FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells
Appelt-Menzel(*), A., Cubukova(*), A., Günther(*), K., Edenhofer(*), F., Piontek(*), J., Krause, G., Stüber(*), T., Walles(*), H., Neuhaus(*), W.; Metzger(*), M.
Stem cell reports, 8:894-906

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Omega cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.

The differentiation and plasticity of Tc17 cells are regulated by CTLA-4-mediated effects on STATs
Arra(*), A., Lingel(*), H., Kuropka, B., Pick(*), J., Schnoeder(*), T., Fischer(*), T., Freund(*), C., Pierau(*), M.; Brunner-Weinzierl(*), M. C.
Oncoimmunology, 6:e1273300

Tags: Mass Spectrometry (Krause, E.)

Abstract: As the blockade of inhibitory surface-molecules such as CTLA-4 on T cells has led to recent advances in antitumor immune therapy, there is great interest in identifying novel mechanisms of action of CD8+ T cells to evoke effective cytotoxic antitumor responses. Using in vitro and in vivo models, we investigated the molecular pathways underlying the CTLA-4-mediated differentiation of IL-17-producing CD8+ T cells (Tc17 cells) that strongly impairs cytotoxicity. Our studies demonstrate that Tc17 cells lacking CTLA-4 signaling have limited production of STAT3-target gene products such as IL-17, IL-21, IL-23R and RORgammat. Upon re-stimulation with IL-12, these cells display fast downregulation of Tc17 hallmarks and acquire Tc1 characteristics such as IFNgamma and TNF-alpha co-expression, which is known to correlate with tumor control. Indeed, upon adoptive transfer, these cells were highly efficient in the antigen-specific rejection of established OVA-expressing B16 melanoma in vivo. Mechanistically, in primary and re-stimulated Tc17 cells, STAT3 binding to the IL-17 promoter was strongly augmented by CTLA-4, associated with less binding of STAT5 and reduced relative activation of STAT1 which is known to block STAT3 activity. Inhibiting CTLA-4-induced STAT3 activity reverses enhancement of signature Tc17 gene products, rendering Tc17 cells susceptible to conversion to Tc1-like cells with enhanced cytotoxic potential. Thus, CTLA-4 critically shapes the characteristics of Tc17 cells by regulating relative STAT3 activation, which provides new perspectives to enhance cytotoxicity of antitumor responses.

NMR Hyperpolarization Techniques of Gases
Barskiy(*), D. A., Coffey(*), A. M., Nikolaou(*), P., Mikhaylov(*), D. M., Goodson(*), B. M., Branca(*), R. T., Lu(*), G. J., Shapiro(*), M. G., Telkki(*), V. V., Zhivonitko(*), V. V., Koptyug(*), I. V., Salnikov(*), O. G., Kovtunov(*), K. V., Bukhtiyarov(*), V. I., Rosen(*), M. S., Barlow(*), M. J., Safavi(*), S., Hall(*), I. P., Schroeder, L.; Chekmenev(*), E. Y.
Chemistry, 23:725-751

Tags: Molecular Imaging (Schröder)

Abstract: Nuclear spin polarization can be significantly increased through the process of hyperpolarization, leading to an increase in the sensitivity of nuclear magnetic resonance (NMR) experiments by 4-8 orders of magnitude. Hyperpolarized gases, unlike liquids and solids, can often be readily separated and purified from the compounds used to mediate the hyperpolarization processes. These pure hyperpolarized gases enabled many novel MRI applications including the visualization of void spaces, imaging of lung function, and remote detection. Additionally, hyperpolarized gases can be dissolved in liquids and can be used as sensitive molecular probes and reporters. This Minireview covers the fundamentals of the preparation of hyperpolarized gases and focuses on selected applications of interest to biomedicine and materials science.

