Joana S. Ferreira

GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome

NMDA receptors play a central role in shaping the strength of synaptic connections throughout development and in mediating synaptic plasticity mechanisms that underlie some forms of learning and memory formation in the CNS. In the hippocampus and the neocortex, GluN1 is combined primarily with GluN2A and GluN2B, which are differentially expressed during development and confer distinct molecular and physiological properties to NMDA receptors. The contribution of each subunit to the synaptic traffic of NMDA receptors and therefore to their role during development and in synaptic plasticity is still controversial. We report a critical role for the GluN2B subunit in regulating NMDA receptor synaptic targeting. In the absence of GluN2B, the synaptic levels of AMPA receptors are increased and accompanied by decreased constitutive endocytosis of GluA1-AMPA receptor. We used quantitative proteomic analysis to identify changes in the composition of postsynaptic densities from GluN2B−/− mouse primary neuronal cultures and found altered levels of several ubiquitin proteasome system components, in particular decreased levels of proteasome subunits. Enhancing the proteasome activity with a novel proteasome activator restored the synaptic levels of AMPA receptors in GluN2B−/− neurons and their endocytosis, revealing that GluN2B-mediated anchoring of the synaptic proteasome is responsible for fine tuning AMPA receptor synaptic levels under basal conditions.

Contactin-associated Protein 1 (Caspr1) Regulates the Traffic and Synaptic Content of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA)-type Glutamate Receptors

Background: The strength of excitatory synapses is determined by the synaptic content of AMPA receptors. Results: We found that Contactin associated protein 1 (Caspr1) binds to AMPA receptors and regulates their neuronal cell surface and synaptic expression. Conclusion: Caspr1 is a binding partner for AMPA receptors, which regulates their traffic and synaptic targeting. Significance: Caspr1 is a new player in plasticity mechanisms in excitatory synapses. Glutamate receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type mediate fast excitatory synaptic transmission in the CNS. Synaptic strength is modulated by AMPA receptor binding partners, which regulate receptor synaptic targeting and functional properties. We identify Contactin-associated protein 1 (Caspr1) as an AMPA receptor interactor. Caspr1 is present in synapses and interacts with AMPA receptors in brain synaptic fractions. Coexpression of Caspr1 with GluA1 increases the amplitude of glutamate-evoked currents. Caspr1 overexpression in hippocampal neurons increases the number and size of synaptic GluA1 clusters, whereas knockdown of Caspr1 decreases the intensity of synaptic GluA1 clusters. Hence, Caspr1 is a regulator of the trafficking of AMPA receptors to synapses.

Glutamate Binding to the GluN2B Subunit Controls Surface Trafficking of N-Methyl-d-aspartate (NMDA) Receptors

Background: Ligand binding is essential for surface delivery of non-NMDA-type glutamate receptors. Results: GluN2B ligand binding site mutants showed reduced surface and synaptic expression correlating with glutamate efficacy. Conclusion: Glutamate binding controls surface delivery of NMDA receptors. Significance: This work extends the receptor classes subject to glutamate regulation of surface delivery and reports parameters controlling synaptic delivery of NMDA receptors critical for neuron function. Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity-dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. Here, we show that glutamate binding to GluN2 is required for trafficking of NMDA receptors to the cell surface. We expressed a panel of GluN2B ligand binding mutants in heterologous cells with GluN1 or in rat cultured neurons and found that surface expression correlates with glutamate efficacy. Such a correlation was found even in the presence of dominant negative dynamin to inhibit endocytosis and surface expression correlated with Golgi localization, indicating differences in forward trafficking. Co-expression of wild type GluN2B did not enhance surface expression of the mutants, suggesting that glutamate must bind to both GluN2 subunits in a tetramer and that surface expression is limited by the least avid of the two glutamate binding sites. Surface trafficking of a constitutively closed cleft GluN2B was indistinguishable from that of wild type, suggesting that glutamate concentrations are typically not limiting for forward trafficking. YFP-GluN2B expressed in hippocampal neurons from GluN2B−/− mice rescued synaptic accumulation at similar levels to wild type. Under these conditions, surface synaptic accumulation of YFP-GluN2B mutants also correlated with apparent glutamate affinity. Altogether, these results indicate that glutamate controls forward trafficking of NMDA receptors to the cell surface and to synapses and raise the intriguing idea that NMDA receptors may be functional at intracellular sites.

