Publications by authors named "Pawel Pelczar"

53 Publications

Neuron-specific spinal cord translatomes reveal a neuropeptide code for mouse dorsal horn excitatory neurons.

Sci Rep 2021 Mar 4;11(1):5232. Epub 2021 Mar 4.

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.

The spinal dorsal horn harbors a sophisticated and heterogeneous network of excitatory and inhibitory neurons that process peripheral signals encoding different sensory modalities. Although it has long been recognized that this network is crucial both for the separation and the integration of sensory signals of different modalities, a systematic unbiased approach to the use of specific neuromodulatory systems is still missing. Here, we have used the translating ribosome affinity purification (TRAP) technique to map the translatomes of excitatory glutamatergic (vGluT2) and inhibitory GABA and/or glycinergic (vGAT or Gad67) neurons of the mouse spinal cord. Our analyses demonstrate that inhibitory and excitatory neurons are not only set apart, as expected, by the expression of genes related to the production, release or re-uptake of their principal neurotransmitters and by genes encoding for transcription factors, but also by a differential engagement of neuromodulator, especially neuropeptide, signaling pathways. Subsequent multiplex in situ hybridization revealed eleven neuropeptide genes that are strongly enriched in excitatory dorsal horn neurons and display largely non-overlapping expression patterns closely adhering to the laminar and presumably also functional organization of the spinal cord grey matter.
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http://dx.doi.org/10.1038/s41598-021-84667-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933427PMC
March 2021

Boundary sequences flanking the mouse tyrosinase locus ensure faithful pattern of gene expression.

Sci Rep 2020 09 23;10(1):15494. Epub 2020 Sep 23.

Department of Molecular and Cellular Biology, National Centre for Biotechnology (CNB-CSIC), Campus de Cantoblanco, Darwin 3, 28049, Madrid, Spain.

Control of gene expression is dictated by cell-type specific regulatory sequences that physically organize the structure of chromatin, including promoters, enhancers and insulators. While promoters and enhancers convey cell-type specific activating signals, insulators prevent the cross-talk of regulatory elements within adjacent loci and safeguard the specificity of action of promoters and enhancers towards their targets in a tissue specific manner. Using the mouse tyrosinase (Tyr) locus as an experimental model, a gene whose mutations are associated with albinism, we described the chromatin structure in cells at two distinct transcriptional states. Guided by chromatin structure, through the use of Chromosome Conformation Capture (3C), we identified sequences at the 5' and 3' boundaries of this mammalian gene that function as enhancers and insulators. By CRISPR/Cas9-mediated chromosomal deletion, we dissected the functions of these two regulatory elements in vivo in the mouse, at the endogenous chromosomal context, and proved their mechanistic role as genomic insulators, shielding the Tyr locus from the expression patterns of adjacent genes.
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http://dx.doi.org/10.1038/s41598-020-72543-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511308PMC
September 2020

Magnetic fields modulate metabolism and gut microbiome in correlation with Pgc-1α expression: Follow-up to an in vitro magnetic mitohormetic study.

FASEB J 2020 08 6;34(8):11143-11167. Epub 2020 Jul 6.

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Exercise modulates metabolism and the gut microbiome. Brief exposure to low mT-range pulsing electromagnetic fields (PEMFs) was previously shown to accentuate in vitro myogenesis and mitochondriogenesis by activating a calcium-mitochondrial axis upstream of PGC-1α transcriptional upregulation, recapitulating a genetic response implicated in exercise-induced metabolic adaptations. We compared the effects of analogous PEMF exposure (1.5 mT, 10 min/week), with and without exercise, on systemic metabolism and gut microbiome in four groups of mice: (a) no intervention; (b) PEMF treatment; (c) exercise; (d) exercise and PEMF treatment. The combination of PEMFs and exercise for 6 weeks enhanced running performance and upregulated muscular and adipose Pgc-1α transcript levels, whereas exercise alone was incapable of elevating Pgc-1α levels. The gut microbiome Firmicutes/Bacteroidetes ratio decreased with exercise and PEMF exposure, alone or in combination, which has been associated in published studies with an increase in lean body mass. After 2 months, brief PEMF treatment alone increased Pgc-1α and mitohormetic gene expression and after >4 months PEMF treatment alone enhanced oxidative muscle expression, fatty acid oxidation, and reduced insulin levels. Hence, short-term PEMF treatment was sufficient to instigate PGC-1α-associated transcriptional cascades governing systemic mitohormetic adaptations, whereas longer-term PEMF treatment was capable of inducing related metabolic adaptations independently of exercise.
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http://dx.doi.org/10.1096/fj.201903005RRDOI Listing
August 2020

Bi-allelic expression of the RyR1 p.A4329D mutation decreases muscle strength in slow-twitch muscles in mice.

