Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Gephyrin

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101672

Synonyms

Synapse and Gephyrin

Large size and polarity of neuronal cell make spatiotemporal segregation of biochemical reactions and signal transduction pathways very important. In addition to the membrane-enclosed organelles, neurons also contain specialized apparatus called synapses for effective communication within the system. The presynaptic terminals containing the relevant neurotransmitter vesicles are opposed by the concomitant receptors for effective signal transduction. Information receiving, interpretation, and storage are facilitated by a resident sub-membranous protein-rich compartment. Such protein-rich compartments are referred to as postsynaptic densities (PSD), which can be a few hundred nanometers in width and ~30 to 50 nm in thickness (Chen et al. 2008).

Since the identification of inhibitory glycine receptor (GlyR) from rat spinal cord, gephyrin has assumed a central role in our current understanding of inhibitory postsynaptic organization and function. As a 93 kDa protein, gephyrin binds to the cytoplasmic domain of GlyR, and its interaction with polymerized tubulin suggested a role for this protein in GlyR anchoring at synaptic sites (Tyagarajan and Fritschy 2014). Immunocytochemistry of developing spinal cord neuron in culture showed co-emergence of GlyR and gephyrin puncta over the developmental period of 3–10 days in vitro (Bechade et al. 1996). However, in the brain, most of the fast inhibitory neurotransmission is facilitated by GABAA receptors (GABAARs). Synaptic GABAARs are composed of pentameric subunits consisting of the γ2 subunit. In the absence of γ2 subunit, GABAARs fail to be transported to synaptic sites, and this is marked by a parallel loss of gephyrin localization at GABAergic postsynaptic site (Tyagarajan and Fritschy 2014). This underscores the interdependence of gephyrin with its concomitant receptor for synaptic recruitment.

Molybdenum Cofactor (MoCo) Synthesis and Gephyrin

Gephyrin deletion leads to MoCo deficiency in mice (Feng et al. 1998) and humans (Reiss et al. 2001). Even though in the past decades research has focused on synapse-specific function of gephyrin, as a multifunctional protein, gephyrin has an evolutionary conserved role in catalyzing MoCo biosynthesis. Functional loss of MoCo biosynthesis can lead to death of the organism due to deficiency in essential metabolic functions. In E.coli the five MoCo-specific operons encode 15 different genes. The MogA and MoeA are separate E.coli proteins that play an essential role during the crucial molybdenum (Mo) insertion (Stallmeyer et al. 1999). The plant counterparts of these two proteins are Cnx1 and CnxE, respectively. The vertebrate Gphn encodes for the MogA-homologous G-domain and MoeA-homologous E-domain while also participating in Mo insertion. Although MogA and MoeA are separate proteins encoded by the E.coli mog locus, protein interaction between them facilitates their functional cooperativity during Mo transfer. In plants, CnxE and Cnx1 are fused into single gene, albeit in reverse order, facilitating Mo transfer via a single protein (see Fig. 1). In the case of Gphn, the N-terminal G-domain and C-terminus E-domain are joined by a long unstructured linker C-domain. Reconstitution studies have shown that mammalian gephyrin can effectively catalyze bacterial and plant MoCo biosynthesis (Stallmeyer et al. 1999). The central role of gephyrin in MoCo synthesis makes it indispensable for an organism’s viability. Hence, loss-of-function gephyrin mutations or structural abnormalities due to post-transcriptional mechanism often prove detrimental to the organism.
Gephyrin, Fig. 1

Gephyrin domains for molybdenum cofactor (MoCo) synthesis and scaffold organization at synapses: (a) Distinct MogA, MoaB and MoeA proteins in E. Coli participate in MoCo synthesis. In plants CnxE and Cnx1 are fused, albeit in reverse order. In Drosophila, rodents and human gephyrin encodes G domain and E domain connected by a central linker. (b) Gephyrin G-domain trimers (yellow) and E-domain dimers allow scaffold organization. Specific PTM on gephyrin regulate its oligomerization properties and scaffold dynamics

Gephyrin Alternative Splicing and Structure

Transcriptional regulation of gene expression allows for modulating mRNA and protein abundance within cells. As a main organizer of inhibitory postsynapse, it was initially assumed that gephyrin promoter activity would regulate synaptic accumulation of gephyrin. However, gephyrin promoter is constitutively active similar to any housekeeping gene (Ramming et al. 1997). A constitutively active gephyrin promoter opened the door for the analysis of other post-transcriptional regulatory mechanism(s) to understand its accumulation at inhibitory synaptic sites. Alternative splicing represents a powerful mechanism for expanding the capacity of genomes to generate molecular diversity. Multiple alterative splice isoforms of gephyrin are expressed in different tissue-specific cells. Alternative splice cassettes are present in all three gephyrin domains (Fritschy et al. 2008). However, given that these cassettes are usually around 35 amino acids, it has been difficult to raise isoform-specific antibodies to fully evaluate gephyrin diversity at inhibitory postsynaptic sites.

