Publications by authors named "Ernst-Martin Füchtbauer"

74 Publications

Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension.

Biotechniques 2020 06 6;68(6):345-348. Epub 2020 May 6.

Department of Molecular Biology & Genetics, Aarhus University, C.F. Møllers Allé 3, 8000 Aarhus C, Denmark.

We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair.
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http://dx.doi.org/10.2144/btn-2019-0104DOI Listing
June 2020

AMPA Induces NO-Dependent cGMP Signals in Hippocampal and Cortical Neurons via L-Type Voltage-Gated Calcium Channels.

Cereb Cortex 2020 04;30(4):2128-2143

Institute of Pharmacology and Toxicology, Ruhr-University Bochum, 44780 Bochum, Germany.

The nitric oxide (NO)/cGMP signaling cascade has an established role in synaptic plasticity. However, with conventional methods, the underlying cGMP signals were barely detectable. Here, we set out to confirm the well-known NMDA-induced cGMP increases, to test the impact of AMPA on those signals, and to identify the relevant phosphodiesterases (PDEs) using a more sensitive fluorescence resonance energy transfer (FRET)-based method. Therefore, a "knock-in" mouse was generated that expresses a FRET-based cGMP indicator (cGi-500) allowing detection of cGMP concentrations between 100 nM and 3 μM. Measurements were performed in cultured hippocampal and cortical neurons as well as acute hippocampal slices. In hippocampal and cortical neurons, NMDA elicited cGMP signals half as high as the ones elicited by exogenous NO. Interestingly, AMPA increased cGMP independently of NMDA receptors and dependent on NO synthase (NOS) activation. NMDA- and AMPA-induced cGMP signals were not additive indicating that both pathways converge on the level of NOS. Accordingly, the same PDEs, PDE1 and PDE2, were responsible for degradation of NMDA- as well as AMPA-induced cGMP signals. Mechanistically, AMPAR induced calcium influx through L-type voltage-gated calcium channels leading to NOS and finally NO-sensitive guanylyl cyclase activation. Our results demonstrate that in addition to NMDA also AMPA triggers endogenous NO formation and hence cGMP production.
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http://dx.doi.org/10.1093/cercor/bhz227DOI Listing
April 2020

Modulation of the fungal mycobiome is regulated by the chitin-binding receptor FIBCD1.

J Exp Med 2019 12 10;216(12):2689-2700. Epub 2019 Oct 10.

Jill Roberts Institute for Research in Inflammatory Bowel Disease, Friedman Center for Nutrition and Inflammation, Joan and Sanford I. Weill Department of Medicine, Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY

Host-microbiota interactions are critical in regulating mammalian health and disease. In addition to bacteria, parasites, and viruses, beneficial communities of fungi (the mycobiome) are important modulators of immune- and tissue-homeostasis. Chitin is a major component of the fungal cell wall, and fibrinogen C containing domain 1 (FIBCD1) is a chitin-binding protein; however, the role of this molecule in influencing host-mycobiome interactions in vivo has never been examined. Here, we identify direct binding of FIBCD1 to intestinal-derived fungi and demonstrate that epithelial-specific expression of FIBCD1 results in significantly reduced fungal colonization and amelioration of fungal-driven intestinal inflammation. Collectively, these results identify FIBCD1 as a previously unrecognized microbial pattern recognition receptor through which intestinal epithelial cells can recognize and control fungal colonization, limit fungal dysbiosis, and dampen intestinal inflammation.
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http://dx.doi.org/10.1084/jem.20182244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888979PMC
December 2019

Mind the gap (junction): cGMP induced by nitric oxide in cardiac myocytes originates from cardiac fibroblasts.

Br J Pharmacol 2019 12 27;176(24):4696-4707. Epub 2019 Dec 27.

Institute of Pharmacology and Toxicology, Ruhr-University Bochum, Bochum, Germany.

Background And Purpose: The intracellular signalling molecule cGMP, formed by NO-sensitive GC (NO-GC), has an established function in the vascular system. Despite numerous reports about NO-induced cGMP effects in the heart, the underlying cGMP signals are poorly characterized.

Experimental Approach: Therefore, we analysed cGMP signals in cardiac myocytes and fibroblasts isolated from knock-in mice expressing a FRET-based cGMP indicator.

Key Results: Whereas in cardiac myocytes, none of the known NO-GC-activating substances (NO, GC activators, and GC stimulators) increased cGMP even in the presence of PDE inhibitors, they induced substantial cGMP increases in cardiac fibroblasts. As cardiac myocytes and fibroblasts are electrically connected via gap junctions, we asked whether cGMP can take the same route. Indeed, in cardiomyocytes co-cultured on cardiac fibroblasts, NO-induced cGMP signals were detectable, and two groups of unrelated gap junction inhibitors abolished these signals.

Conclusion And Implication: We conclude that NO-induced cGMP formed in cardiac fibroblasts enters cardiac myocytes via gap junctions thereby turning cGMP into an intercellular signalling molecule. The findings shed new light on NO/cGMP signalling in the heart and will potentially broaden therapeutic opportunities for cardiac disease.
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http://dx.doi.org/10.1111/bph.14835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965686PMC
December 2019

Isolation of cancer stem cells by selection for miR-302 expressing cells.

PeerJ 2019 26;7:e6635. Epub 2019 Mar 26.

Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.

Background: Cancer stem cells are believed to be a major reason for long-term therapy failure because they are multi-drug resistant and able to rest mitotically inactive in the hypoxic center of tumors. Due to their variable number and their often low proliferation rate, cancer stem cells are difficult to purify in decent quantities and to grow in cell culture systems, where they are easily outcompeted by faster growing more 'differentiated', i.e., less stem cell-like tumor cells.