J Biol Chem 2020 07 4;295(30):10331-10339. Epub 2020 Jun 4.

Department of Biomedicine, Basel University Hospital, Basel, Switzerland

Mutations in the ryanodine receptor 1 () gene are associated with several human congenital myopathies, including the dominantly inherited central core disease and exercise-induced rhabdomyolysis, and the more severe recessive phenotypes, including multiminicore disease, centronuclear myopathy, and congenital fiber type disproportion. Within the latter group, those carrying a hypomorphic mutation in one allele and a missense mutation in the other are the most severely affected. Because of nonsense-mediated decay, most hypomorphic alleles are not expressed, resulting in homozygous expression of the missense mutation allele. This should result in 50% reduced expression of the ryanodine receptor in skeletal muscle, but its observed content is even lower. To study in more detail the biochemistry and pathophysiology of recessive myopathies, here we investigated a mouse model we recently generated by analyzing the effect of bi-allelic mono-allelic expression of the RyR1 p.A4329D mutation. Our results revealed that the expression of two alleles carrying the same mutation or of one allele with the mutation in combination with a hypomorphic allele does not result in functionally equal outcomes and impacts skeletal muscles differently. In particular, the bi-allelic RyR1 p.A4329D mutation caused a milder phenotype than its mono-allelic expression, leading to changes in the biochemical properties and physiological function only of slow-twitch muscles and largely sparing fast-twitch muscles. In summary, bi-allelic expression of the RyR1 p.A4329D mutation phenotypically differs from mono-allelic expression of this mutation in a compound heterozygous carrier.
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http://dx.doi.org/10.1074/jbc.RA120.013846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383393PMC
July 2020

Palmitoylation of BMPR1a regulates neural stem cell fate.

Proc Natl Acad Sci U S A 2019 12 26;116(51):25688-25696. Epub 2019 Nov 26.

Laboratory of Neural Plasticity, Faculties of Medicine and Science, Brain Research Institute, University of Zurich, 8057 Zurich, Switzerland;

Neural stem cells (NSCs) generate neurons and glial cells throughout embryonic and postnatal brain development. The role of S-palmitoylation (also referred to as S-acylation), a reversible posttranslational lipid modification of proteins, in regulating the fate and activity of NSCs remains largely unknown. We used an unbiased screening approach to identify proteins that are S-acylated in mouse NSCs and showed that bone morphogenic protein receptor 1a (BMPR1a), a core mediator of BMP signaling, is palmitoylated. Genetic manipulation of S-acylated sites affects the localization and trafficking of BMPR1a and leads to altered BMP signaling. Strikingly, defective palmitoylation of BMPR1a modulates NSC function within the mouse brain, resulting in enhanced oligodendrogenesis. Thus, we identified a mechanism regulating the behavior of NSCs and provided the framework to characterize dynamic posttranslational lipid modifications of proteins in the context of NSC biology.
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http://dx.doi.org/10.1073/pnas.1912671116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926058PMC
December 2019

Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model.

Mucosal Immunol 2019 07 19;12(4):1013-1024. Epub 2019 May 19.

CSL Behring AG, Wankdorfstrasse 10, 3010, Bern, Switzerland.

Recurrent and persistent airway infections remain prevalent in patients with primary immunodeficiency (PID), despite restoration of serum immunoglobulin levels by intravenous or subcutaneous plasma-derived IgG. We investigated the effectiveness of different human Ig isotype preparations to protect mice against influenza when delivered directly to the respiratory mucosa. Four polyvalent Ig preparations from pooled plasma were compared: IgG, monomeric IgA (mIgA), polymeric IgA-containing IgM (IgAM) and IgAM associated with the secretory component (SIgAM). To evaluate these preparations, a transgenic mouse expressing human FcαRI/CD89 within the myeloid lineage was created. CD89 was expressed on all myeloid cells in the lung and blood except eosinophils, reflecting human CD89 expression. Intranasal administration of IgA-containing preparations was less effective than IgG in reducing pulmonary viral titres after infection of mice with A/California/7/09 (Cal7) or the antigenically distant A/Puerto Rico/8/34 (PR8) viruses. However, IgA reduced weight loss and inflammatory mediator expression. Both IgG and IgA protected mice from a lethal dose of PR8 virus and for mIgA, this effect was partially CD89 dependent. Our data support the beneficial effect of topically applied Ig purified from pooled human plasma for controlling circulating and non-circulating influenza virus infections. This may be important for reducing morbidity in PID patients.
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http://dx.doi.org/10.1038/s41385-019-0167-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746524PMC
July 2019

Fate-Mapping of GM-CSF Expression Identifies a Discrete Subset of Inflammation-Driving T Helper Cells Regulated by Cytokines IL-23 and IL-1β.

