@phdthesis{Fuhl2024,
  author    = {Fuhl, Isabell},
  title     = {Untersuchung der synaptischen Lokalisation des heteromeren Glycin-Rezeptors in einem neuen Mausmodell der \(Startle\) Erkrankung - mit Fokus auf die GlyR-β-Untereinheit -},
  doi       = {10.25972/OPUS-34832},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-348328},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Der Glycin-Rezeptor ist Teil der inhibitorischen liganden-gesteuerten Ionenkan{\"a}le im ZNS und wird am st{\"a}rksten im adulten R{\"u}ckenmark sowie im Hirnstamm exprimiert. In der Nerv-Muskel-Synapse sind GlyR f{\"u}r die rekurrente Hemmung der Motoneuronen wichtig und steuern das Gleichgewicht zwischen Erregung und Hemmung der Muskelzellen. F{\"u}r die glycinerge Neurotransmission sind neben den pr{\"a}synaptischen GlyR 𝛼1 insbesondere postsynaptische GlyR 𝛼1/𝛽 verantwortlich. Durch Mutationen des GlyR entsteht das Erkrankungsbild der Hyperekplexie mit {\"u}bersteigerter Schreckhaftigkeit, Muskelsteifheit und Apnoe. Haupturs{\"a}chlich daf{\"u}r sind Mutationen im GLRA1-Gen. Die shaky Maus stellt ein gutes Modell zur Erforschung dieser seltenen Erkrankung dar. Die shaky Missense-Mutation Q177K in der extrazellul{\"a}ren 𝛽8-𝛽9 Schleife der Glycin- Rezeptor-𝛼1-Untereinheit zeigte strukturell ein gest{\"o}rtes Wasserstoffbr{\"u}ckennetzwerk. Funktionell konnten eingeschr{\"a}nkt leitf{\"a}hige Ionenkan{\"a}le identifiziert werden. Der letale Ph{\"a}notyp {\"a}ußert sich beim homozygoten shaky Tier durch Schrecksymptome mit einem einhergehenden zunehmenden Gewichtsverlust. Die Quantifizierung der Oberfl{\"a}chenexpression deutete auf einen Verlust synaptischer GlyR 𝛼1/𝛽 hin. Aussagen bez{\"u}glich der GlyR-𝛽-Untereinheit, die Teil des synaptischen GlyR Komplexes ist, waren aufgrund fehlender stabiler Antik{\"o}rper bisher nicht m{\"o}glich. Das neuartige KI- Mausmodell Glrb eos exprimiert endogen fluoreszierende 𝛽 -Untereinheiten und erm{\"o}glicht damit erstmalig eine Betrachtung der GlyR- 𝛽-Expression in Tiermodellen der Startle Erkrankung. Ziel dieser Arbeit war es, die Auswirkungen der shaky Mutation auf die Interaktion mit der 𝛽 -Untereinheit und Gephyrin zu erforschen. Daf{\"u}r wurden Markerproteine der glycinergen Synapse in R{\"u}ckenmarksneuronen der Kreuzung Glrb eos x Glra1 sh gef{\"a}rbt und quantifiziert. Die durchgef{\"u}hrte Gewichtsbestimmung der Nachkommen im zeitlichen Verlauf zeigte keinen Einfluss der eingef{\"u}gten mEos4b-Sequenz auf das K{\"o}rpergewicht der Tiere und schließt damit funktionelle Einschr{\"a}nkungen bedingt durch die mEos4b-Sequenz aus. Zur Verst{\"a}rkung des 𝛽 eos-Signals wurde ein Antik{\"o}rper verwendet. Die Quantifizierung der GlyR- 𝛽- Untereinheit an R{\"u}ckenmarksneuronen zeigte f{\"u}r homozygote shaky Tiere im Vergleich zum Wildtyp signifikant reduzierte 𝛽eos Oberfl{\"a}chenexpressionen in Gephyrin Clustern sowie signifikant erniedrigte Kolokalisationen von Gephyrin/𝛼1, 𝛽eos/𝛼1 und 𝛽eos/Gephyrin. Die mutierte GlyR-𝛼1- Untereinheit wurde hingegen vermehrt an der Oberfl{\"a}che in shaky Tieren exprimiert. Die Ergebnisse der R{\"u}ckenmarksschnitte unterst{\"u}tzen diese Befunde aus den Prim{\"a}rneuronen. Die Untersuchung der Pr{\"a}synapse erbrachte f{\"u}r Glrb eos/eos x Glra1 sh/sh eine signifikant verminderte Synapsin und Synapsin/𝛼1 Expression. Die Ergebnisse dieser Arbeit erweitern die Daten fr{\"u}herer Arbeiten zur shaky Maus und zeigen einen starken Verlust synaptischer GlyR 𝛼 1/ 𝛽 an der Oberfl{\"a}che von Motoneuronen. Ein m{\"o}glicher kompensatorischer Versuch durch erh{\"o}hte 𝛼1 Expression bleibt infolge der Funktionsbeeintr{\"a}chtigung dieser mutierten GlyR- 𝛼 1 Rezeptoren erfolglos mit letalem Ausgang. In vorherigen Arbeiten wurde vermutet, dass die Mutation in der extrazellul{\"a}ren Bindungsstelle in der Lage ist, Konformations{\"a}nderungen in die TM3-TM4-Schleifenstruktur zu {\"u}bertragen und dadurch die Gephyrin Bindung und synaptische Verankerung zu st{\"o}ren. Die Daten dieser Arbeit st{\"u}tzen diese Annahme und weisen dar{\"u}ber hinaus auf eine gest{\"o}rte Rezeptorkomplexbindung hin. Die vorliegende Arbeit tr{\"a}gt somit zum besseren Verst{\"a}ndnis der Startle Erkrankung auf synaptischer Ebene bei.},
