Gephyrin expression and clustering affects the size of glutamatergic synaptic contacts

Abstract

We have recently shown that disrupting the expression and post‐synaptic clustering of gephyrin in cultured hippocampal pyramidal cells, by either gephyrin RNAi (RNA interference) or over‐expression of a dominant negative gephyrin‐enhanced green fluorescent protein (EGFP) fusion protein, leads to decreased number of post‐synaptic gephyrin and GABAA receptor clusters and to reduced GABAergic innervation of these cells. On the other hand, increasing gephyrin expression led to a small increase in the number of gephyrin and GABAA receptor clusters and to little or no effect on GABAergic innervation. We are now reporting that altering gephyrin expression and clustering affects the size but not the density of glutamatergic synaptic contacts. Knocking down gephyrin with gephyrin RNAi, or preventing gephyrin clustering by over‐expression of the dominant negative gephyrin‐enhanced green fluorescent protein fusion protein, leads to larger post‐synaptic PSD‐95 clusters and larger pre‐synaptic glutamatergic terminals. On the other hand, over‐expression of gephyrin leads to slightly smaller PSD‐95 clusters and pre‐synaptic glutamatergic terminals. The change in size of PSD‐95 clusters were accompanied by a parallel change in the size of NR2‐NMDA receptor clusters. It is concluded that the levels of expression and clustering of gephyrin, a protein that concentrates at the post‐synaptic complex of the inhibitory synapses, not only has homotypic effects on GABAergic synaptic contacts, but also has heterotypic effects on glutamatergic synaptic contacts. We are proposing that gephyrin is a counterpart of the post‐synaptic glutamatergic scaffold protein PSD‐95 in regulating the number and/or size of the excitatory and inhibitory synaptic contacts.

Gephyrin interacts with the glutamate receptor interacting protein 1 isoforms at GABAergic synapses

“…2004; Li et al 2005b;Lardi-Studler and Fritschy 2007;Yu et al 2007;Yu and De Blas 2008). In this study, we show that GRIP1c4-7 and GRIP1a/b associate and interact directly with gephyrin and that both GRIP1 isoforms colocalize with gephyrin at the GABAergic post-synaptic complex, suggesting that the interaction between gephyrin and GRIP1 plays a role in GABAergic synaptic function.…”

“…Thus, it is unlikely that GRIP1 isoforms are directly involved with the trafficking of GABA A Rs at the GABAergic synapses. Nevertheless, GRIP1a/b and GRIP1c4-7 could indirectly affect the retention of post-synaptic GABA A Rs by being involved in the post-synaptic accumulation of gephyrin at GABAergic synapses, as it has been shown that the post-synaptic clustering of gephyrin is important for the stabilization and retention of several types of GABA A Rs at GABAergic synapses (Essrich et al 1998;Kneussel et al 1999;Levi et al 2004;Jacob et al 2005;Yu et al 2007;Yu and De Blas 2008). The mechanism by which post-synaptic gephyrin stabilizes post-synaptic GABA A Rs is not understood as there is no evidence of direct biochemical interaction between GABA A Rs and gephyrin (Meyer et al 1995;Luscher and Keller 2004;Chen and Olsen 2007).…”

“…Immunofluorescence Double-or triple-label immunofluorescence of neurons (16-18 days in culture) or HEK293 cells was performed as described elsewhere (Christie et al 2002a(Christie et al , 2006Christie and De Blas 2003;Yu et al 2007;Yu and De Blas 2008). Briefly, fixed and Triton X-100-permeabilized cells were incubated with a mixture of primary antibodies followed by incubation with a mixture of secondary antibodies (anti-species specific IgG all made in donkey, Jackson Immunochemicals, West Grove, PA, USA) conjugated to FITC, Texas Red, or aminomethylcoumarin fluorophores.…”

Activity dependent therapies modulate the spinal changes that motoneurons suffer after a peripheral nerve injury

“…In parallel to the Vglut1 decrease, we observed a marked increase in gephyrin immunoreactivity around motoneurons. Gephyrin organizes the postsynaptic density of the inhibitory GABAergic and glycinergic receptors Tretter et al, 2008;Yu and De Blas, 2008), and it is a key inhibitory scaffolding protein. Up-regulation of gephyrin may be a protective action to compensate for the increased excitability observed in motoneurons after axonal injury.…”

Synaptic and nonsynaptic localization of protocadherin‐γC5 in the rat brain

“…Immunofluorescence is blocked by the antigenic peptide. In addition, the clusters show apposition to GABAergic presynaptic glutamate decarboxylase (GAD)-containing terminals (Christie et al, 2002a, 2002b; Christie and De Blas, 2003; Charych et al, 2004a, 2004b; Li et al, 2005a; Serwanski et al, 2006; Li et al, 2007; Yu and De Blas, 2008; Yu et al 2008; Li et al, 2009). …”