Die Forschungsprojekte der Arbeitsgruppe Theuring

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Workflow of 2-DE and MS strategy for identification of proteins and protein species.
2-DE two-dimensional gel electrophoresis, ESI–MS/MS electrospray ionization mass spectrometry,
LTQ-FT-ICR-MS/MS linear ion trap Fourier transform ion cyclotron resonance mass spectrometry,
db database

Project leader: Prof. F. Theuring
Coworkers: Karima Schwab, Boris Neumann, both are PhD students, Souidi, Naima, Diploma student
Funding: Hypatia-Funding, Projektverbund Chancengleichheit für Frauen Charité, PhD-grant
Collaboration: Dr. Christian Scheler, Proteome Factory, Berlin; Dr. P.R. Jungblut, MPI for Infection Biology, Berlin

Cardiovascular diseases (CVD) are the number one cause of morbidity and mortality with an age and sexual divergence. Premenopausal women are at a lower risk for cardiovascular disease as compared to age-matched men, but this risk increases dramatically after menopause, indicating that estrogens may play a protective role. However hormone replacement therapy in humans yielded conflicting results and phytoestrogens such as genistein, widely used in traditional asian medicine, could represent alternative compounds as they are known to exert estrogenic activity and to have beneficial effect on a wide range of cardiovascular parameters.

In the cardiovascular context a disturbed energy metabolism with impaired fatty acid oxidation, ATP synthesis and changing levels of contractile proteins have been observed during diseased conditions. Whereas numerous studies focussed on gene expression analyses at the messenger RNA level, other holistic and undirected techniques, such as proteomics, have been applied to the analysis of CVD. Multiple identifications of a single protein from various spots on 2-DE gels revealed that the suggestion of a single gene or transcript, encoding for a single protein, is obsolete. The diversity in different forms of a protein emerging from one single protein-coding gene promoted the new term of protein species. While genomic and transcriptomic data lack information on protein species in a given tissue, advances in proteome analysis and mass spectrometry enabled the identification and characterization of post-translational modifications (PTMs) in peptides derived from a protein species, which lead to an increasing number of protein species in databases. Furthermore, advances in mass spectrometry allowed the localization of cleavage sites for protein processing, maturation, truncation and degradation. Protein species resulting from such cleavage events play an important role in inflammation, cell degeneration, apoptosis and oncogenesis. In the cardiovascular context, protein species derived from modifications such as acetylation, phosphorylation and cleavage are involved in various processes and disease development.

In this project we analyze the effects of a dietary supplement with the phytoestrogen genistein on the cardiac proteome pattern of young, adult and castrated male and female mice. Our analysis demonstrates considerable changes of the heart proteome with dietary genistein administration for both male and female animals. A changing abundance, of among others metabolic, energetic and contractile proteins, was observed. Initially, we focussed on the identification of PTMs of four selected proteins, using a multiple digestion protocol to enhance sequence coverage (see figure). PTMs were identified by standard NanoLC electrospray ionization ion trap mass spectrometry (nanoLC-ESI-MS/MS) and linear ion trap fourier transform ion cyclotron resonance mass spectrometry (LTQ-FT-ICR-MS/MS) and revealed several modified and truncated species. Protein species resulting from all protein modifications, post-translational chemical modifications and protein truncation are different products of one single gene. These modifications influence subcellular location, degradation, subunit assembly, tertiary structure or enzyme activity and thus protein function.

Therefore, prime importance should be rather given to systematically identify and specify proteins at their species level than to quantify total protein amount. The newly detected protein species were regulated in the myocardium of mice related to age, sex and oral genistein treatment. Therefore those species could be relevant in cardiac disease and should be taken into consideration for the molecular understanding of pathological processes.

Grafik zu Projekt 1
Fig.1: 2D-PAGE of 120 mouse kidney proteins. left: initial sample, mid: flow-through, right: MOAC-eluate. Horizontal: isoelectric focussing (pH 4-10), vertical: SDS-PAGE (approx. 250 kDa - 8 kDa)

Project leader: Prof. F. Theuring
Coworkers: Boris Neumann, Karima Schwab, both are PhD students
Funding: University Research Funding
Collaboration: Dr. Christian Scheler, Proteome Factory, Berlin

Phosphorylation is one of the most prominent post-translational modifications of proteins. Modern methodology utilises mass spectrometry (MS) for the detection and localisation of phosphorylation sites. Essential for the detection of phosphorylation sites in "real-life" samples via common mass spectrometers is an antecedent enrichment of the predominantly low abundant phospho-proteins or –peptides. Various enrichment approaches have been described. While mass spectrometers for the analysis of proteins or peptides permit an indirect detection of phosphorus due to mass shift and neutral loss, the usage of element mass spectrometers allows the direct detection on atomic level with unrivalled detection limits.

