A core aspect of our work concerns the study of the brain at the molecular level, by investigating C-to-U RNA editing and alternative RNA splicing. In particular, we analyze key components of the molecular machine responsible for inhibition of electrical impulses, that are glycine receptors (GlyR) and GABA type A receptors (GABA(A)R) as well as the postsynaptic scaffold protein gephyrin. We elucidate the function of these molecules in physiological and pathophysiological processes on a molecular, cellular and systemic level. Temporal lobe epilepsy (TLE) is a devastating neurodegenerative disease that severely deteriorates life quality due to unpredictable occurrence of seizures and resulting cognitive dysfunction. Moreover, epilepsy patients suffer from severe psychiatric comorbidities including anxiety and depression. Most epilepsy syndromes have no discernable genetic component. This indicates that epileptogenesis is governed by disease-promoting molecular and cellular mechanisms of neuronal plasticity, which may vary from patient to patient and involve cell type-specific mechanisms, resulting in diverse clinical pictures of cryptogenic/idiopathic TLE. Recently, we developed in our lab molecular tools and used advanced chemo-biological approaches to visualize and characterize C-to-U RNA editing in general, and regarding GlyRs in particular, at the single cell level in vitro and in vivo and hence, identify critical cell types with TLE-dependent alterations in RNA editing levels.
In more detail, we identified C-to-U RNA editing of the GlyR as a critical target in the development of neuropsychiatric symptoms of TLE by analyzing hippocampi resected from patients with TLE and assessing their pathophysiological roles in mouse models of the disease. Transgenic animals opened the possibility to study the role of edited GlyR in a neuron type specific way. As part of the VW-Vorab consortium HomeoHIRN we are now addressing the roles of edited GlyR in a neuronal compartment specific way and particularly focus on the possible (patho)physiological dialogue or failure of dialogue between changes in presynaptic excitability and homeostatic regulation by the somatodendritic compartment.
We ask whether there are somatodendritic mechanisms that sense and eventually counteract (or fail to counteract) changes in the output of neurons that are due to changes in presynaptic function due to expression of RNA-edited GlyRs. To achieve this goal, we are developing microfluidic chambers (MFC) for the in vitro cell culture of primary neurons derived from wildtype and our transgenic mice. The microfluidic approach shall allow to combine epifluorescence and confocal imaging as well as super-resolution microscopy with optogenetics, and electrophysiology by microelectrode arrays (MEA) and whole cell patch clamp for analysis of passive membrane properties as well as of various parameters of synaptic connectivity in a compartmentalized environment (see Figure). The MEA will also be used for high-throughput drug screening as it is applicable to any cell type (including fibroblast HEK293 cells expressing selected proteins of interest). Readout is based on fluorescence imaging and stimulation (e.g. optogenetics) and recording of electrical local field potentials. Whole cell patch clamp electrophysiological analyses will complement data sets obtained in a cell type and cell type compartment specific way. Optically accessible Lab-On-Chip systems, that allow experiments with living tissue in microcompartments have been developed in the past by advanced micro/nano fabrication techniques [Mattern et al. 2020, Lorenz et al. 2021]. The MFC to be realized in HomeoHIRN with digital and mask based technologies provides neuronal sub-compartments which are separated by a physical barrier. This barrier can only be passed through microgrooves that guide the growth of individual axons. Due to the combination of larger outer dimensions with much smaller microgroove details, a MFC molding master has to be fabricated using different methods, two-photon-polymerization (2pp) for the microgrooves part and conventional 3d print techniques for the chamber part. Mold replications of the chamber body and of the microgrooves are realized by so-called soft lithography using PDMS. Oxygen plasma bonding is applied to form the combination of both. The MFC is finalized by reversible bonding with a glass slide whereby the microgrooves are capped and optical access for high resolution microscopy is provided. The medium flowing through the different compartments of the chamber will be available for metabolomics and drug screening. In place of a bare glass slide a cover glass equipped with MEA can be bonded to the MFC, which shall be capable of not only recording but also stimulating electrical activities. MEAs are fabricated by lithographic processes in the cleanroom and are adjusted to the microgroove geometries.
We successfully developed different technologies to study RNA editing of specific targets and C-to-U RNA editing in general and at the single cell level and in a compartment-specific way. Our recently developed molecular tools (“RNA editing sensor” [Kankowski et al., 2018] and C-to-U RNA editing inducible protein expression system “CUREIPES” [Schweissthal et al., 2021]) are available to detect and quantitatively measure the activity of C-to-U RNA deaminases that catalyze the RNA editing at the cell type specific level by compartmentalized translocation of fluorescent proteins or onset of fluorescent protein expression through RNA editing. CUREIPES can not only be used as reporter tool but also opens the possibility to express specific proteins of interest in response to increased RNA editing on demand to counteract pathophysiological changes in RNA editing rates. Based on our established novel molecular tools, we currently work on a new system that enables visualization of enzyme activity in real-time directly at the position in the cell (down to the synapse level) where RNA editing occurs. Thus, to take engineering of our molecular tools to the next level in a neuronal compartment-specific way, we are currently also developing this novel tool for the real-time detection of C-to-U RNA editing in a cell compartment-specific way down to the level of single synapses.
Lemmens, V., Thevelein, B., Vella, Y., Kankowski, S., Leonhard, J., Mizuno, H., Rocha, S., Brône, B., Meier, J. C., & Hendrix, J. (2022). Hetero-pentamerization determines mobility and conductance of Glycine receptor α3 splice variants. Cellular and molecular life sciences : CMLS, 79(11), 540. https://doi.org/10.1007/s00018-022-04506-9
Schweissthal B, Brunken K, Brach J, Emde L, Hetsch F, Fricke S, Meier JC. A new triple fluorescence reporter system for discrimination of Apobec1 and Apobec3 C-to-U RNA editing activities and editing-dependent protein expression. bioRxiv 2021; doi: 10.1101/2021.03.03.433736.
Kankowski S, Förstera B, Winkelmann A, Knauff P, Wanker EE, You XA, Semtner M, Hetsch F, Meier JC. A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy. Front Mol Neurosci. 2018 Jan 11; 10:439. doi: 10.3389/fnmol.2017.00439. PMID: 29375302; PMCID: PMC5768626.
Mattern, K., Trotha, J.W., Erfle, P., Köster, R.W., Dietzel, A., NeuroExaminer: an all-glass microfluidic device for whole-brain in vivo imaging in zebrafish (2020) Communications Biology, 3 (1), art. no. 311, DOI: 10.1038/s42003-020-1029-7.
Lorenz, T., Kirschke, M., Ledwig, V., Reichl, S., Dietzel, A., Microfluidic system for in vivo-like drug permeation studies with dynamic dilution profiles (2021) Bioengineering, 8 (5), art. no. 58, DOI: 10.3390/bioengineering8050058.
Prof. Dr. Jochen Meier
Technische Universität Braunschweig
Phone: +49 531 391 3254
Prof. Dr. Andreas Dietzel
Technische Universität Braunschweig
Institute of Microtechnology - IMT
Alte Salzdahlumer Str. 203
Phone: +49 531 391 9760