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Matthias Kneussel - One of the best experts on this subject based on the ideXlab platform.
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design and synthesis of high affinity dimeric inhibitors targeting the interactions between Gephyrin and inhibitory neurotransmitter receptors
Angewandte Chemie, 2014Co-Authors: Hans Michael Maric, Torben J Hausrat, Hermann Schindelin, Matthias Kneussel, Vikram Babu Kasaragod, Linda M Haugaardkedstrom, Kristian StromgaardAbstract:Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between Gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor-derived minimal Gephyrin-binding peptides that displayed exclusive binding towards native Gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220-fold enhancement of the Gephyrin affinity (KD=6.8 nM). In X-ray crystal structures we visualized the simultaneous dimer-to-dimer binding in atomic detail, revealing compound-specific binding modes. Thus, we defined the molecular basis of the affinity-enhancing effect of multivalent Gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the Gephyrin–receptor interplay.
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Gephyrin regulates gabaergic and glutamatergic synaptic transmission in hippocampal cell cultures
Journal of Biological Chemistry, 2011Co-Authors: Zeynep Kasap Varley, Matthias Kneussel, Roberta Antonelli, Rocco Pizzarelli, Enrico Cherubini, Stefka H Stancheva, Paola ZacchiAbstract:Gephyrin is a scaffold protein essential for stabilizing glycine and GABAA receptors at inhibitory synapses. Here, recombinant intrabodies against Gephyrin (scFv-Gephyrin) were used to assess whether this protein exerts a transynaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-Gephyrin-transfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NLG2), a protein that, interacting with neurexins, ensures the cross-talk between the post- and presynaptic sites. Interestingly, hampering Gephyrin function also produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Overexpressing NLG2 in Gephyrin-deprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. Pulldown experiments demonstrated that Gephyrin interacts not only with NLG2 but also with NLG1, the isoform enriched at excitatory synapses. These results suggest a key role of Gephyrin in regulating transynaptic signaling at both inhibitory and excitatory synapses.
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neuronal cotransport of glycine receptor and the scaffold protein Gephyrin
Journal of Cell Biology, 2006Co-Authors: Christoph Maas, Nadia Tagnaouti, Sven Loebrich, Bardo Behrend, Corinna Lappesiefke, Matthias KneusselAbstract:The dynamics of postsynaptic receptor scaffold formation and remodeling at inhibitory synapses remain largely unknown. Gephyrin, which is a multimeric scaffold protein, interacts with cytoskeletal elements and stabilizes glycine receptors (GlyRs) and individual subtypes of γ-aminobutyric acid A receptors at inhibitory postsynaptic sites. We report intracellular mobility of Gephyrin transports packets over time. Gephyrin units enter and exit active synapses within several minutes. In addition to previous reports of GlyR–Gephyrin interactions at plasma membranes, we show cosedimentation and coimmunoprecipitation of both proteins from vesicular fractions. Moreover, GlyR and Gephyrin are cotransported within neuronal dendrites and further coimmunoprecipitate and colocalize with the dynein motor complex. As a result, the blockade of dynein function or dynein–Gephyrin interaction, as well as the depolymerization of microtubules, interferes with retrograde Gephyrin recruitment. Our data suggest a GlyR–Gephyrin–dynein transport complex and support the concept that Gephyrin–motor interactions contribute to the dynamic and activity-dependent rearrangement of postsynaptic GlyRs, a process thought to underlie the regulation of synaptic strength.