Methods: Here we present a proof of principle study based on the idea to select cancer stem cells by means of the expression of a stem cell-specific gene. A selectable egfp-neo coding sequence was inserted in the last exon of the non-coding murine miR-302 host gene. As a stem cell specific regulatory element, 2.1 kb of the genomic region immediately upstream of the miR-302 host gene transcription start site was used. Stable transgenic CJ7 embryonic stem cells were used to induce teratomas.

Results: After three weeks, tumors were removed for analysis and primary cultures were established. Stem cell-like cells were selected from these culture based on G418 selection. When the selection was removed, stem cell morphology and miR-302 expression were rapidly lost, indicating that it was not the original ES cells that had been isolated.

Conclusions: We show the possibility to use drug resistance expressed from a regulatory sequence of a stem cell-specific marker, to isolate and propagate cancer stem cells that otherwise might be hidden in the majority of tumor cells.
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http://dx.doi.org/10.7717/peerj.6635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440458PMC
March 2019

Genome Editing in Mice.

Methods Mol Biol 2019 ;1961:249-254

Department of Molecular Biology and Genetics, Aarhus, Denmark.

Programmable nucleases like CRISPR/Cas9 enable to edit the mouse genome directly in the zygote. Several methods have been successfully used for this. Here we describe injection into one of the pronuclei of the zygote and electroporation of zygotes. Alternative methods will be mentioned.
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http://dx.doi.org/10.1007/978-1-4939-9170-9_15DOI Listing
July 2019

Biochemical and structural analyses suggest that plasminogen activators coevolved with their cognate protein substrates and inhibitors.

J Biol Chem 2019 03 16;294(10):3794-3805. Epub 2019 Jan 16.

From the Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark and

Protein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either co-conservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems.
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http://dx.doi.org/10.1074/jbc.RA118.005419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416416PMC
March 2019

Septins are critical regulators of osteoclastic bone resorption.

Sci Rep 2018 08 29;8(1):13016. Epub 2018 Aug 29.

Department of Clinical Cell Biology, Vejle Hospital/Lillebaelt Hospital, Institute of Regional Health Research, University of Southern Denmark, Beriderbakken 4, 7100, Vejle, Denmark.

Septins are known to play key roles in supporting cytoskeletal stability, vesicular transport, endo-/exocytosis, stabilizing cellular membranes and forming diffusion barriers. Their function in mammalian cells is poorly investigated. The osteoclast offers an interesting tool to investigate septins because all cellular activities septins were reported to be involved in are critical for osteoclasts. However, the existence of septins in osteoclasts has not even been reported. Here we show that the SEPT9 gene and Septin 9 (SEPT9) protein are expressed and synthesized during differentiation of human osteoclasts. Pharmacological stabilization of septin filaments dose dependently inhibits bone resorption of human osteoclasts in vitro suggesting a role for septins in bone resorption. Attesting to this, conditional deletion of Sept9 in mice leads to elevated levels of trabecular bone and diminished femoral growth in vivo. Finally, systematic interrogation of the spatial organization of SEPT9 by confocal microscopy reveals that SEPT9 is closely associated to the structures known to be critical for osteoclast activity. We propose that septins in general and SEPT9 in particular play a previously unappreciated role in osteoclastic bone resorption.
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http://dx.doi.org/10.1038/s41598-018-31159-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115361PMC
August 2018

Islet-Derived eATP Fuels Autoreactive CD8 T Cells and Facilitates the Onset of Type 1 Diabetes.

Diabetes 2018 10 31;67(10):2038-2053. Epub 2018 Jul 31.

Division of Nephrology, Boston Children's Hospital, Harvard Medical School, Boston, MA

Extracellular ATP (eATP) activates T cells by engaging the P2X7R receptor. We identified two loss-of-function P2X7R mutations that are protective against type 1 diabetes (T1D) and thus hypothesized that eATP/P2X7R signaling may represent an early step in T1D onset. Specifically, we observed that in patients with newly diagnosed T1D, P2X7R is upregulated on CD8 effector T cells in comparison with healthy control subjects. eATP is released at high levels by human/murine islets in vitro in high-glucose/inflammatory conditions, thus upregulating P2X7R on CD8 T cells in vitro. P2X7R blockade with oxidized ATP reduces the CD8 T cell-mediated autoimmune response in vitro and delays diabetes onset in NOD mice. Autoreactive CD8 T-cell activation is highly dependent upon eATP/P2X7R-mediated priming, while a novel sP2X7R recombinant protein abrogates changes in metabolism and the autoimmune response associated with CD8 T cells. eATP/P2X7R signaling facilitates the onset of autoimmune T1D by fueling autoreactive CD8 cells and therefore represents a novel targeted therapeutic for the disorder.
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http://dx.doi.org/10.2337/db17-1227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905486PMC
October 2018

The choroid plexus sodium-bicarbonate cotransporter NBCe2 regulates mouse cerebrospinal fluid pH.

J Physiol 2018 10 12;596(19):4709-4728. Epub 2018 Aug 12.

Department of Biomedicine, Health, Aarhus University, Denmark.

Key Points: Normal pH is crucial for proper functioning of the brain, and disorders increasing the level of CO in the blood lead to a decrease in brain pH. CO can easily cross the barriers of the brain and will activate chemoreceptors leading to an increased exhalation of CO . The low pH, however, is harmful and bases such as HCO are imported across the brain barriers in order to normalize brain pH. We show that the HCO transporter NBCe2 in the choroid plexus of the blood-cerebrospinal fluid barrier is absolutely necessary for normalizing CSF pH during high levels of CO . This discovery represents a significant step in understanding the molecular mechanisms behind regulation of CSF pH during acid-base disturbances, such as chronic lung disease.

