ABSTRACT: Platelet-derived growth factor (PDGF) beta receptor activation inhibits N-methyl-d-aspartate (NMDA)-evoked currents in hippocampal and cortical neurons via the activation of phospholipase Cgamma, PKC, the release of intracellular calcium, and a rearrangement of the actin cytoskeleton. In the hippocampus, the majority of NMDA receptors are heteromeric; most are composed of 2 NR1 subunits and 2 NR2A or 2 NR2B subunits. Using NR2B- and NR2A-specific antagonists, we demonstrate that PDGF-BB treatment preferentially inhibits NR2B-containing NMDA receptor currents in CA1 hippocampal neurons and enhances long-term depression in an NR2B subunit-dependent manner. Furthermore, treatment of hippocampal slices or cultures with PDGF-BB decreases the surface localization of NR2B but not of NR2A subunits. PDGFbeta receptors colocalize to a higher degree with NR2B subunits than with NR2A subunits. After neuronal injury, PDGFbeta receptors and PDGF-BB are up-regulated and PDGFbeta receptor activation is neuroprotective against glutamate-induced neuronal damage in cultured neurons. We demonstrate that the neuroprotective effects of PDGF-BB are occluded by the NR2B antagonist, Ro25-6981, and that PDGF-BB promotes NMDA signaling to CREB and ERK1/2. We conclude that PDGFbetaR signaling, by preferentially targeting NR2B receptors, provides an important mechanism for neuroprotection by growth factors in the central nervous system.
Project description:N-Methyl-d-aspartate (NMDA) receptors play critical roles in complex brain functions as well as pathogenesis of neurodegenerative diseases. There are many NMDA isoforms and subunit types that, together with subtype-specific assembly, give rise to significant functional heterogeneity of NMDA receptors. Conventional NMDA receptors are obligatory heterotetramers composed of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits. When individually expressed in heterogeneous cells, most of the NR1 splice variants and the NR2 subunits remain in the endoplasmic reticulum (ER) and do not form homomeric channels. The mechanisms underlying NMDA receptor trafficking and functional expression remain uncertain. Using truncated and chimeric NMDA receptor subunits expressed in heterogeneous cells and hippocampal neurons, together with immunostaining, biochemical, and functional analyses, we found that the NR2A amino-terminal domain (ATD) contains an ER retention signal, which can be specifically masked by the NR1a ATD. Interestingly, no such signal was found in the ATD of the NR2B subunit. We further identified the A2 segment of the NR2A ATD to be the primary determinant of ER retention. These findings indicate that NR2A-containing NMDA receptors may undergo a different ER quality control process from NR2B-containing NMDA receptors.
Project description:The NR2 subunit composition of NMDA receptors (NMDARs) varies during development, and this change is important in NMDAR-dependent signaling. In particular, synaptic NMDAR switch from containing mostly NR2B subunit to a mixture of NR2B and NR2A subunits. The pathways by which neurons differentially traffic NR2A- and NR2B-containing NMDARs are poorly understood. Using single-particle and -molecule approaches and specific antibodies directed against NR2A and NR2B extracellular epitopes, we investigated the surface mobility of native NR2A and NR2B subunits at the surface of cultured neurons. The surface mobility of NMDARs depends on the NR2 subunit subtype, with NR2A-containing NMDARs being more stable than NR2B-containing ones, and NR2A subunit overexpression stabilizes surface NR2B-containing NMDARs. The developmental change in the synaptic surface content of NR2A and NR2B subunits was correlated with a developmental change in the time spent by the subunits within synapses. This suggests that the switch in synaptic NMDAR subtypes depends on the regulation of the receptor surface trafficking.
Project description:N-Methyl-d-aspartate (NMDA) receptors are expressed at excitatory synapses throughout the brain and are essential for neuronal development and synaptic plasticity. Functional NMDA receptors are tetramers, typically composed of NR1 and NR2 subunits (NR2A-D). NR2A and NR2B are expressed in the forebrain and are thought to assemble as diheteromers (NR1/NR2A, NR1/NR2B) and triheteromers (NR1/NR2A/NR2B). NR2A and NR2B contain cytosolic domains that regulate distinct postendocytic sorting events, with NR2A sorting predominantly into the degradation pathway, and NR2B preferentially trafficking through the recycling pathway. However, the interplay between these two subunits remains an open question. We have now developed a novel approach based on the dimeric feature of the alpha- and beta-chains of the human major histocompatibility complex class II molecule. We created chimeras of alpha- and beta-chains with the NR2A and NR2B C termini and evaluated endocytosis of dimers. Like chimeric proteins containing only a single NR2A or NR2B C-terminal domain, major histocompatibility complex class II-NR2A homodimers sort predominantly to late endosomes, whereas NR2B homodimers traffic to recycling endosomes. Interestingly, NR2A/NR2B heterodimers traffic preferentially through the recycling pathway, and NR2B is dominant in regulating dimer trafficking in both heterologous cells and neurons. In addition, the recycling of NR2B-containing NMDARs in wild-type neurons is not significantly different from NR2A(-/-) neurons. These data support a dominant role for NR2B in regulating the trafficking of triheteromeric NMDARs in vivo. Furthermore, our molecular approach allows for the direct and selective evaluation of dimeric assemblies and can be used to define dominant trafficking domains in other multisubunit protein complexes.