  subject      = {Glycinrezeptor},
  language  = {de}
}
@phdthesis{Janzen2022,
  author    = {Janzen, Dieter},
  title     = {Functional analysis of ion channels and neuronal networks in 2D and 3D \(in\) \(vitro\) cell culture models},
  doi       = {10.25972/OPUS-25170},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251700},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {In the central nervous system, excitatory and inhibitory signal transduction processes are mediated by presynaptic release of neurotransmitters, which bind to postsynaptic receptors. Glycine receptors (GlyRs) and GABAA receptors (GABAARs) are ligand-gated ion channels that enable synaptic inhibition. One part of the present thesis elucidated the role of the GlyRα1 β8 β9 loop in receptor expression, localization, and function by means of amino acid substitutions at residue Q177. This residue is underlying a startle disease phenotype in the spontaneous mouse model shaky and affected homozygous animals are dying 4-6 weeks after birth. The residue is located in the β8 β9 loop and thus part of the signal transduction unit essential for proper ion channel function. Moreover, residue Q177 is involved in a hydrogen network important for ligand binding. We observed no difference in ion channel trafficking to the cellular membrane for GlyRα1Q177 variants. However, electrophysiological measurements demonstrated reduced glycine, taurine, and β alanine potency in comparison to the wildtype protein. Modeling revealed that some GlyRα1Q177 variants disrupt the hydrogen network around residue Q177. The largest alterations were observed for the Q177R variant, which displayed similar effects as the Q177K mutation present in shaky mice. Exchange with structurally related amino acids to the original glutamine preserved the hydrogen bond network. Our results underlined the importance of the GlyR β8 β9 loop for proper ion channel gating. GlyRs as well as GABAARs can be modulated by numerous allosteric substances. Recently, we focused on monoterpenes from plant extracts and showed positive allosteric modulation of GABAARs. Here, we focused on the effect of 11 sesquiterpenes and sesquiterpenoids (SQTs) on GABAARs. SQTs are compounds naturally occurring in plants. We tested SQTs of the volatile fractions of hop and chamomile, including their secondary metabolites generated during digestion. Using the patch-clamp technique on transfected cells and neurons, we were able to observe significant GABAAR modulation by some of the compounds analyzed. Furthermore, a possible binding mechanism of SQTs to the neurosteroid binding site of the GABAAR was revealed by modeling and docking studies. We successfully demonstrated GABAAR modulation by SQTs and their secondary metabolites. The second part of the thesis investigated three-dimensional (3D) in vitro cell culture models which are becoming more and more important in different part of natural sciences. The third dimension allows developing of complex models closer to the natural environment of cells, but also requires materials with mechanical and biological properties comparable to the native tissue of the encapsulated cells. This is especially challenging for 3D in vitro cultures of primary neurons and astrocytes as the brain is one of the softest tissues found in the body. Ultra-soft matrices that mimic the neuronal in vivo environment are difficult to handle. We have overcome these challenges using fiber scaffolds created by melt electrowriting to reinforce ultra-soft matrigel. Hence, the scaffolds enabled proper handling of the whole composites and thus structural and functional characterizations requiring movement of the composites to different experimental setups. Using these scaffold-matrigel composites, we successfully established methods necessary for the characterization of neuronal network formation. Before starting with neurons, a mouse fibroblast cell line was seeded in scaffold-matrigel composites and transfected with the GlyR. 3D cultured