Publications by authors named "Birgit Rathkolb"

115 Publications

PAX6 mutation alters circadian rhythm and β cell function in mice without affecting glucose tolerance.

Commun Biol 2020 Oct 30;3(1):628. Epub 2020 Oct 30.

Helmholtz Zentrum München, Institute of Experimental Genetics and German Mouse Clinic, Neuherberg, Germany.

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 mice lack appropriate retinal structures for light perception and do not display normal daily rhythmic changes in energy metabolism. Despite β 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 β cell functional maturity, the Leca2 mutation impacts peripheral metabolism via loss of circadian rhythm, thus revealing pleiotropic effects of PAX6.
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http://dx.doi.org/10.1038/s42003-020-01337-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599253PMC
October 2020

Murine tissue factor disulfide mutation causes a bleeding phenotype with sex specific organ pathology and lethality.

Haematologica 2020 10 1;105(10):2484-2495. Epub 2020 Oct 1.

Department of Cardiology, University Heart Center, University Hospital, Zurich, Switzerland.

Tissue factor is highly expressed in sub-endothelial tissue. The extracellular allosteric disulfide bond Cys186-Cys209 of human tissue factor shows high evolutionary conservation and in vitro evidence suggests that it significantly contributes to tissue factor procoagulant activity. To investigate the role of this allosteric disulfide bond in vivo, we generated a C213G mutant tissue factor mouse by replacing Cys213 of the corresponding disulfide Cys190-Cys213 in murine tissue factor. A bleeding phenotype was prominent in homozygous C213G tissue factor mice. Pre-natal lethality of 1/3rd of homozygous offspring was observed between E9.5 and E14.5 associated with placental hemorrhages. After birth, homozygous mice suffered from bleedings in different organs and reduced survival. Homozygous C213G tissue factor 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 (about 1000-fold). Protein glycosylation was impaired and cell membrane exposure decreased in macrophages in vivo. Single housing of homozygous C213G tissue factor 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 tissue factor allosteric disulfide bond is of crucial importance for normal in vivo expression, post-translational processing and activity of murine tissue factor. Although C213G tissue factor mice do not display the severe embryonic lethality of tissue factor knock-out mice, their postnatal bleeding phenotype emphasizes the importance of fully functional tissue factor for hemostasis.
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http://dx.doi.org/10.3324/haematol.2019.218818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556672PMC
October 2020

METTL6 is a tRNA mC methyltransferase that regulates pluripotency and tumor cell growth.

Sci Adv 2020 Aug 26;6(35):eaaz4551. Epub 2020 Aug 26.

Institute of Functional Epigenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany.

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 in mouse stem cells results in changes in ribosome occupancy and RNA levels, as well as impaired pluripotency. In mice, 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.
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http://dx.doi.org/10.1126/sciadv.aaz4551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449687PMC
August 2020

Physiological relevance of the neuronal isoform of inositol-1,4,5-trisphosphate 3-kinases in mice.

Neurosci Lett 2020 09 25;735:135206. Epub 2020 Jun 25.

Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246, Hamburg, Germany. Electronic address:

Inositol-1,4,5-trisphosphate 3-kinase-A (ITPKA) is the neuronal isoform of ITPKs and exhibits both actin bundling and InsPkinase 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.
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http://dx.doi.org/10.1016/j.neulet.2020.135206DOI Listing
September 2020

Congenic expression of poly-GA but not poly-PR in mice triggers selective neuron loss and interferon responses found in C9orf72 ALS.

Acta Neuropathol 2020 08 19;140(2):121-142. Epub 2020 Jun 19.

German Center for Neurodegenerative Diseases (DZNE), Munich, 81377, Munich, Germany.

