Lengthening Neurogenic Period during Neocortical Development Causes a Hallmark of Neocortex Expansion.

The neocortex is evolutionarily the youngest part of the brain. The human neocortex is about three times bigger than that of our closest living relatives, the chimpanzees. In recent years a research team at the MPI-CBG, led by one of the institute’s founding directors, Wieland Huttner, has explored the cause of the evolutionary expansion of the human neocortex. The neocortex characteristically contains six layers of neurons, and it is primarily the upper layers that have expanded during evolution. Although previous studies of the research group have focused on genes that underlie this expansion, the group also suggested already in 2014, based on the results of a mathematical model, that among the species with highly folded brains the increase in neocortical neuron number can be explained by a longer neurogenic period – the developmental time window during which neocortical neurons are produced.

Stepien BK, Naumann R, Holtz A, Helppi J, Huttner WB, Vaid S. Lengthening Neurogenic Period during Neocortical Development Causes a Hallmark of Neocortex Expansion. Curr Biol. 2020 Aug 29:S0960-9822(20)31236-7

A Rotating Spiral Micromotor for Noninvasive Zygote Transfer.

Embryo transfer (ET) is a decisive step in the in vitro fertilization process. In most cases, the embryo is transferred to the uterus after several days of in vitro culture. Although studies have identified the beneficial effects of ET on proper embryo development in the earlier stages, this strategy is compromised by the necessity to transfer early embryos (zygotes) back to the fallopian tube instead of the uterus, which requires a more invasive, laparoscopic procedure, termed zygote intrafallopian transfer (ZIFT). Magnetic micromotors offer the possibility to mitigate such surgical interventions, as they have the potential to transport and deliver cellular cargo such as zygotes through the uterus and fallopian tube noninvasively, actuated by an externally applied rotating magnetic field. This study presents the capture, transport, and release of bovine and murine zygotes using two types of magnetic micropropellers, helix and spiral. Herein, this is demonstrated with murine oocytes/zygotes as the cargo; this is the first step toward the application of noninvasive, magnetic micromotor-assisted

Schwarz L, Karnaushenko DD, Hebenstreit F, Naumann R, Schmidt OG, Medina-Sánchez M. A Rotating Spiral Micromotor for Noninvasive Zygote Transfer. Adv Sci (Weinh). 2020 Jul 21;7(18):2000843


Novel insights into the genetic background of genetically modified mice.

Unclear or misclassified genetic background of laboratory rodents or a lack of strain awareness causes a number of difficulties in performing or reproducing scientific experiments. Until now, genetic differentiation between strains and substrains of inbred mice has been a challenge. We have developed a screening method for analyzing inbred strains regarding their genetic background. It is based on 240 highly informative short tandem repeat (STR) markers covering the 19 autosomes as well as X and Y chromosomes. Combination of analysis results for presence of known C57BL/6 substrain-specific mutations together with autosomal STR markers and the Y-chromosomal STR-haplotype provides a comprehensive snapshot of the genetic background of mice. In this study, the genetic background of 72 mouse lines obtained from 18 scientific institutions in Germany and Austria was determined. By analyzing only 3 individuals per genetically modified line it was possible to detect mixed genetic backgrounds frequently. In several lines presence of a mispairing Y chromosome was detected. At least every second genetically modified line displayed a mixed genetic background which could lead to unexpected and non-reproducible results, irrespective of the investigated gene of interest.

Dobrowolski P, Fischer M, Naumann R. Novel insights into the genetic background of genetically modified mice. Transgenic Res. 2018 Apr 16. doi: 10.1007/s11248-018-0073-2.


* joint first author # joint corresponding author

Franziska Hettler, Christina Schreck, Sandra Romero Marquez, Thomas Engleitner, Baiba Vilne, Theresa Landspersky, Heike Weidner, Renate Hausinger, Ritu Mishra, Rupert Oellinger, Martina Rauner, Ronald Naumann, Christian Peschel, Florian Bassermann, Roland Rad, Rouzanna Istvanffy, Robert A J Oostendorp
Osteoprogenitor SFRP1 prevents exhaustion of hematopoietic stem cells via PP2A-PR72/130-mediated regulation of p300.
Haematologica, 1-1 (2022)
Remodeling of the bone marrow microenvironment in chronic inflammation and in aging reduces hematopoietic stem cell (HSC) function. To assess the mechanisms of HSC functional decline and find strategies to counteract these, we established a model in which Sfrp1 gene was deleted in Osterix+ osteolineage cells (OS1Δ/Δ mice). HSCs from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the `ROS pathway´ and reduced single cell proliferation. Interestingly, not only was the protein level of Catenin beta-1 (beta-catenin) elevated, but so was its association with the phosphorylated coactivator p300 in the nucleus. Since these two proteins play a key role in promotion of differentiation and senescence, we inhibited in vivo phosphorylation of p300 through PP2APR72/130 by IQ-1 administration in OS1Δ/Δ mice. This treatment not only reduced Catenin beta-1/phospho-p300 association, but also decreased nuclear p300. More importantly, in vivo IQ-1 treatment fully restored HSC repopulating activity of the OS1Δ/Δ mice. Our findings show that osteoprogenitor Sfrp1 is essential for maintaining HSC function. Furthermore, pharmacological downregulation of nuclear Catenin beta-1/phospho-p300 association is a new strategy to restore poor HSC function.

Felipe Mora-Bermúdez, Philipp Kanis, Dominik Macak, Jula Peters, Ronald Naumann, Lei Xing, Mihail Sarov, Sylke Winkler, Christina Eugster Oegema, Christiane Haffner, Pauline Wimberger, Stephan Riesenberg, Tomislav Maricic, Wieland Huttner, Svante Pääbo
Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development.
Sci Adv, 8(30) Art. No. eabn7702 (2022)
Open Access DOI
Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

Tina Schubert, Nicole Reisch, Ronald Naumann, Ilka Reichardt, Dana Landgraf, Friederike Quitter, Shamini Ramkumar Thirumalasetty, Anne-Kristin Heninger, Mihail Sarov, M Peitzsch, Angela Huebner, Katrin Koehler
CYP21A2 Gene Expression in a Humanized 21-Hydroxylase Mouse Model Does Not Affect Adrenocortical Morphology and Function.
J Endocr Soc, 6(6) Art. No. bvac062 (2022)
Open Access DOI
Steroid 21-hydroxylase is an enzyme of the steroid pathway that is involved in the biosynthesis of cortisol and aldosterone by hydroxylation of 17α-hydroxyprogesterone and progesterone at the C21 position. Mutations in CYP21A2, the gene encoding 21-hydroxylase, cause the most frequent form of the autosomal recessive disorder congenital adrenal hyperplasia (CAH). In this study, we generated a humanized 21-hydroxylase mouse model as the first step to the generation of mutant mice with different CAH-causing mutations. We replaced the mouse Cyp21a1 gene with the human CYP21A2 gene using homologous recombination in combination with CRISPR/Cas9 technique. The aim of this study was to characterize the new humanized mouse model. All results described are related to the homozygous animals in comparison with wild-type mice. We show analogous expression patterns of human 21-hydroxylase by the murine promoter and regulatory elements in comparison to murine 21-hydroxylase in wild-type animals. As expected, no Cyp21a1 transcript was detected in homozygous CYP21A2 adrenal glands. Alterations in adrenal gene expression were observed for Cyp11a1, Star, and Cyb11b1. These differences, however, were not pathological. Outward appearance, viability, growth, and fertility were not affected in the humanized CYP21A2 mice. Plasma steroid levels of corticosterone and aldosterone showed no pathological reduction. In addition, adrenal gland morphology and zonation were similar in both the humanized and the wild-type mice. In conclusion, humanized homozygous CYP21A2 mice developed normally and showed no differences in histological analyses, no reduction in adrenal and gonadal gene expression, or in plasma steroids in comparison with wild-type littermates.

Boris Rivkin, Christian Becker, Balram Singh, Azaam Aziz, Farzin Akbar, Aleksandr Egunov, Dmitriy D Karnaushenko, Ronald Naumann, Rudolf Schäfer, Mariana Medina-Sánchez#, Daniil Karnaushenko#, Oliver G. Schmidt#
Electronically integrated microcatheters based on self-assembling polymer films.
Sci Adv, 7(51) Art. No. eabl5408 (2021)
Open Access DOI
Existing electronically integrated catheters rely on the manual assembly of separate components to integrate sensing and actuation capabilities. This strongly impedes their miniaturization and further integration. Here, we report an electronically integrated self-assembled microcatheter. Electronic components for sensing and actuation are embedded into the catheter wall through the self-assembly of photolithographically processed polymer thin films. With a diameter of only about 0.1 mm, the catheter integrates actuated digits for manipulation and a magnetic sensor for navigation and is capable of targeted delivery of liquids. Fundamental functionalities are demonstrated and evaluated with artificial model environments and ex vivo tissue. Using the integrated magnetic sensor, we develop a strategy for the magnetic tracking of medical tools that facilitates basic navigation with a high resolution below 0.1 mm. These highly flexible and microsized integrated catheters might expand the boundary of minimally invasive surgery and lead to new biomedical applications.

Emilie Coppin#, Bala Sai Sundarasetty, Susann Rahmig, Jonas Blume, Nikita A Verheyden, Franz Bahlmann, Sarina Ravens, Undine Schubert, Janine Schmid, Stefan Ludwig, Katharina Geissler, Orlando Guntinas-Lichius, Constantin von Kaisenberg, Tanja Groten, Alexander Platz, Ronald Naumann, Barbara Ludwig, Immo Prinz, Claudia Waskow#, Andreas Krueger#
Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7.
Leukemia, 35(12) 3561-3567 (2021)
Open Access DOI
Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T-cell differentiation remains inefficient. We generated mice expressing human interleukin-7 (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T-cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

Thomas J O'Neill✳︎, Thomas Seeholzer✳︎, Andreas Gewies✳︎, Torben Gehring, Florian Giesert, Isabel Hamp, Carina Graß, Henrik Schmidt, Katharina Kriegsmann, Marie J Tofaute, Katrin Demski, Tanja Poth, Marc Rosenbaum, Theresa Schnalzger, Jurgen Ruland, Martin Göttlicher, Mark Kriegsmann, Ronald Naumann, Vigo Heissmeyer, Oliver Plettenburg, Wolfgang Wurst, Daniel Krappmann
TRAF6 prevents fatal inflammation by homeostatic suppression of MALT1 protease.
Sci Immunol, 6(65) Art. No. eabh2095 (2021)
[Figure: see text].

Hui Wang, Xiaofei Li, Tetsuhiro Kajikawa, Jieun Shin, Jong-Hyung Lim, Ioannis Kourtzelis, Kosuke Nagai, Jonathan Korostoff, Sylvia Grossklaus, Ronald Naumann, Trian Chavakis, George Hajishengallis
Stromal cell-derived DEL-1 inhibits Tfh cell activation and inflammatory arthritis.
J Clin Invest, 131(19) Art. No. e150578 (2021)
The secreted protein developmental endothelial locus 1 (DEL-1) regulates inflammatory cell recruitment and protects against inflammatory pathologies in animal models. Here, we investigated DEL-1 in inflammatory arthritis using collagen-induced arthritis (CIA) and collagen Ab-induced arthritis (CAIA) models. In both models, mice with endothelium-specific overexpression of DEL-1 were protected from arthritis relative to WT controls, whereas arthritis was exacerbated in DEL-1-deficient mice. Compared with WT controls, mice with collagen VI promoter-driven overexpression of DEL-1 in mesenchymal cells were protected against CIA but not CAIA, suggesting a role for DEL-1 in the induction of the arthritogenic Ab response. Indeed, DEL-1 was expressed in perivascular stromal cells of the lymph nodes and inhibited Tfh and germinal center B cell responses. Mechanistically, DEL-1 inhibited DC-dependent induction of Tfh cells by targeting the LFA-1 integrin on T cells. Overall, DEL-1 restrained arthritis through a dual mechanism, one acting locally in the joints and associated with the anti-recruitment function of endothelial cell-derived DEL-1; the other mechanism acting systemically in the lymph nodes and associated with the ability of stromal cell-derived DEL-1 to restrain Tfh responses. DEL-1 may therefore be a promising therapeutic for the treatment of inflammatory arthritis.

