Publications by authors named "Gabriela Salinas"

43 Publications

Regenerative potential of epicardium-derived extracellular vesicles mediated by conserved miRNA transfer.

Cardiovasc Res 2021 Feb 18. Epub 2021 Feb 18.

Department of Physiology, Anatomy and Genetics, British Heart Foundation, Oxbridge Centre of Regenerative Medicine, University of Oxford, UK.

Aims: After a myocardial infarction, the adult human heart lacks sufficient regenerative capacity to restore lost tissue, leading to heart failure progression. Finding novel ways to reprogram adult cardiomyocytes into a regenerative state is a major therapeutic goal. The epicardium, the outermost layer of the heart, contributes cardiovascular cell types to the forming heart and is a source of trophic signals to promote heart muscle growth during embryonic development. The epicardium is also essential for heart regeneration in zebrafish and neonatal mice and can be reactivated after injury in adult hearts to improve outcome. A recently identified mechanism of cell-cell communication and signalling is that mediated by extracellular vesicles (EVs). Here, we aimed to investigate epicardial signalling via EV release in response to cardiac injury and as a means to optimise cardiac repair and regeneration.

Methods And Results: We isolated epicardial EVs from mouse and human sources and targeted the cardiomyocyte population. Epicardial EVs enhanced proliferation in H9C2 cells and in primary neonatal murine cardiomyocytes in vitro and promoted cell cycle re-entry when injected into the injured area of infarcted neonatal hearts. These EVs also enhanced regeneration in cryo-injured engineered human myocardium (EHM) as a novel model of human myocardial injury. Deep RNA-sequencing of epicardial EV cargo revealed conserved microRNAs (miRs) between human and mouse-epicardial-derived exosomes, and the effects on cell cycle re-entry were recapitulated by administration of cargo miR-30a, miR-100, miR-27a and miR-30e to human stem cell-derived cardiomyocytes and cryo-injured EHM constructs.

Conclusion: Here we describe the first characterisation of epicardial EV secretion, which can signal to promote proliferation of cardiomyocytes in infarcted mouse hearts and in a human model of myocardial injury, resulting in enhanced contractile function. Analysis of exosome cargo in mouse and human identified conserved pro-regenerative microRNAs which in combination recapitulated the therapeutic effects of promoting cardiomyocyte proliferation.

Translational Perspective: Coronary artery disease leading to myocardial infarction ("heart attack") is a major cause of death worldwide . After a heart attack, the myocardium is unable to regenerate, therefore, the heart remains permanently scarred and unable to carry out its function, which eventually leads to heart failure. Current therapies are palliative, aimed at unburdening the failing heart, or heart transplantation. Reprogramming adult cardiomyocytes to a proliferative state, via the use of non-inmunogenic EVs is a potential therapeutic approach to restore lost heart muscle, resolve scarring and prevent heart failure progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvab054DOI Listing
February 2021

EVL regulates VEGF receptor-2 internalization and signaling in developmental angiogenesis.

EMBO Rep 2021 Feb 29;22(2):e48961. Epub 2021 Jan 29.

Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany.

Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.201948961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857432PMC
February 2021

Toxicogenomic fin(ger)prints for thyroid disruption AOP refinement and biomarker identification in zebrafish embryos.

Sci Total Environ 2021 Mar 10;760:143914. Epub 2020 Dec 10.

Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany. Electronic address:

Endocrine disruption (ED) can trigger far-reaching effects on environmental populations, justifying a refusal of market approval for chemicals with ED properties. For the hazard assessment of ED effects on the thyroid system, regulatory decisions mostly rely on amphibian studies. Here, we used transcriptomics and proteomics for identifying molecular signatures of interference with thyroid hormone signaling preceding physiological effects in zebrafish embryos. For this, we analyzed the thyroid hormone 3,3',5-triiodothyronine (T3) and the thyroid peroxidase inhibitor 6-propyl-2-thiouracil (6-PTU) as model substances for increased and repressed thyroid hormone signaling in a modified zebrafish embryo toxicity test. We identified consistent gene expression fingerprints for both modes-of-action (MoA) at sublethal test concentrations. T3 and 6-PTU both significantly target the expression of genes involved in muscle contraction and functioning in an opposing fashion, allowing for a mechanistic refinement of key event relationships in thyroid-related adverse outcome pathways in fish. Furthermore, our fingerprints identify biomarker candidates for thyroid disruption hazard screening approaches. Perspectively, our findings will promote the AOP-based development of in vitro assays for thyroidal ED assessment, which in the long term will contribute to a reduction of regulatory animal tests.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scitotenv.2020.143914DOI Listing
March 2021

Long-term effects of empagliflozin on excitation-contraction-coupling in human induced pluripotent stem cell cardiomyocytes.

J Mol Med (Berl) 2020 12 9;98(12):1689-1700. Epub 2020 Oct 9.

Department of Internal Medicine II, University Medical Center Regensburg, Franz-Josef-Strauß-Allee 11, 93053, Regensburg, Germany.