Immunity 2019 05 9;50(5):1289-1304.e6. Epub 2019 May 9.

Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland. Electronic address:

Pathogenic lymphocytes initiate the development of chronic inflammatory diseases. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) (encoded by Csf2) is a key communicator between pathogenic lymphocytes and tissue-invading inflammatory phagocytes. However, the molecular properties of GM-CSF-producing cells and the mode of Csf2 regulation in vivo remain unclear. To systematically study and manipulate GM-CSF cells and their progeny in vivo, we generated a fate-map and reporter of GM-CSF expression mouse strain (FROG). We mapped the phenotypic and functional profile of auto-aggressive T helper (Th) cells during neuroinflammation and identified the signature and pathogenic memory of a discrete encephalitogenic Th subset. These cells required interleukin-23 receptor (IL-23R) and IL-1R but not IL-6R signaling for their maintenance and pathogenicity. Specific ablation of this subset interrupted the inflammatory cascade, despite the unperturbed tissue accumulation of other Th subsets (e.g., Th1 and Th17), highlighting that GM-CSF expression not only marks pathogenic Th cells, but that this subset mediates immunopathology and tissue destruction.
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http://dx.doi.org/10.1016/j.immuni.2019.04.006DOI Listing
May 2019

Maternal overnutrition programs hedonic and metabolic phenotypes across generations through sperm tsRNAs.

Proc Natl Acad Sci U S A 2019 05 6;116(21):10547-10556. Epub 2019 May 6.

Laboratory of Translational Nutrition Biology, Department of Health Sciences and Technology, ETH Zurich, 8603 Schwerzenbach, Switzerland;

There is a growing body of evidence linking maternal overnutrition to obesity and psychopathology that can be conserved across multiple generations. Recently, we demonstrated in a maternal high-fat diet (HFD; MHFD) mouse model that MHFD induced enhanced hedonic behaviors and obesogenic phenotypes that were conserved across three generations via the paternal lineage, which was independent of sperm methylome changes. Here, we show that sperm tRNA-derived small RNAs (tsRNAs) partly contribute to the transmission of such phenotypes. We observe increased expression of sperm tsRNAs in the F1 male offspring born to HFD-exposed dams. Microinjection of sperm tsRNAs from the F1-HFD male into normal zygotes reproduces obesogenic phenotypes and addictive-like behaviors, such as increased preference of palatable foods and enhanced sensitivity to drugs of abuse in the resultant offspring. The expression of several of the differentially expressed sperm tsRNAs predicted targets such as and GRIN3A, which have been implicated in addiction pathology, are altered in the mesolimbic reward brain regions of the F1-HFD father and the resultant HFD-tsRNA offspring. Together, our findings demonstrate that sperm tsRNA is a potential vector that contributes to the transmission of MHFD-induced addictive-like behaviors and obesogenic phenotypes across generations, thereby emphasizing its role in diverse pathological outcomes.
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http://dx.doi.org/10.1073/pnas.1820810116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534971PMC
May 2019

Quantitative RyR1 reduction and loss of calcium sensitivity of RyR1Q1970fsX16+A4329D cause cores and loss of muscle strength.

Hum Mol Genet 2019 09;28(18):2987-2999

Departments of Anaesthesia and Biomedicine, Basel University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland.

Recessive ryanodine receptor 1 (RYR1) mutations cause congenital myopathies including multiminicore disease (MmD), congenital fiber-type disproportion and centronuclear myopathy. We created a mouse model knocked-in for the Q1970fsX16+A4329D RYR1 mutations, which are isogenic with those identified in a severely affected child with MmD. During the first 20 weeks after birth the body weight and the spontaneous running distance of the mutant mice were 20% and 50% lower compared to wild-type littermates. Skeletal muscles from mutant mice contained 'cores' characterized by severe myofibrillar disorganization associated with misplacement of mitochondria. Furthermore, their muscles developed less force and had smaller electrically evoked calcium transients. Mutant RyR1 channels incorporated into lipid bilayers were less sensitive to calcium and caffeine, but no change in single-channel conductance was observed. Our results demonstrate that the phenotype of the RyR1Q1970fsX16+A4329D compound heterozygous mice recapitulates the clinical picture of multiminicore patients and provide evidence of the molecular mechanisms responsible for skeletal muscle defects.
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http://dx.doi.org/10.1093/hmg/ddz092DOI Listing
September 2019

Extrinsic and intrinsic apoptosis activate pannexin-1 to drive NLRP3 inflammasome assembly.

