TY - JOUR A1 - Wieland, Annalena A1 - Strissel, Pamela L. A1 - Schorle, Hannah A1 - Bakirci, Ezgi A1 - Janzen, Dieter A1 - Beckmann, Matthias W. A1 - Eckstein, Markus A1 - Dalton, Paul D. A1 - Strick, Reiner T1 - Brain and breast cancer cells with PTEN loss of function reveal enhanced durotaxis and RHOB dependent amoeboid migration utilizing 3D scaffolds and aligned microfiber tracts JF - Cancers N2 - Background: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures. Methods: 3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology. Results: In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes. Conclusions: This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy. KW - 3D tumor model KW - 3D microfiber KW - amoeboid cell migration KW - brain cancer KW - breast cancer KW - PTEN KW - RHO KW - ROCK KW - durotaxis KW - topotaxis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248443 SN - 2072-6694 VL - 13 IS - 20 ER - TY - JOUR A1 - Janzen, Dieter A1 - Bakirci, Ezgi A1 - Wieland, Annalena A1 - Martin, Corinna A1 - Dalton, Paul D. A1 - Villmann, Carmen T1 - Cortical Neurons form a Functional Neuronal Network in a 3D Printed Reinforced Matrix JF - Advanced Healthcare Materials N2 - Impairments in neuronal circuits underly multiple neurodevelopmental and neurodegenerative disorders. 3D cell culture models enhance the complexity of in vitro systems and provide a microenvironment closer to the native situation than with 2D cultures. Such novel model systems will allow the assessment of neuronal network formation and their dysfunction under disease conditions. Here, mouse cortical neurons are cultured from embryonic day E17 within in a fiber‐reinforced matrix. A soft Matrigel with a shear modulus of 31 ± 5.6 Pa is reinforced with scaffolds created by melt electrowriting, improving its mechanical properties and facilitating the handling. Cortical neurons display enhance cell viability and the neuronal network maturation in 3D, estimated by staining of dendrites and synapses over 21 days in vitro, is faster in 3D compared to 2D cultures. Using functional readouts with electrophysiological recordings, different firing patterns of action potentials are observed, which are absent in the presence of the sodium channel blocker, tetrodotoxin. Voltage‐gated sodium currents display a current–voltage relationship with a maximum peak current at −25 mV. With its high customizability in terms of scaffold reinforcement and soft matrix formulation, this approach represents a new tool to study neuronal networks in 3D under normal and, potentially, disease conditions. KW - 3D electrophysiology KW - 3D neuronal networks KW - cortical neurons KW - melt electrowriting Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215400 VL - 9 IS - 9 ER - TY - JOUR A1 - Kuhlemann, Alexander A1 - Beliu, Gerti A1 - Janzen, Dieter A1 - Petrini, Enrica Maria A1 - Taban, Danush A1 - Helmerich, Dominic A. A1 - Doose, Sören A1 - Bruno, Martina A1 - Barberis, Andrea A1 - Villmann, Carmen A1 - Sauer, Markus A1 - Werner, Christian T1 - Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy JF - Frontiers in Synaptic Neuroscience N2 - Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft. KW - super-resolution microscopy (SRM) KW - click-chemistry KW - dSTORM KW - GABA-A receptor KW - genetic code expansion Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251035 SN - 1663-3563 VL - 13 ER - TY - JOUR A1 - Schaefer, Natascha A1 - Roemer, Vera A1 - Janzen, Dieter A1 - Villmann, Carmen T1 - Impaired Glycine Receptor Trafficking in Neurological Diseases JF - Frontiers in Molecular Neuroscience N2 - Ionotropic glycine receptors (GlyRs) enable fast synaptic neurotransmission in the adult spinal cord and brainstem. The inhibitory GlyR is a transmembrane glycinegated chloride channel. The immature GlyR protein undergoes various processing steps, e.g., folding, assembly, and maturation while traveling from the endoplasmic reticulum to and through the Golgi apparatus, where post-translational modifications, e.g., glycosylation occur. The mature receptors are forward transported via microtubules to the cellular surface and inserted into neuronal membranes followed by synaptic clustering. The normal life cycle of a receptor protein includes further processes like internalization, recycling, and degradation. Defects in GlyR life cycle, e.g., impaired protein maturation and degradation have been demonstrated to underlie pathological mechanisms of various neurological diseases. The neurological disorder startle disease is caused by glycinergic dysfunction mainly due to missense mutations in genes encoding GlyR subunits (GLRA1 and GLRB). In vitro studies have shown that most recessive forms of startle disease are associated with impaired receptor biogenesis. Another neurological disease with a phenotype similar to startle disease is a special form of stiff-person syndrome (SPS), which is most probably due to the development of GlyR autoantibodies. Binding of GlyR autoantibodies leads to enhanced receptor internalization. Here we focus on the normal life cycle of GlyRs concentrating on assembly and maturation, receptor trafficking, post-synaptic integration and clustering, and GlyR internalization/recycling/degradation. Furthermore, this review highlights findings on impairment of these processes under disease conditions such as disturbed neuronal ER-Golgi trafficking as the major pathomechanism for recessive forms of human startle disease. In SPS, enhanced receptor internalization upon autoantibody binding to the GlyR has been shown to underlie the human pathology. In addition, we discuss how the existing mouse models of startle disease increased our current knowledge of GlyR trafficking routes and function. This review further illuminates receptor trafficking of GlyR variants originally identified in startle disease patients and explains changes in the life cycle of GlyRs in patients with SPS with