Claudins are essential for cell shape changes and convergent extension movements during neural tube closure
Baumholtz(*), A. I., Simard(*), A., Nikolopoulou(*), E., Oosenbrug(*), M., Collins(*), M. M., Piontek, A., Krause, G., Piontek(*), J., Greene(*), N. D. E.; Ryan(*), A. K.
Developmental biology, 428:25-38

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: During neural tube closure, regulated changes at the level of individual cells are translated into large-scale morphogenetic movements to facilitate conversion of the flat neural plate into a closed tube. Throughout this process, the integrity of the neural epithelium is maintained via cell interactions through intercellular junctions, including apical tight junctions. Members of the claudin family of tight junction proteins regulate paracellular permeability, apical-basal cell polarity and link the tight junction to the actin cytoskeleton. Here, we show that claudins are essential for neural tube closure: the simultaneous removal of Cldn3, -4 and -8 from tight junctions caused folate-resistant open neural tube defects. Their removal did not affect cell type differentiation, neural ectoderm patterning nor overall apical-basal polarity. However, apical accumulation of Vangl2, RhoA, and pMLC were reduced, and Par3 and Cdc42 were mislocalized at the apical cell surface. Our data showed that claudins act upstream of planar cell polarity and RhoA/ROCK signaling to regulate cell intercalation and actin-myosin contraction, which are required for convergent extension and apical constriction during neural tube closure, respectively.

Post-translational cleavage of Hv1 in human sperm tunes pH- and voltage-dependent gating
Berger(*), T. K., Fusshöller(*), D. M., Goodwin(*), N., Bönigk(*), W., Müller(*), A., Dokani Khesroshahi(*), N., Brenker(*), C., Wachten(*), D., Krause, E., Kaupp(*), U. B.; Strünker(*), T.
J Physiol, 595:1533-1546

Tags: Mass Spectrometry (Krause, E.)

Abstract: KEY POINTS: In human sperm, proton flux across the membrane is controlled by the voltage-gated proton channel Hv1. We show that sperm harbour both Hv1 and an N-terminally cleaved isoform termed Hv1Sper. The pH-control of Hv1Sper and Hv1 is distinctively different. Hv1Sper and Hv1 can form heterodimers that combine features of both constituents. Cleavage and heterodimerization of Hv1 might represent an adaptation to the specific requirements of pH control in sperm. ABSTRACT: In human sperm, the voltage-gated proton channel Hv1 controls the flux of protons across the flagellar membrane. Here, we show that sperm harbour Hv1 and a shorter isoform, termed Hv1Sper. Hv1Sper is generated from Hv1 by removal of 68 amino acids from the N-terminus by post-translational proteolytic cleavage. The pH-dependent gating of the channel isoforms is distinctly different. In both Hv1 and Hv1Sper, the conductance-voltage relationship is determined by the pH difference across the membrane (pH). However, simultaneous changes in intracellular and extracellular pH that leave DeltapH constant strongly shift the activation curve of Hv1Sper but not that of Hv1, demonstrating that cleavage of the N-terminus tunes pH sensing in Hv1. Moreover, we show that Hv1 and Hv1Sper assemble as heterodimers that combine features of both constituents. We suggest that cleavage and heterodimerization of Hv1 represents an adaptation to the specific requirements of pH control in sperm.

Targeting G-protein-coupled receptors by Capture Compound Mass Spectrometry (CCMS) - a case study with sertindole
Blex(*), C., Michaelis(*), S., Schrey(*), A. K., Furkert, J., Eichhorst, J., Bartho(*), K., Quast(*), F. G., Marais(*), A., Hakelberg(*), M., Gruber(*), U., Niquet(*), S., Popp(*), O., Kroll(*), F., Sefkow(*), M., Schülein, R., Mathias(*), D.; Koster(*), H.
Chembiochem, 18:1639-1649

Tags: Protein Trafficking (Schülein), Cellular Imaging (Wiesner/Puchkov)

Abstract: Unbiased chemoproteomic profiling of small molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G-protein coupled receptors (GPCRs). These are hardly tractable by affinity pulldown from lysates. We report a Capture Compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK293 cells. The structure-activity relationship of sertindole for DRD2 binding is reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and CCMS. The activity pattern was rationalized by molecular modelling. The most active CC showed activities very similar to unmodifed sertindole. Well below 100 fmol of DRD2 in living cells used as experiment input were sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions.

