Synaptic Localization Of NMDA Receptors Research Articles

Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

Isoflurane impairs GluN2B-containing NMDA receptors trafficking and cognition via decreasing histone acetylation and EphB2 expression in aged hippocampal neurons.

Perioperative neurocognitive disorders (PND) is a common complication that occurs among elderly patients in the perioperative course. Current clinical evidence has shown that isoflurane exposure could cause cognitive decline, but the exact molecular mechanisms remain unclear. As both NMDARs-dependent synaptic plasticity and histone acetylation play vital roles in processing learning and memory, we postulated that these alternations might occur in the isoflurane-associated PND. Here, we found that isoflurane impaired fear memory in aged mice, decreased GluN2B-containing NMDA receptors phosphorylation and trafficking, as well as the expression of EphB2, a key regulator of synaptic localization of NMDA receptors. We also identified that isoflurane could increase the expression of HDAC2, which was significantly enriched at the ephb2 gene promoter and regulated the transcription of ephb2. Furthermore, we showed that suberoylanilide hydroxamic acid (SAHA), a nonselective HDAC inhibitor or knocking-down HDAC2 rescued the cognitive dysfunction in isoflurane-treated aged mice via increasing acetylation of H3Ac, expression of EphB2 and promoting NMDA receptor trafficking. Collectively, our study highlighted the crucial role of histone posttranslational modifications for EphB2-GluN2B signals in isoflurane-associated PND, and modulating HDAC2 might be a new therapeutic strategy for isoflurane-associated PND.

Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain.

Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function.

Tau-mediated NMDA receptor impairment underlies dysfunction of a selectively vulnerable network in a mouse model of frontotemporal dementia.

Frontotemporal dementia (FTD) is a neurodegenerative behavioral disorder that selectively affects the salience network, including the ventral striatum and insula. Tau mutations cause FTD, but how mutant tau impairs the salience network is unknown. Here, we address this question using a mouse model expressing the entire human tau gene with an FTD-associated mutation (V337M). Mutant, but not wild-type, human tau transgenic mice had aging-dependent repetitive and disinhibited behaviors, with synaptic deficits selectively in the ventral striatum and insula. There, mutant tau depleted PSD-95, resulting in smaller postsynaptic densities and impaired synaptic localization of NMDA receptors (NMDARs). In the ventral striatum, decreased NMDAR-mediated transmission reduced striatal neuron firing. Pharmacologically enhancing NMDAR function with the NMDAR co-agonist cycloserine reversed electrophysiological and behavioral deficits. These results indicate that NMDAR hypofunction critically contributes to FTD-associated behavioral and electrophysiological alterations and that this process can be therapeutically targeted by a Food and Drug Administration-approved drug.

SAP97 directs NMDA receptor spine targeting and synaptic plasticity

SAP97 is a multidomain scaffold protein implicated in the forward trafficking and synaptic localization of NMDA- and AMPA-type glutamate receptors. Alternative splicing of SAP97 transcripts gives rise to palmitoylated αSAP97 and L27-domain containing βSAP97 isoforms that differentially regulate the subsynaptic localization of GluR1 subunits of AMPA receptors. Here, we examined whether SAP97 isoforms regulate the mechanisms underlying long-term potentiation (LTP) and depression (LTD) and find that both α- and β-forms of SAP97 impair LTP but enhance LTD via independent isoform-specific mechanisms. Live imaging of α- and βSAP97 revealed that the altered synaptic plasticity was not due to activity-dependent changes in SAP97 localization or exchange kinetics. However, by recording from pairs of synaptically coupled hippocampal neurons, we show that αSAP97 occludes LTP by enhancing the levels of postsynaptic AMPA receptors, while βSAP97 blocks LTP by reducing the synaptic localization of NMDA receptors. Examination of the surface pools of AMPA and NMDA receptors indicates that αSAP97 selectively regulates the synaptic pool of AMPA receptors, whereas βSAP97 regulates the extrasynaptic pools of both AMPA and NMDA receptors. Knockdown of βSAP97 increases the synaptic localization of both AMPA and NMDA receptors, showing that endogenous βSAP97 restricts glutamate receptor expression at excitatory synapses. This isoform-dependent differential regulation of synaptic versus extrasynaptic pools of glutamate receptors will determine how many receptors are available for the induction and the expression of synaptic plasticity. Our data support a model wherein SAP97 isoforms can regulate the ability of synapses to undergo plasticity by controlling the surface distribution of AMPA and NMDA receptors.

Activity dependent localization of synaptic NMDA receptors in spinal neurons

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.

The Synaptic Localization of NR2B-Containing NMDA Receptors Is Controlled by Interactions with PDZ Proteins and AP-2

The NMDA receptor (NMDAR) is a component of excitatory synapses and a key participant in synaptic plasticity. We investigated the role of two domains in the C terminus of the NR2B subunit--the PDZ binding domain and the clathrin adaptor protein (AP-2) binding motif--in the synaptic localization of NMDA receptors. NR2B subunits lacking functional PDZ binding are excluded from the synapse. Mutations in the AP-2 binding motif, YEKL, significantly increase the number of synaptic receptors and allow the synaptic localization of NR2B subunits lacking PDZ binding. Peptides corresponding to YEKL increase the synaptic response within minutes. In contrast, the NR2A subunit localizes to the synapse in the absence of PDZ binding and is not altered by mutations in its motif corresponding to YEKL of NR2B. This study identifies a dynamic regulation of synaptic NR2B-containing NMDARs through PDZ protein-mediated stabilization and AP-2-mediated internalization that is modulated by phosphorylation by Fyn kinase.

