentration ability, reduced fractional excretion of uric acid, and nephropathological alterations including uromodulin retention in TALH cells, interstitial fibrosis and inflammatory cell infiltrations, tubular atrophy, and occasional glomerulo- und tubulocystic changes, a phenotype highly similar to UAKD in humans. The maturation defect of mutant uromodulin leads to the accumulation of immature uromodulin in the endoplasmic reticulum (ER) and to ER hyperplasia. Further, this study was able to demonstrate for the first time in vivo that the severity of the uromodulin maturation defect as well as onset and speed of progression of renal dysfunction and morphological alterations are strongly dependent on the particular Umod mutation itself and the zygosity status.
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PLoS ONE 8:e78337 (2013)
Uromodulin-associated kidney disease (UAKD) summarizes different clinical features of an autosomal dominant heritable disease syndrome in humans with a proven uromodulin (UMOD) mutation involved. It is often characterized by hyperuricemia, gout, alteration of urine concentrating ability, as well as a variable rate of disease progression inconstantly leading to renal failure and histological alterations of the kidneys. We recently established the two Umod mutant mouse lines UmodC93F and UmodA227T on the C3H inbred genetic background both showing kidney defects analogous to those found in human UAKD patients. In addition, disease symptoms were revealed that were not yet described in other published mouse models of UAKD. To examine if further organ systems and/or metabolic pathways are affected by Umod mutations as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the two mutant mouse lines UmodA227T and UmodC93F in the German Mouse Clinic. Different genotypes as well as different ages were tested. Beside the already published changes in body weight, body composition and bone metabolism, the influence of the Umod mutation on energy metabolism was confirmed. Hematological analysis revealed a moderate microcytic and erythropenic anemia in older Umod mutant mice. Data of the other analyses in 7-10 month-old mutant mice showed single small additional effects.
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Scientific Article
J. Biol. Chem. 288, 16690-16703 (2013)
The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family which bind specifically to nucleosome core particles and affect the structure and function of chromatin, including transcription. Here we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1-/-, Hmgn3-/-, and Hmgn5-/- mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgn-/- lines reveals very little overlap between the genes affected by the specific variants in the different tissues. Pathway analysis revels that loss of an HMGN variant affects subtly the expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner.
Wissenschaftlicher Artikel
Scientific Article
Diabetologia 57, 192-203 (2013)
AIMS/HYPOTHESIS: Diabetic retinopathy is a major complication of type 2 diabetes and the leading cause of blindness in adults of working age. Neuronal defects are known to occur early in disease, but the source of this dysfunction is unknown. The aim of this study was to examine differences in the retinal membrane proteome among non-diabetic mice and mouse models of diabetes either with or without metformin treatment. METHODS: Alterations in the retinal membrane proteome of 10-week-old diabetic db/db mice, diabetic db/db mice orally treated with the anti-hyperglycaemic metformin, and congenic wild-type littermates were examined using label-free mass spectrometry. Pathway enrichment analysis was completed with Genomatix and Ingenuity. Alterations in Slc17a7 mRNA and vesicular glutamate transporter 1 (VGLUT1) protein expression were evaluated using real-time quantitative PCR and immunofluorescence. RESULTS: A total of 98 proteins were significantly differentially abundant between db/db and wild-type animals. Pathway enrichment analysis indicated decreases in levels of proteins related to synaptic transmission and cell signalling. Metformin treatment produced 63 differentially abundant proteins compared with untreated db/db mice, of which only 43 proteins were found to occur in both datasets, suggesting that treatment only partially normalises the alterations induced by diabetes. VGLUT1, which is responsible for loading glutamate into synaptic vesicles, was found to be differentially abundant in db/db mice and was not normalised by metformin. The decrease in Slc17a7/VGLUT1 was confirmed by transcriptomic and immunocytochemical analysis. CONCLUSIONS/INTERPRETATION: These findings expand the knowledge of the protein changes in diabetic retinopathy and suggest that membrane-associated signalling proteins are susceptible to changes that are partially ameliorated by treatment.
Wissenschaftlicher Artikel
Scientific Article
J. Clin. Invest. 123, 3272-3291 (2013)
Aging is a major risk factor for a large number of disorders and functional impairments. Therapeutic targeting of the aging process may therefore represent an innovative strategy in the quest for novel and broadly effective treatments against age-related diseases. The recent report of lifespan extension in mice treated with the FDA-approved mTOR inhibitor rapamycin represented the first demonstration of pharmacological extension of maximal lifespan in mammals. Longevity effects of rapamycin may, however, be due to rapamycin's effects on specific life-limiting pathologies, such as cancers, and it remains unclear if this compound actually slows the rate of aging in mammals. Here, we present results from a comprehensive, large-scale assessment of a wide range of structural and functional aging phenotypes, which we performed to determine whether rapamycin slows the rate of aging in male C57BL/6J mice. While rapamycin did extend lifespan, it ameliorated few studied aging phenotypes. A subset of aging traits appeared to be rescued by rapamycin. Rapamycin, however, had similar effects on many of these traits in young animals, indicating that these effects were not due to a modulation of aging, but rather related to aging-independent drug effects. Therefore, our data largely dissociate rapamycin's longevity effects from effects on aging itself.
Wissenschaftlicher Artikel
Scientific Article
PLoS Biol. 11:e1001506 (2013)
Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.
Wissenschaftlicher Artikel
Scientific Article
Mol. Vis. 19, 877-884 (2013)
PURPOSE: Within a mutagenesis screen, we identified the new mouse mutant Aey80 with small eyes; homozygous mutants were not obtained. The aim of the study was its molecular characterization. METHODS: We analyzed the offspring of paternally N-ethyl-N-nitrosourea (ENU)-treated C3HeB/FeJ mice for dysmorphology parameters, which can be observed with the naked eye. The Aey80 mutant (abnormality of the eye) was further characterized with laser interference biometry, Scheimpflug imaging, and optical coherence tomography. Linkage analysis of the Aey80 mutant was performed using a panel of single nucleotide polymorphisms different among C3HeB/FeJ and C57BL/6J mice. The Aey80 mutation was identified with sequence analysis of the positional candidate gene. RESULTS: We identified a new mutant characterized by an obvious small-eye phenotype; homozygotes are not viable after birth. Embryos at embryonic day 15.5 demonstrate a clear gene-dosage effect: Heterozygotes have small eyes, whereas homozygous mutants do not have eyes. In adult mice, the lenses and the entire eyes of the heterozygous mutants were significantly smaller than those of the wild-types (p<0.01). No other ocular phenotypes were observed; the lenses were fully transparent, and no adhesion to the cornea was observed. The mutation was mapped to chromosome 2; markers between 70.8 MB and 129.5 MB showed significant linkage to the mutation resulting in paired box gene 6 (Pax6) as an excellent candidate gene. We amplified cDNAs from the embryonic eyes and observed an additional band while amplifying the region corresponding to exons 7 and 8. The additional band included an alternative exon of 141 bp, which was associated with a G->A exchange four bases downstream of the end of the alternative exon. The alternative exon in the mutants is predicted to encode 30 novel amino acids and three stop codons. This alternative exon kept the paired domain intact but led to a loss of the homeodomain and the C-terminal proline-serine-threonine (PST) domain. The mutation cosegregated in the mutant line, since all five additional small-eyed mice from this line showed the same mutation. A general polymorphism at the mutated site was excluded with sequence analysis of seven other wild-type mouse strains different from C3HeB/FeJ. CONCLUSIONS: These findings demonstrate a novel allele of the paired box gene 6 (Pax6) that affects lens development in a semidominant manner leading to a classical small-eye phenotype. However, the site of the mutation more than 1 kb downstream of exon 7 and resulting in an alternative exon is quite unusual. It indicates the importance of sequence analysis of cDNA for mutation detection; mutations like this are unlikely to be identified by analyzing genomic sequences only. Moreover, this particular mutation demonstrates how a novel exon can be created by only a single base-pair exchange.