Dipannita Sarkar, Mohammad Shariq, Deepanjali Dwivedi, Nirmal Krishnan, Ronald Naumann, Upinder Singh Bhalla, Hiyaa Singhee Ghosh
Adult brain neurons require continual expression of the schizophrenia-risk gene Tcf4 for structural and functional integrity.
Transl Psychiatry, 11(1) Art. No. 494 (2021)
Open Access DOI
The schizophrenia-risk gene Tcf4 has been widely studied in the context of brain development using mouse models of haploinsufficiency, in utero knockdown and embryonic deletion. However, Tcf4 continues to be abundantly expressed in adult brain neurons where its functions remain unknown. Given the importance of Tcf4 in psychiatric diseases, we investigated its role in adult neurons using cell-specific deletion and genetic tracing in adult animals. Acute loss of Tcf4 in adult excitatory neurons in vivo caused hyperexcitability and increased dendritic complexity of neurons, effects that were distinct from previously observed effects in embryonic-deficiency models. Interestingly, transcriptomic analysis of genetically traced adult-deleted FACS-sorted Tcf4-knockout neurons revealed that Tcf4 targets in adult neurons are distinct from those in the embryonic brain. Meta-analysis of the adult-deleted neuronal transcriptome from our study with the existing datasets of embryonic Tcf4 deficiencies revealed plasma membrane and ciliary genes to underlie Tcf4-mediated structure-function regulation specifically in adult neurons. The profound changes both in the structure and excitability of adult neurons upon acute loss of Tcf4 indicates that proactive regulation of membrane-related processes underlies the functional and structural integrity of adult neurons. These findings not only provide insights for the functional relevance of continual expression of a psychiatric disease-risk gene in the adult brain but also identify previously unappreciated gene networks underpinning mature neuronal regulation during the adult lifespan.

Florian Peters, Sascha Rahn, Marion Mengel, Franka Scharfenberg, Anna Otte, Tomas Koudelka, Erwin F Wagner, F Thomas Wunderlich, Michael Haase, Ronald Naumann, Andreas Tholey, Christoph Becker-Pauly
Syndecan-1 shedding by meprin β impairs keratinocyte adhesion and differentiation in hyperkeratosis.
Matrix Biol, 102 37-69 (2021)
Dysregulation of proteolytic enzymes has huge impact on epidermal homeostasis, which can result in severe pathological conditions such as fibrosis or Netherton syndrome. The metalloprotease meprin β was found to be upregulated in hyperproliferative skin diseases. AP-1 transcription factor complex has been reported to induce Mep1b expression. Since AP-1 and its subunit fos-related antigen 2 (fra-2) are associated with the onset and progression of psoriasis, we wanted to investigate if this could partially be attributed to increased meprin β activity. Here, we demonstrate that fra-2 transgenic mice show increased meprin β expression and proteolytic activity in the epidermis. To avoid influence by other fra-2 regulated genes, we additionally generated a mouse model that enabled tamoxifen-inducible expression of meprin β under the Krt5-promotor to mimic the pathological condition. Interestingly, induced meprin β expression in the epidermis resulted in hyperkeratosis, hair loss and mottled pigmentation of the skin. Employing N-terminomics revealed syndecan-1 as a substrate of meprin β in skin. Shedding of syndecan-1 at the cell surface caused delayed calcium-induced differentiation and impaired adhesion of keratinocytes, which was blocked by the meprin β inhibitor fetuin-B.

Emiel P C van der Vorst✳︎#, Mario A A Pepe✳︎, Linsey J F Peters, Markus Haberbosch, Yvonne Jansen, Ronald Naumann, Georgios T Stathopoulos, Christian Weber, Kiril Bidzhekov#
Transcriptome signature of miRNA-26b KO mouse model suggests novel targets.
BMC Genom Data, 22(1) Art. No. 23 (2021)
Open Access DOI
MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. One of the miRNAs that has been shown to play a role in various pathologies like cancer, neurological disorders and cardiovascular diseases is miRNA-26b. However, these studies only demonstrated rather ambiguous associations without revealing a causal relationship. Therefore, the aim of this study is to establish and validate a mouse model which enables the elucidation of the exact role of miRNA-26b in various pathologies.

Barbara Stepien✳︎, Samir Vaid✳︎#, Ronald Naumann✳︎, Anja Holtz, Wieland Huttner#
Generation of interspecies mouse-rat chimeric embryos by embryonic stem (ES) cell microinjection.
STAR Protoc, 2(2) Art. No. 100494 (2021)
Open Access DOI
Interspecies chimerism is a useful tool to study interactions between cells of different genetic makeup in order to elucidate the mechanisms underlying non-cell-autonomous processes, including evolutionary events. However, generating interspecies chimeras with high efficiency and chimerism level remains challenging. Here, we describe a protocol for generating chimeras between mouse and rat. Donor embryonic stem cells of one species are microinjected into early embryos of the other species (recipient), which are implanted into host foster mothers of the recipient species. For complete details on the use and execution of this protocol, please refer to Stepien et al. (2020).

Boris Rivkin, Christian Becker, Farzin Akbar, Rachappa Ravishankar, Dmitriy D Karnaushenko, Ronald Naumann, Alaleh Mirhajivarzaneh, Mariana Medina-Sánchez#, Daniil Karnaushenko#, Oliver G. Schmidt#
Shape-Controlled Flexible Microelectronics Facilitated by Integrated Sensors and Conductive Polymer Actuators.
Adv Intell Syst, 3(6) Art. No. 2000238 (2021)
Open Access DOI
The next generation of biomedical tools requires reshapeable electronics to closely interface with biological tissues. This will offer unique mechanical properties and the ability to conform to irregular geometries while being robust and lightweight. Such devices can be achieved with soft materials and thin-film structures that are able to reshape on demand. However, reshaping at the submillimeter scale remains a challenging task. Herein, shape-controlled microscale devices are demonstrated that integrate electronic sensors and electroactive polymer actuators. The fast and biocompatible actuators are capable of actively reshaping the device into flat or curved geometries. The curvature and position of the devices are monitored with strain or magnetic sensors. The sensor signals are used in a closed feedback loop to control the actuators. The devices are wafer-scale microfabricated resulting in multiple functional units capable of grasping, holding, and releasing biological tissues, as demonstrated with a neuronal bundle.

Vita Stepanova✳︎, Kaja Ewa Moczulska✳︎, Guido Vacano✳︎, Ilia Kurochkin✳︎, Xiangchun Ju✳︎, Stephan Riesenberg, Dominik Macak, Tomislav Maricic, Linda Dombrowski, Maria Schörnig, Konstantinos Anastassiadis, Oliver Baker, Ronald Naumann, Ekaterina Khrameeva, Anna Vanushkina, Elena Stekolshchikova, Alina Egorova, Anna Tkachev, Randall Mazzarino, Nathan Duval, Dmitri Zubkov, Patrick Giavalisco, Terry G Wilkinson Ii, David Patterson, Philipp Khaitovich, Svante Pääbo
Reduced purine biosynthesis in humans after their divergence from Neandertals.
Elife, 10 Art. No. e58741 (2021)
Open Access DOI
We analyze the metabolomes of humans, chimpanzees and macaques in muscle, kidney and three different regions of the brain. Whereas several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.

Anke Weitzmann, Ronald Naumann, Anne Dudeck, Thomas Zerjatke, Alexander Gerbaulet, Axel Roers
Mast Cells Occupy Stable Clonal Territories in Adult Steady-State Skin.
J Invest Dermatol, 140(12) 2433-2441 (2020)
Mast cells (MCs) are tissue-resident hematopoietic cells intensely studied for their role as effectors in allergic immune responses. Yolk sac-derived embryonic MCs first populate tissues and are later replaced by definitive MCs. We show that definitive MC progenitors expand locally in skin and form clonal colonies that cover stable territories. In MC-deficient skin, colonies grow by proliferation of MCs at the border of the clonal territory. Clonal growth ceases at common borders of neighboring colonies. In steady state, colony self-renewal is independent of bone marrow contribution, and the clonal architecture remains fixed if not disturbed by skin inflammation. Inflammatory cues increase MC density setpoint, stimulating the influx of new progenitors from the bone marrow as well as proliferation of skin-resident cells. The expanding new arrivals disrespect territories of preexisting MC clones. We conclude that during a limited window early in development, definitive MC precursors efficiently enter the skin, expand, and self-maintain, occupying stable territories. In adulthood, circulating progenitors, excluded from steady-state skin, are recruited only into inflamed skin where they clonally expand alongside proliferating skin-resident MCs, disorganizing the original architecture of clonal territories.

Barbara Stepien, Ronald Naumann, Anja Holtz, Jussi Helppi, Wieland Huttner, Samir Vaid
Lengthening Neurogenic Period during Neocortical Development Causes a Hallmark of Neocortex Expansion.
Curr Biol, 30(21) 4227-4237 (2020)
A hallmark of the evolutionary expansion of the neocortex is a specific increase in the number of neurons generated for the upper neocortical layers during development. The cause underlying this increase is unknown. Here, we show that lengthening the neurogenic period during neocortical development is sufficient to specifically increase upper-layer neuron generation. Thus, embryos of mouse strains with longer gestation exhibited a longer neurogenic period and generated more upper-layer, but not more deep-layer, neurons than embryos with shorter gestation. Accordingly, long-gestation embryos showed a greater abundance of neurogenic progenitors in the subventricular zone than short-gestation embryos at late stages of cortical neurogenesis. Analysis of a mouse-rat chimeric embryo, developing inside a rat mother, pointed to factors in the rat environment that influenced the upper-layer neuron generation by the mouse progenitors. Exploring a potential maternal source of such factors, short-gestation strain mouse embryos transferred to long-gestation strain mothers exhibited an increase in the length of the neurogenic period and upper-layer neuron generation. The opposite was the case for long-gestation strain mouse embryos transferred to short-gestation strain mothers, indicating a dominant maternal influence on the length of the neurogenic period and hence upper-layer neuron generation. In summary, our study uncovers a hitherto unknown link between embryonic cortical neurogenesis and the maternal gestational environment and provides experimental evidence that lengthening the neurogenic period during neocortical development underlies a key aspect of neocortical expansion.

Donato Santovito, Virginia Egea, Kiril Bidzhekov, Lucia Natarelli, André Mourão, Xavier Blanchet, Kanin Wichapong, Maria Aslani, Coy Brunßen, Michael Horckmans, Michael Hristov, Arie Geerlof, Esther Lutgens, Mat J A P Daemen, Tilman M Hackeng, Christian Ries, Trian Chavakis, Henning Morawietz, Ronald Naumann, Philipp von Hundelshausen, Sabine Steffens, Johan Duchêne, Remco T A Megens, Michael Sattler, Christian Weber
Autophagy unleashes noncanonical microRNA functions.
Autophagy, 16(12) 2294-2296 (2020)
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression which act by guiding AGO (argonaute) proteins to target RNA transcripts in the RNA-induced silencing complex (RISC). This macromolecular complex includes multiple additional components (e.g., TNRC6A) that allow for interaction with enzymes mediating inhibition of translation or RNA decay. However, miRNAs also reside in low-molecular weight complexes without being engaged in target repression, and their function in this context is largely unknown. Our recent findings show that endothelial cells exposed to protective high-shear stress or MTORC inhibition activate the macroautophagy/autophagy machinery to sustain viability by promoting differential trafficking of MIR126 strands and by enabling unconventional features of MIR126-5p. Whereas MIR126-3p is degraded upon autophagy activation, MIR126-5p interacts with the RNA-binding protein MEX3A to form a ternary complex with AGO2. This complex forms on the autophagosomal surface and facilitates its nuclear localization. Once in the nucleus, MIR126-5p dissociates from AGO2 and establishes aptamer-like interactions with the effector CASP3 (caspase 3). The binding to MIR126-5p prevents dimerization and proper active site formation of CASP3, thus inhibiting proteolytic activity and limiting apoptosis. Disrupting this pathway in vivo by genetic deletion of Mex3a or by specific deficiency of endothelial autophagy aggravates endothelial apoptosis and exacerbates the progression of atherosclerosis. The direct inhibition of CASP3 by MIR126-5p reveals a non-canonical mechanism by which miRNAs can modulate protein function and mediate the autophagy-apoptosis crosstalk.

Lukas Schwarz, Dmitriy D Karnaushenko, Franziska Hebenstreit, Ronald Naumann, Oliver G. Schmidt, Mariana Medina-Sánchez
A Rotating Spiral Micromotor for Noninvasive Zygote Transfer.
Adv Sci (Weinh), 7(18) Art. No. 2000843 (2020)
Open Access DOI
Embryo transfer (ET) is a decisive step in the in vitro fertilization process. In most cases, the embryo is transferred to the uterus after several days of in vitro culture. Although studies have identified the beneficial effects of ET on proper embryo development in the earlier stages, this strategy is compromised by the necessity to transfer early embryos (zygotes) back to the fallopian tube instead of the uterus, which requires a more invasive, laparoscopic procedure, termed zygote intrafallopian transfer (ZIFT). Magnetic micromotors offer the possibility to mitigate such surgical interventions, as they have the potential to transport and deliver cellular cargo such as zygotes through the uterus and fallopian tube noninvasively, actuated by an externally applied rotating magnetic field. This study presents the capture, transport, and release of bovine and murine zygotes using two types of magnetic micropropellers, helix and spiral. Although helices represent an established micromotor architecture, spirals surpass them in terms of motion performance and with their ability to reliably capture and secure the cargo during both motion and transfer between different environments. Herein, this is demonstrated with murine oocytes/zygotes as the cargo; this is the first step toward the application of noninvasive, magnetic micromotor-assisted ZIFT.

