Publications by authors named "Carmen Villmann"

56 Publications

Autoantibodies to cortactin and agrin in sera of patients with myasthenia gravis.

J Neuroimmunol 2021 Jul 30;356:577588. Epub 2021 Apr 30.

University Hospital Würzburg, Department of Neurology, Josef-Schneider-Str. 11, 97080 Würzburg, Germany. Electronic address:

Autoantibodies against agrin and cortactin have been described in patients with myasthenia gravis. To further validate and characterize these autoantibodies, sera and/or plasma exchange material of 135 patients with myasthenia gravis were screened for anti-agrin or anti-cortactin autoantibodies. Autoantibodies against cortactin were detected in three patients and two controls and could be confirmed by cell-based assays using cortactin-transfected human embryonic kidney cells in both controls and one patient, but were not detectable in follow-up sera of the three patients. We did not detect any autoantibodies against agrin. The clinical phenotype of anti-cortactin-positive patients varied, arguing against a relevant pathogenic role.
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http://dx.doi.org/10.1016/j.jneuroim.2021.577588DOI Listing
July 2021

A proline-rich motif in the large intracellular loop of the glycine receptor α1 subunit interacts with the Pleckstrin homology domain of collybistin.

J Adv Res 2021 03 8;29:95-106. Epub 2020 Oct 8.

Department of Biochemistry, German University in Cairo, Egypt.

Introduction: The inhibitory glycine receptor (GlyR), a mediator of fast synaptic inhibition, is located and held at neuronal synapses through the anchoring proteins gephyrin and collybistin. Stable localization of neurotransmitter receptors is essential for synaptic function. In case of GlyRs, only beta subunits were known until now to mediate synaptic anchoring.

Objectives: We identified a poly-proline II helix (PPII) in position 365-373 of the intra-cellular TM3-4 loop of the human GlyRα1 subunit as a novel potential synaptic anchoring site. The potential role of the PPII helix as synaptic anchoring site was tested.

Methods: Glycine receptors and collybistin variants were generated and recombinantly expressed in HEK293 cells and cultured neurons. Receptor function was assessed using patch-clamp electrophysiology, protein-protein interaction was studied using co-immuno-precipitation and pulldown experiments.

Results: Recombinantly expressed collybistin bound to isolated GlyRα1 TM3-4 loops in GST-pulldown assays. When the five proline residues P365A, P366A, P367A, P369A, P373A (GlyRα1) located in the GlyRα1-PPII helix were replaced by alanines, the PPII secondary structure was disrupted. Recombinant GlyRα1 mutant subunits displayed normal cell surface expression and wildtype-like ion channel function, but binding to collybistin was abolished. The GlyRα1-collybistin interaction was independently confirmed by o-immunoprecipitation assays using full-length GlyRα1 subunits. Surprisingly, the interaction was not mediated by the SH3 domain of collybistin, but by its Pleckstrin homology (PH) domain. The mutation GlyRα1, identified in a hyperekplexia patient, is also disrupting the PPII helix, and caused reduced collybistin binding.

Conclusion: Our data suggest a novel interaction between α1 GlyR subunits and collybistin, which is physiologically relevant in vitro and in vivo and may contribute to postsynaptic anchoring of glycine receptors.
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http://dx.doi.org/10.1016/j.jare.2020.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020344PMC
March 2021

C-2-Linked Dimeric Strychnine Analogues as Bivalent Ligands Targeting Glycine Receptors.

J Nat Prod 2021 02 17;84(2):382-394. Epub 2021 Feb 17.

Institute of Pharmacy and Food Chemistry, University of Würzburg, 97074 Würzburg, Germany.

Strychnine is the prototypic antagonist of glycine receptors, a family of pentameric ligand-gated ion channels. Recent high-resolution structures of homomeric glycine receptors have confirmed the presence of five orthosteric binding sites located in the extracellular subunit interfaces of the receptor complex that are targeted by strychnine. Here, we report the synthesis and extensive pharmacological evaluation of bivalent ligands composed of two strychnine pharmacophores connected by appropriate spacers optimized toward simultaneous binding to two adjacent orthosteric sites of homomeric α1 glycine receptors. In all bivalent ligands, the two strychnine units were linked through C-2 by amide spacers of various lengths ranging from 6 to 69 atoms. Characterization of the compounds in two functional assays and in a radioligand binding assay indicated that compound , with a spacer consisting of 57 atoms, may be capable of bridging the homomeric α1 GlyRs by simultaneous occupation of two adjacent strychnine-binding sites. The findings are supported by docking experiments to the crystal structure of the homomeric glycine receptor. Based on its unique binding mode, its relatively high binding affinity and antagonist potency, and its slow binding kinetics, the bivalent strychnine analogue could be a valuable tool to study the functional properties of glycine receptors.
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http://dx.doi.org/10.1021/acs.jnatprod.0c01030DOI Listing
February 2021

Pyridoxal kinase inhibition by artemisinins down-regulates inhibitory neurotransmission.

Proc Natl Acad Sci U S A 2020 12 14;117(52):33235-33245. Epub 2020 Dec 14.

Institute of Structural Biology, Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, 97080 Würzburg, Germany;

The antimalarial artemisinins have also been implicated in the regulation of various cellular pathways including immunomodulation of cancers and regulation of pancreatic cell signaling in mammals. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the postsynaptic scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin B6 (also known as pyridoxal 5'-phosphate [PLP]), in complex with artesunate at 2.4-Å resolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins also interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.
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http://dx.doi.org/10.1073/pnas.2008695117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7777086PMC
December 2020

Melt Electrowritten In Vitro Radial Device to Study Cell Growth and Migration.

Adv Biosyst 2020 10 2;4(10):e2000077. Epub 2020 Sep 2.

Department of Clinical Neurobiology, University Hospital of Würzburg, Versbacherstr. 5, Würzburg, 97078, Germany.

