TY - JOUR A1 - Piro, Inken A1 - Eckes, Anna-Lena A1 - Kasaragod, Vikram Babu A1 - Sommer, Claudia A1 - Harvey, Robert J. A1 - Schaefer, Natascha A1 - Villmann, Carmen T1 - Novel Functional Properties of Missense Mutations in the Glycine Receptor β Subunit in Startle Disease JF - Frontiers in Molecular Neuroscience N2 - Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and β subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR β subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC\(_{50}\) value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR β variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR β subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and β subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR β subunits to gephyrin aggregates. However, a significantly lower number of GlyR β aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR β subunit aggregates was increased in comparison to wild-type β. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR β-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR β M3-M4 loop, we suggest that structural changes within the GlyR β subunit result in differences in GlyR β-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations. KW - glycine receptor KW - hyperekplexia KW - startle disease KW - gephyrin Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246676 SN - 1662-5099 VL - 14 ER - TY - THES A1 - Piro, Inken T1 - Glycinergic dysfunction in Stiff Person Syndrome and hyperekplexia: Investigation of patient specific pathomechanisms T1 - Glyzinerge Dysdunktion in Stiff-Person-Syndrom und Hyperekplexie: Untersuchung Patienten-spezifischer Pathomechanismen N2 - Patients diagnosed with the rare autoimmune disease of Stiff Person Syndrome (SPS) suffer from varying motor symptoms mainly characterized by painful spasms and muscle stiffness. Among patients suffering from Stiff Person spectrum, clinical presentation, course of disease and treatment responses also differ. Regardless of disease severity, which ranges from mild and intermittent motor impairments to the most severe form progressive encephalomyelitis with rigidity and myoclonus (PERM), autoantibodies are the underlying cause. One of the autoantibody targets associated with SPS is the glycine receptor (GlyR). Functional impairment of this protein interferes with inhibitory signal transmission in the central nervous system and subsequently causes motor symptoms. Similar to functional alterations of the GlyR upon autoantibody binding, GlyR function can be altered in patients with mutations in genes encoding GlyR subunits. Such mutations underlie hereditary hyperekplexia. Understanding the GlyR physiology and how different molecular mechanisms contribute to disease pathology is crucial for development of more targeted and effective disease options. Therefore, novel GlyR β subunit mutations identified in hyperekplexia patients were investigated towards their expression, trafficking and receptor function. The findings suggest that impaired recruitment into functional receptors at the synapses might underlie the functional alterations revealed by electrophysiological recordings for most cases. To unravel the autoantibody-related pathology causing the highly diverse clinical appearance of the Stiff Person spectrum, antibody binding abilities were studied. Neutralization assays confirmed that presence of the entire target protein, a sub-domain or a short peptide eliminates the autoantibodies from patient samples. Epitope characterization using residue exchanges within the GlyR in cell-based assays uncovered that GlyR autoantibody epitopes are polyclonal and their combination is patient-specific. Tissue-based binding assays emphasized the high variability in autoantibody distribution within spinal cord and brain sections regardless of the patients’ primary diagnosis. The irregular binding patterns among the patient groups of SPS, PERM, epilepsy and ‘others’ reflected the variation in the symptomatic arrangement. Passive transfer of GlyR autoantibodies from patients with different courses and severity of disease similarly revealed variable effects on murine motor and anxiety-related behavior. The detected small effects on motor function and post-mortem analyses indicate glycinergic disorganization and a possible onset of compensatory mechanisms. Altogether, this study demonstrates that GlyR impairment is patient-specific and of greater variability than expected. N2 - Patienten mit der seltenen Autoimmunerkrankung Stiff Person Syndrom (SPS) leiden unter variierenden motorischen Symptomen, die sich vor allem durch schmerzhafte Spasmen und Steifigkeit der Muskeln auszeichnen. Ebenso unterscheiden sich klinisches Erscheinungsbild, Krankheitsverlauf und Ansprechen auf entsprechende Therapien innerhalb der Gruppe von Patienten, die unter dem Stiff Person-Spektrum leidet. Unabhängig vom Schweregrad, welcher von milden und intermittierenden motorischen Einschränkungen bis hin zur schwerwiegendsten Form, der sogenannten Progressiven Encephalomyelitis mit Rigidität und Myoklonus (PERM) reicht, liegen immer Autoantikörper der Erkrankung zugrunde. Eins der Zielproteine dieser mit SPS assoziierten Autoantikörper ist der Glyzinrezeptor (GlyR). Funktionelle Einschränkungen des Rezeptors stören die inhibitorische Signalübertragung im zentralen Nervensystem, wodurch die motorischen Symptome ausgelöst werden. Ähnlich zur funktionellen Veränderung durch Autoantikörper kann die GlyR-Funktion durch Mutationen in den GlyR-Untereinheiten kodierenden Genen verändert sein. Solche