Neuronal cells exhibit particularly extensive alternative splicing regulation. Independent studies have shed some light into the expression of specific splice cassettes for gephyrin expression and function. The E-domain C7 cassette contains a stop codon and is expressed widely, leading to the expression of truncated gephyrin that is deficient in dimerization, MoCo synthesis, and GlyR synaptic targeting (Fritschy et al. 2008). On the other hand, splice cassettes G2 is selected for expression in non-neuronal cells, perhaps as a mechanism to segregate gephyrin synapse function in neurons from MoCo synthesis function in astrocytes. The identification of C4 cassette in the central linker contains multiple insertions leading to additional splice isoforms of gephyrin (Fritschy et al. 2008).

Alternative splicing can also be spatially and temporally controlled in the brain. Highly selective expression of the KH domain-containing splicing regulators in neuronal subtypes has provided a mechanism for shaping the molecular repertoires of synaptic molecules in neuronal populations in vivo (Iijima et al. 2014). Activity-dependent alternative splicing of synaptic proteins via the activation of splicing regulators also contribute toward synaptic protein diversity (Iijima et al. 2011). Nova has been identified as a neuron-specific splicing factor influencing gephyrin alternative splicing levels, and it represses the expression of C3 cassette in neurons. This suggests a role for C3 cassette expression in non-neuronal tissues (Ule et al. 2003). The presence of C3 cassette reduces gephyrin interaction with GlyR β subunit by an order of magnitude (Fritschy et al. 2008). Hence, regulation of gephyrin alternative splicing could expand the molecular capacity of an essential protein for functional diversity both within and outside synapses.

Gephyrin alternative splicing plays an important role in determining inter- and intramolecular interactions, which in turn contribute toward its synaptic scaffolding property and MoCo synthesis function. The importance of gephyrin trimerization for its scaffolding function came to the fore after the X-ray crystal structure for the N-terminus (2–188 aa) MogA was solved (Sola et al. 2004). Structural similarities seen with X-ray crystal structures of human gephyrin G-domain and plant Cnx1 provided the molecular basis for functional redundancies observed between these well-conserved molecules (Schwarz et al. 2001). Finally, determination of X-ray crystal structure showing gephyrin E-domain monomer interaction with GlyR beta-loop in a “lock and key” manner provided the molecular and structural bases for gephyrin scaffolding at inhibitory synapses (Kim et al. 2006).

Gephyrin Regulation by PTM

It has become increasingly clear that the exquisite anatomical and functional specialization of GABAergic synapses contribute to the integrity of discrete circuits controlling specific brain functions and behavioral states. Molecular diversity at the postsynapse is starting to shed new light into the mechanistic basis for circuit-specific functionality at GABAergic synapses. Given the short time scales within which synapses need to adjust their strength to the incoming signals, cellular pathways have coevolved to facilitate dynamic synaptic adaptations via post-translational modification(s).

Early stages of synapse formation are facilitated by transsynaptic adhesion molecules such as neuroligin (NLGN). Specificity for interaction between NLGN and their presynaptic partner neurexin (NRXN) allows synapse specializations with neurotransmitter release at its matching receptor sites. It has been uncovered that differential association of NLGN isoforms with PSD95 at glutamatergic or gephyrin at inhibitory postsynapse depends on the tyrosine phosphorylation on NLGN at Y782 residue (Giannone et al. 2013). Intriguingly, phosphorylation status of gephyrin can also regulate within a time line of 3 h GABAergic synaptogenesis. One can recapitulate this scenario in vivo and in cultured neurons via either pharmacological blockade of GSK3β pathway or via transgenic gephyrin expression with S270A mutation (Tyagarajan and Fritschy 2014). Receptor tyrosine kinase (RTK) via downstream activation of Ras and Akt pathways can block GSK3β signaling; hence, one can easily envision how crosstalk between two distinct signal transduction pathways that converge on NLGN and gephyrin could facilitate mutual interaction for inhibitory synapse formation. Brain-derived neurotrophic factor (BDNF) signaling, possibly via its cognate receptor TrkB, has been shown to strengthen or weaken GABAergic transmission depending on the time scale of BDNF application (Brünig et al. 2001; Porcher et al. 2011). Hence, in addition to synaptogenesis, RTK signaling can also influence subtle aspects of inhibitory synaptic plasticity via currently unknown downstream signaling.

