@article{WiesslerTalucciPiroetal.2024,
  author    = {Wiessler, Anna-Lena and Talucci, Ivan and Piro, Inken and Seefried, Sabine and H{\"o}rlin, Verena and Baykan, Bet{\"u}l B. and T{\"u}z{\"u}n, Erdem and Schaefer, Natascha and Maric, Hans M. and Sommer, Claudia and Villmann, Carmen},
  title     = {Glycine receptor β-targeting autoantibodies contribute to the pathology of autoimmune diseases},
  series = {Neurology: Neuroimmunology \& Neuroinflammation},
  volume    = {11},
  journal   = {Neurology: Neuroimmunology \& Neuroinflammation},
  number    = {2},
  doi       = {10.1212/NXI.0000000000200187},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-349958},
  year      = {2024},
  abstract  = {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.},
  language  = {en}
}
@article{RauschenbergerPiroKasaragodetal.2023,
  author    = {Rauschenberger, Vera and Piro, Inken and Kasaragod, Vikram Babu and H{\"o}rlin, Verena and Eckes, Anna-Lena and Kluck, Christoph J. and Schindelin, Hermann and Meinck, Hans-Michael and Wickel, Jonathan and Geis, Christian and T{\"u}z{\"u}n, Erdem and Doppler, Kathrin and Sommer, Claudia and Villmann, Carmen},
  title     = {Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation},
  series = {Frontiers in Molecular Neuroscience},
  volume    = {16},
  journal   = {Frontiers in Molecular Neuroscience},
  doi       = {10.3389/fnmol.2023.1089101},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-304206},
  year      = {2023},
  abstract  = {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.},
  language  = {en}
}
@article{PiroEckesKasaragodetal.2021,
  author    = {Piro, Inken and Eckes, Anna-Lena and Kasaragod, Vikram Babu and Sommer, Claudia and Harvey, Robert J. and Schaefer, Natascha and Villmann, Carmen},
  title     = {Novel Functional Properties of Missense Mutations in the Glycine Receptor β Subunit in Startle Disease},
  series = {Frontiers in Molecular Neuroscience},
  volume    = {14},
  journal   = {Frontiers in Molecular Neuroscience},
  issn      = {1662-5099},
  doi       = {10.3389/fnmol.2021.745275},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-246676},
  year      = {2021},
  abstract  = {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.},
  language  = {en}
}