The development of in vitro assays for 3D microenvironments is essential for understanding cell migration processes. A 3D-printed in vitro competitive radial device is developed to identify preferred Matrigel concentration for glioblastoma migration. Melt electrowriting (MEW) is used to fabricate the structural device with defined and intricate radial structures that are filled with Matrigel. Controlling the printing path is necessary to account for the distance lag in the molten jet, the applied electric field, and the continuous direct-writing nature of MEW. Circular printing below a diameter threshold results in substantial inward tilting of the MEW fiber wall. An eight-chamber radial device with a diameter of 9.4 mm is printed. Four different concentrations of Matrigel are dispensed into the radial chambers. Glioblastoma cells are seeded into the center and grow into all chambers within 8 days. The cell spreading area demonstrates that 6 and 8 mg mL of Matrigel are preferred over 2 and 4 mg mL . Furthermore, topographical cues via the MEW fiber wall are observed to promote migration even further away from the cell seeding depot. Previous studies implement MEW to fabricate cell invasive scaffolds whereas here it is applied to 3D-print in vitro tools to study cell migration.
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http://dx.doi.org/10.1002/adbi.202000077DOI Listing
October 2020

Anxiety and Startle Phenotypes in and Mouse Mutants.

Front Mol Neurosci 2020 11;13:152. Epub 2020 Aug 11.

Institute of Clinical Neurobiology, University Hospital, Julius Maximilians University of Würzburg, Würzburg, Germany.

A GWAS study recently demonstrated single nucleotide polymorphisms (SNPs) in the human gene of individuals with a prevalence for agoraphobia. encodes the glycine receptor (GlyRs) β subunit. The identified SNPs are localized within the gene flanking regions (3' and 5' UTRs) and intronic regions. It was suggested that these nucleotide polymorphisms modify GlyRs expression and phenotypic behavior in humans contributing to an anxiety phenotype as a mild form of hyperekplexia. Hyperekplexia is a human neuromotor disorder with massive startle phenotypes due to mutations in genes encoding GlyRs subunits. mutations have been more commonly observed than mutations. If an anxiety phenotype contributes to the hyperekplexia disease pattern has not been investigated yet. Here, we compared two mouse models harboring either a mutation in the murine or gene with regard to anxiety and startle phenotypes. Homozygous animals carrying a point mutation (alanine 52 to serine) displayed abnormally enhanced startle responses. Moreover, mice exhibited significant changes in fear-related behaviors (freezing, rearing and time spent on back) analyzed during the startle paradigm, even in a neutral context. mice exhibit reduced expression levels of the full-length GlyRs β subunit due to aberrant splicing of the gene. Heterozygous animals appear normal without an obvious behavioral phenotype and thus might reflect the human situation analyzed in the GWAS study on agoraphobia and startle. In contrast to mice, heterozygous animals revealed no startle phenotype in a neutral as well as a conditioning context. Other mechanisms such as a modulatory function of the GlyRs β subunit within glycinergic circuits in neuronal networks important for fear and fear-related behavior may exist. Possibly, in human additional changes in fear and fear-related circuits either due to gene-gene interactions e.g., with genes or epigenetic factors are necessary to create the agoraphobia and in particular the startle phenotype.
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http://dx.doi.org/10.3389/fnmol.2020.00152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7433344PMC
August 2020

Antiparanodal antibodies and IgG subclasses in acute autoimmune neuropathy.

Neurol Neuroimmunol Neuroinflamm 2020 09 24;7(5). Epub 2020 Jul 24.

From the Department of Neurology (L.A., A.-M.B., C.S., K.D.), University Hospital of Würzburg; Neuroimmunology Section (A.H., K.-P.W., R.J., F.L.), Institute of Clinical Chemistry, University Hospital of Schleswig-Holstein Campus Kiel; Department of Neurology (P.K.), University Hospital of Magdeburg; and Institute for Clinical Neurobiology (C.V.), University Hospital of Würzburg, Germany.

Objective: To determine whether IgG subclasses of antiparanodal autoantibodies are related to disease course and treatment response in acute- to subacute-onset neuropathies, we retrospectively screened 161 baseline serum/CSF samples and 66 follow-up serum/CSF samples.

Methods: We used ELISA and immunofluorescence assays to detect antiparanodal IgG and their subclasses and titers in serum/CSF of patients with Guillain-Barré syndrome (GBS), recurrent GBS (R-GBS), Miller-Fisher syndrome, and acute- to subacute-onset chronic inflammatory demyelinating polyradiculoneuropathy (A-CIDP). We evaluated clinical data retrospectively.

Results: We detected antiparanodal autoantibodies with a prevalence of 4.3% (7/161), more often in A-CIDP (4/23, 17.4%) compared with GBS (3/114, 2.6%). Longitudinal subclass analysis in the patients with GBS revealed IgG2/3 autoantibodies against Caspr-1 and against anti-contactin-1/Caspr-1, which disappeared at remission. At disease onset, patients with A-CIDP had IgG2/3 anti-Caspr-1 and anti-contactin-1/Caspr-1 or IgG4 anti-contactin-1 antibodies, IgG3 being associated with good response to IV immunoglobulins (IVIg). In the chronic phase of disease, IgG subclass of one patient with A-CIDP switched from IgG3 to IgG4.

Conclusion: Our data (1) confirm and extend previous observations that antiparanodal IgG2/3 but not IgG4 antibodies can occur in acute-onset neuropathies manifesting as monophasic GBS, (2) suggest association of IgG3 to a favorable response to IVIg, and (3) lend support to the hypothesis that in some patients, an IgG subclass switch from IgG3 to IgG4 may be the correlate of a secondary progressive or relapsing course following a GBS-like onset.
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http://dx.doi.org/10.1212/NXI.0000000000000817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413710PMC
September 2020

Glycine Receptor Autoantibodies Impair Receptor Function and Induce Motor Dysfunction.

Ann Neurol 2020 09 20;88(3):544-561. Epub 2020 Jul 20.

Institute for Clinical Neurobiology, University Hospital, Julius Maximilian University of Würzburg, Würzburg, Germany.

Objective: Impairment of glycinergic neurotransmission leads to complex movement and behavioral disorders. Patients harboring glycine receptor autoantibodies suffer from stiff-person syndrome or its severe variant progressive encephalomyelitis with rigidity and myoclonus. Enhanced receptor internalization was proposed as the common molecular mechanism upon autoantibody binding. Although functional impairment of glycine receptors following autoantibody binding has recently been investigated, it is still incompletely understood.