Juliana G. Roscito, Kaushikaram Subramanian, Ronald Naumann, Mihail Sarov, Anna Shevchenko, Aliona Bogdanova, Thomas Kurth, Leo Foerster, Moritz Kreysing, Michael Hiller
Recapitulating evolutionary divergence in a single cis-regulatory element is sufficient to cause expression changes of the lens gene Tdrd7.
Mol Biol Evol, 38(2) 380-392 (2020)
Open Access PDF DOI
Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR; Nannospalax galili) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved non-coding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated evolutionary changes in this element by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor replacement caused a more than two-fold up-regulation of Tdrd7 in the developing lens; however, increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, possibly explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eye-regulatory elements collectively contribute to phenotypic differences.

Marlen Dierich, Alessandro Altoè, Julia Koppelmann, Saskia Evers, Vijay Renigunta, Martin K Schäfer, Ronald Naumann, Sarah Verhulst, Dominik Oliver, Michael G Leitner
Optimized Tuning of Auditory Inner Hair Cells to Encode Complex Sound through Synergistic Activity of Six Independent K+ Current Entities.
Cell Rep, 32(1) Art. No. 107869 (2020)
Open Access DOI
Auditory inner hair cells (IHCs) convert sound vibrations into receptor potentials that drive synaptic transmission. For the precise encoding of sound qualities, receptor potentials are shaped by K+ conductances tuning the properties of the IHC membrane. Using patch-clamp and computational modeling, we unravel this membrane specialization showing that IHCs express an exclusive repertoire of six voltage-dependent K+ conductances mediated by Kv1.8, Kv7.4, Kv11.1, Kv12.1, and BKCa channels. All channels are active at rest but are triggered differentially during sound stimulation. This enables non-saturating tuning over a far larger potential range than in IHCs expressing fewer current entities. Each conductance contributes to optimizing responses, but the combined activity of all channels synergistically improves phase locking and the dynamic range of intensities that IHCs can encode. Conversely, hypothetical simpler IHCs appear limited to encode only certain aspects (frequency or intensity). The exclusive channel repertoire of IHCs thus constitutes an evolutionary adaptation to encode complex sound through multifaceted receptor potentials.

Deepthi Ashokkumar✳︎, Qinyu Zhang✳︎, Christian Much, Anita S. Bledau, Ronald Naumann, Dimitra Alexopoulou, Andreas Dahl, Neha Goveas, Jun Fu, Konstantinos Anastassiadis, A F Stewart#, Andrea Kranz#
MLL4 is required after implantation, whereas MLL3 becomes essential during late gestation.
Development, 147(12) Art. No. dev186999 (2020)
Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of MLL3 and MLL4 mutations in many types of human cancer. Despite this emerging importance, the requirements of these paralogs in mammalian development have only been incompletely reported. Here, we examined the null phenotypes to establish that MLL3 is first required for lung maturation, whereas MLL4 is first required for migration of the anterior visceral endoderm that initiates gastrulation in the mouse. This collective cell migration is preceded by a columnar-to-squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, Mll4 mutants display incompletely penetrant, sex-distorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these paralogs in mouse development argues against their action as general enhancer factors.

Katarina Liedtke, Christina Alter, Anne Günther, Nadine Hövelmeyer, Robert Klopfleisch, Ronald Naumann, F Thomas Wunderlich, Jan Buer, Astrid M Westendorf, Wiebke Hansen
Endogenous CD83 Expression in CD4+ Conventional T Cells Controls Inflammatory Immune Responses.
J Immunol, 204(12) 3217-3226 (2020)
The glycoprotein CD83 is known to be expressed by different immune cells including activated CD4+Foxp3+ regulatory T cells (Tregs) and CD4+Foxp3- conventional T cells. However, the physiological function of endogenous CD83 in CD4+ T cell subsets is still unclear. In this study, we have generated a new CD83flox mouse line on BALB/c background, allowing for specific ablation of CD83 in T cells upon breeding with CD4-cre mice. Tregs from CD83flox/flox/CD4-cretg/wt mice had similar suppressive activity as Tregs from CD83flox/flox/CD4-crewt/wt wild-type littermates, suggesting that endogenous CD83 expression is dispensable for the inhibitory capacity of Tregs. However, CD83-deficient CD4+ conventional T cells showed elevated proliferation and IFN-γ secretion as well as an enhanced capacity to differentiate into Th1 cells and Th17 cells upon stimulation in vitro. T cell-specific ablation of CD83 expression resulted in aggravated contact hypersensitivity reaction accompanied by enhanced CD4+ T cell activation. Moreover, adoptive transfer of CD4+CD45RBhigh T cells from CD83flox/flox/CD4-cretg/wt mice into Rag2-deficient mice elicited more severe colitis associated with increased serum concentrations of IL-12 and elevated CD40 expression on CD11c+ dendritic cells (DCs). Strikingly, DCs from BALB/c mice cocultured with CD83-deficient CD4+ conventional T cells showed enhanced CD40 expression and IL-12 secretion compared with DCs cocultured with CD4+ conventional T cells from CD83flox/flox/CD4-crewt/wt wild-type mice. In summary, these results indicate that endogenous CD83 expression in CD4+ conventional T cells plays a crucial role in controlling CD4+ T cell responses, at least in part, by regulating the activity of CD11c+ DCs.

Donato Santovito, Virginia Egea, Kiril Bidzhekov, Lucia Natarelli, André Mourão, Xavier Blanchet, Kanin Wichapong, Maria Aslani, Coy Brunßen, Michael Horckmans, Michael Hristov, Arie Geerlof, Esther Lutgens, Mat J A P Daemen, Tilman M Hackeng, Christian Ries, Trian Chavakis, Henning Morawietz, Ronald Naumann, Philipp von Hundelshausen, Sabine Steffens, Johan Duchêne, Remco T A Megens, Michael Sattler, Christian Weber
Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.
Sci Transl Med, 12(546) Art. No. eaaz2294 (2020)
MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.

Sören Reinke, Mary Linge, Hans H Diebner, H Luksch, Silke Glage, Anne Gocht, Avril A B Robertson, Matthew A Cooper, Sigrun R Hofmann, Ronald Naumann, Mihail Sarov, Rayk Behrendt, Axel Roers, Frank Pessler, Joachim Roesler, Angela Rösen-Wolff, Stefan Winkler
Non-canonical Caspase-1 Signaling Drives RIP2-Dependent and TNF-α-Mediated Inflammation In Vivo.
Cell Rep, 30(8) 2501-2511 (2020)
Open Access DOI
Pro-inflammatory caspase-1 is a key player in innate immunity. Caspase-1 processes interleukin (IL)-1β and IL-18 to their mature forms and triggers pyroptosis. These caspase-1 functions are linked to its enzymatic activity. However, loss-of-function missense mutations in CASP1 do not prevent autoinflammation in patients, despite decreased IL-1β production. In vitro data suggest that enzymatically inactive caspase-1 drives inflammation via enhanced nuclear factor κB (NF-κB) activation, independent of IL-1β processing. Here, we report two mouse models of enzymatically inactive caspase-1-C284A, demonstrating the relevance of this pathway in vivo. In contrast to Casp1-/- mice, caspase-1-C284A mice show pronounced hypothermia and increased levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 when challenged with lipopolysaccharide (LPS). Caspase-1-C284A signaling is RIP2 dependent and mediated by TNF-α but independent of the NLRP3 inflammasome. LPS-stimulated whole blood from patients carrying loss-of-function missense mutations in CASP1 secretes higher amounts of TNF-α. Taken together, these results reveal non-canonical caspase-1 signaling in vivo.

Jussi Helppi, Ronald Naumann, Oliver Zierau
Phytoestrogen-containing diets offer benefits for mouse embryology but lead to fewer offspring being produced.
Lab Anim, 54(6) 536-545 (2020)
One of the most commonly used protein sources in rodent diets is soy, which is naturally rich in phytoestrogens. Although phytoestrogens have shown potential health benefits in humans, they may also have the ability to disrupt reproduction. Consequently, there has been a tendency to try to exclude them from rodent diets. In the current study, we investigated whether phytoestrogen content in the mouse diet could affect reproduction in mice used as embryo donors. Donor mice (C57BL/6JOlaHsd) were maintained with three different diets: high phytoestrogen (ca. 400 mg/kg genistein), low phytoestrogen (ca. 10 mg/kg genistein) and standard breeding diet (ca. 120 mg/kg genistein). Mice fed a high phytoestrogen diet had a high yield of plugs, embryos, and injectable embryos, as well as producing good quality embryos. Results from donor mice fed a low phytoestrogen diet were consistently but only slightly inferior, whereas mice fed a standard diet performed the poorest. Interestingly, the largest number of born and weaned offspring were observed when recipient females received embryos from the standard diet group. Sperm yield and quality of stud males did not differ between the groups. We surmize that for experimental endpoints requiring fertilized embryos it may be more beneficial to feed mice a diet containing phytoestrogen, but if the goal is to produce transgenic mice, a diet high in phytoestrogen may be inadvisable. In conclusion, care should be taken when selecting a diet for experimental mouse colonies as phytoestrogen could influence the study outcome.

Seungmin Han, Juergen Fink, David J. Jörg, Eunmin Lee, Min Kyu Yum, Lemonia Chatzeli, Sebastian R Merker, Manon Josserand, Teodora Trendafilova, Amanda Andersson-Rolf, Catherine Dabrowska, Hyunki Kim, Ronald Naumann, Ji-Hyun Lee, Nobuo Sasaki, Richard Lester Mort, Onur Basak, Hans Clevers, Daniel E Stange, Anna Philpott, Jong Kyoung Kim, Benjamin D Simons, Bon-Kyoung Koo
Defining the Identity and Dynamics of Adult Gastric Isthmus Stem Cells.
Cell Stem Cell, 25(3) 342-356 (2019)
Open Access DOI
The gastric corpus epithelium is the thickest part of the gastrointestinal tract and is rapidly turned over. Several markers have been proposed for gastric corpus stem cells in both isthmus and base regions. However, the identity of isthmus stem cells (IsthSCs) and the interaction between distinct stem cell populations is still under debate. Here, based on unbiased genetic labeling and biophysical modeling, we show that corpus glands are compartmentalized into two independent zones, with slow-cycling stem cells maintaining the base and actively cycling stem cells maintaining the pit-isthmus-neck region through a process of "punctuated" neutral drift dynamics. Independent lineage tracing based on Stmn1 and Ki67 expression confirmed that rapidly cycling IsthSCs maintain the pit-isthmus-neck region. Finally, single-cell RNA sequencing (RNA-seq) analysis is used to define the molecular identity and lineage relationship of a single, cycling, IsthSC population. These observations define the identity and functional behavior of IsthSCs.

João P L Castro✳︎, Michelle N Yancoskie✳︎, Marta Marchini, Stefanie Belohlavy, Layla Hiramatsu, Marek Kučka, William H Beluch, Ronald Naumann, Isabella Skuplik, John Cobb, Nick H Barton, Campbell Rolian#, Yingguang Frank Chan#
An integrative genomic analysis of the Longshanks selection experiment for longer limbs in mice.
Elife, 8 Art. No. e42014 (2019)
Open Access DOI
Evolutionary studies are often limited by missing data that are critical to understanding the history of selection. Selection experiments, which reproduce rapid evolution under controlled conditions, are excellent tools to study how genomes evolve under selection. Here we present a genomic dissection of the Longshanks selection experiment, in which mice were selectively bred over 20 generations for longer tibiae relative to body mass, resulting in 13% longer tibiae in two replicates. We synthesized evolutionary theory, genome sequences and molecular genetics to understand the selection response and found that it involved both polygenic adaptation and discrete loci of major effect, with the strongest loci tending to be selected in parallel between replicates. We show that selection may favor de-repression of bone growth through inactivating two limb enhancers of an inhibitor, Nkx3-2. Our integrative genomic analyses thus show that it is possible to connect individual base-pair changes to the overall selection response.

Branislav Krljanac, Christoph Schubart, Ronald Naumann, Stefan Wirtz, Stephan Culemann, Gerhard Krönke, David Voehringer
RELMα-expressing macrophages protect against fatal lung damage and reduce parasite burden during helminth infection.
Sci Immunol, 4(35) Art. No. eaau3814 (2019)
Alternatively activated macrophages (AAMs) can contribute to wound healing, regulation of glucose and fat metabolism, resolution of inflammation, and protective immunity against helminths. Their differentiation, tissue distribution, and effector functions are incompletely understood. Murine AAMs express high levels of resistin-like molecule (RELM) α, an effector protein with potent immunomodulatory functions. To visualize RELMα+ macrophages (MΦs) in vivo and evaluate their role in defense against helminths, we generated RELMα reporter/deleter mice. Infection with the helminth Nippostrongylus brasiliensis induced expansion of RELMα+ lung interstitial but not alveolar MΦs in a STAT6-dependent manner. RELMα+ MΦs were required for prevention of fatal lung damage during primary infection. Furthermore, protective immunity was lost upon specific deletion of RELMα+ MΦs during secondary infection. Thus, RELMα reporter/deleter mice reveal compartmentalization of AAMs in different tissues and demonstrate their critical role in resolution of severe lung inflammation and protection against migrating helminths.

