Induced pluripotent stem cell-derived neuronal cells from a sporadic Alzheimer’s disease donor as a model for investigating AD-associated gene regulatory networks

Background Alzheimer’s disease (AD) is a complex, irreversible neurodegenerative disorder. At present there are neither reliable markers to diagnose AD at an early stage nor therapy. To investigate underlying disease mechanisms, induced pluripotent stem cells (iPSCs) allow the generation of patient-derived neuronal cells in a dish. Results In this study, employing iPS technology, we derived and characterized iPSCs from dermal fibroblasts of an 82-year-old female patient affected by sporadic AD. The AD-iPSCs were differentiated into neuronal cells, in order to generate disease-specific protein association networks modeling the molecular pathology on the transcriptome level of AD, to analyse the reflection of the disease phenotype in gene expression in AD-iPS neuronal cells, in particular in the ubiquitin- proteasome system (UPS), and to address expression of typical AD proteins. We detected the expression of p-tau and GSK3B, a physiological kinase of tau, in neuronal cells derived from AD-iPSCs. Treatment of neuronal cells differentiated from AD-iPSCs with an inhibitor of γ-secretase resulted in the down-regulation of p-tau. Transcriptome analysis of AD-iPS derived neuronal cells revealed significant changes in the expression of genes associated with AD and with the constitutive as well as the inducible subunits of the proteasome complex. The neuronal cells expressed numerous genes associated with sub-regions within the brain thus suggesting the usefulness of our in- vitro model. Moreover, an AD-related protein interaction network composed of APP and GSK3B among others could be generated using neuronal cells differentiated from two AD-iPS cell lines. Conclusions Our study demonstrates how an iPSC-based model system could represent (i) a tool to study the underlying molecular basis of sporadic AD, (ii) a platform for drug screening and toxicology studies which might unveil novel therapeutic avenues for this debilitating neuronal disorder

Reciprocal regulation of the cholinic phenotype and epithelial-mesenchymal transition in glioblastoma cells

Glioblastoma (GBM) is the most malignant brain tumor with very limited therapeutic options. Standard multimodal treatments, including surgical resection and combined radio-chemotherapy do not target the most aggressive subtype of glioma cells, brain tumor stem cells (BTSCs). BTSCs are thought to be responsible for tumor initiation, progression, and relapse. Furthermore, they have been associated with the expression of mesenchymal features as a result of epithelial-mesenchymal transition (EMT) thereby inducing tumor dissemination and chemo resistance. Using high resolution proton nuclear magnetic resonance spectroscopy (1H NMR) on GBM cell cultures we provide evidence that the expression of well-known EMT activators of the ZEB, TWIST and SNAI families and EMT target genes N-cadherin and VIMENTIN is associated with aberrant choline metabolism. The cholinic phenotype is characterized by high intracellular levels of phosphocholine and total choline derivatives and was associated with malignancy in various cancers. Both genetic and pharmacological inhibition of the cardinal choline metabolism regulator choline kinase alpha (CHKα) significantly reduces the cell viability, invasiveness, clonogenicity, and expression of EMT associated genes in GBM cells. Moreover, in some cell lines synergetic cytotoxic effects were observed when combining the standard of care chemotherapeutic temozolomide with the CHKα inhibitor V-11-0711. Taken together, specific inhibition of the enzymatic activity of CHKα is a powerful strategy to suppress EMT which opens the possibility to target chemo-resistant BTSCs through impairing their mesenchymal transdifferentiation. Moreover, the newly identified EMT-oncometabolic network may be helpful to monitor the invasive properties of glioblastomas and the success of anti-EMT therapy

Bioaccumulation in aquatic systems: methodological approaches, monitoring and assessment

Bioaccumulation, the accumulation of a chemical in an organism relative to its level in the ambient medium, is of major environmental concern. Thus, monitoring chemical concentrations in biota are widely and increasingly used for assessing the chemical status of aquatic ecosystems. In this paper, various scientific and regulatory aspects of bioaccumulation in aquatic systems and the relevant critical issues are discussed. Monitoring chemical concentrations in biota can be used for compliance checking with regulatory directives, for identification of chemical sources or event related environmental risk assessment. Assessing bioaccumulation in the field is challenging since many factors have to be considered that can effect the accumulation of a chemical in an organism. Passive sampling can complement biota monitoring since samplers with standardised partition properties can be used over a wide temporal and geographical range. Bioaccumulation is also assessed for regulation of chemicals of environmental concern whereby mainly data from laboratory studies on fish bioaccumulation are used. Field data can, however, provide additional important information for regulators. Strategies for bioaccumulation assessment still need to be harmonised for different regulations and groups of chemicals. To create awareness for critical issues and to mutually benefit from technical expertise and scientific findings, communication between risk assessment and monitoring communities needs to be improved. Scientists can support the establishment of new monitoring programs for bioaccumulation, e.g. in the frame of the amended European Environmental Quality Standard Directive

The inducible ubiquitin-proteasome system in neurons under pro-inflammatory conditions