Abstract: The choroid plexus epithelium (CPE) is located in the brain ventricles where it produces the majority of the cerebrospinal fluid (CSF). The hypothesis that normal brain function is sustained by CPE-mediated CSF pH regulation by extrusion of acid-base equivalents was tested by determining the contribution of the electrogenic Na -HCO cotransporter NBCe2 to CSF pH regulation. A novel strain of NBCe2 (Slc4a5) knockout (KO) mice was generated and validated. The base extrusion rate after intracellular alkalization was reduced by 77% in NBCe2 KO mouse CPE cells compared to control mice. NBCe2 KO mice and mice with CPE-targeted NBCe2 siRNA knockdown displayed a reduction in CSF pH recovery during hypercapnia-induced acidosis of approximately 85% and 90%, respectively, compared to control mice. NBCe2 KO did not affect baseline respiration rate or tidal volume, and the NBCe2 KO and wild-type (WT) mice displayed similar ventilatory responses to 5% CO exposure. NBCe2 KO mice were not protected against pharmacological or heating-induced seizure development. In conclusion, we establish the concept that the CPE is involved in the regulation of CSF pH by demonstrating that NBCe2 is necessary for proper CSF pH recovery after hypercapnia-induced acidosis.
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http://dx.doi.org/10.1113/JP275489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166071PMC
October 2018

Mice Knocked Out for the Primary Brain Calcification-Associated Gene Slc20a2 Show Unimpaired Prenatal Survival but Retarded Growth and Nodules in the Brain that Grow and Calcify Over Time.

Am J Pathol 2018 08 25;188(8):1865-1881. Epub 2018 May 25.

Department of Clinical Medicine, Aarhus University, Aarhus, Denmark; Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark. Electronic address:

Brain calcification of especially the basal ganglia characterizes primary familial brain calcification (PFBC). PFBC is a rare neurodegenerative disorder with neuropsychiatric and motor symptoms, and only symptomatic treatment is available. Four PFBC-associated genes are known; approximately 40% of patients carry mutations in the gene SLC20A2, which encodes the type III sodium-dependent inorganic phosphate transporter PiT2. To investigate the role of PiT2 in PFBC development, we studied Slc20a2-knockout (KO) mice using histology, microcomputed tomography, electron microscopy, and energy-dispersive X-ray spectroscopy. Slc20a2-KO mice showed histologically detectable nodules in the brain already at 8 weeks of age, which contained organic material and were weakly calcified. In 15-week-old mice, the nodules were increased in size and number and were markedly more calcified. The major minerals in overt calcifications were Ca and P, but Fe, Zn, and Al were also generally present. Electron microscopy suggested that the calcifications initiate intracellularly, mainly in pericytes and astrocytes. As the calcification grew, they incorporated organic material. Furthermore, endogenous IgG was detected around nodules, suggesting local increased blood-brain barrier permeabilities. Nodules were found in all 8-week-old Slc20a2-KO mice, but no prenatal or marked postnatal lethality was observed. Thus, besides allowing for the study of PFBC development, the Slc20a2-KO mouse is a potential solid preclinical model for evaluation of PFBC treatments.
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http://dx.doi.org/10.1016/j.ajpath.2018.04.010DOI Listing
August 2018

Variable Contribution of TMEM16A to Tone in Murine Arterial Vasculature.

Basic Clin Pharmacol Toxicol 2018 Jul 30;123(1):30-41. Epub 2018 Mar 30.

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

TMEM16A is essential for Ca -activated Cl conductance in vascular smooth muscle. The importance of TMEM16A for agonist-induced vascular constriction and blood pressure control is, however, under debate. Previous studies suggested that TMEM16A might have a complex cellular function beyond being essential for the Ca -activated Cl conductance, for example modulation of Ca channel expression. Mice with constitutive, smooth muscle-specific expression of siRNA directed against Tmem16a (transgenic mice, TG) were generated. Isometric constrictions of isolated aorta, mesenteric, femoral and tail arteries from TG mice were compared with wild-types. Protein expression was analysed by Western blots. Blood pressure and heart rate were studied telemetrically. Significant TMEM16A down-regulation was seen in aorta and tail arteries, while no changes were detected in mesenteric and femoral arteries. Contractile responses of mesenteric and femoral arteries from TG and wild-type mice were not different. Aorta from TG mice showed reduced agonist-induced constriction, while their responses to elevated K were unchanged. Tail arteries from TG mice also constricted less to adrenergic stimulation than wild-types. Surprisingly, tail arteries from TG mice constricted less to elevated K too and were more sensitive to nifedipine-induced relaxation. Consistently, TMEM16A down-regulation in tail arteries was associated with reduction in CACNA1C protein (i.e. vascular L-type Ca channel) expression. No differences in blood pressure and heart rate between the groups were seen. This study suggests a complex contribution of TMEM16A in vascular function. We suggest that TMEM16A modulates arterial contractility, at least in part, indirectly via regulation of CACNA1C expression.
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http://dx.doi.org/10.1111/bcpt.12984DOI Listing
July 2018

Optimized co-transfection of murine embryonic stem cells.

Transgenic Res 2018 02 5;27(1):131-133. Epub 2018 Jan 5.

Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.

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http://dx.doi.org/10.1007/s11248-017-0055-9DOI Listing
February 2018

Plekhg5-regulated autophagy of synaptic vesicles reveals a pathogenic mechanism in motoneuron disease.

Nat Commun 2017 10 30;8(1):678. Epub 2017 Oct 30.

Institute of Clinical Neurobiology, University Hospital Würzburg, 97078, Würzburg, Germany.

