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Kirsten Harvey - One of the best experts on this subject based on the ideXlab platform.
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Table_1_Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability.DOCX
2019Co-Authors: Tzu-ting Chiou, Vera M Kalscheuer, Kirsten Harvey, Philip Long, Alexandra Schumann-gillett, Venkateswarlu Kanamarlapudi, Stefan A. Haas, Megan L. O’mara, Angel L. De Blas, Robert J. HarveyAbstract:The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor Collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR α2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, Collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the Collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant Collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by Collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of Collybistin PH domain phosphoinositide binding.
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missense mutation r338w in arhgef9 in a family with x linked intellectual disability with variable macrocephaly and macro orchidism
Frontiers in Molecular Neuroscience, 2016Co-Authors: Philip Long, Kirsten Harvey, Charles E. Schwartz, Melanie M May, Victoria M James, Simone Granno, John P Johnson, Patrick S Tarpey, Roger E Stevenson, Robert J. HarveyAbstract:Non-syndromal X-linked intellectual disability (NS-XLID) represents a broad group of clinical disorders in which ID is the only clinically consistent manifestation. Although in many cases either chromosomal linkage data or knowledge of the >100 existing XLID genes has assisted mutation discovery, the underlying cause of disease remains unresolved in many families. We report the resolution of a large family (K8010) with NS-XLID, with variable macrocephaly and macro-orchidism. Although a previous linkage study had mapped the locus to Xq12-q21, this region contained too many candidate genes to be analyzed using conventional approaches. However, X-chromosome exome sequencing, bioinformatics analysis and segregation analysis revealed a novel missense mutation (c.1012C>T; p.R338W) in ARHGEF9. This gene encodes Collybistin (CB), a neuronal GDP-GTP exchange factor previously implicated in several cases of XLID, as well as clustering of gephyrin and GABAA receptors at inhibitory synapses. Molecular modeling of the CB R338W substitution revealed that this change results in the substitution of a long electropositive side-chain with a large non-charged hydrophobic side-chain. The R338W change is predicted to result in clashes with adjacent amino acids (K363 and N335) and disruption of electrostatic potential and local folding of the PH domain, which is known to bind phosphatidylinositol-3-phosphate (PI3P/PtdIns-3-P). Consistent with this finding, functional assays revealed that recombinant CB CB2SH3- (R338W) was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Taken together, these results suggest that the R338W mutation in ARHGEF9 is the underlying cause of NS-XLID in this family.
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Supplemental Experimental Procedures Constructs
2015Co-Authors: Ros Poulopoulos, Celine Fuchs, Mrinalini Hoon, Tolga Soykan, Theofilos Papadopoulos, Gayane Aramuni, Guido Meyer, Mingyue Zhang, Ingo Paarmann, Kirsten HarveyAbstract:Flag-CB2SH3 – and Flag-CB2SH3+ were constructed by subcloning of rat Collybistin cDNA sequences into vector pRK5FLAG. CB2SH3–-GFP was subcloned from the HA-tagged Collybistin construct described previously (Kins et al., 2000). The GST-NL2CD construct was created by subcloning the rat NL2 cytoplasmic domain sequence into vector pGEX-4T1. Mutants of the various aforementioned constructs were generated with appropriate oligonucleotide primers following the QuikChange site-directed mutagenesis protocol (Stratagene). cDNA fragments used for the generation of bait constructs were generated by PCR amplification from rat or mouse brain total cDNA (QUICK-clone, Clontech) and cloned in frame with LexA into plexN. Prey constructs designed for expression of fragments were amplified from library derived prey cDNA clones and subcloned into pVP16-3. Sequencing of the full open reading frames was performed to verify constructs in each case. YTH Screen and Assays YTH screening was performed using a pLexN bait construct that expresses Lex
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Cellular/Molecular The GDP–GTP Exchange Factor Collybistin: An Essential Determinant of Neuronal Gephyrin Clustering
