Ontogeny of postsynaptic density proteins at glutamatergic synapses

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Abstract

In glutamatergic synapses, glutamate receptors (GluRs) associate with many other proteins involved in scaffolding and signal transduction. The ontogeny of these postsynaptic density (PSD) proteins involves changes in their composition during development, paralleling changes in GluR type and function. In the CA1 region of the hippocampus, at postnatal day 2 (P2), many synapses already have a distinct PSD. We used immunoblot analysis, subcellular fractionation, and quantitative immunogold electron microscopy to examine the distribution of PSD proteins during development of the hippocampus. Synapses at P2 contained substantial levels of NR1 and NR2B and most GluR-associated proteins, including SAP102, SynGAP, the chain of proteins from GluRs/SAP102 through GKAP/Shank/Homer and metabotropic glutamate receptors, and the adhesion factors, cadherin, catenin, neuroligin, and Nr-CAM. Development was marked by substantial decreases in NR2B and SAP102 and increases in NR2A, PSD-95, AMPA receptors, and CaMKII. Other components showed more moderate changes.

Introduction

Postsynaptic density (PSD) proteins are necessary for excitatory glutamatergic postsynaptic responses, and include scaffolding and functional elements that modulate/mediate signal transduction and trafficking of synaptic proteins (reviewed by Bredt and Nicoll, 2003, Ehrengruber et al., 2004, Sheng and Kim, 2002, Wenthold et al., 2003). In addition to glutamate receptors (GluRs) such as α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptors, numerous other proteins associate with GluRs in the postsynaptic membrane and PSD. GluRs associate via their C-termini with anchoring proteins including membrane-associated guanylate kinases (MAGUKs). The best-studied MAGUKs belong to the PSD-95 group, containing PSD-95, PSD-93, SAP102, and SAP97. Both NMDA and AMPA receptors can bind directly to MAGUKs; AMPA receptors also can bind to them indirectly via stargazin and other transmembrane AMPA receptor regulatory proteins (TARPs). Other associated proteins that affect GluR distribution and function at the synapse include tyrosine receptor kinase B (TrkB), calmodulin-dependent protein kinase II (CaMKII), proteins binding to MAGUKs such as synaptic GTPase-activating protein (SynGAP), spine-associated RapGAP (SPAR), guanylate kinase-associated protein (GKAP), proteins forming links between MAGUKs/GKAP through Shank and Homer dimers to metabotropic glutamate receptors (mGluRs) and receptors regulating internal calcium stores, plus a number of adhesion proteins including the cadherin/catenin complex, neuroligin (which can bind to MAGUKs), and cell-adhesion molecules (CAMs) such as NCAM, L1, and Nr-CAM.

Most studies of these various proteins have concentrated on adult synapses in vivo and developing synapses in vitro, while surprisingly little is known about ontogeny of these proteins in synapses in vivo (see reviews by Garner et al., 2002, Goda and Davis, 2003, Li and Sheng, 2003, Ziv and Garner, 2004). A basic question that needs to be addressed is: Do changes in protein composition of the PSD, during postnatal development, involve mainly changes in amounts of most proteins, or are there radical changes in protein types (e.g., replacement of one receptor subtype for another), as needed during different stages of development? In this study, we examined the developmental changes in GluRs and several associated proteins in synapses. Previous studies showed that (1) NMDA receptors are high from P2-adult, while AMPA receptors increase with age (Petralia et al., 1999), and (2) the NMDA receptor-associated MAGUK, SAP102, decreases with age while other MAGUKs, PSD-95 and PSD-93, increase with age (Sans et al., 2000). Here, we show that those early postnatal synapses that display ultrastructural signs of maturity already have acquired substantial levels of many GluR-associated proteins. Further postnatal development involves major reciprocal switches in levels of similar proteins (SAP102 versus PSD-95; NR2B versus NR2A) and moderate increases or decreases in other proteins. This study provides immunogold data for the hippocampus, showing the developmental switch in NMDA receptor type, and showing that developmental changes occur for other proteins that are associated functionally with these receptors and that contribute to the maturation of learning and memory.

Section snippets

Immunoblots

Immunoblots of the developing hippocampus showed at least 3 trends (Fig. 1): (1) protein already abundant before birth, for the adhesion proteins, N-cadherin, β-catenin, and Nr-CAM, and for Shank and Homer; (2) protein present before birth and abundant by P2, for neuroligin, NR1, NR2B (i.e., as extrapolated from findings for NR2A/B [high] and NR2A [very low]), TrkB, SAP102, and mGluR5; (3) protein increases from little or nothing at P2 to high levels in adult, for NR2A, PSD-95, SAP97, and

Discussion

This study shows that major changes occur in GluR-associated proteins during development of synapses in CA1 stratum radiatum of the hippocampus. Intercellular adhesion proteins showed the greatest abundance in immunoblot in late embryonic and early postnatal hippocampus relative to later ages, of proteins studied here. In addition, a comparison of immature- and mature-appearing synapses at P2, for 4 of the proteins in this study, showed that only the adhesion protein, catenin, was prevalent in

Antibodies and their characterization

Data from 27 antibodies are presented in the immunochemical and immunocytochemical experiments in this study. Of these, the 24 antibodies used for new immunogold experiments carried out in this study have been well-characterized in previous studies, using mainly light microscope level examination and, in some cases, immunogold localization at synapses in adults.

Mouse NR2A and NR2B antibodies (affinity-purified; C-terminal, i.e., recognizes intracellular domain) were characterized in general and

Acknowledgments

We are most grateful to all of those who provided polyclonal antibodies including: NR2A and NR2B (actually the equivalent mouse subunits, GluRɛ1 and GluRɛ2)/Masahiko Watanabe; TrkB/Kuo Wu; Shank and Homer/Paul Worley; GKAP/Morgan Sheng; SPAR/Daniel T.S. Pak; one of the antibodies to SynGAP/Richard L. Huganir; SAP97 and SAP102/Johannes Hell; one antibody to Nr-CAM (837)/Martin Grumet; and another to Nr-CAM as well as a monoclonal to L1/Catherine Faivre-Sarrailh. We also thank Lilly Kan for

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