Anneke Wilharm, Inga Sandrock, Marie Marotel, Abdi Demera, Ronald Naumann, Thierry Walzer, Immo Prinz
Styk1 is specifically expressed in NK1.1+ lymphocytes including NK, γδ T, and iNKT cells in mice, but is dispensable for their ontogeny and function.
Eur J Immunol, 49(5) 686-693 (2019)
Innate T cells, NK cells, and innate-like lymphocytes (ILCs) share transcriptional signatures that translate into overlapping developmental and functional programs. A prominent example for genes that are highly expressed in NK cells but not in ILCs is serine-threonine-tyrosine kinase 1 (Styk1 encoded by Styk1). We found Styk1 to be specifically expressed in lymphocytes positive for Killer cell lectin-like receptor subfamily B, member 1, also known as CD161 or NK1.1, i.e. in NK cell, αβ iNKT, and γδ NKT cell lineages. To investigate the role of Styk1 in the development and function of NK1.1+ innate T-cell subsets, we generated and analyzed a novel Styk1null mutant mouse line. Furthermore, we validated Styk1 expression in γδ NKT cells and in thymic, but not in peripheral invariant αβ iNKT cells through ex vivo analysis of a concomitantly generated transgenic Styk1 reporter mouse line. Despite the very specific expression of Styk1 in NK cells, γδ NKT cells, and thymic αβ iNKT, its absence did not alter homeostasis and function of these lineages. Thus, Styk1 expression is specific for NK cells and selected NK-like innate T-cell subsets, but dispensable for their development and function.

Shashank Rai✳︎, Maryam Arasteh✳︎, Matthew Jefferson✳︎, Timothy Pearson, Yingxue Wang, Weijiao Zhang, Bertalan Bicsak, Devina Divekar, Penny P Powell, Ronald Naumann, Naiara Beraza, Simon R Carding, Oliver Florey, Ulrike Mayer#, Thomas Wileman#
The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3-associated phagocytosis.
Autophagy, 15(4) 599-612 (2019)
Open Access DOI
Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP-/- mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting.

Maarten E Witte, Adrian-Minh Schumacher, Christoph F Mahler, Joerg Bewersdorf, Jonas Lehmitz, Alexander Scheiter, Paula Sánchez, Philip R Williams, Oliver Griesbeck, Ronald Naumann, Thomas Misgeld#, Martin Kerschensteiner#
Calcium Influx through Plasma-Membrane Nanoruptures Drives Axon Degeneration in a Model of Multiple Sclerosis.
Neuron, 101(4) 615-624 (2019)
Open Access DOI
Axon loss determines persistent disability in multiple sclerosis patients. Here, we use in vivo calcium imaging in a multiple sclerosis model to show that cytoplasmic calcium levels determine the choice between axon loss and survival. We rule out the endoplasmic reticulum, glutamate excitotoxicity, and the reversal of the sodium-calcium exchanger as sources of intra-axonal calcium accumulation and instead identify nanoscale ruptures of the axonal plasma membrane as the critical path of calcium entry.

Elisabeth Zinser, Ronald Naumann, Andreas B Wild, Julia Michalski, Andrea Deinzer, Lena Stich, Christine Kuhnt, Alexander Steinkasserer, Ilka Knippertz
Endogenous Expression of the Human CD83 Attenuates EAE Symptoms in Humanized Transgenic Mice and Increases the Activity of Regulatory T Cells.
Front Immunol, 10 Art. No. 1442 (2019)
Open Access DOI
The CD83 is a type I membrane protein and part of the immunoglobulin superfamily of receptors. CD83 is involved in the regulation of antigen presentation and dendritic cell dependent allogeneic T cell proliferation. A soluble form of CD83 inhibits dendritic cell maturation and function. Furthermore, CD83 is expressed on activated B cells, T cells, and in particular on regulatory T cells. Previous studies on murine CD83 demonstrated this molecule to be involved in several immune-regulatory processes, comprising that CD83 plays a key role in the development und function of different immune cells. In order to get further insights into the function of the human CD83 and to provide preclinical tools to guide the function of CD83/sCD83 for therapeutic purposes we generated Bacterial Artificial Chromosomes (BAC) transgenic mice. BACs are excellent tools for manipulating large DNA fragments and are utilized to engineer transgenic mice by pronuclear injection. Two different founders of BAC transgenic mice expressing human CD83 (BAC-hCD83tg mice) were generated and were examined for the hCD83 expression on different immune cells as well as both the in vitro and in vivo role of human CD83 (hCD83) in health and disease. Here, we found the hCD83 molecule to be present on activated DCs, B cells and subtypes of CD4+ T cells. CD8+ T cells, on the other hand, showed almost no hCD83 expression. To address the function of hCD83, we performed in vitro mixed lymphocyte reactions (MLR) as well as suppression assays and we used the in vivo model of experimental autoimmune encephalomyelitis (EAE) comparing wild-type and hCD83-BAC mice. Results herein showed a clearly diminished capacity of hCD83-BAC-derived T cells to proliferate accompanied by an enhanced activation and suppressive activity of hCD83-BAC-derived Tregs. Furthermore, hCD83-BAC mice were found to recover faster from EAE-associated symptoms than wild-type mice, encouraging the relevance also of the hCD83 as a key molecule for the regulatory phenotype of Tregs in vitro and in vivo.

Gulce Itir Percin, Jiri Eitler, Andrea Kranz, Jun Fu, Jeffrey W Pollard, Ronald Naumann, Claudia Waskow
CSF1R regulates the dendritic cell pool size in adult mice via embryo-derived tissue-resident macrophages.
Nat Commun, 9(1) Art. No. 5279 (2018)
Open Access DOI
Regulatory mechanisms controlling the pool size of spleen dendritic cells (DC) remain incompletely understood. DCs are continuously replenished from hematopoietic stem cells, and FLT3-mediated signals cell-intrinsically regulate homeostatic expansion of spleen DCs. Here we show that combining FLT3 and CSF1R-deficiencies results in specific and complete abrogation of spleen DCs in vivo. Spatiotemporally controlled CSF1R depletion reveals a cell-extrinsic and non-hematopoietic mechanism for DC pool size regulation. Lack of CSF1R-mediated signals impedes the differentiation of spleen macrophages of embryonic origin, and the resulted macrophage depletion during development or in adult mice results in loss of DCs. Moreover, embryo-derived macrophages are important for the physiologic regeneration of DC after activation-induced depletion in situ. In summary, we show that the differentiation of DC and their regeneration relies on ontogenetically distinct spleen macrophages, thereby providing a novel regulatory principle that may also be important for the differentiation of other hematopoietic cell types.

Inga Sandrock, Annika Reinhardt, Sarina Ravens, Christoph Binz, Anneke Wilharm, Joana Martins, Linda Oberdörfer, Likai Tan, Stefan Lienenklaus, Baojun Zhang, Ronald Naumann, Yuan Zhuang, Andreas Krueger, Reinhold Förster, Immo Prinz
Genetic models reveal origin, persistence and non-redundant functions of IL-17-producing γδ T cells.
J Exp Med, 215(12) 3006-3018 (2018)
γδ T cells are highly conserved in jawed vertebrates, suggesting an essential role in the immune system. However, γδ T cell-deficient Tcrd-/- mice display surprisingly mild phenotypes. We hypothesized that the lack of γδ T cells in constitutive Tcrd-/- mice is functionally compensated by other lymphocytes taking over genuine γδ T cell functions. To test this, we generated a knock-in model for diphtheria toxin-mediated conditional γδ T cell depletion. In contrast to IFN-γ-producing γδ T cells, IL-17-producing γδ T cells (Tγδ17 cells) recovered inefficiently after depletion, and their niches were filled by expanding Th17 cells and ILC3s. Complementary genetic fate mapping further demonstrated that Tγδ17 cells are long-lived and persisting lymphocytes. Investigating the function of γδ T cells, conditional depletion but not constitutive deficiency protected from imiquimod-induced psoriasis. Together, we clarify that fetal thymus-derived Tγδ17 cells are nonredundant local effector cells in IL-17-driven skin pathology.

Madina Karimova, Oliver Baker, Aylin Camgoz, Ronald Naumann, Frank Buchholz#, Konstantinos Anastassiadis#
A single reporter mouse line for Vika, Flp, Dre, and Cre-recombination.
Sci Rep, 8(1) Art. No. 14453 (2018)
Open Access DOI
Site-specific recombinases (SSR) are utilized as important genome engineering tools to precisely modify the genome of mice and other model organisms. Reporter mice that mark cells that at any given time had expressed the enzyme are frequently used for lineage tracing and to characterize newly generated mice expressing a recombinase from a chosen promoter. With increasing sophistication of genome alteration strategies, the demand for novel SSR systems that efficiently and specifically recombine their targets is rising and several SSR-systems are now used in combination to address complex biological questions in vivo. Generation of reporter mice for each one of these recombinases is cumbersome and increases the number of mouse lines that need to be maintained in animal facilities. Here we present a multi-reporter mouse line for loci-of-recombination (X) (MuX) that streamlines the characterization of mice expressing prominent recombinases. MuX mice constitutively express nuclear green fluorescent protein after recombination by either Cre, Flp, Dre or Vika recombinase, rationalizing the number of animal lines that need to be maintained. We also pioneer the use of the Vika/vox system in mice, illustrating its high efficacy and specificity, thereby facilitating future designs of sophisticated recombinase-based in vivo genome engineering strategies.

Peter Dobrowolski, Melina Fischer, Ronald Naumann
Novel insights into the genetic background of genetically modified mice.
Transgenic Res, 27(3) 265-275 (2018)
Open Access DOI
Unclear or misclassified genetic background of laboratory rodents or a lack of strain awareness causes a number of difficulties in performing or reproducing scientific experiments. Until now, genetic differentiation between strains and substrains of inbred mice has been a challenge. We have developed a screening method for analyzing inbred strains regarding their genetic background. It is based on 240 highly informative short tandem repeat (STR) markers covering the 19 autosomes as well as X and Y chromosomes. Combination of analysis results for presence of known C57BL/6 substrain-specific mutations together with autosomal STR markers and the Y-chromosomal STR-haplotype provides a comprehensive snapshot of the genetic background of mice. In this study, the genetic background of 72 mouse lines obtained from 18 scientific institutions in Germany and Austria was determined. By analyzing only 3 individuals per genetically modified line it was possible to detect mixed genetic backgrounds frequently. In several lines presence of a mispairing Y chromosome was detected. At least every second genetically modified line displayed a mixed genetic background which could lead to unexpected and non-reproducible results, irrespective of the investigated gene of interest.

Julia Brenmoehl, Christina Walz, Marion Spitschak, Elisa Wirthgen, Michael Walz, Martina Langhammer, Armin Tuchscherer, Ronald Naumann, Andreas Hoeflich
Partial phenotype conversion and differential trait response to conditions of husbandry in mice.
J Comp Physiol B , 188(3) 527-539 (2018)
Open Access DOI
Functional genome analysis usually is performed on the level of genotype-phenotype interaction. However, phenotypes also depend on the relations between genomes and environment. In our experimental system, we observed differential response to environmental factors defined by different conditions of husbandry in a semi-barrier unit or in a SPF (specific pathogen free) barrier unit, which resulted in partial reversal of phenotypes previously observed under semi-barrier conditions. To provide an update of basic phenotypes in unselected and randomly mated controls (DUC) and long-term selected DUhTP (Dummerstorf high treadmill performance) mice in the SPF facility, we compared growth parameters, reproductive performance, the accretion of muscle and fat mass, physical activity, and running performance as well as food intake in all experimental groups. For selected parameters, the comparative analysis spans more than 30 generations. In DUC mice, under SPF conditions a more than threefold (P < 0.0001) higher subcutaneous fat mass, higher muscle mass by about 25% (P < 0.0001), but lower epididymal fat mass in DUhTP mice by about 20% (P < 0.0001) were observed. In SPF husbandry, body weight increased to a stronger extent in adult DUC mice (≈ 20%; P < 0.0001) than in DUhTP mice (≈ 8%; P = 0.001). The concentrations of IGF-1 and IGFBPs in the serum as well as the liver weights were similar in all experimental groups, indicating growth effects independent of the somatotropic axis. Under SPF conditions the litter size at birth increased in DUC mice (P < 0.001) but not in DUhTP mice. The differential effect of husbandry on body weights at day 21 and concentrations of triglycerides in the serum of our model were due to the different diets used in the semi-barrier and in the SPF facility. Our results demonstrate differential trait response to environmental factors resulting in partial phenotype conversion in our experimental system. The existence of conditional phenotypes as a result of genotype-environment interactions points to the importance of environmental factors in functional genome analysis.