2013Co-Authors: Kirsten Harvey, Ian C. Duguid, Melissa J. Alldred, Sarah E. Beatty, Hamish Ward, Nicholas H. Keep, Sue E. Lingenfelter, Brian R. Pearce, Johan Lundgren, Michael J. OwenAbstract:Glycine receptors (GlyRs) and specific subtypes of GABA A receptors are clustered at synapses by the multidomain protein gephyrin, which in turn is translocated to the cell membrane by the GDP–GTP exchange factor Collybistin. We report the characterization of several new variants of Collybistin, which are created by alternative splicing of exons encoding an N-terminal src homology 3 (SH3) domain and three alternate C termini (CB1, CB2, and CB3). The presence of the SH3 domain negatively regulates the ability of Collybistin to translocate gephyrin to submembrane microaggregates in transfected mammalian cells. Because the majority of native Collybistin isoforms appear to harbor the SH3 domain, this suggests that Collybistin activity may be regulated by protein–protein interactions at the SH3 domain. We localized the binding sites for Collybistin and the GlyR � subunit to the C-terminal MoeA homology domain of gephyrin and show that multimerization of this domain is required for Collybistin – gephyrin and GlyR – gephyrin interactions. We also demonstrate that gephyrin clustering in recombinant systems and cultured neurons requires both Collybistin – gephyrin interactions and an intact collybisti
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Selective localization of Collybistin at a subset of inhibitory synapses in brain circuits
The Journal of comparative neurology, 2011Co-Authors: Annarita Patrizi, Kirsten Harvey, Laura Viltono, Elena Frola, Robert J. Harvey, Marco Sassoè-pognettoAbstract:Collybistin is a brain-specific guanine nucleotide exchange factor (GEF) that is crucial for the postsynaptic accumulation of gephyrin and γ-aminobutyric acid A receptors (GABAARs) at a specific subset of inhibitory synapses. Our understanding of the in vivo function of Collybistin has been hampered by lack of information about the synaptic localization of this protein in brain circuits. Here we describe the subcellular localization of endogenous Collybistin by using antibodies raised against distinct molecular domains that should recognize the majority of endogenous Collybistin isoforms. We show that Collybistin co-clusters with gephyrin and GABAARs in synaptic puncta and is recruited to postsynaptic specializations early during synapse development. Notably, Collybistin is present in only a subset of gephyrin-positive synapses, with variable co-localization values in different brain regions. Moreover, Collybistin co-localizes with GABAARs containing the α1, α2, or α3 subunits, arguing against a selective association with specific GABAAR subtypes. Surprisingly, we found that Collybistin is expressed only transiently in Purkinje cells, suggesting that in these cerebellar neurons Collybistin plays a selective role during the initial assembly of postsynaptic specializations. These data reveal a remarkable heterogeneity in the organization of GABAergic synapses and provide an anatomical basis for interpreting the variable effects caused by disruption of the Collybistin gene in human X-linked intellectual disability and mouse knockout models. J. Comp. Neurol., 2012. © 2011 Wiley Periodicals, Inc.
Theofilos Papadopoulos - One of the best experts on this subject based on the ideXlab platform.
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Collybistin sh3 protein isoforms are expressed in the rat brain promoting gephyrin and gaba a receptor clustering at gabaergic synapses
Journal of Neurochemistry, 2021Co-Authors: Shanu George, Theofilos Papadopoulos, Tzu-ting Chiou, Celia P. Miralles, John Bear, Michael J. Taylor, Karthik Kanamalla, Christopher D. Fekete, Angel L. De BlasAbstract:Collybistin (CB) is a guanine nucleotide exchange factor (GEF) selectively localized at GABAergic and glycinergic postsynapses. Analysis of mRNA shows that several isoforms of Collybistin are expressed in the brain. Some of the isoforms have a SH3 domain (CBSH3+) and some have no SH3 domain (CBSH3-). The CBSH3+ mRNAs are predominantly expressed over CBSH3-. However, in an immunoblot study of mouse brain homogenates, only CBSH3+ protein isoforms were detected, proposing that CBSH3- protein might not be expressed in the brain. The expression or lack of expression of CBSH3- protein is an important issue because CBSH3- has a strong effect in promoting the postsynaptic clustering of gephyrin and GABA-A receptors (GABAA Rs). Moreover CBSH3- is constitutively active; therefore lower expression of CBSH3- protein might play a relatively stronger functional role than the more abundant but self-inhibited CBSH3+ isoforms, which need to be activated. We are now showing that: (a) CBSH3- protein is expressed in the brain; (b) parvalbumin positive (PV+) interneurons show higher expression of CBSH3- protein than other neurons; (c) CBSH3- is associated with GABAergic synapses in various regions of the brain and (d) knocking down CBSH3- in hippocampal neurons decreases the synaptic clustering of gephyrin and GABAA Rs. The results show that CBSH3- protein is expressed in the brain and that it plays a significant role in the size regulation of the GABAergic postsynapse.