Wissenschaftlicher Artikel
Scientific Article
Mamm. Genome 24, 198-205 (2013)
Spectral domain optical coherence tomography (SD-OCT) has recently been established as a method for in vivo imaging of fundus and retina in the mouse. It enables more effective studies of retinal diseases including investigations of etiopathologic mechanisms. In order to learn more about longitudinal fundus development and to enable recognition of disease-associated irregularities, we performed confocal scanning laser ophthalmoscopy (cSLO) and SD-OCT measurements in the inbred strains C57BL/6J, C3HeB/FeJ, FVB/NCrl, BALB/cByJ, and 129S2/SvJ when they were between 2 and 6 months of age. In general, cSLO and SD-OCT data did not reveal sex-specific or unilateral differences. C3HeB/FeJ and FVB/NCrl mice showed diffuse choroidal dysplasia. Choroidal vein-like structures appeared as dark fundus stripes in C3HeB/FeJ. In FVB/NCrl, fundus fleck accumulation was found. In contrast, only minor time-dependent changes of fundus appearance were observed in C57BL/6J, BALB/cByJ, and 129S2/SvJ. This was also found for individual fundic main blood vessel patterns in all inbred strains. Vessel numbers varied between 6 and 13 in C57BL/6J. This was comparable in most cases. We further found that retinae were significantly thicker in C57BL/6J compared to the other strains. Total retinal thickness generally did not change between 2 and 6 months of age. As a conclusion, our results indicate lifelong pathologic processes in C3HeB/FeJ and FVB/NCrl that affect choroid and orbital tissues. Inbred strains with regular retinal development did not reveal major time-dependent variations of fundus appearance, blood vessel pattern, or retinal thickness. Consequently, progressive changes of these parameters are suitable indicators for pathologic outliers.
Wissenschaftlicher Artikel
Scientific Article
Mamm. Genome 24, 295-302 (2013)
Scheimpflug imaging has recently been established for in vivo imaging of the anterior eye segment and quantitative determination of lens transparency in the mouse. This enables more effective investigations of cataract formation with the mouse model, including longitudinal studies. In order to enable recognition of disease-associated irregularities, we performed Scheimpflug measurements with the common laboratory inbred lines C57BL/6J, C3HeB/FeJ, FVB/NCrl, BALB/cByJ, and 129/SvJ in a period between 2 and 12 months of age. C57BL/6J mice showed lowest mean lens densities during the test period. Progressive cortical lens opacification was generally observed, with the earliest onset in C57BBL/6J, C3HeB/FeJ, and 129/SvJ, between 2 and 6 months after birth. Moreover, lenses of these inbred lines developed nuclear opacities. Calculated mean lens density significantly increased between 6 and 12 months of age in all inbred strains except 129/SvJ. Lens densities (and the corresponding standard deviations) of FVB/NCrl and 129/SvJ increased most likely because of differences in the genetic background. Albinism as confounder might be excluded since the albino Balb/cByJ mice are more similar to the C57BL/6J or C3Heb/FeJ mice. We further identified strain-specific anterior lens opacities (C57BL/6J) and cloudy corneal lesions (C57BL/6J, FVB/NCrl, and BALB/cByJ) at later stages. In conclusion, our results indicate that there are lifelong opacification processes in the mouse lens. The highest lens transparency and a dark coat color, which prevents interference from light reflections, make mice with the C57BL/6J background most suitable for cataract research by Scheimpflug imaging. We show that lens densitometry by Scheimpflug imaging in mouse eyes can resolve differences of less than 1 %, making it possible to detect differences in cataract development in different mouse strains, even if they are small.
Wissenschaftlicher Artikel
Scientific Article
Acta Ophthalmol. 91 (2013)
Purpose To summarize GMC - Eye Screen findings with about 350 tested mouse mutant lines. Methods Eye morphology was analyzed by Slit Lamp Biomicroscopy/Scheimpflug Imaging and Ophthalmoscopy/Optical Coherence Tomography, eye size by Laser Interference Biometry, and the Virtual Optokinetic Drum was used for testing visual ability. Results The GMC is a large-scale phenotyping center where mouse models for human diseases are analyzed in a standardized way. More than 550 parameters are investigated in 14 different areas including behavior, neurology, cardiovascular functionality and vision and eye development. In the past 12 years, we identified eye pathologies in 43 mutant lines of the GMC workflow. The majority of them exhibited irregular eye sizes (72%), followed by morphological changes as corneal and lens opacities (21%) or retinal damages (16%) and moderately reduced visual abilities (7%). Overall phenotype predictions (EuroPhenome database; http://www.europhenome.org/) indicated that 89% of these lines were additionally affected in behavioral and/or neurological parameters, pointing to general relations between eye and brain development. Moreover, 46% showed heart impairments, connecting eye diseases with cardiovascular risks. Our data further proved for 11 gene products a previously unknown participation in ocular processes. These include the chromatin modifiers Asxl1 and Mysm1, the transcription factor Nfya as well as Arvcf, Jmjd5 and Slc20a2 with a role in cell-cell communication, protein hydroxylation and phosphate transport, respectively. Conclusion The GMC - Eye Screen is an effective approach for improving knowledge about ocular genetics and detecting associations with non-ocular disorders.
Meeting abstract
Meeting abstract
Antioxid. Redox Signal. 18, 1418-1427 (2013)
Abstract Significance: Stroke, a leading cause of death and disability, poses a substantial burden for patients, relatives, and our healthcare systems. Only one drug is approved for treating stroke, and more than 30 contraindications exclude its use in 90% of all patients. Thus, new treatments are urgently needed. In this review, we discuss oxidative stress as a pathomechanism of poststroke neurodegeneration and the inhibition of its source, type 4 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4), as a conceptual breakthrough in stroke therapy. Recent Advances: Among potential sources of reactive oxygen species (ROS), the NOXes stand out as the only enzyme family that is solely dedicated to forming ROS. In rodents, three cerebrovascular NOXes exist: the superoxide-forming NOX1 and 2 and the hydrogen peroxide-forming NOX4. Studies using NOX1 knockout mice gave conflicting results, which overall do not point to a role for this isoform. Several reports find NOX2 to be relevant in stroke, albeit to variable and moderate degrees. In our hands, NOX4 is, by far, the major source of oxidative stress and neurodegeneration on ischemic stroke. Critical Issues: We critically discuss the tools that have been used to validate the roles of NOX in stroke. We also highlight the relevance of different animal models and the need for advanced quality control in preclinical stroke research. Future Directions: The development of isoform-specific NOX inhibitors presents a precious tool for further clarifying the role and drugability of NOX homologues. This could pave the avenue for the first clinically effective neuroprotectant applied poststroke, and even beyond this, stroke could provide a proof of principle for antioxidative stress therapy. Antioxid. Redox Signal. 00, 000-000.
Review
Review
Curr. Protoc. Mouse Biol. 3, 69-100 (2013)
Besides hematological analyses, many other parameters, including clinical chemistry and endocrinological values, can be determined from mouse blood samples. For most of these tests, plasma or serum samples are used. Data obtained by these investigations provide indications of genotype effects on metabolism and organ functions. Here we describe in detail the considerations that have to be taken into account to get adequate samples for plasma or serum analyses and the recommended sample processing for different investigations. Furthermore, we describe established methods used in the German Mouse Clinic (GMC) to determine clinical chemical parameters; for more in-depth analysis of specific classes of biomarkers, we provide instructions for ELISAs (sandwich and competitive) as well as LC-MS/MS, focusing on markers associated with bone or steroid metabolism in the mouse as working examples.
Wissenschaftlicher Artikel
Scientific Article
Curr. Protoc. Mouse Biol. 3, 101-119 (2013)
Basic phenotyping of inbred mouse strains and genetically modified mouse models usually includes the determination of blood-based parameters as a diagnostic screen for genotype effects on metabolism and organ function. A broad range of analytes, including hematological parameters, can be reliably determined in mouse blood, if appropriate samples are available. Here we describe recommended techniques for blood collection from mice and the considerations that have to be taken into account to get adequate samples for hematological investigations. Furthermore, we describe established methods used in the German Mouse Clinic (GMC) to determine hematological parameters in the mouse.
