Publications by authors named "Magdalena Laugsch"

13 Publications

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Variants in PRKAR1B cause a neurodevelopmental disorder with autism spectrum disorder, apraxia, and insensitivity to pain.

Genet Med 2021 Apr 8. Epub 2021 Apr 8.

Institute of Human Genetics, Heidelberg University, Heidelberg, Germany.

Purpose: We characterize the clinical and molecular phenotypes of six unrelated individuals with intellectual disability and autism spectrum disorder who carry heterozygous missense variants of the PRKAR1B gene, which encodes the R1β subunit of the cyclic AMP-dependent protein kinase A (PKA).

Methods: Variants of PRKAR1B were identified by single- or trio-exome analysis. We contacted the families and physicians of the six individuals to collect phenotypic information, performed in vitro analyses of the identified PRKAR1B-variants, and investigated PRKAR1B expression during embryonic development.

Results: Recent studies of large patient cohorts with neurodevelopmental disorders found significant enrichment of de novo missense variants in PRKAR1B. In our cohort, de novo origin of the PRKAR1B variants could be confirmed in five of six individuals, and four carried the same heterozygous de novo variant c.1003C>T (p.Arg335Trp; NM_001164760). Global developmental delay, autism spectrum disorder, and apraxia/dyspraxia have been reported in all six, and reduced pain sensitivity was found in three individuals carrying the c.1003C>T variant. PRKAR1B expression in the brain was demonstrated during human embryonal development. Additionally, in vitro analyses revealed altered basal PKA activity in cells transfected with variant-harboring PRKAR1B expression constructs.

Conclusion: Our study provides strong evidence for a PRKAR1B-related neurodevelopmental disorder.
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http://dx.doi.org/10.1038/s41436-021-01152-7DOI Listing
April 2021

Dosage analysis of the 7q11.23 Williams region identifies as a major human gene patterning the modern human face and underlying self-domestication.

Sci Adv 2019 12 4;5(12):eaaw7908. Epub 2019 Dec 4.

Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy.

We undertook a functional dissection of chromatin remodeler BAZ1B in neural crest (NC) stem cells (NCSCs) from a uniquely informative cohort of typical and atypical patients harboring 7q11.23 copy number variants. Our results reveal a key contribution of BAZ1B to NCSC in vitro induction and migration, coupled with a crucial involvement in NC-specific transcriptional circuits and distal regulation. By intersecting our experimental data with new paleogenetic analyses comparing modern and archaic humans, we found a modern-specific enrichment for regulatory changes both in BAZ1B and its experimentally defined downstream targets, thereby providing the first empirical validation of the human self-domestication hypothesis and positioning BAZ1B as a master regulator of the modern human face. In so doing, we provide experimental evidence that the craniofacial and cognitive/behavioral phenotypes caused by alterations of the Williams-Beuren syndrome critical region can serve as a powerful entry point into the evolution of the modern human face and prosociality.
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http://dx.doi.org/10.1126/sciadv.aaw7908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892627PMC
December 2019

mTOR and autophagy pathways are dysregulated in murine and human models of Schaaf-Yang syndrome.

Sci Rep 2019 11 4;9(1):15935. Epub 2019 Nov 4.

Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX, 77030, USA.

MAGEL2 is a maternally imprinted, paternally expressed gene, located in the Prader-Willi region of human chromosome 15. Pathogenic variants in the paternal copy of MAGEL2 cause Schaaf-Yang syndrome (SHFYNG), a neurodevelopmental disorder related to Prader-Willi syndrome (PWS). Patients with SHFYNG, like PWS, manifest neonatal hypotonia, feeding difficulties, hypogonadism, intellectual disability and sleep apnea. However, individuals with SHFYNG have joint contractures, greater cognitive impairment, and higher prevalence of autism than seen in PWS. Additionally, SHFYNG is associated with a lower prevalence of hyperphagia and obesity than PWS. Previous studies have shown that truncating variants in MAGEL2 lead to SHFYNG. However, the molecular pathways involved in manifestation of the SHFYNG disease phenotype are still unknown. Here we show that a Magel2 null mouse model and fibroblast cell lines from individuals with SHFYNG exhibit increased expression of mammalian target of rapamycin (mTOR) and decreased autophagy. Additionally, we show that SHFYNG induced pluripotent stem cell (iPSC)-derived neurons exhibit impaired dendrite formation. Alterations in SHFYNG patient fibroblast lines and iPSC-derived neurons are rescued by treatment with the mTOR inhibitor rapamycin. Collectively, our findings identify mTOR as a potential target for the development of pharmacological treatments for SHFYNG.
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http://dx.doi.org/10.1038/s41598-019-52287-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828689PMC
November 2019

Modeling the Pathological Long-Range Regulatory Effects of Human Structural Variation with Patient-Specific hiPSCs.

Cell Stem Cell 2019 05 11;24(5):736-752.e12. Epub 2019 Apr 11.

Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; Cluster of Excellence Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany; Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), University of Cantabria, Cantabria, Spain. Electronic address:

The pathological consequences of structural variants disrupting 3D genome organization can be difficult to elucidate in vivo due to differences in gene dosage sensitivity between mice and humans. This is illustrated by branchiooculofacial syndrome (BOFS), a rare congenital disorder caused by heterozygous mutations within TFAP2A, a neural crest regulator for which humans, but not mice, are haploinsufficient. Here, we present a BOFS patient carrying a heterozygous inversion with one breakpoint located within a topologically associating domain (TAD) containing enhancers essential for TFAP2A expression in human neural crest cells (hNCCs). Using patient-specific hiPSCs, we show that, although the inversion shuffles the TFAP2A hNCC enhancers with novel genes within the same TAD, this does not result in enhancer adoption. Instead, the inversion disconnects one TFAP2A allele from its cognate enhancers, leading to monoallelic and haploinsufficient TFAP2A expression in patient hNCCs. Our work illustrates the power of hiPSC differentiation to unveil long-range pathomechanisms.
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http://dx.doi.org/10.1016/j.stem.2019.03.004DOI Listing
May 2019

Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs.

Mol Ther 2016 Apr 28;24(4):812-22. Epub 2015 Aug 28.

Stem Cell Engineering, BIOTEC, TU Dresden, Dresden, Germany.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.
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http://dx.doi.org/10.1038/mt.2015.154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886927PMC
April 2016

Imbalance of SMC1 and SMC3 cohesins causes specific and distinct effects.

PLoS One 2013 12;8(6):e65149. Epub 2013 Jun 12.

Institute of Physiological Chemistry, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden, Germany.

SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065149PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680458PMC
January 2014

Generation of inducible immortalized dendritic cells with proper immune function in vitro and in vivo.

PLoS One 2013 23;8(4):e62621. Epub 2013 Apr 23.

Department of Pediatrics, University Clinic Carl Gustav Carus, Technische Universitaet Dresden, Dresden, Germany.

Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. We differentiated bone marrow cells from transgenic mice expressing an inducible SV40 large T-antigen into dendritic cells. When immortalized by dexamethasone and doxycycline, these cells were stable in long-term culture. In the absence of dexamethasone and doxycycline (de-induction), dendritic cells displayed properties of primary cells, characterized by expression of classical dendritic cell surface markers CD11c, CD11b, MHCII, CD40 and CD86. Furthermore, de-induced lipopolysaccharide activated dendritic cells secreted IL-1β, IL-6, TNFα and IL-12. De-induced, Ovalbumin-loaded dendritic cells polarize CD4(+) T cells into Th1, Th17 and Th2 cells, indicating their correct antigen presenting property. Consistent with intratracheal application of Ovalbumin-loaded primary dendritic cells into mice, the application of de-induced dendritic cells resulted in recruitment of lymphocytes to the lungs. In summary, we successfully expanded dendritic cells using conditional immortalization. The generated dendritic cells demonstrate the characteristic immunophenotype of primary dendritic cells and will facilitate further studies on immunomodulatory properties of dendritic cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062621PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633827PMC
November 2013

Lack of functional erythropoietin receptors of cancer cell lines.

Int J Cancer 2008 Mar;122(5):1005-11

Institute of Physiology, University of Luebeck, D-23538 Luebeck, Germany.