Autophagy-mediated degradation of synaptic components maintains synaptic homeostasis but also constitutes a mechanism of neurodegeneration. It is unclear how autophagy of synaptic vesicles and components of presynaptic active zones is regulated. Here, we show that Pleckstrin homology containing family member 5 (Plekhg5) modulates autophagy of synaptic vesicles in axon terminals of motoneurons via its function as a guanine exchange factor for Rab26, a small GTPase that specifically directs synaptic vesicles to preautophagosomal structures. Plekhg5 gene inactivation in mice results in a late-onset motoneuron disease, characterized by degeneration of axon terminals. Plekhg5-depleted cultured motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, which can be rescued by constitutively active Rab26. These findings define a mechanism for regulating autophagy in neurons that specifically targets synaptic vesicles. Disruption of this mechanism may contribute to the pathophysiology of several forms of motoneuron disease.
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http://dx.doi.org/10.1038/s41467-017-00689-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662736PMC
October 2017

Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs.

JCI Insight 2017 10 5;2(19). Epub 2017 Oct 5.

Deparment of Clinical Medicine, Aarhus University, Aarhus, Denmark.

Fibrous cap smooth muscle cells (SMCs) protect atherosclerotic lesions from rupturing and causing thrombosis, while other plaque SMCs may have detrimental roles in plaque development. To gain insight into recruitment of different plaque SMCs, we mapped their clonal architecture in aggregation chimeras of eGFP+Apoe-/- and Apoe-/- mouse embryos and in mice with a mosaic expression of fluorescent proteins in medial SMCs that were rendered atherosclerotic by PCSK9-induced hypercholesterolemia. Fibrous caps in aggregation chimeras were found constructed from large, endothelial-aligned layers of either eGFP+ or nonfluorescent SMCs, indicating substantial clonal expansion of a few cells. Similarly, plaques in mice with SMC-restricted Confetti expression showed oligoclonal SMC populations with little intermixing between the progeny of different medial SMCs. Phenotypes comprised both ACTA2+ SMCs in the cap and heterogeneous ACTA2- SMCs in the plaque interior, including chondrocyte-like cells and cells with intracellular lipid and crystalline material. Fibrous cap SMCs were invariably arranged in endothelium-aligned clonal sheets, confirming results in the aggregation chimeras. Analysis of the clonal structure showed that a low number of local medial SMCs partake in atherosclerosis and that single medial SMCs can produce several different SMC phenotypes in plaque. The combined results show that few medial SMCs proliferate to form the entire phenotypically heterogeneous plaque SMC population in murine atherosclerosis.
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http://dx.doi.org/10.1172/jci.insight.95890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841865PMC
October 2017

Constitutive transgene expression of Stem Cell Antigen-1 in the hair follicle alters the sensitivity to tumor formation and progression.

Stem Cell Res 2017 08 8;23:109-118. Epub 2017 Jul 8.

Department of Clinical Genetics, Aarhus University Hospital, Brendstrupgaardsvej 21C, 8200 Aarhus N, Denmark. Electronic address:

The cell surface protein Stem Cell Antigen-1 (Sca-1) marks stem or progenitor cells in several murine tissues and is normally upregulated during cancer development. Although the specific function of Sca-1 remains unknown, Sca-1 seems to play a role in proliferation, differentiation and cell migration in a number of tissues. In the skin epithelium, Sca-1 is highly expressed in the interfollicular epidermis but is absent in most compartments of the hair follicle; however, the function of Sca-1 in the skin has not been investigated. To explore the role of Sca-1 in normal and malignant skin development we generated transgenic mice that express Sca-1 in the hair follicle stem cells that are normally Sca-1 negative. Development of hair follicles and interfollicular epidermis appeared normal in Sca-1 mutant mice; however, follicular induction of Sca-1 expression in bulge region and isthmus stem cells reduced the overall yield of papillomas in a chemical carcinogenesis protocol. Despite that fewer papillomas developed in transgenic mice a higher proportion of the papillomas underwent malignant conversion. These findings suggest that overexpression of Sca-1 in the hair follicle stem cells contributes at different stages of tumour development. In early stages, overexpression of Sca-1 decreases tumour formation while at later stages overexpression of Sca-1 seems to drive tumours towards malignant progression.
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http://dx.doi.org/10.1016/j.scr.2017.07.002DOI Listing
August 2017

High folic acid diet enhances tumour growth in PyMT-induced breast cancer.

Br J Cancer 2017 Mar 2;116(6):752-761. Epub 2017 Feb 2.

Department of Molecular Biology and Genetics, Aarhus University, Aarhus C 8000, Denmark.

Background: The B-vitamin folate is among the most studied bioactive food compound, and a dietary intake meeting the daily requirements has been found to reduce the risk of cancer and cardiovascular diseases as well as preventing neural tube defects during fetal development. Several countries have therefore introduced dietary fortification with folic acid. However, clinical and animal studies suggest that folic acid has a dual role in cancer development.

Methods: During the period of initial tumour progression, MMTV-PyMT (MMTV-polyoma virus middle T) transgenic mice were fed with normal diet and high folic acid diet.

Results: We found that PyMT-induced breast tumours highly express the cancer-specific folate receptor (FR), a feature they share with several human epithelial cancers in which expression of FRα correlates with tumour grade. Mice receiving a high folic acid diet displayed a significantly increased tumour volume compared with mice receiving normal diet. In the largest tumours, only found in mice on high folic acid diet, STAT3 was activated. In primary cells from PyMT tumours, STAT3 was activated upon treatment with folic acid in culture.