Although gephyrin has been long predicted to be phosphoprotein, it is only in the past decade that studies of gephyrin PTM have shed new light into the mechanistic basis underlying GABAergic and glycinergic synaptic plasticity. Pin1-dependent gephyrin phosphorylation was shown to play an important role for GlyR interaction and synaptic accumulation (Tyagarajan and Fritschy 2014). Subsequently, an independent study identified a role for protein kinase C in phosphorylating GlyR β subunit (S403), which causes reduced binding affinity for gephyrin, leading to decreased synaptic accumulation of GlyR. The phosphorylation-dependent interaction of GlyR with gephyrin is also linked to its higher membrane diffusion dynamics (Choquet and Triller 2013). Gephyrin phospho-peptide analysis using protein isolated from insect Sf9 cells, HEK293, or rodent brain homogenate identified several novel phosphorylation sites on the protein (Tyagarajan and Fritschy 2014). Of these various phosphorylation sites, only few have been functionally characterized so far. Ser270 regulation by GSK3β has been shown to modulate gephyrin cluster density and GABAergic mIPSC frequency. Similarly, Ser268 regulation by ERK1/ERK2 modulates gephyrin scaffold size via its susceptibility to cleavage by cysteine protease calpain (Tyagarajan and Fritschy 2014).

Rapid change in neuronal function and neural circuits controls our capacity to learn and remember; at the same time, stable maintenance of brain circuit output is integral to our daily experiences. It has become clear that homeostatic signaling mechanisms are inherently encoded within the central and peripheral nervous system to maintain functional integrity of information encoding. In spite of recent progress in our understanding of gephyrin regulation, molecular basis for activity-dependent adaptations at GABAergic synapses has been unclear. Enhancing glutamatergic transmission via NMDA receptors (NMDARs) induces rapid adaptations at GABAergic terminals, which can be observed by dynamic increase in gephyrin cluster size and density during the 24 h period. This change returns to baseline at 48 h post-stimulation. Excitation-dependent changes in GABAergic inhibition can be blocked using a single CaMKIIα-insensitive gephyrin mutation (S305A) (Flores et al. 2015). In a complementary study using a chemical paradigm for long-term potentiation of inhibition (iLTP), it was shown that gephyrin scaffold is recruited from extrasynaptic areas via CaMKIIα-dependent phosphorylation of GABAAR-β3-Ser(383) (Petrini et al. 2014). These results demonstrate that signaling pathways that converge onto gephyrin scaffold can also act on GABAAR subunits, perhaps influencing gephyrin conformation and GABAAR interaction. Evidence for dynamic gephyrin scaffolding has shed fresh light into plasticity adaptations at GABAergic postsynapse replacing the static self-aggregation model for gephyrin clustering in neurons (Tyagarajan and Fritschy 2014; Villa et al. 2016).

Early attempts to explain gephyrin recruitment at synaptic locations postulated a role for receptor-mediated membrane depolarization leading to calcium-facilitated aggregation of gephyrin scaffolds at synaptic locations (Kneussel and Betz 2000). Subsequent discovery of ArhGEF9 or collybistin (CB), which contains a pleckstrin homology (PH) domain for membrane anchoring and a DBL homology domain (DH) for small GTPase Cdc42 activation, led to further refinement of this model. Generation of ArhGEF9 KO mice confirmed the importance of CB in gephyrin scaffold recruitment for GABAergic synapse function (Tyagarajan and Fritschy 2014). Interaction of CB with phosphatidylinositol 3-monophosphate (PI3P) and phosphatidylinositol 4-monophosphate (PI4P) has been demonstrated (Ludolphs et al. 2016). Gephyrin itself has been shown to be lipid modified via palmitoylation at Cys212 and Cys284, offering a much simpler explanation for gephyrin membrane recruitment. Gephyrin palmitoylation is facilitated by the palmitoyl transferase DHHC-12, which in turn facilitates GABAergic transmission via membrane stabilization of gephyrin scaffold (Dejanovic et al. 2014). Hence, in addition to kinase pathways, lipid signaling is also an important regulator at GABAergic synapses.