The SGLT2 inhibitor empagliflozin improved cardiovascular outcomes in patients with diabetes. As the cardiac mechanisms remain elusive, we investigated the long-term effects (up to 2 months) of empagliflozin on excitation-contraction (EC)-coupling in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) in a blinded manner. IPSC from 3 donors, differentiated into pure iPSC-CM (4 differentiations), were treated with a clinically relevant concentration of empagliflozin (0.5 μmol/l) or vehicle control. Treatment, data acquisition, and analysis were conducted externally blinded. Epifluorescence microscopy measurements in iPSC-CM showed that empagliflozin has neutral effects on Ca transient amplitude, diastolic Ca levels, Ca transient kinetics, or sarcoplasmic Ca load after 2 weeks or 8 weeks of treatment. Confocal microscopy determining possible effects on proarrhythmogenic diastolic Ca release events showed that in iPSC-CM, Ca spark frequency and leak was not altered after chronic treatment with empagliflozin. Finally, in patch-clamp experiments, empagliflozin did not change action potential duration, amplitude, or resting membrane potential compared with vehicle control after long-term treatment. Next-generation RNA sequencing (NGS) and mapped transcriptome profiles of iPSC-CMs untreated and treated with empagliflozin for 8 weeks showed no differentially expressed EC-coupling genes. In line with NGS data, Western blots indicate that empagliflozin has negligible effects on key EC-coupling proteins. In this blinded study, direct treatment of iPSC-CM with empagliflozin for a clinically relevant duration of 2 months did not influence cardiomyocyte EC-coupling and electrophysiology. Therefore, it is likely that other mechanisms independent of cardiomyocyte EC-coupling are responsible for the beneficial treatment effect of empagliflozin. KEY MESSAGES: This blinded study investigated the clinically relevant long-term effects (up to 2 months) of empagliflozin on cardiomyocyte excitation-contraction (EC)-coupling. Human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) were used to study a human model including a high repetition number of experiments. Empagliflozin has neutral effects on cardiomyocyte Ca transients, sarcoplasmic Ca load, and diastolic sarcoplasmic Ca leak. In patch-clamp experiments, empagliflozin did not change the action potential. Next-generation RNA sequencing, mapped transcriptome profiles, and Western blots of iPSC-CM untreated and treated with empagliflozin showed no differentially expressed EC-coupling candidates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00109-020-01989-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679329PMC
December 2020

Motor neuron translatome reveals deregulation of SYNGR4 and PLEKHB1 in mutant TDP-43 amyotrophic lateral sclerosis models.

Hum Mol Genet 2020 Sep;29(16):2647-2661

Neuronal Translational Control Research Group, Center for Molecular Neurobiology Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg 20251, Germany.

Amyotrophic lateral sclerosis (ALS) is an incurable neurological disease with progressive loss of motor neuron (MN) function in the brain and spinal cord. Mutations in TARDBP, encoding the RNA-binding protein TDP-43, are one cause of ALS, and TDP-43 mislocalization in MNs is a key pathological feature of >95% of ALS cases. While numerous studies support altered RNA regulation by TDP-43 as a major cause of disease, specific changes within MNs that trigger disease onset remain unclear. Here, we combined translating ribosome affinity purification (TRAP) with RNA sequencing to identify molecular changes in spinal MNs of TDP-43-driven ALS at motor symptom onset. By comparing the MN translatome of hTDP-43A315T mice to littermate controls and to mice expressing wild type hTDP-43, we identified hundreds of mRNAs that were selectively up- or downregulated in MNs. We validated the deregulated candidates Tex26, Syngr4, and Plekhb1 mRNAs in an independent TRAP experiment. Moreover, by quantitative immunostaining of spinal cord MNs, we found corresponding protein level changes for SYNGR4 and PLEKHB1. We also observed these changes in spinal MNs of an independent ALS mouse model caused by a different patient mutant allele of TDP-43, suggesting that they are general features of TDP-43-driven ALS. Thus, we identified SYNGR4 and PLEKHB1 to be deregulated in MNs at motor symptom onset in TDP-43-driven ALS models. This spatial and temporal pattern suggests that these proteins could be functionally important for driving the transition to the symptomatic phase of the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddaa140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530531PMC
September 2020

Intronic CRISPR Repair in a Preclinical Model of Noonan Syndrome-Associated Cardiomyopathy.

Circulation 2020 Sep 6;142(11):1059-1076. Epub 2020 Jul 6.

Clinic for Cardiology and Pneumology (U.H., M.K., L.R., R.H., G.H., L.C.).

Background: Noonan syndrome (NS) is a multisystemic developmental disorder characterized by common, clinically variable symptoms, such as typical facial dysmorphisms, short stature, developmental delay, intellectual disability as well as cardiac hypertrophy. The underlying mechanism is a gain-of-function of the RAS-mitogen-activated protein kinase signaling pathway. However, our understanding of the pathophysiological alterations and mechanisms, especially of the associated cardiomyopathy, remains limited and effective therapeutic options are lacking.

Methods: Here, we present a family with two siblings displaying an autosomal recessive form of NS with massive hypertrophic cardiomyopathy as clinically the most prevalent symptom caused by biallelic mutations within the leucine zipper-like transcription regulator 1 (). We generated induced pluripotent stem cell-derived cardiomyocytes of the affected siblings and investigated the patient-specific cardiomyocytes on the molecular and functional level.

Results: Patients' induced pluripotent stem cell-derived cardiomyocytes recapitulated the hypertrophic phenotype and uncovered a so-far-not-described causal link between LZTR1 dysfunction, RAS-mitogen-activated protein kinase signaling hyperactivity, hypertrophic gene response and cellular hypertrophy. Calcium channel blockade and MEK inhibition could prevent some of the disease characteristics, providing a molecular underpinning for the clinical use of these drugs in patients with NS, but might not be a sustainable therapeutic option. In a proof-of-concept approach, we explored a clinically translatable intronic CRISPR (clustered regularly interspaced short palindromic repeats) repair and demonstrated a rescue of the hypertrophic phenotype.

Conclusions: Our study revealed the human cardiac pathogenesis in patient-specific induced pluripotent stem cell-derived cardiomyocytes from NS patients carrying biallelic variants in and identified a unique disease-specific proteome signature. In addition, we identified the intronic CRISPR repair as a personalized and in our view clinically translatable therapeutic strategy to treat NS-associated hypertrophic cardiomyopathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.119.044794DOI Listing
September 2020

IgG Fc sialylation is regulated during the germinal center reaction following immunization with different adjuvants.

J Allergy Clin Immunol 2020 09 21;146(3):652-666.e11. Epub 2020 May 21.

Laboratories of Immunology and Antibody Glycan Analysis, Institute for Nutritional Medicine, University of Lübeck and University Medical Center Schleswig-Holstein, Lübeck, Germany; Laboratory of Tolerance and Autoimmunity at the German Rheumatism Research Center, a Leibniz Institute, Berlin, Germany; Airway Research Center North, University of Lübeck, German Center for Lung Research, Lübeck, Germany. Electronic address:

Background: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects.

Objective: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants.

Methods: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns.

Results: Different adjuvants induce distinct IgG GC B-cell responses with specific transcriptomes and expression levels of the α2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3 follicular helper T (T) cell-inducing cytokine IL-6, here in particular induced by water-in-oil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-γ T cells, IL-6/IL-23-dependent IL-17A T cells, and high ratios of T cells to Foxp3 follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor.

Conclusion: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2020.04.059DOI Listing
September 2020

De novo mutations in FBRSL1 cause a novel recognizable malformation and intellectual disability syndrome.

Hum Genet 2020 Nov 18;139(11):1363-1379. Epub 2020 May 18.

Institute of Human Genetics, University Medical Center Göttingen, Heinrich-Düker-Weg 12, 37073, Göttingen, Germany.

We report truncating de novo variants in specific exons of FBRSL1 in three unrelated children with an overlapping syndromic phenotype with respiratory insufficiency, postnatal growth restriction, microcephaly, global developmental delay and other malformations. The function of FBRSL1 is largely unknown. Interestingly, mutations in the FBRSL1 paralogue AUTS2 lead to an intellectual disability syndrome (AUTS2 syndrome). We determined human FBRSL1 transcripts and describe protein-coding forms by Western blot analysis as well as the cellular localization by immunocytochemistry stainings. All detected mutations affect the two short N-terminal isoforms, which show a ubiquitous expression in fetal tissues. Next, we performed a Fbrsl1 knockdown in Xenopus laevis embryos to explore the role of Fbrsl1 during development and detected craniofacial abnormalities and a disturbance in neurite outgrowth. The aberrant phenotype in Xenopus laevis embryos could be rescued with a human N-terminal isoform, while the long isoform and the N-terminal isoform containing the mutation p.Gln163* isolated from a patient could not rescue the craniofacial defects caused by Fbrsl1 depletion. Based on these data, we propose that the disruption of the validated N-terminal isoforms of FBRSL1 at critical timepoints during embryogenesis leads to a hitherto undescribed complex neurodevelopmental syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00439-020-02175-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519918PMC
November 2020

Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing.

Cell Death Differ 2020 Oct 27;27(10):2810-2827. Epub 2020 Apr 27.

Department of Neurology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075, Göttingen, Germany.

Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genes Kif1b and Ddit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41418-020-0543-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493890PMC
October 2020

Diversity of Clinically Relevant Outcomes Resulting from Hypofractionated Radiation in Human Glioma Stem Cells Mirrors Distinct Patterns of Transcriptomic Changes.

Cancers (Basel) 2020 Mar 1;12(3). Epub 2020 Mar 1.

Laboratory for Experimental Neurooncology, Clinic for Neurosurgery, Johannes Gutenberg University Medical Centre, 55131 Mainz, Germany.

Hypofractionated radiotherapy is the mainstay of the current treatment for glioblastoma. However, the efficacy of radiotherapy is hindered by the high degree of radioresistance associated with glioma stem cells comprising a heterogeneous compartment of cell lineages differing in their phenotypic characteristics, molecular signatures, and biological responses to external signals. Reconstruction of radiation responses in glioma stem cells is necessary for understanding the biological and molecular determinants of glioblastoma radioresistance. To date, there is a paucity of information on the longitudinal outcomes of hypofractionated radiation in glioma stem cells. This study addresses long-term outcomes of hypofractionated radiation in human glioma stem cells by using a combinatorial approach integrating parallel assessments of the tumor-propagating capacity, stemness-associated properties, and array-based profiling of gene expression. The study reveals a broad spectrum of changes in the tumor-propagating capacity of glioma stem cells after radiation and finds association with proliferative changes at the onset of differentiation. Evidence is provided that parallel transcriptomic patterns and a cumulative impact of pathways involved in the regulation of apoptosis, neural differentiation, and cell proliferation underly similarities in tumorigenicity changes after radiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12030570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139840PMC
March 2020

Ammonium acts systemically while nitrate exerts an additional local effect on Medicago truncatula nodules.

Plant Sci 2020 Mar 19;292:110383. Epub 2019 Dec 19.

Departamento de Producción Agrícola, Facultad de Ciencias Agrarias, Universidad de Talca, Casilla 747, Talca, Chile. Electronic address:

Symbiotic nitrogen fixation (SNF) has a high energetic cost for legume plants; legumes thus reduce SNF when soil N is available. The present study aimed to increase our understanding regarding the impacts of the two principal forms of available N in soils (ammonium and nitrate) on SNF. We continuously measured the SNF of Medicago truncatula under controlled conditions. This permitted nodule sampling for comparative transcriptome profiling at points connected to the nodules' reaction following ammonium or nitrate applications. The N component of both ions systemically induced a rhythmic pattern of SNF, while the activity in control plants remained constant. This rhythmic activity reduced the per-day SNF. The nitrate ion had additional local effects; the more pronounced were a strong downregulation of leghaemoglobin, nodule cysteine-rich (NCR) peptides and nodule-enhanced nicotianamine synthase (neNAS). The neNAS has proven to be of importance for nodule functioning. Although other physiological impacts of nitrate on nodules were observed (e.g. nitrosylation of leghaemoglobin), the main effect was a rapid ion-specific and organ-specific change in gene expression levels. Contrastingly, during the first hours after ammonium applications, the transcriptome remained virtually unaffected. Therefore, nitrate-induced genes could be key for increasing the nitrate tolerance of SNF.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.plantsci.2019.110383DOI Listing
March 2020

Increased presence and differential molecular imprinting of transit amplifying cells in psoriasis.

J Mol Med (Berl) 2020 01 12;98(1):111-122. Epub 2019 Dec 12.

Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology, Charité-Universitätsmedizin, Charitéplatz 1, Berlin, Germany.

Psoriasis is a very common chronic inflammatory skin disease characterized by epidermal thickening and scaling resulting from keratinocyte hyperproliferation and impaired differentiation. Pathomechanistic studies in psoriasis are often limited by using whole skin tissue biopsies, neglecting their stratification and cellular diversity. This study aimed at characterizing epidermal alterations in psoriasis at the level of keratinocyte populations. Epidermal cell populations were purified from skin biopsies of psoriasis patients and healthy donors using a novel cell type-specific approach. Molecular characterization of the transit-amplifying cells (TAC), the key players of epidermal renewal, was performed using immunocytofluorescence-technique and integrated multiscale-omics analyses. Already TAC from non-lesional psoriatic skin showed altered methylation and differential expression in 1.7% and 1.0% of all protein-coding genes, respectively. In psoriatic lesions, TAC were strongly expanded showing further increased differentially methylated (10-fold) and expressed (22-fold) genes numbers. Importantly, 17.2% of differentially expressed genes were associated with respective gene methylations. Compared with non-lesional TAC, pathway analyses revealed metabolic alterations as one feature predominantly changed in TAC derived from active psoriatic lesions. Overall, our study showed stage-specific molecular alterations, allows new insights into the pathogenesis, and implies the involvement of epigenetic mechanisms in lesion development in psoriasis. KEY MESSAGES: Transit amplifying cell (TAC) numbers are highly increased in psoriatic lesions Psoriatic TAC show profound molecular alterations & stage-specific identity TAC from unaffected areas already show first signs of molecular alterations Lesional TAC show a preference in metabolic-related alterations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00109-019-01860-3DOI Listing
January 2020

CRISPR-Mediated Activation of Endogenous Gene Expression in the Postnatal Heart.

Circ Res 2020 01 15;126(1):6-24. Epub 2019 Nov 15.

From the Institute of Pharmacology and Toxicology (E.S., L.M.I., C.N., W.-H.Z., L.C.Z.), University Medical Center Goettingen, Georg-August University, Germany.

Rationale: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far.

Objective: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting and loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription.

Methods And Results: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected , which when overexpressed, led to hypertrophy and heart failure, and , which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting , we could enhance its transcription significantly, although is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects.

Conclusions: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.118.314522DOI Listing
January 2020

Nuclear Pre-snRNA Export Is an Essential Quality Assurance Mechanism for Functional Spliceosomes.

Cell Rep 2019 06;27(11):3199-3214.e3

Abteilung für Molekulare Genetik, Institut für Mikrobiologie und Genetik, Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August Universität Göttingen, Göttingen, Germany. Electronic address:

Removal of introns from pre-mRNAs is an essential step in eukaryotic gene expression, mediated by spliceosomes that contain snRNAs as key components. Although snRNAs are transcribed in the nucleus and function in the same compartment, all except U6 shuttle to the cytoplasm. Surprisingly, the physiological relevance for shuttling is unclear, in particular because the snRNAs in Saccharomyces cerevisiae were reported to remain nuclear. Here, we show that all yeast pre-snRNAs including U6 undergo a stepwise maturation process after nuclear export by Mex67 and Xpo1. Sm- and Lsm-ring attachment occurs in the cytoplasm and is important for the snRNA re-import, mediated by Cse1 and Mtr10. Finally, nuclear pre-snRNA cleavage and trimethylation of the 5'-cap finalizes shuttling. Importantly, preventing pre-snRNAs from being exported or processed results in faulty spliceosome assembly and subsequent genome-wide splicing defects. Thus, pre-snRNA export is obligatory for functional splicing and resembles an essential evolutionarily conserved quality assurance step.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.05.031DOI Listing
June 2019

Bassoon proteinopathy drives neurodegeneration in multiple sclerosis.

Nat Neurosci 2019 06 22;22(6):887-896. Epub 2019 Apr 22.

Institut für Neuroimmunologie und Multiple Sklerose, Zentrum für Molekulare Neurobiologie Hamburg, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

Multiple sclerosis (MS) is characterized by inflammatory insults that drive neuroaxonal injury. However, knowledge about neuron-intrinsic responses to inflammation is limited. By leveraging neuron-specific messenger RNA profiling, we found that neuroinflammation leads to induction and toxic accumulation of the synaptic protein bassoon (Bsn) in the neuronal somata of mice and patients with MS. Neuronal overexpression of Bsn in flies resulted in reduction of lifespan, while genetic disruption of Bsn protected mice from inflammation-induced neuroaxonal injury. Notably, pharmacological proteasome activation boosted the clearance of accumulated Bsn and enhanced neuronal survival. Our study demonstrates that neuroinflammation initiates toxic protein accumulation in neuronal somata and advocates proteasome activation as a potential remedy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41593-019-0385-4DOI Listing
June 2019

The IL-1 Pathway Is Hyperactive in Hidradenitis Suppurativa and Contributes to Skin Infiltration and Destruction.

J Invest Dermatol 2019 06 5;139(6):1294-1305. Epub 2018 Dec 5.

Interdisciplinary Group Molecular Immunopathology, Dermatology/Medical Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Psoriasis Research and Treatment Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. Electronic address:

Hidradenitis suppurativa (HS) (also designated acne inversa) is a chronic inflammatory disease characterized by painful purulent skin lesions and progressive destruction of skin architecture. Despite the high burden for the patients, pathogenetic pathways underlying HS alterations remain obscure. When we examined the HS cytokine pattern, IL-1β turned out to be a highly prominent cytokine, overexpressed even compared with psoriatic lesions. Analyses of IL-1β-induced transcriptome in various cell types showed overlapping profiles, with upregulations of molecules causing immune cell infiltration and extracellular matrix degradation, and of specific cytokines including IL-6, IL-32, and IL-36. Matching cellular IL-1 receptor levels, dermal fibroblasts showed both the strongest and broadest IL-1β response, which was not clearly shared or strengthened by other cytokines. The IL-1β signature was specifically present in HS lesions and could be reversed by application of IL-1 receptor antagonist. Search for blood parameters associated with IL-1β pathway activity in HS identified serum amyloid A, which was synergistically induced by IL-1β and IL-6 in hepatocytes. Consequently, strongly elevated blood serum amyloid A levels in HS correlated positively with the extent of inflammatory skin alterations. In summary, the IL-1β pathway represents a pathogenetic cascade, whose activity may be therapeutically targeted and monitored by blood SAA levels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jid.2018.11.018DOI Listing
June 2019

PI3K: A master regulator of brain metastasis-promoting macrophages/microglia.