Expansion of a (GC) repeat in C9orf72 causes 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 in C9orf72 cases. 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 in C9orf72 disease and contributes to TDP-43 abnormalities and neuron loss selectively in disease-relevant regions.
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http://dx.doi.org/10.1007/s00401-020-02176-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360660PMC
August 2020

Functional changes of the liver in the absence of growth hormone (GH) action - Proteomic and metabolomic insights from a GH receptor deficient pig model.

Mol Metab 2020 06 18;36:100978. Epub 2020 Mar 18.

Laboratory for Functional Genome Analysis, (LAFUGA), Gene Center, LMU Munich, 81377 Munich, Germany; Institute of Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, 81377 Munich, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molmet.2020.100978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184181PMC
June 2020

A comprehensive and comparative phenotypic analysis of the collaborative founder strains identifies new and known phenotypes.

Mamm Genome 2020 02 14;31(1-2):30-48. Epub 2020 Feb 14.

German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstrasse 1, 85764, Neuherberg, Germany.

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.
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http://dx.doi.org/10.1007/s00335-020-09827-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060152PMC
February 2020

Irp2 regulates insulin production through iron-mediated Cdkal1-catalyzed tRNA modification.

Nat Commun 2020 01 15;11(1):296. Epub 2020 Jan 15.

Department of Medicine, Division of Hematology, University of Utah, Salt Lake City, UT, 84112, USA.

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 β cells. This impairs Fe-S cluster biosynthesis, reducing the function of Cdkal1, an Fe-S cluster enzyme that catalyzes methylthiolation of tA37 in tRNA to mstA37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes mstA37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2 β 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.
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http://dx.doi.org/10.1038/s41467-019-14004-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962211PMC
January 2020

In-depth phenotyping reveals common and novel disease symptoms in a hemizygous knock-in mouse model (Mut-ko/ki) of mut-type methylmalonic aciduria.

Biochim Biophys Acta Mol Basis Dis 2020 03 23;1866(3):165622. Epub 2019 Nov 23.

Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland; radiz - Rare Disease Initiative Zurich, Clinical Research Priority Program for Rare Diseases, University of Zurich, Zurich, Switzerland; Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland. Electronic address:

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-carnitine (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.
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http://dx.doi.org/10.1016/j.bbadis.2019.165622DOI Listing
March 2020

Mild maternal hyperglycemia in transgenic pigs causes impaired glucose tolerance and metabolic alterations in neonatal offspring.

Dis Model Mech 2019 08 12;12(8). Epub 2019 Aug 12.

Chair for Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, 81377 Munich, Germany.

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 transgenic pigs, which are a novel genetically modified large-animal model expressing mutant insulin (INS) C93S in pancreatic β-cells. This mutation results in impaired glucose tolerance, mild fasting hyperglycemia and insulin resistance during late pregnancy. Compared with offspring from wild-type 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 lyso-phosphatidylcholines) and α-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.
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http://dx.doi.org/10.1242/dmm.039156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737953PMC
August 2019

Glutathione peroxidase 4 and vitamin E control reticulocyte maturation, stress erythropoiesis and iron homeostasis.

Haematologica 2020 04 27;105(4):937-950. Epub 2019 Jun 27.

Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), München, Germany

Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation 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 elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the gene in mice failed to provide evidence for this hypothesis. 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 , we asked whether ablation of 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 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 extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. -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 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation.
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http://dx.doi.org/10.3324/haematol.2018.212977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109755PMC
April 2020

Multi-omics insights into functional alterations of the liver in insulin-deficient diabetes mellitus.

Mol Metab 2019 08 4;26:30-44. Epub 2019 Jun 4.

Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, 81377 Munich, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany; Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, 81377 Munich, Germany; Center for Innovative Medical Models (CiMM), LMU Munich, 85764 Oberschleißheim, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molmet.2019.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667734PMC
August 2019

Low catalytic activity is insufficient to induce disease pathology in triosephosphate isomerase deficiency.

J Inherit Metab Dis 2019 09 11;42(5):839-849. Epub 2019 Jun 11.