Methods: A cell-based assay was used for positive sample evaluation. Glycine receptor function was assessed by electrophysiological recordings and radioligand binding assays. The in vivo passive transfer of patient autoantibodies was done using the zebrafish animal model.

Results: Glycine receptor function as assessed by glycine dose-response curves showed significantly decreased glycine potency in the presence of patient sera. Upon binding of autoantibodies from 2 patients, a decreased fraction of desensitized receptors was observed, whereas closing of the ion channel remained fast. The glycine receptor N-terminal residues A to G were mapped as a common epitope of glycine receptor autoantibodies. An in vivo transfer into the zebrafish animal model generated a phenotype with disturbed escape behavior accompanied by a reduced number of glycine receptor clusters in the spinal cord of affected animals.

Interpretation: Autoantibodies against the extracellular domain mediate alterations of glycine receptor physiology. Moreover, our in vivo data demonstrate that the autoantibodies are a direct cause of the disease, because the transfer of human glycine receptor autoantibodies to zebrafish larvae generated impaired escape behavior in the animal model compatible with abnormal startle response in stiff-person syndrome or progressive encephalitis with rigidity and myoclonus patients. ANN NEUROL 2020;88:544-561.
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http://dx.doi.org/10.1002/ana.25832DOI Listing
September 2020

Comparison of biophysical properties of α1β2 and α3β2 GABAA receptors in whole-cell patch-clamp electrophysiological recordings.

PLoS One 2020 1;15(6):e0234080. Epub 2020 Jun 1.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

In the present study we have characterized the biophysical properties of wild-type (WT) α1β2 and α3β2 GABAA receptors and probed the molecular basis for the observed differences. The activation and desensitization behavior and the residual currents of the receptors expressed in HEK293 cells were determined in whole-cell patch clamp recordings. Kinetic parameters of α1β2 and α3β2 activation differed significantly, with α1β2 and α3β2 exhibiting rise times (10-90%) of 24 ± 2 ms and 51 ± 7 ms, respectively. In contrast, the two receptors exhibited largely comparable desensitization behavior with decay currents that could be fitted to exponential functions with two or three components. Most notably, the two receptor compositions displayed different degrees of desentization, with the residual currents of α1β2 and α3β2 constituting 34 ± 2% and 21 ± 2% of the peak current, respectively. The respective contributions of the extracellular domains and the transmembrane/intracellular domains of the α-subunit to these physiological profiles were next assessed in recordings from cells expressing αβ2 receptors comprising chimeric α-subunits. The rise times displayed by α1ECD/α3TMDβ2 and α3ECD/α1TMDβ2 receptors were intermediate to those of WT α1β2 and WT α3β2, and the distribution of the different components of the current decays exhibited by the two chimeric receptors followed the same pattern as the two WT receptors. The residual current exhibited by α1ECD/α3TMDβ2 (23 ± 3%) was similar to that of α3β2 but significantly different from that of α1β2, whereas the residual current displayed by α3ECD/α1TMDβ2 (27 ± 2%) was intermediate to and did not differ significantly from either of the WT receptors. This points to molecular differences in the transmembrane/intracellular domains of the α-subunit as the main determinants of the observed differences in receptor physiology between α1β2 and α3β2 receptors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234080PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263626PMC
August 2020

A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition.

J Neurosci 2020 06 30;40(25):4954-4969. Epub 2020 Apr 30.

Institute for Clinical Neurobiology, University Hospital, Julius-Maximilians-University of Würzburg, 97070 Würzburg, Germany

Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels. We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.
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http://dx.doi.org/10.1523/JNEUROSCI.2490-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326357PMC
June 2020

Cortical Neurons form a Functional Neuronal Network in a 3D Printed Reinforced Matrix.

Adv Healthc Mater 2020 05 17;9(9):e1901630. Epub 2020 Mar 17.

Institute for Clinical Neurobiology, University Hospital Würzburg, Versbacherstr. 5, Würzburg, 97078, Germany.

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.
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http://dx.doi.org/10.1002/adhm.201901630DOI Listing
May 2020

Anti-pan-neurofascin IgG3 as a marker of fulminant autoimmune neuropathy.

Neurol Neuroimmunol Neuroinflamm 2019 09 16;6(5). Epub 2019 Aug 16.

From the Department of Neurology (H.S., A.M.B., L.A., B.F., A.P., C.S., K.D.), University Hospital Würzburg; Institute of Clinical Neuroimmunology (A.V., E.M.), Biomedical Center, University Hospitals, Ludwig-Maximilians-Universität München, Planegg-Martinsried; Universitätsklinikum Schleswig-Holstein Campus Kiel (A.H., F.L.), Neuroimmunology Section, Institute of Clinical Chemistry, Kiel/Lübeck; Department of Neurology (F.G.), University Hospital Halle; Department of Neurology (C.D., F.B.), University Hospital Mainz, Mainz; University Hospital Aachen (J.W.), Institute of Neuropathology, Aachen; Department of Neurology (T.H., P.K.), University Hospital Magdeburg; Institute for Pharmacology and Toxicology (C.S.), Otto-von-Guericke University; German Center for Neurodegenerative Diseases (P.K.), Magdeburg; Institute for Clinical Neurobiology (C.V.), University Hospital Würzburg; Department of Neurology (F.L.), Universitätsklinikum Schleswig-Holstein, Kiel, Germany; and Research Center for Translational Medicine (A.V), Koç University, Istanbul, Turkey.

Objective: To identify and characterize patients with autoantibodies against different neurofascin (NF) isoforms.

Methods: Screening of a large cohort of patient sera for anti-NF autoantibodies by ELISA and further characterization by cell-based assays, epitope mapping, and complement binding assays.

Results: Two different clinical phenotypes became apparent in this study: The well-known clinical picture of subacute-onset severe sensorimotor neuropathy with tremor that is known to be associated with IgG4 autoantibodies against the paranodal isoform NF-155 was found in 2 patients. The second phenotype with a dramatic course of disease with tetraplegia and almost locked-in syndrome was associated with IgG3 autoantibodies against nodal and paranodal isoforms of NF in 3 patients. The epitope against which these autoantibodies were directed in this second phenotype was the common Ig domain found in all 3 NF isoforms. In contrast, anti-NF-155 IgG4 were directed against the NF-155-specific Fn3Fn4 domain. The description of a second phenotype of anti-NF-associated neuropathy is in line with some case reports of similar patients that were published in the last year.