Wissenschaftlicher Artikel
Scientific Article
Am. J. Pathol. 183, 352-368 (2013)
Within the Munich, Germany, N-ethyl-N-nitrosourea mouse mutagenesis program, we isolated a dominant Jak1 mouse model resembling phenotypic characteristics related to autoimmune disease. Chromosomal sequencing revealed a new Jak1 (p.Ser645Pro) point mutation at the conserved serine of the pseudokinase domain, corresponding to a somatic human mutation (p.Ser646Phe) inducing a constitutive activation of the Janus kinase (JAK)/STAT pathway. Morphologically, all Jak1(S645P+/-) mice showed a progressive structural deterioration of ears starting at the age of 4 months, with mononuclear cell infiltration into the dermis. Female mutant mice, in particular, developed severe skin lesions in the neck from 7 months of age. The IHC analysis of these lesions showed an activation of Stat3 downstream to Jak1(S645P) and elevated tissue levels of IL-6. Histopathological analysis of liver revealed a nodular regenerative hyperplasia. In the spleen, the number of Russell bodies was doubled, correlating with significant increased levels of all immunoglobulin isotypes and anti-DNA antibodies in serum. Older mutant mice developed thrombocytopenia and altered microcytic red blood cell counts. Jak1(S645P+/-) mice showed phenotypes related to impaired bone metabolism as increased carboxy-terminal collagen cross-link-1 levels and alkaline phosphatase activities in plasma, hypophosphatemia, and strongly decreased bone morphometric values. Taken together, Jak1(S645P+/-) mice showed an increased activation of the IL-6-JAK-STAT pathway leading to a systemic lupus erythematosus-like phenotype and offering a new valuable tool to study the role of the JAK/STAT pathway in disease development.
Wissenschaftlicher Artikel
Scientific Article
Histol. Histopathol. 28, 79-88 (2013)
The mouse is the most commonly used animal for modelling human disease. New approaches for generating genetically manipulated mouse models to represent human disease, as well as target the function of specific genes, has increased the importance of mice in biomedical science. For the correct interpretation of alterations in mouse phenotype the basic morphology of background mouse strains must be known. Despite on-going efforts to create publicly available baseline phenotypic data, the information concerning spontaneous lesions in wild-type mice is incomplete and scattered so far, and further studies are needed. We addressed this problem by screening haematoxylin-eosin stained sections of brain, reproductive organs, urinary bladder, kidney, thyroid, parathyroid, heart, lung, spleen, thymus, lymph nodes, adrenal glands, stomach, intestine, liver, skin and pancreas of six commonly used inbred mouse strains (C57BL6/J, C57BL6/NTac, C3HeB/FeJ, BALB/cByJ, 129P2/OlaHsd and FVB/N) for inherent spontaneous morphological lesions. Interesting spontaneous phenotypes were seen in morphology of the liver, pancreas, adrenal glands, lungs, intestines and heart. In conclusion, care should be taken when choosing the background mouse strain for genetic manipulations, since different mouse strains harbour different inherent lesions that can affect the function of targeted genes, interpretation of results and translation of results to model human disease.
Wissenschaftlicher Artikel
Scientific Article
J. Immunol. 191, 3913-3921 (2013)
Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF-α or IL-6 and might thus contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-α receptor- and IFN regulatory factor (Irf)9-dependent manner. Also, LPS induced secretion of IL-6 and TNF-α by bone marrow-derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-α and reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-β mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-α and Il-6 secretion but not Tnf-α and Il-6 mRNA expression levels were reduced. Thus, IFN-stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock.
Wissenschaftlicher Artikel
Scientific Article
Genome Biol. 14:R82 (2013)
BACKGROUND: The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as The International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms. RESULTS: We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems. CONCLUSIONS: Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.
Wissenschaftlicher Artikel
Scientific Article
Eur. Heart J. 34, 1058 (2013)
Meeting abstract
Meeting abstract
Mamm. Genome 24, 333-348 (2013)
βB2-crystallin (gene symbol: Crybb2/CRYBB2) was first described as a structural protein of the ocular lens. This gene, however, is also expressed in several regions of the mammalian brain, although its function in this organ remains entirely unknown. To unravel some aspects of its function in the brain, we combined behavioral, neuroanatomical, and physiological analyses in a novel Crybb2 mouse mutant, O377. Behavioral tests with male O377 mutants revealed altered sensorimotor gating, suggesting modified neuronal functions. Since these mouse mutants also displayed reduced hippocampal size, we concentrated further investigations on the hippocampus. Free intracellular Ca2+ levels were increased and apoptosis was enhanced in the hippocampus of O377 mutants. Moreover, the expression of the gene encoding calpain 3 (gene symbol Capn3) was elevated and the expression of genes coding for the NMDA receptor subunits was downregulated. Additionally, the number of parvalbumin-positive interneurons was decreased in the hippocampus but not in the cortex of the mutants. High-speed voltage-sensitive dye imaging demonstrated an increased translation of input-to-output neuronal activity in the dentate gyrus of this Crybb2 mutant. These results point to an important function of βB2-crystallin in the hippocampal network. They indicate pleiotropic effects of mutations in the Crybb2 gene, which previously had been considered to be specific to the ocular lens. Moreover, our results are the first to demonstrate that βB2-crystallin has a role in hippocampal function and behavioral phenotypes. This model can now be further explored by future experiments.
Wissenschaftlicher Artikel
Scientific Article
PLoS ONE 8:e61406 (2013)
Background: Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11th 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. Methodology/Principal Findings: 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. Conclusion/Significance: 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.
Wissenschaftlicher Artikel
Scientific Article
2012
Mamm. Genome 23, 600-610 (2012)
Two large-scale phenotyping efforts, the European Mouse Disease Clinic (EUMODIC) and the Wellcome Trust Sanger Institute Mouse Genetics Project (SANGER-MGP), started during the late 2000s with the aim to deliver a comprehensive assessment of phenotypes or to screen for robust indicators of diseases in mouse mutants. They both took advantage of available mouse mutant lines but predominantly of the embryonic stem (ES) cells resources derived from the European Conditional Mouse Mutagenesis programme (EUCOMM) and the Knockout Mouse Project (KOMP) to produce and study 799 mouse models that were systematically analysed with a comprehensive set of physiological and behavioural paradigms. They captured more than 400 variables and an additional panel of metadata describing the conditions of the tests. All the data are now available through EuroPhenome database ( www.europhenome.org ) and the WTSI mouse portal ( http://www.sanger.ac.uk/mouseportal/ ), and the corresponding mouse lines are available through the European Mouse Mutant Archive (EMMA), the International Knockout Mouse Consortium (IKMC), or the Knockout Mouse Project (KOMP) Repository. Overall conclusions from both studies converged, with at least one phenotype scored in at least 80 % of the mutant lines. In addition, 57 % of the lines were viable, 13 % subviable, 30 % embryonic lethal, and 7 % displayed fertility impairments. These efforts provide an important underpinning for a future global programme that will undertake the complete functional annotation of the mammalian genome in the mouse model.
Wissenschaftlicher Artikel
Scientific Article
J. Nat. Med. 18, 1847-1856 (2012)
We report the development of a new combinatorial approach that allows for peptide-mediated selective tissue targeting of nuclear hormone pharmacology while eliminating adverse effects in other tissues. Specifically, we report the development of a glucagon-like peptide-1 (GLP-1)-estrogen conjugate that has superior sex-independent efficacy over either of the individual hormones alone to correct obesity, hyperglycemia and dyslipidemia in mice. The therapeutic benefits are driven by pleiotropic dual hormone action to improve energy, glucose and lipid metabolism, as shown by loss-of-function models and genetic action profiling. Notably, the peptide-based targeting strategy also prevents hallmark side effects of estrogen in male and female mice, such as reproductive endocrine toxicity and oncogenicity. Collectively, selective activation of estrogen receptors in GLP-1-targeted tissues produces unprecedented efficacy to enhance the metabolic benefits of GLP-1 agonism. This example of targeting the metabolic syndrome represents the discovery of a new class of therapeutics that enables synergistic co-agonism through peptide-based selective delivery of small molecules. Although our observations with the GLP-1-estrogen conjugate justify translational studies for diabetes and obesity, the multitude of other possible combinations of peptides and small molecules may offer equal promise for other diseases.