Reggie-1 and reggie-2 (flotillins) participate in Rab11a-dependent cargo trafficking, spine synapse formation and LTP-related AMPA receptor (GluA1) surface exposure in mouse hippocampal neurons
Bodrikov(*), V., Pauschert(*), A., Kochlamazashvili, G.; Stuermer(*), C. A.
Exp Neurol, 289:31-45

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Reggie-1 and -2 (flotillins) reside at recycling vesicles and promote jointly with Rab11a the targeted delivery of cargo. Recycling is essential for synapse formation suggesting that reggies and Rab11a may regulate the development of spine synapses. Recycling vesicles provide cargo for dendritic growth and recycle surface glutamate receptors (AMPAR, GluA) for long-term potentiation (LTP) induced surface exposure. Here, we show reduced number of spine synapses and impairment of an in vitro correlate of LTP in hippocampal neurons from reggie-1 k.o. (Flot2-/-) mice maturating in culture. These defects apparently result from reduced trafficking of PSD-95 revealed by live imaging of 10 div reggie-1 k.o. (Flot2-/-) neurons and likely impairs co-transport of cargo destined for spines: N-cadherin and the glutamate receptors GluA1 and GluN1. Impaired cargo trafficking and fewer synapses also emerged in reggie-1 siRNA, reggie-2 siRNA, and reggie-1 and -2 siRNA-treated neurons and was in siRNA and k.o. neurons rescued by reggie-1-EGFP and CA-Rab11a-EGFP. While correlative expressional changes of specific synapse proteins were observed in reggie-1 k.o. (Flot2-/-) brains in vivo, this did not occur in neurons maturating in vitro. Our work suggests that reggie-1 and reggie-2 function at Rab11a recycling containers in the transport of PSD-95, N-cadherin, GluA1 and GluN1, and promote (together with significant signaling molecules) spine-directed trafficking, spine synapse formation and the in vitro correlate of LTP.

Oxidative inactivation of the endogenous antioxidant protein DJ-1 by the food contaminants 3-MCPD and 2-MCPD
Buhrke(*), T., Voss(*), L., Briese(*), A., Stephanowitz, H., Krause, E., Braeuning(*), A.; Lampen(*), A.
Archives of toxicology,

Tags: Mass Spectrometry (Krause, E.)

Abstract: 3-Chloro-1,2-propanediol (3-MCPD) and 2-chloro-1,3-propanediol (2-MCPD) are heat-induced food contaminants being present either as free substances or as fatty acid esters in numerous foods. 3-MCPD was classified to be possibly carcinogenic to humans (category 2B) with kidney and testis being the primary target organs according to animal studies. A previous 28-day oral feeding study with rats revealed that the endogenous antioxidant protein DJ-1 was strongly deregulated at the protein level in kidney, liver, and testis of the experimental animals that had been treated either with 3-MCPD, 2-MCPD or their dipalmitate esters. Here we show that this deregulation is due to the oxidation of a conserved, redox-active cysteine residue (Cys106) of DJ-1 to a cysteine sulfonic acid which is equivalent to loss of function of DJ-1. Irreversible oxidation of DJ-1 is associated with a number of oxidative stress-related diseases such as Parkinson, cancer, and type II diabetes. It is assumed that 3-MCPD or 2-MCPD do not directly oxidize DJ-1, but that these substances induce the formation of reactive oxygen species (ROS) which in turn trigger DJ-1 oxidation. The implications of 3-MCPD/2-MCPD-mediated ROS formation in vivo for the ongoing risk assessment of these compounds as well as the potential of oxidized DJ-1 to serve as a novel effect biomarker for 3-MCPD/2-MCPD toxicity are being discussed.