respect to structural and functional consequences at the receptor level. KW - glycine receptor KW - startle disease KW - autoimmune antibodies KW - protein maturation KW - trafficking pathways Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227531 VL - 11 IS - 291 ER - TY - JOUR A1 - Milanos, Sinem A1 - Elsharif, Shaimaa A. A1 - Janzen, Dieter A1 - Buettner, Andrea A1 - Villmann, Carmen T1 - Metabolic Products of Linalool and Modulation of GABA\(_{A}\) Receptors JF - Frontiers in Chemistry N2 - Terpenoids are major subcomponents in aroma substances which harbor sedative physiological potential. We have demonstrated that various monoterpenoids such as the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon overexpression of inhibitory α1β2 GABA\(_{A}\) receptors in various expression systems. However, in plants or humans, i.e., following intake via inhalation or ingestion, linalool undergoes metabolic modifications including oxygenation and acetylation, which may affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the modulatory potential of linalool derivatives at α1β2γ2 GABA\(_{A}\) receptors upon transient overexpression. Following receptor expression control, electrophysiological recordings in a whole cell configuration were used to determine the chloride influx upon co-application of GABA EC\(_{10-30}\) together with the modulatory substance. Our results show that only oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated linalool derivatives resulted in non-significant changes of GABAergic currents. We can conclude that metabolism of linalool reduces its positive allosteric potential at GABAA receptors compared to the significant potentiation effects of the parent molecule linalool itself. KW - Cys-loop receptor KW - GABA\(_{A}\) KW - receptor KW - linalool KW - linalyl acetate KW - oxygenation KW - patch-clamp Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170779 VL - 5 IS - 46 ER - TY - JOUR A1 - Janzen, Dieter A1 - Bakirci, Ezgi A1 - Faber, Jessica A1 - Andrade Mier, Mateo A1 - Hauptstein, Julia A1 - Pal, Arindam A1 - Forster, Leonard A1 - Hazur, Jonas A1 - Boccaccini, Aldo R. A1 - Detsch, Rainer A1 - Teßmar, Jörg A1 - Budday, Silvia A1 - Blunk, Torsten A1 - Dalton, Paul D. A1 - Villmann, Carmen T1 - Reinforced Hyaluronic Acid-Based Matrices Promote 3D Neuronal Network Formation JF - Advanced Healthcare Materials N2 - 3D neuronal cultures attempt to better replicate the in vivo environment to study neurological/neurodegenerative diseases compared to 2D models. A challenge to establish 3D neuron culture models is the low elastic modulus (30–500 Pa) of the native brain. Here, an ultra-soft matrix based on thiolated hyaluronic acid (HA-SH) reinforced with a microfiber frame is formulated and used. Hyaluronic acid represents an essential component of the brain extracellular matrix (ECM). Box-shaped frames with a microfiber spacing of 200 µm composed of 10-layers of poly(ɛ-caprolactone) (PCL) microfibers (9.7 ± 0.2 µm) made via melt electrowriting (MEW) are used to reinforce the HA-SH matrix which has an elastic modulus of 95 Pa. The neuronal viability is low in pure HA-SH matrix, however, when astrocytes are pre-seeded below this reinforced construct, they significantly support neuronal survival, network formation quantified by neurite length, and neuronal firing shown by Ca\(^{2+}\) imaging. The astrocyte-seeded HA-SH matrix is able to match the neuronal viability to the level of Matrigel, a gold standard matrix for neuronal culture for over two decades. Thus, this 3D MEW frame reinforced HA-SH composite with neurons and astrocytes constitutes a reliable and reproducible system to further study brain diseases. KW - 3D model systems KW - melt electrowriting KW - cortical neurons KW - astrocytes KW - Ca\(^{2+}\)-Imaging KW - hyaluronic acid Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318682 VL - 11 IS - 21 ER - TY - JOUR A1 - Janzen, Dieter A1 - Slavik, Benedikt A1 - Zehe, Markus A1 - Sotriffer, Christoph A1 - Loos, Helene M. A1 - Buettner, Andrea A1 - Villmann, Carmen T1 - Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric potential and affect inhibitory GABA\(_{A}\) receptor function in vitro JF - Journal of Neurochemistry N2 - Naturally occurring compounds such as sesquiterpenes and sesquiterpenoids (SQTs) have been shown to modulate GABA\(_{A}\) receptors (GABA\(_{A}\)Rs). In this study, the modulatory potential of 11 SQTs at GABA\(_{A}\)Rs was analyzed to characterize their potential neurotropic activity. Transfected HEK293 cells and primary hippocampal neurons were functionally investigated using electrophysiological whole-cell recordings. Significantly different effects of β-caryophyllene and α-humulene, as well as their respective derivatives β-caryolanol and humulol, were observed in the HEK293 cell system. In neurons, the concomitant presence of phasic and tonic GABA\(_{A}\)R configurations accounts for differences in receptor modulation by SQTs. The in vivo presence of the γ\(_{2}\) and δ subunits is important for SQT modulation. While phasic GABA\(_{A}\) receptors in hippocampal neurons exhibited significantly altered GABA-evoked current amplitudes in the presence of humulol and guaiol, negative allosteric potential at recombinantly expressed α\(_{1}\)β\(_{2}\)γ\(_{2}\) receptors was only verified for humolol. Modeling and docking studies provided support for the binding of SQTs to the neurosteroid-binding site of the GABA\(_{A}\)R localized between transmembrane segments 1 and 3 at the (\(^{+}\)α)-(\(^{-}\)α) interface. In sum, differences in the modulation of GABA\(_{A}\)R isoforms between SQTs were identified. Another finding is that our results provide an indication that nutritional digestion affects the neurotropic potential of natural compounds. KW - allosteric modulation KW - GABA\(_{A}\) receptor KW - patch clamp recording Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259546 VL - 159 IS - 1 ER -