NMDA Receptor Trafficking Research Articles

Regulation of NMDA glutamate receptor functions by the GluN2 subunits.

The N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate the flux of calcium (Ca2+ ) into the post-synaptic compartment. Ca2+ influx subsequently triggers the activation of various intracellular signalling cascades that underpin multiple forms of synaptic plasticity. Functional NMDARs are assembled as heterotetramers composed of two obligatory GluN1 subunits and two GluN2 or GluN3 subunits. Four different GluN2 subunits (GluN2A-D) are present throughout the central nervous system; however, they are differentially expressed, both developmentally and spatially, in a cell- and synapse-specific manner. Each GluN2 subunit confers NMDARs with distinct ion channel properties and intracellular trafficking pathways. Regulated membrane trafficking of NMDARs is a dynamic process that ultimately determines the number of NMDARs at synapses, and is controlled by subunit-specific interactions with various intracellular regulatory proteins. Here we review recent progress made towards understanding the molecular mechanisms that regulate the trafficking of GluN2-containing NMDARs, focusing on the roles of several key synaptic proteins that interact with NMDARs via their carboxyl termini.

An autism-associated mutation in GluN2B prevents NMDA receptor trafficking and interferes with dendrite growth.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders with multiple genetic associations. Analysis of de novo mutations identified GRIN2B, which encodes the GluN2B subunit of NMDA receptors, as a gene linked to ASDs with high probability. However, the mechanisms by which GRIN2B mutations contribute to ASD pathophysiology are not understood. Here, we investigated the cellular phenotypes induced by a human mutation that is predicted to truncate GluN2B within the extracellular loop. This mutation abolished NMDA-dependent Ca2+ influx. Mutant GluN2B co-assembled with GluN1 but was not trafficked to the cell surface or dendrites. When mutant GluN2B was expressed in developing cortical neurons, dendrites appeared underdeveloped, with shorter and fewer branches, while spine density was unaffected. Mutant dendritic arbors were often dysmorphic, displaying abnormal filopodial-like structures. Interestingly, dendrite maldevelopment appeared when mutant GluN2B was expressed on a wild-type background, reflecting the disease given that individuals are heterozygous for GRIN2B mutations. Restoring the fourth transmembrane domain and cytoplasmic tail did not rescue the phenotypes. Finally, abnormal development was not accompanied by reduced mTOR signaling. These data suggest that mutations in GluN2B contribute to ASD pathogenesis by disrupting dendrite development.

N-glycosylation regulates the trafficking, surface mobility and function of GluN3A-containing NMDA receptors

N-glycosylation regulates the trafficking, surface mobility and function of GluN3A-containing NMDA receptors

TRPV1 channel contributes to remifentanil-induced postoperative hyperalgesia via regulation of NMDA receptor trafficking in dorsal root ganglion.

BackgroundRemifentanil is widely used in general anesthesia due to its reliability and rapid onset. However, remifentanil-induced postoperative hyperalgesia might be a challenge nowadays. Accumulating evidence suggests that the transient receptor potential vanilloid 1 (TRPV1) was involved in the development of neuropathic pain and hyperalgesia. However, the contribution of TRPV1 in modulating remifentanil-induced postoperative hyperalgesia is still unknown. The aim of this study is the contribution of TRPV1 to the surface expression of N-methyl-d-aspartate (NMDA) receptors in remifentanil-induced postoperative hyperalgesia.MethodsThe hot plate test and the Von Frey test were performed to evaluate thermal and mechanical hyperalgesia. Capsazepine (CPZ) was administrated intrathecally to confirm our results. TRPV1, NMDA receptors, CaMKII (calcium/calmodulin-dependent kinase II), and protein kinase C (PKC) in the dorsal root ganglion (DRG) were detected by Western blotting. Immunofluorescence assay was applied to analyze the distribution of TRPV1 and the relationship between TRPV1 and NMDA receptor subunit 1 (NR1).ResultsRemifentanil-induced both thermal and mechanical postoperative hyperalgesia. Here, we found the membrane trafficking of NR1, possibly due to the activation of TRPV1 in DRG neurons after remifentanil infusion. Furthermore, intrathecal injection of CPZ was able to relieve remifentanil-induced postoperative hyperalgesia according to a behavioral test and CPZ confirmed that TRPV1 is involved in NR1 trafficking. In addition, CaMKII/PKC but not protein kinase A (PKA) contributed to remifentanil-induced postoperative hyperalgesia.ConclusionOur study demonstrates that TRPV1 receptors are involved in remifentanil-induced postoperative hyperalgesia. TRPV1 contributes to the persistence of remifentanil-induced postoperative hyperalgesia through the trafficking of NMDA receptors via the activation of CaMKII-PKC signaling pathways in DRG neurons.