Conclusions: Our results offer a novel molecular explanation for folic acid-induced growth of existing tumours.
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http://dx.doi.org/10.1038/bjc.2017.11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355920PMC
March 2017

The Schizophrenia-Associated BRD1 Gene Regulates Behavior, Neurotransmission, and Expression of Schizophrenia Risk Enriched Gene Sets in Mice.

Biol Psychiatry 2017 07 15;82(1):62-76. Epub 2016 Sep 15.

Departments of Biomedicine, Aarhus; iSEQ, Centre for Integrative Sequencing, Aarhus; iPSYCH, The Lundbeck Foundation Initiative for Integrative Psychiatric Research, Aarhus. Electronic address:

Background: The schizophrenia-associated BRD1 gene encodes a transcriptional regulator whose comprehensive chromatin interactome is enriched with schizophrenia risk genes. However, the biology underlying the disease association of BRD1 remains speculative.

Methods: This study assessed the transcriptional drive of a schizophrenia-associated BRD1 risk variant in vitro. Accordingly, to examine the effects of reduced Brd1 expression, we generated a genetically modified Brd1 mouse and subjected it to behavioral, electrophysiological, molecular, and integrative genomic analyses with focus on schizophrenia-relevant parameters.

Results: Brd1 mice displayed cerebral histone H3K14 hypoacetylation and a broad range of behavioral changes with translational relevance to schizophrenia. These behaviors were accompanied by striatal dopamine/serotonin abnormalities and cortical excitation-inhibition imbalances involving loss of parvalbumin immunoreactive interneurons. RNA-sequencing analyses of cortical and striatal micropunches from Brd1 and wild-type mice revealed differential expression of genes enriched for schizophrenia risk, including several schizophrenia genome-wide association study risk genes (e.g., calcium channel subunits [Cacna1c and Cacnb2], cholinergic muscarinic receptor 4 [Chrm4)], dopamine receptor D [Drd2], and transcription factor 4 [Tcf4]). Integrative analyses further found differentially expressed genes to cluster in functional networks and canonical pathways associated with mental illness and molecular signaling processes (e.g., glutamatergic, monoaminergic, calcium, cyclic adenosine monophosphate [cAMP], dopamine- and cAMP-regulated neuronal phosphoprotein 32 kDa [DARPP-32], and cAMP responsive element binding protein signaling [CREB]).

Conclusions: Our study bridges the gap between genetic association and pathogenic effects and yields novel insights into the unfolding molecular changes in the brain of a new schizophrenia model that incorporates genetic risk at three levels: allelic, chromatin interactomic, and brain transcriptomic.
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http://dx.doi.org/10.1016/j.biopsych.2016.08.037DOI Listing
July 2017

Cognitive deficits caused by a disease-mutation in the α3 Na(+)/K(+)-ATPase isoform.

Sci Rep 2016 08 23;6:31972. Epub 2016 Aug 23.

Aarhus University, Department of Biomedicine, DK-8000 Aarhus, Denmark.

The Na(+)/K(+)-ATPases maintain Na(+) and K(+) electrochemical gradients across the plasma membrane, a prerequisite for electrical excitability and secondary transport in neurons. Autosomal dominant mutations in the human ATP1A3 gene encoding the neuron-specific Na(+)/K(+)-ATPase α3 isoform cause different neurological diseases, including rapid-onset dystonia-parkinsonism (RDP) and alternating hemiplegia of childhood (AHC) with overlapping symptoms, including hemiplegia, dystonia, ataxia, hyperactivity, epileptic seizures, and cognitive deficits. Position D801 in the α3 isoform is a mutational hotspot, with the D801N, D801E and D801V mutations causing AHC and the D801Y mutation causing RDP or mild AHC. Despite intensive research, mechanisms underlying these disorders remain largely unknown. To study the genotype-to-phenotype relationship, a heterozygous knock-in mouse harboring the D801Y mutation (α3(+/D801Y)) was generated. The α3(+/D801Y) mice displayed hyperactivity, increased sensitivity to chemically induced epileptic seizures and cognitive deficits. Interestingly, no change in the excitability of CA1 pyramidal neurons in the α3(+/D801Y) mice was observed. The cognitive deficits were rescued by administration of the benzodiazepine, clonazepam, a GABA positive allosteric modulator. Our findings reveal the functional significance of the Na(+)/K(+)-ATPase α3 isoform in the control of spatial learning and memory and suggest a link to GABA transmission.
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http://dx.doi.org/10.1038/srep31972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4994072PMC
August 2016

Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System.

PLoS One 2016 19;11(8):e0161471. Epub 2016 Aug 19.

Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus C, Denmark.

Recombinase mediated cassette exchange (RMCE) is a powerful tool for targeted insertion of transgenes. Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increased efficiency of integration of the genetic element of interest (GEI) at a single docking site, lack of bacterial backbone at the docking site both before and after GEI integration, removal of selection and visual markers initially present at the docking site upon GEI integration and the possibility to validate GEI integration by loss of a red fluorescence reporter. Moreover, the RMCE-in cell lines are compatible with GEI donors used for the Flp-in system. We demonstrate a three-step procedure for generating RMCE-in cell lines, (I) RMCE-in transposon and SB10 transposase transfection, (II) clone isolation, and (III) selecting single integrated clones with highest RFP level, which could in principle be used to turn any cell line into an RMCE-in cell line. The RMCE-in system was used as a proof of concept to produce three new RMCE-in cell lines using HEK293, HeLa, and murine embryonic stem (mES) cells. The established RMCE-in cell lines and vector are freely available from the ATCC cell bank and Addgene respectively.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161471PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991790PMC
July 2017

C4.4A gene ablation is compatible with normal epidermal development and causes modest overt phenotypes.

Sci Rep 2016 05 12;6:25833. Epub 2016 May 12.

The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark.