cells displayed high viability, could be immunocytochemically stained, and electrophysiologically analyzed. In a follow-up study, primary mouse cortical neurons in fiber-reinforced matrigel were grown for up to 21 days in vitro. Neurons displayed high viability, and quantification of neurite lengths and synapse density revealed a fully formed neuronal network already after 7 days in 3D culture. Calcium imaging and patch clamp experiments demonstrated spontaneous network activity, functional voltage-gated sodium channels as well as action potential firing. By combining ultra-soft hydrogels with fiber scaffolds, we successfully created a cell culture model suitable for future work in the context of cell-cell interactions between primary cells of the brain and tumor cells, which will help to elucidate the molecular pathology of aggressive brain tumors and possibly other disease mechanisms.},
  subject      = {Zellkultur},
  language  = {en}
}
@phdthesis{Rauschenberger2021,
  author    = {Rauschenberger, Vera},
  title     = {Stiff-person syndrome - Pathophysiological mechanisms of glycine receptor autoantibodies},
  doi       = {10.25972/OPUS-20958},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-209588},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The Stiff-person syndrome (SPS) is a rare autoimmune disease that is characterized by symptoms including stiffness in axial and limb muscles as well as painful spasms. Different variants of SPS are known ranging from moderate forms like the stiff-limb syndrome to the most severe form progressive encephalomyelitis with rigidity and myoclonus (PERM). SPS is elicited by autoantibodies that target different pre- or postsynaptic proteins. The focus of the present work is on autoantibodies against the glycine receptor (GlyR). At start of the present thesis, as main characteristic of the GlyR autoantibody pathology, receptor cross-linking followed by enhanced receptor internalization and degradation via the lysosomal pathway was described. If binding of autoantibodies modulates GlyR function and therefore contributes to the GlyR autoantibody pathology has not yet been investigated. Moreover, not all patients respond well to plasmapheresis or other treatments used in the clinic. Relapses with even higher autoantibody titers regularly occur. In the present work, further insights into the disease pathology of GlyRα autoantibodies were achieved. We identified a common GlyRα1 autoantibody epitope located in the far N-terminus including amino acids A1-G34 which at least represent a part of the autoantibody epitope. This part of the receptor is easily accessible for autoantibodies due to its location at the outermost surface of the GlyRα1 extracellular domain. It was further investigated if the glycosylation status of the GlyR interferes with autoantibody binding. Using a GlyRα1 de-glycosylation mutant exhibited that patient autoantibodies are able to detect the de-glycosylated GlyRα1 variant as well. The direct modulation of the GlyR analyzed by electrophysiological recordings demonstrated functional alterations of the GlyR upon autoantibody binding. Whole cell patch clamp recordings revealed that autoantibodies decreased the glycine potency, shown by increased EC50 values. Furthermore, an influence on the desensitization behavior of the receptor was shown. The GlyR autoantibodies, however, had no impact on the binding affinity of glycine. These issues can be explained by the localization of the GlyR autoantibody epitope. The determined epitope has been exhibited to influence GlyR desensitization upon binding of allosteric modulators and differs from the orthosteric binding site for glycine, which is localized much deeper in the structure at the interface between two adjacent subunits. To neutralize GlyR autoantibodies, two different methods have been carried out. Transfected HEK293 cells expressing GlyRα1 and ELISA plates coated with the GlyRα1 extracellular domain were used to efficiently neutralize the autoantibodies. Finally, the successful passive transfer of GlyRα1 autoantibodies into zebrafish larvae and mice was shown. The autoantibodies detected their target in spinal cord and brain regions rich in GlyRs of zebrafish and mice. A passive transfer of human GlyRα autoantibodies to zebrafish larvae generated an impaired escape behavior in the animals compatible with the abnormal startle response in SPS or PERM patients.},