Characterization of alternatively spliced isoforms of AMPA receptor subunits encoding truncated receptors

Glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type play an important role in synaptic plasticity and contribute to cell death under excitotoxic conditions. AMPA receptors form heterotetramers of four homologous subunits (GluR1-4), which exist in two functionally different isoforms, flip and flop, generated by alternative splicing. We identified transcripts for alternatively spliced isoforms of AMPA receptor subunits which lack both the flip and the flop exons, in hippocampal and retinal cultures. These transcripts originate AMPA receptor subunits lacking the flip/flop cassette, the fourth transmembrane domain and the intracellular C-terminus. Truncated GluR1 associates with full-length GluR1 and exerts a dominant negative effect, giving rise to non-functional receptors. Moreover, truncated GluR1 reaches the cell surface, but is not efficiently targeted to the synapse. Hippocampal neuronal transfection with truncated GluR1 resulted in a significant reduction in apoptotic neuronal death triggered by toxic concentrations of glutamate. Furthermore, mRNA coding for the truncated subunits is consistently detected in some regions of the brain in epileptic rats and in hippocampal neurons submitted to toxic concentrations of glutamate. The existence of truncated AMPA receptor subunits may constitute an intrinsic neuroprotective mechanism.

Activity and protein kinase C regulate synaptic accumulation of NMDA receptors independently of GluN1 splice variant

NMDA receptors are calcium-permeable ionotropic receptors that detect coincident glutamate binding and membrane depolarization and are essential for many forms of synaptic plasticity in the mammalian brain. The obligatory GluN1 subunit of NMDA receptors is alternatively spliced at multiple sites, generating forms that vary in N-terminal N1 and C-terminal C1, C2, and C2′ cassettes. Based on expression of GluN1 constructs in heterologous cells and in wild type neurons, the prevalent view is that the C-terminal cassettes regulate synaptic accumulation and its modulation by homeostatic activity blockade and by protein kinase C (PKC). Here, we tested the role of GluN1 splicing in regulated synaptic accumulation of NMDA receptors by lentiviral expression of individual GluN1 splice variants in hippocampal neurons cultured from GluN1 (−/−) mice. High efficiency transduction of GluN1 at levels similar to endogenous was achieved. Under control conditions, the C2′ cassette mediated enhanced synaptic accumulation relative to the alternate C2 cassette, whereas the presence or absence of N1 or C1 had no effect. Surprisingly all GluN1 splice variants showed >2-fold increased synaptic accumulation with chronic blockade of NMDA receptor activity. Furthermore, in this neuronal rescue system, all GluN1 splice variants were equally rapidly dispersed upon activation of PKC. These results indicate that the major mechanisms mediating homeostatic synaptic accumulation and PKC dispersal of NMDA receptors occur independently of GluN1 splice isoform.

Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

The subunit composition of synaptic NMDA receptors (NMDAR), such as the relative content of GluN2A- and GluN2B-containing receptors, greatly influences the glutamate synaptic transmission. Receptor co-agonists, glycine and D-serine, have intriguingly emerged as potential regulators of the receptor trafficking in addition to their requirement for its activation. Using a combination of single-molecule imaging, biochemistry and electrophysiology, we show that glycine and D-serine relative availability at rat hippocampal glutamatergic synapses regulate the trafficking and synaptic content of NMDAR subtypes. Acute manipulations of co-agonist levels, both ex vivo and in vitro, unveil that D-serine alter the membrane dynamics and content of GluN2B-NMDAR, but not GluN2A-NMDAR, at synapses through a process requiring PDZ binding scaffold partners. In addition, using FRET-based FLIM approach, we demonstrate that D-serine rapidly induces a conformational change of the GluN1 subunit intracellular C-terminus domain. Together our data fuels the view that the extracellular microenvironment regulates synaptic NMDAR signaling. DOI: http://dx.doi.org/10.7554/eLife.25492.001

Multiple domains in the C-terminus of NMDA receptor GluN2B subunit contribute to neuronal death following in vitro ischemia.

Global cerebral ischemia induces selective degeneration of specific subsets of neurons throughout the brain, particularly in the hippocampus and cortex. One of the major hallmarks of cerebral ischemia is excitotoxicity, characterized by overactivation of glutamate receptors leading to intracellular Ca(2+) overload and ultimately neuronal demise. N-methyl-d-aspartate receptors (NMDARs) are considered to be largely responsible for excitotoxic injury due to their high Ca(2+) permeability. In the hippocampus and cortex, these receptors are most prominently composed of combinations of two GluN1 subunits and two GluN2A and/or GluN2B subunits. Due to the controversy regarding the differential role of GluN2A and GluN2B subunits in excitotoxic cell death, we investigated the role of GluN2B in the activation of pro-death signaling following an in vitro model of global ischemia, oxygen and glucose deprivation (OGD). For this purpose, we used GluN2B(-/-) mouse cortical cultures and observed that OGD-induced damage was reduced in these neurons, and partially prevented in wild-type rat neurons by a selective GluN2B antagonist. Notably, we found a crucial role of the C-terminal domain of the GluN2B subunit in triggering excitotoxic signaling. Indeed, expression of YFP-GluN2B C-terminus mutants for the binding sites to post-synaptic density protein 95 (PSD95), Ca(2+)-calmodulin kinase IIα (CaMKIIα) or clathrin adaptor protein 2 (AP2) failed to mediate neuronal death in OGD conditions. We focused on the GluN2B-CaMKIIα interaction and found a determinant role of this interaction in OGD-induced death. Inhibition or knock-down of CaMKIIα exerted a neuroprotective effect against OGD-induced death, whereas overexpression of this kinase had a detrimental effect. Importantly, in comparison with neurons overexpressing wild-type CaMKIIα, neurons overexpressing a mutant form of the kinase (CaMKII-I205K), unable to interact with GluN2B, were partially protected against OGD-induced damage. Taken together, our results identify crucial determinants in the C-terminal domain of GluN2B subunits in promoting neuronal death in ischemic conditions. These mechanisms underlie the divergent roles of the GluN2A- and GluN2B-NMDARs in determining neuronal fate in cerebral ischemia.