C4.4A is a modular glycolipid-anchored Ly6/uPAR/α-neurotoxin multidomain protein that exhibits a prominent membrane-associated expression in stratified squamous epithelia. C4.4A is also expressed in various solid cancer lesions, where high expression levels often are correlated to poor prognosis. Circumstantial evidence suggests a role for C4.4A in cell adhesion, migration, and invasion, but a well-defined biological function is currently unknown. In the present study, we have generated and characterized the first C4.4A-deficient mouse line to gain insight into the functional significance of C4.4A in normal physiology and cancer progression. The unchallenged C4.4A-deficient mice were viable, fertile, born in a normal Mendelian distribution and, surprisingly, displayed normal development of squamous epithelia. The C4.4A-deficient mice were, nonetheless, significantly lighter than littermate controls predominantly due to differences in fat mass. Congenital C4.4A deficiency delayed migration of keratinocytes enclosing incisional skin wounds in male mice. In chemically induced bladder carcinomas, C4.4A deficiency attenuated the incidence of invasive lesions despite having no effect on total tumour burden. This new C4.4A-deficient mouse line provides a useful platform for future studies on functional aspects of C4.4A in tumour cell invasion in vivo.
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http://dx.doi.org/10.1038/srep25833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864438PMC
May 2016

Prior renovascular hypertension does not predispose to atherosclerosis in mice.

Atherosclerosis 2016 06 24;249:157-63. Epub 2016 Mar 24.

Department of Clinical Medicine, Aarhus University, Denmark; Department of Cardiology, Aarhus University Hospital, Denmark.

Background: Hypertension is a major risk factor for development of atherosclerotic cardiovascular disease (ASCVD). Although lowering blood pressure with antihypertensive drugs reduces the increased risk of ASCVD, residual increased risk still remains, suggesting that hypertension may cause chronic changes that promote atherosclerosis. Thus, we tested the hypothesis that hypertension increases the susceptibility to atherosclerosis in mice even after a period of re-established normotension.

Methods: We used the 2-kidney, 1-clip (2K1C) technique to induce angiotensin-driven renovascular hypertension, and overexpression of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene to cause severe hypercholesterolemia and atherosclerosis.

Results: First, we performed 2K1C (n = 8) or sham surgery (n = 9) in PCSK9 transgenic mice before they were fed a high fat diet for 14 weeks. As expected, 2K1C did not affect cholesterol levels, but induced cardiac hypertrophy and significantly increased the atherosclerotic lesion area compared to sham mice (1.8 fold, p < 0.01). Next, we performed 2K1C (n = 13) or sham surgery (n = 14) in wild-type mice but removed the clipped/sham-operated kidney after 10 weeks to eliminate hypertension, and subsequently induced hypercholesterolemia by way of adeno-associated virus-mediated hepatic gene transfer of PCSK9 combined with high-fat diet. After 14 weeks of hypercholesterolemia, atherosclerotic lesion areas were not significantly different in mice with or without prior 2K1C hypertension (0.95 fold, p = 0.35).

Conclusion: Renovascular hypertension in mice does not induce pro-atherogenic changes that persist beyond the hypertensive phase. These results indicate that hypertension only promotes atherogenesis when coinciding temporally with hypercholesterolemia.
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http://dx.doi.org/10.1016/j.atherosclerosis.2016.03.030DOI Listing
June 2016

Glutamate-system defects behind psychiatric manifestations in a familial hemiplegic migraine type 2 disease-mutation mouse model.

Sci Rep 2016 Feb 25;6:22047. Epub 2016 Feb 25.

Aarhus University, Department of Biomedicine, DK-8000 Aarhus, Denmark.

Migraine is a complex brain disorder, and understanding the complexity of this prevalent disease could improve quality of life for millions of people. Familial Hemiplegic Migraine type 2 (FHM2) is a subtype of migraine with aura and co-morbidities like epilepsy/seizures, cognitive impairments and psychiatric manifestations, such as obsessive-compulsive disorder (OCD). FHM2 disease-mutations locate to the ATP1A2 gene encoding the astrocyte-located α2-isoform of the sodium-potassium pump (α2Na(+)/K(+)-ATPase). We show that knock-in mice heterozygous for the FHM2-associated G301R-mutation (α2(+/G301R)) phenocopy several FHM2-relevant disease traits e.g., by mimicking mood depression and OCD. In vitro studies showed impaired glutamate uptake in hippocampal mixed astrocyte-neuron cultures from α2(G301R/G301R) E17 embryonic mice, and moreover, induction of cortical spreading depression (CSD) resulted in reduced recovery in α2(+/G301R) male mice. Moreover, NMDA-type glutamate receptor antagonists or progestin-only treatment reverted specific α2(+/G301R) behavioral phenotypes. Our findings demonstrate that studies of an in vivo relevant FHM2 disease knock-in mouse model provide a link between the female sex hormone cycle and the glutamate system and a link to co-morbid psychiatric manifestations of FHM2.
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http://dx.doi.org/10.1038/srep22047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766516PMC
February 2016

MFAP4 Promotes Vascular Smooth Muscle Migration, Proliferation and Accelerates Neointima Formation.

Arterioscler Thromb Vasc Biol 2016 Jan 12;36(1):122-33. Epub 2015 Nov 12.