Project description:In cultured spinal neurons, NMDA receptors are absent from excitatory synapses under basal conditions, but can be made to appear at excitatory synapses following blockade of excitatory synaptic activity. The activity dependent synaptic localization of NMDA receptors is critically dependent on both the gradual, global accumulation of the NR2A and NR2B subunits and on a rapid, surface redistribution phase that is primed by the accumulation of NR2A and NR2B and inhibited by synaptic activity. Global changes in NR2A and NR2B accumulation and heterogeneous increases in synaptic NMDA receptor localization can also result from inhibitors of proteasomal processing, from manipulations of proteasomal subunit composition and from media conditioned by neurons undergoing synaptic scaling. While proteasomal processing is a mechanism shared with AMPA receptor scaling in cultured spinal neurons, diffusible factors, heterogeneity, and a rapid surface redistribution phase appear to be unique to activity dependent synaptic NMDA receptor localization.
Project description:NMDA receptors (NMDARs) are important for neuronal development and circuit formation. The NMDAR subunits NR2A and NR2B are biophysically distinct and differentially expressed during development but their individual contribution to structural plasticity is unknown. Here we test whether NR2A and NR2B subunits have specific functions in the morphological development of tectal neurons in living Xenopus tadpoles. We use exogenous subunit expression and endogenous subunit knockdown to shift synaptic NMDAR composition toward NR2A or NR2B, as shown electrophysiologically. We analyzed the dendritic arbor structure and found evidence for both overlapping and distinct functions of NR2A and NR2B in dendritic development. Control neurons develop regions of high local branch density in their dendritic arbor, which may be important for processing topographically organized inputs. Exogenous expression of either NR2A or NR2B decreases local branch clusters, indicating a requirement for both subunits in dendritic arbor development. Knockdown of endogenous NR2A reduces local branch clusters, whereas knockdown of NR2B has no effect on branch clustering. Analysis of the underlying branch dynamics shows that exogenous NR2B-expressing neurons are more dynamic than control or exogenous NR2A-expressing neurons, demonstrating subunit-specific regulation of branch dynamics. Visual experience-dependent increases in dendritic arbor growth rate seen in control neurons are blocked in both exogenous NR2A- and NR2B-expressing neurons. These experiments indicate that NR2A and NR2B have subunit-specific properties in dendritic arbor development, but also overlapping functions, indicating a requirement for both subunits in neuronal development.
Project description:<h4>Background</h4>NMDA-type glutamate receptors (NMDARs) are major contributors to long-term potentiation (LTP), a form of synaptic plasticity implicated in the process of learning and memory. These receptors consist of calcium-permeating NR1 and multiple regulatory NR2 subunits. A majority of studies show that both NR2A and NR2B-containing NMDARs can contribute to LTP, but their unique contributions to this form of synaptic plasticity remain poorly understood.<h4>Methodology/principal findings</h4>In this study, we show that NR2A and NR2B-containing receptors promote LTP differently in the CA1 hippocampus of 1-month old mice, with the NR2A receptors functioning through Ras-GRF2 and its downstream effector, Erk Map kinase, and NR2B receptors functioning independently of these signaling molecules.<h4>Conclusions/significance</h4>This study demonstrates that NR2A-, but not NR2B, containing NMDA receptors induce LTP in pyramidal neurons of the CA1 hippocampus from 1 month old mice through Ras-GRF2 and Erk. This difference add new significance to the observation that the relative levels of these NMDAR subtypes is regulated in neurons, such that NR2A-containing receptors become more prominent late in postnatal development, after sensory experience and synaptic activity.
Project description: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.