At synaptic sites nitric oxide (NO) is an important retrograde signaling molecule involved in diverse neurotransmission systems. The generation of NO at the postsynaptic sites often leads to S-nitrosylation of specific target proteins leading to adaptations that accompany presynaptic changes in neurotransmitter release. Gephyrin interacts with nNOS and is a novel substrate for S-nitrosylation. Gephyrin S-nitrosylation reduces scaffold size at GABAergic postsynapse (Dejanovic and Schwarz 2014). More recently, two novel modifications, namely, SUMOylation and acetylation, have also been identified on gephyrin. SUMO1 conjugation at K148 and SUMO2 conjugation at K724, in conjunction to K-Ac at K666 and phosphorylation at S268, are thought to regulate gephyrin scaffolding. Transgenic expression of single SUMO1 or SUMO2 point mutant eGFP-gephyrin into Gabra2 KO neurons was sufficient to rescue gephyrin scaffolding in vivo. This identified a role for α2 containing GABAARs in modulating the SUMO pathway upstream of the phosphorylation and acetylation pathways for gephyrin scaffold recruitment (Ghosh et al. 2016).

Gephyrin interacts with diverse functional class of proteins (see Table 1 below). Functional role for many of these interaction proteins at inhibitory synapses remain unknown. However, the identification of these proteins opens up an interesting question. Glutamatergic postsynapse is organized via the presence of well-conserved PDZ motif on PSD95 and its interaction partners, but at inhibitory postsynapse, gephyrin and its interaction partners lack canonical protein motif. Hence, how is multi-molecular protein complexes assembled at GABAergic postsynapse. Given the diversity of post-translational modifications on gephyrin, it appears that GABAergic synapses have coopted for a more archaic system of signaling pathways to organize and regulate gephyrin and its interaction partners for a dynamic synapse.
Gephyrin, Table 1

Gephyrin interaction partners

 

Interacting site

Binding domain

Publication

Synaptic partners

   

GABAAR α1 subunit

Cytoplasmic domain

E

Mukherjee J et al., J Neurosci. 2011

GABAAR α2 subunit

FNIVGTTYPI in the M3–M4 loop

E

Tretter et al., J Biol Chem. 2011

GABAAR α3 subunit

FNIVGTTYPI in the M3–M4 loop

E

Tretter et al., J Biol Chem. 2011

GABAAR β2 subunit

 

E

Kowalczyk et al., Eur J Neurosci. 2013

GABAAR β3 subunit

 

E

Kowalczyk et al., Eur J Neurosci. 2013

GlyR β subunit

Cytoplasmic domain

E

Rees et al., J Biol Chem. 2003

Collybistin

GEF

C/E

Harvey et al., J Neurosci. 2004

GIT1/betaPIX complex

Unknown

Unknown

Smith et al., Cell Report 2014

SRGAP2

SH3 domain

E (PFLP motif)

Fossati et al., Neuron 2016. Okada et al., 2011

Neuroligin 2

Cytoplasmic domain

E

Poulopoulos et al., Neuron 2009

IQSEC3 (SynArfGEF)

aa 101–200 and aa 636–1194

G

Um et al., JBC 2016

Cytoskeleton partners

GABARAP

aa 36–117

C

Kneussel et al., PNAS 2000

Dlc1/Dlc2

Unknown

C

Fuhrmann et al., J Neurosci. 2002

KIF5

Unknown

Unknown

Rathgeber et al., Eur J Cell Biol 2015

Profilin

G-actin- and PIP2-binding site

E

Giesemann et al., J Neurosci. 2003

Mena/VASP

Unknown

E

Giesemann et al., J Neurosci. 2003

Tubulin

Unknown

C

Kirsch et al., JBC 1991

Others

RAFT1 (FRAP, mTOR)

aa 1010–1128

C (aa 209–685)

Sabatini et al., Science 1999

GRIP1

PDZ domain

Unknown

Yu et al., J Neurochem 2010

HSC70

Unknown

G

Machado et al., J Neurosci. 2011

GAP43

Unknown

Unknown

Wang et al., Mol Cell Biol 2015

Post-translational modification

Phosphorylation

 

Sites

 

CaMKIIa

S305

Flores et al., PNAS 2015

PKA

S303

Flores et al., PNAS 2015

ERK

S268

Tyagarajan et al., JBC 2013

GSK3b

S270

Tyagarajan et al., PNAS 2011

Cdk5

S270

Kuhse et al., JBC 2012

Pin1

S188, S194, S200, S319, T337

Zita et al., EMBO 2007

PKC

S403

 