EMBO J 2019 05 22;38(10). Epub 2019 Mar 22.

Department of Biochemistry, University of Lausanne, Epalinges, Switzerland

Pyroptosis is a form of lytic inflammatory cell death driven by inflammatory caspase-1, caspase-4, caspase-5 and caspase-11. These caspases cleave and activate the pore-forming protein gasdermin D (GSDMD) to induce membrane damage. By contrast, apoptosis is driven by apoptotic caspase-8 or caspase-9 and has traditionally been classified as an immunologically silent form of cell death. Emerging evidence suggests that therapeutics designed for cancer chemotherapy or inflammatory disorders such as SMAC mimetics, TAK1 inhibitors and BH3 mimetics promote caspase-8 or caspase-9-dependent inflammatory cell death and NLRP3 inflammasome activation. However, the mechanism by which caspase-8 or caspase-9 triggers cell lysis and NLRP3 activation is still undefined. Here, we demonstrate that during extrinsic apoptosis, caspase-1 and caspase-8 cleave GSDMD to promote lytic cell death. By engineering a novel D88A knock-in mouse, we further demonstrate that this proinflammatory function of caspase-8 is counteracted by caspase-3-dependent cleavage and inactivation of GSDMD at aspartate 88, and is essential to suppress GSDMD-dependent cell lysis during caspase-8-dependent apoptosis. Lastly, we provide evidence that channel-forming glycoprotein pannexin-1, but not GSDMD or GSDME promotes NLRP3 inflammasome activation during caspase-8 or caspase-9-dependent apoptosis.
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http://dx.doi.org/10.15252/embj.2019101638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6517827PMC
May 2019

The transcription factor Duxbl mediates elimination of pre-T cells that fail β-selection.

J Exp Med 2019 03 14;216(3):638-655. Epub 2019 Feb 14.

Developmental and Molecular Immunology, Department of Biomedicine, University of Basel, Basel, Switzerland

T cell development is critically dependent on successful rearrangement of antigen-receptor chains. At the β-selection checkpoint, only cells with a functional rearrangement continue in development. However, how nonselected T cells proceed in their dead-end fate is not clear. We identified low CD27 expression to mark pre-T cells that have failed to rearrange their β-chain. Expression profiling and single-cell transcriptome clustering identified a developmental trajectory through β-selection and revealed specific expression of the transcription factor Duxbl at a stage of high recombination activity before β-selection. Conditional transgenic expression of Duxbl resulted in a developmental block at the DN3-to-DN4 transition due to reduced proliferation and enhanced apoptosis, whereas RNA silencing of Duxbl led to a decrease in apoptosis. Transcriptome analysis linked Duxbl to elevated expression of the apoptosis-inducing Oas/RNaseL pathway. RNaseL deficiency or sustained Bcl2 expression led to a partial rescue of cells in Duxbl transgenic mice. These findings identify Duxbl as a regulator of β-selection by inducing apoptosis in cells with a nonfunctional rearrangement.
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http://dx.doi.org/10.1084/jem.20181444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400535PMC
March 2019

Quantitative reduction of RyR1 protein caused by a single-allele frameshift mutation in RYR1 ex36 impairs the strength of adult skeletal muscle fibres.

Hum Mol Genet 2019 06;28(11):1872-1884

Departments of Anaesthesia and Biomedicine, Basel University Hospital, Hebelstrasse, Basel, Switzerland.

