Pin1-dependent signalling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction.
Ontology highlight
ABSTRACT:
SUBMITTER: Antonelli R
PROVIDER: S-EPMC4197815 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Gephyrin is a scaffold protein essential for stabilizing glycine and GABA(A) receptors at inhibitory synapses. Here, recombinant intrabodies against gephyrin (scFv-gephyrin) were used to assess whether this protein exerts a transynaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrin-transfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NLG2), a protein that, interacting with neurexins, ensures the cross-talk between the post- and presynaptic sites. Interestingly, hampering gephyrin function also produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Overexpressing NLG2 in gephyrin-deprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. Pulldown experiments demonstrated that gephyrin interacts not only with NLG2 but also with NLG1, the isoform enriched at excitatory synapses. These results suggest a key role of gephyrin in regulating transynaptic signaling at both inhibitory and excitatory synapses.
| S-EPMC3121485 | biostudies-literature
Project description:Gephyrin, the principal scaffolding protein at inhibitory synapses, is essential for postsynaptic clustering of glycine and GABA type A receptors (GABA(A)Rs). Gephyrin cluster formation, which determines the strength of GABAergic transmission, is modulated by interaction with signaling proteins and post-translational modifications. Here, we show that gephyrin was found to be associated with neuronal nitric oxide synthase (nNOS), the major source of the ubiquitous and important signaling molecule NO in brain. Furthermore, we identified that gephyrin is S-nitrosylated in vivo. Overexpression of nNOS decreased the size of postsynaptic gephyrin clusters in primary hippocampal neurons. Conversely, inhibition of nNOS resulted in a loss of S-nitrosylation of gephyrin and the formation of larger gephyrin clusters at synaptic sites, ultimately increasing the number of cell surface expressed synaptic GABA(A)Rs. In conclusion, S-nitrosylation of gephyrin is important for homeostatic assembly and plasticity of GABAergic synapses.
| S-EPMC6608267 | biostudies-literature
Project description:GABAA receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the brain via synergistic association with the postsynaptic scaffolding protein gephyrin and its interaction partners. However, unlike their counterparts at glutamatergic synapses, gephyrin and its binding partners lack canonical protein interaction motifs; hence, the molecular basis for gephyrin scaffolding has remained unclear. In this study, we identify and characterize two new posttranslational modifications of gephyrin, SUMOylation and acetylation. We demonstrate that crosstalk between SUMOylation, acetylation and phosphorylation pathways regulates gephyrin scaffolding. Pharmacological intervention of SUMO pathway or transgenic expression of SUMOylation-deficient gephyrin variants rescued gephyrin clustering in CA1 or neocortical neurons of Gabra2-null mice, which otherwise lack gephyrin clusters, indicating that gephyrin SUMO modification is an essential determinant for scaffolding at GABAergic synapses. Together, our results demonstrate that concerted modifications on a protein scaffold by evolutionarily conserved yet functionally diverse signalling pathways facilitate GABAergic transmission.
| S-EPMC5103071 | biostudies-literature
Project description:Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3? (GSK3?) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3? inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca(2+)-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.
| S-EPMC3017200 | biostudies-literature
Project description:Growth-associated protein 43 (GAP43) is known to regulate axon growth, but whether it also plays a role in synaptogenesis remains unclear. Here, we found that GAP43 regulates the aggregation of gephyrin, a pivotal protein for clustering postsynaptic GABA(A) receptors (GABA(A)Rs), in developing cortical neurons. Pharmacological blockade of either protein kinase C (PKC) or neuronal activity increased both GAP43-gephyrin association and gephyrin misfolding-induced aggregation, suggesting the importance of PKC-dependent regulation of GABAergic synapses. Furthermore, we found that PKC phosphorylation-resistant GAP43(S41A), but not PKC phosphorylation-mimicking GAP43(S41D), interacted with cytosolic gephyrin to trigger gephyrin misfolding and its sequestration into aggresomes. In contrast, GAP43(S41D), but not GAP43(S41A), inhibited the physiological aggregation/clustering of gephyrin, reduced surface GABA(A)Rs under physiological conditions, and attenuated gephyrin misfolding under transient oxygen-glucose deprivation (tOGD) that mimics pathological neonatal hypoxia. Calcineurin-mediated GAP43 dephosphorylation that accompanied tOGD also led to GAP43-gephyrin association and gephyrin misfolding. Thus, PKC-dependent phosphorylation of GAP43 plays a critical role in regulating postsynaptic gephyrin aggregation in developing GABAergic synapses.
| S-EPMC4405633 | biostudies-literature
Project description:Collybistin (CB) is a guanine-nucleotide-exchange factor (GEF) selectively activating Cdc42. CB mutations cause X-linked mental retardation due to defective clustering of gephyrin, a postsynaptic protein associated with both glycine and GABA(A) receptors. Using a combination of biochemistry and cell biology we provide novel insights into the roles of the CB2 splice variants, CB2(SH3+) and CB2(SH3-), and their substrate, Cdc42, in regulating gephyrin clustering at GABAergic synapses. Transfection of Myc-tagged CB2(SH3+) and CB2(SH3-) into cultured neurons revealed strong, but distinct, effects promoting postsynaptic gephyrin clustering, denoting mechanistic differences in their function. In addition, overexpression of constitutively active or dominant-negative Cdc42 mutants identified a new function of Cdc42 in regulating the shape and size of postsynaptic gephyrin clusters. Using biochemical assays and native brain tissue, we identify a direct interaction between gephyrin and Cdc42, independent of its activation state. Finally, our data show that CB2(SH3-), but not CB2(SH3+), can form a ternary complex with gephyrin and Cdc42, providing a biochemical substrate for the distinct contribution of these CB isoforms in gephyrin clustering at GABAergic postsynaptic sites. Taken together, our results identify CB and Cdc42 as major regulators of GABAergic postsynaptic densities.