Project description:Activation of NMDA receptors (NMDAR) in the hippocampus is essential for the formation of contextual and trace memory. However, the role of individual NMDAR subunits in the molecular mechanisms contributing to these memory processes is not known. Here we demonstrate, using intrahippocampal injection of subunit-selective compounds, that the NR2A-preferring antagonist impaired contextual and trace fear conditioning as well as learning-induced increase of the nuclear protein c-Fos. The NR2B-specific antagonist, on the other hand, selectively blocked trace fear conditioning without affecting c-Fos levels. Studies with cultured primary hippocampal neurons, further showed that synaptic and extrasynaptic NR2A and NR2B differentially regulate the extracellular signal-regulated kinase 1 and 2/mitogen- and stress-activated protein kinase 1 (ERK1/2/MSK1)/c-Fos pathway. Activation of the synaptic population of NMDAR induced cytosolic, cytoskeletal, and perinuclear phosphorylation of ERK1/2 (pERK1/2). The nuclear propagation of pERK1/2 signals, revealed by upregulation of the downstream nuclear targets pMSK1 and c-Fos, was blocked by a preferential NR2A but not by a specific NR2B antagonist. Conversely, activation of total (synaptic and extrasynaptic) NMDAR engaged receptors with NR2B subunits, and resulted in membrane retention of pERK1/2 without inducing pMSK1 and c-Fos. Stimulation of extrasynaptic NMDAR alone was consistently ineffective at activating ERK signaling. The discrete contribution of synaptic and total NR2A- and NR2B-containing NMDAR to nuclear transmission vs. membrane retention of ERK signaling may underlie their specific roles in the formation of contextual and trace fear memory.
Project description:BACKGROUND: The N-methyl-D-aspartate receptors are key mediators of excitatory transmission and are implicated in many forms of synaptic plasticity. These receptors are heterotetrameres consisting of two obligatory NR1 and two regulatory subunits, usually NR2A or NR2B. The NR2B subunits are abundant in the early postnatal brain, while the NR2A/NR2B ratio increases during early postnatal development. This shift is driven by NMDA receptor activity. A functional interplay of the Low Density Lipoprotein Receptor Related Protein 1 (LRP1) NMDA receptor has already been reported. Such abilities as interaction of LRP1 with NMDA receptor subunits or its important role in tPa-mediated NMDA receptor signaling were already demonstrated. Moreover, mice harboring a conditional neuronal knock-out mutation of the entire Lrp1 gene display NMDA-associated behavioral changes. However, the exact role of LRP1 on NMDA receptor function remains still elusive. RESULTS: To provide a mechanistic explanation for such effects we investigated whether an inactivating knock-in mutation into the NPxY2 motif of LRP1 might influence the cell surface expression of LRP1 and NMDA receptors in primary cortical neurons. Here we demonstrate that a knock-in into the NPxY2 motif of LRP1 results in an increased surface expression of LRP1 and NR2B NMDA receptor subunit due to reduced endocytosis rates of LRP1 and the NR2B subunit in primary neurons derived from LRP1?NPxY2 animals. Furthermore, we demonstrate an altered phosphorylation pattern of S1480 and Y1472 in the NR2B subunit at the surface of LRP1?NPxY2 neurons, while the respective kinases Fyn and casein kinase II are not differently regulated compared with wild type controls. Performing co-immunoprecipitation experiments we demonstrate that binding of LRP1 to NR2B might be linked by PSD95, is phosphorylation dependent and this regulation mechanism is impaired in LRP1?NPxY2 neurons. Finally, we demonstrate hyperactivity and changes in spatial and reversal learning in LRP1?NPxY2 mice, confirming the mechanistic interaction in a physiological readout. CONCLUSIONS: In summary, our data demonstrate that LRP1 plays a critical role in the regulation of NR2B expression at the cell surface and may provide a mechanistic explanation for the behavioral abnormalities detected in neuronal LRP1 knock-out animals reported earlier.
Project description:Diphtheria toxin-mediated, acute ablation of hypothalamic neurons expressing agouti-related protein (AgRP) in adult mice leads to anorexia and starvation within 7 d that is caused by hyperactivity of neurons within the parabrachial nucleus (PBN). Because NMDA glutamate receptors are involved in various synaptic plasticity-based behavioral modifications, we hypothesized that modulation of the NR2A and NR2B subunits of the NMDA receptor in PBN neurons could contribute to the anorexia phenotype. We observed by Western blot analyses that ablation of AgRP neurons results in enhanced expression of NR2B along with a modest suppression of NR2A. Interestingly, systemic administration of LiCl in a critical time window before AgRP neuron ablation abolished the anorectic response. LiCl treatment suppressed NR2B levels in the PBN and ameliorated the local Fos induction that is associated with anorexia. This protective role of LiCl on feeding was blunted in vagotomized mice. Chronic infusion of RO25-6981, a selective NR2B inhibitor, into the PBN recapitulated the role of LiCl in maintaining feeding after AgRP neuron ablation. We suggest that the accumulation of NR2B subunits in the PBN contributes to aphagia in response to AgRP neuron ablation and may be involved in other forms of anorexia.