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- andGluN2B-NMDARs, with nooverall change inNMDAR current amplitude, allowed bi-directional tuning of synaptic LTP. Thus,GluN2A- andGluN2B-NMDAR nanoscale organizations aredifferentially regulated and seem to involve distinct signaling complexes during synaptic adaptation.

Parkinson\u2019s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

BackgroundParkinson’s disease (PD)-associated E3 ubiquitin ligase Parkin is enriched at glutamatergic synapses, where it ubiquitinates multiple substrates, suggesting that its mutation/loss-of-function could contribute to the etiology of PD by disrupting excitatory neurotransmission. Here, we evaluate the impact of four common PD-associated Parkin point mutations (T240M, R275W, R334C, G430D) on glutamatergic synaptic function in hippocampal neurons.ResultsWe find that expression of these point mutants in cultured hippocampal neurons from Parkin-deficient and Parkin-null backgrounds alters NMDA and AMPA receptor-mediated currents and cell-surface levels and prevents the induction of long-term depression. Mechanistically, we demonstrate that Parkin regulates NMDA receptor trafficking through its ubiquitination of GluN1, and that all four mutants are impaired in this ubiquitinating activity. Furthermore, Parkin regulates synaptic AMPA receptor trafficking via its binding and retention of the postsynaptic scaffold Homer1, and all mutants are similarly impaired in this capacity.ConclusionOur findings demonstrate that pathogenic Parkin mutations disrupt glutamatergic synaptic transmission in hippocampal neurons by impeding NMDA and AMPA receptor trafficking. Such effects may contribute to the pathophysiology of PD in PARK2 patients.

Postsynaptic RIM1 modulates synaptic function by facilitating membrane delivery of recycling NMDARs in hippocampal neurons

NMDA receptors (NMDARs) are crucial for excitatory synaptic transmission and synaptic plasticity. The number and subunit composition of synaptic NMDARs are tightly controlled by neuronal activity and sensory experience, but the molecular mechanism mediating NMDAR trafficking remains poorly understood. Here, we report that RIM1, with a well-established role in presynaptic vesicle release, also localizes postsynaptically in the mouse hippocampus. Postsynaptic RIM1 in hippocampal CA1 region is required for basal NMDAR-, but not AMPA receptor (AMPAR)-, mediated synaptic responses, and contributes to synaptic plasticity and hippocampus-dependent memory. Moreover, RIM1 levels in hippocampal neurons influence both the constitutive and regulated NMDAR trafficking, without affecting constitutive AMPAR trafficking. We further demonstrate that RIM1 binds to Rab11 via its N terminus, and knockdown of RIM1 impairs membrane insertion of Rab11-positive recycling endosomes containing NMDARs. Together, these results identify a RIM1-dependent mechanism critical for modulating synaptic function by facilitating membrane delivery of recycling NMDARs.

N-Glycosylation Regulates the Trafficking and Surface Mobility of GluN3A-Containing NMDA Receptors.