| S-EPMC3148131 | biostudies-literature
Project description:Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transsynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.
| S-EPMC2464357 | biostudies-literature
Project description:Spinal cord neurons release glycine and GABA and accumulate glycine receptors (GlyRs) and GABA(A) receptors in the same postsynaptic densities. In contrast, supramedullar neurons prefer GABA as a neurotransmitter and exclude GlyRs from postsynaptic anchoring. The general aim of the present study was to elucidate the mechanisms underlying transmitter-appropriate receptor accumulation at inhibitory synapses. Specifically, we intended to clarify the molecular basis for the prohibition of GlyR accumulation in the postsynaptic densities of GABAergic synapses. A green fluorescent protein (GFP)-tagged gephyrin-binding loop of the GlyR beta subunit (GFP::betaL) was used as a surrogate for full-length receptors to characterize the GlyR binding capacity of postsynaptic gephyrins in transfected neurons. Both in spinal cord neurons (SCNs) and hippocampal neurons (HNs) GFP::betaL distribution displayed transmitter specificity; i.e., postsynaptic accumulation of GFP::betaL was high opposite terminals able to release glycine and low opposite purely GABAergic terminals. When comparing SCN and HN cultures we found that the level of mRNA coding for gephyrin splice variants containing the cassette C5 (C5-gephyrins) was significantly higher in HNs. In HNs depleted of C5-gephyrins, both GFP::betaL and endogenous GlyRs accumulated at postsynaptic GABAergic sites. Accordingly in SCNs, GFP-tagged C5-gephyrin displayed a preferential postsynaptic accumulation opposite GABAergic synapses. Comparison of glycinergic, mixed, and GABAergic synapses in SCNs showed that the degree of GlyR accumulation was inversely related to the amount of postsynaptic C5-gephyrin. These results identify the C5 splice variant of gephyrin as a factor regulating the transmitter-appropriate degree of GlyR accumulation at inhibitory synapses.
| S-EPMC6730342 | biostudies-literature
Project description:Postsynaptic scaffolding proteins regulate coordinated neurotransmission by anchoring and clustering receptors and adhesion molecules. Gephyrin is the major instructive molecule at inhibitory synapses, where it clusters glycine as well as major subsets of GABA type A receptors (GABAARs). Here, we identified palmitoylation of gephyrin as an important mechanism of strengthening GABAergic synaptic transmission, which is regulated by GABAAR activity. We mapped palmitoylation to Cys212 and Cys284, which are critical for both association of gephyrin with the postsynaptic membrane and gephyrin clustering. We identified DHHC-12 as the principal palmitoyl acyltransferase that palmitoylates gephyrin. Furthermore, gephyrin pamitoylation potentiated GABAergic synaptic transmission, as evidenced by an increased amplitude of miniature inhibitory postsynaptic currents. Consistently, inhibiting gephyrin palmitoylation either pharmacologically or by expression of palmitoylation-deficient gephyrin reduced the gephyrin cluster size. In aggregate, our study reveals that palmitoylation of gephyrin by DHHC-12 contributes to dynamic and functional modulation of GABAergic synapses.
| S-EPMC4099074 | biostudies-literature
Project description:Anxiety disorders are the most common mental illness in the U.S. and are estimated to consume one-third of the country's mental health spending. Although anxiolytic therapies are available, many patients exhibit treatment-resistance, relapse, or substantial side effects. An urgent need exists to explore the underlying mechanisms of chronic anxiety and to develop alternative therapies. Presently, we identified dihydromyricetin (DHM), a flavonoid that has anxiolytic properties in a mouse model of isolation-induced anxiety. Socially isolated mice demonstrated increased anxiety levels and reduced exploratory behavior measured by elevated plus-maze and open-field tests. Socially isolated mice showed impaired GABAergic neurotransmission, including reduction in GABAA receptor-mediated extrasynaptic tonic currents, as well as amplitude and frequency of miniature inhibitory postsynaptic currents measured by whole-cell patch-clamp recordings from hippocampal slices. Furthermore, intracellular ATP levels and gephyrin expression decreased in anxious animals. DHM treatment restored ATP and gephyrin expression, GABAergic transmission and synaptic function, as well as decreased anxiety-like behavior. Our findings indicate broader roles for DHM in anxiolysis, GABAergic neurotransmission, and synaptic function. Collectively, our data suggest that reduction in intracellular ATP and gephyrin contribute to the development of anxiety, and represent novel treatment targets. DHM is a potential candidate for pharmacotherapy for anxiety disorders.
| S-EPMC7364153 | biostudies-literature