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Gephyrin interacts with dynein light chains 1 and 2 components of motor protein complexes
The Journal of Neuroscience, 2002Co-Authors: Jens C. Fuhrmann, Joachim Kirsch, Heinrich Betz, Antoine Triller, Stefan Kins, Philippe Rostaing, Oussama El Far, Morgan Sheng, Matthias KneusselAbstract:The clustering of glycine receptors and major subtypes of GABAA receptors at inhibitory synapses is mediated by the tubulin-binding protein Gephyrin. In an attempt to identify additional components of inhibitory postsynaptic specializations, we performed a yeast two-hybrid screen using Gephyrin as bait. Multiple positive clones encoded either the dynein light chain-1 (Dlc-1), also known as dynein LC8 and protein inhibitor of neuronal nitric oxide synthase, or its homolog Dlc-2. Dlc-1 protein bound efficiently to Gephyrin in in vitro binding assays and colocalized with Gephyrin during coexpression in HEK293 cells. The binding site for Dlc was mapped to a fragment of 63 amino acids within the central linker domain of Gephyrin. In hippocampal neurons, endogenous Dlc protein was enriched at synaptic sites identified by synaptophysin and Gephyrin immunostaining. Immunoelectron microscopy in spinal cord sections revealed Dlc immunoreactivity at the edges of postsynaptic differentiations, in close contact with cytoskeletal structures and at the periphery of the Golgi apparatus. Because Dlc-1 and Dlc-2 have been described as stoichiometric components of cytoplasmic dynein and myosin-Va complexes, our results suggest that motor proteins are involved in the subcellular localization of Gephyrin.
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identification of a Gephyrin binding motif in the gdp gtp exchange factor collybistin
Biological Chemistry, 2001Co-Authors: Y. Grosskreutz, Achim Hermann, Stefan Kins, Jens C. Fuhrmann, Matthias KneusselAbstract:The brain-specific GDP/GTP exchange factor collybistin interacts with the receptor-anchoring protein Gephyrin and activates the Rho-like GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous Gephyrin aggregates which cluster with hetero-oligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with Gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind Gephyrin upon coexpression in heterologous cells, in GST pull-down assays and in the yeast two-hybrid system. Site-directed mutagenesis revealed polar amino acid residues as essential determinants of Gephyrin binding. Furthermore, in vitro Gephyrin bound simultaneously to both collybistin and the GlyR beta-subunit binding motif. Our data are consistent with collybistin-Gephyrin interactions occuring during inhibitory postsynaptic membrane formation.
Theofilos Papadopoulos - One of the best experts on this subject based on the ideXlab platform.
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the α3 subunit of gabaa receptors promotes formation of inhibitory synapses in the absence of collybistin
Journal of Biological Chemistry, 2021Co-Authors: Sven Wagner, Nils Brose, Jeongseop Rhee, Choongku Lee, Lucia Rojas, Christian G Specht, Theofilos PapadopoulosAbstract:Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects—or synaptopathies—are at the basis of many neurological and psychiatric disorders. Collybistin (CB), a brain-specific guanine nucleotide exchange factor, is essential for the formation of γ-aminobutyric acidergic (GABAergic) postsynapses in defined regions of the mammalian forebrain, including the hippocampus and basolateral amygdala. This process depends on a direct interaction of CB with the scaffolding protein Gephyrin, which leads to the redistribution of Gephyrin into submembranous clusters at nascent inhibitory synapses. Strikingly, synaptic clustering of Gephyrin and GABAA type A receptors (GABAARs) in several brain regions, including the cerebral cortex and certain thalamic areas, is unperturbed in CB-deficient mice, indicating that the formation of a substantial subset of inhibitory postsynapses must be controlled by Gephyrin-interacting proteins other than CB. Previous studies indicated that the α3 subunit of GABAARs (GABAAR-α3) binds directly and with high affinity to Gephyrin. Here, we provide evidence (i) that a homooligomeric GABAAR-α3A343W mutant induces the formation of submembranous Gephyrin clusters independently of CB in COS-7 cells, (ii) that Gephyrin clustering is unaltered in the neuronal subpopulations endogenously expressing the GABAAR-α3 in CB-deficient brains, and (iii) that exogenous expression of GABAAR-α3 partially rescues impaired Gephyrin clustering in CB-deficient hippocampal neurons. Our results identify an important role of GABAAR-α3 in promoting Gephyrin-mediated and CB-independent formation of inhibitory postsynapses.