Activity and Protein Kinase C Regulate Synaptic Accumulation of N-Methyl-D-aspartate (NMDA) Receptors Independently of GluN1 Splice Variant

NMDA receptors are calcium-permeable ionotropic receptors that detect coincident glutamate binding and membrane depolarization and are essential for many forms of synaptic plasticity in the mammalian brain. The obligatory GluN1 subunit of NMDA receptors is alternatively spliced at multiple sites, generating forms that vary in N-terminal N1 and C-terminal C1, C2, and C2′ cassettes. Based on expression of GluN1 constructs in heterologous cells and in wild type neurons, the prevalent view is that the C-terminal cassettes regulate synaptic accumulation and its modulation by homeostatic activity blockade and by protein kinase C (PKC). Here, we tested the role of GluN1 splicing in regulated synaptic accumulation of NMDA receptors by lentiviral expression of individual GluN1 splice variants in hippocampal neurons cultured from GluN1 (−/−) mice. High efficiency transduction of GluN1 at levels similar to endogenous was achieved. Under control conditions, the C2′ cassette mediated enhanced synaptic accumulation relative to the alternate C2 cassette, whereas the presence or absence of N1 or C1 had no effect. Surprisingly all GluN1 splice variants showed >2-fold increased synaptic accumulation with chronic blockade of NMDA receptor activity. Furthermore, in this neuronal rescue system, all GluN1 splice variants were equally rapidly dispersed upon activation of PKC. These results indicate that the major mechanisms mediating homeostatic synaptic accumulation and PKC dispersal of NMDA receptors occur independently of GluN1 splice isoform.

Single nanoparticle tracking of N-methyl-d-aspartate receptors in cultured and intact brain tissue

Abstract. Recent developments in single-molecule imaging have revealed many biological mechanisms, providing high spatial and temporal resolution maps of molecular events. In neurobiology, these techniques unveiled that plasma membrane neurotransmitter receptors and transporters laterally diffuse at the surface of cultured brain cells. The photostability of bright nanoprobes, such as quantum dots (QDs), has given access to neurotransmitter receptor tracking over long periods of time with a high spatial resolution. However, our knowledge has been restricted to cultured systems, i.e., neurons and organotypic slices, therefore lacking several aspects of the intact brain rheology and connectivity. Here, we used QDs to track single glutamatergic N-methyl-d-aspartate receptors (NMDAR) in acute brain slices. By delivering functionalized nanoparticles in vivo through intraventricular injections to rats expressing genetically engineered-tagged NMDAR, we successfully tracked the receptors in native brain tissue. Comparing NMDAR tracking to different classical brain preparations (acute brain slices, cultured organotypic brain slices, and cultured neurons) revealed that the surface diffusion properties shared several features and are also influenced by the nature of the extracellular environment. Together, we describe the experimental procedures to track plasma membrane NMDAR in dissociated and native brain tissue, paving the way for investigations aiming at characterizing receptor diffusion biophysics in intact tissue and exploring the physiopathological roles of receptor surface dynamics.

Differential Nanoscale Topography and Functional Role of GluN2-NMDA Receptor Subtypes at Glutamatergic Synapses

NMDA receptors (NMDARs) play key roles in the use-dependent adaptation of glutamatergic synapses underpinning memory formation. In the forebrain, these plastic processes involve the varied contributions of GluN2A- and GluN2B-containing NMDARs that have different signaling properties. Although the molecular machinery of synaptic NMDAR trafficking has been under scrutiny, the postsynaptic spatial organization of these two receptor subtypes has remained elusive. Here, we used super-resolution imaging of NMDARs in rat hippocampal synapses to unveil the nanoscale topography of native GluN2A- and GluN2B-NMDARs. Both subtypes were found to be organized in separate nanodomains that vary over the course of development. Furthermore, GluN2A- and GluN2B-NMDAR nanoscale organizations relied on distinct regulatory mechanisms. Strikingly, the selective rearrangement of GluN2A- andxa0GluN2B-NMDARs, with noxa0overall change inxa0NMDAR current amplitude, allowed bi-directional tuning of synaptic LTP. Thus,xa0GluN2A- andxa0GluN2B-NMDAR nanoscale organizations arexa0differentially regulated and seem to involve distinct signaling complexes during synaptic adaptation.