Publications by authors named "Jan Rozman"

95 Publications

Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Nature 2021 Mar 25. Epub 2021 Mar 25.

European Commission, Joint Research Centre, Ispra, Italy.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-19. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-19. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.
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http://dx.doi.org/10.1038/s41586-021-03461-yDOI Listing
March 2021

Author Correction: Endogenous FGF21-signaling controls paradoxical obesity resistance of UCP1-deficient mice.

Nat Commun 2021 Mar 16;12(1):1804. Epub 2021 Mar 16.

Institute for Diabetes and Obesity, Helmholtz Diabetes Center, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), 85764, Neuherberg, Germany.

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http://dx.doi.org/10.1038/s41467-021-22119-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966759PMC
March 2021

Bispecific antibody prevents SARS-CoV-2 escape and protects mice from disease.

bioRxiv 2021 Jan 22. Epub 2021 Jan 22.

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19) . We developed a bispecific, IgG1-like molecule based on two antibodies derived from COVID-19 convalescent donors, C121 and C135 . CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, completely prevents S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 recognizes a broad panel of RBD variants and neutralizes SARS-CoV-2 and the escape mutants generated by the single monoclonals at sub-nanomolar concentrations. In a novel model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.
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http://dx.doi.org/10.1101/2021.01.22.427567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836104PMC
January 2021

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

PLoS Genet 2020 12 28;16(12):e1009190. Epub 2020 Dec 28.

Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1009190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822523PMC
December 2020

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

Structure of a DNA G-Quadruplex Related to Osteoporosis with a G-A Bulge Forming a .

Molecules 2020 Oct 21;25(20). Epub 2020 Oct 21.

Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.

Bone remodeling is a fine-tuned process principally regulated by a cascade triggered by interaction of receptor activator of NF-κB (RANK) and RANK ligand (RANKL). Excessive activity of the gene leads to increased bone resorption and can influence the incidence of osteoporosis. Although much has been learned about the intracellular signals activated by RANKL/RANK complex, significantly less is known about the molecular mechanisms of regulation of expression. Here, we report on the structure of an unprecedented DNA G-quadruplex, well-known secondary structure-mediated gene expression regulator, formed by a G-rich sequence found in the regulatory region of a gene. Solution-state NMR structural study reveals the formation of a three-layered parallel-type G-quadruplex characterized by an unique features, including a G-A bulge. Although a guanine within a G-tract occupies glycosidic conformation, bulge-forming residues arrange in a conformation to facilitate partial 5/6-ring stacking, typical of G-quadruplex structures with parallel G-tracts orientation. Such distinctive structural features protruding from the core of the structure can represent a novel platform for design of highly specific ligands with anti-osteoporotic function. Additionally, our study suggests that the expression of gene may be regulated by putative folding of its G-rich region into non-B-DNA structure(s).
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http://dx.doi.org/10.3390/molecules25204867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588008PMC
October 2020

Type 2 diabetes risk gene Dusp8 regulates hypothalamic Jnk signaling and insulin sensitivity.

J Clin Invest 2020 11;130(11):6093-6108

Neurobiology of Stress Resilience, Max Planck Institute of Psychiatry, Munich, Germany.

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.
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http://dx.doi.org/10.1172/JCI136363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598066PMC
November 2020

Collective cell mechanics of epithelial shells with organoid-like morphologies.

Nat Commun 2020 07 30;11(1):3805. Epub 2020 Jul 30.

Jožef Stefan Institute, Jamova 39, Ljubljana, 1000, Slovenia.

The study of organoids, artificially grown cell aggregates with the functionality and small-scale anatomy of real organs, is one of the most active areas of research in biology and biophysics, yet the basic physical origins of their different morphologies remain poorly understood. Here, we propose a mechanistic theory of epithelial shells which resemble small-organoid morphologies. Using a 3D surface tension-based vertex model, we reproduce the characteristic shapes from branched and budded to invaginated structures. We find that the formation of branched morphologies relies strongly on junctional activity, enabling temporary aggregations of topological defects in cell packing. To elucidate our numerical results, we develop an effective elasticity theory, which allows one to estimate the apico-basal polarity from the tissue-scale modulation of cell height. Our work provides a generic interpretation of the observed epithelial shell morphologies, highlighting the role of physical factors such as differential surface tension, cell rearrangements, and tissue growth.
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http://dx.doi.org/10.1038/s41467-020-17535-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393134PMC
July 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.

Department of Neurology, Friedrich-Baur-Institute, Klinikum Der Ludwig-Maximilians-Universität München, Ziemssenstr. 1a, 80336, Munich, 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

Endogenous FGF21-signaling controls paradoxical obesity resistance of UCP1-deficient mice.

Nat Commun 2020 01 31;11(1):624. Epub 2020 Jan 31.

