PSD95 Suppresses Dendritic Arbor Development in Mature Hippocampal Neurons by Occluding the Clustering of NR2B-NMDA Receptors

Fernando J Bustos,Martin Montecino,Marnie Phillips,Nibaldo C Inestrosa,Martha Constantine-Paton,Matias Campos,Lorena Varela-Nallar,Berta Henriquez,Carlos Opazo,Brigitte Van Zundert,Luis G Aguayo,Mohammed Akaaboune

doi:10.1371/journal.pone.0094037

Abstract

Considerable evidence indicates that the NMDA receptor (NMDAR) subunits NR2A and NR2B are critical mediators of synaptic plasticity and dendritogenesis; however, how they differentially regulate these processes is unclear. Here we investigate the roles of the NR2A and NR2B subunits, and of their scaffolding proteins PSD-95 and SAP102, in remodeling the dendritic architecture of developing hippocampal neurons (2–25 DIV). Analysis of the dendritic architecture and the temporal and spatial expression patterns of the NMDARs and anchoring proteins in immature cultures revealed a strong positive correlation between synaptic expression of the NR2B subunit and dendritogenesis. With maturation, the pruning of dendritic branches was paralleled by a strong reduction in overall and synaptic expression of NR2B, and a significant elevation in synaptic expression of NR2A and PSD95. Using constructs that alter the synaptic composition, we found that either over-expression of NR2B or knock-down of PSD95 by shRNA-PSD95 augmented dendritogenesis in immature neurons. Reactivation of dendritogenesis could also be achieved in mature cultured neurons, but required both manipulations simultaneously, and was accompanied by increased dendritic clustering of NR2B. Our results indicate that the developmental increase in synaptic expression of PSD95 obstructs the synaptic clustering of NR2B-NMDARs, and thereby restricts reactivation of dendritic branching. Experiments with shRNA-PSD95 and chimeric NR2A/NR2B constructs further revealed that C-terminus of the NR2B subunit (tail) was sufficient to induce robust dendritic branching in mature hippocampal neurons, and suggest that the NR2B tail is important in recruiting calcium-dependent signaling proteins and scaffolding proteins necessary for dendritogenesis.

Highlights

Before the dendritic arbor stabilizes in the mature CNS and dendritic spines are formed to allow communication between neurons, large-scale neuronal morphological changes occur during the first weeks of postnatal development that include growth of dendritic branches followed by elimination of excessive and mis-targeted branches [1]
We provide evidence that developmental increases in synaptic expression of PSD95 obstruct the clustering of NR2B-NMDA receptor (NMDAR) at postsynaptic sites, thereby restricting the reactivation of dendritic branching
We show that knock-down of PSD95 results in increased clustering of exogenous NR2B, and restores the dendritic branching capacity of mature neurons to a state that is normally seen only in early development (7 DIV)

Summary

Introduction

Before the dendritic arbor stabilizes in the mature CNS and dendritic spines are formed to allow communication between neurons, large-scale neuronal morphological changes occur during the first weeks of postnatal development that include growth of dendritic branches followed by elimination (pruning) of excessive and mis-targeted branches [1]. We do know from comparative studies that PSD95 and SAP102 can have distinctly different binding partners, spine distribution, mobility, and temporal expression patterns [4,10,12,13,14]; but see [15]). These and other studies led us to hypothesize that synaptic insertion of PSD95/NR2A-NMDARs complexes during development displaces SAP102/NR2BNMDARs complexes, and thereby limits plasticity [4]. Use of NMDAR chimera constructs further indicates that the C-terminal of NR2B (tail) is critical in dendritogenesis

Results

Discussion

Experimental Procedures