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IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3) Interacts with Gephyrin to Promote Inhibitory Synapse Formation
'American Society for Biochemistry & Molecular Biology (ASBMB)', 2018Co-Authors: Gayoung Choii, Theofilos Papadopoulos, Sangmin Jeon, Takuma Mori, Taesun Yoo, Dongseok Park, Dongwook Kim, Hyeyeon Kang, Yeunkum LeeAbstract:Gephyrin is a central scaffold protein that mediates development, function, and plasticity of mammalian inhibitory synapses by interacting with various inhibitory synaptic proteins. Here, we show that IQSLC3, a guanine nucleotide exchange factor for ARE6, directly interacts with Gephyrin, an interaction that is critical for the inhibitory synapse localization of IQSEC3. Overexpression of IQSEC3 increases inhibitory, but not excitatory, synapse density in a guanine nucleotide exchange factor activity-dependent manner. Conversely, knockdown of IQSEC3 decreases size of Gephyrin cluster without altering Gephyrin puncta density. Collectively, these data reveal that IQSEC3 acts together with Gephyrin to regulate inhibitory synapse development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.1551sciescopu
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endosomal phosphatidylinositol 3 phosphate promotes Gephyrin clustering and gabaergic neurotransmission at inhibitory postsynapses
Journal of Biological Chemistry, 2017Co-Authors: Theofilos Papadopoulos, Nils Brose, Hong Jun Rhee, Devaraj Subramanian, Foteini Paraskevopoulou, Rainer Mueller, Carsten Schultz, Jeongseop RheeAbstract:Abstract The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein Gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, time-lapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/sorting endosomes regulates the postsynaptic clustering of Gephyrin and GABAA receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of Gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cb-mediated recruitment of Gephyrin and GABAA receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations.
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iq motif and sec7 domain containing protein 3 iqsec3 interacts with Gephyrin to promote inhibitory synapse formation
Journal of Biological Chemistry, 2016Co-Authors: Gayoung Choii, Theofilos Papadopoulos, Sangmin Jeon, Takuma Mori, Taesun Yoo, Yeunkum Lee, Eunjoon KimAbstract:Gephyrin is a central scaffold protein that mediates development, function, and plasticity of mammalian inhibitory synapses by interacting with various inhibitory synaptic proteins. Here, we show that IQSEC3, a guanine nucleotide exchange factor for ARF6, directly interacts with Gephyrin, an interaction that is critical for the inhibitory synapse localization of IQSEC3. Overexpression of IQSEC3 increases inhibitory, but not excitatory, synapse density in a guanine nucleotide exchange factor activity-dependent manner. Conversely, knockdown of IQSEC3 decreases size of Gephyrin cluster without altering Gephyrin puncta density. Collectively, these data reveal that IQSEC3 acts together with Gephyrin to regulate inhibitory synapse development.
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A conformational switch in collybistin determines the differentiation of inhibitory postsynapses
The EMBO journal, 2014Co-Authors: Tolga Soykan, Daniela Schneeberger, Giancarlo Tria, Claudia N. Buechner, Nicole Bader, Dmitri I. Svergun, Ingrid Tessmer, Alexandros Poulopoulos, Theofilos Papadopoulos, Frederique VaroqueauxAbstract:The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein Gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin-based protein interaction network that controls the Gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links Gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin-2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans-synaptic neuroligin-dependent adhesion and phosphoinositide signaling with Gephyrin recruitment, the collybistin-based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.
Heinrich Betz - One of the best experts on this subject based on the ideXlab platform.
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Gephyrin interacts with dynein light chains 1 and 2 components of motor protein complexes
The Journal of Neuroscience, 2002Co-Authors: Jens C. Fuhrmann, Joachim Kirsch, Heinrich Betz, Antoine Triller, Stefan Kins, Philippe Rostaing, Oussama El Far, Morgan Sheng, Matthias KneusselAbstract:The clustering of glycine receptors and major subtypes of GABAA receptors at inhibitory synapses is mediated by the tubulin-binding protein Gephyrin. In an attempt to identify additional components of inhibitory postsynaptic specializations, we performed a yeast two-hybrid screen using Gephyrin as bait. Multiple positive clones encoded either the dynein light chain-1 (Dlc-1), also known as dynein LC8 and protein inhibitor of neuronal nitric oxide synthase, or its homolog Dlc-2. Dlc-1 protein bound efficiently to Gephyrin in in vitro binding assays and colocalized with Gephyrin during coexpression in HEK293 cells. The binding site for Dlc was mapped to a fragment of 63 amino acids within the central linker domain of Gephyrin. In hippocampal neurons, endogenous Dlc protein was enriched at synaptic sites identified by synaptophysin and Gephyrin immunostaining. Immunoelectron microscopy in spinal cord sections revealed Dlc immunoreactivity at the edges of postsynaptic differentiations, in close contact with cytoskeletal structures and at the periphery of the Golgi apparatus. Because Dlc-1 and Dlc-2 have been described as stoichiometric components of cytoplasmic dynein and myosin-Va complexes, our results suggest that motor proteins are involved in the subcellular localization of Gephyrin.