Glia 2018 11 25;66(11):2438-2455. Epub 2018 Oct 25.

Department of Internal Medicine III, University Hospital Regensburg, Regensburg, Germany.

Mutations and activation of the PI3K signaling pathway in breast cancer cells have been linked to brain metastases. However, here we describe that in some breast cancer brain metastases samples the protein expression of PI3K signaling components is restricted to the metastatic microenvironment. In contrast to the therapeutic effects of PI3K inhibition on the breast cancer cells, the reaction of the brain microenvironment is less understood. Therefore we aimed to quantify the PI3K pathway activity in breast cancer brain metastasis and investigate the effects of PI3K inhibition on the central nervous system (CNS) microenvironment. First, to systematically quantify the PI3K pathway activity in breast cancer brain metastases, we performed a prospective biomarker study using a reverse phase protein array (RPPA). The majority, namely 30 out of 48 (62.5%) brain metastatic tissues examined, revealed high PI3K signaling activity that was associated with a median overall survival (OS) of 9.41 months, while that of patients, whose brain metastases showed only moderate or low PI3K activity, amounted to only 1.93 and 6.71 months, respectively. Second, we identified PI3K as a master regulator of metastasis-promoting macrophages/microglia during CNS colonization; and treatment with buparlisib (BKM120), a pan-PI3K Class I inhibitor with a good blood-brain-barrier penetrance, reduced their metastasis-promoting features. In conclusion, PI3K signaling is active in the majority of breast cancer brain metastases. Since PI3K inhibition does not only affect the metastatic cells but also re-educates the metastasis-promoting macrophages/microglia, PI3K inhibition may hold considerable promise in the treatment of brain metastasis and the respective microenvironment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.23485DOI Listing
November 2018

Immortalization of common marmoset monkey fibroblasts by piggyBac transposition of hTERT.

PLoS One 2018 27;13(9):e0204580. Epub 2018 Sep 27.

Platform Degenerative Diseases, German Primate Center- Leibniz Institute for Primate Research, Göttingen, Germany.

Following a certain type-specific number of mitotic divisions, terminally differentiated cells undergo proliferative senescence, thwarting efforts to expand different cell populations in vitro for the needs of scientific research or medical therapies. The primary cause of this phenomenon is the progressive shortening of the telomeres and the subsequent activation of cell cycle control pathways leading to a block of cell proliferation. Restoration of telomere length by transgenic expression of telomerase reverse transcriptase (TERT) usually results in bypassing of the replicative senescence and ultimately in cell immortalization. To date, there have not been any reports regarding immortalization of cells from common marmoset (Callithrix jacchus), an important non-human primate model for various human diseases, with the use of exogenous human TERT (hTERT). In this study, marmoset fibroblasts were successfully immortalized with transposon-integrated transgenic hTERT and expanded in vitro for over 500 population doublings. Calculation of population doubling levels (PDL) showed that the derived hTERT-transgenic lines had significantly higher proliferation potential than the wild-type fibroblasts, which reached only a maximum of 46 doublings. However, the immortalized cells exhibited differences in the morphology compared with the control fibroblasts and transcriptome analysis also revealed changes in the gene expression patterns. Finally, the karyotypes of all hTERT-transgenic cell lines showed various aberrations such as presence of extra Chromosome 17, isochromosome 21q, or tetraploidy. By single-cell expansion of the least affected monoclonal immortalized line, one sub-clonal line with normal karyotype was established, suggesting the possibility to derive immortal marmoset cells with normal karyotypes. The results of this study are an important step towards the development and optimization of methods for the production of immortalized cells from common marmoset monkeys.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204580PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160115PMC
March 2019

Neurons under T Cell Attack Coordinate Phagocyte-Mediated Synaptic Stripping.

Cell 2018 10 30;175(2):458-471.e19. Epub 2018 Aug 30.

Department of Pathology and Immunology, University of Geneva, 1211 Geneva, Switzerland; Division of Clinical Pathology, Geneva University Hospital, 1211 Geneva, Switzerland. Electronic address:

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8 T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8 T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen's encephalitis patients. In this devastating CD8 T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8 T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2018.07.049DOI Listing
October 2018

Deep phenotyping of human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes.

JCI Insight 2018 06 21;3(12). Epub 2018 Jun 21.

Clinic for Cardiology and Pneumology, University Medical Center Göttingen (UMG), Göttingen, Germany.

Generation of homogeneous populations of subtype-specific cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) and their comprehensive phenotyping is crucial for a better understanding of the subtype-related disease mechanisms and as tools for the development of chamber-specific drugs. The goals of this study were to apply a simple and efficient method for differentiation of iPSCs into defined functional CM subtypes in feeder-free conditions and to obtain a comprehensive understanding of the molecular, cell biological, and functional properties of atrial and ventricular iPSC-CMs on both the single-cell and engineered heart muscle (EHM) level. By a stage-specific activation of retinoic acid signaling in monolayer-based and well-defined culture, we showed that cardiac progenitors can be directed towards a highly homogeneous population of atrial CMs. By combining the transcriptome and proteome profiling of the iPSC-CM subtypes with functional characterizations via optical action potential and calcium imaging, and with contractile analyses in EHM, we demonstrated that atrial and ventricular iPSC-CMs and -EHM highly correspond to the atrial and ventricular heart muscle, respectively. This study provides a comprehensive understanding of the molecular and functional identities characteristic of atrial and ventricular iPSC-CMs and -EHM and supports their suitability in disease modeling and chamber-specific drug screening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.99941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124434PMC
June 2018

Expression of the DNA-Binding Factor TOX Promotes the Encephalitogenic Potential of Microbe-Induced Autoreactive CD8 T Cells.