Conclusions: Our results indicate that anti-pan-NF-associated neuropathy differs from anti-NF-155-associated neuropathy, and epitope and subclass play a major role in the pathogenesis and severity of anti-NF-associated neuropathy and should be determined to correctly classify patients, also in respect to possible differences in therapeutic response.
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http://dx.doi.org/10.1212/NXI.0000000000000603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705632PMC
September 2019

11-Aminostrychnine and -(Strychnine-11-yl)propionamide: Synthesis, Configuration, and Pharmacological Evaluation at Glycine Receptors.

J Nat Prod 2019 08 6;82(8):2332-2336. Epub 2019 Aug 6.

Institute of Pharmacy and Food Chemistry , University of Würzburg , 97074 Würzburg , Germany.

(11)-11-Aminostrychnine () and -[(11)-strychnine-11-yl]propionamide () were synthesized and characterized as antagonists of homomeric α1 and heteromeric α1β glycine receptors in a functional fluorescence-based assay and a patch-clamp assay and in radioligand binding studies. The absolute configuration at C-11 of was determined based on vicinal coupling constants and NOESY data. Docking experiments to the orthosteric binding site of the α3 glycine receptor showed a binding mode of compound analogous to that of strychnine, explaining its high antagonistic potency. The findings identify the C-11 amide function of strychnine as a suitable linker group for the future development of dimeric strychnine analogues targeting glycine receptors. The findings extend the SAR of strychnine at glycine receptors.
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http://dx.doi.org/10.1021/acs.jnatprod.9b00180DOI Listing
August 2019

The P429L loss of function mutation of the human glycine transporter 2 associated with hyperekplexia.

Eur J Neurosci 2019 12 5;50(12):3906-3920. Epub 2019 Sep 5.

Institute for Clinical Neurobiology, Julius-Maximilians-University of Würzburg, Würzburg, Germany.

Glycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. We characterised the GlyT2 variant at the molecular, cellular and protein level. Functionality was determined by glycine uptake assays. Homology modelling revealed that the mutation localises to α-helix 5, presumably disrupting the integrity of this α-helix. GlyT2 shows protein trafficking through various intracellular compartments to the cellular surface. However, the protein expression at the whole cell level was significantly reduced. Although present at the cellular surface, GlyT2 demonstrated a loss of protein function. Coexpression of the mutant with the wild-type protein, reflecting the situation in the parents, did not affect transporter function, thus explaining their non-symptomatic phenotype. Nevertheless, when the mutant was expressed in excess compared with the wild-type protein, glycine uptake was significantly reduced. Thus, these data demonstrate that the proline residue at position 429 is structurally important for the correct formation of α-helix 5. The failure in functionality of the mutated GlyT2 is most probably due to structural changes localised in close proximity to the sodium-binding site of the transporter.
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http://dx.doi.org/10.1111/ejn.14533DOI Listing
December 2019

Anti-CNTN1 IgG3 induces acute conduction block and motor deficits in a passive transfer rat model.

J Neuroinflammation 2019 Apr 5;16(1):73. Epub 2019 Apr 5.

Department of Neurology, University Hospital Würzburg, Josef-Schneider-Str. 11, 97080, Würzburg, Germany.

Background: Autoantibodies against the paranodal protein contactin-1 have recently been described in patients with severe acute-onset autoimmune neuropathies and mainly belong to the IgG4 subclass that does not activate complement. IgG3 anti-contactin-1 autoantibodies are rare, but have been detected during the acute onset of disease in some cases. There is evidence that anti-contactin-1 prevents adhesive interaction, and chronic exposure to anti-contactin-1 IgG4 leads to structural changes at the nodes accompanied by neuropathic symptoms. However, the pathomechanism of acute onset of disease and the pathogenic role of IgG3 anti-contactin-1 is largely unknown.

Methods: In the present study, we aimed to model acute autoantibody exposure by intraneural injection of IgG of patients with anti-contacin-1 autoantibodies to Lewis rats. Patient IgG obtained during acute onset of disease (IgG3 predominant) and IgG from the chronic phase of disease (IgG4 predominant) were studied in comparison.

Results: Conduction blocks were measured in rats injected with the "acute" IgG more often than after injection of "chronic" IgG (83.3% versus 35%) and proved to be reversible within a week after injection. Impaired nerve conduction was accompanied by motor deficits in rats after injection of the "acute" IgG but only minor structural changes of the nodes. Paranodal complement deposition was detected after injection of the "acute IgG". We did not detect any inflammatory infiltrates, arguing against an inflammatory cascade as cause of damage to the nerve. We also did not observe dispersion of paranodal proteins or sodium channels to the juxtaparanodes as seen in patients after chronic exposure to anti-contactin-1.

Conclusions: Our data suggest that anti-contactin-1 IgG3 induces an acute conduction block that is most probably mediated by autoantibody binding and subsequent complement deposition and may account for acute onset of disease in these patients. This supports the notion of anti-contactin-1-associated neuropathy as a paranodopathy with the nodes of Ranvier as the site of pathogenesis.
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http://dx.doi.org/10.1186/s12974-019-1462-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450014PMC
April 2019

Elucidating the Molecular Basis for Inhibitory Neurotransmission Regulation by Artemisinins.

Neuron 2019 02 28;101(4):673-689.e11. Epub 2019 Jan 28.