The main focus of the project is the adaption of enrichment and detection schemes to the mouse kidney proteome. Figure 1 shows 2D-gels of mouse kidney proteins that were subject to phosphoprotein enrichment by metal oxide affinity chromatography (MOAC). The protein population on the eluate gel is significantly different and exhibits a pI-shift which can be estimated due to the adopted character of the negative phosphate moiety. Proteins were identified and characterised by nanoLC-ESI-MS/MS (electrospray ionisation). Analysis by element ICP-MS (inductively couple plasma) showed comprehensible differences in the amount of atomic phosphorus 31P in the samples from the three gels. Current focus is on the further method development towards absolute 31P quantification within liquid or gel based samples.

Project leader: Prof. F. Theuring
Co-workers: Nicolas Vignon-Zellweger, Karima Schwab, both are PhD students
Funding: Marie Curie Host Fellowship for ES-Training (CARDIOVASC) to N. V-Z and F.T.) and DFG (TH 466/7-1)
Collaboration: Prof. B. Hocher, CCR; Dr. J.P.Stasch, Bayer Schering Pharma, Wuppertal

The intact endothelium produces a variety of vasoactive substances. Important among those is endothelin-1 (ET-1), a potent vasoconstrictor and mitogen in vivo and in vitro. The nitric oxide (NO) system is considered as the natural functional counterpart of the ET system, thus contributing to the subtle balance of vascular tone. The clinical relevance of this delicate interplay has been acknowledged because of its implication in many cardiovascular diseases such as pulmonary arterial hypertension, systemic hypertension, and coronary artery disease. However, the underlying molecular mechanisms remain to be fully clarified.

ET-1 overexpressing transgenic mice develop a sclerotic and/or fibrotic renal, pulmonary and myocardial phenotype. Surprisingly the ET+/+ mice remain normotensive. This lack of hypertension is believed to be the consequence of a compensatory effect of the nitric oxide system. To disrupt this compensatory effect we decided to crossbreed ET+/+ mice and eNOS knockout mice to generate the four different genotypes: ET+/+, eNOS-/-, ET+/+eNOS-/-, and WT.

The crossbred animals (ET+/+eNOS-/-) develop significantly high systolic blood pressure compared to WT mice and eNOS-/- mice. Furthermore, at the age of nine month , the eNOS-/-, but not the ET+/+eNOS-/- mice, are characterized by diastolic dysfunction. These findings suggested that transgenic overexpression of ET‐1 on an eNOS‐/‐ background could be beneficial for diastolic functions.

In order to identify the underlying mechanisms leading to this phenotype, molecular, histological, physiological and protein chemical methods were employed. In particular to analyze for the complete cardiac proteome of three months old animals high resolution two Dimensional Gel Electrophoresis coupled to mass spectrometry was performed. We could demonstrate that the cardiac proteome of the three different genotypes compared to WT animals resulted in prominent changes of the cardiac protein abundance.

The proteomics study revealed that transgenic overexpression of ET‐1, with or without eNOS, led to a higher abundance of proteins regulating oxidative stress indicating that, in contrast to eNOS‐/‐ animals, ET+/+ and ET+/+eNOS‐/‐ mice developed molecular mechanisms limiting oxidative damages. Moreover, diastolic dysfunction observed in eNOS‐/‐ mice may be explained by the differential abundance of proteins involved in the contractile machinery. Overexpression of ET‐1 in eNOS‐/‐ mice restored these changes and may have thereby benefited the cardiac functions. Finally, this study indicated that a shift from fatty acid to glucose metabolism, considered as cardioprotective, may have occurred to a greater extent in crossbred animals than in eNOS‐/‐ mice.

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Whole mount preparations of wild type and ET-1 transgenic mammary glands from lactation day 3 demonstrating lactational incompetence in the ET-1 transgenic mammary glands.
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Grafik 2

Project leader: Prof. F. Theuring, Dr. A. Fischer
Coworkers: N. Gül, PhD student
Funding: Charité-Stipend

Endothelin 1 (ET-1) is a small vasoactive peptide having wide physiological effects on vascular homeostasis and a variety of physiological and pathophysiological processes unrelated to cardiovascular physiology. It exerts its effect by binding to two distinct G Protein Coupled Receptors (GPCR). In addition to the classical GPCR signaling pathways, these receptors are also able to activate structurally unrelated receptors such as those belonging to the receptor tyrosine kinase (RTK) family in what is termed receptor transactivation.

