Acute Inactivation of PSD-95 Destabilizes AMPA Receptors at Hippocampal Synapses

Guillermo A Yudowski,Kurt W Marek,David S Bredt,Olav Olsen,Hillel Adesnik,Jean-Pierre Mothet

doi:10.1371/journal.pone.0053965

Abstract

Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.

Highlights

Chemical synapses are highly specialized sites for communication between neurons
Expression of Kv1.4, PSD95 or postsynaptic density (PSD)-95-4c alone resulted in a perinuclear localization pattern for each protein (Fig. 1B)
Live cell analysis in COS cells co-expressing RFP-PSD-95-wt and Kv 1.4-CFP did not show redistribution of clusters after FlAsH incubation and illumination, demonstrating the specificity of the redistribution effect to the tetracysteine-containing construct (Figure S1). Taken together these results demonstrate that the PSD-95-4c forms clusters with Kv1.4 similar to those observed with the wild type PSD-95 and this localization is ablated in the PSD-95 tetracysteine-containing motif after FlAsH incubation and illumination

Summary

Introduction

Vesicles filled with neurotransmitter aggregate at the nerve terminal active zone. Neurotransmitter receptors and signaling molecules align with sites of presynaptic vesicle release in an electron-dense specialization, the postsynaptic density (PSD). Multivalent scaffolding proteins assemble the neurotransmitter receptors and signaling molecules into organized signal transduction pathways. One major family of postsynaptic scaffolding molecules is the membrane associated guanylate kinase proteins (MAGUKs). MAGUKs share a common core domain organization, consisting of three N-terminal PSD-95/Discs large/zona occludens-1 (PDZ) domains, a Src homology (SH3) domain, and a guanylate kinase (GK) domain [1]. PDZ domains often bind to the C-terminal tails of receptors, channels, and adhesion molecules [2,3,4]. The SH3 and GK domains bind receptors as well as other cytoplasmic molecules such as GKAP, MAP1A and SPAR1 [6]

Methods

Results

Conclusion