Acetylation

   
  

K666

Ghosh et al. 2016

SUMOylation

   

SUMO1

K148, K326, K645

Ghosh et al. 2016

SUMO2

K724

Ghosh et al. 2016

S-nitrosylation

   
 

nNOS

E domain

Dejanovic and Schwarz, J Neurosci. 2014

Palmitoylation

   
 

DHHC-12

Cys212, Cys208

Dejanovic et al. 2014

Gephyrin-Interacting Proteins

Several gephyrin-interacting proteins have been identified over the years (see Table 1). The characterization of gephyrin-interacting proteins was thought to provide an understanding into the mechanistic basis for inhibitory synapse formation and maintenance. Of the various gephyrin-interacting proteins that are described in literature, some of them play an essential role in GABAergic synapse formation and maintenance. For example, ArhGEF9 encodes CB, only RhoGEF identified for GABAergic synapse regulation (Kins et al. 2000). CB-deficient neurons show a distinct loss of gephyrin clustering and impaired GABAergic transmission (Papadopoulos et al. 2007). Further, CB plays an essential role in gephyrin scaffold recruitment at GABAergic synapses via its interaction with Cdc42 and gephyrin (Tyagarajan and Fritschy 2014).

Rho family GAPs [guanosine triphosphatase (GTPase)-activating proteins] negatively regulate Rho family GTPase activity and control cytoskeletal dynamics. The specificity of such GAPs for cellular GTPases and spatial-temporal distribution within neurons in turn are controlled by their interactions with diverse cellular substrates. SRGAP2 interacts with gephyrin via its SH3 domain (Okada et al. 2011). Recently, it was demonstrated that SRGAP2 activates Rho-Rac1 downstream to modulate the levels of both excitatory and inhibitory synapse density via specific interactions with both homer and gephyrin, respectively (Fossati et al. 2016). Hence, it is likely that additional RhoGEFs are operational at GABAergic synapses.

Interaction between NRX1β and NLGN1 can induce early recruitment of the PSD95 at glutamatergic synapses. Similarly, NLGN2 that has been shown to be specific for inhibitory postsynapses activates CB via gephyrin interaction for GABAergic synapse formation. NLGN2 interaction with CB guides membrane recruitment of gephyrin scaffold (Tyagarajan and Fritschy 2014). Mammalian target of rapamycin (mTOR) is a kinase that is important for dendritic protein synthesis via the activation of downstream kinase pathways (Tang et al. 2002). mTOR has been shown to interact with gephyrin, but mechanistic implications for this interaction have remained unclear (Tyagarajan and Fritschy 2014). A recent study linked EphA7 signaling to mTOR activation and demonstrated that mTOR activation leads to loss in gephyrin binding. Interestingly, in the same study, EphA7-dependent mTOR activation was shown to enhance mTOR interaction with CB. Hence, RTK signaling via EphA7 receptor signaling can regulate binding affinities between gephyrin interaction proteins at GABAergic postsynapse. Biochemical changes in protein affinities can influence connectivity between neuronal subpopulation as shown by the loss of GABAergic basket cell innervations onto proximal dendrites of principal neurons when Eph7A signaling is disrupted (Beuter et al. 2016).

Structural configurations and biochemical evidence offer necessary understanding for a strong gephyrin interaction with GlyR β subunit. However, gephyrin interaction with GABAAR has been elusive due to low-affinity interaction between them. It is only recently that direct interaction between gephyrin- and GABAAR-specific subunits (α1, α2, α3, β2, and β3) have emerged (see Table 1). The first small angle X-ray structure of full-length gephyrin shows gephyrin as a dynamic molecule present in both closed and open conformations (see Fig. 2; Sander et al. 2013). Gephyrin interaction with GlyR β subunit occurs already along the secretory pathway and is co-transported to glycinergic synapses (Tyagarajan and Fritschy 2014). However, at GABAergic synapses, gephyrin is recruited from a cytoplasmic pool after receptor assembly. While super-resolution microscopy study has predicted 5,000–10,000 gephyrin molecules/μm2 with a stoichiometry of 1:1 between gephyrin molecules and receptor-binding sites (Tyagarajan and Fritschy 2014), it is possible that gephyrin open and close structural states determine interaction with GABAAR subunits.
Gephyrin, Fig. 2