Erythropoietin (Epo) therapy reduces red cell transfusion requirements and improves the quality of life of anemic cancer patients receiving chemotherapy. However, there is concern that Epo may promote tumor growth. We investigated by real-time RT-PCR, immunofluorescence microscopy, Western blotting and cell growth analysis whether human cancer cell lines (SH-SY5Y, MCF7, HepG2, U2-OS, HeLa, HEK293T, RCC4, HCT116, 7860wt and SW480) possess functional Epo receptors (EpoR). We detected EpoR mRNA in all cell lines. Neither hypoxia nor Epo treatment altered the level of EpoR mRNA expression. Four commonly used commercial antibodies proved to be unsuitable for immunoblot procedures because they cross-reacted with several proteins unrelated with EpoR. Depending on the antibody used, EpoR was localized to the plasma membrane, the cytoplasm or the nucleus. Experiments with small interfering RNA showed that EpoR protein was not expressed by the tumor cells except by UT7/Epo leukemia cells, which served as an EpoR positive control line, and by cells transfected with the human EpoR gene. Apart from UT7/Epo, none of the tumor cell lines responded to Epo treatment with phosphorylation of signaling molecules or with cell proliferation.
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http://dx.doi.org/10.1002/ijc.23201DOI Listing
March 2008

Human hair follicles are an extrarenal source and a nonhematopoietic target of erythropoietin.

FASEB J 2007 Oct 31;21(12):3346-54. Epub 2007 May 31.

Department of Dermatology, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany.

Erythropoietin primarily serves as an essential growth factor for erythrocyte precursor cells. However, there is increasing evidence that erythropoietin (EPO)/EPO receptor (EPO-R) signaling operates as a potential tissue-protective system outside the bone marrow. Arguing that growing hair follicles (HF) are among the most rapidly proliferating tissues, we have here explored whether human HFs are sources of EPO and targets of EPO-R-mediated signaling. Human scalp skin and microdissected HFs were assessed for EPO and EPO-R expression, and the effects of EPO on organ-cultured HFs were assessed in the presence/absence of a classical apoptosis-inducing chemotherapeutic agent. Here, we show that human scalp HFs express EPO on the mRNA and protein level in situ, up-regulate EPO transcription under hypoxic conditions, and express transcripts for EPO-R and the EPO-stimulatory transcriptional cofactor hypoxia-inducible factor-1alpha. Although EPO does not significantly alter human hair growth in vitro, it significantly down-regulates chemotherapy-induced intrafollicular apoptosis and changes the gene expression program of the HFs. The current study points to intriguing targets of EPO beyond the erythropoietic system: human HFs are an extrarenal site of EPO production and an extrahematopoietic site of EPO-R expression. They may recruit EPO/EPO-R signaling e.g., for modulating HF apoptosis under conditions of hypoxia and chemotherapy-induced stress.
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http://dx.doi.org/10.1096/fj.07-8628comDOI Listing
October 2007

Differences in maternal supply and early development of closely related nematode species.

Int J Dev Biol 2004 Sep;48(7):655-62

Zoologisches Institut, Universitat Koln, Koln, Germany.

Comparative analyses revealed considerable differences in embryonic pattern formation and cell-specification between Caenorhabditis elegans and Acrobeloides nanus, members of two neighboring nematode clades. While C.elegans develops very rapidly, A. nanus needs 4-5 times as long. To investigate whether differences during early embryogenesis could be related to developmental tempo, we studied three more slowly developing representatives of the genus Rhabditis, thus close relatives of C.elegans. Besides differences in body size and mode of reproduction, they differ from C.elegans in the order of cleavages, germline behavior and requirement for early zygotic transcription, showing evident similarities to A. nanus. The distinct variations in cell-cycle rhythms and arrest after inhibition of transcription appear to reflect a species-specific interplay in the timing between exhausting maternal supplies and making available newly transcribed gene products. Looking for the reversal of cleavage polarity in the germline present in C.elegans but not in A. nanus, two of the studied species express this distinct feature only in a later cell generation. We found that a C.elegans mutant in the mes-1 gene shows a similar deviation. Concerning specification of the gut cell lineage and the potential to compensate for lost cells, the three tested Rhabditis species behave less regulatively, like C.elegans; in contrast to A. nanus, the gut precursor EMS requires an inductive signal from the germline cell P2 and an experimentally eliminated EMS cell is not replaced by a neighboring blastomere. In conclusion, embryogenesis of the examined Rhabditis species includes features of both the fast-developing C. elegans and the slow-developing A. nanus.
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http://dx.doi.org/10.1387/ijdb.031758mlDOI Listing
September 2004

Heterodimerization of substance P and mu-opioid receptors regulates receptor trafficking and resensitization.