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Gephyrin independent clustering of postsynaptic gaba a receptor subtypes
Molecular and Cellular Neuroscience, 2001Co-Authors: Matthias Kneussel, Guoping Feng, Joshua R. Sanes, Johann Helmut Brandstatter, Bruno Gasnier, Heinrich BetzAbstract:Gephyrin has been shown to be essential for the synaptic localization of the inhibitory glycine receptor and major GABA(A) receptor (GABA(A)R) subtypes. However, in retina certain GABA(A)R subunits are found at synaptic sites in the absence of Gephyrin. Here, we quantitatively analyzed GABA(A)R alpha1, alpha2, alpha3, alpha5, beta2/3, and gamma2 subunit immunoreactivities in spinal cord sections derived from wild-type and Gephyrin-deficient (geph -/-) mice. The punctate staining of GABA(A)R alpha1 and alpha5 subunits was unaltered in geph -/- mice, whereas the numbers of alpha2-, alpha3-, beta2/3-, and gamma2-subunit-immunoreactive synaptic sites were significantly or even strikingly reduced in the mutant animals. Immunostaining with an antibody specific for the vesicular inhibitory amino acid transporter revealed that the number of inhibitory presynaptic terminals is unaltered upon Gephyrin deficiency. These data show that in addition to Gephyrin other clustering proteins must exist that mediate the synaptic localization of selected GABA(A)R subtypes.
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diversity and phylogeny of Gephyrin tissue specific splice variants gene structure and sequence similarities to molybdenum cofactor synthesizing and cytoskeleton associated proteins
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Markus Ramming, Heinrich Betz, Achim Hermann, Stefan Kins, Nikos Werner, Joachim KirschAbstract:Gephyrin is essential for both the postsynaptic localization of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (Moco) in different peripheral organs. Several alternatively spliced Gephyrin transcripts have been identified in rat brain that differ in their 5′ coding regions. Here, we describe Gephyrin splice variants that are differentially expressed in non-neuronal tissues and different regions of the adult mouse brain. Analysis of the murine Gephyrin gene indicates a highly mosaic organization, with eight of its 29 exons corresponding to the alternatively spliced regions identified by cDNA sequencing. The N- and C-terminal domains of Gephyrin encoded by exons 3–7 and 16–29, respectively, display sequence similarities to bacterial, invertebrate, and plant proteins involved in Moco biosynthesis, whereas the central exons 8, 13, and 14 encode motifs that may mediate oligomerization and tubulin binding. Our data are consistent with Gephyrin having evolved from a Moco biosynthetic protein by insertion of protein interaction sequences.
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the gamma aminobutyric acid type a receptor gabaar associated protein gabarap interacts with Gephyrin but is not involved in receptor anchoring at the synapse
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Matthias Kneussel, Heinz Wässle, Richard W. Olsen, Jens C. Fuhrmann, Hongbing Wang, Silke Haverkamp, Heinrich BetzAbstract:γ-Aminobutyric acid type A receptors (GABAARs) are ligand-gated chloride channels that exist in numerous distinct subunit combinations. At postsynaptic membrane specializations, different GABAAR isoforms colocalize with the tubulin-binding protein Gephyrin. However, direct interactions of GABAAR subunits with Gephyrin have not been reported. Recently, the GABAAR-associated protein GABARAP was found to bind to the γ2 subunit of GABAARs. Here we show that GABARAP interacts with Gephyrin in both biochemical assays and transfected cells. Confocal analysis of neurons derived from wild-type and Gephyrin-knockout mice revealed that GABARAP is highly enriched in intracellular compartments, but not at Gephyrin-positive postsynaptic membrane specializations. Our data indicate that GABARAP–Gephyrin interactions are not important for postsynaptic GABAAR anchoring but may be implicated in receptor sorting and/or targeting mechanisms. Consistent with this idea, a close homolog of GABARAP, p16, has been found to function as a late-acting intra-Golgi transport factor.