  subject      = {Glycinrezeptor},
  language  = {en}
}
@phdthesis{Mohsen2017,
  author    = {Mohsen, Amal Mahmoud Yassin},
  title     = {Structure Activity Relationships of Monomeric and Dimeric Strychnine Analogs as Ligands Targeting Glycine Receptors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142228},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The inhibitory glycine receptors are one of the major mediators of rapid synaptic inhibition in the mammalian brainstem, spinal cord and higher brain centres. They are ligand-gated ion channels that are mainly involved in the regulation of motor functions. Dysfunction of the receptor is associated with motor disorders such as hypereklepxia or some forms of spasticity. GlyR is composed of two glycosylated integral membrane proteins α and β and a peripheral membrane protein of gephyrin. Moreover, there are four known isoforms of the α-subunit (α1-4) of GlyR while there is a single β-subunit. Glycine receptors can be homomeric including α subunits only or heteromeric containing both α and β subunits. To date, strychnine is the ligand that has the highest affinity as glycine receptor ligand. It acts as a competitive antagonist of glycine that results in the inhibition of Cl- ions permeation and consequently reducing GlyR-mediated inhibition. For a long time, the details of the molecular mechanism of GlyRs inactivation by strychnine were insufficient due to the lack of high-resolution structures of the receptor. Only homology models based on structures of other cys-loop receptors have been available. Recently, 3.0 {\AA} X-ray structure of the human glycine receptor- α3 homopentamer in complex with strychnine, as well as electro cryo-microscopy structures of the zebra fish α1 GlyR in complex with strychnine and glycine were published. Such information provided detailed insight into the molecular recognition of agonists and antagonists and mechanisms of GlyR activation and inactivation. Very recently, a series of dimeric strychnine analogs obtained by diamide formation of two molecules of 2-aminostrychnine with diacids of different chain length was pharmacologically evaluated at human α1 and α1β glycine receptors. None of the dimeric analogs was superior to strychnine. The present work focused on the extension of the structure-activity relationships of strychnine derivatives at glycine receptors All the synthesized compounds were pharmacologically evaluated at human α1 and α1β glycine receptors in a functional FLIPRTM assay and the most potent analogs were pharmacologically evaluated in a whole cell patch-clamp assay and in [3H]strychnine binding studies. It was reported that 11-(E)-isonitrosostrychnine displayed a 2-times increased binding to both α1 and α1β glycine receptors which prompted us to choose the hydroxyl group as a suitable attachment point to connect two 11-(E)-isonitrosostrychnine molecules using a spacer. In order to explore the GlyR pocket tolerance for oxime extension, a series of oxime ethers with different spacer lengths and sterical/lipophilic properties were synthesized biologically evaluated. Among all the oxime ethers, methyl, allyl and propagyl oxime ethers were the most potent antagonists displaying IC50 values similar to that of strychnine. These findings indicated that strychnine binding site at GlyRs comprises an additional small lipophilic pocket located in close proximity to C11 of strychnine and the groups best accommodated in this pocket are (E)-allyl and (E)-propagyl oxime ethers. Moreover, 11-aminostrychnine, and the corresponding propionamide were prepared and pharmacologically evaluated to examine the amide function at C11 as potential linker. A series of dimeric strychnine analogs designed by linking two strychnine molecules through amino groups