J Biol Chem 2003 Dec 7;278(51):51630-7. Epub 2003 Oct 7.

Department of Pharmacology and Toxicology, Otto-von-Guericke University, 39120 Magdeburg, Germany.

The micro-opioid receptor (MOR1) and the substance P receptor (NK1) coexist and functionally interact in nociceptive brain regions; however, a molecular basis for this interaction has not been established. Using coimmunoprecipitation and bioluminescence resonance energy transfer (BRET), we show that MOR1 and NK1 can form heterodimers in HEK 293 cells coexpressing the two receptors. Although NK1-MOR1 heterodimerization did not substantially change the ligand binding and signaling properties of these receptors, it dramatically altered their internalization and resensitization profile. Exposure of the NK1-MOR1 heterodimer to the MOR1-selective ligand [D-Ala2,Me-Phe4,Gly5-ol]enkephalin (DAMGO) promoted cross-phosphorylation and cointernalization of the NK1 receptor. Conversely, exposure of the NK1-MOR1 heterodimer to the NK1-selective ligand substance P (SP) promoted cross-phosphorylation and cointernalization of the MOR1 receptor. In cells expressing MOR1 alone, beta-arrestin directs the receptors to clathrin-coated pits, but does not internalize with the receptor. In cells expressing NK1 alone, beta-arrestin internalizes with the receptor into endosomes. Interestingly, in cells coexpressing MOR1 and NK1 both DAMGO and SP induced the recruitment of beta-arrestin to the plasma membrane and cointernalization of NK1-MOR1 heterodimers with beta-arrestin into the same endosomal compartment. Consequently, resensitization of MOR1-dependent receptor functions was severely delayed in coexpressing cells as compared with cells expressing MOR1 alone. Together, our findings indicate that MOR1 by virtue of its physical interaction with NK1 is sequestered via an endocytotic pathway with delayed recycling and resensitization kinetics.
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http://dx.doi.org/10.1074/jbc.M307095200DOI Listing
December 2003

Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization.

J Biol Chem 2002 May 14;277(22):19762-72. Epub 2002 Mar 14.

Department of Pharmacology and Toxicology, Otto-von-Guericke University, 39120 Magdeburg, Germany.

Heterodimerization has been shown to modulate the ligand binding, signaling, and trafficking properties of G protein-coupled receptors. However, to what extent heterodimerization may alter agonist-induced phosphorylation and desensitization of these receptors has not been documented. We have recently shown that heterodimerization of sst(2A) and sst(3) somatostatin receptors results in inactivation of sst(3) receptor function (Pfeiffer, M., Koch, T., Schröder, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., Höllt, V., and Schulz, S. (2001) J. Biol. Chem. 276, 14027-14036). Here we examine dimerization of the sst(2A) somatostatin receptor and the mu-opioid receptor, members of closely related G protein-coupled receptor families. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and MOR1 in human embryonic kidney 293 cells. Unlike heteromeric assembly of sst(2A) and sst(3), sst(2A)-MOR1 heterodimerization did not substantially alter the ligand binding or coupling properties of these receptors. However, exposure of the sst(2A)-MOR1 heterodimer to the sst(2A)-selective ligand L-779,976 induced phosphorylation, internalization, and desensitization of sst(2A) as well as MOR1. Similarly, exposure of the sst(2A)-MOR1 heterodimer to the mu-selective ligand [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin induced phosphorylation and desensitization of both MOR1 and sst(2A) but not internalization of sst(2A). Cross-phosphorylation and cross-desensitization of the sst(2A)-MOR1 heterodimer were selective; they were neither observed with the sst(2A)-sst(3) heterodimer nor with the endogenously expressed lysophosphatidic acid receptor. Heterodimerization may thus represent a novel regulatory mechanism that could either restrict or enhance phosphorylation and desensitization of G protein-coupled receptors.
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http://dx.doi.org/10.1074/jbc.M110373200DOI Listing
May 2002