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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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neuroligin 2 dependent conformational activation of Collybistin reconstituted in supported hybrid membranes
Journal of Biological Chemistry, 2020Co-Authors: Jonas Schafer, Theofilos Papadopoulos, Lucas Forster, Ingo Mey, Nils Brose, Claudia SteinemAbstract:The assembly of the postsynaptic transmitter sensing machinery at inhibitory nerve cell synapses requires the intimate interplay between cell adhesion proteins, scaffold and adaptor proteins, and γ-aminobutyric acid (GABA) or glycine receptors. We developed an in vitro membrane system to reconstitute this process, to identify the essential protein components, and to define their mechanism of action, with a specific focus on the mechanism by which the cytosolic C terminus of the synaptic cell adhesion protein Neuroligin-2 alters the conformation of the adaptor protein Collybistin-2 and thereby controls Collybistin-2-interactions with phosphoinositides (PtdInsPs) in the plasma membrane. Supported hybrid membranes doped with different PtdInsPs and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) to allow for the specific adsorption of the His6-tagged intracellular domain of Neuroligin-2 (His-cytNL2) were prepared on hydrophobically functionalized silicon dioxide substrates via vesicle spreading. Two different Collybistin variants, the WT protein (CB2SH3) and a mutant that adopts an intrinsically 'open' and activated conformation (CB2SH3/W24A-E262A), were bound to supported membranes in the absence or presence of His-cytNL2. The corresponding binding data, obtained by reflectometric interference spectroscopy, show that the interaction of the C terminus of Neuroligin-2 with Collybistin-2 induces a conformational change in Collybistin-2 that promotes its interaction with distinct membrane PtdInsPs.
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A GTPase-induced switch in phospholipid affinity of Collybistin contributes to synaptic gephyrin clustering
Journal of cell science, 2020Co-Authors: Markus Kilisch, Simone Mayer, Miso Mitkovski, Heiko Roehse, Jennifer Hentrich, Blanche Schwappach, Theofilos PapadopoulosAbstract:Synaptic transmission between neurons relies on the exact spatial organization of postsynaptic transmitter receptors, which are recruited and positioned by dedicated scaffolding and regulatory proteins. At GABAergic synapses, the regulatory protein Collybistin (Cb, also known as ARHGEF9) interacts with small GTPases, cell adhesion proteins and phosphoinositides to recruit the scaffolding protein gephyrin and GABAA receptors to nascent synapses. We dissected the interaction of Cb with the small Rho-like GTPase TC10 (also known as RhoQ) and phospholipids. Our data define a protein-lipid interaction network that controls the clustering of gephyrin at synapses. Within this network, TC10 and monophosphorylated phosphoinositides, particulary phosphatidylinositol 3-phosphate (PI3P), provide a coincidence detection platform that allows the accumulation and activation of Cb in endomembranes. Upon activation, TC10 induces a phospholipid affinity switch in Cb, which allows Cb to specifically interact with phosphoinositide species present at the plasma membrane. We propose that this GTPase-based regulatory switch mechanism represents an important step in the process of tethering of Cb-dependent scaffolds and receptors at nascent postsynapses.
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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.
Robert J. Harvey - One of the best experts on this subject based on the ideXlab platform.
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Table_1_Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability.DOCX
2019Co-Authors: Tzu-ting Chiou, Vera M Kalscheuer, Kirsten Harvey, Philip Long, Alexandra Schumann-gillett, Venkateswarlu Kanamarlapudi, Stefan A. Haas, Megan L. O’mara, Angel L. De Blas, Robert J. HarveyAbstract:The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor Collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR α2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, Collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the Collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant Collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by Collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of Collybistin PH domain phosphoinositide binding.
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missense mutation r338w in arhgef9 in a family with x linked intellectual disability with variable macrocephaly and macro orchidism
Frontiers in Molecular Neuroscience, 2016Co-Authors: Philip Long, Kirsten Harvey, Charles E. Schwartz, Melanie M May, Victoria M James, Simone Granno, John P Johnson, Patrick S Tarpey, Roger E Stevenson, Robert J. HarveyAbstract:Non-syndromal X-linked intellectual disability (NS-XLID) represents a broad group of clinical disorders in which ID is the only clinically consistent manifestation. Although in many cases either chromosomal linkage data or knowledge of the >100 existing XLID genes has assisted mutation discovery, the underlying cause of disease remains unresolved in many families. We report the resolution of a large family (K8010) with NS-XLID, with variable macrocephaly and macro-orchidism. Although a previous linkage study had mapped the locus to Xq12-q21, this region contained too many candidate genes to be analyzed using conventional approaches. However, X-chromosome exome sequencing, bioinformatics analysis and segregation analysis revealed a novel missense mutation (c.1012C>T; p.R338W) in ARHGEF9. This gene encodes Collybistin (CB), a neuronal GDP-GTP exchange factor previously implicated in several cases of XLID, as well as clustering of gephyrin and GABAA receptors at inhibitory synapses. Molecular modeling of the CB R338W substitution revealed that this change results in the substitution of a long electropositive side-chain with a large non-charged hydrophobic side-chain. The R338W change is predicted to result in clashes with adjacent amino acids (K363 and N335) and disruption of electrostatic potential and local folding of the PH domain, which is known to bind phosphatidylinositol-3-phosphate (PI3P/PtdIns-3-P). Consistent with this finding, functional assays revealed that recombinant CB CB2SH3- (R338W) was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Taken together, these results suggest that the R338W mutation in ARHGEF9 is the underlying cause of NS-XLID in this family.
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Selective localization of Collybistin at a subset of inhibitory synapses in brain circuits
The Journal of comparative neurology, 2011Co-Authors: Annarita Patrizi, Kirsten Harvey, Laura Viltono, Elena Frola, Robert J. Harvey, Marco Sassoè-pognettoAbstract:Collybistin is a brain-specific guanine nucleotide exchange factor (GEF) that is crucial for the postsynaptic accumulation of gephyrin and γ-aminobutyric acid A receptors (GABAARs) at a specific subset of inhibitory synapses. Our understanding of the in vivo function of Collybistin has been hampered by lack of information about the synaptic localization of this protein in brain circuits. Here we describe the subcellular localization of endogenous Collybistin by using antibodies raised against distinct molecular domains that should recognize the majority of endogenous Collybistin isoforms. We show that Collybistin co-clusters with gephyrin and GABAARs in synaptic puncta and is recruited to postsynaptic specializations early during synapse development. Notably, Collybistin is present in only a subset of gephyrin-positive synapses, with variable co-localization values in different brain regions. Moreover, Collybistin co-localizes with GABAARs containing the α1, α2, or α3 subunits, arguing against a selective association with specific GABAAR subtypes. Surprisingly, we found that Collybistin is expressed only transiently in Purkinje cells, suggesting that in these cerebellar neurons Collybistin plays a selective role during the initial assembly of postsynaptic specializations. These data reveal a remarkable heterogeneity in the organization of GABAergic synapses and provide an anatomical basis for interpreting the variable effects caused by disruption of the Collybistin gene in human X-linked intellectual disability and mouse knockout models. J. Comp. Neurol., 2012. © 2011 Wiley Periodicals, Inc.
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Differential regulation of the postsynaptic clustering of γ-aminobutyric acid type A (GABAA) receptors by Collybistin isoforms.
The Journal of biological chemistry, 2011Co-Authors: Tzu-ting Chiou, Kirsten Harvey, Robert J. Harvey, Bevan Bonhomme, Hongbing Jin, Celia P. Miralles, Haiyan Xiao, Stefano Vicini, Angel L. De BlasAbstract:Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and γ-aminobutyric acid type A (GABAA) receptors at GABAergic synapses in hippocampus and amygdala. Four Collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all Collybistin isoforms (CB2SH3+, CB2SH3−, CB3SH3+, and CB3SH3−) target to and concentrate at GABAergic postsynapses. Moreover, in non-transfected neurons, Collybistin concentrates at GABAergic synapses. Hippocampal neurons co-transfected with CB2SH3− and gephyrin developed very large postsynaptic gephyrin and GABAA receptor clusters (superclusters). This effect was accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents. Co-transfection with CB2SH3+ and gephyrin induced the formation of many (supernumerary) non-synaptic clusters. Transfection with gephyrin alone did not affect cluster number or size, but gephyrin potentiated the clustering effect of CB2SH3− or CB2SH3+. Co-transfection with CB2SH3− or CB2SH3+ and gephyrin did not affect the density of presynaptic GABAergic terminals contacting the transfected cells, indicating that Collybistin is not synaptogenic. Nevertheless, the synaptic superclusters induced by CB2SH3− and gephyrin were accompanied by enlarged presynaptic GABAergic terminals. The enhanced clustering of gephyrin and GABAA receptors induced by Collybistin isoforms was not accompanied by enhanced clustering of neuroligin 2. Moreover, during the development of GABAergic synapses, the clustering of gephyrin and GABAA receptors preceded the clustering of neuroligin 2. We propose a model in which the SH3− isoforms play a major role in the postsynaptic accumulation of GABAA receptors and in GABAergic synaptic strength.
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complex role of Collybistin and gephyrin in gabaa receptor clustering
Journal of Biological Chemistry, 2010Co-Authors: Leila Saiepour, Celine Fuchs, Annarita Patrizi, Robert J. Harvey, Marco Sassoepognetto, Kirsten HarveyAbstract:Gephyrin and Collybistin are key components of GABA(A) receptor (GABA(A)R) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA(A)R subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA(A)R α2 and α3 subunit intracellular M3-M4 domain (but not α1, α4, α5, α6, β1-3, or γ1-3) with gephyrin. Curiously, GABA(A)R α2, but not α3, binds to both gephyrin and Collybistin using overlapping sites. The reciprocal binding sites on gephyrin for Collybistin and GABA(A)R α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA(A)R α3 interacts with gephyrin, GABA(A)R α2, Collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA(A)R α2 and Collybistin or GABA(A)R α2 and gephyrin are strengthened in the presence of gephyrin or Collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA(A)R α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA(A)R α2 is capable of "activating " Collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA(A)R α2-Collybistin interaction was disrupted by a pathogenic mutation in the Collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA(A)R and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of Collybistin with α2 subunit containing GABA(A)Rs, but not GlyRs or other GABA(A)R subtypes, we propose that the Collybistin-gephyrin complex has an intimate role in the clustering of GABA(A)Rs containing the α2 subunit.
Angel L. De Blas - One of the best experts on this subject based on the ideXlab platform.
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selective overexpression of Collybistin in mouse hippocampal pyramidal cells enhances gabaergic neurotransmission and protects against ptz induced seizures
eNeuro, 2021Co-Authors: Shanu George, Shaun James, Angel L. De BlasAbstract:Collybistin (CB) is a rho guanine exchange factor found at GABAergic and glycinergic postsynapses that interacts with the inhibitory scaffold protein, gephyrin, and induces accumulation of gephyrin and GABAA receptors (GABAARs) to the postsynapse. We have previously reported that the isoform without the src homology 3 (SH3) domain, CBSH3-, is particularly active in enhancing the GABAergic postsynapse in both cultured hippocampal neurons as well as in cortical pyramidal neurons after chronic in vivo expression in in utero electroporated (IUE) rats. Deficiency of CB in knockout mice results in absence of gephyrin and gephyrin-dependent GABAARs at postsynaptic sites in several brain regions, including hippocampus. In the present study, we have generated an adeno-associated virus (AAV) that expresses CBSH3- in a cre-dependent manner. Using male and female VGLUT1-IRES-cre or VGAT-IRES-cre mice, we explore the effect of overexpression of CBSH3- in hippocampal pyramidal cells or hippocampal interneurons. The results show that: 1) the accumulation of gephyrin and GABAARs at inhibitory postsynapses in hippocampal pyramidal neurons or interneurons can be enhanced by CBSH3- overexpression, 2) overexpression of CBSH3- in hippocampal pyramidal cells can enhance the strength of inhibitory neurotransmission, and 3) these enhanced inhibitory synapses provide protection against PTZ-induced seizures. The results indicate that this AAV vector carrying CBSH3- can be used for in vivo enhancement of GABAergic synaptic transmission in selected target neurons in the brain. Significance statement Excessive or imbalanced excitation in the hippocampus can result in acute or chronic pathological conditions, such as seizures, epilepsy, and learning impairments. It is therefore important to uncover target genes that can be manipulated to restore or prevent this imbalance. This study uses a novel adeno-associated virus to express Collybistin SH3- in select cells of the hippocampus. We have found that overexpression of this protein enhances GABAergic inhibitory synaptic transmission. The results also bring attention to Collybistin as a possible target for therapeutic intervention aimed to restore the balance between excitation and inhibition.