The Molecular Biology of Metabolism Laboratory, Francis Crick Institute, London, UK.

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. TPI 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. TPI 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.
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http://dx.doi.org/10.1002/jimd.12105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887927PMC
September 2019

A mouse model for intellectual disability caused by mutations in the X-linked 2'‑O‑methyltransferase Ftsj1 gene.

Biochim Biophys Acta Mol Basis Dis 2019 09 14;1865(9):2083-2093. Epub 2018 Dec 14.

Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, University Medicine Greifswald, Felix-Hausdorff-Strasse 8, 17489 Greifswald, Germany.

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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http://dx.doi.org/10.1016/j.bbadis.2018.12.011DOI Listing
September 2019

Dilution correction for dynamically influenced urinary analyte data.

Anal Chim Acta 2018 Nov 28;1032:18-31. Epub 2018 Jul 28.

Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Germany; Institute of Epidemiology, Helmholtz Zentrum München, Germany; German Center for Diabetes Research (DZD), München, Germany.

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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http://dx.doi.org/10.1016/j.aca.2018.07.068DOI Listing
November 2018

Night Shift Work Affects Urine Metabolite Profiles of Nurses with Early Chronotype.

Metabolites 2018 Aug 21;8(3). Epub 2018 Aug 21.

Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, 85764 München-Neuherberg, Germany.

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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http://dx.doi.org/10.3390/metabo8030045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161245PMC
August 2018

The heterozygous R155C VCP mutation: Toxic in humans! Harmless in mice?

Biochem Biophys Res Commun 2018 09 9;503(4):2770-2777. Epub 2018 Aug 9.

Institute of Neuropathology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, 91054, Erlangen, Germany. Electronic address:

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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http://dx.doi.org/10.1016/j.bbrc.2018.08.038DOI Listing
September 2018

Streptozotocin-induced β-cell damage, high fat diet, and metformin administration regulate Hes3 expression in the adult mouse brain.

Sci Rep 2018 07 27;8(1):11335. Epub 2018 Jul 27.

Department of Medicine, Technische Universität Dresden, Dresden, Germany.

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 β-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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http://dx.doi.org/10.1038/s41598-018-29434-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063949PMC
July 2018

Metabolic syndrome and extensive adipose tissue inflammation in morbidly obese Göttingen minipigs.

Mol Metab 2018 10 28;16:180-190. Epub 2018 Jun 28.

Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, Feodor-Lynen-Str. 25, 81377, Munich, Germany; Center for Innovative Medical Models (CiMM), Department of Veterinary Sciences, LMU Munich, Hackerstr. 27, 85764, Oberschleißheim, Germany; German Center for Diabetes Research (DZD), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany; Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377, Munich, Germany.

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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http://dx.doi.org/10.1016/j.molmet.2018.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6157610PMC
October 2018

Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver.

Mol Metab 2018 05 15;11:113-128. Epub 2018 Mar 15.

Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany; Center for Innovative Medical Models (CiMM), Department of Veterinary Sciences, LMU Munich, Hackerstr. 27, 85764 Oberschleißheim, Germany; Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama, Kawasaki, 214-8571, Japan; German Center for Diabetes Research (DZD), Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany. Electronic address:

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 1-bp 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.
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http://dx.doi.org/10.1016/j.molmet.2018.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6001387PMC
May 2018

Laboratory mouse housing conditions can be improved using common environmental enrichment without compromising data.

PLoS Biol 2018 04 16;16(4):e2005019. Epub 2018 Apr 16.

German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany.

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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http://dx.doi.org/10.1371/journal.pbio.2005019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922977PMC
April 2018

Defective immuno- and thymoproteasome assembly causes severe immunodeficiency.

Sci Rep 2018 04 13;8(1):5975. Epub 2018 Apr 13.

Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Trogerstr. 30, 81675, Munich, Germany.