Wissenschaftlicher Artikel
Scientific Article
In: Suhre, K.* [Eds.]: Genetics Meets Metabolomics: from Experiment to Systems Biology. Springer, 2012. 85-106
The mouse is widely considered as a toolbox for modeling human diseases: mice are easy to handle and breed, there exist inbred strains, and the mouse genome sequence is available. Mutant mouse lines can be generated by different technologies, and standardized phenotyping of these mutant mouse lines produces a huge amount of valuable data. Useful resources for the scientific community are archives of mutant lines and strains as well as databases delivering information about the mouse lines and their availability. The phenotypic characterization of mutant mouse lines is the bottleneck within the pipeline from the generation via phenotyping to archiving of mutant mouse lines. Mouse clinics generate large data sets by the standardized, comprehensive phenotypic characterization of mutant mouse lines. There is a portfolio of phenotyping protocols available for a broad spectrum of disease areas that is considered as an international standard. For the investigation of human diseases like diabetes, obesity or the metabolic syndrome, metabolic tests to analyze mutant mouse lines become increasingly important. In this respect, challenge experiments have become the major focus to induce disease phenotypes in mutant mice that would remain undiscovered without the environmental challenges. These experimental setups reflect human conditions, where genetic predisposition and the environmental factors originating from different life style act together and enhance each other.
Eur. J. Oral Sci. 120, 269-277 (2012)
We analyzed two mutant mouse lines, ATE1 and ATE2, that carry point mutations in the enamelin gene which result in premature stop codons in exon 8 and exon 7, respectively. Both mutant lines show amelogenesis imperfecta. To establish the effect of mutations within the enamelin gene on different organs, we performed a systematic, standardized phenotypic analysis of both mutant lines in the German Mouse Clinic. In addition to the initially characterized tooth phenotype that is present in both mutant lines, we detected effects of enamelin mutations on bone and energy metabolism, as well as on clinical chemical and hematological parameters. These data raise the hypothesis that enamelin defects have pleiotropic effects on organs other than the teeth.
Wissenschaftlicher Artikel
Scientific Article
Mamm. Genome 23, 611-622 (2012)
Under the label of the German Mouse Clinic (GMC), a concept has been developed and implemented that allows the better understanding of human diseases on the pathophysiological and molecular level. This includes better understanding of the crosstalk between different organs, pleiotropy of genes, and the systemic impact of envirotypes and drugs. In the GMC, experts from various fields of mouse genetics and physiology, in close collaboration with clinicians, work side by side under one roof. The GMC is an open-access platform for the scientific community by providing phenotypic analysis in bilateral collaborations ("bottom-up projects") and as a partner and driver in international large-scale biology projects ("top-down projects"). Furthermore, technology development is a major topic in the GMC. Innovative techniques for primary and secondary screens are developed and implemented into the phenotyping pipelines (e.g., detection of volatile organic compounds, VOCs).
Wissenschaftlicher Artikel
Scientific Article
PLoS Genet. 8:e1002711 (2012)
PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix.
Wissenschaftlicher Artikel
Scientific Article
BMC Neurosci. 13:61 (2012)
BACKGROUND: The role played by adult neurogenesis in anxiety is not clear. A recent study revealed a surprising positive correlation between increased anxiety and elevated neurogenesis following chronic voluntary wheel running and multiple behavioural testing in mice, suggesting that adult hippocampal neurogenesis is involved in the genesis of anxiety. To exclude the possible confounding effect of multiple testing that may have occurred in the aforementioned study, we assessed (1) the effects of mouse voluntary wheel running (14 vs. 28 days) on anxiety in just one behavioural test; the open field, and (2), using different markers, proliferation, differentiation, survival and maturation of newly born neurons in the dentate gyrus immediately afterwards. Effects of wheel running on anxiety-related behaviour were confirmed in a separate batch of animals tested in another test of anxiety, the light/dark box test. RESULTS: Running altered measures of locomotion and exploration, but not anxiety-related behaviour in either test. 14 days running significantly increased proliferation, and differentiation and survival were increased after both running durations. 28 day running mice also exhibited an increased rate of maturation. Furthermore, there was a significant positive correlation between the amount of proliferation, but not maturation, and anxiety measures in the open field of the 28 day running mice. CONCLUSIONS: Overall, this evidence suggests that without repeated testing, newly born mature neurons may not be involved in the genesis of anxiety per se.
Wissenschaftlicher Artikel
Scientific Article
Diabetes akt. 10, 106-110 (2012)
By the establishment of the new networks of six German Centres for Health Research (DZG) the German Federal Ministry of Education and Research aims to create excellent research conditions for combating the most widespread diseases. In the German Center of Diabetes Research (DZD) the best researchers from universities and research institutions work side by side on improved prevention and innovative therapies for patients with an elevated risk or suffering from diabetes mellitus.
Wissenschaftlicher Artikel
Scientific Article
FASEB J. 26, 5081-5091 (2012)
The hepatic phosphatidylcholine (PC) transporter ATP-binding cassette (ABC) B4 flops PC from hepatocytes into bile, and its dysfunction causes chronic cholestasis and fibrosis. Because a nuclear receptor-dependent PC pathway has been determined to exert antidiabetic effects, we now analyzed the role of ABCB4 in glucose metabolism. We bred congenic Abcb4-knockout (Abcb4(-/-)) mice on the fibrosis-susceptible BALB/cJ background. Knockout mice and wild-type controls were phenotyped by measuring plasma glucose concentrations, intraperitoneal glucose tolerance, hepatic RNA expression profiles, and liver histology. In addition, 4 procholestatic ABCB4 gene variants were correlated with blood glucose levels in 682 individuals from 2 independent European cohorts. Systemic glucose levels differ significantly between Abcb4(-/-) mice and wild-type controls, and knockout mice display improved glucose tolerance with significantly lower area under the curve values on intraperitoneal glucose challenge. Of note, hepatic expression of the antidiabetic nuclear receptor 5A2 (LRH-1) is induced consistently in Abcb4(-/-) mice, and its specific rare PC ligands are detected in liver by mass spectrometry imaging. In humans, serum glucose levels are associated significantly with the common ABCB4 variant c.711A>T. In summary, ABCB4 might play a critical role in glucose homeostasis in mice and humans. We speculate that the effects could be mediated via LRH-1-dependent PC pathways.-Hochrath, K., Krawczyk, M., Goebel, R., Langhirt, M., Rathkolb, B., Micklich, K., Rozman, J., Horsch, M., Beckers, J., Klingenspor, M., Fuchs, H., Gailus-Durner, V., Wolf, E., Acalovschi, M., Volmer, D. A., Hrabě de Angelis, M., Lammert, F. The hepatic phosphatidylcholine transporter ABCB4 as modulator of glucose homeostasis.
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Scientific Article
FASEB J. 26, 2916-2930 (2012)
Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Institute of Experimental Genetics, Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced P(enh) and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (-14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.-Hüttemann, M., Lee, I., Gao, X., Pecina, P., Pecinova, A., Liu, J., Aras, S., Sommer, N., Sanderson, T. H., Tost, M., Neff, F., Aguilar-Pimentel, J. A., Becker, L., Naton, B., Rathkolb, B., Rozman, J., Favor, J., Hans, W., Prehn, C., Puk, O., Schrewe, A., Sun, M., Höfler, H., Adamski, J., Bekeredjian, R., Graw, J., Adler, T., Busch, D. H., Klingenspor, M., Klopstock, T., Ollert, M., Wolf, E., Fuchs, H., Gailus-Durner, V., Hrabě de Angelis, M., Weissmann, N., Doan, J. W., Bassett, D. J. P., Grossman, L. I. Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology.
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Acta Neuropathol. 124, 187-197 (2012)
Aging and neurodegeneration are often accompanied by a functionally impaired ubiquitin-proteasome system (UPS). In tauopathies and polyglutamine diseases, a mutant form of ubiquitin B (UBB(+1)) accumulates in disease-specific aggregates. UBB(+1) mRNA is generated at low levels in vivo during transcription from the ubiquitin B locus by molecular misreading. The resulting mutant protein has been shown to inhibit proteasome function. To elucidate causative effects and neuropathological consequences of UBB(+1) accumulation, we used a UBB(+1) expressing transgenic mouse line that models UPS inhibition in neurons and exhibits behavioral phenotypes reminiscent of Alzheimer's disease (AD). In order to reveal affected organs and functions, young and aged UBB(+1) transgenic mice were comprehensively phenotyped for more than 240 parameters. This revealed unexpected changes in spontaneous breathing patterns and an altered response to hypoxic conditions. Our findings point to a central dysfunction of respiratory regulation in transgenic mice in comparison to wild-type littermate mice. Accordingly, UBB(+1) was strongly expressed in brainstem regions of transgenic mice controlling respiration. These regions included, e.g., the medial part of the nucleus of the tractus solitarius and the lateral subdivisions of the parabrachial nucleus. In addition, UBB(+1) was also strongly expressed in these anatomical structures of AD patients (Braak stage #6) and was not expressed in non-demented controls. We conclude that long-term UPS inhibition due to UBB(+1) expression causes central breathing dysfunction in a transgenic mouse model of AD. The UBB(+1) expression pattern in humans is consistent with the contribution of bronchopneumonia as a cause of death in AD patients.