Institute for Diabetes and Obesity, Helmholtz Diabetes Center, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), 85764, Neuherberg, Germany.

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.
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http://dx.doi.org/10.1038/s41467-019-14069-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994690PMC
January 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

High-throughput discovery of genetic determinants of circadian misalignment.

PLoS Genet 2020 01 13;16(1):e1008577. Epub 2020 Jan 13.

SKL of Pharmaceutical Biotechnology and Model Animal Research Center, Collaborative Innovation Center for Genetics and Development, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, China.

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.
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http://dx.doi.org/10.1371/journal.pgen.1008577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980734PMC
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

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.

Max Planck Institute for Molecular Genetics, Berlin, Germany.

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.

Institute of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilians-University München, Feodor-Lynen Str. 25, 81377 München, 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

Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy.

EMBO Mol Med 2019 01;11(1)

Folkhälsan Research Center, Helsinki, Finland

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 AOX transgene into RC complex III (cIII)-deficient 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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http://dx.doi.org/10.15252/emmm.201809456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328925PMC
January 2019

Adenine-Driven Structural Switch from a Two- to Three-Quartet DNA G-Quadruplex.

Angew Chem Int Ed Engl 2018 11 17;57(47):15395-15399. Epub 2018 Oct 17.

Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 9, Ljubljana, Slovenia.

A G-rich sequence found in the regulatory region of the RANKL gene, which is associated with homeostasis of bone metabolism, folds into a two-quartet basket-type G-quadruplex stabilized by A⋅G⋅A and G⋅G⋅G base-triads. Perusal of local structural features together with G/A-to-T modifications uncovered the critical role of A5 for the formation of a distinct antiparallel two-quartet topology and not the three-quartet topology that would be expected based on the sequence with four GGG-tracts alone. The structural changes induced by the A5-to-T5 modification include a switch in orientation and relative positions of G-strands that together with anti to syn reorientation of G12 provide insights into the complexity of the interactions that influence the folding of G-rich DNA. Understanding the impact of loop residues on the stability and formation of G-quadruplexes advances our knowledge and ability to predict structures adopted by G-rich sequences, which are involved in regulatory mechanisms in the cell, and may also facilitate drug design.
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http://dx.doi.org/10.1002/anie.201809328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563693PMC
November 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

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

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

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

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

Monterotondo Mouse Clinic, Italian National Research Council (CNR), Institute of Cell Biology and Neurobiology, Adriano Buzzati-Traverso Campus, Via E. Ramarini 32, Monterotondo Scalo, RM, 00015, Italy.

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

Noncanonical thyroid hormone signaling mediates cardiometabolic effects in vivo.

Proc Natl Acad Sci U S A 2017 12 11;114(52):E11323-E11332. Epub 2017 Dec 11.

Department of Endocrinology, Diabetes and Metabolism, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;

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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http://dx.doi.org/10.1073/pnas.1706801115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748168PMC
December 2017

The Role of Eif6 in Skeletal Muscle Homeostasis Revealed by Endurance Training Co-expression Networks.

Cell Rep 2017 Nov;21(6):1507-1520

Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK. Electronic address:

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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http://dx.doi.org/10.1016/j.celrep.2017.10.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695912PMC
November 2017

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.

Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Trogerstr. 30, 81675 Munich, Germany.

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

A systemic view on the distribution of diet-derived methanol and hepatic acetone in mice.

J Breath Res 2017 12 6;12(1):017102. Epub 2017 Dec 6.

Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Centre for Environmental Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Munich, Germany. German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Centre for Environmental Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Munich, Germany. German Centre for Diabetes Research (DZD), Germany.

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 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, feces 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. Cecum content especially was identified as a likely source of systemic methanol, whereas the 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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http://dx.doi.org/10.1088/1752-7163/aa8a15DOI Listing
December 2017

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.

Research Group Experimental Neuropsychopharmacology, Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, 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

Metformin causes a futile intestinal-hepatic cycle which increases energy expenditure and slows down development of a type 2 diabetes-like state.

Mol Metab 2017 07 5;6(7):737-747. Epub 2017 May 5.

Institute of Vegetative Physiology, Medical Faculty, University of Köln, 50931 Köln, Germany.

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-C to glucose-1,6-C 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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http://dx.doi.org/10.1016/j.molmet.2017.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5485244PMC
July 2017

Fgf9 Mutation Alters Information Processing and Social Memory in Mice.

Mol Neurobiol 2018 Jun 10;55(6):4580-4595. Epub 2017 Jul 10.

Institutes of Developmental Genetics, Helmholtz Zentrum München, Neuherberg, Germany.

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 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 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 mutant mice as a valuable tool to study the predictive etiological aspects.
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http://dx.doi.org/10.1007/s12035-017-0659-3DOI Listing
June 2018