Structure of gephyrin: (a) Structures of the full length gephyrin in extended (red) and compact (blue) conformations derived from the small angle X-ray scattering (SAXS) data. (b) Cartoon representation of the crystal structure of the N-terminal G domain (PDB:1JLJ), where one monomer is represented in red and other two in black. (c) Cartoon representation of the crystal structure of the C-terminal dimeric E-domain (PBD: 5ERQ), where one of the monomers is colored according to four subdomains (subdomain I- red, subdomain II- green, subdomain III-yellow and subdomain IV-blue) and other monomer is shown in black color. Image courtesy: Prof. Hermann Schindelin University of Würzburg

Gephyrin and Human Diseases

Gphn-associated human mutations are rare, which could be due to the importance of glycinergic transmission in breathing, suckling, and swallowing behaviors. Mutations in GlyR β subunit or mutations within any of the diverse gephyrin interaction proteins could also contribute toward lethality of Gphn KO neonatal mouse pups (Grosskreutz et al. 2003). However, identified human mutations within the described protein repertoire do not lead to a loss in glycinergic neurotransmission, but Gphn mutation causes severe glycinergic synapse deficit. This could offer a possible explanation for why human mutations associated with GlyR subunits, glycine transporters, or collybistin causes hyperekplexia (Harvey et al. 2008).

Defective gephyrin alternative splicing in neurons has been reported for human patients with temporal lobe epilepsy. The splicing defect within the G-domain negatively impairs scaffolding at synaptic sites leading to inhibitory neurotransmission defect. Interestingly, the observed splicing defect leading to GABAergic synapse impairment is not due to genetic mutations but exon skipping caused by cellular stress (Förstera et al. 2010). Hence, gephyrin splicing regulation offers a novel therapeutic window for intervention in similar pathologies. Unlinked human subjects with autism spectrum disorder, schizophrenia or seizures contain rare de novo or inherited hemizygous microdeletions overlapping exons of GPHN at chromosome 14q23.3. These Gphn microdeletions occur within the G-domain leading to scaffold loss at inhibitory postsynaptic sites (Lionel et al. 2013).

De novo missense mutation (G375D) in the gephyrin E-domain has been associated with epileptic encephalopathy resembling Dravet syndrome (Dejanovic et al. 2015). Unlike exon skipping or microdeletions within the G-domain, this de novo mutation did not disrupt protein folding but in turn acted as dominant negative mutation influencing GABAAR accumulation at synaptic sites (Dejanovic et al. 2015). A functional loss in MoCo synthesis was also observed with the G375D gephyrin mutation, suggesting that the scaffolding may not be completely uncoupled from the MoCo synthesis function of gephyrin.

Gephyrin cluster regulation by signal transduction pathways also offer potential new windows for therapeutic intervention. For example, gephyrin phosphorylation at Ser268 and Ser270 is also relevant in the context of bipolar disorder. Lithium chloride (LiCl), a drug commonly used to treat bipolar patients, induces plasticity adaptations at GABAergic synapses via the modulation of Ser268 and Ser270 sites on gephyrin. It has been known for some time now that specific bipolar patients are resistant to LiCl treatment. Hence, it will be interesting to examine whether bipolar patients resistant to LiCl treatment also harbor specific gephyrin polymorphisms at Ser268 and Ser270 residues (Tyagarajan and Fritschy 2014).

Summary

Inhibitory neurotransmission plays a fundamental role for normal brain function by controlling neuronal excitability, dendritic integration, and spike-timing, as well as neuronal synchronization and generation of oscillations. One can envision how gephyrin scaffolds that act as signaling hubs can integrate signals from diverse neurotransmitter systems to adjust the strength of inhibitory transmission. Furthermore, diverse signaling pathways that converge on gephyrin scaffold show striking homologies with the “histone code” regulating chromatin function. Understanding the “gephyrin code” could offer a glimpse into the dynamic landscape of intracellular signaling networks can in turn influence network function. The core message from these studies is that studying similar integrated signaling hubs could be relevant within the context of immunology and cancer research or even for the design of self-assembling scaffolds for nanotechnology applications.