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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http://dx.doi.org/10.1038/s41598-018-24199-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899138PMC
April 2018

Epigenetic alterations in longevity regulators, reduced life span, and exacerbated aging-related pathology in old father offspring mice.

Proc Natl Acad Sci U S A 2018 03 21;115(10):E2348-E2357. Epub 2018 Feb 21.

Molecular and Cellular Cognition Lab, German Center for Neurodegenerative Diseases (DZNE), 53127 Bonn, Germany;

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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http://dx.doi.org/10.1073/pnas.1707337115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877957PMC
March 2018

Epigallocatechin gallate (EGCG) reduces the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) transgenic mice.

Sci Rep 2018 01 18;8(1):1116. Epub 2018 Jan 18.

Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany.

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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http://dx.doi.org/10.1038/s41598-017-18807-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773570PMC
January 2018

Identification of genetic elements in metabolism by high-throughput mouse phenotyping.

Nat Commun 2018 01 18;9(1):288. Epub 2018 Jan 18.

German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.

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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http://dx.doi.org/10.1038/s41467-017-01995-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773596PMC
January 2018

Faim2 contributes to neuroprotection by erythropoietin in transient brain ischemia.

J Neurochem 2018 05 20;145(3):258-270. Epub 2018 Feb 20.

Department of Neurology, RWTH Aachen University, Aachen, Germany.

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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http://dx.doi.org/10.1111/jnc.14296DOI Listing
May 2018

Understanding gene functions and disease mechanisms: Phenotyping pipelines in the German Mouse Clinic.

Behav Brain Res 2018 10 29;352:187-196. Epub 2017 Sep 29.

German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter-Landstr. 1, 85764 Neuherberg, Germany; German Center for Diabetes Research (DZD), Ingolstädter-Landstr. 1, 85764 Neuherberg, Germany; Chair of Experimental Genetics, School of Life Science Weihenstephan, Technische Universität München, Alte Akademie 8, 85354 Freising, Germany. Electronic address:

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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http://dx.doi.org/10.1016/j.bbr.2017.09.048DOI Listing
October 2018

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

J Biomed Sci 2017 Aug 17;24(1):57. Epub 2017 Aug 17.

Chair for Molecular Animal Breeding and Biotechnology, and Laboratory for Functional Genome Analysis, Gene Center, LMU Munich, 81377, Munich, Germany.

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 thereby affecting the functional BTB domain of the protein. This line is the first mouse model harboring a Kctd1 mutation. Kctd1 homozygous mutant mice die perinatally. Standardized, systemic phenotypic analysis of Kctd1 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 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 heterozygous mutants as compared to wild-type controls.

Conclusions: In summary, the main alteration of the Kctd1 heterozygous mutants consists in kidney dysfunction. Additional analyses in 9-21 week-old heterozygous mutants revealed only few minor effects.
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http://dx.doi.org/10.1186/s12929-017-0365-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559776PMC
August 2017

Every-other-day feeding extends lifespan but fails to delay many symptoms of aging in mice.

Nat Commun 2017 07 24;8(1):155. Epub 2017 Jul 24.

DZNE, German Center for Neurodegenerative Diseases, Ludwig-Erhard-Allee 2, 53175, Bonn, Germany.

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.
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http://dx.doi.org/10.1038/s41467-017-00178-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5537224PMC
July 2017

The Munich MIDY Pig Biobank - A unique resource for studying organ crosstalk in diabetes.

Mol Metab 2017 08 13;6(8):931-940. Epub 2017 Jun 13.

Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, and Center for Innovative Medical Models (CiMM), LMU Munich, Feodor-Lynen-Str. 25, D-81377 Munich, Germany; German Center for Diabetes Research (DZD), Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany; Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Feodor-Lynen-Str. 25, D-81377 Munich, Germany. Electronic address:

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 transgenic pigs, a model of mutant 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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http://dx.doi.org/10.1016/j.molmet.2017.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518720PMC
August 2017