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Scientific Article
PLoS ONE 7:e44609 (2012)
The mammalian Interferon induced transmembrane protein 1 (Ifitm1) gene was originally identified as a member of a gene family highly inducible by type I and type II interferons. Based on expression analyses, it was suggested to be required for normal primordial germ cell migration. The knockdown of Ifitm1 in mouse embryos provided evidence for a role in somitogenesis. We generated the first targeted knockin allele of the Ifitm1 gene to systematically reassess all inferred functions. Sperm motility and the fertility of male and female mutant mice are as in wild type littermates. Embryonic somites and the adult vertebral column appear normal in homozygous Ifitm1 knockout mice, demonstrating that Ifitm1 is not essential for normal segmentation of the paraxial mesoderm. Proportions of leucocyte subsets, including granulocytes, monocytes, B-cells, T-cells, NK-cells, and NKT-cells, are unchanged in mutant mice. Based on a normal immune response to Listeria monocytogenes infection, there is no evidence for a dysfunction in downstream IFNγ signaling in Ifitm1 mutant mice. Expression from the Ifitm1 locus from E8.5 to E14.5 is highly dynamic. In contrast, in adult mice, Ifitm1 expression is highly restricted and strong in the bronchial epithelium. Intriguingly, IFITM1 is highly overexpressed in tumor epithelia cells of human squamous cell carcinomas and in adenocarcinomas of NSCLC patients. These analyses underline the general importance of targeted in vivo studies for the functional annotation of the mammalian genome. The first comprehensive description of the Ifitm1 expression pattern provides a rational basis for the further examination of Ifitm1 gene functions. Based on our data, the fact that IFITM1 can function as a negative regulator of cell proliferation, and because the gene maps to chromosome band 11p15.5, previously associated with NSCLC, it is likely that IFITM1 in man has a key role in tumor formation.
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J. Amer. Assoc. Lab. Anim. Sci. 51, 418-435 (2012)
Collecting and analyzing available information on the building plans, concepts, and workflow from existing animal facilities is an essential prerequisite for most centers that are planning and designing the construction of a new animal experimental research unit. Here, we have collected and analyzed such information in the context of the European project Infrafrontier, which aims to develop a common European infrastructure for high-throughput systemic phenotyping, archiving, and dissemination of mouse models. A team of experts visited 9 research facilities and 3 commercial breeders in Europe, Canada, the United States, and Singapore. During the visits, detailed data of each facility were collected and subsequently represented in standardized floor plans and descriptive tables. These data showed that because the local needs of scientists and their projects, property issues, and national and regional laws require very specific solutions, a common strategy for the construction of such facilities does not exist. However, several basic concepts were apparent that can be described by standardized floor plans showing the principle functional units and their interconnection. Here, we provide detailed information of how individual facilities addressed their specific needs by using different concepts of connecting the principle units. Our analysis likely will be valuable to research centers that are planning to design new mouse phenotyping and archiving facilities.
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J. Comp. Neurol. 520, 3150-3180 (2012)
The corticotropin-releasing hormone (CRH) and its type 1 receptor (CRHR1) play a central role in coordinating the endocrine, autonomic, and behavioral responses to stress. A prerequisite to functionally dissect the complexity of the CRH/CRHR1 system is to unravel the identity of CRHR1-expressing neurons and their connectivities. Therefore, we used a knockin approach to genetically label CRHR1-expressing cells with a tau-lacZ (tZ) reporter gene. The distribution of neurons expressing β-galactosidase in the brain and the relative intensity of labeling is in full accordance with previously described Crhr1 mRNA expression. Combining the microtubule-binding properties of TAU with the Cre-loxP system allowed to direct the β-galactosidase to proximal dendrites, and in particular to axons. Thereby, we were able to visualize projections of CRHR1 neurons such as glutamatergic and dopaminergic afferent connections of the striatum and GABAergic CRHR1-expressing neurons located within its patch compartment. In addition, the tZ reporter gene revealed novel details of CRHR1 expression in the spinal cord, skin, and eye. CRHR1 expression in the retina prompted the identification of a new physiological role of CRHR1 related to the visual system. Besides its reporter properties, this novel CRHR1 allele comprises the possibility to conditionally restore or delete CRHR1 via Flp and Cre recombinase, respectively. Finally, the allele is suitable for further manipulations of the CRHR1 locus by recombinase-mediated cassette exchange. Taken together, this novel mouse allele will significantly facilitate the neuroanatomical analysis of CRHR1 circuits and opens up new avenues to address CRHR1 function in more detail.
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Scientific Article
PLoS ONE 7:e41537 (2012)
To date, genome-wide association studies (GWAS) have identified at least 32 novel loci for obesity and body mass-related traits. However, the causal genetic variant and molecular mechanisms of specific susceptibility genes in relation to obesity are yet to be fully confirmed and characterised. Here, we examined whether the candidate gene NEGR1 encoding the neuronal growth regulator 1, also termed neurotractin or Kilon, accounts for the obesity association. To characterise the function of NEGR1 for body weight control in vivo, we generated two novel mutant mouse lines, including a constitutive NEGR1-deficient mouse line as well as an ENU-mutagenised line carrying a loss-of-function mutation (Negr1-I87N) and performed metabolic phenotypic analyses. Ablation of NEGR1 results in a small but steady reduction of body mass in both mutant lines, accompanied with a small reduction in body length in the Negr1-I87N mutants. Magnetic resonance scanning reveals that the reduction of body mass in Negr1-I87N mice is due to a reduced proportion of lean mass. Negr1-I87N mutants display reduced food intake and physical activity while normalised energy expenditure remains unchanged. Expression analyses confirmed the brain-specific distribution of NEGR1 including strong expression in the hypothalamus. In vitro assays show that NEGR1 promotes cell-cell adhesion and neurite growth of hypothalamic neurons. Our results indicate a role of NEGR1 in the control of body weight and food intake. This study provides evidence that supports the link of the GWAS candidate gene NEGR1 with body weight control.
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Scientific Article
PLoS Genet. 8:e1002568 (2012)
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.
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Scientific Article
PLoS ONE 7:e50818 (2012)
Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220(+) bone marrow cells, CD19(-) thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis.
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Scientific Article
Mamm. Genome 23, 416-430 (2012)
Metabolic bone disorders arise as primary diseases or may be secondary due to a multitude of organ malfunctions. Animal models are required to understand the molecular mechanisms responsible for the imbalances of bone metabolism in disturbed bone mineralization diseases. Here we present the isolation of mutant mouse models for metabolic bone diseases by phenotyping blood parameters that target bone turnover within the large-scale genome-wide Munich ENU Mutagenesis Project. A screening panel of three clinical parameters, also commonly used as biochemical markers in patients with metabolic bone diseases, was chosen. Total alkaline phosphatase activity and total calcium and inorganic phosphate levels in plasma samples of F1 offspring produced from ENU-mutagenized C3HeB/FeJ male mice were measured. Screening of 9,540 mice led to the identification of 257 phenodeviants of which 190 were tested by genetic confirmation crosses. Seventy-one new dominant mutant lines showing alterations of at least one of the biochemical parameters of interest were confirmed. Fifteen mutations among three genes (Phex, Casr, and Alpl) have been identified by positional-candidate gene approaches and one mutation of the Asgr1 gene, which was identified by next-generation sequencing. All new mutant mouse lines are offered as a resource for the scientific community.
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Scientific Article
PLoS ONE 7:e38310 (2012)
Cln3(Delta ex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Delta ex7/8) mice. Homozygous Cln3(Delta ex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Delta ex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post- receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12- 13 week old homozygous Cln3(Delta ex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Delta ex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Delta ex7/8) mice, and male homozygotes had a relative T- cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Delta ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Delta ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Delta ex7/8) mice that merit further study for JNCL biomarker development.