Institute of Structural Biology, Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany. Electronic address:

The frontline anti-malarial drug artemisinin and its derivatives have also been implicated in modulating multiple mammalian cellular pathways, including the recent identification of targeting γ-aminobutyric acid type A receptor (GABAR) signaling in the pancreas. Their molecular mechanism of action, however, remains elusive. Here, we present crystal structures of gephyrin, the central organizer at inhibitory postsynapses, in complex with artesunate and artemether at 1.5-Å resolution. These artemisinins target the universal inhibitory neurotransmitter receptor-binding epitope of gephyrin, thus inhibiting critical interactions between gephyrin and glycine receptors (GlyRs) as well as GABARs. Electrophysiological recordings reveal a significant inhibition of gephyrin-mediated neurotransmission by artemisinins. Furthermore, clustering analyses in primary neurons demonstrate a rapid inhibition and a time-dependent regulation of gephyrin and GABAR cluster parameters. Our data not only provide a comprehensive model for artemisinin-mediated modulation of inhibitory neurotransmission but also establish artemisinins as potential lead compounds to pharmacologically interfere with this process.
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http://dx.doi.org/10.1016/j.neuron.2019.01.001DOI Listing
February 2019

3D Electrophysiological Measurements on Cells Embedded within Fiber-Reinforced Matrigel.

Adv Healthc Mater 2019 03 13;8(5):e1801226. Epub 2019 Jan 13.

Institute for Clinical Neurobiology, University Hospital Würzburg, Versbacherstr. 5, 97078, Würzburg, Germany.

2D electrophysiology is often used to determine the electrical properties of neurons. In the brain however, neurons form extensive 3D networks. Thus, performing electrophysiology in a 3D environment provides a closer situation to the physiological condition and serves as a useful tool for various applications in the field of neuroscience. In this study, 3D electrophysiology is established within a fiber-reinforced matrix to enable fast readouts from transfected cells, which are often used as model systems for 2D electrophysiology. Using melt electrowriting (MEW) of scaffolds to reinforce Matrigel, 3D electrophysiology is performed on a glycine receptor-transfected Ltk-11 mouse fibroblast cell line. The glycine receptor is an inhibitory ion channel associated when mutated with impaired neuromotor behavior. The average thickness of the MEW scaffold is 141.4 ± 5.7 µm, using 9.7 ± 0.2 µm diameter fibers, and square pore spacings of 100, 200, and 400 µm. For the first time, the electrophysiological characterization of glycine receptor-transfected cells is demonstrated with respect to agonist efficacy and potency in a 3D matrix. With the MEW scaffold reinforcement not interfering with the electrophysiological measurement, this approach can now be further adapted and developed for different kinds of neuronal cultures to study and understand pathological mechanisms under disease conditions.
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http://dx.doi.org/10.1002/adhm.201801226DOI Listing
March 2019

Impaired Glycine Receptor Trafficking in Neurological Diseases.

Front Mol Neurosci 2018 21;11:291. Epub 2018 Aug 21.

Institute for Clinical Neurobiology, Julius-Maximilians-University Würzburg, Würzburg, Germany.

Ionotropic glycine receptors (GlyRs) enable fast synaptic neurotransmission in the adult spinal cord and brainstem. The inhibitory GlyR is a transmembrane glycine-gated 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 ( and ). 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.
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http://dx.doi.org/10.3389/fnmol.2018.00291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110938PMC
August 2018

Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Mouse Model of Startle Disease.

Front Mol Neurosci 2018 24;11:167. Epub 2018 May 24.

Institute for Clinical Neurobiology, Julius-Maximilians-University of Würzburg, Würzburg, Germany.

Mutations in GlyR α1 or β subunit genes in humans and rodents lead to severe startle disease characterized by rigidity, massive stiffness and excessive startle responses upon unexpected tactile or acoustic stimuli. The recently characterized startle disease mouse mutant carries a missense mutation (Q177K) in the β8-β9 loop within the large extracellular N-terminal domain of the GlyR α1 subunit. This results in a disrupted hydrogen bond network around K177 and faster GlyR decay times. Symptoms in mice start at postnatal day 14 and increase until premature death of homozygous mice around 4-6 weeks after birth. Here we investigate the functional effects of the Q177K mutation using behavioral analysis coupled to protein biochemistry and functional assays. Western blot analysis revealed GlyR α1 subunit expression in wild-type and animals around postnatal day 7, a week before symptoms in mutant mice become obvious. Before 2 weeks of age, homozygous mice appeared healthy and showed no changes in body weight. However, analysis of gait and hind-limb clasping revealed that motor coordination was already impaired. Motor coordination and the activity pattern at P28 improved significantly upon diazepam treatment, a pharmacotherapy used in human startle disease. To investigate whether functional deficits in glycinergic neurotransmission are present prior to phenotypic onset, we performed whole-cell recordings from hypoglossal motoneurons (HMs) in brain stem slices from wild-type and mice at different postnatal stages. homozygotes showed a decline in mIPSC amplitude and frequency at P9-P13, progressing to significant reductions in mIPSC amplitude and decay time at P18-24 compared to wild-type littermates. Extrasynaptic GlyRs recorded by bath-application of glycine also revealed reduced current amplitudes in mice compared to wild-type neurons, suggesting that presynaptic GlyR function is also impaired. Thus, a distinct, but behaviorally ineffective impairment of glycinergic synapses precedes the symptoms onset in mice. These findings extend our current knowledge on startle disease in the mouse model in that they demonstrate how the progression of GlyR dysfunction causes, with a delay of about 1 week, the appearance of disease symptoms.
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http://dx.doi.org/10.3389/fnmol.2018.00167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992992PMC
May 2018

PKA and PKC Modulators Affect Ion Channel Function and Internalization of Recombinant Alpha1 and Alpha1-Beta Glycine Receptors.

Front Mol Neurosci 2018 14;11:154. Epub 2018 May 14.

Department of Biochemistry, German University in Cairo, New Cairo, Egypt.