Identification of a Novel Benzimidazole Pyrazolone Scaffold That Inhibits KDM4 Lysine Demethylases and Reduces Proliferation of Prostate Cancer Cells
Carter(*), D. M., Specker, E., Przygodda, J., Neuenschwander, M., von Kries, J. P., Heinemann(*), U., Nazare, M.; Gohlke(*), U.
SLAS discovery, 22:801-812

Tags: Screening Unit (von Kries), Medicinal Chemistry (Nazare)

Abstract: Human lysine demethylase (KDM) enzymes (KDM1-7) constitute an emerging class of therapeutic targets, with activities that support growth and development of metastatic disease. By interacting with and co-activating the androgen receptor, the KDM4 subfamily (KDM4A-E) promotes aggressive phenotypes of prostate cancer (PCa). Knockdown of KDM4 expression or inhibition of KDM4 enzyme activity reduces the proliferation of PCa cell lines and highlights inhibition of lysine demethylation as a possible therapeutic method for PCa treatment. To address this possibility, we screened the ChemBioNet small molecule library for inhibitors of the human KDM4E isoform and identified several compounds with IC50 values in the low micromolar range. Two hits, validated as active by an orthogonal enzyme-linked immunosorbent assay, displayed moderate selectivity toward the KDM4 subfamily and exhibited antiproliferative effects in cellular models of PCa. These compounds were further characterized by their ability to maintain the transcriptionally silent histone H3 tri-methyl K9 epigenetic mark at subcytotoxic concentrations. Taken together, these efforts identify and validate a hydroxyquinoline scaffold and a novel benzimidazole pyrazolone scaffold as tractable for entry into hit-to-lead chemical optimization campaigns.

Gamma oscillations organize top-down signalling to hypothalamus and enable food seeking
Carus-Cadavieco, M., Gorbati, M., Ye(*), L., Bender, F., van der Veldt, S., Kosse(*), C., Borgers(*), C., Lee(*), S. Y., Ramakrishnan(*), C., Hu, Y., Denisova, N., Ramm, F., Volitaki, E., Burdakov(*), D., Deisseroth(*), K., Ponomarenko, A.; Korotkova, T.
Nature, 542:232-236

Tags: Behavioral Neurodynamics (Korotkova/Ponomarenko)

Abstract: Both humans and animals seek primary rewards in the environment, even when such rewards do not correspond to current physiological needs. An example of this is a dissociation between food-seeking behaviour and metabolic needs, a notoriously difficult-to-treat symptom of eating disorders. Feeding relies on distinct cell groups in the hypothalamus, the activity of which also changes in anticipation of feeding onset. The hypothalamus receives strong descending inputs from the lateral septum, which is connected, in turn, with cortical networks, but cognitive regulation of feeding-related behaviours is not yet understood. Cortical cognitive processing involves gamma oscillations, which support memory, attention, cognitive flexibility and sensory responses. These functions contribute crucially to feeding behaviour by unknown neural mechanisms. Here we show that coordinated gamma (30-90 Hz) oscillations in the lateral hypothalamus and upstream brain regions organize food-seeking behaviour in mice. Gamma-rhythmic input to the lateral hypothalamus from somatostatin-positive lateral septum cells evokes food approach without affecting food intake. Inhibitory inputs from the lateral septum enable separate signalling by lateral hypothalamus neurons according to their feeding-related activity, making them fire at distinct phases of the gamma oscillation. Upstream, medial prefrontal cortical projections provide gamma-rhythmic inputs to the lateral septum; these inputs are causally associated with improved performance in a food-rewarded learning task. Overall, our work identifies a top-down pathway that uses gamma synchronization to guide the activity of subcortical networks and to regulate feeding behaviour by dynamic reorganization of functional cell groups in the hypothalamus.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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