Alina Neunkirchner, Bernhard Kratzer, Cordula Köhler, Ursula Smole, Lukas F Mager, Klaus G Schmetterer, Doris Trapin, Victoria Leb-Reichl, Edward Rosloniec, Ronald Naumann, Lukas Kenner, Beatrice Jahn-Schmid, Barbara Bohle, Rudolf Valenta, Winfried F Pickl
Genetic restriction of antigen-presentation dictates allergic sensitization and disease in humanized mice.
EBioMedicine, 31 66-78 (2018)
Open Access DOI
Immunoglobulin(Ig)E-associated allergies result from misguided immune responses against innocuous antigens. CD4+ T lymphocytes are critical for initiating and perpetuating that process, yet the crucial factors determining whether an individual becomes sensitized towards a given allergen remain largely unknown.

Stefano Lazzarano, Marek Kučka, João P L Castro, Ronald Naumann, Paloma Medina, Michael N C Fletcher, Rebecka Wombacher, Joost Gribnau, Tino Hochepied, Marc Van Montagu, Claude Libert, Yingguang Frank Chan
Genetic mapping of species differences via in vitro crosses in mouse embryonic stem cells.
Proc Natl Acad Sci U.S.A., 115(14) 3680-3685 (2018)
Open Access DOI
Discovering the genetic changes underlying species differences is a central goal in evolutionary genetics. However, hybrid crosses between species in mammals often suffer from hybrid sterility, greatly complicating genetic mapping of trait variation across species. Here, we describe a simple, robust, and transgene-free technique to generate "in vitro crosses" in hybrid mouse embryonic stem (ES) cells by inducing random mitotic cross-overs with the drug ML216, which inhibits the DNA helicase Bloom syndrome (BLM). Starting with an interspecific F1 hybrid ES cell line between theMus musculuslaboratory mouse andMus spretus(∼1.5 million years of divergence), we mapped the genetic basis of drug resistance to the antimetabolite tioguanine to a single region containing hypoxanthine-guanine phosphoribosyltransferase (Hprt) in as few as 21 d through "flow mapping" by coupling in vitro crosses with fluorescence-activated cell sorting (FACS). We also show how our platform can enable direct study of developmental variation by rederiving embryos with contribution from the recombinant ES cell lines. We demonstrate how in vitro crosses can overcome major bottlenecks in mouse complex trait genetics and address fundamental questions in evolutionary biology that are otherwise intractable through traditional breeding due to high cost, small litter sizes, and/or hybrid sterility. In doing so, we describe an experimental platform toward studying evolutionary systems biology in mouse and potentially in human and other mammals, including cross-species hybrids.

Katharina S Schneider, Christina J Groß, Roland F Dreier, Benedikt S Saller, Ritu Mishra, Oliver Gorka, Rosalie Heilig, Etienne Meunier, Mathias S Dick, Tamara Ćiković, Jan Sodenkamp, Guillaume Médard, Ronald Naumann, Jurgen Ruland, Bernhard Kuster, Petr Broz, Olaf Groß
The Inflammasome Drives GSDMD-Independent Secondary Pyroptosis and IL-1 Release in the Absence of Caspase-1 Protease Activity.
Cell Rep, 21(13) 3846-3859 (2017)
Open Access DOI
Inflammasomes activate the protease caspase-1, which cleaves interleukin-1β and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1C284A, we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1C284A, we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis.

Sara Carvalhal, Michelle Stevense, Katrin Koehler, Ronald Naumann, Angela Huebner, Rolf Jessberger, Eric R Griffis
ALADIN is required for the production of fertile mouse oocytes.
Mol Biol Cell, 28(19) 2470-2478 (2017)
Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell's center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.

Sabine Stopp, Marco Gründl, Marc Fackler, Jonas Malkmus, Marina Leone, Ronald Naumann, Stefan Frantz, Elmar Wolf, Björn von Eyss, Felix B Engel, Stefan Gaubatz
Deletion of Gas2l3 in mice leads to specific defects in cardiomyocyte cytokinesis during development.
Proc Natl Acad Sci U.S.A., 114(30) 8029-8034 (2017)
GAS2L3 is a recently identified cytoskeleton-associated protein that interacts with actin filaments and tubulin. The in vivo function of GAS2L3 in mammals remains unknown. Here, we show that mice deficient in GAS2L3 die shortly after birth because of heart failure. Mammalian cardiomyocytes lose the ability to proliferate shortly after birth, and further increase in cardiac mass is achieved by hypertrophy. The proliferation arrest of cardiomyocytes is accompanied by binucleation through incomplete cytokinesis. We observed that GAS2L3 deficiency leads to inhibition of cardiomyocyte proliferation and to cardiomyocyte hypertrophy during embryonic development. Cardiomyocyte-specific deletion of GAS2L3 confirmed that the phenotype results from the loss of GAS2L3 in cardiomyocytes. Cardiomyocytes from Gas2l3-deficient mice exhibit increased expression of a p53-transcriptional program including the cell cycle inhibitor p21. Furthermore, loss of GAS2L3 results in premature binucleation of cardiomyocytes accompanied by unresolved midbody structures. Together these results suggest that GAS2L3 plays a specific role in cardiomyocyte cytokinesis and proliferation during heart development.

Jussi Helppi, Ronald Naumann, Marianne Asikainen, Jaakko Mononen, Oliver Zierau
Novel Bedding Material Results in Poor Pregnancy Rate with CD-1 Female Mice Used as Fosters for Producing Transgenic Mice
Scand J Lab Anim Sci, 43(3) 1-5 (2017)
Open Access PDF
The impact of a novel bedding material (cotton cloth) on the reproductive performance (pregnancy rate and production of offspring) was studied in foster females used for producing transgenic mice. Embryos injected with DNA were transferred to pseudo-pregnant foster females housed under standard conditions (aspen bedding and nesting material). After embryo transfer, mice were divided between the experimental group (AGREBE cotton cloth) and control group (aspen bedding and nesting material). Pregnant mice were observed at day 15 after the transfer and the number of offspring was recorded on post-natal days 3 and 21. Altogether 116 foster mice were used as embryo recipients. Significantly more pregnancies were observed in the control group versus the experimental group: 43% and 19% of foster mice, respectively. Informal interviews with animal caretakers revealed a general dislike towards the cotton cloth (dirtier cages, mice often found on the plastic cage surface, difficult husbandry routines). The cotton cloth showed major signs of wear and tear after only a few weeks of usage. In conclusion, this study with female mice demonstrated that a cotton cloth cannot be recommended as a sole replacement for bedding and nesting material.

Philipp von Hundelshausen, Stijn M Agten, Veit Eckardt, Xavier Blanchet, Martin M Schmitt, Hans Ippel, Carlos Neideck, Kiril Bidzhekov, Julian Leberzammer, Kanin Wichapong, Alexander Faussner, Maik Drechsler, Jochen Grommes, Johanna P van Geffen, He Li, Almudena Ortega-Gomez, Remco T A Megens, Ronald Naumann, Ingrid Dijkgraaf, Gerry A F Nicolaes, Yvonne Döring, Oliver Soehnlein, Esther Lutgens, Johan W M Heemskerk, Rory R Koenen, Kevin H Mayo, Tilman M Hackeng, Christian Weber
Chemokine interactome mapping enables tailored intervention in acute and chronic inflammation.
Sci Transl Med, 9(384) Art. No. eaah6650 (2017)
Chemokines orchestrate leukocyte trafficking and function in health and disease. Heterophilic interactions between chemokines in a given microenvironment may amplify, inhibit, or modulate their activity; however, a systematic evaluation of the chemokine interactome has not been performed. We used immunoligand blotting and surface plasmon resonance to obtain a comprehensive map of chemokine-chemokine interactions and to confirm their specificity. Structure-function analyses revealed that chemokine activity can be enhanced by CC-type heterodimers but inhibited by CXC-type heterodimers. Functional synergism was achieved through receptor heteromerization induced by CCL5-CCL17 or receptor retention at the cell surface via auxiliary proteoglycan binding of CCL5-CXCL4. In contrast, inhibitory activity relied on conformational changes (in CXCL12), affecting receptor signaling. Obligate CC-type heterodimers showed high efficacy and potency and drove acute lung injury and atherosclerosis, processes abrogated by specific CCL5-derived peptide inhibitors or knock-in of an interaction-deficient CXCL4 variant. Atheroprotective effects of CCL17 deficiency were phenocopied by a CCL5-derived peptide disrupting CCL5-CCL17 heterodimers, whereas a CCL5 α-helix peptide mimicked inhibitory effects on CXCL12-driven platelet aggregation. Thus, formation of specific chemokine heterodimers differentially dictates functional activity and can be exploited for therapeutic targeting.

Tatiana Pushkarsky, Evgeny Shilov, Natalya Kruglova, Ronald Naumann, Beda Brichacek, Lucas Jennelle, Dmitri Sviridov, Andrey Kruglov, Sergei Nedospasov, Michael I Bukrinsky
Short Communication: Accumulation of Neutral Lipids in Liver and Aorta of Nef-Transgenic Mice.
AIDS Res Hum Retroviruses, 33(1) 57-60 (2017)
HIV-infected individuals are at high risk of developing atherosclerosis and cardiovascular disease, in part, due to HIV-induced impairment of cholesterol metabolism. In vitro studies demonstrated that HIV-1 protein Nef inhibits activity of ABCA1, the main cellular cholesterol transporter, leading to cholesterol accumulation in macrophages and conversion of these cells into foam cells, characteristic for atherosclerosis. However, the mechanisms of Nef-mediated effects on cholesterol metabolism in vivo are not well characterized. In this study, we generated Nef-transgenic mice and evaluated the accumulation of neutral lipids in liver and aorta of these animals. Nef expression was low in all transgenic mice, with some mice carrying the Nef transgene, but not expressing the Nef RNA. Using Oil Red O staining, we demonstrated increased levels of neutral lipids in liver and aorta of mice expressing Nef relative to transgenic animals, with no detectable Nef expression or control wild-type mice. These results provide direct evidence that Nef promotes cholesterol deposition in tissues.

Katrin Peschke, Martin Achleitner, Kathrin Frenzel, Alexander Gerbaulet, Servi Remzi Ada, Nicolas Zeller, Stefan Lienenklaus, Mathias Lesche, Claire Poulet, Ronald Naumann, Andreas Dahl, Ursula Ravens, Claudia Günther, Werner Müller, Klaus-Peter Knobeloch, Marco Prinz, Axel Roers, Rayk Behrendt
Loss of Trex1 in Dendritic Cells Is Sufficient To Trigger Systemic Autoimmunity.
J Immunol, 197(6) 2157-2166 (2016)
Defects of the intracellular enzyme 3' repair exonuclease 1 (Trex1) cause the rare autoimmune condition Aicardi-Goutières syndrome and are associated with systemic lupus erythematosus. Trex1(-/-) mice develop type I IFN-driven autoimmunity, resulting from activation of the cytoplasmic DNA sensor cyclic GMP-AMP synthase by a nucleic acid substrate of Trex1 that remains unknown. To identify cell types responsible for initiation of autoimmunity, we generated conditional Trex1 knockout mice. Loss of Trex1 in dendritic cells was sufficient to cause IFN release and autoimmunity, whereas Trex1-deficient keratinocytes and microglia produced IFN but did not induce inflammation. In contrast, B cells, cardiomyocytes, neurons, and astrocytes did not show any detectable response to the inactivation of Trex1. Thus, individual cell types differentially respond to the loss of Trex1, and Trex1 expression in dendritic cells is essential to prevent breakdown of self-tolerance ensuing from aberrant detection of endogenous DNA.

Jussi Helppi, Dora Schreier, Ronald Naumann, Oliver Zierau
Mouse reproductive fitness is maintained up to an ambient temperature of 28℃ when housed in individually-ventilated cages.
Lab Anim, 50(4) 254-263 (2016)
Production of genetically-modified mice is strongly dependent on environmental conditions. Mice are commonly housed at 22℃, which is significantly lower than their thermoneutral zone. But, when given a choice, mice often seem to prefer higher ambient temperatures. In the current study we investigated the effect of higher ambient temperature on the production of transgenic mice, with emphasis on embryo and sperm yield and quality. Mice (C57BL/6JOlaHsd) were housed under four different ambient temperatures (22, 25, 28 and 30℃). Female mice were superovulated, and mated with males. As indicators for reproductive fitness, the success of the mating was observed, including embryo yield and quality, as well as sperm count, motility and progressivity. Female mice were found to produce high amounts of high quality embryos from 22 to 28℃. Sperm count dropped continuously from 22 to 30℃, but sperm motility and progressivity remained high from 22 to 28℃. We conclude that mice can be housed at significantly higher temperatures than is commonly recommended without compromising embryo production and quality, or sperm quality. These results could lead to fundamental changes in how mouse facilities are built and operated - especially in warmer climates whereby energy consumption and therefore costs could be significantly reduced.