Glycine receptors (GlyRs) are important mediators of fast inhibitory neurotransmission in the mammalian central nervous system. Their function is controlled by multiple cellular mechanisms, including intracellular regulatory processes. Modulation of GlyR function by protein kinases has been reported for many cell types, involving different techniques, and often yielding contradictory results. Here, we studied the effects of protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) on glycine induced currents in HEK293 cells expressing human homomeric α1 and heteromeric α1-β GlyRs using whole-cell patch clamp techniques as well as internalization assays. In whole-cell patch-clamp measurements, modulators were applied in the intracellular buffer at concentrations between 0.1 μM and 0.5 μM. EC of glycine increased upon application of the protein kinase activators Forskolin and phorbol-12-myristate-13-acetate (PMA) but decreased in the presence of the PKC inhibitor Staurosporine aglycon and the PKA inhibitor H-89. Desensitization of recombinant α1 receptors was significantly increased in the presence of Forskolin. Staurosporine aglycon, on the other hand decreased desensitization of heteromeric α1-β GlyRs. The time course of receptor activation was determined for homomeric α1 receptors and revealed two simultaneous effects: cells showed a decrease of EC after 3-6 min of establishing whole-cell configuration. This effect was independent of protein kinase modulators. All modulators of PKA and PKC, however, produced an additional shift of EC, which overlay and eventually exceeded the cells intrinsic variation of EC. The effect of kinase activators was abolished if the corresponding inhibitors were co-applied, consistent with PKA and PKC directly mediating the modulation of GlyR function. Direct effects of PKA- and PKC-modulators on receptor expression on transfected HEK cells were monitored within 15 min of drug application, showing a significant increase of receptor internalization with PKA and PKC activators, while the corresponding inhibitors had no significant effect on receptor surface expression or internalization. Our results confirm the observation that phosphorylation via PKA and PKC has a direct effect on the GlyR ion channel complex and plays an important role in the fine-tuning of glycinergic signaling.
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http://dx.doi.org/10.3389/fnmol.2018.00154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961436PMC
May 2018

Structural changes at the myrtenol backbone reverse its positive allosteric potential into inhibitory GABAA receptor modulation.

Biol Chem 2018 05;399(6):549-563

Institute for Clinical Neurobiology, Julius-Maximilians-Universität Würzburg, Versbacherstr. 5, D-97078 Würzburg, Germany.

GABAA receptors are ligand-gated anion channels that form pentameric arrangements of various subunits. Positive allosteric modulators of GABAA receptors have been reported as being isolated either from plants or synthesized analogs of known GABAA receptor targeting drugs. Recently, we identified monoterpenes, e.g. myrtenol as a positive allosteric modulator at α1β2 GABAA receptors. Here, along with pharmacophore-based virtual screening studies, we demonstrate that scaffold modifications of myrtenol resulted in the loss of modulatory activity. Two independent approaches, fluorescence-based compound analysis and electrophysiological recordings in whole-cell configurations were used for analysis of transfected cells. C-atoms 1 and 2 of the myrtenol backbone were identified as crucial to preserve positive allosteric potential. A modification at C-atom 2 and lack of the hydroxyl group at C-atom 1 exhibited significantly reduced GABAergic currents at α1β2, α1β2γ, α2β3, α2β3γ and α4β3δ receptors. This effect was independent of the γ2 subunit. A sub-screen with side chain length and volume differences at the C-atom 1 identified two compounds that inhibited GABAergic responses but without receptor subtype specificity. Our combined approach of pharmacophore-based virtual screening and functional readouts reveals that side chain modifications of the bridged six-membered ring structure of myrtenol are crucial for its modulatory potential at GABAA receptors.
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http://dx.doi.org/10.1515/hsz-2017-0262DOI Listing
May 2018

The GlyR Extracellular β8-β9 Loop - A Functional Determinant of Agonist Potency.

Front Mol Neurosci 2017 9;10:322. Epub 2017 Oct 9.

Institute for Clinical Neurobiology, University of Würzburg, Würzburg, Germany.

Ligand-binding of Cys-loop receptors results in rearrangements of extracellular loop structures which are further translated into the tilting of membrane spanning helices, and finally opening of the ion channels. The cryo-EM structure of the homopentameric α1 glycine receptor (GlyR) demonstrated an involvement of the extracellular β8-β9 loop in the transition from ligand-bound receptors to the open channel state. Recently, we identified a functional role of the β8-β9 loop in a novel startle disease mouse model . The mutation of residue GlyRα1 to lysine present in mice resulted in reduced glycine potency, reduced synaptic expression, and a disrupted hydrogen network at the structural level around position GlyRα1. Here, we investigated the role of amino acid volume, side chain length, and charge at position Q177 to get deeper insights into the functional role of the β8-β9 loop. We used a combined approach of expression analysis, functional electrophysiological recordings, and GlyR modeling to describe the role of Q177 for GlyR ion channel function. GlyRα1 variants do not disturb ion channel transport to the cellular surface of transfected cells, neither in homomeric nor in heteromeric GlyR configurations. The EC values were increased for all GlyRα1 variants in comparison to the wild type. The largest decrease in glycine potency was observed for the variant GlyRα1. Potencies of the partial agonists β-alanine and taurine were also reduced. Our data are further supported by homology modeling. The GlyRα1 variant does not form hydrogen bonds with the surrounding network of residue Q177 similar to the substitution with a basic lysine present in the mouse mutant . Among all investigated Q177 mutants, the neutral exchange of glutamine to asparagine as well as the introduction of the closely related amino acid glutamic acid preserve the hydrogen bond network. Introduction of amino acids with small side chains or larger volume resulted in a loss of their hydrogen bonds to neighboring residues. The β8-β9 loop is thus an important structural and functional determinant of the inhibitory GlyR.
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http://dx.doi.org/10.3389/fnmol.2017.00322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640878PMC
October 2017

Disruption of a Structurally Important Extracellular Element in the Glycine Receptor Leads to Decreased Synaptic Integration and Signaling Resulting in Severe Startle Disease.

J Neurosci 2017 08 19;37(33):7948-7961. Epub 2017 Jul 19.