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Scientific Article
Mamm. Genome 23, 259-269 (2012)
Mice genetically engineered to express human FcRn are valuable models for the evaluation of therapeutic antibodies in the context of human FcRn in vivo. However, only limited clinical chemistry information on these mouse strains is available. Thus, we have compared 30 clinical chemical parameters of C57BL/6J wild-type mice, murine FcRn-knockout mice, and two human FcRn transgenic mouse strains expressing human FcRn in the absence of murine FcRn. Since FcRn-mediated recycling prevents albumin and IgG from intracellular degradation, significant differences for both proteins were observed in the murine FcRn-knockout mice. Mice lacking FcRn show lower IgG and albumin levels compared to wild-type mice. The most prominent differences in clinical chemical parameters can be explained by secondary effects of the altered albumin levels of murine FcRn-knockout mice on liver metabolism, as similar tendencies have been observed in analbuminemic Nagase rats and hypoalbuminemic human patients, showing an overall increased liver metabolism. Both human FcRn transgenic strains show clinical chemical parameters similar to those found for wild-type mice, with the exception of endogenous IgG levels, which are greatly reduced in these mice.
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Hum. Mol. Genet. 21, 3535-3545 (2012)
Osteogenesis imperfecta (OI) is an inherited connective tissue disorder with skeletal dysplasia of varying severity, predominantly caused by mutations in the collagen I genes (COL1A1/COL1A2). Extraskeletal findings such as cardiac and pulmonary complications are generally considered to be significant secondary features. Aga2, a murine model for human OI, was systemically analyzed in the German Mouse Clinic by means of in vivo and in vitro examinations of the cardiopulmonary system, to identify novel mechanisms accounting for perinatal lethality. Pulmonary and, especially, cardiac fibroblast of perinatal lethal Aga2/+ animals display a strong down-regulation of Col1a1 transcripts in vivo and in vitro, resulting in a loss of extracellular matrix integrity. In addition, dysregulated gene expression of Nppa, different types of collagen and Agt in heart and lung tissue support a bone-independent vicious cycle of heart dysfunction, including hypertrophy, loss of myocardial matrix integrity, pulmonary hypertension, pneumonia and hypoxia leading to death in Aga2. These murine findings are corroborated by a pediatric OI cohort study, displaying significant progressive decline in pulmonary function and restrictive pulmonary disease independent of scoliosis. Most participants show mild cardiac valvular regurgitation, independent of pulmonary and skeletal findings. Data obtained from human OI patients and the mouse model Aga2 provide novel evidence for primary effects of type I collagen mutations on the heart and lung. The findings will have potential benefits of anticipatory clinical exams and early intervention in OI patients.
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FASEB J. 26, 4418-4428 (2012)
Mutations in the SRGAP3 gene residing on chromosome 3p25 have previously been associated with intellectual disability. Genome-wide association studies have also revealed SRGAP3, together with genes from the same cellular network, as risk genes for schizophrenia. SRGAP3 regulates cytoskeletal dynamics through the RHO protein RAC1. RHO proteins are known to be involved in cytoskeletal reorganization during brain development to control processes such as synaptic plasticity. To elucidate the importance of SRGAP3 in brain development, we generated Srgap3-knockout mice. Ten percent of these mice developed a hydrocephalus and died before adulthood. Surviving mice showed various neuroanatomical changes, including enlarged lateral ventricles, white matter tracts, and dendritic spines together with molecular changes, including an increased basal activity of RAC1. Srgap3(-/-) mice additionally exhibited a complex behavioral phenotype. Behavioral studies revealed an impaired spontaneous alternation and social behavior, while long-term memory was unchanged. The animals also had tics. Lower locomotor activity was observed in male Srgap3(-/-) only. Srgap3(-/-) mice showed increased methylphenidate stimulation in males and an impaired prepulse inhibition in females. Together, the results show neurodevelopmental aberration in Srgap3(-/-) mice, with many of the observed phenotypes matching several schizophrenia-related intermediate phenotypes. Mutations of SRGAP3 may thus contribute to various neurodevelopmental disorders.
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In: Ruf, T.* ; Bieber, C.* ; Arnold, W.* ; Millesi, E.* [Eds.]: Living in a Seasonal World. Berlin, Heidelberg: Springer, 2012. 459-469
Spontaneous daily torpor as well as cold- and fasting-induced torpor is observed in many mammals and birds. The frequency and intensity of torpor are largely influenced by well characterized environmental conditions. The inherited component of phenotypic variation in body temperature regulation is, however, only poorly understood. The identification of mutations affecting physiological body temperature regulation may elucidate novel molecular mechanisms contributing to metabo- and thermoregulatory behavior. Therefore, we monitored rectal body temperature (T b) in six inbred mouse strains (129/SvJ, AKR/J, C57BL/6J, C57BL/6N, Balb/cJ, and SWR/J) and in 146 mutant mouse lines and age-matched controls. Measurements were taken under ad libitum conditions and in response to either food restriction or food deprivation. Inbred mice showed considerable strain-specific differences in T b under ad libitum feeding as well as after food restriction and deprivation. T b was on average ≈0.4°C higher in females as compared to males in all inbred lines. The thermoregulatory response to food restriction reflected a trade-off between changes in T b and body mass with the least body mass loss in mice exhibiting the largest drop in T b. In mutant mouse lines under ad libitum feeding T b of 36 out of 146 was significantly higher (15 lines) or lower (21 lines) as compared with controls. In response to food restriction and deprivation, 13 mutant mouse lines with augmented T b reduction were found two of which showed rectal T b lower than 31°C. In conclusion, T b under ad libitum as well as in response to food deprivation is an important indicator for genotype-related differences on systemic functions. Depending on the test conditions 25–32% of all mutant lines showed significant alterations in T b.
2011
Arthritis Rheum. 63, 1301-1311 (2011)
OBJECTIVE: It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS: In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS: A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION: These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.
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Methods 53, 120-135 (2011)
Model organisms like the mouse are important tools to learn more about gene function in man. Within the last 20years many mutant mouse lines have been generated by different methods such as ENU mutagenesis, constitutive and conditional knock-out approaches, knock-down, introduction of human genes, and knock-in techniques, thus creating models which mimic human conditions. Due to pleiotropic effects, one gene may have different functions in different organ systems or time points during development. Therefore mutant mouse lines have to be phenotyped comprehensively in a highly standardized manner to enable the detection of phenotypes which might otherwise remain hidden. The German Mouse Clinic (GMC) has been established at the Helmholtz Zentrum München as a phenotyping platform with open access to the scientific community (www.mousclinic.de; [1]). The GMC is a member of the EUMODIC consortium which created the European standard workflow EMPReSSslim for the systemic phenotyping of mouse models (http://www.eumodic.org/[2]).
Review
Review
Part. Fibre Toxicol. 8:30 (2011)
BACKGROUND: Obesity can be linked to disease risks such as diabetes and cardiovascular disorders, but recently, the adipose tissue (AT) macrophage also emerges as actively participating in inflammation and immune function, producing pro- and anti-inflammatory factors. Connections between the AT and chronic lung diseases, like emphysema and asthma and a protective role of adipocyte-derived proteins against acute lung injury were suggested.In this study we addressed the question, whether a diet challenge increases the inflammatory response in the alveolar and the blood compartment in response to carbon nanoparticles (CNP), as a surrogate for ambient/urban particulate air pollutants METHODS: Mice were fed a high caloric carbohydrate-rich (CA) or a fat-rich (HF) diet for six weeks and were compared to mice kept on a purified low fat (LF) diet, respectively. Bronchoalveolar lavage (BAL) and blood samples were taken 24 h after intratracheal CNP instillation and checked for cellular and molecular markers of inflammation. RESULTS AND DISCUSSION: The high caloric diets resulted in distinct effects when compared with LF mice, respectively: CA resulted in increased body and fat mass without affecting blood cellular immunity. Conversely, HF activated the blood system, increasing lymphocyte and neutrophil counts, and resulted in slightly increased body fat content. In contrast to higher pro-inflammatory BAL Leptin in CA and HF mice, on a cellular level, both diets did not lead to an increased pro-inflammatory basal status in the alveolar compartment per se, nor did result in differences in the particle-triggered response. However both diets resulted in a disturbance of the alveolar capillary barrier as indicated by enhanced BAL protein and lactate-dehydrogenase concentrations. Systemically, reduced serum Adiponectin in HF mice might be related to the observed white blood cell increase. CONCLUSION: The increase in BAL pro-inflammatory factors in high caloric groups and reductions in serum concentrations of anti-inflammatory factors in HF mice, clearly show diet-specific effects, pointing towards augmented systemic inflammatory conditions. Our data suggest that extended feeding periods, leading to manifest obesity, are necessary to generate an increased susceptibility to particle-induced lung inflammation; although the diet-challenge already was efficient in driving pro-inflammatory systemic events.