Immunity 2018 05;48(5):937-950.e8

Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland; Division of Clinical Pathology, Geneva University Hospital, Geneva, Switzerland. Electronic address:

Infections are thought to trigger CD8 cytotoxic T lymphocyte (CTL) responses during autoimmunity. However, the transcriptional programs governing the tissue-destructive potential of CTLs remain poorly defined. In a model of central nervous system (CNS) inflammation, we found that infection with lymphocytic choriomeningitis virus (LCMV), but not Listeria monocytogenes (Lm), drove autoimmunity. The DNA-binding factor TOX was induced in CTLs during LCMV infection and was essential for their encephalitogenic properties, and its expression was inhibited by interleukin-12 during Lm infection. TOX repressed the activity of several transcription factors (including Id2, TCF-1, and Notch) that are known to drive CTL differentiation. TOX also reduced immune checkpoint sensitivity by restraining the expression of the inhibitory checkpoint receptor CD244 on the surface of CTLs, leading to increased CTL-mediated damage in the CNS. Our results identify TOX as a transcriptional regulator of tissue-destructive CTLs in autoimmunity, offering a potential mechanistic link to microbial triggers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2018.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040915PMC
May 2018

Epithelial-Mesenchymal Transition during Metastasis of HPV-Negative Pharyngeal Squamous Cell Carcinoma.

Biomed Res Int 2018 6;2018:7929104. Epub 2018 Mar 6.

Department of Otorhinolaryngology, University Medical Center Göttingen, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37099 Göttingen, Germany.

In epithelial tumors, a shift towards a mesenchymal phenotype has been associated with increased invasiveness and metastasis. It is assumed that this phenomenon plays a major role in disease progression and ultimately prognosis. This study investigated epithelial-mesenchymal transition (EMT) in human papillomavirus- (HPV-) negative pharyngeal squamous cell carcinoma. Tissue was obtained from one hypopharyngeal primary tumor and a regional lymph node metastasis during surgery with curative intention. A cell culture was established from the primary tumor and mesenchymal growth conditions were emulated. Gene expression profiling was performed (Human 8 × 60 K design array, Agilent Technologies) and EMT was assessed by a gene set (MSigDB: M5930, Hallmark_epithelial_mesenchymal_transition), applying gene set expression analysis (GSEA). Immunohistochemical staining and flow cytometry of CD44 and E-cadherin were compared in primary tumor, metastasis, and cell cultures. Primary tumor and metastasis were highly positive for CD44. A loss of E-cadherin occurred in the metastasis. Flow cytometry showed the appearance of a population without E-cadherin in spheroid colonies. In GSEA, the EMT phenotype was enriched in the primary tumor compared to metastasis and cell cultures (FDR < 25%, < 5%). EMT showed variable expression during metastasis. It may thereby be a dynamic state in HPV-negative pharyngeal squamous cell carcinoma that is active only during the process of metastasis itself. Thereby, the primary tumor as well as the metastasis may exhibit fewer EMT properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2018/7929104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859914PMC
September 2018

Effects of repeated long-term psychosocial stress and acute cannabinoid exposure on mouse corticostriatal circuitries: Implications for neuropsychiatric disorders.

CNS Neurosci Ther 2018 06 31;24(6):528-538. Epub 2018 Jan 31.

Department of Psychiatry and Psychotherapy, University of Göttingen, Göttingen, Germany.

Introduction: Vulnerability to psychiatric manifestations is achieved by the influence of genetic and environment including stress and cannabis consumption. Here, we used a psychosocial stress model based on resident-intruder confrontations to study the brain corticostriatal-function, since deregulation of corticostriatal circuitries has been reported in many psychiatric disorders. CB receptors are widely expressed in the central nervous system and particularly, in both cortex and striatum brain structures.

Aims And Methods: The investigation presented here is addressed to assess the impact of repeated stress following acute cannabinoid exposure on behavior and corticostriatal brain physiology by assessing mice behavior, the concentration of endocannabinoid and endocannabinoid-like molecules and changes in the transcriptome.

Results: Stressed animals urinated frequently; showed exacerbated scratching activity, lower striatal N-arachidonylethanolamine (AEA) levels and higher cortical expression of cholinergic receptor nicotinic alpha 6. The cannabinoid agonist WIN55212.2 diminished locomotor activity while the inverse agonist increased the distance travelled in the center of the open field. Upon CB activation, N-oleoylethanolamide and N-palmitoylethanolamide, two AEA congeners that do not interact directly with cannabinoid receptors, were enhanced in the striatum. The co-administration with both cannabinoids induced an up-regulation of striatal FK506 binding protein 5. The inverse agonist in controls reversed the effects of WIN55212.2 on motor activity. When Rimonabant was injected under stress, the cortical levels of 2-arachidonoylglycerol were maximum. The agonist and the antagonist influenced the cortical expression of cholinergic receptor nicotinic alpha 6 and serotonin transporter neurotransmitter type 4 in opposite directions, while their co-administration tended to produce a null effect under stress.

Conclusions: The endocannabinoid system had a direct effect on serotoninergic neurotransmission and glucocorticoid signaling. Cholinergic receptor nicotinic alpha-6 was shown to be deregulated in response to stress and following synthetic cannabinoid drugs thus could confer vulnerability to cannabis addiction and psychosis. Targeting the receptors of endocannabinoids and endocannabinoid-like mediators might be a valuable option for treating stress-related neuropsychiatric symptoms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cns.12810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969305PMC
June 2018

Hepatic gene therapy rescues high-fat diet responses in circadian mutant mice.