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Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of Gephyrin
Nature Neuroscience, 2000Co-Authors: Stefan Kins, Heinrich Betz, Joachim KirschAbstract:The formation of postsynaptic GABA_A and glycine receptor clusters requires the receptor-associated peripheral membrane protein Gephyrin. Here we describe two splice variants of a novel Gephyrin-binding protein, termed collybistin I and II, which belong to the family of dbl-like GDP/GTP exchange factors (GEFs). Co-expression of collybistin II with Gephyrin induced the formation of submembrane Gephyrin aggregates that accumulate hetero-oligomeric glycine receptors. Our data suggest that collybistin II regulates the membrane deposition of Gephyrin by activating a GTPase of the Rho/Rac family. Therefore, this protein may be an important determinant of inhibitory postsynaptic membrane formation and plasticity.
Borislav Dejanovic - One of the best experts on this subject based on the ideXlab platform.
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simultaneous impairment of neuronal and metabolic function of mutated Gephyrin in a patient with epileptic encephalopathy
Embo Molecular Medicine, 2015Co-Authors: Borislav Dejanovic, Tania Djemie, Nora Grunewald, Arvid Suls, Vanessa Kress, Florian Hetsch, Dana Craiu, Matthew Zemel, Padhraig Gormley, Candace T MyersAbstract:Synaptic inhibition is essential for shaping the dynamics of neuronal networks, and aberrant inhibition plays an important role in neurological disorders. Gephyrin is a central player at inhibitory postsynapses, directly binds and organizes GABAA and glycine receptors (GABAARs and GlyRs), and is thereby indispensable for normal inhibitory neurotransmission. Additionally, Gephyrin catalyzes the synthesis of the molybdenum cofactor (MoCo) in peripheral tissue. We identified a de novo missense mutation (G375D) in the Gephyrin gene (GPHN) in a patient with epileptic encephalopathy resembling Dravet syndrome. Although stably expressed and correctly folded, Gephyrin‐G375D was non‐synaptically localized in neurons and acted dominant‐negatively on the clustering of wild‐type Gephyrin leading to a marked decrease in GABAAR surface expression and GABAergic signaling. We identified a decreased binding affinity between Gephyrin‐G375D and the receptors, suggesting that Gly375 is essential for Gephyrin–receptor complex formation. Surprisingly, Gephyrin‐G375D was also unable to synthesize MoCo and activate MoCo‐dependent enzymes. Thus, we describe a missense mutation that affects both functions of Gephyrin and suggest that the identified defect at GABAergic synapses is the mechanism underlying the patient's severe phenotype.
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Palmitoylation of Gephyrin controls receptor clustering and plasticity of GABAergic synapses.
PLOS Biology, 2014Co-Authors: Borislav Dejanovic, Marcus Semtner, Silvia Ebert, Franziska Neuser, Tobias Lamkemeyer, Jochen C Meier, Bernhard Luscher, Guenter SchwarzAbstract:Postsynaptic scaffolding proteins regulate coordinated neurotransmission by anchoring and clustering receptors and adhesion molecules. Gephyrin is the major instructive molecule at inhibitory synapses, where it clusters glycine as well as major subsets of GABA type A receptors (GABAARs). Here, we identified palmitoylation of Gephyrin as an important mechanism of strengthening GABAergic synaptic transmission, which is regulated by GABAAR activity. We mapped palmitoylation to Cys212 and Cys284, which are critical for both association of Gephyrin with the postsynaptic membrane and Gephyrin clustering. We identified DHHC-12 as the principal palmitoyl acyltransferase that palmitoylates Gephyrin. Furthermore, Gephyrin pamitoylation potentiated GABAergic synaptic transmission, as evidenced by an increased amplitude of miniature inhibitory postsynaptic currents. Consistently, inhibiting Gephyrin palmitoylation either pharmacologically or by expression of palmitoylation-deficient Gephyrin reduced the Gephyrin cluster size. In aggregate, our study reveals that palmitoylation of Gephyrin by DHHC-12 contributes to dynamic and functional modulation of GABAergic synapses.
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Exonic microdeletions of the Gephyrin gene impair GABAergic synaptic inhibition in patients with idiopathic generalized epilepsy.