N-methyl-D-aspartate receptors (NMDARs) play critical roles in both excitatory neurotransmission and synaptic plasticity. NMDARs containing the nonconventional GluN3A subunit have different functional properties compared to receptors comprised of GluN1/GluN2 subunits. Previous studies showed that GluN1/GluN2 receptors are regulated by N-glycosylation; however, limited information is available regarding the role of N-glycosylation in GluN3A-containing NMDARs. Using a combination of microscopy, biochemistry, and electrophysiology in mammalian cell lines and rat hippocampal neurons, we found that two asparagine residues (N203 and N368) in the GluN1 subunit and three asparagine residues (N145, N264 and N275) in the GluN3A subunit are required for surface delivery of GluN3A-containing NMDARs. Furthermore, deglycosylation and lectin-based analysis revealed that GluN3A subunits contain extensively modified N-glycan structures, including hybrid/complex forms of N-glycans. We also found (either using a panel of inhibitors or by studying human fibroblasts derived from patients with a congenital disorder of glycosylation) that N-glycan remodeling is not required for the surface delivery of GluN3A-containing NMDARs. Finally, we found that the surface mobility of GluN3A-containing NMDARs in hippocampal neurons is increased following incubation with 1-deoxymannojirimycin (DMM, an inhibitor of the formation of the hybrid/complex forms of N-glycans) and decreased in the presence of specific lectins. These findings provide new insight regarding the mechanisms by which neurons can regulate NMDAR trafficking and function.

Abstract P2-04-02: Comparison of DNA methylation patterns in normal breast tissue from women with and without breast cancer

Abstract BACKGROUND: Increasing evidence suggests that epigenetic mechanisms play critical roles in the development of breast cancer. However, precise DNA methylation signatures associated with breast cancer susceptibility remain unknown. We sought to compare DNA methylation changes in the normal breast tissue of women with and without breast cancer to identify patterns of aberrant DNA methylation in women with breast cancer. METHODS:Samples of normal breast tissue were collected from four cohorts of women: age < 50 years with and without breast cancer, and age ≥50 years with and without breast cancer. Normal breast tissue from healthy women was obtained from the Komen Tissue Bank at IU Simon Cancer Center and from women presenting for reduction mammoplasty at Yale New Haven Hospital. Normal breast tissue from women with breast cancer was obtained from patients undergoing adjuvant total mastectomy at Yale Breast Center. DNA was extracted using Qiagen AllPrep Universal kit. Raw data files in idat format were imported to Partek Genomics Suite 6.6 for normalization and differential methylation analysis. Raw intensities were normalized using With Array Normalization (SWAN) method. Principal component analysis (PCA) were performed as quality control. Differentially methylated loci (DML) between control and breast cancer groups were detected when False discovery rate (FDR) < 0.05 and fold change > 1.5. Functional enrichment analysis of genes with DML in the gene body were conducted using METACORE™. Pathways with FDR < 0.05 were selected. RESULTS: Ninety-three normal breast tissue samples from 89 subjects were analyzed (breast cancer=40, unaffected=53). Comparison of DNA methylation patterns between women with and without breast cancer revealed 200 DMLs. The majority of DMLs (186) were hyper-methylated in breast cancer patients, and 48 DMLs locate in enhancers of genes. 170 DMLs locate in 134 genes, enriched in two pathways: (1) Cell adhesion_Endothelial cell contacts by junctional mechanisms, and (2) Neurophysiological process_Constitutive and regulated NMDA receptor trafficking. Genes associated with cell adhesion and cell contacts included: ACTN2, GJA4, GJA7 and MAGI1. Two hyper-methylated loci were found in enhancers of ACTN2. In addition, one hyper-methylated locus in GJA4, one hyper-methylated and one hypo-methylated loci in GJA7, and two hyper-methylated loci in MAGI1 were detected in breast cancer patients. Genes associated with NMDA receptor trafficking include: TPK1, ADCY4 and LIN7C. One and two loci were found in TPK1 and ADCY4, respectively, that were hyper-methylated in normal breast tissue from cancer patients in the gene body, while a hypo-methylated locus in breast cancer patients was identified in LIN7C. CONCLUSIONS: Comparison of DNA methylation patterns of normal breast tissue from women with and without breast cancer reveal specific mechanistic pathways and genes that are differentially methylated in women with breast cancer. DNA methylation of normal breast tissue deserves further study as a potential biomarker for breast cancer risk stratification and may lend new insight into mechanisms of breast cancer development. Citation Format: Hofstatter EW, Zhu Y, Horvath S, Chagpar AB, Wali VB, Bossuyt V, Storniolo AM, Hatzis C, Patwardhan G, Von Wahlde M-K, Butler M, Epstein L, Stavris K, Sturrock T, Au A, Kwei S, Pusztai L. Comparison of DNA methylation patterns in normal breast tissue from women with and without breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-04-02.