Mol Metab 2017 06 29;6(6):512-523. Epub 2017 Mar 29.

Circadian Rhythms Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Objective: Circadian gene mutant mice show dampened 24-h feeding rhythms and an increased sensitivity to high-fat diet (HFD) feeding. Restricting HFD access to the dark phase counteracts its obesogenic effect in wild-type mice. The extent to which altered feeding rhythms are causative for the obesogenic phenotype of mutant mice, however, remains unknown.

Methods: Metabolic parameters of wild-type (WT) and mutant mice (MT) were investigated under and nighttime restricted HFD feeding. Liver circadian clock function was partially rescued by hydrodynamic tail vein delivery of WT- DNA vectors in mutant mice and transcriptional, metabolic, endocrine and behavioral rhythms studied.

Results: Nighttime-restricted feeding restored food intake, but not body weight regulation in MT mice under HFD, suggesting -dependent metabolic dysregulation downstream of circadian appetite control. Liver-directed gene therapy partially restored liver circadian oscillator function and transcriptome regulation without affecting centrally controlled circadian behaviors. Under HFD, MT mice with partially restored liver clock function (MT-LR) showed normalized body weight gain, rescued 24-h food intake rhythms, and WT-like energy expenditure. This was associated with decreased nighttime leptin and daytime ghrelin levels, reduced hepatic lipid accumulation, and improved glucose tolerance. Transcriptome analysis revealed that hepatic rescue in MT mice affected a range of metabolic pathways.

Conclusion: Liver gene therapy improves resistance against HFD-induced metabolic impairments in mice with circadian clock disruption. Restoring or stabilizing liver clock function might be a promising target for therapeutic interventions in obesity and metabolic disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molmet.2017.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444075PMC
June 2017

The adaptation of colorectal cancer cells when forming metastases in the liver: expression of associated genes and pathways in a mouse model.

BMC Cancer 2017 05 19;17(1):342. Epub 2017 May 19.

Department of General, Visceral and Paediatric Surgery, University Medical Centre, Georg - August - University Goettingen, Göttingen, Germany.

Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death in men and women. Systemic disease with metastatic spread to distant sites such as the liver reduces the survival rate considerably. The aim of this study was to investigate the changes in gene expression that occur on invasion and expansion of CRC cells when forming metastases in the liver.

Methods: The livers of syngeneic C57BL/6NCrl mice were inoculated with 1 million CRC cells (CMT-93) via the portal vein, leading to the stable formation of metastases within 4 weeks. RNA sequencing performed on the Illumina platform was employed to evaluate the expression profiles of more than 14,000 genes, utilizing the RNA of the cell line cells and liver metastases as well as from corresponding tumour-free liver.

Results: A total of 3329 differentially expressed genes (DEGs) were identified when cultured CMT-93 cells propagated as metastases in the liver. Hierarchical clustering on heat maps demonstrated the clear changes in gene expression of CMT-93 cells on propagation in the liver. Gene ontology analysis determined inflammation, angiogenesis, and signal transduction as the top three relevant biological processes involved. Using a selection list, matrix metallopeptidases 2, 7, and 9, wnt inhibitory factor, and chemokine receptor 4 were the top five significantly dysregulated genes.

Conclusion: Bioinformatics assists in elucidating the factors and processes involved in CRC liver metastasis. Our results support the notion of an invasion-metastasis cascade involving CRC cells forming metastases on successful invasion and expansion within the liver. Furthermore, we identified a gene expression signature correlating strongly with invasiveness and migration. Our findings may guide future research on novel therapeutic targets in the treatment of CRC liver metastasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12885-017-3342-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437520PMC
May 2017

The β-catenin/CBP-antagonist ICG-001 inhibits pediatric glioma tumorigenicity in a Wnt-independent manner.

Oncotarget 2017 Apr;8(16):27300-27313

Division of Pediatric Hematology and Oncology, Department of Child and Adolescent Health, University Medical Center Goettingen, Goettingen, Germany.

Pediatric high-grade gliomas (pedHGG) belong to the most aggressive cancers in children with a poor prognosis due to a lack of efficient therapeutic strategies. The β-catenin/Wnt-signaling pathway was shown to hold promising potential as a treatment target in adult high-grade gliomas by abrogating tumor cell invasion and the acquisition of stem cell-like characteristics. Since pedHGG differ from their adult counterparts in genetically and biologically we aimed to investigate the effects of β-catenin/Wnt-signaling pathway-inhibition by the β-catenin/CBP antagonist ICG-001 in pedHGG cell lines. In contrast to adult HGG, pedHGG cells displayed minimal detectable canonical Wnt-signaling activity. Nevertheless, low doses of ICG-001 inhibited cell migration/invasion, tumorsphere- and colony formation, proliferation in vitro as well as tumor growth in vivo/ovo, suggesting that ICG-001 affects pedHGG tumor cell characteristics independent of β-catenin/Wnt-signaling. RNA-sequencing analyses support a Wnt/β-catenin-independent effect of ICG-001 on target gene transcription, revealing strong effects on genes involved in cellular metabolic/biosynthetic processes and cell cycle progression. Among these, high mRNA expression of cell cycle regulator JDP2 was found to confer a better prognosis for pedHGG patients. In conclusion, ICG-001 might offer an effective treatment option for pedHGG patients functioning to regulate cell phenotype and gene expression programs in absence of Wnt/β-catenin signaling-activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.15934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432336PMC
April 2017

Histone deacetylase inhibitor MS-275 augments expression of a subset of IFN-γ-regulated genes in Toxoplasma gondii-infected macrophages but does not improve parasite control.

Exp Parasitol 2017 Sep 9;180:45-54. Epub 2017 Feb 9.

