04-901 Sigma-AldrichAnti-NR2A Antibody, clone A12W, rabbit monoclonal

The N-methyl-D-aspartate (NMDA) receptor has long been associated with learning and memory processes as well as diseased states, particularly in schizophrenia (SZ). Additionally, SZ is increasingly recognized as a neurodevelopmental disorder with cognitive impairments often preceding the onset of psychosis. However, the cause of these cognitive deficits and what initiates the pathological process is unknown. Growing evidence has implicated the glutamate system and, in particular, N-methyl-D-aspartate receptor (NMDAR) dysfunction in the pathophysiology of SZ. Yet, the vast majority of SZ-related research has focused on NMDAR function in adults leaving the role of NMDARs during development uncharacterized. We used the prenatal methylazoxymethanol acetate (MAM, E17) exposure model to determine the alterations of NMDAR protein levels and function, as well as associated cognitive deficits during development. We found that MAM-exposed animals have significantly altered NMDAR protein levels and function in the juvenile and adolescent hippocampus. Furthermore, these changes are associated with learning and memory deficits in the Morris Water Maze. Thus, in the prenatal MAM-exposure SZ model, NMDAR expression and function is altered during the critical period of hippocampal development. These changes may be involved in disease initiation and cognitive impairment in the early stage of SZ.

Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. All three ionotropic glutamate subfamilies (i.e. AMPA-type, kainate-type, and NMDA-type) assemble as tetramers of four homologous subunits. There is good evidence that both heteromeric AMPA and kainate receptors have a 2:2 subunit stoichiometry and an alternating subunit arrangement. Recent studies based on presumed structural homology have indicated that NMDA receptors adopt the same arrangement. Here, we use atomic force microscopy imaging of receptor-antibody complexes to show that whereas the GluA1/GluA2 AMPA receptor assembles with an alternating (i.e. 1/2/1/2) subunit arrangement, the GluN1/GluN2A NMDA receptor adopts an adjacent (i.e. 1/1/2/2) arrangement. We conclude that the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. This surprising finding necessitates a reassessment of the assembly of these important receptors.

NMDA receptors are widely expressed in the central nervous system and play a major role in excitatory synaptic transmission and plasticity. Here, we used atomic force microscopy (AFM) imaging to visualize activation-induced structural changes in the GluN1/GluN2A NMDA receptor reconstituted into a lipid bilayer. In the absence of agonist, AFM imaging revealed two populations of particles with heights above the bilayer surface of 8.6 and 3.4 nm. The taller, but not the shorter, particles could be specifically decorated by an anti-GluN1 antibody, which recognizes the S2 segment of the agonist-binding domain, indicating that the two populations represent the extracellular and intracellular regions of the receptor, respectively. In the presence of glycine and glutamate, there was a reduction in the height of the extracellular region to 7.3 nm. In contrast, the height of the intracellular domain was unaffected. Fast-scan AFM imaging combined with UV photolysis of caged glutamate permitted the detection of a rapid reduction in the height of individual NMDA receptors. The reduction in height did not occur in the absence of the co-agonist glycine or in the presence of the selective NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid, indicating that the observed structural change was caused by receptor activation. These results represent the first demonstration of an activation-induced effect on the structure of the NMDA receptor at the single-molecule level. A change in receptor size following activation could have important functional implications, in particular by affecting interactions between the NMDA receptor and its extracellular synaptic partners.

Membrane-associated guanylate kinases (MAGUKs), including SAP102, PSD-95, PSD-93, and SAP97, are scaffolding proteins for ionotropic glutamate receptors at excitatory synapses. MAGUKs play critical roles in synaptic plasticity; however, details of signaling roles for each MAGUK remain largely unknown. Here we report that SAP102 regulates cortical synapse development through the EphB and PAK signaling pathways. Using lentivirus-delivered shRNAs, we found that SAP102 and PSD-95, but not PSD-93, are necessary for excitatory synapse formation and synaptic AMPA receptor (AMPAR) localization in developing mouse cortical neurons. SAP102 knockdown (KD) increased numbers of elongated dendritic filopodia, which is often observed in mouse models and human patients with mental retardation. Further analysis revealed that SAP102 coimmunoprecipitated the receptor tyrosine kinase EphB2 and RacGEF Kalirin-7 in neonatal cortex, and SAP102 KD reduced surface expression and dendritic localization of EphB. Moreover, SAP102 KD prevented reorganization of actin filaments, synapse formation, and synaptic AMPAR trafficking in response to EphB activation triggered by its ligand ephrinB. Last, p21-activated kinases (PAKs) were downregulated in SAP102 KD neurons. These results demonstrate that SAP102 has unique roles in cortical synapse development by mediating EphB and its downstream PAK signaling pathway. Both SAP102 and PAKs are associated with X-linked mental retardation in humans; thus, synapse formation mediated by EphB/SAP102/PAK signaling in the early postnatal brain may be crucial for cognitive development.

Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth.

The ability of dendritic spines to change size and shape rapidly is critical in modulating synaptic strength; these morphological changes are dependent upon rearrangements of the actin cytoskeleton. Kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor localized to the postsynaptic density (PSD), modulates dendritic spine morphology in vitro and in vivo. Kal7 activates Rac and interacts with several PSD proteins, including PSD-95, DISC-1, AF-6, and Arf6. Mice genetically lacking Kal7 (Kal7(KO)) exhibit deficient hippocampal long-term potentiation (LTP) as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7(KO) mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7(KO) animals have decreased levels of NR2B-dependent NMDA receptor currents in cortical pyramidal neurons as well as a specific deficit in cell surface expression of NR2B. Additionally, we demonstrate that the genotypic differences in conditioned place preference and passive avoidance learning seen in Kal7(KO) mice are abrogated when animals are treated with an NR2B-specific antagonist during conditioning. Finally, we identify a stable interaction between the pleckstrin homology domain of Kal7 and the juxtamembrane region of NR2B preceding its cytosolic C-terminal domain. Binding of NR2B to a protein that modulates the actin cytoskeleton is important, as NMDA receptors require actin integrity for synaptic localization and function. These studies demonstrate a novel and functionally important interaction between the NR2B subunit of the NMDA receptor and Kalirin, proteins known to be essential for normal synaptic plasticity.