Here we characterized a mouse model knocked-in for a frameshift mutation in RYR1 exon 36 (p.Gln1970fsX16) that is isogenic to that identified in one parent of a severely affected patient with recessively inherited multiminicore disease. This individual carrying the RYR1 frameshifting mutation complained of mild muscle weakness and fatigability. Analysis of the RyR1 protein content in a muscle biopsy from this individual showed a content of only 20% of that present in a control individual. The biochemical and physiological characteristics of skeletal muscles from RyR1Q1970fsX16 heterozygous mice recapitulates that of the heterozygous parent. RyR1 protein content in the muscles of mutant mice reached 38% and 58% of that present in total muscle homogenates of fast and slow muscles from wild-type (WT) littermates. The decrease of RyR1 protein content in total homogenates is not accompanied by a decrease of Cav1.1 content, whereby the Cav1.1/RyR1 stoichiometry ratio in skeletal muscles from RyR1Q1970fsX16 heterozygous mice is lower compared to that from WT mice. Electron microscopy (EM) revealed a 36% reduction in the number/area of calcium release units accompanied by a 2.5-fold increase of dyads (triads that have lost one junctional sarcoplasmic reticulum element); both results suggest a reduction of the RyR1 arrays. Compared to WT, muscle strength and depolarization-induced calcium transients in RyR1Q1970fsX16 heterozygous mice muscles were decreased by 20% and 15%, respectively. The RyR1Q1970fsX16 mouse model provides mechanistic insight concerning the phenotype of the parent carrying the RYR1 ex36 mutation and suggests that in skeletal muscle fibres there is a functional reserve of RyR1.
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http://dx.doi.org/10.1093/hmg/ddz025DOI Listing
June 2019

Primed Track, high-fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins.

Elife 2019 01 21;8. Epub 2019 Jan 21.

Department for Biosystems Science and Engineering (D-BSSE), ETH Zurich, Basel, Switzerland.

Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescence labeling techniques is complicated by increasing cell density and rapid embryo rotation, which hampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging data sets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population in order to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP-labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift, reduce overall data size, and accomplish high-fidelity lineage tracing even for increased imaging time intervals - addressing major concerns in the field of volumetric embryo imaging.
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http://dx.doi.org/10.7554/eLife.44491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340703PMC
January 2019

Transgenerational transmission of hedonic behaviors and metabolic phenotypes induced by maternal overnutrition.

Transl Psychiatry 2018 10 12;8(1):195. Epub 2018 Oct 12.

Laboratory of Translational Nutrition Biology, Department of Health Sciences and Technology, ETH Zurich, 8603, Schwerzenbach, Switzerland.

Maternal overnutrition has been associated with increased susceptibility to develop obesity and neurological disorders later in life. Most epidemiological as well as experimental studies have focused on the metabolic consequences across generations following an early developmental nutritional insult. Recently, it has been shown that maternal high-fat diet (HFD) affects third-generation female body mass via the paternal lineage. We showed here that the offspring born to HFD ancestors displayed addictive-like behaviors as well as obesity and insulin resistance up to the third generation in the absence of any further exposure to HFD. These findings, implicate that the male germ line is a major player in transferring phenotypic traits. These behavioral and physiological alterations were paralleled by reduced striatal dopamine levels and increased dopamine 2 receptor density. Interestingly, by the third generation a clear gender segregation emerged, where females showed addictive-like behaviors while male HFD offspring showed an obesogenic phenotype. However, methylome profiling of F1 and F2 sperm revealed no significant difference between the offspring groups, suggesting that the sperm methylome might not be the major carrier for the transmission of the phenotypes observed in our mouse model. Together, our study for the first time demonstrates that maternal HFD insult causes sustained alterations of the mesolimbic dopaminergic system suggestive of a predisposition to develop obesity and addictive-like behaviors across multiple generations.
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http://dx.doi.org/10.1038/s41398-018-0243-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185972PMC
October 2018

A Transgenic MMTV-Flippase Mouse Line for Molecular Engineering in Mammary Gland and Breast Cancer Mouse Models.

J Mammary Gland Biol Neoplasia 2019 03 12;24(1):39-45. Epub 2018 Sep 12.

Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058, Basel, Switzerland.

Genetically engineered mouse models have become an indispensable tool for breast cancer research. Combination of multiple site-specific recombination systems such as Cre/loxP and Flippase (Flp)/Frt allows for engineering of sophisticated, multi-layered conditional mouse models. Here, we report the generation and characterization of a novel transgenic mouse line expressing a mouse codon-optimized Flp under the control of the mouse mammary tumor virus (MMTV) promoter. These mice show robust Flp-mediated recombination in luminal mammary gland and breast cancer cells but no Flp activity in non-mammary tissues, with the exception of limited activity in salivary glands. These mice provide a unique tool for studying mammary gland biology and carcinogenesis in mice.
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http://dx.doi.org/10.1007/s10911-018-9412-4DOI Listing
March 2019

Publisher Correction: Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

Nat Med 2018 Nov;24(11):1777

Institute of Food, Nutrition and Health, ETH Zurich, Schwerzenbach, Switzerland.