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J. Biol. Chem. 286, 18614-18622 (2011)
ADAR2, an RNA editing enzyme that converts specific adenosines to inosines in certain pre-mRNAs, often leading to amino acid substitutions in the encoded proteins, is mainly expressed in brain. Of all ADAR2-mediated edits, a single one in the pre-mRNA of the AMPA receptor subunit GluA2 is essential for survival. Hence, early postnatal death of mice lacking ADAR2 is averted when the critical edit is engineered into both GluA2 encoding Gria2 alleles. Adar2(-/-)/Gria2(R/R) mice display normal appearance and life span, but the general phenotypic effects of global lack of ADAR2 have remained unexplored. Here we have employed the Adar2(-/-)/Gria2(R/R) mouse line, and Gria2(R/R) mice as controls, to study the phenotypic consequences of loss of all ADAR2-mediated edits except the critical one in GluA2. Our extended phenotypic analysis covering ∼320 parameters identified significant changes related to absence of ADAR2 in behavior, hearing ability, allergy parameters and transcript profiles of brain.
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Pain 152, 2384-2398 (2011)
The perception of pain is initiated by the transduction of noxious stimuli through specialized ion channels and receptors expressed by primary nociceptive neurons. The molecular mechanisms that orchestrate the expression and function of ion channels relevant for pain processing are poorly understood. We demonstrate here a central role of the transcription factor Smad-interacting protein 1 (Sip1/Zfhx1b/Zeb2), a 2-handed zinc finger DNA-binding protein with essential functions in neural crest and forebrain development, in controlling nociceptive neuron excitability and pain sensitivity. Mutant mice lacking 1 Zfhx1b allele displayed decreased thermal pain responses, whereas mechanical pain was unaffected. In parallel, repetitive firing of capsaicin/heat-sensitive nociceptive DRG neurons was markedly impaired. Analysis of the voltage-gated currents underlying repetitive firing revealed a significant increase in persistent sodium currents and a reduction in delayed rectifier potassium currents. Modeling experiments in conjunction with experimental results suggest that these changes cause a depolarization-induced block of action potential propagation past the DRG axon T-junction. These data suggest that Sip1 controls the transduction properties of heat-sensitive primary sensory neurons and thus thermal pain sensitivity in a novel manner via coordinated changes in DRG-neuron voltage-gated ion channels.
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Blood 117, 519-529 (2011)
Fas Ligand (FasL) not only induces apoptosis in Fas receptor-bearing target cells, it is also able to transmit signals into the FasL-expressing cell via its intracellular domain (ICD). Recently, we described a Notch-like proteolytic processing of FasL that leads to the release of the FasL intracellular domain (ICD) into the cytoplasm and subsequent translocation into the nucleus where it may influence gene transcription. To study the molecular mechanism underlying such reverse FasL signaling in detail and to analyze its physiological importance in vivo, we established a knockout/knockin mouse model in which wildtype FasL was replaced with a deletion mutant lacking the ICD. Our results demonstrate that FasL ICD signaling impairs activation-induced proliferation in B and T cells by diminishing phosphorylation of PLCγ, PKC and ERK1/2. We also demonstrate that the FasL ICD interacts with the transcription factor Lymphoid-enhancer binding factor-1 (Lef-1) and inhibits Lef-1-dependent transcription. In vivo, plasma cell numbers, generation of germinal center B cells and, consequently, production of antigen-specific IgM antibodies in response to immunization with T cell-dependent (TD) or T cell-independent (TI) antigen are negatively affected in presence of the FasL ICD, suggesting that FasL reverse signaling participates in negative fine-tuning of certain immune responses.
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Nat. Commun. 2:395 (2011)
High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions.
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Scientific Article
J. Cell Sci. 124, 1245-1255 (2011)
MIM/MTSS1 is a tissue-specific regulator of plasma membrane dynamics, whose altered expression levels have been linked to cancer metastasis. MIM deforms phosphoinositide-rich membranes through its I-BAR domain and interacts with actin monomers through its WH2 domain. Recent work proposed that MIM also potentiates Sonic hedgehog (Shh)-induced gene expression. Here, we generated MIM mutant mice and found that full-length MIM protein is dispensable for embryonic development. However, MIM-deficient mice displayed a severe urinary concentration defect caused by compromised integrity of kidney epithelia intercellular junctions, which led to bone abnormalities and end-stage renal failure. In cultured kidney epithelial (MDCK) cells, MIM displayed dynamic localization to adherens junctions, where it promoted Arp2/3-mediated actin filament assembly. This activity was dependent on the ability of MIM to interact with both membranes and actin monomers. Furthermore, results from the mouse model and cell culture experiments suggest that full-length MIM is not crucial for Shh signaling, at least during embryogenesis. Collectively, these data demonstrate that MIM modulates interplay between the actin cytoskeleton and plasma membrane to promote the maintenance of intercellular contacts in kidney epithelia.
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Scientific Article
Eur. J. Endocrinol. 165, 555-561 (2011)
Context Thyroid hormone transport across the plasma membrane depends on transmembrane transport proteins, including monocarboxylate transporter 8 (MCT8). Mutations in MCT8 (or SLC16A2) lead to a severe form of X-linked psychomotor retardation, which is characterised by elevated plasma triiodothyronine (T(3)) and low/normal thyroxine (T(4)). MCT8 contributes to hormone release from the thyroid gland. Objective To characterise the potential impact of MCT8-deficiency on thyroid morphology in a patient and in Mct8-deficient mice. Design Thyroid morphology in a patient carrying the A224V mutation was followed by ultrasound imaging for over 10 years. After thyroidectomy, a histopathological analysis was carried out. The findings were compared with histological analyses of mouse thyroids from the Mct8(-/y) model. Results We show that an inactivating mutation in MCT8 leads to a unique, progressive thyroid follicular pathology in a patient. After thyroidectomy, histological analysis revealed gross morphological changes, including several hyperplastic nodules, microfollicular areas with stromal fibrosis and a small focus of microfollicular structures with nuclear features reminiscent of papillary thyroid carcinoma (PTC). These findings are supported by an Mct8-null mouse model in which we found massive papillary hyperplasia in 6- to 12-month-old mice and nuclear features consistent with PTC in almost 2-year-old animals. After complete thyroidectomy and substitution with levothyroxine (l-T(4)), the preoperative, inadequately low T(4) and free T(4) remained, while increasing the l-T(4) dosage led to T(3) serum concentrations above the normal range. Conclusions Our results implicate peripheral deiodination in the peculiar hormonal constellation of MCT8-deficient patients. Other MCT8-deficient patients should be closely monitored for potential thyroid abnormalities.
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Scientific Article
2010
Am. J. Respir. Crit. Care Med. 181, 7-16 (2010)
RATIONALE: Studies in humans and rodents have indicated a causative role for CD8(+) T cells in IgE-mediated allergic inflammation, but their function is still controversial. OBJECTIVES: To analyze the role of allergen-specific CD8(+) T cells during the development of allergic airway inflammation in two parallel but diverging outcome models. METHODS: We used H2-Kb SIINFEKL (OVA(257-264)) multimers to analyze induction, natural distribution, and phenotype of allergen-specific CD8(+) T cells in a murine C57BL/6 model of ovalbumin (OVA)-induced allergic airway inflammation using low-dose or high-dose OVA sensitization. MEASUREMENTS AND MAIN RESULTS: The low-dose protocol was characterized by a significant induction of total and OVA-specific IgE, eosinophilic airway inflammation, IL-4 levels in bronchoalveolar lavage fluid. And significant alterations in lung function. The high dose protocol was characterized by a significant reduction of the allergic phenotype. Using OVA(257-264) H2-Kb multimers, we observed lung and airway infiltrating OVA-specific CD8(+) T cells showing an effector/effector-memory phenotype. The high-dose protocol caused significantly higher infiltration of allergen-specific CD8(+) cells to the airways and enhanced their cytotoxicity. Adoptive transfer with CD8(+) T cells from transgenic OT-I mice to TAP1(-/-) or wild-type mice showed their migration to the lungs and TAP1-dependent proliferation after OVA-aerosol exposure. TAP1(-/-) mice defective in CD8(+) T cells showed exacerbated symptoms in the low-dose sensitization model. CONCLUSIONS: Allergen-specific CD8(+) T cells seem to protect from allergic inflammation in the lungs. Their number, which is dependent on the sensitization dose, appears to be a critical predictor for the severity of the allergic phenotype.