Deregulation of this transactivation as induced by overexpression, amplification or mutation of critical pathway elements and autocrine stimulation through aberrant growth factor loops is frequently linked to hyperproliferative diseases.

The aim of this project is to further characterize the crosstalk between these two systems in the mammary epithelium in order to elucidate its contribution to mammary gland physiology and tumorigenesis. This is achieved by the molecular and morphological characterization of transgenic mice overexpressing ET-1 employing current state of the art techniques. Furthermore, an in vitro system was established employing mammary epithelial cell lines. First results suggest that endothelin is able to activate the EGFR in vivo and in vitro and that this transactivation is accompanied by impaired lactational competence in ET-1 transgenic animals as well as an upregulation of the EGFR ligand amphiregulin and ADAM 17. To further characterize the molecular events involved in this project, functional experiments employing various inhibitor compounds will be performed in order to identify the signalling pathways mediating the observed effects.

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Adalimumab prevents barrier disruption by the proinflammatory cytokines TNFα and IFNγ. T-84 intestinal epithelial cells were treated with TNFα and IFNγ or a combination of both cytokines and the therapeutic anti-TNFα antibody adalimumab. The tight junction components occludin and ZO-1 were visualized by immuno-staining.

Project leader: Dr. A. Fischer, Prof. F. Theuring
Coworkers: M. Gluth, PhD thesis, C. Tanneberger. Technician
Funding: University Research Funding, Industry Funding
Collaboration: Prof. D.C. Baumgart, Dr. U-F. Pape - Division of Hepatology and Gatroenterology CVK, Charité

The intestinal barrier constitutes the largest mucosal surface of the human body, separating the highly antigenic environment of the intestinal lumen from the milieu intérieur. Perturbations of this barrier have long been recognized as key features in inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, but have also been found in celiac disease, graft-versus-host disease and food allergies. In addition, structural components of the epithelial tight junctions have been identified as primary targets for various pathogenic bacteria-derived toxins. Pharmacological strategies to modulate the permeability of intestinal tight junctions therefore represent an attractive approach to improve the management of these disorders.

Using Caco-2 and T-84 monolayers cultivated on semipermeable filter supports as a model system of the intestinal epithelial barrier, our work focuses on the analysis of the impact of proinflammatory cytokines such as TNFα or IFNγ on barrier function. Using specific inhibitor compounds, we aim at characterizing signaling pathways responsible for the deleterious effects of these cytokines in order to identify potential targets for the treatment of inflammatory bowel diseases. In addition, compounds well established in the treatment of these disorders such as glucocorticoid hormones and anti-TNFα antibodies are evaluated in terms of their effect on barrier function. By further characterizing the mechanisms regulating paracellular permeability in the intestine, these  studies  might  provide a basis for developing new pharmacological approaches to modulate barrier function.

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Dose- and time-dependend induction of RhoA expression
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Immunofluorescent staining of RhoA expression (red) and MLC phosphorylation (green) after doxycycline withdrawal in Caco-2 intestinal epithelial cells.

Project leader: Dr. A. Fischer, Prof. F. Theuring
Coworkers: M. Gluth, PhD thesis, C. Tanneberger. Technician
Funding: University Research Funding
Collaboration: Prof. J. Turner, University of Chicago, USA

Among signaling pathways associated with the tight junction, the small GTPase RhoA has been shown to be of pivotal importance to the control of cell proliferation, migration and differentiation. Previous studies have demonstrated that expression of either constitutively active or dominant negative RhoA results in reduced tight junction barrier function in cultured MDCK monolayers. We have shown that modulation of signaling pathways downstream of RhoA regulates tight junction function in the mammary epithelium, both in vivo and in vitro. However, the precise mechanisms by which RhoA controls barrier function have not been defined.

Therefore, our work focuses on the characterization of RhoA and one of its downstream targets, the protein kinase PKN in order to elucidate molecular events involved in the RhoA-mediated regulation of epithelial tight junction permeability.

By employing an inducible expression system as an in vitro barrier model, the effects of RhoA and PKN can be studied at various developmental stages, enabling us to delineate their contribution to the regulation of tight junction function. Functional observations are complemented by the analysis of changes in the expression and localization of key tight junction components and the characterization of the molecular cross-talk to other signaling pathways. As Rho GTPases are pivotal in the pathogenesis of a variety of diseases including arterial and pulmonary hypertension, myocardial hypertrophy and vasospastic angina as well as tumorigenesis and metastasis, our work might contribute to a better understanding of the signaling pathways downstream of these molecules.