Role of α7nAChR-NMDAR in sevoflurane-induced memory deficits in the developing rat hippocampus.

Detrimental effects of volatile anaesthetics, including sevoflurane, on the structure and function of the developing brain have been reported. The internalization of N-methyl-D-aspartate receptors (NMDARs) contributes to anaesthetic neurotoxicity. Both nicotinic acetylcholine receptors (nAChRs) and NMDAR play a critical role in the development of the nervous system. Moreover, nAChR can interact with NMDAR, and previous studies have demonstrated modulation of NMDAR by nAChR. In our study, we used an α7 nicotinic acetylcholine receptor (α7nAChR) agonist and α7nAChR antagonist to explore the role of α7nAChR and NMDAR in sevoflurane-induced long-term effects on memory and dendritic spine both in vivo and in vitro. The results revealed that the activation of α7nAChR attenuated the development of sevoflurane-induced memory deficit and dendritic spine changes, which might be by regulating NR2B-containing NMDAR trafficking from the intracellular pool to the cell surface pool in the hippocampus. Moreover, we demonstrated that α7nAChR could regulate NR2B-containing NMDAR via Src-family tyrosine kinase (SFK). Thus, our current study indicates that the trafficking of NR2B-containing NMDAR is regulated by α7nAChR via SFK in neonatal rat hippocampus, which may be secondary to sevoflurane-induced cognitive deficits in the developing hippocampus.

Acute Amino Acid d-Serine Administration, Similar to Ketamine, Produces Antidepressant-like Effects through Identical Mechanisms

d-Serine is an amino acid and can work as an agonist at the glycine sites of N-methyl-d-aspartate receptor (NMDAR). Interestingly, both types of glutamatergic modulators, NMDAR enhancers and blockers, can improve depression through common targets, namely alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionaic acid receptors (AMPARs) and mammalian target of rapamycin (mTOR). To elucidate the cellular signaling pathway underlying this counterintuitive observation, we activated NMDARs in rats by using d-serine. Saline, ketamine (NMDAR antagonist), and desipramine (tricyclic antidepressant) were used as controls. The antidepressant-like effects of all agents were evaluated using the forced swim test. The activation of the AMPAR-mTOR signaling pathway, release of brain-derived neurotrophic factor (BDNF), and alteration of AMPAR and NMDAR trafficking in the hippocampus of rats were examined. A single high dose of d-serine exerted an antidepressant-like effect that was mediated by rapid AMPAR-induced mTOR signaling pathway and increased BDNF proteins, identical to that of ketamine. Furthermore, in addition to the increased protein kinase A phosphorylation of the AMPAR subunit GluR1 (an indicator of AMPAR insertion in neurons), treatment with individual optimal doses of d-serine and ketamine also increased adaptin β2-NMDAR association (an indicator of the intracellular endocytic machinery and subsequent internalization of NMDARs). Desipramine did not influence these processes. Our study is the first to demonstrate an association between d-serine and ketamine; following adaptative regulation of AMPAR and NMDAR may lead to common changes of them. These findings provide novel targets for safer antidepressant agents with mechanisms similar to those of ketamine.

