PSD95 suppresses dendritic arbor development in mature hippocampal neurons by occluding the clustering of NR2B-NMDA receptors.
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.
Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago, Chile; Faculty of Biological Science, Universidad de Concepción, Concepción, Chile; FONDAP Center for Genome Regulation, Santiago, Chile.
Fernando J
FJ
Bustos
Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago, Chile; Department of Molecular and Cellular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile.
Lorena
L
Varela-Nallar
Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago, Chile.
Matias
M
Campos
Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago, Chile.
Berta
B
Henriquez
McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
Marnie
M
Phillips
Faculty of Biological Science, Universidad de Concepción, Concepción, Chile.
Carlos
C
Opazo
Faculty of Biological Science, Universidad de Concepción, Concepción, Chile.
Luis G
LG
Aguayo
Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago, Chile; FONDAP Center for Genome Regulation, Santiago, Chile.
Martin
M
Montecino
McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
Martha
M
Constantine-Paton
Department of Molecular and Cellular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile.
Nibaldo C
NC
Inestrosa
Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago, Chile.
Brigitte
B
van Zundert
D000818:Animals; D003712:Dendrites / Q000378:metabolism*; D000076122:Disks Large Homolog 4 Protein; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D006624:Hippocampus / Q000166:cytology* / Q000196:embryology / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D061251:Primary Cell Culture; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits / Q000235:genetics; D017966:Pyramidal Cells / Q000378:metabolism*; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C121001:NR2B NMDA receptor; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate