ABSTRACT Gephyrin is a central element that anchors, clusters and stabilizes glycine and γ-aminobutyric acid type A receptors at inhibitory synapses of the mammalian brain. It self-assembles

into a hexagonal lattice and interacts with various inhibitory synaptic proteins. Intriguingly, the clustering of gephyrin, which is regulated by multiple posttranslational modifications,

is critical for inhibitory synapse formation and function. In this review, we summarize the basic properties of gephyrin and describe recent findings regarding its roles in inhibitory

synapse formation, function and plasticity. We will also discuss the implications for the pathophysiology of brain disorders and raise the remaining open questions in this field. SIMILAR

Open access 17 September 2024 INTRODUCTION Synapses, which are the fundamental information-processing units of neural circuits, form the basis for all brain functions by controlling the

excitation-to-inhibition balance. Synapses form via a coordinated process that orchestrates (sequentially or in parallel) a variety of dynamically regulated cellular events. Various

molecular and cellular mechanisms underlying synapse formation have been elucidated. For example, multiple synaptic adhesion molecules have been shown to initiate physical contact between

axons and dendrites, recruit other synaptic molecules (e.g., scaffolding proteins) to assemble nascent synaptic sites and specify their properties to organize functional synapses (reviewed

in refs 1, 2, 3). Numerous studies have examined the development of excitatory synapse, and the identification of inhibitory synapse components has accelerated in recent years. This has

allowed researchers to establish a conceptual framework for the organization of inhibitory synapse development. Postsynaptic scaffolding molecules are key components in the organization of

functional synapses. They ensure the accurate accumulation of neurotransmitter receptors in precise apposition to presynaptic release sites, as this is required for reliable synaptic

transmission. Scaffolding molecules also interact with cytoskeletal anchoring elements to provide a physical platform for maintaining receptors at synapses, and regulate downstream signaling

pathways to adjust the molecular composition of the postsynaptic devices necessary to sustain synaptic plasticity. Gephyrin is the most extensively studied scaffold responsible for

organizing the inhibitory postsynaptic density, which is essential for clustering of glycine and γ-aminobutyric acid type A (GABAA) receptors, inhibitory synaptic transmission and long-term

potentiation (reviewed in refs 4, 5, 6). Since the discovery of gephyrin in 1982 by Heinrich Betz’s group,7 its biochemical and cellular properties have been extensively studied. The protein

has been shown to bind to various classes of inhibitory synaptic molecules, thereby governing the dynamic processes of inhibitory synapse formation, function and plasticity. Although

significant progress has been made in elucidating the role of gephyrin at inhibitory synapses, many unanswered questions remain. In the current review, we describe the domain structure,

expression profile and trafficking of gephyrin, and link this information to its synaptic role. We then describe various interacting partners of gephyrin and their roles in inhibitory

synapse development. Finally, we discuss the possible implications of gephyrin dysfunctions in some neurological disorders. Owing to space constraints, we focus on recent advances on

gephyrin and its interacting proteins, and we also cite classic papers where needed. Other excellent reviews provide detailed discussions regarding the historical context of gephyrin.8, 9

DOMAIN STRUCTURE Gephyrin was originally purified as a 93-kDa protein that associated with glycine receptors (GlyRs) and copurified with polymerized tubulin.10 Thus, it was thought to be an

anchoring protein of GlyRs. In vertebrates, gephyrin is composed of three major distinct domains: the G, C and E domains (Figure 1). Molecular cloning revealed that gephyrin shows similarity

to a group of invertebrate proteins that are essential to synthesize a cofactor required for the activity of molybdenum.11 The G and E domains of gephyrin are homologous to the _Escherichia

coli_ enzymes MogA and MoeA, respectively, both of which are involved in molybdenum cofactor biosynthesis, as demonstrated by crystal structure analysis of the trimeric N-terminal domain of

gephyrin.12 The C domain of gephyrin contains numerous residues that act as sites for posttranslational modification and binding to other synaptic proteins, such as GABAA

receptor-associated protein (GABARAP) and dynein light chains 1 and 2.8 Thus, the C domain is believed to be critical for the functions of gephyrin. However, other domains also appear to be

required for gephyrin function. For example, individual gephyrin E domain monomers are bound by the β-loop of the GlyR β-subunit, where a serine residue within the binding motif acts as a

phosphorylation site that controls the binding affinity to gephyrin.13, 14, 15 Moreover, vertebrate-specific sequences in the E domain have been shown to regulate cytosolic aggregation and

postsynaptic receptor clustering. The enzymatic and cytosolic aggregations of gephyrin were found to be independent,16 and crystal structure analysis of individual gephyrin domains revealed

that dimerization of gephyrin E domains and trimerization of gephyrin G domains triggered the formation of multimeric, hexagonal aggregates.17 The multimeric conformations of gephyrin are

thought to help position it beneath the plasma membrane, where it forms an anchor for inhibitory receptors.18 However, the structure for full-length gephyrin protein has not yet been solved,

in part because certain properties of the C domain complicate the crystallization of the full-length protein. The gephyrin transcript is subject to extensive alternative splicing10, 19, 20

in all three domains. Each domain contains at least three variable expression cassettes, providing a regulatory mechanism through which the signaling properties of specific synapses may be

altered. We do not yet know how the alternative splicing of gephyrin is regulated. However insertion of the C3 cassette (corresponding to exon 9) was reported to be regulated by Nova

proteins, and the presence of Nova in neurons has been correlated with alternative exon skipping in gephyrin.21, 22 Interestingly, the C3 cassette-containing splice variant of gephyrin

predominates in the mouse liver,23 indicating that the mechanisms of gephyrin splicing may be tissue-specific. The function of the C4d cassette (corresponding to exon 11) has been studied in

the greatest detail. Although it is located in the N-terminal domain of gephyrin, the presence of C4d abolishes GlyR binding.24 A recent biochemical study demonstrated that different splice

variants of gephyrin can form hexameric complexes that differ in their stabilities.25 However, additional systematic analyses are needed to explore the significances of other gephyrin

splicing variants in various contexts. EXPRESSION AND LOCALIZATION Northern blot analysis identified gephyrin mRNA transcripts in various tissues.10, 26 Intriguingly, a high diversity of

gephyrin transcripts was observed in the skeletal muscle, heart, liver and brain, all of which showed distinct patterns. This observation is in accord with the notion that gephyrin has an

important role in both synaptic and metabolic pathways in the nervous system. _In situ_ hybridization analyses with exon-specific probes revealed that certain gephyrin transcripts are

predominantly expressed in specific tissues.26 For example, gephyrin transcripts containing the C2 cassette (corresponding to exon 3) were abundantly expressed at synapses, whereas those

containing the C3 and C4 cassettes (corresponding to exon 11) were not;27 see also Fritschy _et al._8 for revised gephyrin splicing cassette nomenclature. Immunohistochemical analyses have

shown that gephyrin proteins are primarily localized to GABAergic and glycinergic synapses (but not glutamatergic synapses) in various brain regions.28, 29, 30, 31, 32, 33 Strikingly, the

inhibitory synaptic localization of gephyrin was not found to be affected by the depolymerizations of microtubules or actin, suggesting that its targeting mechanism is

cytoskeleton-independent.34 Recently, quantitative nanoscopy was used to calculate the number of gephyrin molecules and inhibitory synaptic receptor binding sites.35 Purely GABAergic

synapses were found to harbor ~40–500 gephyrin molecules and 30–200 GABAA receptors organized in microclusters. In contrast, spinal cord synapses, which contain both GlyRs and GABAA

receptors, showed a twofold higher packing density of gephyrin, suggesting that the binding to GlyRs affected the compactness of the hexagonal gephyrin lattice.35 Moreover, although the

phosphorylation of gephyrin is known to modulate its binding affinity, clustering and receptor association, it is not yet known whether increased gephyrin cluster stability correlates with

tighter packaging within the lattice.35 Overall, these data suggest that gephyrin is tightly packed beneath the GlyR-containing postsynaptic membrane. CLUSTERING Immunostaining of cultured

neurons with an anti-gephyrin antibody revealed the existence of discrete puncta that could be categorized as large (0.4–10.0 μm2) or small (<0.2 μm2).28 The large puncta were restricted

to inhibitory synaptic sites, whereas the small puncta were found in extrasynaptic sites, suggesting that high local concentrations of self-assembled gephyrin molecules (i.e., clusters) are

required to stabilize synaptic sites.28 Neuron-specific molecular mechanisms selectively target and cluster gephyrin molecules at inhibitory postsynaptic sites, while preventing gephyrin

from self-aggregating at extrasynaptic sites (indeed, gephyrin forms intracellular microaggregates when overexpressed in non-neuronal cells).36 More recently, specific splice variants of

collybistin were shown to promote gephyrin clustering in collaboration with Cdc42 via phosphoinositide-mediated membrane anchoring (refs 37, 38, 39; see ‘Interacting proteins of gephyrin’

subsection for more details). At inhibitory synapses, molecular diversity arises from heterogeneity in the GABAA receptor subunit composition. It can be also derived from the subcellular

localization of inhibitory synapses on perisoma, dendrites or axon initial segment, as each of these cellular compartments provides a distinct microenvironment where specific inhibitory

synapses are localized. Diverse mechanisms underlying gephyrin clustering may provide additional layer to the complex mode of inhibitory synapse diversity. POSTTRANSLATIONAL MODIFICATIONS

Historically, the posttranslational modifications of gephyrin were not thought to be functionally significant, perhaps because the protein was considered to be a structural component of the

inhibitory synapse. However, gephyrin has been identified as a phosphoprotein when copurified with the GlyR (which has kinase activity),40 and mass spectrometric analyses of rat and mouse

brains revealed that gephyrin has 22 phosphorylation sites, most of which are located within the C domain (except for threonine 324, which lies in the E domain25, 41, 42, 43) (Figure 1).

These modifications might induce conformational changes by affecting the structure of the C domain or the neighboring G and E domains, thereby altering the clustering, trafficking and

binding properties of gephyrin. Interestingly, lithium chloride (an inhibitor of glycogen synthase kinase-β (GSK3β)) enhances gephyrin clustering in cultured neurons, and it has been

speculated that the mood-stabilizing effect of lithium may be mediated at least partly via this mechanism.44 Serine 268 of gephyrin is phosphorylated by extracellular signal-regulated

kinases 1 and 2; this synergistically alters gephyrin clustering and alters the amplitude and frequency of miniature inhibitory postsynaptic currents43 (Figure 1). In addition,

cyclin-dependent kinase 5 mediates the collybistin-dependent phosphorylation of Ser270, suggesting that multiple signaling pathways converge at this residue of gephyrin.42 It is less clear

how gephyrin is dephosphorylated by phosphatases. Puzzlingly, one study showed that protein phosphatase 1 directly interacted with gephyrin and that gephyrin clustering was decreased by the

application of broad-spectrum phosphatase inhibitors,45 whereas two other studies conversely found that the dephosphorylation of gephyrin _increased_ gephyrin clustering.43, 44 In this

context, it seems noteworthy that acute application of tumor necrosis factor-α downregulates inhibitory synaptic transmission via the protein phosphatase 1-dependent trafficking of GABAA

receptors.46 In addition to phosphorylation, gephyrin is subject to palmitoylation47 (Figure 1). Gephyrin was initially postulated as a potential substrate for palmitoyl acyltransferase,48

and later work confirmed that cysteine 212 and cysteine 284 are palmitoylated by the enzyme, Asp-His-His-Cys (DHHC)-12, which is localized to the Golgi apparatus and dendritic shaft, and

directly interacts with gephyrin.47 The DHHC-12-mediated palmitoylation of gephyrin, which is regulated by GABAA receptor activity, is essential for the postsynaptic membrane association and

clustering of gephyrin, and has been shown to increase inhibitory synaptic transmission.47 It is possible that the palmitoylation of gephyrin may indirectly impact its phosphorylation

status, emphasizing the potential complexity of the mechanisms that form the basis for gephyrin clustering. Gephyrin was found to interact directly with and be subject to proteolysis by the

Ca2+-dependent cysteine protease, calpain-1, suggesting that this may be a gephyrin turnover mechanism.44 Intriguingly, gephyrin contains two PEST sequences that may be exposed by a

conformational change upon its phosphorylation by GSK3β, perhaps facilitating the calpain-1-mediated degradation of gephyrin.44 INTERACTING PROTEINS OF GEPHYRIN Most of the known

gephyrin-binding proteins have been primarily identified by yeast two-hybrid screens. Although some of the interactions have been validated _in vivo_, their significances remain largely

unknown (see Table 1 and Figures 1 and 2). GLYR Β-SUBUNIT The GlyR is a ligand-gated chloride channel protein that was identified as the first binding protein of gephyrin, as an affinity

chromatography study showed that the two could be copurified in stoichiometric amounts.7 The cytoplasmic loop of the GlyR β-subunit was later shown to bind to the E domain dimerization

interface of gephyrin.49, 50 Coexpression of GlyR with gephyrin was found to change the ligand-binding affinities of human embryonic kidney 293 cells.51 Treatment of rat spinal neurons with

gephyrin antisense oligonucleotides was shown to block the formation of GlyR clusters in the neuronal membrane, suggesting that gephyrin is required to anchor GlyR in postsynaptic membrane

specializations.52 Moreover, treatment with a competitive GlyR antagonist (strychnine) or L-type Ca2+ channel blockers inhibited the accumulation of GlyR and gephyrin at postsynaptic

membranes in cultured rat spinal neurons, suggesting that the activation of GlyR is necessary for the clustering of gephyrin and GlyR.53, 54 These notions were later validated _in vivo_ by

studies showing that GlyR clustering was disrupted in gephyrin-null mice.55, 56 GlyR clusters form quickly and reversibly via a two-step process wherein gephyrin-independent cell surface

clustering is followed by gephyrin-mediated postsynaptic accumulation.24 In the latter process, gephyrin breaks the equilibrium between the diffusive and confined states of GlyR to stabilize

it at submembranous sites.57 Moreover, the oligomerization of gephyrin has been shown to modulate the lateral diffusion of GlyRs.58 GABAA RECEPTOR Α-SUBUNITS Although gephyrin was observed

to be involved in GABAA receptor clustering _in vivo_, up until recently it was unclear whether this clustering activity was mediated by a direct interaction with gephyrin.59, 60 For

example, a biochemical study failed to demonstrate a direct interaction between GABAA β-subunits (β1–β3 subunits) and gephyrin,49 and gephyrin-independent mechanisms were identified as

underlying the postsynaptic clustering of certain GABAA subtypes.61 Therefore, it was surprising when Tretter _et al._62 reported that a 10-amino-acid hydrophobic motif within the

intracellular domain of the GABAA α2-subunit was responsible for the inhibitory synaptic accumulation of GABAA receptors and underwent direct binding to gephyrin. The authors further showed

that GABAA receptor subtypes containing the α2-subunit require gephyrin binding for their inhibitory synaptic targeting.62 Subsequent studies then identified motifs in the GABAA receptor α3-

and α1-subunits that were responsible for binding to the E domain of gephyrin.60, 63 Interestingly, some of the gephyrin residues that are key to the binding of GABAA receptor subtypes

containing the α2- and α3-subunits are also important for binding to GlyRs (albeit with different binding affinities).63 The clustering of some GABAA receptor subtypes is reportedly

dependent on gephyrin,64, 65 but future work is needed to determine systematically whether other GABAA receptor subtypes bind to gephyrin. DLC-1 AND DLC-2 Dynein light chain-1 (Dlc-1; also

known as dynein LC8) and its homolog, Dlc-2, were identified as binding proteins of gephyrin in a yeast two-hybrid screen.66 Dlc-1 and -2 are subunits of the cytoplasmic dynein motor

protein, which mediates the movement of cargo proteins toward the minus ends of microtubule tracks. A central fragment of gephyrin (63 amino acids corresponding to amino acids 181–243) was

found to be sufficient to bind Dlc-1/2, and the expression of a Dlc-1/2-binding defective gephyrin construct in cultured neurons had no effect on the synaptic localization of gephyrin,

suggesting that the inhibitory synaptic targeting of gephyrin does not require the Dlc-binding domain.66 This interaction supports the ideas that motor proteins are involved in the

intracellular trafficking of gephyrin to the submembranous compartment, and that gephyrin clustering may be regulated in a highly dynamic manner. However, the precise functional significance

of this interaction is not yet understood in detail. GABAA RECEPTOR-ASSOCIATED PROTEIN GABARAP was originally isolated as a binding partner of the GABAA receptor γ2-subunit in a yeast

two-hybrid screen, and was suggested to have a role in the targeting and clustering of GABAA receptors.67 A later study showed that GABARAP directly interacts with gephyrin.68 Strikingly,

GABARAP is enriched in intracellular compartments but not gephyrin-positive membrane compartments, indicating that the interaction of GABARAP with gephyrin is not critical for postsynaptic

GABAR clustering.68 Instead, the interaction of GABARAP with _N_-ethylmaleimide-sensitive factor appears to have a role in the intracellular transport of GABAA receptors.69 In

GABARAP-deficient mice, however, the total number of GABAA receptors was unaffected,70 indicating that GABARAP is not essential for GABAA receptor targeting. The exact cellular roles of

GABARAP are currently unknown, but recent sequence analyses have suggested that there are many GABARAP-like proteins constituting a GABARAP-like family across evolution.71 Some of these

GABARAP-like proteins have been linked to ubiquitination, vesicular transport events, apoptosis and autophagy. GABARAP itself interacts with multiple proteins in addition to the GABAA

receptor, gephyrin and _N_-ethylmaleimide-sensitive factor, but the functional significances of these interactions are largely unclear at this point. Therefore, further research is needed to

elucidate the roles of GABARAP in various contexts at inhibitory synapses. COLLYBISTIN Collybistin (also called ARHDH9) was also identified as a gephyrin-interacting protein in a yeast

two-hybrid screen.72 Based on cDNA cloning and sequence analysis, collybistin was revealed as a neuron-specific dbl-like GDP/GTP exchange factor characterized by three functional domains: an

Src homology 3 (SH3) domain; a DBL homology domain for the small GTPase, Cdc42; and a pleckstrin homology domain.72 The DBL homology domain of collybistin directly binds to the C-terminal

domain of gephyrin.37, 73 Collybistin is mainly expressed in the brain,37, 72, 74 and alternative splicing generates three isoforms (collybistin I, II and III; differing only in their C

termini and further specifies two different collybistin II variants (CB2SH3+ and CB2SH3−; differing in their SH3 domain usage).37, 73 Importantly, the coexpression of collybistin II and

gephyrin in non-neuronal cells was shown to induce submembranous collybistin/gephyrin microaggregates that also accumulated hetero-oligomeric GlyRs.72 However, CB2SH3+ is inactive in

dendritic gephyrin clustering, indicating that the alternative splicing of collybistin has functional importance in the development of inhibitory synapse (Harvey _et al._;37 also Tyagarajan

_et al._ and Korber _et al._39, 75) for the involvement of Cdc42 in regulating the clustering of gephyrin). Mutation of glycine 55 to alanine (G55A) in the SH3 domain of collybistin has been

linked to hyperekplexia and epilepsy, reinforcing the importance of this domain of collybistin in its dendritic trafficking and association with gephyrin. However, it should be noted that

most of the variants of collybistin found to date in rodents and humans contain an SH3 domain that inhibits their membrane-targeting ability,37 hinting that additional factor(s) are likely

to activate collybistin by relieving this SH3 domain-mediated inhibition. Neuroligin-2 (NL-2) and the Rho-like GTPase, TC10, have been shown to have this role by interacting with the

collybistin SH3 domain.76, 77 Furthermore, a recent structural study demonstrated that the collybistins can adopt open and closed conformations that act as switchable adaptors, and that NL-2

favors the open conformation by competing with a closed-conformation-favoring intramolecular interaction in collybistin.78 The proposed functions of collybistin were demonstrated _in vivo_:

collybistin-deficient mice displayed region-specific reductions in synaptic gephyrin clustering, reduced clustering of the GABAA receptor γ2-subunit, decreased GABAergic synaptic

transmission, impaired hippocampal synaptic plasticity (i.e., increased long-term potentiation and decreased long-term depression) and impaired spatial learning.79 Similar functional

alterations were reproduced in conditional collybistin-knockout mice.80 Moreover, increased granule cell excitability, somatic GABAergic network inhibition and impaired induction of LTP in

the dentate gyrus of the hippocampus were observed in collybistin-null mice,81 suggesting that collybistin deficiency alters GABAergic inhibition, network excitability and synaptic

plasticity in certain brain areas. Strikingly, collybistin-deficient mice did not exhibit deficits in glycinergic synaptic transmission, suggesting that collybistin is dispensable for

gephyrin-mediated GlyR clustering in glycinergic synapses, but is required for the gephyrin-mediated clustering of certain GABAA receptors at inhibitory postsynaptic sites.75, 82, 83, 84

PROFILIN AND MENA A study seeking the linkage between gephyrin and microfilament identified profilin, Mena and G-actin as gephyrin-binding proteins.85 Endogenous profilin and Mena were found

to colocalize with gephyrin in the inhibitory synapses of spinal cord neurons, and were enriched in gephyrin-rich domains of the neuronal plasma membrane.85 Gephyrin and profilin 1 were

shown to form a complex,86 but via an unexpected binding mode that required the E domain of gephyrin and the actin/PIP2-binding site of profilins (Giesemann _et al._,85 but also see Bausen

_et al._87). A study examining the potential impact of these multipartite interactions on the function of gephyrin showed that cytochalasin D (an actin-depolymerizing agent) had differential

effects on gephyrin clusters that were lost following cytochalasin D treatment of immature cultures but not following the same treatment of more mature cultures.87 These data suggest that

the formation of a complex between gephyrin and profilin/Mena may contribute to actin cytoskeleton-dependent inhibitory synapse organization. However, further studies are required to fully

confirm this hypothesis. PEPTIDYL-PROLYL ISOMERASE NIMA-INTERACTING PROTEIN 1 Peptidyl-prolyl isomerase NIMA-interacting protein 1 (Pin1) specifically binds phosphorylated serine or

threonine residues immediately preceding proline residues, and promotes the _cis/trans_ isomerization of peptide bonds.88, 89 These conformational changes profoundly affect on the function

of Pin1 substrates by modulating their catalytic activity, phosphorylation status, protein interactions and/or protein stability.90 Gephyrin undergoes proline-directed phosphorylation and

interacts with Pin1 via a large region that encompasses the E domain and part of the C domain, eliciting conformational changes in gephyrin.91 The ability of gephyrin to interact with GlyR

was significantly decreased in Pin1-deficient mice, reducing the number of GlyR clusters.91 Recently, the inhibitory synaptic adhesion molecule, NL-2, was also shown to be a substrate for

Pin1,92 which negatively regulates the interaction of gephyrin with NL-2 by phosphorylating the serine 714 residue. In addition, the absence of Pin1 was found to promote the formation of

NL-2/gephyrin complexes, enrich GABAA receptors at inhibitory synaptic sites and inhibit synaptic transmission.92 These data suggest a model wherein the Pin1-mediated phosphorylation of NL-2

drives conformational changes that detach gephyrin from NL-2. RAPAMYCIN AND FKBP12 TARGET 1 Rapamycin and FKBP12 target 1 (RAFT1; also called FRAP or mTOR) was found to interact with

gephyrin in a yeast two-hybrid screen.93 The association of gephyrin with RAFT1 was shown to be required for RAFT1-mediated signaling,93 and mutations in the minimal gephyrin-binding domains

of RAFT1 were found to prevent the activations of p70S6K and the eukaryotic initiation factor eIF-4E binding-protein, 4E-BP1.93 However, the functional significance of this interaction has

not yet been clearly defined in the context of gephyrin functions at inhibitory synapses. NEUROLIGIN-2 NL-2 is a neuronal transmembrane protein that was identified as the first inhibitory

synapse-specific adhesion molecule.94 NL-2 interacts with presynaptic neurexins (as do the other neuroligins) and functionally validates inhibitory synapse development in an

activity-dependent manner.76, 95, 96, 97, 98 It is not yet clear how NL-2 is specifically targeted to inhibitory synaptic sites, but studies have shown that GABAA receptors are dispensable

for the inhibitory synaptic targeting of NL-2.99 In addition, NL-2, but not other neuroligin paralogs, has been shown to directly interact with gephyrin through a conserved tyrosine residue

(Tyr770) that helps recruit gephyrin to NL-2 clusters in cultured neurons.76, 100 Moreover, NL-2 activates the collybistin-mediated targeting of gephyrin to the plasma membrane in

non-neuronal cells. Intriguingly, NL-2 was found to be required for gephyrin to be recruited to the perisomatic site but not to the dendritic shaft.76 In addition, deletion of NL-2 was shown

to affect both GABAergic and glycinergic synaptic transmission.76 Taken together, these data suggest a model for inhibitory postsynaptic assembly whereby the cytoplasmic

gephyrin–collybistin complexes are transiently recruited to the plasma membrane sites of NL-2 accumulation via the NL-2/gephyrin interaction, and then other postsynaptic components are

further recruited to help construct the functional inhibitory synapse. SYNAPTIC FUNCTIONS OF GEPHYRIN Gephyrin is believed to act as a scaffold at inhibitory synapses, in a manner analogous

to that of the prototypic excitatory synaptic scaffold, PSD-95. The best-known function of gephyrin is to bring the inhibitory synaptic receptors and to stabilize them at the inhibitory

synapses (Figure 2). Therefore, gephyrin might act as a hub protein, integrating various synaptic activities and signals and adjusting inhibitory synaptic transmission by reorganizing the

properties of GABAA and GlyRs (Figure 2). However, as discussed above, gephyrin interacts with multiple classes of proteins, implying that it may have broader roles in neurons. Notably,

gephyrin interacts with NL-2 and collybistin, suggesting that it may be critical for the maturation or maintenance of inhibitory synapses. As gephyrin undergoes various posttranslational

modifications that regulate its clustering at inhibitory synapses, studies aimed at deciphering how and when such posttranslational modifications are regulated will help us understand the

synaptic functions of this protein. Gephyrin may also act as a sensor that adjusts inhibitory synaptic transmission in response to changes in global network activity, and the alteration of

this function appears to activate various signaling cascades. Finally, gephyrin may bind to neuroligin-1 (a paralog of NL-2 that is important for excitatory synaptic function) when it is

dephosphorylated at a unique tyrosine residue (Tyr782), and this interaction may be modulated by the binding of neurexin-1β.101 Thus, gephyrin is subject to ligand-induced _trans_-synaptic

adhesion signaling, which controls the balance between excitation and inhibition. It is possible that gephyrin uses alternative pathways to mediate the development of inhibitory synapses.

For example, Slitrk3, IgSF9b and Calsyntenin-3 were recently reported to be specifically localized at inhibitory synapses.102, 103, 104, 105 Future work is warranted to determine whether

gephyrin interacts with these adhesion molecules to govern inhibitory synapse formation and function; exploration of this possibility will help address the diverse roles of gephyrin in

organizing the development of inhibitory synapses. Super-resolution imaging has revealed the existence of distinct subsynaptic domains, where NL-2 and gephyrin are coupled to constitute one

domain, whereas IgSF9 is indirectly linked to NL-2 via another domain.105 It would be informative to use similar approaches to examine systematically the localization of other ‘endogenous’

inhibitory synapse proteins, such as collybistin, Slitrk3 and MDGA1106. Gephyrin may also be linked to certain forms of synaptic plasticity at inhibitory synapses, such as those that involve

rearrangement of the postsynaptic molecular components of GABAergic synapses (e.g., GABAA receptors, gephyrin and other structural proteins).6 Indeed, CaMKII-dependent phosphorylation of

the GABAA receptor β3-subunit promotes the recruitment of gephyrin from extrasynaptic sites during postsynaptic LTP of inhibition.6 Impairment of gephyrin assembly blocks chemically induced

postsynaptic LTP of inhibition and the accumulation/confinement of GABAA receptors at inhibitory synapses. These results strongly indicate that the immobilization (or clustering) of gephyrin

at synapses and the subsequent immobilization of receptors are crucial for postsynaptic LTP of inhibition. The recent development of a new tool, called ‘fibronectin intrabodies generated

with mRNA display (FingRs)’ should allow us to better visualize the dynamic localization of inhibitory synaptic molecules in living neurons, allowing them to be implicated in various forms

of inhibitory synaptic plasticity.107 DISEASE IMPLICATIONS OF GEPHYRIN Some neurological disorders have been linked to gephyrin dysfunctions (e.g., stiff-person syndrome (Moersch–Woltman),

hyperekplexia, temporal lobe epilepsy, molybdenum cofactor deficiency, autism and schizophrenia).108, 109, 110, 111, 112, 113 These diseases may be partly attributed to defects in the

glycinergic system, which can be easily understood given the role of gephyrin in GlyR clustering. Gephyrin levels are reduced in the brains of patients suffering from Alzheimer’s disease,

but its direct involvement in this disorder has not yet been fully explored.114, 115 Intriguingly, temporal lobe epilepsy has been linked to abnormal operation of the gephyrin splicing

machinery.113 More specifically, cellular stresses (e.g., alkalosis) increase the proportions of gephyrin splice variants lacking exons encoding the G domain, potentially altering the

activity of gephyrin clustering.116 These results suggest that targeting the alternative splicing of gephyrin could provide a possible therapeutic strategy against the above-listed diseases.

Modulation of gephyrin clustering could also be a therapeutic target, as restoration of the excitatory–inhibitory balance could ameliorate a subset of neuropsychiatric disorders associated

with gephyrin dysfunctions. Chronic treatments with lithium and valproic acid have been widely used to treat epilepsy and bipolar disorder.117 Intriguingly, gephyrin clustering is enhanced

by lithium treatment, which acts on GSK3β.44 This emphasizes the potential benefits of targeting gephyrin when seeking to design effective new drugs for mood disorders, including bipolar

disorder. CONCLUSIONS It is increasingly clear that gephyrin is crucial for the structure, function and plasticity of inhibitory synapses, and that it may be highly relevant to neural

circuits that undergo various forms of inhibition _in vivo_. However, we still do not fully understand the precise roles of gephyrin at GABAergic and glycinergic synapses. Although a dozen

gephyrin-binding proteins (see Figure 2 and Table 1) have been identified to date, most of them have been discovered by yeast two-hybrid screens, which are not exhaustive in isolating

physiologically relevant binding partners. In the future, advanced proteomics should be used to identify additional binding proteins and gain additional insights into the synaptic roles of

gephyrin. We need a more detailed mechanistic understanding of how inhibitory receptors are dynamically modulated with respect to postsynaptic specializations, and the physiological

significance of the posttranslational modification of gephyrin should be broadly examined in conjunction with the known roles of other inhibitory synaptic molecules. Obviously, these future

studies will bring us ever closer to understanding the pathological mechanisms underlying various brain disorders caused by dysfunctions of inhibitory synaptic molecules. REFERENCES *

Missler M, Sudhof TC, Biederer T . Synaptic cell adhesion. _Cold Spring Harb Pespect Biol_ 2012; 4: a005694. Google Scholar  * Ko J . The leucine-rich repeat superfamily of synaptic adhesion

molecules: LRRTMs and Slitrks. _Mol Cells_ 2012; 34: 335–340. Article  CAS  PubMed  PubMed Central  Google Scholar  * Um JW, Ko J . LAR-RPTPs: synaptic adhesion molecules that shape synapse

development. _Trends Cell Biol_ 2013; 23: 465–475. Article  CAS  PubMed  Google Scholar  * Tretter V, Mukherjee J, Maric HM, Schindelin H, Sieghart W, Moss SJ . Gephyrin, the enigmatic

organizer at GABAergic synapses. _Front Cell Neurosci_ 2012; 6: 23. Article  CAS  PubMed  PubMed Central  Google Scholar  * Tyagarajan SK, Fritschy JM . Gephyrin: a master regulator of

neuronal function? _Nat Rev Neurosci_ 2014; 15: 141–156. Article  CAS  PubMed  Google Scholar  * Petrini EM, Ravasenga T, Hausrat TJ, Iurilli G, Olcese U, Racine V _et al_. Synaptic

recruitment of gephyrin regulates surface GABAA receptor dynamics for the expression of inhibitory LTP. _Nat Commun_ 2014; 5: 3921. Article  CAS  PubMed  Google Scholar  * Pfeiffer F, Graham

D, Betz H . Purification by affinity chromatography of the glycine receptor of rat spinal cord. _J Biol Chem_ 1982; 257: 9389–9393. CAS  PubMed  Google Scholar  * Fritschy JM, Harvey RJ,

Schwarz G . Gephyrin: where do we stand, where do we go? _Trends Neurosci_ 2008; 31: 257–264. Article  CAS  PubMed  Google Scholar  * Tyagarajan SK, Fritschy JM . GABA(A) receptors, gephyrin

and homeostatic synaptic plasticity. _J Physiol_ 2010; 588: 101–106. Article  CAS  PubMed  Google Scholar  * Prior P, Schmitt B, Grenningloh G, Pribilla I, Multhaup G, Beyreuther K _et al_.

Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein. _Neuron_ 1992; 8: 1161–1170. Article  CAS  PubMed  Google Scholar  *

Kamdar KP, Shelton ME, Finnerty V . The _Drosophila_ molybdenum cofactor gene cinnamon is homologous to three _Escherichia coli_ cofactor proteins and to the rat protein gephyrin. _Genetics_

1994; 137: 791–801. CAS  PubMed  PubMed Central  Google Scholar  * Sola M, Kneussel M, Heck IS, Betz H, Weissenhorn W . X-ray crystal structure of the trimeric N-terminal domain of

gephyrin. _J Biol Chem_ 2001; 276: 25294–25301. Article  CAS  PubMed  Google Scholar  * Bedet C, Bruusgaard JC, Vergo S, Groth-Pedersen L, Eimer S, Triller A _et al_. Regulation of gephyrin

assembly and glycine receptor synaptic stability. _J Biol Chem_ 2006; 281: 30046–30056. Article  CAS  PubMed  Google Scholar  * Kim EY, Schrader N, Smolinsky B, Bedet C, Vannier C, Schwarz G

_et al_. Deciphering the structural framework of glycine receptor anchoring by gephyrin. _EMBO J_ 2006; 25: 1385–1395. Article  CAS  PubMed  PubMed Central  Google Scholar  * Specht CG,

Grunewald N, Pascual O, Rostgaard N, Schwarz G, Triller A . Regulation of glycine receptor diffusion properties and gephyrin interactions by protein kinase C. _EMBO J_ 2011; 30: 3842–3853.

Article  CAS  PubMed  PubMed Central  Google Scholar  * Lardi-Studler B, Smolinsky B, Petitjean CM, Koenig F, Sidler C, Meier JC _et al_. Vertebrate-specific sequences in the gephyrin

E-domain regulate cytosolic aggregation and postsynaptic clustering. _J Cell Sci_ 2007; 120: 1371–1382. Article  CAS  PubMed  Google Scholar  * Kneussel M, Betz H . Clustering of inhibitory

neurotransmitter receptors at developing postsynaptic sites: the membrane activation model. _Trends Neurosci_ 2000; 23: 429–435. Article  CAS  PubMed  Google Scholar  * Sola M, Bavro VN,

Timmins J, Franz T, Ricard-Blum S, Schoehn G _et al_. Structural basis of dynamic glycine receptor clustering by gephyrin. _EMBO J_ 2004; 23: 2510–2519. Article  CAS  PubMed  PubMed Central

  Google Scholar  * Smolinsky B, Eichler SA, Buchmeier S, Meier JC, Schwarz G . Splice-specific functions of gephyrin in molybdenum cofactor biosynthesis. _J Biol Chem_ 2008; 283:

17370–17379. Article  CAS  PubMed  Google Scholar  * Paarmann I, Schmitt B, Meyer B, Karas M, Betz H . Mass spectrometric analysis of glycine receptor-associated gephyrin splice variants. _J

Biol Chem_ 2006; 281: 34918–34925. Article  CAS  PubMed  Google Scholar  * Ule J, Jensen KB, Ruggiu M, Mele A, Ule A, Darnell RB . CLIP identifies Nova-regulated RNA networks in the brain.

_Science_ 2003; 302: 1212–1215. Article  CAS  PubMed  Google Scholar  * Licatalosi DD, Mele A, Fak JJ, Ule J, Kayikci M, Chi SW _et al_. HITS-CLIP yields genome-wide insights into brain

alternative RNA processing. _Nature_ 2008; 456: 464–469. Article  CAS  PubMed  PubMed Central  Google Scholar  * Nawrotzki R, Islinger M, Vogel I, Volkl A, Kirsch J . Expression and

subcellular distribution of gephyrin in non-neuronal tissues and cells. _Histochem Cell Biol_ 2012; 137: 471–482. Article  CAS  PubMed  Google Scholar  * Meier J, De Chaldee M, Triller A,

Vannier C . Functional heterogeneity of gephyrins. _Mol Cell Neurosci_ 2000; 16: 566–577. Article  CAS  PubMed  Google Scholar  * Herweg J, Schwarz G . Splice-specific glycine receptor

binding, folding, and phosphorylation of the scaffolding protein gephyrin. _J Biol Chem_ 2012; 287: 12645–12656. Article  CAS  PubMed  PubMed Central  Google Scholar  * Ramming M, Kins S,

Werner N, Hermann A, Betz H, Kirsch J . Diversity and phylogeny of gephyrin: tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing

and cytoskeleton-associated proteins. _Proc Natl Acad Sci USA_ 2000; 97: 10266–10271. Article  CAS  PubMed  PubMed Central  Google Scholar  * Kirsch J, Malosio ML, Wolters I, Betz H .

Distribution of gephyrin transcripts in the adult and developing rat brain. _Eur J Neurosci_ 1993; 5: 1109–1117. Article  CAS  PubMed  Google Scholar  * Craig AM, Banker G, Chang W, McGrath

ME, Serpinskaya AS . Clustering of gephyrin at GABAergic but not glutamatergic synapses in cultured rat hippocampal neurons. _J Neurosci_ 1996; 16: 3166–3177. Article  CAS  PubMed  PubMed

Central  Google Scholar  * Giustetto M, Kirsch J, Fritschy JM, Cantino D, Sassoe-Pognetto M . Localization of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb

of the rat. _J Comp Neurol_ 1998; 395: 231–244. Article  CAS  PubMed  Google Scholar  * Sassoe-Pognetto M, Panzanelli P, Sieghart W, Fritschy JM . Colocalization of multiple GABA(A)

receptor subtypes with gephyrin at postsynaptic sites. _J Comp Neurol_ 2000; 420: 481–498. Article  CAS  PubMed  Google Scholar  * Zucker CL . Localization of gephyrin and glycine receptor

subunit immunoreactivity in the rabbit retina. _Vis Neurosci_ 1998; 15: 389–395. Article  CAS  PubMed  Google Scholar  * Waldvogel HJ, Baer K, Snell RG, During MJ, Faull RL, Rees MI .

Distribution of gephyrin in the human brain: an immunohistochemical analysis. _Neuroscience_ 2003; 116: 145–156. Article  CAS  PubMed  Google Scholar  * Studler B, Sidler C, Fritschy JM .

Differential regulation of GABA(A) receptor and gephyrin postsynaptic clustering in immature hippocampal neuronal cultures. _J Comp Neurol_ 2005; 484: 344–355. Article  CAS  PubMed  Google

Scholar  * Allison DW, Chervin AS, Gelfand VI, Craig AM . Postsynaptic scaffolds of excitatory and inhibitory synapses in hippocampal neurons: maintenance of core components independent of

actin filaments and microtubules. _J Neurosci_ 2000; 20: 4545–4554. Article  CAS  PubMed  PubMed Central  Google Scholar  * Specht CG, Izeddin I, Rodriguez PC, El Beheiry M, Rostaing P,

Darzacq X _et al_. Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites. _Neuron_ 2013; 79: 308–321. Article  CAS  PubMed  Google Scholar  *

Kirsch J, Kuhse J, Betz H . Targeting of glycine receptor subunits to gephyrin-rich domains in transfected human embryonic kidney cells. _Mol Cell Neurosci_ 1995; 6: 450–461. Article  CAS 

PubMed  Google Scholar  * Harvey K, Duguid IC, Alldred MJ, Beatty SE, Ward H, Keep NH _et al_. The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin

clustering. _J Neurosci_ 2004; 24: 5816–5826. Article  CAS  PubMed  PubMed Central  Google Scholar  * Reddy-Alla S, Schmitt B, Birkenfeld J, Eulenburg V, Dutertre S, Bohringer C _et al_.

PH-domain-driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering. _Eur J Neurosci_ 2010; 3: 1173–1184. Article  Google Scholar  * Tyagarajan

SK, Ghosh H, Harvey K, Fritschy JM . Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses. _J Cell Sci_ 2011;

124: 2786–2796. Article  CAS  PubMed  PubMed Central  Google Scholar  * Langosch D, Hoch W, Betz H . The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor

are phosphorylated by an endogenous protein kinase. _FEBS Lett_ 1992; 298: 113–117. Article  CAS  PubMed  Google Scholar  * Zacchi P, Antonelli R, Cherubini E . Gephyrin phosphorylation in

the functional organization and plasticity of GABAergic synapses. _Front Cell Neurosci_ 2014; 8: 103. Article  PubMed  PubMed Central  CAS  Google Scholar  * Kuhse J, Kalbouneh H,

Schlicksupp A, Mukusch S, Nawrotzki R, Kirsch J . Phosphorylation of gephyrin in hippocampal neurons by cyclin-dependent kinase CDK5 at Ser-270 is dependent on collybistin. _J Biol Chem_

2012; 287: 30952–30966. Article  CAS  PubMed  PubMed Central  Google Scholar  * Tyagarajan SK, Ghosh H, Yevenes GE, Imanishi SY, Zeilhofer HU, Gerrits B _et al_. Extracellular

signal-regulated kinase and glycogen synthase kinase 3beta regulate gephyrin postsynaptic aggregation and GABAergic synaptic function in a calpain-dependent mechanism. _J Biol Chem_ 2013;

288: 9634–9647. Article  CAS  PubMed  PubMed Central  Google Scholar  * Tyagarajan SK, Ghosh H, Yevenes GE, Nikonenko I, Ebeling C, Schwerdel C _et al_. Regulation of GABAergic synapse

formation and plasticity by GSK3beta-dependent phosphorylation of gephyrin. _Proc Natl Acad Sci USA_ 2011; 108: 379–384. Article  CAS  PubMed  Google Scholar  * Bausen M, Weltzien F, Betz H,

O'Sullivan GA . Regulation of postsynaptic gephyrin cluster size by protein phosphatase 1. _Mol Cell Neurosci_ 2010; 44: 201–209. Article  CAS  PubMed  Google Scholar  * Pribiag H,

Stellwagen D . TNF-alpha downregulates inhibitory neurotransmission through protein phosphatase 1-dependent trafficking of GABA(A) receptors. _J Neurosci_ 2013; 33: 15879–15893. Article  CAS

  PubMed  PubMed Central  Google Scholar  * Dejanovic B, Semtner M, Ebert S, Lamkemeyer T, Neuser F, Luscher B _et al_. Palmitoylation of gephyrin controls receptor clustering and plasticity

of GABAergic synapses. _PLoS Biol_ 2014; 12: e1001908. Article  PubMed  PubMed Central  CAS  Google Scholar  * Kang R, Wan J, Arstikaitis P, Takahashi H, Huang K, Bailey AO _et al_. Neural

palmitoyl-proteomics reveals dynamic synaptic palmitoylation. _Nature_ 2008; 456: 904–909. Article  CAS  PubMed  PubMed Central  Google Scholar  * Meyer G, Kirsch J, Betz H, Langosch D .

Identification of a gephyrin binding motif on the glycine receptor beta subunit. _Neuron_ 1995; 15: 563–572. Article  CAS  PubMed  Google Scholar  * Schrader N, Kim EY, Winking J, Paulukat

J, Schindelin H, Schwarz G . Biochemical characterization of the high affinity binding between the glycine receptor and gephyrin. _J Biol Chem_ 2004; 279: 18733–18741. Article  CAS  PubMed 

Google Scholar  * Takagi T, Pribilla I, Kirsch J, Betz H . Coexpression of the receptor-associated protein gephyrin changes the ligand binding affinities of alpha 2 glycine receptors. _FEBS

Lett_ 1992; 303: 178–180. Article  CAS  PubMed  Google Scholar  * Kirsch J, Wolters I, Triller A, Betz H . Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal

neurons. _Nature_ 1993; 366: 745–748. Article  CAS  PubMed  Google Scholar  * Kirsch J, Betz H . Glycine-receptor activation is required for receptor clustering in spinal neurons. _Nature_

1998; 392: 717–720. Article  CAS  PubMed  Google Scholar  * Levi S, Logan SM, Tovar KR, Craig AM . Gephyrin is critical for glycine receptor clustering but not for the formation of

functional GABAergic synapses in hippocampal neurons. _J Neurosci_ 2004; 24: 207–217. Article  CAS  PubMed  PubMed Central  Google Scholar  * Feng G, Tintrup H, Kirsch J, Nichol MC, Kuhse J,

Betz H _et al_. Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity. _Science_ 1998; 282: 1321–1324. Article  CAS  PubMed  Google Scholar  * Fischer F,

Kneussel M, Tintrup H, Haverkamp S, Rauen T, Betz H _et al_. Reduced synaptic clustering of GABA and glycine receptors in the retina of the gephyrin null mutant mouse. _J Comp Neurol_ 2000;

427: 634–648. Article  CAS  PubMed  Google Scholar  * Meier J, Vannier C, Serge A, Triller A, Choquet D . Fast and reversible trapping of surface glycine receptors by gephyrin. _Nat

Neurosci_ 2001; 4: 253–260. Article  CAS  PubMed  Google Scholar  * Calamai M, Specht CG, Heller J, Alcor D, Machado P, Vannier C _et al_. Gephyrin oligomerization controls GlyR mobility and

synaptic clustering. _J Neurosci_ 2009; 29: 7639–7648. Article  CAS  PubMed  PubMed Central  Google Scholar  * Kneussel M, Hermann A, Kirsch J, Betz H . Hydrophobic interactions mediate

binding of the glycine receptor beta-subunit to gephyrin. _J Neurochem_ 1999; 72: 1323–1326. Article  CAS  PubMed  Google Scholar  * Mukherjee J, Kretschmannova K, Gouzer G, Maric HM,

Ramsden S, Tretter V _et al_. The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor alpha1 subunit to gephyrin. _J Neurosci_ 2011; 31:

14677–14687. Article  CAS  PubMed  PubMed Central  Google Scholar  * Kneussel M, Brandstatter JH, Gasnier B, Feng G, Sanes JR, Betz H . Gephyrin-independent clustering of postsynaptic

GABA(A) receptor subtypes. _Mol Cell Neurosci_ 2001; 17: 973–982. Article  CAS  PubMed  Google Scholar  * Tretter V, Jacob TC, Mukherjee J, Fritschy JM, Pangalos MN, Moss SJ . The clustering

of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor alpha 2 subunits to gephyrin. _J Neurosci_ 2008; 28: 1356–1365. Article  CAS  PubMed 

PubMed Central  Google Scholar  * Tretter V, Kerschner B, Milenkovic I, Ramsden SL, Ramerstorfer J, Saiepour L _et al_. Molecular basis of the gamma-aminobutyric acid A receptor alpha3

subunit interaction with the clustering protein gephyrin. _J Biol Chem_ 2011; 286: 37702–37711. Article  CAS  PubMed  PubMed Central  Google Scholar  * Luscher B, Keller CA . Regulation of

GABAA receptor trafficking, channel activity, and functional plasticity of inhibitory synapses. _Pharmacol Ther_ 2004; 102: 195–221. Article  CAS  PubMed  Google Scholar  * Alldred MJ,

Mulder-Rosi J, Lingenfelter SE, Chen G, Luscher B . Distinct gamma2 subunit domains mediate clustering and synaptic function of postsynaptic GABAA receptors and gephyrin. _J Neurosci_ 2005;

25: 594–603. Article  CAS  PubMed  PubMed Central  Google Scholar  * Fuhrmann JC, Kins S, Rostaing P, El Far O, Kirsch J, Sheng M _et al_. Gephyrin interacts with dynein light chains 1 and

2, components of motor protein complexes. _J Neurosci_ 2002; 22: 5393–5402. Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang H, Bedford FK, Brandon NJ, Moss SJ, Olsen RW .

GABA(A)-receptor-associated protein links GABA(A) receptors and the cytoskeleton. _Nature_ 1999; 397: 69–72. Article  CAS  PubMed  Google Scholar  * Kneussel M, Haverkamp S, Fuhrmann JC,

Wang H, Wassle H, Olsen RW _et al_. The gamma-aminobutyric acid type A receptor (GABAAR)-associated protein GABARAP interacts with gephyrin but is not involved in receptor anchoring at the

synapse. _Proc Natl Acad Sci USA_ 2000; 97: 8594–8599. Article  CAS  PubMed  PubMed Central  Google Scholar  * Kittler JT, Rostaing P, Schiavo G, Fritschy JM, Olsen R, Triller A _et al_. The

subcellular distribution of GABARAP and its ability to interact with NSF suggest a role for this protein in the intracellular transport of GABA(A) receptors. _Mol Cell Neurosci_ 2001; 18:

13–25. Article  CAS  PubMed  Google Scholar  * O'Sullivan GA, Kneussel M, Elazar Z, Betz H . GABARAP is not essential for GABA receptor targeting to the synapse. _Eur J Neurosci_ 2005;

22: 2644–2648. Article  PubMed  Google Scholar  * Mohrluder J, Schwarten M, Willbold D . Structure and potential function of gamma-aminobutyrate type A receptor-associated protein. _FEBS J_

2009; 276: 4989–5005. Article  PubMed  CAS  Google Scholar  * Kins S, Betz H, Kirsch J . Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin. _Nat

Neurosci_ 2000; 3: 22–29. Article  CAS  PubMed  Google Scholar  * Grosskreutz Y, Hermann A, Kins S, Fuhrmann JC, Betz H, Kneussel M . Identification of a gephyrin-binding motif in the

GDP/GTP exchange factor collybistin. _Biol Chem_ 2001; 382: 1455–1462. Article  CAS  PubMed  Google Scholar  * Kneussel M, Engelkamp D, Betz H . Distribution of transcripts for the

brain-specific GDP/GTP exchange factor collybistin in the developing mouse brain. _Eur J Neurosci_ 2001; 13: 487–492. Article  CAS  PubMed  Google Scholar  * Korber C, Richter A, Kaiser M,

Schlicksupp A, Mukusch S, Kuner T _et al_. Effects of distinct collybistin isoforms on the formation of GABAergic synapses in hippocampal neurons. _Mol Cell Neurosci_ 2012; 50: 250–259.

Article  PubMed  CAS  Google Scholar  * Poulopoulos A, Aramuni G, Meyer G, Soykan T, Hoon M, Papadopoulos T _et al_. Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory

synapses through gephyrin and collybistin. _Neuron_ 2009; 63: 628–642. Article  CAS  PubMed  Google Scholar  * Mayer S, Kumar R, Jaiswal M, Soykan T, Ahmadian MR, Brose N _et al_.

Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission. _Proc Natl Acad Sci USA_ 2013; 110: 20795–20800. Article  CAS 

PubMed  PubMed Central  Google Scholar  * Soykan T, Schneeberger D, Tria G, Buechner C, Bader N, Svergun D _et al_. A conformational switch in collybistin determines the differentiation of

inhibitory postsynapses. _EMBO J_ 2014; 33: 2113–2133. Article  CAS  PubMed  PubMed Central  Google Scholar  * Papadopoulos T, Korte M, Eulenburg V, Kubota H, Retiounskaia M, Harvey RJ _et

al_. Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice. _EMBO J_ 2007; 26: 3888–3899. Article  CAS  PubMed  PubMed Central  Google

Scholar  * Papadopoulos T, Eulenburg V, Reddy-Alla S, Mansuy IM, Li Y, Betz H . Collybistin is required for both the formation and maintenance of GABAergic postsynapses in the hippocampus.

_Mol Cell Neurosci_ 2008; 39: 161–169. Article  CAS  PubMed  Google Scholar  * Jedlicka P, Papadopoulos T, Deller T, Betz H, Schwarzacher SW . Increased network excitability and impaired

induction of long-term potentiation in the dentate gyrus of collybistin-deficient mice _in vivo_. _Mol Cell Neurosci_ 2009; 41: 94–100. Article  CAS  PubMed  Google Scholar  * Saiepour L,

Fuchs C, Patrizi A, Sassoe-Pognetto M, Harvey RJ, Harvey K . Complex role of collybistin and gephyrin in GABAA receptor clustering. _J Biol Chem_ 2010; 285: 29623–29631. Article  CAS  PubMed

  PubMed Central  Google Scholar  * Chiou TT, Bonhomme B, Jin H, Miralles CP, Xiao H, Fu Z _et al_. Differential regulation of the postsynaptic clustering of gamma-aminobutyric acid type A

(GABAA) receptors by collybistin isoforms. _J Biol Chem_ 2011; 286: 22456–22468. Article  CAS  PubMed  PubMed Central  Google Scholar  * Papadopoulos T, Soykan T . The role of collybistin in

gephyrin clustering at inhibitory synapses: facts and open questions. _Front Cell Neurosci_ 2011; 5: 11. Article  CAS  PubMed  PubMed Central  Google Scholar  * Giesemann T, Schwarz G,

Nawrotzki R, Berhorster K, Rothkegel M, Schluter K _et al_. Complex formation between the postsynaptic scaffolding protein gephyrin, profilin, and Mena: a possible link to the microfilament

system. _J Neurosci_ 2003; 23: 8330–8339. Article  CAS  PubMed  PubMed Central  Google Scholar  * Mammoto A, Sasaki T, Asakura T, Hotta I, Imamura H, Takahashi K _et al_. Interactions of

drebrin and gephyrin with profilin. _Biochem Biophys Res Commun_ 1998; 243: 86–89. Article  CAS  PubMed  Google Scholar  * Bausen M, Fuhrmann JC, Betz H, O'Sullivan GA . The state of

the actin cytoskeleton determines its association with gephyrin: role of ena/VASP family members. _Mol Cell Neurosci_ 2006; 31: 376–386. Article  CAS  PubMed  Google Scholar  * Lu KP, Hanes

SD, Hunter T . A human peptidyl-prolyl isomerase essential for regulation of mitosis. _Nature_ 1996; 380: 544–547. Article  CAS  PubMed  Google Scholar  * Shen M, Stukenberg PT, Kirschner

MW, Lu KP . The essential mitotic peptidyl-prolyl isomerase Pin1 binds and regulates mitosis-specific phosphoproteins. _Genes Dev_ 1998; 12: 706–720. Article  CAS  PubMed  PubMed Central 

Google Scholar  * Wulf G, Finn G, Suizu F, Lu KP . Phosphorylation-specific prolyl isomerization: is there an underlying theme? _Nat Cell Biol_ 2005; 7: 435–441. Article  CAS  PubMed  Google

Scholar  * Zita MM, Marchionni I, Bottos E, Righi M, Del Sal G, Cherubini E _et al_. Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine

receptors function. _EMBO J_ 2007; 26: 1761–1771. Article  PubMed  CAS  Google Scholar  * Antonelli R, Pizzarelli R, Pedroni A, Fritschy JM, Del Sal G, Cherubini E _et al_. Pin1-dependent

signalling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction. _Nat Commun_ 2014; 5: 5066. Article  CAS  PubMed  Google Scholar  * Sabatini DM, Barrow

RK, Blackshaw S, Burnett PE, Lai MM, Field ME _et al_. Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling. _Science_ 1999; 284: 1161–1164. Article  CAS  PubMed 