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An increase of tau aggregates in cortical sections of transgenic mice is observed with increasing age (left: 6 mo old, right 12 mo old)
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Fig2 Projekt 7

Project leader: Prof. F. Theuring
Coworkers: Dr. S. Dietze, scientist; M. Magbagbeolu, B. Seelhorst, both are Technicians; V. Melis, PhD guest; K. Schwab, scientist; A. Thoma, Animal Care Taker
Funding: TauRx Therapeutics, WisTa Laboratories, Singapore
Collaboration: Prof. C.M. Wischik, University of Aberdeen, Scotland

Tau transgenic mouse lines had been established to further analyse the functional role this protein and its aggregates, the so-called tau tangles play in clinical dementia, i.e. Alzheimer's Disease and to test putative new drug candidates in a preclinical setting to fight this neurodegenerative and terminal brain disease. In a most fruitful scientific collaboration with Prof. Wischik's group from the University of Aberdeen for the last 12 years these mice had been pivotal in establishing a relevant transgenic tau-based Alzheimer mouse model. Several dozens of newly discovered and synthesized drug candidates had been tested in vivo for their activity in reducing tau pathology and to enhance cognitive behaviour and motor skills in the drug-treated animals.

By teaming up with TauRx Pharma - a Singapore-based company spun out of the University of Aberdeen and the Charité - it is now our great pleasure to reveal a major breakthrough in the treatment of Alzheimer's disease. A total of 321 patients with mild and moderate Alzheimer's Disease were treated in a phase II clinical trial with Rember® a novel form of methylthioninium chloride (MTC). Patients receiving the study treatment experienced an 81% reduction in cognitive decline over one year, and did not experience a significant decline in their mental function over 24 months. In addition patients had repeated brain scans at the start of the study and after 25 weeks. These showed that the treatment effect was greatest in the memory-critical brain regions where the density of Alzheimer tangles is greatest. In the control group, there was a significant decline from the starting score in cognitive testing and on brain scans.

By employing our transgenic mice a group of second-generation rember® derivatives had been discovered and successfully tested. These compounds have the same mechanism of action as rember® acting as Tau Aggregation Inhibitors, with potential utility in the treatment of Alzheimer's disease and other neurodegenerative disorders. TauRx has now initiated preparations for Phase 3 studies in mild and moderate AD, and also in orphan indications such as Fronto-Temporal Dementia and provisionally Progressive Supranuclear Palsy.

These data underline the importance of generating relevant transgenic mouse models and their use in identification and validation of new drug candidates for human diseases, which then are planned to enter into clinical testing.

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Hippocampal sections of wildtype (left) and transgenic mouse (right) brains exhibiting prominent α-synuclein staining
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Fig2 Projekt 8

Project leader: Prof. F. Theuring
Coworkers: Dr. S. Dietze, scientist; M. Magbagbeolu, B. Seelhorst, both are Technicians; A. Thoma, Animal Care Taker; Dr. C. Zabke, scientist
Funding: WisTa Laboratories, Singapore
Collaboration: Prof. C.M. Wischik, University of Aberdeen, Scotland

Parkinson's disease (PD) is a common human neurodegenerative movement disorder and affects 1% of the elderly population. PD is neuropathologically characterized by a marked and progressive degeneration of dopaminergic neurons and by the presence of fibrillar cytoplasmic inclusions (Lewy bodies [LBs]) and dystrophic neurites (Lewy neurites [LNs]) in the substantia nigra and other regions of the brain. Although the loss of dopamine neurons is certainly related to the major clinical symptoms of PD, the causes and the pathogenesis of this multifactorial disease as well as that of related "synucleinopathies" are still largely unknown.

The major components of both LBs and LNs are fibrillar aggregates of α-synuclein. α Synuclein is a widely expressed, neuronal presynaptic protein that appears to play a role in membrane-associated processes and synaptic plasticity and has been linked to learning and development processes. While the mechanism(s) of formation of LBs and LNs and their association with PD are yet not understood, several lines of evidence suggest that α synuclein fibrillization is associated with PD and that α-synuclein fibrillization causes toxicity. Thus the inhibition or reversal of synuclein aggregation is believed to be of therapeutic benefit.

The development of drugs that prevent this aggregation form the basis for the scientific rationale for this project. The aim is to model the molecular processes of α-synuclein aggregation in an animal model to test and evaluate new therapeutic drug candidates for PD.

Therefore we generated α-synuclein transgenic mice. These mice are currently being characterized in more detail and will be used to study basic mechanisms of the pathogenesis underlying PD. Most importantly, these mice will then be used to screen for α-synuclein aggregation inhibitors to facilitate the development of a new therapeutic intervention for this disease.