Qian Li, Song Guo, Xi Jiang, Jaroslaw Bryk, Ronald Naumann, Wolfgang Enard, Masaru Tomita, Masahiro Sugimoto, Philipp Khaitovich, Svante Pääbo
Mice carrying a human GLUD2 gene recapitulate aspects of human transcriptome and metabolome development.
Proc Natl Acad Sci U.S.A., 113(19) 5358-5363 (2016)
Open Access PDF DOI
Whereas all mammals have one glutamate dehydrogenase gene (GLUD1), humans and apes carry an additional gene (GLUD2), which encodes an enzyme with distinct biochemical properties. We inserted a bacterial artificial chromosome containing the human GLUD2 gene into mice and analyzed the resulting changes in the transcriptome and metabolome during postnatal brain development. Effects were most pronounced early postnatally, and predominantly genes involved in neuronal development were affected. Remarkably, the effects in the transgenic mice partially parallel the transcriptome and metabolome differences seen between humans and macaques analyzed. Notably, the introduction of GLUD2 did not affect glutamate levels in mice, consistent with observations in the primates. Instead, the metabolic effects of GLUD2 center on the tricarboxylic acid cycle, suggesting that GLUD2 affects carbon flux during early brain development, possibly supporting lipid biosynthesis.

Gesa I Albert, Christoph Schell, Karin M Kirschner, Sebastian Schäfer, Ronald Naumann, Alexandra Müller, Oliver Kretz, Benno Kuropka, Mathias Girbig, Norbert Hübner, Eberhard Krause, Holger Scholz, Tobias B Huber, Klaus-Peter Knobeloch, Christian Freund
The GYF domain protein CD2BP2 is critical for embryogenesis and podocyte function.
J Mol Cell Biol, 7(5) 402-414 (2015)
Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome. The adaptor protein CD2BP2, originally identified as a binding partner of the adhesion molecule CD2, is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine (GYF) domain to co-localize with spliceosomal proteins. So far, its function in vertebrates is unknown. Using conditional gene targeting in mice, we show that CD2BP2 is crucial for embryogenesis, leading to growth retardation, defects in vascularization, and premature death at embryonic day 10.5 when absent. Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins. At the molecular level, we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2. Given the strong expression of CD2BP2 in podocytes of the kidney, we use selective depletion of CD2BP2, in combination with next-generation sequencing, to monitor changes in exon usage of genes critical for podocyte functions, including VEGF and actin regulators. CD2BP2-depleted podocytes display foot process effacement, and cause proteinuria and ultimately lethal kidney failure in mice. Collectively, our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity.

Marta Florio, Mareike Albert, Elena Taverna, Takashi Namba, Holger Brandl, Eric Lewitus, Christiane Haffner, Alex Sykes, Fong Kuan Wong, Jula Peters, E. Guhr, Sylvia Klemroth, Kay Prüfer, Janet Kelso, Ronald Naumann, Ina Nüsslein, Andreas Dahl, Robert Lachmann, Svante Pääbo, Wieland B. Huttner
Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion.
Science, 347(6229) 1465-1470 (2015)
Evolutionary expansion of the human neocortex reflects increased amplification of basal progenitors in the subventricular zone, producing more neurons during fetal corticogenesis. In this work, we analyze the transcriptomes of distinct progenitor subpopulations isolated by a cell polarity-based approach from developing mouse and human neocortex. We identify 56 genes preferentially expressed in human apical and basal radial glia that lack mouse orthologs. Among these, ARHGAP11B has the highest degree of radial glia-specific expression. ARHGAP11B arose from partial duplication of ARHGAP11A (which encodes a Rho guanosine triphosphatase-activating protein) on the human lineage after separation from the chimpanzee lineage. Expression of ARHGAP11B in embryonic mouse neocortex promotes basal progenitor generation and self-renewal and can increase cortical plate area and induce gyrification. Hence, ARHGAP11B may have contributed to evolutionary expansion of human neocortex.

Lars Ketscher, Ronny Hannß, David J Morales, Anja Basters, Susana Guerra, Tobias Goldmann, Annika Hausmann, Marco Prinz, Ronald Naumann, Andrew Pekosz, Olaf Utermöhlen, Deborah J Lenschow, Klaus-Peter Knobeloch
Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance.
Proc Natl Acad Sci U.S.A., 112(5) 1577-1582 (2015)
Protein modification by the ubiquitin-like protein ISG15 is an interferon (IFN) effector system, which plays a major role in antiviral defense. ISG15 modification is counteracted by the isopeptidase USP18, a major negative regulator of IFN signaling, which was also shown to exert its regulatory function in an isopeptidase-independent manner. To dissect enzymatic and nonenzymatic functions of USP18 in vivo, we generated knock-in mice (USP18(C61A/C61A)) expressing enzymatically inactive USP18. USP18(C61A/C61A) mice displayed increased levels of ISG15 conjugates, validating that USP18 is a major ISG15 isopeptidase in vivo. Unlike USP18(-/-) mice, USP18(C61A/C61A) animals did not exhibit morphological abnormalities, fatal IFN hypersensitivity, or increased lethality, clearly showing that major USP18 functions are unrelated to its protease activity. Strikingly, elevated ISGylation in USP18(C61A/C61A) mice was accompanied by increased viral resistance against vaccinia virus and influenza B virus infections. Enhanced resistance upon influenza B infection in USP18(C61A/C61A) mice was completely reversed in USP18(C61A/C61A) mice, which additionally lack ISG15, providing evidence that the observed reduction in viral titers is ISG15 dependent. These results suggest that increasing ISGylation by specific inhibition of USP18 protease activity could constitute a promising antiviral strategy with only a minimal risk of severe adverse effects.

Catherine Diamante Sorbara, Nicolai Wagner, Anne Ladwig, Ivana Nikić, Doron Merkler, Tatjana Kleele, Petar Marinković, Ronald Naumann, Leanne Godinho, Florence M Bareyre, Derron Bishop, Thomas Misgeld, Martin Kerschensteiner
Pervasive Axonal Transport Deficits in Multiple Sclerosis Models.
Neuron, 84(6) 1183-1190 (2014)
Impaired axonal transport can contribute to axon degeneration and has been described in many neurodegenerative diseases. Multiple sclerosis (MS) is a common neuroinflammatory disease, which is characterized by progressive axon degeneration-whether, when, and how axonal transport is affected in this condition is unknown. Here we used in vivo two-photon imaging to directly assay transport of organelles and the stability of microtubule tracks in individual spinal axons in mouse models of MS. We found widespread transport deficits, which preceded structural alterations of axons, cargos, or microtubules and could be reversed by acute anti-inflammatory interventions or redox scavenging. Our study shows that acute neuroinflammation induces a pervasive state of reversible axonal dysfunction, which coincides with acute disease symptoms. Moreover, perpetuated transport dysfunction, as we found in a model of progressive MS, led to reduced distal organelle supply and could thus contribute to axonal dystrophy in advanced stages of the disease.

Michael O Breckwoldt, Franz M J Pfister, Peter M Bradley, Petar Marinković, Philip R Williams, Monika S Brill, Barbara Plomer, Anja Schmalz, Daret K St Clair, Ronald Naumann, Oliver Griesbeck, Markus Schwarzländer, Leanne Godinho, Florence M Bareyre, Tobias P Dick, Martin Kerschensteiner, Thomas Misgeld
Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo.
Nat Med, 20(5) 555-560 (2014)
Mitochondrial redox signals have a central role in neuronal physiology and disease. Here we describe a new optical approach to measure fast redox signals with single-organelle resolution in living mice that express genetically encoded redox biosensors in their neuronal mitochondria. Moreover, we demonstrate how parallel measurements with several biosensors can integrate these redox signals into a comprehensive characterization of mitochondrial function. This approach revealed that axonal mitochondria undergo spontaneous 'contractions' that are accompanied by reversible redox changes. These contractions are amplified by neuronal activity and acute or chronic neuronal insults. Multiparametric imaging reveals that contractions constitute respiratory chain-dependent episodes of depolarization coinciding with matrix alkalinization, followed by uncoupling. In contrast, permanent mitochondrial damage after spinal cord injury depends on calcium influx and mitochondrial permeability transition. Thus, our approach allows us to identify heterogeneity among physiological and pathological redox signals, correlate such signals to functional and structural organelle dynamics and dissect the underlying mechanisms.

Hannah Rabenstein, Anna Behrendt, Joachim W Ellwart, Ronald Naumann, Marion Horsch, Johannes Beckers, Reinhard Obst
Differential kinetics of antigen dependency of CD4+ and CD8+ T cells.
J Immunol, 192(8) 3507-3517 (2014)
Ag recognition via the TCR is necessary for the expansion of specific T cells that then contribute to adaptive immunity as effector and memory cells. Because CD4+ and CD8+ T cells differ in terms of their priming APCs and MHC ligands we compared their requirements of Ag persistence during their expansion phase side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal mouse T cells were thus analyzed after transient and continuous TCR signals. Following equally strong stimulation, CD4+ T cell proliferation depended on prolonged Ag presence, whereas CD8+ T cells were able to divide and differentiate into effector cells despite discontinued Ag presentation. CD4+ T cell proliferation was neither affected by Th lineage or memory differentiation nor blocked by coinhibitory signals or missing inflammatory stimuli. Continued CD8+ T cell proliferation was truly independent of self-peptide/MHC-derived signals. The subset divergence was also illustrated by surprisingly broad transcriptional differences supporting a stronger propensity of CD8+ T cells to programmed expansion. These T cell data indicate an intrinsic difference between CD4+ and CD8+ T cells regarding the processing of TCR signals for proliferation. We also found that the presentation of a MHC class II-restricted peptide is more efficiently prolonged by dendritic cell activation in vivo than a class I bound one. In summary, our data demonstrate that CD4+ T cells require continuous stimulation for clonal expansion, whereas CD8+ T cells can divide following a much shorter TCR signal.

Derek Spieler, Maria Kaffe, Franziska Knauf, José Bessa, Juan J Tena, Florian Giesert, Barbara Schormair, Erik Tilch, Hyun-Ok Kate Lee, Marion Horsch, Darina Czamara, Nazanin Karbalai, Christine von Toerne, Melanie Waldenberger, Christian Gieger, Peter Lichtner, Melina Claussnitzer, Ronald Naumann, Bertram Müller-Myhsok, Miguel Torres, Lillian Garrett, Jan Rozman, Martin Klingenspor, Valérie Gailus-Durner, Helmut Fuchs, Martin Hrabe de Angelis, Johannes Beckers, Sabine M Hölter, Thomas Meitinger, Stefanie M Hauck, Helmut Laumen, Wolfgang Wurst, Fernando Casares, Jose Luis Gómez-Skarmeta, Juliane Winkelmann
Restless Legs Syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon.
Genome Res, 24(4) 592-603 (2014)
Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS.

Konstantin Neumann, Mercedes Castiñeiras-Vilariño, Ulrike Höckendorf, Nicole Hannesschläger, Simone Lemeer, Danny Kupka, Svenia Meyermann, Maciej Lech, Hans-Joachim Anders, Bernhard Kuster, Dirk H Busch, Andreas Gewies, Ronald Naumann, Olaf Groß, Jurgen Ruland
Clec12a Is an Inhibitory Receptor for Uric Acid Crystals that Regulates Inflammation in Response to Cell Death.
Immunity, 40(3) 389-399 (2014)
Recognition of cell death by the innate immune system triggers inflammatory responses. However, how these reactions are regulated is not well understood. Here, we identify the inhibitory C-type lectin receptor Clec12a as a specific receptor for dead cells. Both human and mouse Clec12a could physically sense uric acid crystals (monosodium urate, MSU), which are key danger signals for cell-death-induced immunity. Clec12a inhibited inflammatory responses to MSU in vitro, and Clec12a-deficient mice exhibited hyperinflammatory responses after being challenged with MSU or necrotic cells and after radiation-induced thymocyte killing in vivo. Thus, we identified a negative regulatory MSU receptor that controls noninfectious inflammation in response to cell death that has implications for autoimmunity and inflammatory disease.

Andreas Begitt, Mathias Droescher, Thomas Meyer, Christoph D Schmid, Michelle Baker, Filipa Antunes, Markus R Owen, Ronald Naumann, Thomas Decker, Uwe Vinkemeier
STAT1-cooperative DNA binding distinguishes type 1 from type 2 interferon signaling.
Nat Immunol, 15(2) 168-176 (2014)
STAT1 is an indispensable component of a heterotrimer (ISGF3) and a STAT1 homodimer (GAF) that function as transcription regulators in type 1 and type 2 interferon signaling, respectively. To investigate the importance of STAT1-cooperative DNA binding, we generated gene-targeted mice expressing cooperativity-deficient STAT1 with alanine substituted for Phe77. Neither ISGF3 nor GAF bound DNA cooperatively in the STAT1F77A mouse strain, but type 1 and type 2 interferon responses were affected differently. Type 2 interferon-mediated transcription and antibacterial immunity essentially disappeared owing to defective promoter recruitment of GAF. In contrast, STAT1 recruitment to ISGF3 binding sites and type 1 interferon-dependent responses, including antiviral protection, remained intact. We conclude that STAT1 cooperativity is essential for its biological activity and underlies the cellular responses to type 2, but not type 1 interferon.