References

  1. Bechade C, Colin I, Kirsch J, Betz H, Triller A. Expression of glycine receptor α subunits and gephyrin in cultured spinal neurons. Eur J Neurosci. Blackwell Publishing Ltd1996;8(2):429–35.PubMedCrossRefGoogle Scholar
  2. Beuter S, Ardi Z, Horovitz O, Wuchter J, Keller S, Saha R, et al. Receptor tyrosine kinase EphA7 is required for interneuron connectivity at specific subcellular compartments of granule cells. Sci Rep. 2016;6:29710.PubMedCrossRefPubMedCentralGoogle Scholar
  3. Brünig I, Penschuck S, Berninger B, Benson JA, Fritschy JM. BDNF reduces miniature inhibitory postsynaptic currents by rapid downregulation of GABAA receptor surface expression. Eur J Neurosci [Internet]. 2001;7(7):1320–8 .Available from: http://dx.doi.org/10.1046/j.0953-816x.2001.01506.x
  4. Chen X, Winters C, Azzam R, Li X, Galbraith JA, Leapman RD, et al. Organization of the core structure of the postsynaptic density. Proc Natl Acad Sci. 2008;105(11):4453–8.PubMedCrossRefPubMedCentralGoogle Scholar
  5. Choquet D, Triller A. The dynamic synapse. Neuron. 2013;80(3):691–703.PubMedCrossRefGoogle Scholar
  6. Dejanovic B, Schwarz G. Neuronal nitric oxide synthase-dependent S-nitrosylation of gephyrin regulates gephyrin clustering at GABAergic synapses. J Neurosci. 2014;34(23):7763–8.PubMedCrossRefGoogle Scholar
  7. Dejanovic B, Semtner M, Ebert S, Lamkemeyer T, Neuser F, Lüscher B, et al. Palmitoylation of gephyrin controls receptor clustering and plasticity of GABAergic synapses. PLoS Biol. 2014;12(7):e1001908.PubMedCrossRefPubMedCentralGoogle Scholar
  8. Dejanovic B, Djémié T, Grünewald N, Suls A, Kress V, Hetsch F, et al. Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy. EMBO Mol Med. 2015;7(12):1580–94.PubMedCrossRefPubMedCentralGoogle Scholar
  9. 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(5392):1321–4.PubMedCrossRefGoogle Scholar
  10. Förstera B, Belaidi AA, Jüttner R, Bernert C, Tsokos M, Lehmann T-N, et al. Irregular RNA splicing curtails postsynaptic gephyrin in the cornu ammonis of patients with epilepsy. Brain. 2010;133(Pt 12):3778–94.PubMedCrossRefGoogle Scholar
  11. Fossati M, Pizzarelli R, Schmidt ER, Kupferman JV, Stroebel D, Polleux F, et al. SRGAP2 and its human-specific paralog co-regulate the development of excitatory and inhibitory synapses. Neuron. 2016;91(2):356–69.PubMedCrossRefPubMedCentralGoogle Scholar
  12. Fritschy J-M, Harvey RJ, Schwarz G. Gephyrin: where do we stand, where do we go? Trends Neurosci. 2008;31(5):257–64.PubMedCrossRefGoogle Scholar
  13. Ghosh H, Auguadri L, Battaglia S, Simone Thirouin Z, Zemoura K, Messner S, Acuña MA, Wildner H, Yévenes GE, Dieter A, Kawasaki H, O Hottiger M, Zeilhofer HU, Fritschy JM, Tyagarajan SK. Several posttranslational modifications act in concert to regulate gephyrin scaffolding and GABAergic transmission. Nat Commun. 2016;7:13365.PubMedCrossRefPubMedCentralGoogle Scholar
  14. Giannone G, Mondin M, Grillo-Bosch D, Tessier B, Saint-Michel E, Czöndör K, et al. Neurexin-1β binding to neuroligin-1 triggers the preferential recruitment of PSD-95 versus gephyrin through tyrosine phosphorylation of neuroligin-1. Cell Rep. 2013;3(6):1996–2007.PubMedCrossRefGoogle Scholar
  15. Grosskreutz Y, Betz H, Kneussel M. Rescue of molybdenum cofactor biosynthesis in gephyrin-deficient mice by a Cnx1 transgene. Biochem Biophys Res Commun. 2003;301(2):450–5.PubMedCrossRefGoogle Scholar
  16. Iijima T, Wu K, Witte H, Hanno-Iijima Y, Glatter T, Richard S, et al. SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1. Cell. 2011;147(7):1601–14.PubMedCrossRefPubMedCentralGoogle Scholar
  17. Iijima T, Iijima Y, Witte H, Scheiffele P. Neuronal cell type-specific alternative splicing is regulated by the KH domain protein SLM1. J Cell Biol. 2014;204(3):331–42.PubMedCrossRefPubMedCentralGoogle Scholar