Institute for Medical Microbiology, University Medical Center, Georg-August-University, Kreuzbergring 57, 37075 Göttingen, Germany. Electronic address:

Toxoplasma gondii is a ubiquitous apicomplexan parasite of mammals and birds and an important pathogen of humans. IFN-γ is the major mediator of host resistance against T. gondii but intriguingly, parasite-infected host cells including macrophages are severely impaired to respond to IFN-γ due to defective transcriptional activation of target genes. Here, we tested the possibility that the impaired responsiveness of T. gondii-infected macrophages to IFN-γ can be restored by inhibiting histone deacetylases (HDACs) using the class I-specific inhibitor MS-275. Treatment of RAW264.7 cells with MS-275 indeed increased MHC class II surface expression in infected and non-infected cells and largely abolished the inhibition of IFN-γ-regulated MHC class II expression exerted by T. gondii. Genome-wide transcriptome profiling revealed that MS-275 increased mean mRNA levels of IFN-γ-regulated genes particularly in non-infected macrophages. Transcript levels of 33% of IFN-γ secondary response genes but only those of a few primary response genes were also increased by MS-275 in T. gondii-infected cells. Importantly, the unresponsiveness of parasite-infected cells to IFN-γ was however not abolished by MS-275. Furthermore, MS-275 also up-regulated several anti-inflammatory cytokines or signaling molecules in T. gondii-infected macrophages. It additionally regulated expression of more than 2500 genes in non-infected macrophages expression of which was surprisingly counteracted by prior infection with T. gondii. FACS analysis and immunofluorescence microscopy revealed that MS-275 did not considerably diminish the number of parasite-positive cells or the intracellular replication in macrophages stimulated or not with IFN-γ. Thus, a supportive therapy using MS-275 appears inappropriate for treatment of toxoplasmosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exppara.2017.02.011DOI Listing
September 2017

mRNA quality control is bypassed for immediate export of stress-responsive transcripts.

Nature 2016 Dec 12;540(7634):593-596. Epub 2016 Dec 12.

Abteilung für Molekulare Genetik, Institut für Mikrobiologie und Genetik, Göttinger Zentrum für Molekulare Biowissenschaften, Georg-August Universität Göttingen, Göttingen, Germany.

Cells grow well only in a narrow range of physiological conditions. Surviving extreme conditions requires the instantaneous expression of chaperones that help to overcome stressful situations. To ensure the preferential synthesis of these heat-shock proteins, cells inhibit transcription, pre-mRNA processing and nuclear export of non-heat-shock transcripts, while stress-specific mRNAs are exclusively exported and translated. How cells manage the selective retention of regular transcripts and the simultaneous rapid export of heat-shock mRNAs is largely unknown. In Saccharomyces cerevisiae, the shuttling RNA adaptor proteins Npl3, Gbp2, Hrb1 and Nab2 are loaded co-transcriptionally onto growing pre-mRNAs. For nuclear export, they recruit the export-receptor heterodimer Mex67-Mtr2 (TAP-p15 in humans). Here we show that cellular stress induces the dissociation of Mex67 and its adaptor proteins from regular mRNAs to prevent general mRNA export. At the same time, heat-shock mRNAs are rapidly exported in association with Mex67, without the need for adapters. The immediate co-transcriptional loading of Mex67 onto heat-shock mRNAs involves Hsf1, a heat-shock transcription factor that binds to heat-shock-promoter elements in stress-responsive genes. An important difference between the export modes is that adaptor-protein-bound mRNAs undergo quality control, whereas stress-specific transcripts do not. In fact, regular mRNAs are converted into uncontrolled stress-responsive transcripts if expressed under the control of a heat-shock promoter, suggesting that whether an mRNA undergoes quality control is encrypted therein. Under normal conditions, Mex67 adaptor proteins are recruited for RNA surveillance, with only quality-controlled mRNAs allowed to associate with Mex67 and leave the nucleus. Thus, at the cost of error-free mRNA formation, heat-shock mRNAs are exported and translated without delay, allowing cells to survive extreme situations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature20572DOI Listing
December 2016

Pre-infection transcript levels of FAM26F in peripheral blood mononuclear cells inform about overall plasma viral load in acute and post-acute phase after simian immunodeficiency virus infection.

J Gen Virol 2016 Dec 18;97(12):3400-3412. Epub 2016 Oct 18.

Deutsches Primatenzentrum GmbH, Leibniz-Institut für Primatenforschung, Unit of Infection Models, Göttingen, Germany.

CD8+ cells from simian immunodeficiency virus (SIV)-infected long-term non-progressors and some uninfected macaques can suppress viral replication in vitro without killing the infected cells. The aim of this study was to identify factors responsible for non-cytolytic viral suppression by transcriptional profiling and to investigate their potential impact on SIV replication. Results of microarray experiments and further validation with cells from infected and uninfected macaques revealed that FAM26F RNA levels distinguished CD8+ cells of controllers and non-controllers (P=0.001). However, FAM26F was also expressed in CD4+ T-cells and B-cells. FAM26F expression increased in lymphocytes after in vitro IFN-γ treatment on average 40-fold, and ex vivo FAM26F RNA levels in peripheral blood mononuclear cells correlated with plasma IFN-γ but not with IFN-α. Baseline FAM26F expression appeared to be stable for months, albeit the individual expression levels varied up to tenfold. Investigating its role in SIV-infection revealed that FAM26F was upregulated after infection (P<0.0008), but did not directly correlate with viral load in contrast to MX1 and CXCL10. However, pre-infection levels of FAM26F correlated inversely with overall plasma viral load (AUC) during the acute and post-acute phases of infection (e.g. AUC weeks post infection 0-8; no AIDS vaccine: P<0.0001, Spearman rank correlation coefficient (rs)=-0.89, n=16; immunized with an AIDS vaccine: P=0.033, rs=-0.43; n=25). FAM26F transcript levels prior to infection can provide information about the pace and strength of the antiviral immune response during the early stage of infection. FAM26F expression represented, in our experiments, one of the earliest prognostic markers, and could supplement major histocompatibility complex (MHC)-typing to predict disease progression before SIV-infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jgv.0.000632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203675PMC
December 2016