In the version of this article originally published, the bars in the mean temperature graph in Fig. 1a were incorrectly aligned. The left-most bar should have been aligned with the Apr label on the projected month of conception axis. The error has been corrected in the print, PDF and HTML versions of this article.
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http://dx.doi.org/10.1038/s41591-018-0163-yDOI Listing
November 2018

Author Correction: Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

Nat Med 2018 Nov;24(11):1776

Institute of Food, Nutrition and Health, ETH Zurich, Schwerzenbach, Switzerland.

In the version of this article originally published, the months on the axis labeled projected month of conception in Fig. 1a were out of order. April and March should have been the first and last months listed, respectively. The error has been corrected in the print, PDF and HTML versions of this article.
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http://dx.doi.org/10.1038/s41591-018-0162-zDOI Listing
November 2018

TRPC1 regulates brown adipose tissue activity in a PPARγ-dependent manner.

Am J Physiol Endocrinol Metab 2018 11 10;315(5):E825-E832. Epub 2018 Jul 10.

Center for Transgenic Models, University of Basel , Basel , Switzerland.

Brown adipose tissue (BAT) has the unique ability to convert energy stored in the form of triglycerides into heat. This property makes BAT a target tissue to increase energy expenditure and improve systemic metabolic control. TRPC1 is a founding member of the TRP protein family that also includes several temperature sensitive channels. We show that TRPC1 is highly expressed in all adipocyte depots including BAT and that Trpc1-deficient mice are prone to weight gain and manifest reduced metabolic control. We also demonstrate that knockdown of TRPC1 in cultured brown adipocytes leads to a downregulation of several metabolic genes, including UCP1 and PPARγ, as well as upregulation of a BAT-specific thermosensitive channel TRPV2, ultimately resulting in impaired respiratory function. We also show that TRPC1 is a possible target of PPARγ, suggesting that TRPC1 is a downstream component of a mechanism that translates metabolic or environmental stimuli into output in the form of BAT activity. Better understanding of the possible role of TRPC1 and other TRP channels in body temperature regulation and BAT function may help us to develop obesity therapies based on BAT activation.
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http://dx.doi.org/10.1152/ajpendo.00170.2017DOI Listing
November 2018

Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

Nat Med 2018 09 9;24(9):1372-1383. Epub 2018 Jul 9.

Institute of Food, Nutrition and Health, ETH Zurich, Schwerzenbach, Switzerland.

Recent research has focused on environmental effects that control tissue functionality and systemic metabolism. However, whether such stimuli affect human thermogenesis and body mass index (BMI) has not been explored. Here we show retrospectively that the presence of brown adipose tissue (BAT) and the season of conception are linked to BMI in humans. In mice, we demonstrate that cold exposure (CE) of males, but not females, before mating results in improved systemic metabolism and protection from diet-induced obesity of the male offspring. Integrated analyses of the DNA methylome and RNA sequencing of the sperm from male mice revealed several clusters of co-regulated differentially methylated regions (DMRs) and differentially expressed genes (DEGs), suggesting that the improved metabolic health of the offspring was due to enhanced BAT formation and increased neurogenesis. The conclusions are supported by cell-autonomous studies in the offspring that demonstrate an enhanced capacity to form mature active brown adipocytes, improved neuronal density and more norepinephrine release in BAT in response to cold stimulation. Taken together, our results indicate that in humans and in mice, seasonal or experimental CE induces an epigenetic programming of the sperm such that the offspring harbor hyperactive BAT and an improved adaptation to overnutrition and hypothermia.
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http://dx.doi.org/10.1038/s41591-018-0102-yDOI Listing
September 2018

LPS targets host guanylate-binding proteins to the bacterial outer membrane for non-canonical inflammasome activation.

EMBO J 2018 03 19;37(6). Epub 2018 Feb 19.

Focal Area Infection Biology, Biozentrum, University of Basel, Basel, Switzerland

Pathogenic and commensal Gram-negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase-11 non-canonical inflammasome. Here, we show that OMV-induced inflammasome activation requires TLR4-TRIF signaling, the production of type I interferons, and the action of guanylate-binding proteins (GBPs), both in macrophages and Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram-negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell-autonomous immunity and innate immunity.
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http://dx.doi.org/10.15252/embj.201798089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852652PMC
March 2018

Pramel7 mediates ground-state pluripotency through proteasomal-epigenetic combined pathways.

Nat Cell Biol 2017 Jul 12;19(7):763-773. Epub 2017 Jun 12.