Neurobiology of Disease, 2014Co-Authors: Borislav Dejanovic, Dennis Lal, Claudia B. Catarino, Sita Arjune, Abdel A. Belaidi, Holger Trucks, Christian Vollmar, Rainer Surges, Wolfram S. Kunz, Susanne MotamenyAbstract:Gephyrin is a postsynaptic scaffolding protein, essential for the clustering of glycine and γ-aminobutyric acid type-A receptors (GABAARs) at inhibitory synapses. An impairment of GABAergic synaptic inhibition represents a key pathway of epileptogenesis. Recently, exonic microdeletions in the Gephyrin (GPHN) gene have been associated with neurodevelopmental disorders including autism spectrum disorder, schizophrenia and epileptic seizures. Here we report the identification of novel exonic GPHN microdeletions in two patients with idiopathic generalized epilepsy (IGE), representing the most common group of genetically determined epilepsies. The identified GPHN microdeletions involve exons 5-9 (Δ5-9) and 2-3 (Δ2-3), both affecting the Gephyrin G-domain. Molecular characterization of the GPHN Δ5-9 variant demonstrated that it perturbs the clustering of regular Gephyrin at inhibitory synapses in cultured mouse hippocampal neurons in a dominant-negative manner, resulting in a significant loss of γ2-subunit containing GABAARs. GPHN Δ2-3 causes a frameshift resulting in a premature stop codon (p.V22Gfs*7) leading to haplo-insufficiency of the gene. Our results demonstrate that structural exonic microdeletions affecting the GPHN gene constitute a rare genetic risk factor for IGE and other neuropsychiatric disorders by an impairment of the GABAergic inhibitory synaptic transmission.
Jean-marc Fritschy - One of the best experts on this subject based on the ideXlab platform.
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V5-CB1 splice isoforms enhance eGFP-Gephyrin clustering along proximal-distal axis.
2017Co-Authors: Claire De Groot, Cornelia Schwerdel, Amalia Floriou-servou, Yuan-chen Tsai, Simon Früh, Manuela Kohler, Georgia Parkin, Giovanna Bosshard, Kai Kaila, Jean-marc FritschyAbstract:(A) Cartoon of various CB isoforms and deletion mutations used in the current study. (B-F) 8+7 DIV neurons co-transfected with either V5-CB1SH3+, V5-CB1SH3-, V5-CB2SH3+ or V5-CB2SH3- isoform (blue) and eGFP-Gephyrin (green) and stained for presynaptic marker VGAT (red). Alterations in the morphology of eGFP-Gephyrin synaptic clusters was observed. (B-F, lower panels) zoom of dendritic proximal or distal dendritic segment showing eGFP-Gephyrin clustering and vGAT co-localization. The localization of eGFP-Gephyrin clusters with γ2 GABAAR subunit was also observed. (G-I) Quantification of eGFP-Gephyrin synaptic cluster density per 20 μm dendrite (DIV 8+7) at proximal and distal dendritic segments of neurons co-expressing eGFP-Gephyrin and mCherry-CB1/2 isoforms. Scale bar 10μm and 5 μm. Statistical analysis for cluster density; One-way ANOVA, Bonferonni post-hoc test, p = 0.85; size analysis: Kruskal-Wallis non parametric test, Dunn's multiple comparison test p
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eGFP-CB1SH3- can rescue Gephyrin scaffolding in Gabra2 KO neurons.
2017Co-Authors: Claire De Groot, Cornelia Schwerdel, Amalia Floriou-servou, Yuan-chen Tsai, Simon Früh, Manuela Kohler, Georgia Parkin, Giovanna Bosshard, Kai Kaila, Jean-marc FritschyAbstract:(A) Morphology of retrovirus eGFP with proximal and distal dendritic sites. Right panels; WT and Gabra2 KO neuron expressing eGFP with Gephyrin and α1 GABAAR staining. Arrows show reduced Gephyrin clustering and increased α1 GABAAR clusters in Gabra2 KO neurons. (B-B’) α1 GABAAR cluster density in proximal dendritic segments show significant increase in Gabra2 KO cells at 14 dpi, 28 dpi, but not 42 dpi. Similarly, α1 GABAAR cluster density in distal dendritic segments are increased Gabra2 KO cells at 28 dpi, but not 14 dpi and 42 dpi. (C-C’) Gephyrin cluster density in proximal and distal dendritic segments show significant reduction in Gabra2 KO cells at 14 dpi, 28 dpi and 42 dpi. (D-D’) Gephyrin clustering at both proximal and distal dendritic segments is restored in Gabra2 KO neurons upon eGFP-CB1SH3- overexpression. Rescue of Gephyrin clustering does not influence α1 GABAAR cluster density in Gabra2 KO neurons.