Google Scholar  * Varoqueaux F, Jamain S, Brose N . Neuroligin 2 is exclusively localized to inhibitory synapses. _Eur J Cell Biol_ 2004; 83: 449–456. Article  CAS  PubMed  Google Scholar  *

Chih B, Engelman H, Scheiffele P . Control of excitatory and inhibitory synapse formation by neuroligins. _Science_ 2005; 307: 1324–1328. Article  CAS  PubMed  Google Scholar  * Graf ER,

Zhang X, Jin SX, Linhoff MW, Craig AM . Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. _Cell_ 2004; 119: 1013–1026. Article  CAS  PubMed

  PubMed Central  Google Scholar  * Chubykin AA, Atasoy D, Etherton MR, Brose N, Kavalali ET, Gibson JR _et al_. Activity-dependent validation of excitatory versus inhibitory synapses by

neuroligin-1 versus neuroligin-2. _Neuron_ 2007; 54: 919–931. Article  CAS  PubMed  PubMed Central  Google Scholar  * Sudhof TC . Neuroligins and neurexins link synaptic function to

cognitive disease. _Nature_ 2008; 455: 903–911. Article  PubMed  PubMed Central  CAS  Google Scholar  * Patrizi A, Scelfo B, Viltono L, Briatore F, Fukaya M, Watanabe M _et al_. Synapse

formation and clustering of neuroligin-2 in the absence of GABAA receptors. _Proc Natl Acad Sci USA_ 2008; 105: 13151–13156. Article  CAS  PubMed  PubMed Central  Google Scholar  * Kang Y,

Ge Y, Cassidy RM, Lam V, Luo L, Moon KM _et al_. A combined transgenic proteomic analysis and regulated trafficking of neuroligin-2. _J Biol Chem_ 2014; 289: 29350–29364. Article  CAS 

PubMed  PubMed Central  Google Scholar  * Giannone G, Mondin M, Grillo-Bosch D, Tessier B, Saint-Michel E, Czondor K _et al_. Neurexin-1beta binding to neuroligin-1 triggers the preferential

recruitment of PSD-95 versus gephyrin through tyrosine phosphorylation of neuroligin-1. _Cell Rep_ 2013; 3: 1996–2007. Article  CAS  PubMed  Google Scholar  * Yim YS, Kwon Y, Nam J, Yoon

HI, Lee K, Kim DG _et al_. Slitrks control excitatory and inhibitory synapse formation with LAR receptor protein tyrosine phosphatases. _Proc Natl Acad Sci USA_ 2013; 110: 4057–4062. Article

  CAS  PubMed  PubMed Central  Google Scholar  * Takahashi H, Katayama K, Sohya K, Miyamoto H, Prasad T, Matsumoto Y _et al_. Selective control of inhibitory synapse development by

Slitrk3-PTPdelta trans-synaptic interaction. _Nat Neurosci_ 2012; 15: 389–398. Article  CAS  PubMed  PubMed Central  Google Scholar  * Um JW, Pramanik G, Ko JS, Song MY, Lee D, Kim H _et

al_. Calsyntenins function as synaptogenic adhesion molecules in concert with neurexins. _Cell Rep_ 2014; 6: 1096–1109. Article  CAS  PubMed  PubMed Central  Google Scholar  * Woo J, Kwon

SK, Nam J, Choi S, Takahashi H, Krueger D _et al_. The adhesion protein IgSF9b is coupled to neuroligin 2 via S-SCAM to promote inhibitory synapse development. _J Cell Biol_ 2013; 201:

929–944. Article  CAS  PubMed  PubMed Central  Google Scholar  * Lee K, Kim Y, Lee SJ, Qiang Y, Lee D, Lee HW _et al_. MDGAs interact selectively with neuroligin-2 but not other neuroligins

to regulate inhibitory synapse development. _Proc Natl Acad Sci USA_ 2013; 110: 336–341. Article  CAS  PubMed  Google Scholar  * Gross GG, Junge JA, Mora RJ, Kwon HB, Olson CA, Takahashi TT

_et al_. Recombinant probes for visualizing endogenous synaptic proteins in living neurons. _Neuron_ 2013; 78: 971–985. Article  CAS  PubMed  PubMed Central  Google Scholar  * Butler MH,

Hayashi A, Ohkoshi N, Villmann C, Becker CM, Feng G _et al_. Autoimmunity to gephyrin in Stiff-Man syndrome. _Neuron_ 2000; 26: 307–312. Article  CAS  PubMed  Google Scholar  * Reiss J,

Johnson JL . Mutations in the molybdenum cofactor biosynthetic genes MOCS1, MOCS2, and GEPH. _Hum Mutat_ 2003; 21: 569–576. Article  CAS  PubMed  Google Scholar  * Reiss J, Lenz U,

Aquaviva-Bourdain C, Joriot-Chekaf S, Mention-Mulliez K, Holder-Espinasse M . A GPHN point mutation leading to molybdenum cofactor deficiency. _Clin Genet_ 2011; 80: 598–599. Article  CAS 

PubMed  Google Scholar  * Rees MI, Harvey K, Ward H, White JH, Evans L, Duguid IC _et al_. Isoform heterogeneity of the human gephyrin gene (GPHN), binding domains to the glycine receptor,

and mutation analysis in hyperekplexia. _J Biol Chem_ 2003; 278: 24688–24696. Article  CAS  PubMed  Google Scholar  * Fang M, Shen L, Yin H, Pan YM, Wang L, Chen D _et al_. Downregulation of

gephyrin in temporal lobe epilepsy neurons in humans and a rat model. _Synapse_ 2011; 65: 1006–1014. Article  CAS  PubMed  Google Scholar  * Lionel AC, Vaags AK, Sato D, Gazzellone MJ,

Mitchell EB, Chen HY _et al_. Rare exonic deletions implicate the synaptic organizer Gephyrin (GPHN) in risk for autism, schizophrenia and seizures. _Hum Mol Genet_ 2013; 22: 2055–2066.

Article  CAS  PubMed  Google Scholar  * Agarwal S, Tannenberg RK, Dodd PR . Reduced expression of the inhibitory synapse scaffolding protein gephyrin in Alzheimer's disease. _J

Alzheimers Dis_ 2008; 14: 313–321. Article  CAS  PubMed  Google Scholar  * Hales CM, Rees H, Seyfried NT, Dammer EB, Duong DM, Gearing M _et al_. Abnormal gephyrin immunoreactivity

associated with Alzheimer disease pathologic changes. _J Neuropathol Exp Neurol_ 2013; 72: 1009–1015. Article  CAS  PubMed  Google Scholar  * Forstera B, Belaidi AA, Juttner R, Bernert C,

Tsokos M, Lehmann TN _et al_. Irregular RNA splicing curtails postsynaptic gephyrin in the cornu ammonis of patients with epilepsy. _Brain_ 2010; 133: 3778–3794. Article  PubMed  Google

Scholar  * Wang CC, Chen PS, Hsu CW, Wu SJ, Lin CT, Gean PW . Valproic acid mediates the synaptic excitatory/inhibitory balance through astrocytes—a preliminary study. _Prog

Neuropsychopharmacol Biol Psychiatry_ 2012; 37: 111–120. Article  PubMed  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the Yonsei University

Future-leading Research Initiative of 2014 (to JK), and in part by Brain Korea 21 (BK21) PLUS program. GC is a fellowship awardee by BK21 PLUS program. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea Gayoung Choii & Jaewon Ko * Department of Psychiatry, Yonsei

University College of Medicine, Seoul, Korea Jaewon Ko Authors * Gayoung Choii View author publications You can also search for this author inPubMed Google Scholar * Jaewon Ko View author

publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Jaewon Ko. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Choii, G., Ko, J. Gephyrin: a central GABAergic synapse organizer. _Exp Mol Med_ 47, e158 (2015). https://doi.org/10.1038/emm.2015.5 Download

citation * Received: 09 November 2014 * Accepted: 18 December 2014 * Published: 17 April 2015 * Issue Date: April 2015 * DOI: https://doi.org/10.1038/emm.2015.5 SHARE THIS ARTICLE Anyone

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