POSH participates in epileptogenesis by increasing the surface expression of the NMDA receptor: a promising therapeutic target for epilepsy

ABSTRACTObjectives: Plenty of SH3 (POSH) was originally found to be a key regulator of neuronal apoptosis, axon outgrowth, and neuronal migration. However, the role of POSH in epilepsy has not been reported.Methods: We investigated the expression of POSH in patients with intractable temporal epilepsy (TLE) and in a kainic acid (KA)-induced mouse model, and then we performed behavioral, electrophysiological and biochemical analyses after the lentivirus (LV)-mediated knockdown or overexpression of POSH in the KA-induced model.Results: POSH overexpression shortened the latency of seizure onset, increased the frequency of spontaneous recurrent seizures, and increased the frequency of electrical epileptic discharges, while POSH knockdown had contrasting effects. Whole-cell patch-clamp recordings confirmed that POSH overexpression and knockdown were associated with increased and decreased miniature excitatory postsynaptic currents (mEPSCs) and N-methyl-D-aspartate receptor (NMDAR)-mediated currents, respectively. Finally, co-immunoprecipitation showed that POSH and NMDA receptor subunit 1 (NMDAR1) precipitated with each other, and western blot analysis revealed that the surface expression of NMDAR1 was altered in the hippocampus of epileptic mice.Conclusion: These results show that POSH plays a critical role in the progression of epileptic seizures via NMDAR trafficking and suggest that the protein is a novel target for the treatment of human epilepsy.

A Wnt/Calcium Signaling Cascade Regulates Neuronal Excitability and Trafficking of NMDARs

Wnt signaling controls multiple biological process, particularly the embryonic development of metazoans. Sustained expression of Wnt signaling components in the mature mammalian CNS and their apparent deregulation in certain neuropathologies suggest that it also plays a part beyond embryonic development to regulate normal brain function. Wedescribe a noncanonical Wnt/Ca2+ signaling cascade that regulates the electrophysiological intrinsic properties of rat neurons, resulting in sustained membrane depolarization and the mobilization of Ca2+ from internal stores. These effects require tyrosine kinase-like orphan receptor 2 (RoR2), activation of PLC, and voltage-gated Ca2+ channels. Activation of this signaling cascade then promotes surface expression of N-methyl-D-aspartate receptors (NMDARs) through a SNARE-dependent mechanism. This neuronal Wnt/Ca2+ signaling pathway represents a mechanism for Wnt ligands to regulate normal brain processes in the mature animal and provides a framework for understanding how alterations in this pathway may contribute to the etiology of psychiatric disorders where NMDARs are compromised.

Trafficking patterns of NMDA and GABAA receptors in a Mg2+-free cultured hippocampal neuron model of status epilepticus

An altered pattern of receptor trafficking is one of the pathophysiologic mechanisms of status epilepticus (SE). The gradual internalization of GABAA receptors (GABARs) occurs in both in vitro and in vivo models of SE and is thought to be a cause of decreased GABAergic inhibition. Unlike GABARs, little is known about alterations in NMDA receptor (NMDAR) trafficking during SE, even though increased activity of NMDARs is indispensable for the induction and maintenance of SE. Therefore, we aimed to simultaneously investigate the changes in the trafficking patterns of GABARs and NMDARs in an in vitro cultured hippocampal neuron model of SE. For induction of epileptiform discharges, hippocampal neurons were exposed to external medium without Mg2+. Biotinylation assay and immunofluorescence staining for GABAR β2,3 and NMDAR NR1 subunits were performed to quantify and visualize surface GABARs and NMDARs, respectively. The frequency of spontaneous action potentials increased more than 4-fold after Mg2+-free induction. The level of surface GABARs decreased over time after Mg2+-free induction, dropping to approximately 50% of control levels an hour after Mg2+-free induction. By contrast, the trafficking of NMDARs to the surface was enhanced after a slight time lag, increasing by 30% of control levels an hour after Mg2+-free induction. Our data showed the changes of both NMDAR and GABAR trafficking during prolonged SE induced by a Mg2+-free extracellular environment and confirmed that this in vitro SE model is suitable for examining alterations in the receptor trafficking pattern by prolonged seizure activity. These results suggest that targeting of surface NMDAR could be a promising method in controlling benzodiazepine-resistant SE.