Department of Trauma Surgery, Center for Clinical Research, University Hospital Zurich, University of Zurich, Sternwartstrasse 14, CH-8091 Zurich, Switzerland.

Naive pluripotency is established in preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of naive pluripotency. 2i culture has optimized this state, leading to a gene signature and DNA hypomethylation closely comparable to preimplantation epiblast, the developmental ground state. Here we show that Pramel7 (PRAME-like 7), a protein highly expressed in the inner cell mass (ICM) but expressed at low levels in ESCs, targets for proteasomal degradation UHRF1, a key factor for DNA methylation maintenance. Increasing Pramel7 expression in serum-cultured ESCs promotes a preimplantation epiblast-like gene signature, reduces UHRF1 levels and causes global DNA hypomethylation. Pramel7 is required for blastocyst formation and its forced expression locks ESCs in pluripotency. Pramel7/UHRF1 expression is mutually exclusive in ICMs whereas Pramel7-knockout embryos express high levels of UHRF1. Our data reveal an as-yet-unappreciated dynamic nature of DNA methylation through proteasome pathways and offer insights that might help to improve ESC culture to reproduce in vitro the in vivo ground-state pluripotency.
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http://dx.doi.org/10.1038/ncb3554DOI Listing
July 2017

Dysregulation of the Cytokine GM-CSF Induces Spontaneous Phagocyte Invasion and Immunopathology in the Central Nervous System.

Immunity 2017 02;46(2):245-260

Institute of Experimental Immunology, Inflammation Research, University of Zurich, 8057 Zurich, Switzerland. Electronic address:

Chronic inflammatory diseases are influenced by dysregulation of cytokines. Among them, granulocyte macrophage colony stimulating factor (GM-CSF) is crucial for the pathogenic function of T cells in preclinical models of autoimmunity. To study the impact of dysregulated GM-CSF expression in vivo, we generated a transgenic mouse line allowing the induction of GM-CSF expression in mature, peripheral helper T (Th) cells. Antigen-independent GM-CSF release led to the invasion of inflammatory myeloid cells into the central nervous system (CNS), which was accompanied by the spontaneous development of severe neurological deficits. CNS-invading phagocytes produced reactive oxygen species and exhibited a distinct genetic signature compared to myeloid cells invading other organs. We propose that the CNS is particularly vulnerable to the attack of monocyte-derived phagocytes and that the effector functions of GM-CSF-expanded myeloid cells are in turn guided by the tissue microenvironment.
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http://dx.doi.org/10.1016/j.immuni.2017.01.007DOI Listing
February 2017

Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein.

PLoS One 2017 16;12(2):e0170503. Epub 2017 Feb 16.

Institute of Neuropathology, University Hospital of Zurich, University of Zurich, Schmelzbergstrasse 12, Zurich, Switzerland.

Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrPSc) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrPSc does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrPSc. Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2-α2 loop of the cellular prion protein (PrPC). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrPC but expressing MoPrP169F generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP169F prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP169F prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP169F prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2-α2 loop of PrPC affects the sensitivity of PrPSc to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrPC can be linked to a physicochemical property of the corresponding PrPSc.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170503PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313174PMC
August 2017

Modeling the ferrochelatase c.315-48C modifier mutation for erythropoietic protoporphyria (EPP) in mice.

Dis Model Mech 2017 03 12;10(3):225-233. Epub 2017 Jan 12.

Institute of Laboratory Medicine, Municipal Hospital Triemli, Zürich 8063, Switzerland

Erythropoietic protoporphyria (EPP) is caused by deficiency of ferrochelatase (FECH), which incorporates iron into protoporphyrin IX (PPIX) to form heme. Excitation of accumulated PPIX by light generates oxygen radicals that evoke excessive pain and, after longer light exposure, cause ulcerations in exposed skin areas of individuals with EPP. Moreover, ∼5% of the patients develop a liver dysfunction as a result of PPIX accumulation. Most patients (∼97%) have a severe mutation (Mut) to an intronic polymorphism (c.315-48C), which reduces ferrochelatase synthesis by stimulating the use of an aberrant 3' splice site 63 nt upstream of the normal site for exon 4. In contrast, with the predominant c.315-48T allele, the correct splice site is mostly used, and individuals with a T/Mut genotype do not develop EPP symptoms. Thus, the C allele is a potential target for therapeutic approaches that modify this splicing decision. To provide a model for pre-clinical studies of such approaches, we engineered a mouse containing a partly humanized gene with the c.315-48C polymorphism. F1 hybrids obtained by crossing these mice with another inbred line carrying a severe mutation (named m1Pas) show a very strong EPP phenotype that includes elevated PPIX in the blood, enlargement of liver and spleen, anemia, as well as strong pain reactions and skin lesions after a short period of light exposure. In addition to the expected use of the aberrant splice site, the mice also show a strong skipping of the partly humanized exon 3. This will limit the use of this model for certain applications and illustrates that engineering of a hybrid gene may have unforeseeable consequences on its splicing.
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http://dx.doi.org/10.1242/dmm.027755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374324PMC
March 2017