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Scientific Article
J. Neurosci. 30, 9103-9116 (2010)
Urocortin 3 (UCN3) is strongly expressed in specific nuclei of the rodent brain, at sites distinct from those expressing urocortin 1 and urocortin 2, the other endogenous ligands of corticotropin-releasing hormone receptor type 2 (CRH-R2). To determine the physiological role of UCN3, we generated UCN3-deficient mice, in which the UCN3 open reading frame was replaced by a tau-lacZ reporter gene. By means of this reporter gene, the nucleus parabrachialis and the premammillary nucleus were identified as previously unknown sites of UCN3 expression. Additionally, the introduced reporter gene enabled the visualization of axonal projections of UCN3-expressing neurons from the superior paraolivary nucleus to the inferior colliculus and from the posterodorsal part of the medial amygdala to the principal nucleus of the bed nucleus of the stria terminalis, respectively. The examination of tau-lacZ reporter gene activity throughout the brain underscored a predominant expression of UCN3 in nuclei functionally connected to the accessory olfactory system. Male and female mice were comprehensively phenotyped but none of the applied tests provided indications for a role of UCN3 in the context of hypothalamic-pituitary-adrenocortical axis regulation, anxiety- or depression-related behavior. However, inspired by the prevalent expression throughout the accessory olfactory system, we identified alterations in social discrimination abilities of male and female UCN3 knock-out mice that were also present in male CRH-R2 knock-out mice. In conclusion, our results suggest a novel role for UCN3 and CRH-R2 related to the processing of social cues and to the establishment of social memories.
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Vortrag: EVER-Meeting (Eurpean Vision and Eye Researchers), 6.-9. Oktober 2010, Kreta. (2010)
Purpose: Systematic analysis of the ocular defects in a new mouse mutant carrying a Bmpr1b mutation. Method: The Bmpr1b mutants were analyzed by slit lamp, funduscopy, laser interference biometry, electroretinography, virtual drum, histology and in-situ hybridization. Results: The new Bmpr1b mutant is characterized by a T->G transversion in the splice donor site of exon 10. The mutants show irregular forelimb and hindlimb morphology, brachydactyly, irregular cartilage development and alterations in the reproductive system. The lens is clear, but in homozygous mutants, an enlarged optic disc was observed by funduscopy. The eyes are slightly larger, which is mainly due to an increased aqueous humor. Electroretinography demonstrated a more rapid decline of the b-wave indicating a less efficient transmission of electric stimuli within the retina. Moreover, homozygous mutant mice show also almost no response to moving stripes as compared to the wild type mice. Histological analysis indicated dramatic changes in the optic nerve head. Analysis of the embryonic development of the eye showed that the expression area of Pax6 expands posterior, and concomitantly, Pax2 expression (which is regular at embryonic day 11.5) is repressed at embryonic days 15.5 and 17.5 at the optic nerve head. Conclusion: The T-G mutation in the splice donor site of exon 10 of the Bmpr1b gene leads to a recessive hypoplasia of the optic nerve head, which is mainly caused by repression of Pax2 expression in the late phase of embryogenesis.
Am. J. Physiol.-Renal Physiol. 298, F1405-F1415 (2010)
The bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter NKCC2, located in the thick ascending limb of Henle's loop, plays a critical role in the kidney's ability to concentrate urine. In humans, loss-of-function mutations of the solute carrier family 12 member 1 gene (SLC12A1), coding for NKCC2, cause type I Bartter syndrome, which is characterized by prenatal onset of a severe polyuria, salt-wasting tubulopathy, and hyperreninemia. In this study, we describe a novel chemically induced, recessive mutant mouse line termed Slc12a1(I299F) exhibiting late-onset manifestation of type I Bartter syndrome. Homozygous mutant mice are viable and exhibit severe polyuria, metabolic alkalosis, marked increase in plasma urea but close to normal creatininemia, hypermagnesemia, hyperprostaglandinuria, hypotension,, and osteopenia. Fractional excretion of urea is markedly decreased. In addition, calcium and magnesium excretions are more than doubled compared with wild-type mice, while uric acid excretion is twofold lower. In contrast to hyperreninemia present in human disease, plasma renin concentration in homozygotes is not increased. The polyuria observed in homozygotes may be due to the combination of two additive factors, a decrease in activity of mutant NKCC2 and an increase in medullary blood flow, due to prostaglandin-induced vasodilation, that impairs countercurrent exchange of urea in the medulla. In conclusion, this novel viable mouse line with a missense Slc12a1 mutation exhibits most of the features of type I Bartter syndrome and may represent a new model for the study of this human disease.
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PLoS Biol. 8:e1000479 (2010)
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.
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Genes Brain Behav. 9, 305-317 (2010)
Loss of function of DJ-1 (PARK7) is associated with autosomal recessive early-onset Parkinson's disease (PD), one of the major age-related neurological diseases. In this study, we extended former studies on DJ-1 knockout mice by identifying subtle morphological and behavioural phenotypes. The DJ-1 gene trap-induced null mutants exhibit less dopamine-producing neurons in the ventral tegmental area (VTA). They also exhibit slight changes in behaviour, i.e. diminished rearing behaviour and impairments in object recognition. Furthermore, we detected subtle phenotypes, which suggest that these animals compensate for the loss of DJ-1. First, we found a significant upregulation of mitochondrial respiratory enzyme activities, a mechanism known to protect against oxidative stress. Second, a close to significant increase in c-Jun N-terminal kinase 1 phosphorylation in old DJ-1-deficient mice hints at a differential activation of neuronal cell survival pathways. Third, as no change in the density of tyrosine hydroxylase (TH)-positive terminals in the striatum was observed, the remaining dopamine-producing neurons likely compensate by increasing axonal sprouting. In summary, the present data suggest that DJ-1 is implicated in major non-motor symptoms of PD appearing in the early phases of the disease-such as subtle impairments in motivated behaviour and cognition-and that under basal conditions the loss of DJ-1 is compensated.
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Scientific Article
Mamm. Genome 21, 13-27 (2010)
A new spontaneous mouse mutant was characterized by closed eyelids at weaning and without apparent eyes (provisional gene name, eyeless; provisional gene symbol, eyl). The mutation follows a recessive pattern of inheritance and was mapped to the region of chromosome 19 containing Pitx3. Genetic complementation tests using Pitx3 ( ak/+ ) mice confirmed eyl as a new allele of Pitx3 (Pitx3 ( eyl )). Sequencing of the Pitx3 gene in eyl mutants identified an inserted G after cDNA position 416 (416insG; exon 4). The shifted open reading frame is predicted to result in a hybrid protein still containing the Pitx3 homeobox, but followed by 121 new amino acids. The novel Pitx3 ( eyl/eyl ) mutants expressed ophthalmological and brain defects similar to Pitx3 ( ak/ak ) mice: microphthalmia or anophthalmia and loss of dopamine neurons of the substantia nigra. In addition, we observed in the homozygous eyeless mutants increased extramedullary hematopoiesis in the spleen, frequently liver steatosis, and reduced body weight. There were also several behavioral changes in the homozygous mutants, including reduced forelimb grip strength and increased nociception. In addition to these alterations in both sexes, we observed in female Pitx3 ( eyl/eyl ) mice increased anxiety-related behavior, reduced locomotor activity, reduced object exploration, and increased social contacts; however, we observed decreased anxiety-related behavior and increased arousal in males. Most of these defects identified in the new Pitx3 mutation are observed in Parkinson patients, making the Pitx3 ( eyl ) mutant a valuable new model. It is the first mouse mutant carrying a point mutation within the coding region of Pitx3.
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EMBO J. 29, 2421-2432 (2010)
Despite extensive investigations of Cbl-interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85(Deltaex2)) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85(Deltaex2) animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post-synaptic compartment of striatal neurons in which it co-clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85(Deltaex2) mice.
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