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pin1 dependent signalling negatively affects gabaergic transmission by modulating neuroligin2 Gephyrin interaction
Nature Communications, 2014Co-Authors: Roberta Antonelli, Jean-marc Fritschy, Rocco Pizzarelli, Andrea Pedroni, Giannino Del Sal, Enrico Cherubini, Paola ZacchiAbstract:The cell adhesion molecule Neuroligin2 (NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein Gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis-trans isomerase Pin1. This signalling cascade negatively regulates NL2's ability to interact with Gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of NL2, Gephyrin and GABAA receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1-/-) associated with an increase in amplitude of spontaneous GABAA-mediated post-synaptic currents. Our results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/Gephyrin interaction.
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extracellular signal regulated kinase and glycogen synthase kinase 3β regulate Gephyrin postsynaptic aggregation and gabaergic synaptic function in a calpain dependent mechanism
Journal of Biological Chemistry, 2013Co-Authors: Shiva K. Tyagarajan, Himanish Ghosh, Gonzalo E Yevenes, Hanns Ulrich Zeilhofer, Bertran Gerrits, Susumu Y Imanishi, Jean-marc FritschyAbstract:Molecular mechanisms of plasticity at GABAergic synapses are currently poorly understood. To identify signaling cascades that converge onto GABAergic postsynaptic density proteins, we performed MS analysis using Gephyrin isolated from rat brain and identified multiple novel phosphorylation and acetylation residues on Gephyrin. Here, we report the characterization of one of these phosphoresidues, Ser-268, which when dephosphorylated leads to the formation of larger postsynaptic scaffolds. Using a combination of mutagenesis, pharmacological treatment, and biochemical assays, we identify ERK as the kinase phosphorylating Ser-268 and describe a functional interaction between residues Ser-268 and Ser-270. We further demonstrate that alterations in Gephyrin clustering via ERK modulation are reflected by amplitude and frequency changes in miniature GABAergic postsynaptic currents. We unravel novel mechanisms for activity- and ERK-dependent calpain action on Gephyrin, which are likely relevant in the context of cellular signaling affecting GABAergic transmission and homeostatic synaptic plasticity in pathology.
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Collybistin splice variants differentially interact with Gephyrin and Cdc42 to regulate Gephyrin clustering at GABAergic synapses.
Journal of Cell Science, 2011Co-Authors: Shiva K. Tyagarajan, Himanish Ghosh, Jean-marc FritschyAbstract:Collybistin (CB) is a guanine-nucleotide-exchange factor (GEF) selectively activating Cdc42. CB mutations cause X-linked mental retardation due to defective clustering of Gephyrin, a postsynaptic protein associated with both glycine and GABAA receptors. Using a combination of biochemistry and cell biology we provide novel insights into the roles of the CB2 splice variants, CB2SH3+ and CB2SH3−, and their substrate, Cdc42, in regulating Gephyrin clustering at GABAergic synapses. Transfection of Myc-tagged CB2SH3+ and CB2SH3− into cultured neurons revealed strong, but distinct, effects promoting postsynaptic Gephyrin clustering, denoting mechanistic differences in their function. In addition, overexpression of constitutively active or dominant-negative Cdc42 mutants identified a new function of Cdc42 in regulating the shape and size of postsynaptic Gephyrin clusters. Using biochemical assays and native brain tissue, we identify a direct interaction between Gephyrin and Cdc42, independent of its activation state. Finally, our data show that CB2SH3−, but not CB2SH3+, can form a ternary complex with Gephyrin and Cdc42, providing a biochemical substrate for the distinct contribution of these CB isoforms in Gephyrin clustering at GABAergic postsynaptic sites. Taken together, our results identify CB and Cdc42 as major regulators of GABAergic postsynaptic densities.