Simultaneous triple therapy for the treatment of status epilepticus

Early maladaptive internalization of synaptic GABAA receptors (GABAAR) and externalization of NMDA receptors (NMDAR) may explain the time-dependent loss of potency of standard anti-epileptic drugs (AED) in refractory status epilepticus (SE). We hypothesized that correcting the effects of changes in GABAAR and NMDAR would terminate SE, even when treatment is delayed 40 minutes. SE was induced in adult Sprague-Dawley rats with a high dose of lithium and pilocarpine. The GABAAR agonist midazolam, the NMDAR antagonist ketamine and the AED valproate were injected 40 min after SE onset in combination or as monotherapy. The midazolam-ketamine-valproate combination was more efficient than triple-dose midazolam, ketamine or valproate monotherapy or higher-dose dual therapy in reducing several parameters of SE severity. Triple therapy also reduced SE-induced acute neuronal injury and spatial memory deficits. In addition, simultaneous triple therapy was more efficient than sequential triple therapy: giving the three drugs simultaneously was more efficient at stopping seizures than the standard practice of giving them sequentially. Furthermore, midazolam-ketamine-valproate therapy suppressed seizures far better than the midazolam-fosphenytoin-valproate therapy, which follows evidence-based AES guidelines. These results show that a treatment aimed at correcting maladaptive GABAAR and NMDAR trafficking can reduce the severity of SE and its long-term consequences.

A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that are crucial for neuronal development and higher cognitive processes. NMDAR dysfunction is involved in a variety of neurological and psychiatric diseases; however, the mechanistic link between the human pathology and NMDAR dysfunction is poorly understood. Rare missense variants within NMDAR subunits have been identified in numerous patients with mental or neurological disorders. We specifically focused on the GluN2B NMDAR subunit, which is highly expressed in the hippocampus and cortex throughout development. We analyzed several variants located in the GluN2B C terminus and found that three variants in patients with autism (S1415L) or schizophrenia (L1424F and S1452F) (S1413L, L1422F, and S1450F in rodents, respectively) displayed impaired binding to membrane-associated guanylate kinase (MAGUK) proteins. In addition, we observed a deficit in surface expression for GluN2B S1413L. Furthermore, there were fewer dendritic spines in GluN2B S1413L-expressing neurons. Importantly, synaptic NMDAR currents in neurons transfected with GluN2B S1413L in GluN2A/B-deficient mouse brain slices revealed only partial rescue of synaptic current amplitude. Functional properties of GluN2B S1413L in recombinant systems revealed no change in receptor properties, consistent with synaptic defects being the result of reduced trafficking and targeting of GluN2B S1413L to the synapse. Therefore, we find that GluN2B S1413L displays deficits in NMDAR trafficking, synaptic currents, and spine density, raising the possibility that this mutation may contribute to the phenotype in this autism patient. More broadly, our research demonstrates that the targeted study of certain residues in NMDARs based on rare variants identified in patients is a powerful approach to studying receptor function.SIGNIFICANCE STATEMENT We have used a "bedside-to-bench" approach to investigate the functional regulation of NMDA receptors (NMDARs). Using information from deep sequencing of patients with neurological or psychiatric disorders, we investigated missense variants identified in the intracellular C-terminal domain of the GluN2B NMDAR subunit. We found several variants that displayed altered properties. In particular, one variant identified in a patient with autism, human GluN2B S1415L, displayed reduced surface expression and binding to PSD-95. Furthermore expression of GluN2B S1415L (S1413L in mouse) showed a deficit in rescue of synaptic NMDAR currents and fewer dendritic spines, consistent with other reports of spine abnormalities being associated with autism. More broadly, we demonstrate that using patient data is an effective approach to probing the structure/function relationship of NMDARs.

Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency \u2013 molecular profiling and functional rescue

Mutations in the N-methyl-D-aspartate receptor (NMDAR) gene GRIN2A cause epilepsy-aphasia syndrome (EAS), a spectrum of epileptic, cognitive and language disorders. Using bioinformatic and patient data we shortlisted 10 diverse missense mutations for characterisation. We used high-throughput calcium-flux assays and patch clamp recordings of transiently transfected HEK-293 cells for electrophysiological characterization, and Western blotting and confocal imaging to assay expression and surface trafficking. Mutations P79R, C231Y, G483R and M705V caused a significant reduction in glutamate and glycine agonist potency, whilst D731N was non-responsive. These mutants, along with E714K, also showed significantly decreased total protein levels and trafficking to the cell surface, whilst C436R was not trafficked at all. Crucially this reduced surface expression did not cause the reduced agonist response. We were able to rescue the phenotype of P79R, C231Y, G483R and M705V after treatment with a GluN2A-selective positive allosteric modulator. With our methodology we were not able to identify any functional deficits in mutations I814T, D933N and N976S located between the glutamate-binding domain and C-terminus. We show GRIN2A mutations affect the expression and function of the receptor in different ways. Careful molecular profiling of patients will be essential for future effective personalised treatment options.

Prevention of Remifentanil Induced Postoperative Hyperalgesia by Dexmedetomidine via Regulating the Trafficking and Function of Spinal NMDA Receptors as well as PKC and CaMKII Level In Vivo and In Vitro

Remifentanil-induced secondary hyperalgesia has been demonstrated in both animal experiments and clinical trials. Enhancement of N-methyl-D-aspartate (NMDA) receptor trafficking as well as protein kinase C (PKC) and calmodulin-dependent protein kinase II (CaMKII) have been reported to be involved in the induction and maintenance of central sensitization. In the current study, it was demonstrated that dexmedetomidine could prevent remifentanil-induced hyperalgesia (RIH) via regulating spinal NMDAR-PKC-Ca2+/ CaMKII pathway in vivo and in vitro. We firstly investigated the effect of dexmedetomidine, a highly selective α2-adrenergic receptor agonist, on mechanical and thermal hyperalgesia using a rat model of RIH. NMDA receptor subunits (NR1, NR2A and NR2B) expression and membrane trafficking as well as PKC and CaMKII expression in spinal cord L4–L5 segments were measured by Western blot analysis. The expression of NMDA receptor subunits (NR1, NR2A and NR2B) were also detected by immunohistochemistry. Further more, the effect of dexmedetomidine on NMDA receptor current amplitude and frequency in spinal cord slices were investigated by whole-cell patch-clamp recording. We found that remifentail infusion at 1.2 μg.kg−1.min−1 for 90 min caused mechanical and thermal hyperalgesia, up-regulated NMDA receptor subunits NR1 and NR2B expression in both membrane fraction and total lysate as well as increased PKC and CaMKII expression in spinal cord dorsal horn. Subcutaneously injection of dexmedetomidine at the dose of 50 μg/kg at 30 min before plantar incision significantly attenuated remifentanil-induced mechanical and thermal hyperalgesia from 2 h to 48 h after infusion, and this was associated with reversal of up-regulated NR1 and NR2B subunits in both membrane fraction and total lysate as well as increased PKC and CaMKII expression in spinal cord dorsal horn. Furthermore, remifentanil incubation increased amplitude and frequency of NMDA receptor-induced current in dorsal horn neurons, which was dose-dependently attenuated by dexmedetomidine. These results suggest that dexmedetomidine can significantly ameliorate RIH via modulating the expression, membrane trafficking and function of NMDA receptors as well as PKC and CaMKII level in spinal dorsal horn, which present useful insights into the mechanistic action of dexmedetomidine as a potential anti-hyperalgesic agents for treating RIH.

Making of a Synapse: Recurrent Roles of Drebrin A at Excitatory Synapses Throughout Life.