Institute of Clinical Neurobiology, Julius-Maximilians-University of Würzburg, 97078 Würzburg, Germany,

Functional impairments or trafficking defects of inhibitory glycine receptors (GlyRs) have been linked to human hyperekplexia/startle disease and autism spectrum disorders. We found that a lack of synaptic integration of GlyRs, together with disrupted receptor function, is responsible for a lethal startle phenotype in a novel spontaneous mouse mutant , caused by a missense mutation, Q177K, located in the extracellular β8-β9 loop of the GlyR α1 subunit. Recently, structural data provided evidence that the flexibility of the β8-β9 loop is crucial for conformational transitions during opening and closing of the ion channel and represents a novel allosteric binding site in Cys-loop receptors. We identified the underlying neuropathological mechanisms in male and female mice through a combination of protein biochemistry, immunocytochemistry, and both and in electrophysiology. Increased expression of the mutant GlyR α1 subunit was not sufficient to compensate for a decrease in synaptic integration of α1β GlyRs. The remaining synaptic heteromeric α1β GlyRs had decreased current amplitudes with significantly faster decay times. This functional disruption reveals an important role for the GlyR α1 subunit β8-β9 loop in initiating rearrangements within the extracellular-transmembrane GlyR interface and that this structural element is vital for inhibitory GlyR function, signaling, and synaptic clustering. GlyR dysfunction underlies neuromotor deficits in startle disease and autism spectrum disorders. We describe an extracellular GlyR α1 subunit mutation (Q177K) in a novel mouse startle disease mutant Structural data suggest that during signal transduction, large transitions of the β8-β9 loop occur in response to neurotransmitter binding. Disruption of the β8-β9 loop by the Q177K mutation results in a disruption of hydrogen bonds between Q177 and the ligand-binding residue R65. Functionally, the Q177K change resulted in decreased current amplitudes, altered desensitization decay time constants, and reduced GlyR clustering and synaptic strength. The GlyR β8-β9 loop is therefore an essential regulator of conformational rearrangements during ion channel opening and closing.
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http://dx.doi.org/10.1523/JNEUROSCI.0009-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559766PMC
August 2017

Metabolic Products of Linalool and Modulation of GABA Receptors.

Front Chem 2017 21;5:46. Epub 2017 Jun 21.

Institute of Clinical Neurobiology, Julius-Maximilians-University of WürzburgWürzburg, Germany.

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 upon overexpression of inhibitory α1β2 GABA 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 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 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 GABA receptors compared to the significant potentiation effects of the parent molecule linalool itself.
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http://dx.doi.org/10.3389/fchem.2017.00046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478857PMC
June 2017

The role of charged residues in independent glycine receptor folding domains for intermolecular interactions and ion channel function.

J Neurochem 2017 07 22;142(1):41-55. Epub 2017 May 22.

Institute for Clinical Neurobiology, Julius-Maximilians-University of Würzburg, Würzburg, Germany.

Glycine receptor (GlyR) truncations in the intracellular TM3-4 loop, documented in patients suffering from hyperekplexia and in the mouse mutant oscillator, lead to non-functionality of GlyRs. The missing part that contains the TM3-4 loop, TM4 and C-terminal sequences is essential for pentameric receptor arrangements. In vitro co-expressions of GlyRα1-truncated N-domains and C-domains were able to restore ion channel function. An ionic interaction between both domains was hypothesized as the underlying mechanism. Here, we analysed the proposed ionic interaction between GlyR N- and C-domains using C-terminal constructs with either positively or negatively charged N-termini. Charged residues at the N-terminus of the C-domain did interfere with receptor surface expression and ion channel function. In particular, presence of negatively charged residues at the N-terminus led to significantly decreased ion channel function. Presence of positive charges resulted in reduced maximal currents possibly as a result of repulsion of both domains. If the C-domain was tagged by a myc-epitope, low maximal current amplitudes were detected. Intrinsic charges of the myc-epitope and charged N-terminal ends of the C-domain most probably induce intramolecular interactions. These interactions might hinder the close proximity of C-domains and N-domains, which is a prerequisite for functional ion channel configurations. The remaining basic subdomains close to TM3 and 4 were sufficient for domain complementation and functional ion channel formation. Thus, these basic subdomains forming α-helical elements or an intracellular portal represent attractants for incoming negatively charged chloride ions and interact with the phospholipids thereby stabilizing the GlyR in a conformation that allows ion channel opening.
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http://dx.doi.org/10.1111/jnc.14049DOI Listing
July 2017

Oxime Ethers of (E)-11-Isonitrosostrychnine as Highly Potent Glycine Receptor Antagonists.

J Nat Prod 2016 Dec 14;79(12):2997-3005. Epub 2016 Dec 14.

Faculty of Pharmacy and Biotechnology, The German University in Cairo , New Cairo City, 11835 Cairo, Egypt.

A series of (E)-11-isonitrosostrychnine oxime ethers, 2-aminostrychnine, (strychnine-2-yl)propionamide, 18-oxostrychnine, and N-propylstrychnine bromide were synthesized and evaluated pharmacologically at human α1 and α1β glycine receptors in a functional fluorescence-based and a whole-cell patch-clamp assay and in [H]strychnine binding studies. 2-Aminostrychnine and the methyl, allyl, and propargyl oxime ethers were the most potent α1 and α1β antagonists in the series, displaying IC values similar to those of strychnine at the two receptors. Docking experiments to the strychnine binding site of the crystal structure of the α3 glycine receptor indicated the same orientation of the strychnine core for all analogues. For the most potent oxime ethers, the ether substituent was accommodated in a lipophilic receptor binding pocket. The findings identify the oxime hydroxy group as a suitable attachment point for linking two strychnine pharmacophores by a polymethylene spacer and are, therefore, important for the design of bivalent ligands targeting glycine receptors.
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http://dx.doi.org/10.1021/acs.jnatprod.6b00479DOI Listing
December 2016

Auto-antibodies to contactin-associated protein 1 (Caspr) in two patients with painful inflammatory neuropathy.

Brain 2016 Oct 29;139(Pt 10):2617-2630. Epub 2016 Jul 29.