  18. 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(6):1385–95.PubMedCrossRefPubMedCentralGoogle Scholar
  19. Kins S, Betz H, Kirsch J. Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin. Nat Neurosci. 2000;3(1):22–9.PubMedCrossRefGoogle Scholar
  20. Kneussel M, Betz H. Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: the membrane activation model. Trends Neurosci. 2000;23(9):429–35.PubMedCrossRefGoogle Scholar
  21. 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. Oxford University Press 2013;22(10):2055–66.PubMedCrossRefGoogle Scholar
  22. Ludolphs M, Schneeberger D, Soykan T, Schäfer J, Papadopoulos T, Brose N, et al. Specificity of collybistin-phosphoinositide interactions: impact of the individual protein domains. J Biol Chem. 2016;291(1):244–54.PubMedCrossRefGoogle Scholar
  23. Okada H, Uezu A, Mason FM, Soderblom EJ, Moseley MA, Soderling SH. SH3 domain-based phototrapping in living cells reveals Rho family GAP signaling complexes. Sci Signal. 2011;4(201):rs13–3.Google Scholar
  24. Papadopoulos T, Korte M, Eulenburg V, Kubota H, Retiounskaia M, Harvey RJ, Harvey K, O’Sullivan GA, Laube B, Hülsmann S, Geiger JR, Betz H. Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice. EMBO J. 2007;26(17):3888–99.PubMedCrossRefPubMedCentralGoogle Scholar
  25. 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.Google Scholar
  26. Porcher C, Hatchett C, Longbottom RE, McAinch K, Sihra TS, Moss SJ, et al. Positive feedback regulation between -aminobutyric acid type a (GABAA) receptor signaling and brain-derived neurotrophic factor (BDNF) release in developing neurons. J Biol Chem. 2011;286(24):21667–77.PubMedCrossRefPubMedCentralGoogle Scholar
  27. Ramming M, Betz H, Kirsch J. Analysis of the promoter region of the murine gephyrin gene. FEBS Lett. 1997;405(2):137–40.PubMedCrossRefGoogle Scholar
  28. Reiss J, Gross-Hardt S, Christensen E, Schmidt P, Mendel RR, Schwarz G. A mutation in the gene for the neurotransmitter receptor-clustering protein gephyrin causes a novel form of molybdenum cofactor deficiency. Am J Hum Genet. 2001;68(1):208–13.PubMedCrossRefGoogle Scholar
  29. Sander B, Tria G, Shkumatov AV, Kim EY, Grossmann JG, Tessmer I, et al. Structural characterization of gephyrin by AFM and SAXS reveals a mixture of compact and extended states. Acta Crystallogr D Biol Crystallogr. 2013;69(10):2050–60.PubMedCrossRefGoogle Scholar
  30. Schwarz G, Schrader N, Mendel RR, Hecht H-J, Schindelin H. Crystal structures of human gephyrin and plant Cnx1 G domains: comparative analysis and functional implications. J Mol Biol. 2001;312(2):405–18.PubMedCrossRefGoogle Scholar
  31. 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(13):2510–9.PubMedCrossRefPubMedCentralGoogle Scholar
  32. Stallmeyer B, Schwarz G, Schulze J, Nerlich A, Reiss J, Kirsch J, et al. The neurotransmitter receptor-anchoring protein gephyrin reconstitutes molybdenum cofactor biosynthesis in bacteria, plants, and mammalian cells. Proc Natl Acad Sci U S A. 1999;96(4):1333–8.PubMedCrossRefPubMedCentralGoogle Scholar
  33. Tyagarajan SK, Fritschy J-M. Gephyrin: a master regulator of neuronal function? Nat Rev Neurosci. 2014;15(3):141–56.PubMedCrossRefGoogle Scholar
  34. Ule J, Jensen KB, Ruggiu M, Mele A, Ule A, Darnell RB. CLIP identifies Nova-regulated RNA networks in the brain. Science. 2003;302(5648):1212–5.PubMedCrossRefGoogle Scholar
  35. Villa KL, Berry KP, Subramanian J, Cha JW, WC O, Kwon H-B, et al. Inhibitory synapses are repeatedly assembled and removed at persistent sites in vivo. Neuron. 2016;89(4):756–69.PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Institute of Pharmacology and Toxicology, Center for Neuroscience Zurich (ZNZ)University of ZurichZurichSwitzerland