Strictly co-isogenic C57BL/6J-Prnp-/- mice: A rigorous resource for prion science.

J Exp Med 2016 Mar 29;213(3):313-27. Epub 2016 Feb 29.

Institute of Neuropathology, University Hospital of Zurich, 8091 Zurich, Switzerland

Although its involvement in prion replication and neurotoxicity during transmissible spongiform encephalopathies is undisputed, the physiological role of the cellular prion protein (PrP(C)) remains enigmatic. A plethora of functions have been ascribed to PrP(C) based on phenotypes of Prnp(-/-) mice. However, all currently available Prnp(-/-) lines were generated in embryonic stem cells from the 129 strain of the laboratory mouse and mostly crossed to non-129 strains. Therefore, Prnp-linked loci polymorphic between 129 and the backcrossing strain resulted in systematic genetic confounders and led to erroneous conclusions. We used TALEN-mediated genome editing in fertilized mouse oocytes to create the Zurich-3 (ZH3) Prnp-ablated allele on a pure C57BL/6J genetic background. Genomic, transcriptional, and phenotypic characterization of Prnp(ZH3/ZH3) mice failed to identify phenotypes previously described in non-co-isogenic Prnp(-/-) mice. However, aged Prnp(ZH3/ZH3) mice developed a chronic demyelinating peripheral neuropathy, confirming the crucial involvement of PrP(C) in peripheral myelin maintenance. This new line represents a rigorous genetic resource for studying the role of PrP(C) in physiology and disease.
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http://dx.doi.org/10.1084/jem.20151610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813672PMC
March 2016

Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis.

Mol Cell 2015 Nov 5;60(4):611-25. Epub 2015 Nov 5.

Medical Research Council Clinical Sciences Centre (MRC CSC), Faculty of Medicine, Imperial College London, London W12 0NN, UK. Electronic address:

The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.
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http://dx.doi.org/10.1016/j.molcel.2015.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672152PMC
November 2015

The Cytokine GM-CSF Drives the Inflammatory Signature of CCR2+ Monocytes and Licenses Autoimmunity.

Immunity 2015 Sep 1;43(3):502-14. Epub 2015 Sep 1.

Institute of Experimental Immunology, University of Zürich, Zürich 8057, Switzerland. Electronic address:

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine produced by auto-reactive T helper (Th) cells that initiate tissue inflammation. Multiple cell types can sense GM-CSF, but the identity of the pathogenic GM-CSF-responsive cells is unclear. By using conditional gene targeting, we systematically deleted the GM-CSF receptor (Csf2rb) in specific subpopulations throughout the myeloid lineages. Experimental autoimmune encephalomyelitis (EAE) progressed normally when either classical dendritic cells (cDCs) or neutrophils lacked GM-CSF responsiveness. The development of tissue-invading monocyte-derived dendritic cells (moDCs) was also unperturbed upon Csf2rb deletion. Instead, deletion of Csf2rb in CCR2(+)Ly6C(hi) monocytes phenocopied the EAE resistance seen in complete Csf2rb-deficient mice. High-dimensional analysis of tissue-infiltrating moDCs revealed that GM-CSF initiates a combination of inflammatory mechanisms. These results indicate that GM-CSF signaling controls a pathogenic expression signature in CCR2(+)Ly6C(hi) monocytes and their progeny, which was essential for tissue damage.
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http://dx.doi.org/10.1016/j.immuni.2015.08.010DOI Listing
September 2015

Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR-Cas9-mediated mutagenesis.

Nucleic Acids Res 2015 May 20;43(10):4855-67. Epub 2015 Apr 20.

Department of Molecular and Cellular Biology, National Centre for Biotechnology (CNB-CSIC), Campus Cantoblanco, Darwin 3, 28049 Madrid, Spain CIBERER-ISCIII, Madrid, Spain

Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5' upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.
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http://dx.doi.org/10.1093/nar/gkv375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446435PMC
May 2015