Mature excitatory synapses are composed of more than 1500 proteins postsynaptically and hundreds more that operate presynaptically. Among them, drebrin is an F-actin-binding protein that increases noticeably during juvenile synaptogenesis. Electron microscopic analysis reveals that drebrin is highly enriched specifically on the postsynaptic side of excitatory synapses. Since dendritic spines are structures specialized for excitatory synaptic transmission, the function of drebrin was probed by analyzing the ultrastructural characteristics of dendritic spines of animals with genetic deletion of drebrin A (DAKO), the adult isoform of drebrin. Electron microscopic analyses revealed that these brains are surprisingly intact, in that axo-spinous synaptic junctions are well-formed and not significantly altered in number. This normal ultrastructure may be because drebrin E, the alternate embryonic isoform, compensates for the genetic deletion of drebrin A. However, DAKO results in the loss of homeostatic plasticity of N-methyl-D-aspartate receptors (NMDARs). The NMDAR activation-dependent trafficking of the NR2A subunit-containing NMDARs from dendritic shafts into spine head cytoplasm is greatly diminished within brains of DAKO. Conversely, within brains of wild-type rodents, spines respond to NMDAR blockade with influx of F-actin, drebrin A, and NR2A subunits of NMDARs. These observations indicate that drebrin A facilitates the trafficking of NMDAR cargos in an F-actin-dependent manner to mediate homeostatic plasticity. Analysis of the brains of transgenic mice used as models of Alzheimer's disease (AD) reveals that the loss of drebrin from dendritic spines predates the emergence of synaptic dysfunction and cognitive impairment, suggesting that this form of homeostatic plasticity contributes toward cognition. Two studies suggest that the nature of drebrin's interaction with NMDARs is dependent on the receptor's subunit composition. Drebrin A can be found co-clustering with NR2B-containing NMDARs at the plasma membrane, while NR2A-containing NMDARs co-traffic into the spine cytoplasm but do not co-cluster at the plasma membrane. Most recently, we encountered a physiological condition that supports this idea. When adolescent female rats are reared under a condition of restricted food access and ad libitum wheel access, they paradoxically become excessive runners, choosing to run, even during the limited hours of food availability. This behavioral pattern is termed activity-based anorexia (ABA) and has served as an animal model for anorexia nervosa. Those animals that exhibit the greatest ABA vulnerability, in that they lose the most amount of body weight and run with greatest exuberance to the point of risking their lives, exhibit the highest levels of NR2B-NMDARs and drebrin at the postsynaptic membrane of hippocampal pyramidal neurons. Those animals that exhibit the greatest resilience to ABA, in that they run minimally under such condition, thereby losing minimal amount of weight, exhibit the highest level of NR2A-NMDARs in the spine cytoplasm and lowest levels of drebrin at the postsynaptic membrane. This pattern suggests that drebrin has dual roles: retention of NR2A-NMDARs in the reserve pool and trafficking of NR2B-NMDARs to the postsynaptic membrane, ultimately contributing to an individual's reactivity to stress. Altogether, these observations indicate that drebrin is a protein that is important for synaptic plasticity and deserves the attention of neuroscientists studying the neurobiological basis of cognition and stress reactivity.

Blocking the Interaction between EphB2 and ADDLs by a Small Peptide Rescues Impaired Synaptic Plasticity and Memory Deficits in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is an age-dependent neurodegenerative disorder and amyloid β-derived diffusible ligands (ADDLs) play a key role in triggering the early cognitive deficits that constitute AD. ADDLs may bind EphB2 and alter NMDA receptor trafficking and synaptic plasticity. Here, we identified the interaction sites of the EphB2 FN domain with ADDLs for the first time to develop a small (10 aa) peptide (Pep63) capable of blocking the EphB2-ADDL interaction. We found that Pep63 not only rescued the ADDL-induced depletion of EphB2 and GluN2B-containing NMDA receptors from the neuronal surface in cultured hippocampal neurons, but also improved impaired memory deficits in APPswe/PS1dE9 (APP/PS1) transgenic mice. Our results suggest that blocking the EphB2-ADDL interaction with Pep63 may be a promising strategy for AD treatment.