Several posttranslational modifications act in concert to regulate gephyrin scaffolding and GABAergic transmission

Abstract

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.

RhoGEF9 splice isoforms influence neuronal maturation and synapse formation downstream ofα2 GABAAreceptors

“…1B-F). Independent studies have demonstrated that alterations in postsynaptic gephyrin scaffolds are reflected by corresponding changes in GABAergic transmission [20][21][22]. Here, we confirmed that eGFP-gephyrin clusters along the proximal-distal axis of dendrites are associated with GABA A Rs by co-labeling for the g2 GABA A R subunit ( Fig.…”

“…a2 GABA A R can directly interact with CB and facilitate gephyrin scaffold recruitment at GABAergic postsynaptic sites [12,28]. Hence, we examined in Gabra2 KO mice, where we have reported deficit in gephyrin scaffolding both in hippocampal formation and neocortex [20,27], whether CB overexpression can compensate for the absence of the GABA A R in facilitating gephyrin clustering. We quantified gephyrin cluster density in the first 40 µm segment of dendrite from the soma and found a significant reduction in GCS from Gabra2 KO compared to the WT mice.…”

Extranuclear SUMOylation in Neurons

“…Indeed, it remains possible that the reduction in SUMO1 conjugation observed in these mice may be compensated for by SUMO2/3 conjugation, as occurs in SUMO1 knockout mice [64] however, this possibility was not examined in the recent study [13]. We also note that the original studies reporting SUMOylation of synapsin1a [20], gephyrin [36], GluK2 [23], syntaxin1a [19], RIM1α [16] and mGluR7 [41] investigated the functional effects of SUMOylation. Thus, in addition to demonstrating SUMOylation biochemically, those studies demonstrated that SUMOylation affects the target functionally, something which was not addressed in the recent study questioning the functional role of SUMOylation at synapses.…”

“…Gephyrin is part of a scaffolding protein complex that is required for postsynaptic clustering of GABAA receptors. Gephyrin is SUMOylated and it has been reported that its deSUMOylation promotes the formation of postsynaptic clusters via cross-talk between phosphorylation and acetylation of gephyrin [36].…”

“…Notwithstanding these findings arising from many different labs, two related studies using a His6-HA-SUMO1 knock-in mouse model concluded that SUMO1-ylation of synaptic proteins does not occur to any significant extent [12,13]. In the more recent paper of the two, SUMOylation of seven previously identified synaptic SUMO1 substrates was investigated -synapsin1a [20], gephyrin [36], GluK2 [23], syntaxin1a [19], RIM1α [16], mGluR7 [41], and synaptotagmin1 [17] and no noticeable His6-HA-SUMO1-ylation of any of these proteins was detected in the His6-HA-SUMO1 knock-in mice [13]. Moreover, using subcellular fractionation and immunocytochemical staining, the authors similarly did not detect SUMO1ylation in synaptic fractions, or colocalisation of anti-SUMO1 signal with synaptic markers.…”