Eine wichtige Funktion des Ubiquitin-Proteasom Systmes (UPS) ist die Generierung von Peptiden, die von dem Molekül MHC I auf der Zelloberfläche den CD8+ T-Lymphozyten präsentiert werden. Zahlreiche Untersuchungen dokumentieren, dass ein gestörtes UPS mit vielen neurodegenerativen Erkrankungen einhergeht. Unter inflammatorischen Prozessen findet durch einen Austausch der katalytisch-aktiven konstitutiven gegen die katalytisch-aktiven induzierbaren Untereinheiten die Bildung des Immunoproteasoms (iP) statt. Das iP bewirkt eine effizientere Peptid-Generierung und eine erhöhte Protein- Abbau-Rate. In neuralen Zellen wurde dieser induzierbare Proteasom-Komplex vor allem bei neurodegenerativen Erkrankungen beobachtet. Dennoch ist die Funktion des iP in Zellen des zentralen Nervensystems (ZNS) bislang ungeklärt. Fokus dieser Arbeit war das Immunoproteasom in neuralen Zellen in vitro und in vivo unter physiologischen und pathophysiologischen Bedingungen. In der experimentellen autoimmunen Encephalomyelitis erkrankten iP-defiziente Tiere im Vergleich zu den Wildtyp-Mäusen früher und schwerer. Während des inflammatorischen Prozesses im ZNS konnte eine iP-Synthese in Gliazellen, jedoch nicht in Neuronen beobachtet werden. Das Fehlen eines iP-Komplexes führte in den Zellen des ZNS zu einer Akkumulation von Poly-Ubiquitin- Konjugaten, und in der Folge zur Apoptose und einem verstärkten pro- inflammatorischen Stimulus. Die in vitro-Experimente mit neuronalen Zellkulturen (HT-22, neurale differenzierte Vorläuferzellen, primäre neuronale Mischkultur) zeigten, dass Neurone auch unter physiologischen Bedingungen keinen iP-Komplex synthetisieren. Die in vivo-Daten konnten dadurch bestätigt werden, dass das pro-inflammatorische Zytokin Interferon-gamma (IFNg) nur in den Gliazellen der primären Kulturen die de novo-Synthese des iPs induzierte. Es zeigte sich, dass die Bindungsstelle des Interferon-regulierenden Faktors 1 im neuronalen Promotor der für die Assemblierung und proteolytischen Aktivität des iPs essentiellen Untereinheit LMP7 methyliert vorliegt. In den primären, reifen Neuronen konnte eine Induktion der LMP7-Untereinheit in Abhängigkeit der Aktionspotential-Blockade mit dem Zytokin IFNg gezeigt werden. Überraschender-weise konnte in isolierten, primären Neuronen eine Korrelation zwischen der Oberflächen-expression des MHC I-Moleküls und der iP-Synthese beobachtet werden. Außerdem konnte eine Beteiligung des iPs an der synaptischen Plastizität dokumentiert werden. Die in dieser Arbeit vorgestellten Daten zeigen erstmalig, dass unter physiologischen und pathophysiologischen Bedingungen die Expression des Immunoproteasoms in Neuronen epigenetisch und in Abhängigkeit von der elektrischen Aktivität reguliert wird, und die wichtige Funktion des Immunoproteasoms während einer neurodegenerativen Erkrankungen daher auf seine Rolle in Gliazellen zurückzuführen ist.The proteasome, one crucial component of the ubiquitin-proteasome system (UPS), assists as an ATP-dependent multi-catalytic protease in different essential processes of eukaryotic cells. The generation of peptides, which are presented on the cell-surface to CD8+ t-lymphocytes, is one of the main functions of the proteasome. A dysfunction of the UPS is connected to various neurodegenerative diseases. Under inflammatory stimuli the exchange of the catalytically active subunits, which transforms the constitutive proteasome into the inducible proteasome, results in the de novo synthesis of the immunoproteasome (iP). The iP implicates a more efficient peptide-generation for the MHC I complex caused by a higher protein-turnover. The inducible proteasome is known to be expressed in neural cells especially in neurodegenerative diseases. The precise function, however, has not been revealed so far in the cells of the central nervous system (CNS). Main focus of the investigations was to define the role of the iP in neural cells in in vitro and in vivo experiments under physiological and pathophysiological conditions like neuroinflammation. By the induction of experimental autoimmune encephalomyelitis in iP-deficient mice an earlier onset and higher severity of the disease in these animals was discovered. The de novo-synthesis of the iP was only observed in glia-cells but not in neurons during pro-inflammatory processes in the CNS. Animals without an iP-complex accumulated more poly- ubiquitin conjugates resulting in apoptosis and enhanced pro-inflammatory stimuli. In vitro experiments revealed that neuronal cells (HT-22, differentiated neural progenitor cells and primary neurons) do not synthesize the iP-complex under physiological conditions and by induction of the iP with the pro-inflammatory cytokine interferon-gamma (IFNg) in glia-cells the in vivo data was confirmed. Furthermore it was shown that the interferon regulatory factor 1 binding site of the neuronal promoter of the LMP7 subunit, which is essential for the assembly and the proteolytic activity of the iP, is methylated. Interestingly, inhibition of action potential combined with IFNg treatment lead to the synthesis of the iP-complex in electrically active neurons. In addition a correlation between the neuronal surface expression of the MHC I molecule and the de novo synthesis of the iP was also determined in isolated primary neurons. Aside from that the involvement of the inducible proteasome-complex in the synaptic plasticity was observed. IP-deficient neural cells were characterized by a diminished electrical activity under pro- inflammatory stimuli and an enhanced chemical long-term depression. This work shows for the first time, that in neurons the expression of the immuno- proteasome is regulated by epigenetic factors and electrical activity and that the important function of the iP during neurodegenerative diseases is attributed to its crucial role in glia-cells

Lymphoblast-derived integration-free ISRM-CON9 iPS cell line from a 75 year old female

Human lymphoblast cells were used to generate integration-free induced pluripotent stem cells (iPSCs) employing episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. The derived iPSCs were defined as pluripotent based on (i) expression of pluripotency-associated markers, (ii) embryoid body-based differentiation into cell types representative of the three germ layers and (iii) the similarity between the transcriptomes of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.95