1 Department of Neurology, University of Würzburg, Germany.

Auto-antibodies against the paranodal proteins neurofascin-155 and contactin-1 have recently been described in patients with chronic inflammatory demyelinating polyradiculoneuropathy and are associated with a distinct clinical phenotype and response to treatment. Contactin-associated protein 1 (Caspr, encoded by CNTNAP1) is a paranodal protein that is attached to neurofascin-155 and contactin-1 (CNTN1) but has not yet been identified as a sole antigen in patients with inflammatory neuropathies. In the present study, we screened a cohort of 35 patients with chronic inflammatory demyelinating polyradiculoneuropathy (age range 20-80, 10 female, 25 male) and 22 patients with Guillain-Barré syndrome (age range 17-86, eight female, 14 male) for autoantibodies against paranodal antigens. We identified two patients, one with chronic inflammatory demyelinating polyradiculoneuropathy and one with Guillain-Barré syndrome, with autoantibodies against Caspr by binding assays using Caspr transfected human embryonic kidney cells and murine teased fibres. IgG3 was the predominant autoantibody subclass in the patient with Guillain-Barré syndrome, IgG4 was predominant in the patient with chronic inflammatory demyelinating polyradiculoneuropathy. Accordingly, complement deposition after binding to HEK293 cells was detectable in the patient with IgG3 autoantibodies only, not in the patient with IgG4. Severe disruption of the paranodal and nodal architecture was detectable in teased fibres of the sural nerve biopsy and in dermal myelinated fibres, supporting the notion of the paranodes being the site of pathology. Deposition of IgG at the paranodes was detected in teased fibre preparations of the sural nerve, further supporting the pathogenicity of anti-Caspr autoantibodies. Pain was one of the predominant findings in both patients, possibly reflected by binding of patients' IgG to TRPV1 immunoreactive dorsal root ganglia neurons. Our results demonstrate that the paranodal protein Caspr constitutes a new antigen that leads to autoantibody generation as part of the novel entity of neuropathies associated with autoantibodies against paranodal proteins.
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http://dx.doi.org/10.1093/brain/aww189DOI Listing
October 2016

Three-Step Test System for the Identification of Novel GABA Receptor Modulating Food Plants.

Plant Foods Hum Nutr 2016 Dec;71(4):355-360

Food Chemistry Unit, Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Schuhstr. 19, 91052, Erlangen, Germany.

Potentiation of γ-amino butyric acid (GABA)-induced GABA receptor (GABAR) activation is a common pathway to achieve sedative, sleep-enhancing, anxiolytic, and antidepressant effects. Presently, a three-component test system was established for the identification of novel GABAR modulating food plants. In the first step, potentiation of GABA-induced response of the GABAR was analysed by two-electrode voltage clamp (TEVC) for activity on human α1β2-GABAR expressed in Xenopus laevis oocytes. Positively tested food plants were then subjected to quantification of GABA content by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) to exclude test foods, which evoke a TEVC-response by endogenous GABA. In the third step, specificity of GABA-modulating activity was assessed by TEVC analysis of Xenopus laevis oocytes expressing the homologous glycine receptor (GlyR). The three-component test was then applied to screen 10 aqueous extracts of food plants for their GABAR activity. Thus, hop cones (Humulus lupulus) and Sideritis sipylea were identified as the most potent specific GABAR modulators eliciting significant potentiation of the current by 182 ± 27 and 172 ± 19 %, respectively, at the lowest concentration of 0.5 μg/mL. The extracts can now be further evaluated by in vivo studies and by structural evaluation of the active components.
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http://dx.doi.org/10.1007/s11130-016-0566-1DOI Listing
December 2016

The Intracellular Loop of the Glycine Receptor: It's not all about the Size.

Front Mol Neurosci 2016 3;9:41. Epub 2016 Jun 3.

Institute of Clinical Neurobiology, University of Würzburg Würzburg, Germany.

The family of Cys-loop receptors (CLRs) shares a high degree of homology and sequence identity. The overall structural elements are highly conserved with a large extracellular domain (ECD) harboring an α-helix and 10 β-sheets. Following the ECD, four transmembrane domains (TMD) are connected by intracellular and extracellular loop structures. Except the TM3-4 loop, their length comprises 7-14 residues. The TM3-4 loop forms the largest part of the intracellular domain (ICD) and exhibits the most variable region between all CLRs. The ICD is defined by the TM3-4 loop together with the TM1-2 loop preceding the ion channel pore. During the last decade, crystallization approaches were successful for some members of the CLR family. To allow crystallization, the intracellular loop was in most structures replaced by a short linker present in prokaryotic CLRs. Therefore, no structural information about the large TM3-4 loop of CLRs including the glycine receptors (GlyRs) is available except for some basic stretches close to TM3 and TM4. The intracellular loop has been intensively studied with regard to functional aspects including desensitization, modulation of channel physiology by pharmacological substances, posttranslational modifications, and motifs important for trafficking. Furthermore, the ICD interacts with scaffold proteins enabling inhibitory synapse formation. This review focuses on attempts to define structural and functional elements within the ICD of GlyRs discussed with the background of protein-protein interactions and functional channel formation in the absence of the TM3-4 loop.
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http://dx.doi.org/10.3389/fnmol.2016.00041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891346PMC
June 2016

The terpenoids Myrtenol and Verbenol act on δ subunit-containing GABAA receptors and enhance tonic inhibition in dentate gyrus granule cells.

Neurosci Lett 2016 08 14;628:91-7. Epub 2016 Jun 14.

Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany. Electronic address:

Sideritis plants and their extracts have been used in traditional medicine as sedatives, anxiolytics and anticonvulsant agents. Pinenes are the most prevalent of the volatile aroma components in Siderites extracts and the pinene metabolites myrtenol and verbenol have been identified as the most potent positive allosteric modulators of synaptic GABAA receptors composed of α1β2 and α1β2γ2 subunits. In view of their therapeutic spectrum, we wondered whether these two terpenoids would also augment tonic GABA currents mediated by extrasynaptic GABAA receptors containing the δ subunit. When we expressed α4β2δ receptors in HEK293 cells, we found that co-application of myrtenol or verbenol enhanced whole-cell current responses to GABA by up to 100%. Consistent with their effects on heterologous α1β2γ2 receptors, we found that myrtenol and verbenol, when co-applied with GABA via local perfusion, increased the amplitude and area of miniature inhibitory postsynaptic potentials (mIPSCs) recorded in whole-cell voltage-clamp recordings from granule cells in the dentate gyrus of mouse hippocampal brain slices. In addition, co-application of terpenoids with GABA was also able to enhance tonic GABA current, measured from the change in baseline current and current noise, compared to GABA perfusion alone. Our results suggest that myrtenol and verbenol act as positive allosteric modulators at synaptic and extrasynaptic GABAA receptors, thereby augmenting phasic and tonic GABAergic inhibition. Thus, our study reveals an important pharmacological and therapeutic target of bicyclic monoterpenoids.
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http://dx.doi.org/10.1016/j.neulet.2016.06.027DOI Listing
August 2016