Tatjana Kleele, Petar Marinković, Philip R Williams, Sina Stern, Emily E Weigand, Peter Engerer, Ronald Naumann, Jana Hartmann, Rosa M Karl, Frank Bradke, Derron Bishop, Jochen Herms, Arthur Konnerth, Martin Kerschensteiner, Leanne Godinho, Thomas Misgeld
An assay to image neuronal microtubule dynamics in mice.
Nat Commun, 5 Art. No. 4827 (2014)
Open Access DOI
Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.

Raymond Behrendt, Tina Schumann, Alexander Gerbaulet, Laura A Nguyen, Nadja Schubert, Dimitra Alexopoulou, Ursula Berka, Stefan Lienenklaus, Katrin Peschke, Kathrin Gibbert, Sabine Wittmann, Dirk Lindemann, Siegfried Weiss, Andreas Dahl, Ronald Naumann, Ulf Dittmer, Baek Kim, Werner Mueller, Thomas Gramberg, Axel Roers
Mouse SAMHD1 has antiretroviral activity and suppresses a spontaneous cell-intrinsic antiviral response.
Cell Rep, 4(4) 689-696 (2013)
Open Access DOI
Aicardi-Goutières syndrome (AGS), a hereditary autoimmune disease, clinically and biochemically overlaps with systemic lupus erythematosus (SLE) and, like SLE, is characterized by spontaneous type I interferon (IFN) production. The finding that defects of intracellular nucleases cause AGS led to the concept that intracellular accumulation of nucleic acids triggers inappropriate production of type I IFN and autoimmunity. AGS can also be caused by defects of SAMHD1, a 3' exonuclease and deoxynucleotide (dNTP) triphosphohydrolase. Human SAMHD1 is an HIV-1 restriction factor that hydrolyzes dNTPs and decreases their concentration below the levels required for retroviral reverse transcription. We show in gene-targeted mice that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells. Importantly, the absence of SAMHD1 triggered IFN-β-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types indicative of spontaneous IFN production. SAMHD1-deficient mice may be instrumental for elucidating the mechanisms that trigger pathogenic type I IFN responses in AGS and SLE.

Rashim Pal Singh, Kristin Franke, Joanna Kalucka, Soulafa Mamlouk, Antje Muschter, Agnieszka Gembarska, Tatyana Grinenko, Carsten Willam, Ronald Naumann, Konstantinos Anastassiadis, A Francis Stewart, Stefan Bornstein, Trian Chavakis, Georg Breier, Claudia Waskow, Ben Wielockx
HIF prolyl hydroxylase 2 (PHD2) is a critical regulator of hematopoietic stem cell maintenance during steady-state and stress.
Blood, 121(26) 5158-5166 (2013)
Hypoxia is a prominent feature in the maintenance of hematopoietic stem cell (HSC) quiescence and multipotency. Hypoxia-inducible factor (HIF) prolyl hydroxylase domain proteins (PHDs) serve as oxygen sensors and may therefore regulate this system. Here, we describe a mouse line with conditional loss of HIF prolyl hydroxylase 2 (PHD2) in very early hematopoietic precursors that results in self-renewal of multipotent progenitors under steady-state conditions in a HIF1α- and SMAD7-dependent manner. Competitive bone marrow (BM) transplantations show decreased peripheral and central chimerism of PHD2-deficient cells but not of the most primitive progenitors. Conversely, in whole BM transfer, PHD2-deficient HSCs replenish the entire hematopoietic system and display an enhanced self-renewal capacity reliant on HIF1α. Taken together, our results demonstrate that loss of PHD2 controls the maintenance of the HSC compartment under physiological conditions and causes the outcompetition of PHD2-deficient hematopoietic cells by their wild-type counterparts during stress while promoting the self-renewal of very early hematopoietic progenitors.

Jörg Schönheit, Christiane Kuhl, Marie Luise Gebhardt, Francisco Fernández Klett, Pia Riemke, Marina Scheller, Gang Huang, Ronald Naumann, Achim Leutz, Carol Stocking, Josef Priller, Miguel A Andrade-Navarro, Frank Rosenbauer
PU.1 level-directed chromatin structure remodeling at the Irf8 gene drives dendritic cell commitment.
Cell Rep, 3(5) 1617-1628 (2013)
Open Access DOI
Dendritic cells (DCs) are essential regulators of immune responses; however, transcriptional mechanisms that establish DC lineage commitment are poorly defined. Here, we report that the PU.1 transcription factor induces specific remodeling of the higher-order chromatin structure at the interferon regulatory factor 8 (Irf8) gene to initiate DC fate choice. An Irf8 reporter mouse enabled us to pinpoint an initial progenitor stage at which DCs separate from other myeloid lineages in the bone marrow. In the absence of Irf8, this progenitor undergoes DC-to-neutrophil reprogramming, indicating that DC commitment requires an active, Irf8-dependent escape from alternative myeloid lineage potential. Mechanistically, myeloid Irf8 expression depends on high PU.1 levels, resulting in local chromosomal looping and activation of a lineage- and developmental-stage-specific cis-enhancer. These data delineate PU.1 as a concentration-dependent rheostat of myeloid lineage selection by controlling long-distance contacts between regulatory elements and suggest that specific higher-order chromatin remodeling at the Irf8 gene determines DC differentiation.

Natalia Zietara, Marcin Łyszkiewicz, Katrin Witzlau, Ronald Naumann, Robert Hurwitz, Jörg Langemeier, Jens Bohne, Inga Sandrock, Matthias Ballmaier, Siegfried Weiss, Immo Prinz, Andreas Krueger
Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells.
Proc Natl Acad Sci U.S.A., 110(18) 7407-7412 (2013)
T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4(+)CD8(+) double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1(-/-) mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.

Tomislav Maricic, Viola Günther, Oleg Georgiev, Sabine Gehre, Marija Curlin, Christiane Schreiweis, Ronald Naumann, Hernán A Burbano, Matthias Meyer, Carles Lalueza-Fox, Marco de la Rasilla, Antonio Rosas, Srecko Gajovic, Janet Kelso, Wolfgang Enard, Walter Schaffner, Svante Pääbo
A Recent Evolutionary Change Affects a Regulatory Element in the Human FOXP2 Gene.
Mol Biol Evol, 30(4) 844-852 (2013)
The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

Mariya Rostovskaya, Ronald Naumann, Jun Fu, Mandy Obst, Doris Mueller, A Francis Stewart, Konstantinos Anastassiadis
Transposon mediated BAC transgenesis via pronuclear injection of mouse zygotes.
Genesis, 51(2) 135-141 (2013)
Pronuclear microinjection of bacterial artificial chromosomes (BACs) is the preferred way to generate transgenic mice because the transgene accurately recapitulates expression of the endogenous gene. However, the method is demanding and the integrity and copy number of the BAC transgene is difficult to control. Here, we describe a simpler pronuclear injection method that relies on transposition to introduce full-length BACs into the mouse genome. The bacterial backbone of a hPAX6-GFP reporter BAC was retrofitted with PiggyBac transposon inverted terminal repeats and co-injected with PiggyBac transposase mRNA. Both the frequency of transgenic founders as well as intact, full-length, single copy integrations were increased. Transposition was determined by a rapid PCR screen for a transpositional signature and confirmation by splinkerette sequencing to show that theBACs were integrated as a single copy either in one or two different genomic sites. BAC transposons displayed improved functional accuracy over random integrants as evaluated by expression of the hPAX6-GFP reporter in embryonic neural tube and absence of ectopic expression. This method involves less work to achieve increased frequencies of both transgenesis and single copy, full-length integrations. These advantages are not only relevant to rodents but also for transgenesis in all systems. genesis 51:135-141, 2013. © 2012 Wiley Periodicals, Inc.

Bjoern Hiller, Martin Achleitner, Silke Glage, Ronald Naumann, Raymond Behrendt, Axel Roers
Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity.
J Exp Med, 209(8) 1419-1426 (2012)
Ribonucleases H (RNases H) are endonucleases which cleave the RNA moiety of RNA/DNA hybrids. Their function in mammalian cells is incompletely understood. RNase H2 mutations cause Aicardi-Goutières syndrome, an inflammatory condition clinically overlapping with lupus erythematosus. We show that RNase H2 is essential in mouse embryonic development. RNase H2-deficient cells proliferated slower than control cells and accumulated in G2/M phase due to chronic activation of a DNA damage response associated with an increased frequency of single-strand breaks, increased histone H2AX phosphorylation, and induction of p53 target genes, most prominently the cyclin-dependent kinase inhibitor 1 encoding cell cycle inhibitor p21. RNase H2-deficient cells featured an increased genomic ribonucleotide load, suggesting that unrepaired ribonucleotides trigger the DNA damage response in these cells. Collectively, we show that RNase H2 is essential to remove ribonucleotides from the mammalian genome to prevent DNA damage.

Jens Haas, Sarina Ravens, Sandra Düber, Inga Sandrock, Linda Oberdörfer, Elham Kashani, Vijaykumar Chennupati, Lisa Föhse, Ronald Naumann, Siegfried Weiss, Andreas Krueger, Reinhold Förster, Immo Prinz
Development of Interleukin-17-Producing γδ T Cells Is Restricted to a Functional Embryonic Wave.
Immunity, 37(1) 48-59 (2012)
γδ T cells are an important innate source of interleukin-17 (IL-17). In contrast to T helper 17 (Th17) cell differentiation, which occurs in the periphery, IL-17-producing γδ T cells (γδT17 cells) are probably committed during thymic development. To study when γδT17 cells arise during ontogeny, we used TcrdH2BeGFP reporter mice to monitor T cell receptor (TCR) rearrangement and IL-17 production in the embryonic thymus. We observed that several populations such as innate lymphoid cells and early T cell precursors were able to produce IL-17 prior to (and thus independent of) TCR recombination. γδT17 cells were absent after transplantation of IL-17-sufficient bone marrow into mice lacking both Il17a and Il17f. Also, γδT17 cells were not generated after genetic restoration of defective Rag1 function in adult mice. Together, these data suggested that these cells developed exclusively before birth and subsequently persisted in adult mice as self-renewing, long-lived cells.

Maria Arantzazu Sanchez-Fernandez, Silvia Sbacchi, Miguel Correa-Tapia, Ronald Naumann, Julia Klemm, Pierre Chambon, Samiya Al-Robaiy, Manfred Blessing, Bernard Hoflack
Transgenic mice for a tamoxifen-induced, conditional expression of the cre recombinase in osteoclasts.
PLoS ONE, 7(5) Art. No. e37592 (2012)
Open Access PDF DOI
Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood.

Ursula Ehmer, Sandra Kalthoff, Bastian Fakundiny, Brigitte Pabst, Nicole Freiberg, Ronald Naumann, Michael P Manns, Christian P Strassburg
Gilbert syndrome redefined: A complex genetic haplotype influences the regulation of glucuronidation.
Hepatology, 55(6) 1912-1921 (2012)
Gilbert syndrome (GS) is characterized by intermittent unconjugated hyperbilirubinemia without structural liver damage affecting about 10% of the white population. In GS the UGT1A1*28 variant reduces bilirubin conjugation by 70% and is associated with irinotecan and protease inhibitor side effects. Aim of this study was to characterize potential in vivo consequences of UGT1A gene variability in GS. Three hundred GS patients (UGT1A1*28 homozygous) and 249 healthy blood donors (HBD) were genotyped for UGT1A (UGT1A1*28, UGT1A3-66 T>C, UGT1A6*3a, UGT1A7*3) and transporter SNPs (SCLO1B1 p.V174A, SCLO1B1 p.N130D, ABCC2 p.I1324I, ABCC2 -24 UTR) using TaqMan-5'-nuclease-assays. A humanized transgenic UGT1A-SNP and corresponding wild-type mouse model were established carrying the GS-associated UGT1A variant haplotype. UGT1A transcript and protein expression, and transcriptional activation were studied in vivo. Homozygous UGT1A1*28 GS individuals were simultaneously homozygous for UGT1A3-66 T>C (91%), UGT1A6*2a (77%), and UGT1A7*3 (77%). Seventy-six percent of GS and only 9% of HBD were homozygous for the variant haplotype spanning 4 UGT1A genes. SCLO1B1 and ABCC2 single nucleotide polymorphisms (SNPs) showed no differences. In transgenic humanized UGT1A SNP and wild-type mice this UGT1A haplotype led to lower UGT1A mRNA expression and UGT1A protein synthesis. UGT1A transcriptional activation by dioxin, phenobarbital and endotoxin was significantly reduced in SNP mice. CONCLUSION: Our data redefine the genetic basis behind GS. In vivo data studying the genotype present in 76% of GS individuals suggest that transcription and transcriptional activation of glucuronidation genes responsible for conjugation and detoxification is directly affected leading to lower responsiveness. This study suggests that GS should be considered as a potential risk factor for drug toxicity. (HEPATOLOGY 2011.).