Alterations in NMDA receptor expression during retinal degeneration in the RCS rat.

To determine how a progressive loss of photoreceptor cells and the concomitant loss of glutamatergic input to second-order neurons can affect inner-retinal signaling, glutamate receptor expression was analyzed in the Royal College of Surgeons (RCS) rat, an animal model of retinitis pigmentosa. Immunohistochemistry was performed on retinal sections of RCS rats and congenic controls between postnatal (P) day 3 and the aged adult (up to P350) using specific antibodies against N-methyl-D-aspartate (NMDA) subunits. All NMDA subunits (NR1, NR2A-2D) were expressed in control and dystrophic retinas at all ages, and distinct patterns of labeling were found in horizontal cells, subpopulations of amacrine cells and ganglion cells, as well as in the outer and inner plexiform layer (IPL). NRI immunoreactivity in the inner plexiform layer of adult control retinas was concentrated in two distinct bands, indicating a synaptic localization of NMDA receptors in the OFF and ON signal pathways. In the RCS retina, these bands of NRI immunoreactivity in the IPL were much weaker in animals older than P40. In parallel, NR2B immunoreactivity in the outer plexiform layer (OPL) of RCS rats was always reduced compared to controls and vanished between P40 and P120. The most striking alteration observed in the degenerating retina, however, was a strong expression of NRI immunoreactivity in Müller cell processes in the inner retina which was not observed in control animals and which was present prior to any visible sign of photoreceptor degeneration. The results suggest functional changes in glutamatergic receptor signaling in the dystrophic retina and a possible involvement of Müller cells in early processes of this disease.

Microtubule binding by CRIPT and its potential role in the synaptic clustering of PSD-95.

CRIPT is a postsynaptic protein that binds selectively to the third PDZ domain (PDZ3) of PSD-95. Here we show that CRIPT also binds directly to microtubules, thereby linking PSD-95 to the microtubule cytoskeleton. Disrupting the CRIPT-PSD-95 interaction in cultured hippocampal neurons with a PDZ3-specific peptide prevented the association of PSD-95 with microtubules and inhibited the synaptic clustering of PSD-95, chapsyn-110/PSD-93 and GKAP (a PSD-95-binding protein). However, the number of synapses and the synaptic clustering of NMDA receptors were unaffected, suggesting that PSD-95-family proteins are not essential for the maintenance of synapses and the synaptic localization of NMDA receptors.

Heterogeneity in the Molecular Composition of Excitatory Postsynaptic Sites during Development of Hippocampal Neurons in Culture

To determine their roles in the assembly of glutamatergic postsynaptic sites, we studied the distributions of NMDA- and AMPA-type glutamate receptors; the NMDA receptor-interacting proteins alpha-actinin-2, PSD-95, and chapsyn; and the PSD-95-associated protein GKAP during the development of hippocampal neurons in culture. NMDA receptors first formed nonsynaptic proximal dendrite shaft clusters within 2-5 d. AMPA receptors were diffuse at this stage and began to cluster on spines at 9-10 d. NMDA receptor clusters remained partially nonsynaptic and mainly distinct from AMPA receptor clusters until after 3 weeks in culture, when the two began to colocalize at spiny synaptic sites. Thus, the localization of NMDA and AMPA receptors must be regulated by different mechanisms. alpha-Actinin-2 colocalized with the NMDA receptor only at spiny synaptic clusters, but not at shaft nonsynaptic or synaptic clusters, suggesting a modulatory role in the anchoring of NMDA receptor at spines. PSD-95, chapsyn, and GKAP were present at some, but not all, nonsynaptic NMDA receptor clusters during the first 2 weeks, indicating that none is essential for NMDA receptor cluster formation. When NMDA receptor clusters became synaptic, PSD-95 and GKAP were always present, consistent with an essential function in synaptic localization of NMDA receptors. Furthermore, PSD-95 and GKAP clustered opposite presynaptic terminals several days before either NMDA or AMPA receptors clustered at these presumptive postsynaptic sites. These results suggest that synapse development proceeds by formation of a postsynaptic scaffold containing PSD-95 and GKAP in concert with presynaptic vesicle clustering, followed by regulated attachment of glutamate receptor subtypes to this scaffold.

Activity Regulates the Synaptic Localization of the NMDA Receptor in Hippocampal Neurons

We describe here a novel effect of activity on the subcellular distribution of NMDA receptors in hippocampal neurons in culture. In spontaneously active neurons, NMDA receptors were clustered at a few synaptic and nonsynaptic sites. Chronic blockade of NMDA receptor activity induced a 380% increase in the number of NMDA receptor clusters and a shift to a more synaptic distribution. This effect was reversible. The distributions of the presynaptic marker synaptophysin, the AMPA-type glutamate receptor subunit GluR1, and the putative NMDA receptor clustering protein PSD-95 were not affected by blockade. Regulation of the synaptic localization of NMDA receptors by activity may define a novel mechanism by which input controls a neuron's ability to modify its synapses.