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correction to recruitment of plasma membrane gaba a receptors by submembranous gephyrin Collybistin clusters
Cellular and Molecular Neurobiology, 2021Co-Authors: Shanu George, Tzu-ting Chiou, Karthik Kanamalla, Angel L. De BlasAbstract:The original version of this article unfortunately contained editorial errors in supplementary material.
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recruitment of plasma membrane gaba a receptors by submembranous gephyrin Collybistin clusters
Cellular and Molecular Neurobiology, 2021Co-Authors: Shanu George, Tzu-ting Chiou, Karthik Kanamalla, Angel L. De BlasAbstract:It has been shown that subunit composition is the main determinant of the synaptic or extrasynaptic localization of GABAA receptors (GABAARs). Synaptic and extrasynaptic GABAARs are involved in phasic and tonic inhibition, respectively. It has been proposed that synaptic GABAARs bind to the postsynaptic gephyrin/Collybistin (Geph/CB) lattice, but not the typically extrasynaptic GABAARs. Nevertheless, there are no studies of the direct binding of various types of GABAARs with the submembranous Geph/CB lattice in the absence of other synaptic proteins, some of which are known to interact with GABAARs. We have reconstituted GABAARs of various subunit compositions, together with the Geph/CB scaffold, in HEK293 cells, and have investigated the recruitment of surface GABAARs by submembranous Geph/CB clusters. Results show that the typically synaptic α1β3γ2 GABAARs were trapped by submembranous Geph/CB clusters. The α5β3γ2 GABAARs, which are both synaptic and extrasynaptic, were also trapped by Geph/CB clusters. Extrasynaptic α4β3δ GABAARs consistently showed little or no trapping by the Geph/CB clusters. However, the extrasynaptic α6β3δ, α1β3, α6β3 (and less α4β3) GABAARs were highly trapped by the Geph/CB clusters. AMPA and NMDA glutamate receptors were not trapped. The results suggest: (I) in the absence of other synaptic molecules, the Geph/CB lattice has the capacity to trap not only synaptic but also several typically extrasynaptic GABAARs; (II) the Geph/CB lattice is important but does not play a decisive role in the synaptic localization of GABAARs; and (III) in neurons there must be mechanisms preventing the trapping of several typically extrasynaptic GABAARs by the postsynaptic Geph/CB lattice.
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Collybistin sh3 protein isoforms are expressed in the rat brain promoting gephyrin and gaba a receptor clustering at gabaergic synapses
Journal of Neurochemistry, 2021Co-Authors: Shanu George, Theofilos Papadopoulos, Tzu-ting Chiou, Celia P. Miralles, John Bear, Michael J. Taylor, Karthik Kanamalla, Christopher D. Fekete, Angel L. De BlasAbstract:Collybistin (CB) is a guanine nucleotide exchange factor (GEF) selectively localized at GABAergic and glycinergic postsynapses. Analysis of mRNA shows that several isoforms of Collybistin are expressed in the brain. Some of the isoforms have a SH3 domain (CBSH3+) and some have no SH3 domain (CBSH3-). The CBSH3+ mRNAs are predominantly expressed over CBSH3-. However, in an immunoblot study of mouse brain homogenates, only CBSH3+ protein isoforms were detected, proposing that CBSH3- protein might not be expressed in the brain. The expression or lack of expression of CBSH3- protein is an important issue because CBSH3- has a strong effect in promoting the postsynaptic clustering of gephyrin and GABA-A receptors (GABAA Rs). Moreover CBSH3- is constitutively active; therefore lower expression of CBSH3- protein might play a relatively stronger functional role than the more abundant but self-inhibited CBSH3+ isoforms, which need to be activated. We are now showing that: (a) CBSH3- protein is expressed in the brain; (b) parvalbumin positive (PV+) interneurons show higher expression of CBSH3- protein than other neurons; (c) CBSH3- is associated with GABAergic synapses in various regions of the brain and (d) knocking down CBSH3- in hippocampal neurons decreases the synaptic clustering of gephyrin and GABAA Rs. The results show that CBSH3- protein is expressed in the brain and that it plays a significant role in the size regulation of the GABAergic postsynapse.
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Table_1_Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability.DOCX
2019Co-Authors: Tzu-ting Chiou, Vera M Kalscheuer, Kirsten Harvey, Philip Long, Alexandra Schumann-gillett, Venkateswarlu Kanamarlapudi, Stefan A. Haas, Megan L. O’mara, Angel L. De Blas, Robert J. HarveyAbstract:The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor Collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR α2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, Collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the Collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant Collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by Collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of Collybistin PH domain phosphoinositide binding.
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Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS.
The Journal of biological chemistry, 2015Co-Authors: Michaela Ludolphs, Tolga Soykan, Daniela Schneeberger, Theofilos Papadopoulos, Jonas Schafer, Nils Brose, Hermann Schindelin, Claudia SteinemAbstract:The regulatory protein Collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally "opens" CB2SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.
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Supplemental Experimental Procedures Constructs
2015Co-Authors: Ros Poulopoulos, Celine Fuchs, Mrinalini Hoon, Tolga Soykan, Theofilos Papadopoulos, Gayane Aramuni, Guido Meyer, Mingyue Zhang, Ingo Paarmann, Kirsten HarveyAbstract:Flag-CB2SH3 – and Flag-CB2SH3+ were constructed by subcloning of rat Collybistin cDNA sequences into vector pRK5FLAG. CB2SH3–-GFP was subcloned from the HA-tagged Collybistin construct described previously (Kins et al., 2000). The GST-NL2CD construct was created by subcloning the rat NL2 cytoplasmic domain sequence into vector pGEX-4T1. Mutants of the various aforementioned constructs were generated with appropriate oligonucleotide primers following the QuikChange site-directed mutagenesis protocol (Stratagene). cDNA fragments used for the generation of bait constructs were generated by PCR amplification from rat or mouse brain total cDNA (QUICK-clone, Clontech) and cloned in frame with LexA into plexN. Prey constructs designed for expression of fragments were amplified from library derived prey cDNA clones and subcloned into pVP16-3. Sequencing of the full open reading frames was performed to verify constructs in each case. YTH Screen and Assays YTH screening was performed using a pLexN bait construct that expresses Lex
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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.
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Collybistin activation by gtp tc10 enhances postsynaptic gephyrin clustering and hippocampal gabaergic neurotransmission
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Simone Mayer, Tolga Soykan, Nils Brose, Rohit Kumar, Mamta Jaiswal, Mohammad Reza Ahmadian, Jeongseop Rhee, Theofilos PapadopoulosAbstract:In many brain regions, gephyrin and GABAA receptor clustering at developing inhibitory synapses depends on the guanine nucleotide exchange factor Collybistin (Cb). The vast majority of Cb splice variants contain an autoinhibitory src homology 3 domain, and several synaptic proteins are known to bind to this SH3 domain and to thereby activate gephyrin clustering. However, many functional GABAergic synapses form independently of the known Cb-activating proteins, indicating that additional Cb activators must exist. Here we show that the small Rho-like GTPase TC10 stimulates Cb-dependent gephyrin clustering by binding in its active, GTP-bound state to the pleckstrin homology domain of Cb. Overexpression of a constitutively active TC10 variant in neurons causes an increase in the density of synaptic gephyrin clusters and mean miniature inhibitory postsynaptic current amplitudes, whereas a dominant negative TC10 variant has opposite effects. The enhancement of Cb-induced gephyrin clustering by GTP-TC10 does not depend on the guanine nucleotide exchange activity of Cb but involves an interaction that resembles reported interactions of other small GTPases with their effectors. Our data indicate that GTP-TC10 activates the major src homology 3 domain-containing Cb variants by relieving autoinhibition and thus define an alternative GTPase-driven signaling pathway in the genesis of inhibitory synapses.
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The role of Collybistin in gephyrin clustering at inhibitory synapses: facts and open questions.
Frontiers in cellular neuroscience, 2011Co-Authors: Theofilos Papadopoulos, Tolga SoykanAbstract:Collybistin (Cb) is a brain-specific GDP/GTP-exchange factor, which interacts with the inhibitory receptor anchoring protein gephyrin. Data from mice carrying an inactivated Cb gene indicate that Cb is required for the formation and maintenance of gephyrin and gephyrin-dependent GABAA receptor (GABAAR) clusters at inhibitory postsynapses in selected regions of the mammalian forebrain. However, important aspects of how Cb’s GDP/GTP exchange activity, structure and regulation contribute to gephyrin and GABAAR clustering, as well as its role in synaptic plasticity, remain poorly understood. Here we review the current state of knowledge about Cb’s function and address open questions concerning its contribution to synapse formation, maintenance, plasticity and adaptive changes in response to altered network activity.