Wei Liu, Min Zi, Ronald Naumann, Susanne Ulm, Jiawei Jin, Domenico M Taglieri, Sukhpal Prehar, Junhong Gui, Hoyee Tsui, Rui-Ping Xiao, Ludwig Neyses, R John Solaro, Yunbo Ke, Elizabeth J Cartwright, Ming Lei, Xin Wang
Pak1 as a Novel Therapeutic Target for Antihypertrophic Treatment in the Heart.
Circulation, 124(24) 2702-2715 (2011)
Background-Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. Although many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy, the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study, we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator that antagonizes cardiac hypertrophy.Methods and Results-Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs) or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an antihypertrophic role. To investigate the in vivo role of Pak1 in the heart, we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1(cko)). When subjected to 2 weeks of pressure overload, Pak1(cko) mice developed greater cardiac hypertrophy with attendant blunting of JNK activation compared with controls, and these knockout mice underwent the transition into heart failure when prolonged stress was applied. Chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1(cko) mice. Moreover, we discovered that the Pak1 activator FTY720, a sphingosine-like analog, was able to prevent pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. Meanwhile, FTY720 failed to exert such an effect on Pak1(cko) mice, suggesting that the antihypertrophic effect of FTY720 likely acts through Pak1 activation.Conclusions-These results, for the first time, establish Pak1 as a novel antihypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.

Sabine Schramm, Johanna Fraune, Ronald Naumann, Abrahan Hernandez-Hernandez, Christer Höög, Howard J Cooke, Manfred Alsheimer, Ricardo Benavente
A novel mouse synaptonemal complex protein is essential for loading of central element proteins, recombination, and fertility.
PLoS Genet, 7(5) Art. No. e1002088 (2011)
Open Access DOI
The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE-specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE-specific proteins, which in turn would promote synapsis between homologous chromosomes.

Alexander Gerbaulet, Claudia Wickenhauser, Julia Scholten, Katrin Peschke, Sebastian Drube, Hans-Peter Horny, Thomas Kamradt, Ronald Naumann, Werner Müller, Thomas Krieg, Claudia Waskow, Karin Hartmann, Axel Roers
Mast cell hyperplasia, B cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit.
Blood, 117(6) 2012-2021 (2011)
Signaling through the receptor tyrosine kinase kit controls proliferation and differentiation of hematopoietic precursor cells and mast cells. Somatic point mutations of the receptor that constitutively activate kit signaling are associated with mastocytosis and various hematopoietic malignancies. We generated a Cre/loxP-based bacterial artificial chromosome (BAC) transgenic mouse model that allows conditional expression of a kit gene carrying the kitD814V mutation (the murine homolog of the most common mutation in human mastocytosis, kitD816V) driven by the kit promoter. Expression of the mutant kit in adult mice caused severe mastocytosis with 100 % penetrance at young age frequently associated with additional hematopoietic (mostly B lineage-derived) neoplasms and focal colitis. Restriction of transgene expression to mature mast cells resulted in a similar mast cell disease developing with slower kinetics. Embryonic expression led to a hyperproliferative dysregulation of the erythroid lineage with a high rate of perinatal lethality. In addition, most adult animals developed colitis associated with mucosal mast cell accumulation. Our findings demonstrate that the effects of constitutive kit signaling critically depend on the developmental stage and the state of differentiation of the cell hit by the gain of function mutation.

Julia Elisabeth Pulverer, Ulfert Rand, Stefan Lienenklaus, Daniela Kugel, Natalia Zietara, Georg Kochs, Ronald Naumann, Siegfried Weiss, Peter Staeheli, Hansjörg Hauser#, Mario Köster#
Temporal and spatial resolution of type I and III interferon responses in vivo.
J Virol, 84(17) 8626-8638 (2010)
Although the action of interferons (IFNs) has been extensively studied in vitro, limited information is available on the spatial and temporal activation pattern of IFN-induced genes in vivo. We created BAC transgenic mice expressing firefly luciferase under transcriptional control of the Mx2 gene promoter. Expression of the reporter with regard to onset and kinetics of induction parallels that of Mx2 and is thus a hallmark for the host response. Substantial constitutive expression of the reporter gene was observed in the liver and most other tissues of transgenic mice, whereas this expression was strongly reduced in animals lacking functional type I IFN receptors. As expected, the reporter gene was induced not only in response to type I (alpha and beta) and type III (lambda) IFNs but also in response to a variety of IFN inducers such as double-stranded RNA, lipopolysaccharide (LPS), and viruses. In vivo IFN subtypes show clear differences with respect to their kinetics of action and to their spatial activation pattern: while the type I IFN response was strong in liver, spleen, and kidney, type III IFN reactivity was most prominent in organs with mucosal surfaces. Infection of reporter mice with virus strains that differ in their pathogenicity shows that the IFN response is significantly altered in the strength of IFN action at sites which are not primarily infected as well as by the onset and duration of gene induction.

Jeremy N. Pulvers, Jaroslaw Bryk, Jennifer L. Fish, Michaela Wilsch-Bräuninger, Yoko Arai, Dora Schreier, Ronald Naumann, Jussi Helppi, Bianca Habermann, Johannes Vogt, Robert Nitsch, Attila Toth, Wolfgang Enard, Svante Pääbo, Wieland B. Huttner
Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline.
Proc Natl Acad Sci U.S.A., 107(38) 16595-16600 (2010)
Mutations in ASPM (abnormal spindle-like microcephaly associated) cause primary microcephaly in humans, a disorder characterized by a major reduction in brain size in the apparent absence of nonneurological anomalies. The function of the Aspm protein in neural progenitor cell expansion, as well as its localization to the mitotic spindle and midbody, suggest that it regulates brain development by a cell division-related mechanism. Furthermore, evidence that positive selection affected ASPM during primate evolution has led to suggestions that such a function changed during primate evolution. Here, we report that in Aspm mutant mice, truncated Aspm proteins similar to those causing microcephaly in humans fail to localize to the midbody during M-phase and cause mild microcephaly. A human ASPM transgene rescues this phenotype but, interestingly, does not cause a gain of function. Strikingly, truncated Aspm proteins also cause a massive loss of germ cells, resulting in a severe reduction in testis and ovary size accompanied by reduced fertility. These germline effects, too, are fully rescued by the human ASPM transgene, indicating that ASPM is functionally similar in mice and humans. Our findings broaden the spectrum of phenotypic effects of ASPM mutations and raise the possibility that positive selection of ASPM during primate evolution reflects its function in the germline.

Caspar Ohnmacht✳︎, Christian Schwartz✳︎, Marc Panzer, Isabell Schiedewitz, Ronald Naumann, David Voehringer
Basophils Orchestrate Chronic Allergic Dermatitis and Protective Immunity against Helminths.
Immunity, 33(3) 364-374 (2010)
Basophils are associated with T helper 2 (Th2) cell-polarized immune responses such as allergic disorders or helminth infections. To directly address the role of basophils for type 2 immunity, we generated transgenic mice with constitutive and selective deletion of basophils. Differentiation and accumulation of Th2 cells, induction of eosinophilia, and increase in serum IgE or IgG1 induced by allergens or by infection with the helminth Nippostrongylus brasiliensis appeared to be basophil independent. Further, basophils were not required for passive IgE- or IgG1-mediated systemic anaphylaxis. However, basophils were essential for IgE-meditated chronic allergic dermatitis and for protection against secondary infection with N. brasiliensis. These results demonstrate that basophils play an important role for protective immunity against helminths and orchestrate chronic allergic inflammation, whereas primary Th2 cell responses can operate efficiently in the absence of this cell type.

Andrea Kranz, Jun Fu, Kristin Duerschke, Stefanie Weidlich, Ronald Naumann, A. Francis Stewart#, Konstantinos Anastassiadis#
An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase.
Genesis, 48(8) 512-520 (2010)
Recently a codon improved version of the Flpe site specific recombinase, termed Flpo, was reported as having greatly improved performance in mammalian cell applications. However the degree of improvement could not be estimated because essentially no Flpe activity was observed. Here we compare Flpe and Flpo accurately in a mammalian cell assay to estimate that Flpo is about five times more active than Flpe and similar to Cre and Dre. Consequently we generated a Flpo deleter mouse line from the JM8 C57Bl/6 ES cells used in the EUCOMM and KOMP systematic knock-out programs. In breeding experiments we show that the Flpo deleter delivers complete recombination using alleles that are incompletely recombined by a commonly used Flpe deleter. This indicates that the Flpo deleter is more efficient. (c) 2010 Wiley-Liss, Inc.

Susann Becker✳︎, Elke Wandel✳︎, Manja Wobus, Rick Schneider, Salah Amasheh, Doreen Sittig, Christiane Kerner, Ronald Naumann, Joerg Hamann, Gabriela Aust
Overexpression of CD97 in intestinal epithelial cells of transgenic mice attenuates colitis by strengthening adherens junctions.
PLoS ONE, 5(1) Art. No. e8507 (2010)
Open Access PDF DOI
The adhesion G-protein-coupled receptor CD97 is present in normal colonic enterocytes but overexpressed in colorectal carcinoma. To investigate the function of CD97 in colorectal carcinogenesis, transgenic Tg(villin-CD97) mice overexpressing CD97 in enterocytes were generated and subjected to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated tumorigenesis. Unexpectedly, we found a CD97 cDNA copy number-dependent reduction of DSS-induced colitis in Tg compared to wild-type (WT) mice that was confirmed by applying a simple DSS protocol. Ultrastructural analysis revealed that overexpression of CD97 strengthened lateral cell-cell contacts between enterocytes, which, in contrast, were weakened in CD97 knockout (Ko) mice. Transepithelial resistance was not altered in Tg and Ko mice, indicating that tight junctions were not affected. In Tg murine and normal human colonic enterocytes as well as in colorectal cell lines CD97 was localized preferentially in E-cadherin-based adherens junctions. CD97 overexpression upregulated membrane-bound but not cytoplasmic or nuclear beta-catenin and reduced phospho-beta-catenin, labeled for degradation. This was associated with inactivation of glycogen synthase kinase-3beta (GSK-3beta) and activation of Akt. In summary, CD97 increases the structural integrity of enterocytic adherens junctions by increasing and stabilizing junctional beta-catenin, thereby regulating intestinal epithelial strength and attenuating experimental colitis.

Daniel Teupser✳︎, Daniel Kretzschmar✳︎, Carsten Tennert, Ralph Burkhardt, Ronald Naumann, Albrecht E Sippel, Joachim Thiery, Wolfgang Wilfert, Dörte Fengler
Effect of macrophage overexpression of murine liver X receptor-alpha (LXR-alpha) on atherosclerosis in LDL-receptor deficient mice.
Arterioscler Thromb Vasc Biol, 28(11) 2009-2015 (2008)
Background- The nuclear liver X receptor-alpha (LXR-alpha) has been implicated in the regulation of intracellular cholesterol homeostasis, inflammatory response, and atherosclerosis susceptibility. The aim of the present study was to test whether transgenic expression of LXR-alpha might affect these mechanisms and result in a reduction of atherosclerosis. METHODS AND RESULTS: We generated mice with macrophage overexpression of mouse LXR-alpha, evidenced by significantly elevated expression levels of LXR-target genes (ABCA1, ABCG1) in these cells. For atherosclerosis studies, mice were crossed onto the LDL-receptor deficient background. Plasma lipids and lipoproteins as well as liver triglycerides were not significantly different between transgenic animals and nontransgenic controls. However, lesion area at the brachiocephalic artery (BCA) was significantly reduced (-83%, P=0.02) in male LXR-alpha transgenic mice. This was associated with a significantly increased cholesterol efflux to acceptor-free media (+24%, P=0.002) and ApoA1 containing media (+20%, P<0.0001) as well as reduced lipopolysaccharide (LPS)-induced NO-release from macrophages of transgenic animals, providing a potential mechanism for the reduction of atherosclerosis. CONCLUSIONS: Our data show for the first time that transgenic overexpression of LXR-alpha in macrophages has significant antiatherogenic properties. We conclude that overexpression of LXR-alpha in macrophages might be useful as a therapeutic principle for the prevention of atherosclerosis.

Ina Poser, Mihail Sarov, James R A Hutchins, Jean-Karim Hériché, Yusuke Toyoda, Andrei I. Pozniakovsky, Daniela Weigl, Anja Nitzsche, Björn Hegemann, Alexander W. Bird, Laurence Pelletier, Ralf Kittler, Sujun Hua, Ronald Naumann, Martina Augsburg, Martina M Sykora, Helmut Hofemeister, Youming Zhang, Kim Nasmyth, Kevin P White, Steffen Dietzel, Karl Mechtler, Richard Durbin, A. Francis Stewart, Jan-Michael Peters, Frank Buchholz, Anthony A. Hyman
BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.
Nat Methods, 5(5) 409-415 (2008)
The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.

Kristina Vintersten, Giuseppe Testa, Ronald Naumann, Konstantinos Anastassiadis, A. Francis Stewart
Bacterial artificial chromosome transgenesis through pronuclear injection of fertilized mouse oocytes
In: Innate immunity. (Eds.) Jonathan Ewbank Methods in molecular biology, 415.,Totowa, USA,Humana Press (2008),83-100 Ch. 5