Gephyrin clustering is required for the stability of GABAergic synapses

Abstract

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 transynaptic 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.

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

“…We favor the first explanation, because the decreased density of synaptic ␥2-GABA A R clusters after knocking down septin 11 with shRNAs was not accompanied by parallel changes in the density of PSD-95 clusters or vGlut1 puncta. Moreover, we and others have shown that in these hippocampal cultures, interfering with the postsynaptic clustering of GABA A Rs or gephyrin in the postsynaptic cells leads to decreased GABAergic innervation of these cells (27,31,65). Therefore, if septin 11 clusters are involved in the assembly and/or maintenance of the postsynaptic GABAergic complex, their disruption would lead to decreased GABAergic innervation.…”

“…3A). A DNA oligonucleotide encoding both arms of the shRNA was annealed with the corresponding reverse DNA strand, and the double-stranded DNA was ligated between the BbsI and XbaI sites of mU6pro vector (30,31). The control shRNAs were generated by introducing three point mutations in the sense and antisense arms of the corresponding shRNA (supplemental Fig.…”

γ-Aminobutyric Acid Type A (GABAA) Receptor α Subunits Play a Direct Role in Synaptic Versus Extrasynaptic Targeting

“…Gephyrin and GABA A -R Targeting-Synaptic GABA A -Rs are thought to be first inserted to extrasynaptic membranes and then laterally diffused into postsynaptic sites, where they are stabilized by the scaffolding protein complex (40,50,67,68). Both knock-out and knockdown of gephyrin expression disrupted the clustering of a major subset of synaptic GABA A -Rs and resulted in decreased GABAergic neurotransmission (28,40,50,69,70).…”

“…Both knock-out and knockdown of gephyrin expression disrupted the clustering of a major subset of synaptic GABA A -Rs and resulted in decreased GABAergic neurotransmission (28,40,50,69,70). However, not all GABA A -R clusters are dependent on gephyrin (70,71).…”

“…The HA tag was inserted between the signal peptide and the N terminus of the mature protein. The gephyrin-GFP construct encodes human gephyrin with GFP fused to the C terminus of gephyrin (40). The collybistin constructs encode two isoforms of human collybistin: CB3 SH3ϩ /hPEM2 SH3ϩ containing the SH3 domain and CB3 SH3Ϫ /hPEM2 SH3Ϫ lacking the SH3 domain (41).…”