Mutationen verursachen hereditäre Hyperekplexie. Ein gutes Verständnis der GlyR-Physiologie sowie der Pathomechanismen, die zur Erkrankung führen, ist entscheidend für die Entwicklung gezielter und effektiver Therapieansätze. Aus diesem Grund wurden neuartige Mutationen der GlyR β-Untereinheit, die in Hyperekplexie-Patienten identifiziert wurden, hinsichtlich ihrer Expression, ihres Transports durch die Zelle und ihres Beitrags zur Rezeptorfunktion untersucht. Die Ergebnisse suggerieren, dass eine verminderte Rekrutierung der mutierten GlyR-Untereinheit in funktionsfähige Rezeptoren an der Synapse den funktionellen Veränderungen zugrunde liegen könnte. Diese wurden in elektrophysiologischen Messungen für die meisten der untersuchten Mutationen detektiert. Um die Autoantikörper assoziierte Pathologie zu verstehen, welche das stark diverse klinische Erscheinungsbild des Stiff-Person-Spektrums hervorruft, wurden die Bindungseigenschaften der Antikörper genauer untersucht. Neutralisierungsversuche zeigten, dass die Anwesenheit des gesamten Zielproteins, einer enthaltenen Domäne oder nur eines kurzen Peptids ausreicht um die Antikörper aus einer Probe zu eliminieren. Gleichzeitig zeigte die Epitop-Charakterisierung in zellbasierten Experimenten mit Austausch einzelner Aminosäurereste im GlyR, dass die Epitope polyklonal und patientenspezifisch sind. Gewebebasierte Bindungsversuche offenbarten dementsprechend eine hohe Variabilität der Autoantikörper-Verteilung in Rückenmarks- und Gehirnschnitten unabhängig von der primären Diagnose der entsprechenden Patienten. Die ungleichmäßigen Bindungsmuster der Autoantikörper von Patientengruppen mit SPS-, PERM, Epilepsie- oder anderen Diagnosen spiegelten die Varianz der Symptomkombination wider. Ebenso verursachte passiver Transfer der GlyR-Autoantikörper von Patienten mit unterschiedlichem Verlauf und Schweregrad der Erkrankung variierende Effekte auf motorisches Verhalten und Angst-Verhalten in Mäusen. Die detektierten unterschwelligen Effekte in den Verhaltenstests und post mortem-Untersuchungen deuten auf glyzinerge Desorganisation und einen möglichen Kompensationsmechanismus hin. Insgesamt demonstriert die vorliegende Studie, dass Beeinträchtigungen des GlyR patientenspezifisch und somit vielseitiger sind als vermutet. KW - Glycinrezeptor KW - Bewegungsstörung KW - Stiff Person Syndrome KW - Hyperekplexia KW - Startle disease KW - Glycinergic dysfunction KW - autoimmune encephalitis KW - in vivo passive transfer KW - autoantibodies KW - epitope mapping KW - Autoaggressionskrankheit KW - Autoimmunkrankheit Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-327465 ER - TY - JOUR A1 - Wiessler, Anna-Lena A1 - Talucci, Ivan A1 - Piro, Inken A1 - Seefried, Sabine A1 - Hörlin, Verena A1 - Baykan, Betül B. A1 - Tüzün, Erdem A1 - Schaefer, Natascha A1 - Maric, Hans M. A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine receptor β–targeting autoantibodies contribute to the pathology of autoimmune diseases JF - Neurology: Neuroimmunology & Neuroinflammation N2 - Background and Objectives Stiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyRα subunit–binding autoantibodies with subsequent changes in function and surface numbers were reported. GlyR autoantibodies have also been described in patients with focal epilepsy. Autoimmune reactivity against the GlyRβ subunits has not yet been shown. Autoantibodies against GlyRα1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyRβ making it not unlikely that GlyRβ-specific autoantibody (aAb) exist and contribute to the disease pathology. Methods In this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyRβ. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyRβ binding and define aAb binding to distinct protein regions. Preadsorption approaches of aAbs using living cells and the purified extracellular receptor domain were further used. Finally, functional consequences for inhibitory neurotransmission upon GlyRβ aAb binding were resolved by whole-cell patch-clamp recordings. Results Among 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyRβ aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyRβ colocalized with the scaffold protein gephyrin independent of the presence of GlyRα1. At the functional level, binding of GlyRβ aAb from both patients to its target impair glycine efficacy. Discussion Our study establishes GlyRβ as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyRα1-positive sera, which alter glycine potency, aAbs against GlyRβ impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyRβ aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization. KW - autoantibody (aAb) KW - glycine receptor (GlyR) KW - stiff-person syndrome (SPS) KW - clinical neurology KW - movement disorders KW - progressive encephalitis with rigidity and myoclonus (PERM) Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349958 VL - 11 IS - 2 ER - TY - JOUR A1 - Rauschenberger, Vera A1 - Piro, Inken A1 - Kasaragod, Vikram Babu A1 - Hörlin, Verena A1 - Eckes, Anna-Lena A1 - Kluck, Christoph J. A1 - Schindelin, Hermann A1 - Meinck, Hans-Michael A1 - Wickel, Jonathan A1 - Geis, Christian A1 - Tüzün, Erdem A1 - Doppler, Kathrin A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation JF - Frontiers in Molecular Neuroscience N2 - Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor’s glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera. KW - glycine receptor KW - autoantibodies KW - glycosylation KW - extracellular domain KW - adsorption Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304206 VL - 16 ER -