From the Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark (A.S., B.P., L.E.H., K.K., G.B.K., H.W.-J., J.B.M., K.K.-M., L.K.D., P.B.L.H., J.S., U.H, G.L.S.); Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark (E.-M.F.); Department of Pathology, Odense University Hospital, Odense, Denmark (O.N.); Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany (C.W., J.H., M.O.); Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany (C.W., J.H., M.O.); REBIRTH Cluster of Excellence, Hannover, Germany (C.W., J.H., M.O.); German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany (B.R., A.S., V.G.-D., H.F., M.H.d.A.); Institute of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilians-University München, Munich, Germany (B.R, E.W.); Division of Cardiology, Department of Medicine III, University of Heidelberg, Heidelberg, Germany (A.S., R.B.); Chair of Experimental Genetics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, Freising-Weihenstephan, Munich, Germany (M.H.d.A.); Cardiovascular Research Unit, Viborg Hospital, Viborg, Denmark (J.S.L.); and Department of Cardiothoracic and Vascular Surgery, Center of Individualized Medicine in Arterial Diseases (CIMA), Odense University Hospital, Odense, Denmark (J.S.L.).

Objective: Arterial injury stimulates remodeling responses that, when excessive, lead to stenosis. These responses are influenced by integrin signaling in vascular smooth muscle cells (VSMCs). Microfibrillar-associated protein 4 (MFAP4) is an integrin ligand localized to extracellular matrix fibers in the vascular wall. The role of MFAP4 in vascular biology is unknown. We aimed to test the hypothesis that MFAP4 would enhance integrin-dependent VSMC activation.

Approach And Results: We produced Mfap4-deficient (Mfap4(-/-)) mice and performed carotid artery ligation to explore the role of MFAP4 in vascular biology in vivo. Furthermore, we investigated the effects of MFAP4 in neointimal formation ex vivo and in primary VSMC and monocyte cultures in vitro. When challenged with carotid artery ligation, Mfap4(-/-) mice exhibited delayed neointimal formation, accompanied by early reduction in the number of proliferating medial and neointimal cells, as well as infiltrating leukocytes. Delayed neointimal formation was associated with decreased cross-sectional area of ligated Mfap4(-/-) carotid arteries resulting in lumen narrowing 28 days after ligation. MFAP4 blockade prohibited the formation of neointimal hyperplasia ex vivo. Moreover, we demonstrated that MFAP4 is a ligand for integrin αVβ3 and mediates VSMC phosphorylation of focal adhesion kinase, migration, and proliferation in vitro. MFAP4-dependent VSMC activation was reversible by treatment with MFAP4-blocking antibodies and inhibitors of focal adhesion kinase and downstream kinases. In addition, we showed that MFAP4 promotes monocyte chemotaxis in integrin αVβ3-dependent manner.

Conclusions: MFAP4 regulates integrin αVβ3-induced VSMC proliferation and migration, as well as monocyte chemotaxis, and accelerates neointimal hyperplasia after vascular injury.
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http://dx.doi.org/10.1161/ATVBAHA.115.306672DOI Listing
January 2016

Distinct Functions for Anterograde and Retrograde Sorting of SORLA in Amyloidogenic Processes in the Brain.

J Neurosci 2015 Sep;35(37):12703-13

Max Delbrueck Center for Molecular Medicine, D-13125 Berlin, Germany and

Unlabelled: SORLA is a neuronal sorting receptor implicated both in sporadic and familial forms of AD. SORLA reduces the amyloidogenic burden by two mechanisms, either by rerouting internalized APP molecules from endosomes to the trans-Golgi network (TGN) to prevent proteolytic processing or by directing newly produced Aβ to lysosomes for catabolism. Studies in cell lines suggested that the interaction of SORLA with cytosolic adaptors retromer and GGA is required for receptor sorting to and from the TGN. However, the relevance of anterograde or retrograde trafficking for SORLA activity in vivo remained largely unexplored. Here, we generated mouse models expressing SORLA variants lacking binding sites for GGA or retromer to query this concept in the brain. Disruption of retromer binding resulted in a retrograde-sorting defect with accumulation of SORLA in endosomes and depletion from the TGN, and in an overall enhanced APP processing. In contrast, disruption of the GGA interaction did not impact APP processing but caused increased brain Aβ levels, a mechanism attributed to a defect in anterograde lysosomal targeting of Aβ. Our findings substantiated the significance of adaptor-mediated sorting for SORLA activities in vivo, and they uncovered that anterograde and retrograde sorting paths may serve discrete receptor functions in amyloidogenic processes.

Significance Statement: SORLA is a sorting receptor that directs target proteins to distinct intracellular compartments in neurons. SORLA has been identified as a genetic risk factor for sporadic, but recently also for familial forms of AD. To confirm the relevance of SORLA sorting for AD processes in the brain, we generated mouse lines, which express trafficking mutants instead of the wild-type form of this receptor. Studying neuronal activities in these mutant mice, we dissected distinct trafficking routes for SORLA guided by two cytosolic adaptors termed GGA and retromer. We show that these sorting pathways serve discrete functions in control of amyloidogenic processes and may represent unique therapeutic targets to interfere with specific aspects of neurodegenerative processes in the diseased brain.
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http://dx.doi.org/10.1523/JNEUROSCI.0427-15.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795211PMC
September 2015

The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses.