in position 11 with diacids were synthesized and tested in binding studies and functional assays at human α1 and α1β glycine receptors. The synthesized bivalent ligands were designed to bind simultaneously to two α-subunits of the pentameric glycine receptors causing a possibly stronger inhibition than the monomeric strychnine. However, all the bivalent derivatives showed no significant difference in potency compared to strychnine. When comparing the reference monomeric propionamide containing ethylene spacer to the dimeric ligand containing butylene spacer, a 3-fold increase in potency was observed. Since the dimer containing (CH2)10 spacer length was found to be equipotent to strychnine, it is assumed that one molecule of strychnine binds to the receptor and the 'additional' strychnine molecule in the dimer probably protrudes from the orthosteric binding sites of the receptor.},
  subject      = {Strychnin},
  language  = {en}
}
@phdthesis{Langlhofer2016,
  author    = {Langlhofer, Georg},
  title     = {{\"U}ber die Bedeutung intrazellul{\"a}rer Subdom{\"a}nen des Glycinrezeptors f{\"u}r die Kanalfunktion},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140249},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Der zur Familie der pentameren ligandengesteuerten Ionenkan{\"a}le zugeh{\"o}rige Glycinrezeptor (GlyR) ist ein wichtiger Vermittler synaptischer Inhibition im Zentralnervensystem von S{\"a}ugetieren. GlyR-Mutationen f{\"u}hren zur neurologischen Bewegungsst{\"o}rung Hyperekplexie. Aufgrund fehlender struktureller Daten ist die intrazellul{\"a}re Loop-Struktur zwischen den Transmembransegmenten 3 und 4 (TM3-4 Loop) eine weitgehend unerforschte Dom{\"a}ne des GlyR. Innerhalb dieser Dom{\"a}ne wurden Rezeptortrunkierungen sowie Punktmutationen identifiziert. Rezeptortrunkierung geht mit Funktionslosigkeit einher, welche jedoch durch Koexpression des fehlenden Sequenzabschnitts zum Teil wiederhergestellt werden kann. Innerhalb dieser Arbeit wurde die Interaktion zwischen trunkierten, funktionslosen GlyR und sukzessiv verk{\"u}rzten Komplementationskonstrukten untersucht. Dabei wurden als Minimaldom{\"a}nen f{\"u}r die Interaktion das C-terminalen basische Motive des TM3-4 Loops, die TM4 sowie der extrazellul{\"a}re C-Terminus identifiziert. Die R{\"u}ckkreuzung transgener M{\"a}use, die das Komplementationskonstrukt iD-TM4 unter Kontrolle des GlyR-Promotors exprimierten, mit der oscillator-Maus spdot, die einen trunkierten GlyR exprimiert und 3 Wochen nach der Geburt verstirbt, hatte aufgrund fehlender Proteinexpression keinen Effekt auf die Letalit{\"a}t der Mutation. Des Weiteren wurde die Bedeutsamkeit der Integrit{\"a}t beider basischer Motive 316RFRRKRR322 und 385KKIDKISR392 im TM3-4 Loop in Kombination mit der Loop-L{\"a}nge f{\"u}r die Funktionalit{\"a}t und das Desensitisierungsverhalten des humanen GlyRα1 anhand von chim{\"a}ren Rezeptoren identifiziert. Eine bisher unbekannte Patientenmutation P366L innerhalb des TM3-4 Loops wurde mit molekularbiologischen, biochemischen und elektrophysiologischen Methoden charakterisiert. Es wurde gezeigt, dass die mutierten Rezeptorkomplexe in vitro deutlich reduzierte Glycin-induzierte Maximalstr{\"o}me sowie eine beschleunigte Schließkinetik aufweisen. P366L hat im Gegensatz zu bereits charakterisierten Hyperekplexiemutationen innerhalb des TM3-4 Loops keinen Einfluss auf die Biogenese des Rezeptors. P366 ist Teil einer m{\"o}glichen Poly-Prolin-Helix, die eine Erkennungssequenz f{\"u}r SH3-Dom{\"a}nen darstellt. Ein potenzieller Interaktionspartner des TM3-4 Loops des GlyRα1 ist Collybistin, welches eine wichtige Rolle bei der synaptischen Rezeptorintegration spielt und die Verbindung zum Zytoskelett vermittelt. An der inhibitorischen Synapse verursacht P366L durch die Reduzierung postsynaptischer Chloridstr{\"o}me, das beschleunigte Desensitisierungsverhalten des GlyRα1 sowie ein ver{\"a}ndertes Interaktionsmotiv St{\"o}rungen der glycinergen Transmission, die zur Auspr{\"a}gung ph{\"a}notypischer Symptome der Hyperekplexie f{\"u}hren.},
  subject      = {Glycinrezeptor},
  language  = {de}
}