Genes Dev 2015 Mar;29(5):538-54

The Eunice Kennedy Shriver National Institute of Child Health and Human Development, The National Institutes of Health, Bethesda, Maryland 20892, USA;

Retroviruses have been invading mammalian germlines for millions of years, accumulating in the form of endogenous retroviruses (ERVs) that account for nearly one-tenth of the mouse and human genomes. ERVs are epigenetically silenced during development, yet the cellular factors recognizing ERVs in a sequence-specific manner remain elusive. Here we demonstrate that ZFP809, a member of the Krüppel-associated box zinc finger protein (KRAB-ZFP) family, initiates the silencing of ERVs in a sequence-specific manner via recruitment of heterochromatin-inducing complexes. ZFP809 knockout mice display highly elevated levels of ZFP809-targeted ERVs in somatic tissues. ERV reactivation is accompanied by an epigenetic shift from repressive to active histone modifications but only slight destabilization of DNA methylation. Importantly, using conditional alleles and rescue experiments, we demonstrate that ZFP809 is required to initiate ERV silencing during embryonic development but becomes largely dispensable in somatic tissues. Finally, we show that the DNA-binding specificity of ZFP809 is evolutionarily conserved in the Muroidea superfamily of rodents and predates the endogenization of retroviruses presently targeted by ZFP809 in Mus musculus. In sum, these data provide compelling evidence that ZFP809 evolved to recognize foreign DNA and establish histone modification-based epigenetic silencing of ERVs.
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http://dx.doi.org/10.1101/gad.252767.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358406PMC
March 2015

Stanniocalcin-2 inhibits mammalian growth by proteolytic inhibition of the insulin-like growth factor axis.

J Biol Chem 2015 Feb 22;290(6):3430-9. Epub 2014 Dec 22.

From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark

Mammalian stanniocalcin-2 (STC2) is a secreted polypeptide widely expressed in developing and adult tissues. However, although transgenic expression in mice is known to cause severe dwarfism, and targeted deletion of STC2 causes increased postnatal growth, its precise biological role is still unknown. We found that STC2 potently inhibits the proteolytic activity of the growth-promoting metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). Proteolytic inhibition requires covalent binding of STC2 to PAPP-A and is mediated by a disulfide bond, which involves Cys-120 of STC2. Binding of STC2 prevents PAPP-A cleavage of insulin-like growth factor-binding protein (IGFBP)-4 and hence release within tissues of bioactive IGF, required for normal growth. Concordantly, we show that STC2 efficiently inhibits PAPP-A-mediated IGF receptor signaling in vitro and that transgenic mice expressing a mutated variant of STC2, STC2(C120A), which is unable to inhibit PAPP-A, grow like wild-type mice. Our work identifies STC2 as a novel proteinase inhibitor and a previously unrecognized extracellular component of the IGF system.
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http://dx.doi.org/10.1074/jbc.M114.611665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4319012PMC
February 2015

Lack of the serum- and glucocorticoid-inducible kinase SGK1 improves muscle force characteristics and attenuates fibrosis in dystrophic mdx mouse muscle.

Pflugers Arch 2015 Sep 14;467(9):1965-74. Epub 2014 Nov 14.

Institute of Pathophysiology, University Medicine Greifswald, Greifswalder Str. 11c, 17495, Karlsburg, Germany.

Duchenne muscular dystrophy (DMD) is a human genetic disease characterized by fibrosis and severe muscle weakness. Currently, there is no effective treatment available to prevent progressive fibrosis in skeletal muscles. The serum- and glucocorticoid-inducible kinase SGK1 regulates a variety of physiological functions and participates in fibrosis stimulation. Here, we investigated whether SGK1 influences structure, function and/or fibrosis of the muscles from the mdx mouse, an animal model for DMD. As expected, mdx muscles showed the typical pathological features of muscular dystrophy including fiber size variations, central nuclei of muscle fibers, fibrosis in the diaphragm, and force reduction by 30-50 %. Muscles from sgk1 (-/-) mice were histologically overall intact and specific force was only slightly reduced compared to wild-type muscles. Surprisingly, soleus and diaphragm muscles of mdx/sgk1 (-/-) mice displayed forces close to wild-type levels. Most muscle fibers of the double mutants contained central nuclei, but fibrosis was not observed in any of the tested limb and diaphragm muscles. We conclude that the sole lack of SGK1 in mouse muscle does not lead to pronounced changes in muscle structure and function. However, dystrophin-deficient mdx muscle seems to benefit from SGK1 deficiency. SGK1 appears to be an important enzyme in the process of fibrotic remodeling and subsequent weakness of dystrophin-deficient mouse muscle.
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http://dx.doi.org/10.1007/s00424-014-1645-5DOI Listing
September 2015

SorCS2 regulates dopaminergic wiring and is processed into an apoptotic two-chain receptor in peripheral glia.

Neuron 2014 Jun;82(5):1074-87

The Lundbeck Foundation Research Center MIND, Department of Biomedicine, Aarhus University, Vennelyst Boulevard 4, 8000 C Aarhus, Denmark; Danish Research Institute of Translational Neuroscience DANDRITE Nordic-EMBL Partnership, Department of Biomedicine, Aarhus University, Vennelyst Boulevard 4, 8000 C Aarhus, Denmark; Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA. Electronic address:

Balancing trophic and apoptotic cues is critical for development and regeneration of neuronal circuits. Here we identify SorCS2 as a proneurotrophin (proNT) receptor, mediating both trophic and apoptotic signals in conjunction with p75(NTR). CNS neurons, but not glia, express SorCS2 as a single-chain protein that is essential for proBDNF-induced growth cone collapse in developing dopaminergic processes. SorCS2- or p75(NTR)-deficient in mice caused reduced dopamine levels and metabolism and dopaminergic hyperinnervation of the frontal cortex. Accordingly, both knockout models displayed a paradoxical behavioral response to amphetamine reminiscent of ADHD. Contrary, in PNS glia, but not in neurons, proteolytic processing produced a two-chain SorCS2 isoform that mediated proNT-dependent Schwann cell apoptosis. Sciatic nerve injury triggered generation of two-chain SorCS2 in p75(NTR)-positive dying Schwann cells, with apoptosis being profoundly attenuated in Sorcs2(-/-) mice. In conclusion, we have demonstrated that two-chain processing of SorCS2 enables neurons and glia to respond differently to proneurotrophins.
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http://dx.doi.org/10.1016/j.neuron.2014.04.022DOI Listing
June 2014